abstracts · abucejo-ladesma e1, simoes e4, lupisan s2, sombrero l 2, quiambao b , lucero m2, arcay...

33ISPPD-4 (4th International Symposium on Pneumococci and Pneumococcal Diseases), Helsinki, Finland, May 9–13, 2004

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34 ISPPD-4 (4th International Symposium on Pneumococci and Pneumococcal Diseases), Helsinki, Finland, May 9–13, 2004

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The role of pneumococcal vaccination in preventing illness and death frompneumonia and achieving child survival goals

*El Arifeen, S

ICDDR,B: Centre for Heath and Population Research(Cholera Hospital)P.O. Box 128Dhaka [email protected]

Pneumococcal outbreaks

*Butler, J

Centers for Disease Control and Prevention4055 Tudor Centre DrAnchorage, Alaska 99508UNITED [email protected]


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Indirect Effect of Childhood Heptavalent Pneumococcal Conjugate Vaccina-tion on Adult Carriage of Streptococcus pneumoniae: A Community Study

Hammitt LL, Bruden D, Butler JC, Hurlburt D, Parkinson A, Baggett H, HennessyT, Arctic Investigations Program, Centers for Disease Control and Prevention,Anchorage, Alaska

Aims: In 2001, heptavalent pneumococcal conjugate vaccine (PCV7) was intro-duced in Alaska for children <5 years old. PCV7 use in children could affect trans-mission of Streptococcus pneumoniae (SP) to adults; this may account for declin-ing rates of adult disease. To determine changes in SP colonization, we conductedcommunity-wide nasopharyngeal (NP) carriage surveys.Methods: NP swabs were collected in Alaska villages (population: 3,900) annu-ally from 1998 through 2003. Swabs were plated onto gentamicin blood agar platesand incubated at 37º C. Pneumococcal isolates were confirmed by optochin andbile solubility testing; serotype was determined by the Quellung reaction. Trendsin PCV7 use and SP carriage were analyzed for children <5 years and adults �20years.Results: Overall, SP carriage was determined for 55% of village residents. Per-centages of children aged <5 years who were up-to-date for PCV7 were 21% in2001, 68% in 2002, and 71% in 2003. Among carriers, the proportion carrying aPCV7 serotype declined from 1998-2000 to 2003, in both children <5 years (55%to 10%, p<0.0001) and adults �20 years (27% to 5%, p<0.0001). The decline inadults occurred in individuals living in households with and without children <5years (31% to 7%, p<0.0001, and 22% to 4%, p<0.0001, respectively). Adultsliving with children aged <5 years were more likely to carry a PCV7 serotype thanadults not living with children aged <5 years (OR 2.03, 95% CI:1.40-2.94). Adultsliving in households where at least one child was up-to-date were less likely tocarry PCV7 serotypes than adults in households where no children were up-to-date (OR 0.54, 95% CI:0.29-0.96).Conclusions: These findings provide an explanation for the declining rates ofadult pneumococcal disease through decreased transmission of PCV7 serotypesfrom vaccinated children to adults.

Surveillance of invasive Streptococcus pneumoniae disease in South Africa in2003.

Quan V1, Soma K1, von Gottberg A1, de Gouveia L1, Madhi SA1, Schuchat A2,Klugman KP1,3 and the national surveillance network. 1RMPRU (MRC/WITS/NICD), Johannesburg, South Africa; 2Centers for Disease Control and 3Depart-ment of International Health, Emory University, Atlanta, Georgia, USA

Aim: To describe the burden of invasive pneumococcal disease (IPD) in South Af-rica in 2003.Methods: IPD isolates are referred to a central laboratory in a national surveillanceprogramme. Population data were obtained from Statistics South Africa. Addi-tional data from sentinel sites were collected.Results: By 1 December 2003 2378 episodes had been reported to the central ref-erence laboratory. Of these, 1266/2338 (54%) occurred in males and 592/681 (87%)patients were HIV-seropositive. Disease occurred most commonly in the <5-year-old (772/2378, 33%) and the 25- to 44-year-old age groups (740/2378, 31%). HIVinfection was documented in 206 of 248 cases of IPD (83%) and 248/268 (93%) inthese age groups respectively, P=0.002. The estimated incidence rate by provincefor all ages ranged from 0.9/100000 (Eastern Cape) to 17/100000 (Gauteng). Thenational estimated incidence rate in children under 5 years of age was 17.4 per100000, (in Gauteng, 60/100000; and in the Western Cape, 32/100000). At ChrisHani Baragwanath Hospital (CHBH) the incidence in all ages was 69/100 000 andfor <5-year-olds 170/100000. The HIV seroprevalence (2002 national antenatalsurvey) in Gauteng was 32% and in the Western Cape was 12%. National esti-mated incidence of meningitis in children <5 years old was 3.7/100000, rates inGauteng and in Western Cape were 11/100000 and 6.3/100000 respectively; and atCHBH it was 23/100000. Comparing 2 academic hospitals: the number of bloodcultures taken per total admissions in 1 year in the Eastern Cape hospital was 548/13549 (4%), and in the Gauteng hospital it was 31768/67486 (47%).Conclusions: The estimated incidence of IPD reflects an underestimation of bur-den of disease, with variable blood culture practices probably being one of themajor determinants. Improving clinical and site-specific data collection will im-prove interpreting the surveillance data.


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Invasive pneumococcal disease in HIV-AIDS: Has introduction of 7-valent pneu-mococcal conjugate vaccine (PCV-7) reduced HIV-related disease burden in theUS?

Schuchat A1, Flannery B1, Heffernan R2, Hadler J2 Harrison L3, Reingold A4,Gallagher K1, Whitney CG, ABCs Team. CDC, Atlanta1 , Connecticut EmergingInfections Program (EIP)2, MD EIP3, CA EIP4, USA.

Aims: To evaluate indirect effects of PCV-7 introduction in children on incidenceof invasive pneumococcal disease (IPD) in HIV-infected adultsMethods: Pop.-based surveillance was conducted by the Active Bacterial Core sur-veillance (ABCs) in: CT; San Francisco County, CA; and 6 counties in MD. IPDwas defined as pneumococcus isolated from a sterile site in a resident 18-64 y.o.with HIV/AIDS noted on the hospital chart. Data on 18-64 y.o. alive with AIDScame state AIDS programs. The ratio of IPD adults with HIV/AIDS to the AIDSpop. was compared for July 1997 – June 2000 (“Pre-PCV7”) vs. July 2001 – June2003 (PCV-7 era) by chi-square.Results: From July 1997-June 2003, 1,250 IPD cases in HIV-infected adults oc-curred. The ratio of HIV/AIDS-associated IPD to the AIDS pop. averaged 1091/105 pre-PCV7 vs. 922/105 in the PCV7–era (p =0.009). The ratio in males fell 24%from 933/105AIDS persons in 1997-2000 to 710/105 in the PCV-7 era (p<0.001),while no significant decline occurred in females (1730/105 to 1711/105, p=0.92).HIV/AIDS-associated IPD in whites fell 27% (pre-PCV7: 622/105 v. PCV7-era:452/105, p=0.02), but declined only 10% among black HIV/AIDS patients (pre-PCV7: 1690/105 v PCV-7 era 1500/105, p=.12). Vaccine type (VT) IPD ratios per105 declined more in males (535 to 264, -51%) and whites (369 to 162, -56%) vs.females (922 to 619, -33%) and blacks (967 to 513, -47%) while nonVT IPDincreases were greatest in females (551 to 857, +64%) and blacks (435 to 821,+89%) vs. males (240 to 354, +47%) and whites (169 to 239, +41%).Conclusions: PCV-7 in children led to declining IPD in adults with HIV/AIDS,with benefits greatest among whites and males. Replacement by nonVT strainsblunted overall benefit, especially in females.

The serogroup/type (SGT)-specific invasive disease potential of Streptococcuspneumoniae (Sp) is temporally and geographically stable in children

Brueggemann, AB1, Peto, TEA1, Crook, DW1, Butler, JC2, Kristinsson, KG3 andSpratt, BG4. Univ of Oxford, Oxford, United Kingdom1; Centers for Disease Con-trol and Prevention, Anchorage, Alaska, USA2; Landspitali Univ Hospital, Reykja-vik, Iceland3; Imperial College London, London, United Kingdom4.

Aims: A meta-analysis study design was used to analyze data sets of well-matchedsamples of invasive and carriage isolates ofSp recovered from young children, to determine whether the invasive disease po-tential differs for each serotype, and if SGT-specific invasiveness has changed overtime or differs geographically.Methods: 8 datasets from 1975-2002 were analyzed, including Sp studies fromlow- and high-income countries. SGT-specific odds ratios (ORs) for invasive dis-ease were calculated for each individual study and as a pooled estimate, using thehighly invasive serotype 14 as the reference group. Only serogroups 6 & 7 showedsignificant heterogeneity in the pooled OR estimates; the heterogeneity in serogroup6 was resolved by calculating ORs for serotypes 6A and 6B separately, but this wasnot possible for serogroup 7.Results: SGTs 1, 4, 5, 7, 14 & 18 were the most invasive, with pooled ORs (rela-tive to serotype 14) ranging from 0.6 – 4.6, whereas 3, 6A, 6B, 15, 19 & 23 werethe least invasive, with pooled ORs of only 0.1 – 0.4. There was a significant in-verse correlation between invasive disease potential and carriage prevalence forthe serotypes in this meta-analysis, implying that the most invasive SGTs were theleast commonly carried and the most frequently carried were the least likely tocause invasive disease. There was no evidence of any temporal changes or majorgeographical differences in SGT-specific invasive disease potential.Conclusions: There were significant differences in the invasive disease potentialof individual SGTs. Changes in the Sp population as a result of the use of Spconjugate vaccines (e.g. serotype replacement) are of concern, and this methodol-ogy may be helpful in understanding ecological changes in Sp resulting from theselective pressure of vaccine use and provide guidance for future vaccine design.

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Bacterial Meningitis in Children 2-59 Months of Age Admitted to a TertiaryHospital in Central Philippines

Abucejo-Ladesma E1, Simoes E4, Lupisan S2, Sombrero L2, Quiambao B2, LuceroM2, Arcay J1, Galia E1, Riley I3, Herva E5, Ruutu P5 for the ARIVAC Consortium1Gov. Celestino Gallares Memorial Hospital, Bohol, the Philippines, 2Research In-stitute for Tropical Medicine, Manila, the Philippines, 3University of Queensland,Australia, 4University of Colorado, USA5National Public Health Institute, Helsinki and Oulu, Finland

Aim: To describe the clinical and laboratory profile as well as outcome of bacterialmeningitis in children 2 to 59 months old at a tertiary hospital in the Philippines.Methods: Children 2 to 59 months old fulfilling defined criteria of suspect menin-gitis were prospectively enrolled in an etiology study of serious infections at theGallares Hospital, a tertiary general hospital in Central Philippines from April 1994to May 2000 using a guideline based on a combination of clinical signs and symp-toms. On admission, blood and CSF were drawn for culture using standard tech-niques, total CSF cell count and latex agglutination tests.Results: 734 patients fulfilled the criteria for suspect meningitis. All had bloodcultures among whom 477 (65%) had CSF samples. Bacterial etiology was foundin 57 (7.7%). The most common bacterial pathogens were H. influenzae type b(Hib) (N=24; 3.3%) and S. pneumoniae (Pnc) (8; 1.0%). 22 (92%) of Hib infec-tions and 6 (75%) of Pnc infections were in children less than 1 year old. Fifteen(26%) of the cases with bacterial meningitis died. All strains of S. pneumoniae andH. influenzae type b were sensitive to chloramphenicol, cotrimoxazole and ampi-cillin.Conclusion: S. pneumoniae and H. influenzae type b are the most common etiologicagents of bacterial meningitis, and occur especially in children less than 1 year old.Etiologic agents were susceptible to the currently recommended antimicrobial agents.

Effect of a Seven Valent Pneumococcal Conjugated (PCV7) Vaccine in Childrenwith Sickle Cell Disease (SCD).

Adamkiewicz TV, Silk B, Howgate J, Platt A., Eckman J. Farley, M.M. Emory Uni-versity, Atlanta VAMC, Georgia Emerging Infections Program, Atlanta, GA. USA.

Aims: To assess the impact of the introduction of PCV7 vaccine on children withSCD.Methods: Invasive Pneumococcal infections (PI) in patients (pts) with SCD fol-lowed (DOB >12/31/1989) were identified between 1996-2002 through the Geor-gia Emerging Infections/ABCs surveillance network. Results: 48 episodes of PIsoccurred in 40 out of 802 pts with hemoglobin (HB) SS or SC between 1996-2002.PCV7 vaccine history was known for 616 of 781 pts followed after vaccine licensure[46 % (n=268) had � 2 PCV7 doses, 21 % (n=129) had � 3 doses, 9 % (n=54) �4 doses]; 23-valent Polysaccharide vaccine preceded PCV7 in > 53 % of cases(n=328). PCV7 serotypes were present in serotyped isolates as follows: 67% (23/34) pre-licensure; 100% (8/8) post licensure in pts not vaccinated with PCV7 and0% (0/2) in pts vaccinated with PCV7. In pts aged 0 to 5, the rate of PIs in 2001-2002 was significant lower compared to the pre-licensure era [HB SS: 96-99, n=307,597 pt years, 20 PIs, 3.4 PIs /100 pt years; 2001-2002,n=277, 356 pt years, 4 PIs,1.1 PIs /100 pt years; % Reduction (PR): 66.5%, 95% CI: 1.9 to 88.5; P=0.036; HBSC: 96-99, n=149, 272 pt years, 9 PIs, 3.3 PIs/100 pt years; 2001-2002, n=134, 170pt years, 0 PIs; PR: 100%; P=0.015]. After 1999, pts with HB SS and SC 0 to 5years old were significantly less likely to be infected after receiving the 1st PCV7dose (prior to 1st dose, n=295, 207 pt years, 6 PIs, 2.9 PIs /100 pt years; after 1stdose, n=330, 495 pt years, 3 PIs, 0.6 PIs /100 pt years; PR: 79.1%, 95% CI:16.4 to95.8;P=0.023). In a case control model, pts infected with vaccine serotypes wereage matched with 14 non-infected SCD controls actively followed at the time of theinfection. Vaccination status at the time of infection was significantly different be-tween cases and controls [cases: no PCV7=8(100%); controls: no PCV7=53 (47%),�1 PCV7 dose =37 (33%); unknown PCV7 status =22 (20%); P=0.012].Conclusion: A significant decline in PIs was noted in children with SCD after PCV7licensure, despite variability in vaccine schedule administration and may reflectboth PCV7 effectiveness in this population and herd immunity.

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Descriptive epidemiology of pneumonia and pneumococcal disease among adultsin The GambiaAdegbola R1, Watt J2, Ameyaw S1, Hill P1, Levine O2, Van Beneden C3, Corrah T1.Medical Research Council Laboratories, The Gambia1, Johns Hopkins School ofPublic Health, Baltimore, MD2, Centres for Disease Control and Prevention, At-lanta, GA3, USA.Aim: The objective of this study was to determine the frequency of pneumonia (ofany aetiology) and pneumococcal disease among adults admitted to the MedicalResearch Council (MRC) hospital in The Gambia.Methods: The hospital database was reviewed to identify adult (�13 years) in-patients with a discharge diagnosis consistent with pneumonia, meningitis, or in-vasive pneumococcal disease (IPD). Clinical, laboratory and radiological data wereused to identify cases meeting predetermined criteria.Results: There were a total of 3,397 adult admissions between January 2000 andAugust 2003. Of these, 1453 cases (42.8%) were identified as possible pneumo-nia, meningitis, or IPD based on discharge diagnosis. A total of 1065 (73.3%)clinical records were retrieved for review, of which 261 (24.5%) met inclusioncriteria; 243 pneumonia cases and 18 meningitis cases were identified. The meanage of patients was 40.1 years, (range 13-75). The male: female ratio was 1.7:1 andthe mean length of hospitalisation was 4.8 days for pneumonia and 5 days formeningitis. Forty-two (17.3%) of the pneumonia cases had positive pneumococcalcultures from blood, pleural or lung tap. Nine (50%) of the meningitis cases wereconfirmed as pneumococcal by culture or latex agglutination. The mortality for in-patient pneumonia cases was 6.5% while that of meningitis cases was 22.2%. Forty-six (17.6%) of the study cases were confirmed HIV positive, while 8 (3.3%) pneu-monia cases had sputum positive tuberculosis. Thirty-seven cases had more thanone admission and of these, 29 (78.4%) were HIV positive.Conclusions: Pneumonia is a relatively common cause of admissions among adultsin The Gambia accounting for approximately 10% of hospitalisation at MRC Hos-pital. Meningitis is less common but associated with a four-fold greater case-fatal-ity ratio. Streptococcus pneumoniae (pneumococcus) was a common cause of pneu-monia based on blood culture results. The proportion of pneumonia caused bypneumococcus is likely to be substantially higher as blood culture is an insensitivetest. Recurrent episodes of pneumonia and meningitis were a feature mainly ofHIV infected patients.

A novel PCR-Restriction Fragment Length Polymorphism method to deter-mine serotype or serogroup of Streptococcus pneumoniae

Batt SL, Charalambous BM, McHugh TD and Gillespie SHMedical Microbiology, Royal Free and University College Medical School, Lon-don, NW3 2PF

Aim: To design a long-range PCR and RFLP based method for serotyping S.pneumoniae to make the technique, which is generally confined to reference labo-ratories as purchasing pneumococcal antisera is a huge investment, more accessi-ble to developing countries where it is rarely performed at all.Method: This paper describes a long-range PCR which amplifies the entire capsu-lation locus between dexB and aliA. Amplicons are digested to produce serotypespecific patterns.Result: We have shown using 55 epidemiologically unrelated strains that the pat-terns agree with 90-100% similarity for the same serotype or serogroup. Prospec-tive testing of 85 isolates of unknown serotype confirmed reliable serotype attribu-tion and serotype profiles are reproducible on repeated testing.Conclusion: Once our database contains all 90 serotypes, this technique should befully portable, cheap and useful in any laboratory with sufficient molecular expe-rience.


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Population genetics of penicillin intermediate resistant isolates of Streptococ-cus pneumoniae from a semi-closed community in northern Tanzania

Batt SL, Charalambous BM, McHugh TD and Gillespie SHMedical Microbiology, Royal Free and University College Medical School, Lon-don, NW3 2PF

Aims: Penicillin resistance in S. pneumoniae is increasing globally and is thoughtto be dominated by the spread of successful clones. This work aimed to examinethe penicillin intermediate strains collected from children in northern Tanzaniausing PCR and RFLP and to see whether resistance has resulted from local recom-bination of PBP genes or has been driven by the introduction of internationallyrecognised resistant clones.Method: Penicillin intermediate resistant isolates were collected from a semi-closedcommunity in northern Tanzania during a prospective study on the affect of com-munity treatment with Azithromycin for Trachoma.Results: Out of 382 samples collected 69 were intermediate for penicillin and theremaining 313 were sensitive. PBP2B and PBP2X PCRs were performed for thepenicillin intermediate strains and the amplimers digested with HinfI and DdeI.Using Bionumerics the degree of clustering was calculated for the four digestionsand mean values were 74%, 25%, 29% and 22% for PBP2B HinfI, PBP2B DdeI,PBP2X HinfI and PBP2X DdeI respectively. Using PBP genotype and serotypealmost all intermediate resistant strains were distinct.Conclusion: These data suggest a highly polymictic population with respect toPBP genotypes and that resistance to penicillin is evolving locally. This is in con-trast to the common assumption that recent emergence of penicillin resistance isfrequently the result of donation of partial PBP genes from already resistant iso-lates and the spread of internationally recognised multi-drug resistant strains isfacilitating and accelerating this. This community may potentially offer an oppor-tunity for studying the evolution of penicillin resistance in S. pneumoniae as itemerges independently of fully penicillin resistant strains.

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Invasive Pneumococcal Disease (IPD) Epidemiology and 23-Valent Pneumo-coccal Polysaccharide Vaccine (PPV-23) Effectiveness in Alaska Native andAmerican Indian (AN) Adults in Alaska

Butler JC1, Singleton R1,2, Bulkow L1, Hurlburt D1, O’Brien KL3, Hennessy TW1,Parkinson AJ1. CDC, Anchorage1, Alaska Native Tribal Health Consortium, An-chorage2, Johns Hopkins University, Baltimore3

Aims: ANs have age-adjusted invasive pneumococcal disease rates 2-3-fold greaterthan that of non-Native Alaskans. AN adults with underlying conditions and thoseaged �55 yrs routinely receive PPV-23. To characterize IPD epidemiology andPPV-23 effectiveness in AN adults, we reviewed all cases of IPD reported throughongoing, state-wide, laboratory-based surveillance in Alaska during 1986 through2000.Methods: Sterile site isolates submitted through surveillance were serotyped byQuellung reaction. Detailed clinical information and vaccine histories were col-lected for IPD cases occurring in AN adults by medical record review. Vaccineeffectiveness (VE) was estimated by comparing the proportion of cases caused byPPV-23 serotypes among vaccinated and unvaccinated persons (indirect cohortmethod).Results: 394 cases (44.5 cases/100,000/year) occurred in 374 ANs aged 20-96 yrs(median 48 yrs; 36.0% aged �55 yrs). Underlying conditions included heavy etha-nol use (61.9%), smoking (57.6%), and chronic lung diseases (31.0%). For 307persons with a serotyped isolate from a first IPD episode, 91 (29.6%) had receivedPPV-23 a median of 2.6 years before onset; 78% vaccinated <5 years before onset.PPV-23 serotypes infected 87.9% of vaccinated and 96.3% of unvaccinated per-sons. Overall VE = 72% (95% confidence interval [CI]:21% to 90%) but declinedwith increasing age; for persons �55 years, VE = –54% (95% CI: <0% to 72%).VE = 86% (95% CI: 45% to 97%) for persons who used ethanol heavily, 86%(95% CI: 37% to 97%) for smokers, and 74% (95% CI:–202% to 100%) for thosewith chronic lung diseases.Conclusions: Conditions associated with increased risk of IPD in other populationsare common among AN adults with IPD and may contribute to high rates of infec-tion. PPV-23 is effective for preventing IPD in AN adults with certain underlyingconditions but VE was not confirmed among ANs aged 55 years and older.

Predictors of early onset of pneumococcal carriage in Aboriginal and non-Aboriginal children in arid Western Australia

Carville KS1,2, Lehmann D1, Firth M1, Bowman J3, Riley TV3,4, Elsbury D1, StokesA1, Finucane J1, Monck R 1 for the Kalgoorlie Otitis Media Research Team. TelethonInstitute for Child Health Research, Perth1, National Centre for Epidemiology &Population Health, Australian National University2, Division of Microbiology &Infectious Diseases, PathCentre, Perth3, Department of Microbiology, Universityof Western Australia4, Australia.

Aims: To evaluate predictors of carriage of pneumococci (Pnc) in Aboriginal andnon-Aboriginal children <3 months of age who are enrolled in a cohort study in-vestigating causal pathways to otitis media.Methods: 413 nasopharyngeal aspirates (NPAs) were collected from 78 Aborigi-nal and 157 non-Aboriginal children at 1-3 weeks and 6-8 weeks of age. NPAswere cultured and predictors of Pnc carriage were evaluated (a) for each follow-upage group by logistic regression and (b) for combined <3-month data using gener-alised estimating equations to account for repeated measures. Variables includedgender, maternal age, education, seasonality, passive smoking, exclusivebreastfeeding, crowding, and those cited below.Results: Aboriginality, age group, the presence of other children in the house andconcurrent carriage of Moraxella catarrhalis (Mc) and Haemophilus influenzae(Hi) were independently associated with carriage of Pnc. When data were split byAboriginality, predictors of Pnc carriage in Aboriginal children were: simultane-ous Mc carriage (odds ratio, OR=2.5) and presence of �2 older children in thehouse (OR=3.1). In 6-8-week-old non-Aboriginal children, any number of oldersiblings in the house (OR=33.3), maternal employment (OR=3.2) and smoking inpregnancy (OR=3.3) were predictors of Pnc carriage.Conclusions: Predictors of early onset of Pnc carriage may differ between Aborigi-nal and non-Aboriginal children. Some predictors may be proxy measures, such asmaternal employment which could be a proxy for day care.


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Epidemiology of invasive pneumococcal infections in Sardinian children

Castiglia P1, Gallisai D2, Sotgiu G1, Maida A1 & Group for Sourveillance on pneu-mococcal infections. Institute of Hygiene1, Institute of Pediatrics2-University ofSassari

Aims: Data on pneumococcal meningitis are available on Italian children (1.4 per105), but not on bacteremic pneumococcal conditions. Purpose of the study was todescribe, in an area considered at low risk for this kind of infections, the epidemi-ology of invasive pneumococcal infections in children, 0-5 years old.Methods: Active surveillance trough sentinel pediatrics was conducted to identifyall cases of invasive pneumococcal infections in children in the District of Sassari(Northern Sardinia - Italy). Eligible case definition comprised: a) in absence of adefined localised infection (except pneumonia), - fever >38°C for 48 h, or - twoconsecutive febrile episodes > 38°C, or - WBC counts >15.000/ml; b) clinical orradiological defined pneumonia; c) post-surgical infectious disease; d) febrile epi-sode in neutropenic surgical patient. Case definition comprised isolation of S.pneumoniae from blood: two blood samples at 20' one from the other were ad-vised. Pneumococcal carriage was investigated too.Results: The paper reports preliminary results of a 64 weeks period on 7940 per-son-years. In that period 410 eligible cases arose, of which only 6.5% with bothsamples of blood and 59.2% with at least a rhinopharyngeal specimen. Six caseswere microbiologically confirmed, of which only four satisfied age and residencecriteria. The incidence of invasive pneumococcal infections was 50.4/100,000/year(CI95%: 18.9-134.2). Rhinopharyngeal carriage of Streptococcus pneumoniae byeligible cases (19.2%; CI95%: 16.7-21.7%) did not differ from other studies.Conclusions: Invasive pneumococcal infections in Sardinian children under 5 yearsold seems to be not less frequent than in other European populations. Active sur-veillance on eligible cases by means of blood sampling was able to elicit this kindof infections. These results enlighten the opportunity of vaccination of children upto 5 years.

Impact of age and HIV infection status on differential vaccine coverage ofpneumococcal conjugate vaccines (PncCV) in African children hospitalisedwith invasive pneumococcal disease (IPD).

Cutland C 1, Madhi SA1, von Gottberg A1, Wasas A1, Rafundisani T1, de GouveiaL1, Klugman K1, 2 .1 Respiratory and Meningeal Pathogens Research Unit (MRC/ WITS/ NICD), Jo-hannesburg, South Africa; 2Department of International Health, Emory Univer-sity, Atlanta, Georgia, USA

Aims: To determine the impact of age, disease entity and HIV infection status onthe potential coverage of a 7 (serogroups 4, 6, 9, 14, 18, 19 and 23), 9 (additionalserogroups 1 and 5) and an 11-valent (additional serogroups 3 and 7) conjugatevaccine in South African children.Methods: Children treated for IPD at Chris Hani Baragwanath Hospital between1997 and 2002 were enrolled prospectively. Children who received a 9-valent PncCVduring this period were excluded from analysis.Results: A total of 694 children were enrolled, 56% of whom were male and 75%(444/591) of who were HIV infected. Overall the potential vaccine coverage forthe 7, 9 and 11-valent vaccines were 78%, 88% and 90% respectively for childrenof all age groups. Vaccine coverage for the 7,9 and 11 valent vaccines among HIVinfected children was 85%, 89% and 91% (p=0.02) and 65%, 85% and 88% forHIV uninfected children. (p<0.001)Among HIV uninfected children, vaccine coverage at ages<2, 2-5 and >=5 yearsfor the 7, 9 and 11- valent vaccines were 69%, 83% and 87% (p=0.04); 71%, 90%and 90% (p=0.15) and 44%, 88% and 88% (p=0.0002).Conclusion: 9- and 11-valent PncCV have significantly more coverage againstIPD than 7-valent PnCV in HIV-uninfected and older children.


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Determination of Pediatric Emergency Room (PER) visits and hospitaliza-tion for community-acquired alveolar pneumonia (CAAP) in children, usingthe WHO standardization of interpretation of chest radiographs (WHO-SICR)

Dagan R1, Givon-Lavi N1, Bar-Ziv J2, Greenberg D1. Ben-Gurion Univ, Beer-Sheva1,the Hebrew Univ, Jerusalem, Israel2.

Aims: Currently, etiology of CAAP in children is only rarely determined. A WHOworking group has standardized radiological diagnosis of CAAP (WHO/V&B/01.35, 2001), attempting to characterize radiologically CAAP that will be reducedby pneumococcal vaccine. Clinical and epidemiological characteristics of CAAPdefined by WHO-SICR in children <5 yrs living in southern Israel are being stud-ied prospectively.Methods: A single medical center provides all services to the Negev region insouthern Israel. In the present study, all chest radiographs (CXRs) from children<5 yrs are being read according to the WHO-SICR and categorized to: 1) end-point consolidation (EPC); 2) non-EPC; and 3) no pneumonia. Demographic andclinical variable are compared between EPC and non-EPC. Results from the first18 mos of the study started in Nov/ 01 are presented.Results: Of 9,802 CXR pairs (anteroposterior and lateral), 1,525 (16%) episodes(defined as �1 visits within 1 mo) were EPC and 215 (2%) were non-EPC CAAP.The 2 categories differed significantly both epidemiologically and clinically: Themean age (m) in EPC and non-EPC was 21.3 and 19.2, respect. (P=0.057). Therespective figures for mean WBC counts (x103/mm3), highest temp recorded andCRP concentration (mg/liter) were 19.8 vs. 14.2; 39.4 vs. 39.0; and 68 vs. 32(P<0.001 for all). 24% and 50% had 0

2 saturation <94 in the EPC and non-EPC

groups, respect. (P<.001). 8% of all children born in southern Israel presented tothe PER with CAAP before age 5 yrs (61% of whom < 2yrs). The respectivefigure for hospitalization were 4% and 73%.Conclusions: 1) CAAP as determined by WHO-SICR is a distinct clinical entitywith features suggesting high prevalence of bacterial etiology compared with non-EPC. However, considerable overlap exists between those two entities; 2) In south-ern Israel, a high incidence of PER visits and hospitalizations due to CAAP werefound, particularly <2 yrs of age.

Are all cases of community-acquired alveolar pneumonia (CAAP) in childrendetermined by the WHO standardization of interpretation of chest radiographs(WHO-SICR) similar?

Dagan R1, Greenberg D1, Givon-Lavi N1, Bar-Ziv J2. Ben-Gurion Univ, Beer-Sheva1;the Hebrew Univ, Jerusalem, Israel2.

Aims: When using WHO-SICR (WHO/V&B/0.1.35, 2001), radiologically-definedCAAP (defined as end-point consolidation; EPC) is epidemiologically and clini-cally distinct from non-EPC pneumonia (Dagan, ISPPD 4, 2004). We attemptedto determine whether additional subclassification of EPC to an even more obviousECP (EPC-MAL) can further dtermine a distinct epidemiologic and clinical entity.Methods: This is a prospective ongoing study. Results from the first 18 months(Nov/01 through Apr/03) were analyzed so far. EPC-MAL was defined as EPCthat produced a dense opacity involving one or more segments of the lung, withone or more of the following: Well-defined margins; presence of airbronchogram;or silhouette sign. In the present study, 1,443 pairs of anteroposterior and lateralchest radiographs (CXRs) of EPC, each representing one episode, were read sepa-rately by 2 pediatricians. Inter-reader disagreement was 13%, in which case theCXRs were subject to additional readings.Results: A total of 1,233 EPC-MAL, 210 EPC-nonMAL were analyzed. In addi-tion, 215 were non-EPC. The mean age (m) in EPC-MAL, was 21.8 m vs. EPC-nonMAL and non-EPC (18.7 and 19.2 resp; P<0.001). The respective figures formean highest recorded temp. (ºC) were 39.5, 39.3 and 39.0 (P<0.001), for meanWBC count (x103/mm3) 20.1, 17.6 and 14.2 resp. (P<0.001); for mean CRP con-centration (mg/liter) 67, 58 and 32, resp. (P<0.001). The ratios of incidence ofEPC-MAL to EPC-nonMAL in the fall, winter, summer and spring were 3.6, 5.8,9.6 and 7.5 resp. (P<0.01).Conclusions: Although both EPC-MAL and EPC-nonMAL could be differenti-ated from non-EPC cases, EPC-MAL is a distinct entity compared to EPC-nonMAL,appearing in older children, with a relative higher incidence in summer and fall,and higher rate of findings suggesting bacterial infections than in EPC-nonMAL.


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EPI-24

The association of nasopharyngeal (NP) S. pneumoniae (Pnc) serotypes withcommunity-acquired alveolar pneumonia (CAAP) determined by WHO stand-ardization of interpretation of chest radiographs in children (WHO-SICR)

Dagan R1, Givon-Lavi N1, Bar-Ziv J2 and Porat N1, Greenberg D1. Ben-GurionUniv, Beer-Sheva1; the Hebrew Univ, Jerusalem, Israel2.

Aims: Since the first step in all Pnc infections is NP colonization, we attempted tocharacterize its pattern during CAAP in children aged <5 yrs.Methods: End-point consolidation (EPC) was determined by the WHO-SICR crite-ria (WHO/V&B/0.1.35, 2001) and further subdivided to a more obvious EPC (EPC-MAL) and other EPC (EPC-nonMAL) (Dagan et al. ISPPD-4). NP Pnc cultureswere obtained within 24 h from presentation. Controls were seasonally, ethnicallyand age matched healthy children.Results: 389 EPC-MAL, 46 EPC-nonMAL and 833 controls were enrolled. NPcultures were positive in 65%, 57% and 74%, respect. NP colonization differed byentity, age and ethnicity. Among Bedouins, for ages < 5, 6-35 and > 35 mos, Pncserotypes (ST) included in the 7-valent vaccine (VT7), were found in 28%, 40%and 27% respect. of EPC-MAL, vs. 24%, 35% and 19%, respect. of controls. AmongJews, the respective figures were 9%, 29%, and 17% vs. 6%, 16% and 3% (P< 0.05for age >5 m). For ST 1+5, the figures for both populations grouped for ages <5, 6-35 and >35 mos were 0%, 7% and 8% in EPC-MAL, 0%, 0% and 0% for EPC-nonMAL and 0%, 0.5% and 1% for controls, with no difference between Jews andBedouins (P< 0.05 EPC-MAL vs. controls for age >5 mos). Non-VT7 ST (exclud-ing VT7-related and ST 1+5) were more common in controls.Conclusions: 1) In Jews, VT7 were more common in EPC-MAL (living conditionsof developed populations) compared with controls; 2) In Bedouins (living condi-tions of developing populations), this trend was only in children aged >35 mos; 3)ST 1+5 were markedly increased in EPC-MAL in both populations compared toEPC-nonMAL and controls, suggesting that a vaccine with ST 1+5 is important inCAAP prevention: 4) Since ST included in the 9-valent vaccine is absent in > 50%of patients, effectiveness > 30% in prevention of CAAP by the vaccine seems unre-alistic.

Increased Streptococcus pneumoniae surveillance in Latin America, SIREVA-Vigía Group, 1999-2002.

Di Fabio JL1 and the SIREVA-Vigía Latin America Surveillance Group2. Pan Ameri-can Health Organization, USA1, and National Reference Public Health Laborato-ries in Latin American countries2.

Aim: Since 1993 the PAHO has coordinated a surveillance network with NationalReference Laboratories of Latin American countries aimed at monitoring capsulartypes and antimicrobial susceptibility of S. pneumoniae. We present data from 15countries: Argentina, Bolivia, Brazil, Chile, Colombia, Cuba, Dominican Repub-lic, Ecuador, Mexico, Nicaragua, Panama, Paraguay, Peru, Uruguay and Venezuela.Methods: Capsular typing was performed by Quellung reaction, and penicillin andcefotaxime MICs were determined according to current NCCLS guidelines. Thequality control system for result validation was developed by the National Centerfor Streptococcus, Canada, and by 3 sub-regional laboratories in Brazil, Colombiaand Mexico.Results: From 1999-2002, 8,047 invasive pneumococcal isolates were collected frompneumonia (47%), meningitis (43%) and other clinical diagnosis (10%); 60% werefrom children <6 years old. The most frequent serotypes from children (81.0%), indescending order, were: 14(32.7%), 6A/6B(13.3%), 1(7.0%), 5(6.7%), 18C(5.4%),23F(5.1%), 19F(4.6%), 19A(3.3%), and 7F(2.9%). Among isolates from patients6 y ears old, the most frequent types were: 1(10.0%), 14(8.0%), 6A/B(6.0%),

3(5.3%), 5(4.5%), 18C(4.5%), 23F(4.2%), 19F(4.1%) and 7F(3.9%), representing54.8% of the isolates. Reduced susceptibility to penicillin was detected in 36.3% ofisolates from children <6, 20.3% with intermediate and 16.0% with full resistance.Conclusion: Comparison between these data and those from the previous period(1993-1998) shows no significant differences in the seroprevalence, even though 8countries have been added to the pneumococcal network. Significant increases inpenicillin resistance (from 28.6% to 36.9%, in 3.2% for intermediate and 5.1% forfull resistance) were observed in countries that participated in both surveillanceperiods.


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EPI-26

IQ in survivors of Pneumococcal meningitis: case-control study

El Bashir H1, Viner R.M2, Christie D2, Coen P.G1, Knox K3, Legood R3, Booy R1.Queen Mary’s School of Medicine and Dentistry at Barts and The London, Univer-sity of London1; University College London2; University of Oxford, Oxford3, UK

Aims: Streptococcus pneumoniae remains an important childhood pathogen world-wide. Multivalent pneumococcal vaccines are becoming available. However, dataneeded to inform vaccine introduction debates concerning the neuropsychologicalsequelae of pneumococcal meningitis (PM) are lacking. We examined the long-term effects of PM on verbal (IQ

V), performance (IQ

P) and full-scale intelligence

quotients (IQFS

).Methods: A matched retrospective case-control study: 114 cases drawn from twoprospectively collected cohorts of UK children aged 0-14 years at onset of illness(now 1-10 years after PM) and 104 controls (siblings & neighbourhood controls).Outcomes measured through supervised questionnaire and age-appropriate neuro-psychological tests conducted by trained researchers.Results: Cases: median age at assessment 5.5 years, 33% female; median age at PM9 months. Mean IQ

FS in controls was normal, making them an appropriate com-

parison group. Mean IQFs

in cases was 6 points lower than controls (p<0.01). Caseswere 4 times more likely to have an IQ below 85 compared to controls (p<0.05).Longer time since PM was associated with lower IQ: IQ

FS declined by 1 point per

year (p<0.01) & IQP by 2 points per year (p<0.001). Younger age at PM was associ-

ated with lower IQV (p <0.05).

Conclusions: IQFs

is significantly lower by nearly 0.5 SD in survivors of PM. IQV

but not IQP was also significantly lower in cases. Younger age at diagnosis and

longer time since illness were independently associated with greater reduction inIQ. Modelling of the economic costs of the neuropsychological & educational se-quelae of PM is needed to inform vaccine debates.

Epidemiology of invasive pneumococcal and non-typhi salmonella infections inGambia

Enwere G, for Gambian pneumococcal vaccine trial group.

Introduction: Non-typhoidal salmonella infection (NTS) is increasingly recognised asan important cause of mortality and morbidity in African children. We compared thecharacteristics of children from whom pneumococci were isolated with those withNTS infection, obtained in the ongoing 9-valent pneumococcal conjugate vaccine trial.Method: Children are investigated on an outpatient basis if they have fever (�38°C)and signs of acute lower respiratory infection (ALRI). Additional investigations aredone for in-patients if they have signs of meningitis or fever (�38°C) with no localisedinfection.Results: 306 organisms were isolated from 4542 children. One hundred and seven(35%) of the organisms were streptococcus pneumoniae, isolated from 95 children,and 79 (25.8%) were NTS. Most of the organisms were from blood cultures, although11 of the pneumococci but no NTS isolates were obtained from lung aspirates. Chil-dren with pneumococcal infection were more likely than those with NTS infection tobe in-patients (81% vs 50%, p=0.002), and to be younger (median age 10.2 vs 11months, P=0.09). The pneumococcus was isolated more in the dry season (64.2%),while 69.6% of NTS isolates were obtained in the wet seasons. Children with pneumo-cocci were significantly more likely to have cough, fast breathing, difficult breathing,reduced feeding, grunting, sub-costal recession and initial diagnosis of WHO definedALRI. On the other hand children with NTS infection were more likely to have diar-rhoea, a diagnosis of malaria and to have parasites on blood films. Two children withpneumococci (non with NTS) presented with wheezing. Fifty-four of the 73 childrenwith pneumococci, but only 1 of 18 children with NTS isolates who had a chest x-raytaken had a lobar consolidation (WHO defined endpoint) (p=0.001). The case fatalityrate for the children with pneumococci was 10% while that for NTS infection was5.7% (p=0.3).Conclusion: Clinical signs and chest x-ray indicative of ALRI occur more frequently inchildren with pneumococcal infection than those with NTS infection. In a rural area ofThe Gambia, pneumococcal bacteraemia had a worse outcome than NTS bacteraemia.


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EPI-28

A pneumococcal burden rapid assessment tool: where to start?

Flannery B1, Scott A2, English M2, Watt J3, Levine O3, Schuchat A1, Feikin D1.CDC, Atlanta, USA1; Wellcome Trust/KEMRI, Kilifi, Kenya2; Johns Hopkins Uni-versity, Baltimore, USA3.

Background: When considering introduction of pneumococcal conjugate vaccines,local disease burden data will be most persuasive for health officials. Few develop-ing countries have the resources to conduct prospective surveillance for invasivedisease. For those settings, we are developing a Rapid Assessment Tool (RAT) thatuses locally-available data to estimate the vaccine-preventable burden of pneumo-coccal disease.Aims: To assess availability of data on endpoints related to pneumococcal diseasein children <5 years.Methods: We visited a rural district hospital and an urban provincial hospital inwestern Kenya. We reviewed admission registers, patient charts, laboratory andradiology logbooks, and quarterly disease summary sheets. Numbers of cases byICD-10 coded discharge diagnosis were calculated from summary sheets for hospi-tal admissions for children 0-4 years. Government census data were used to calcu-late incidence rates.Results: Neither hospital routinely conducted blood or CSF culture. Pediatric chestx-rays were infrequently obtained. Patient charts contained insufficient informa-tion to retrospectively determine criteria used for discharge diagnoses. Accordingto MOH quarterly summary statistics, at the district hospital, pneumonia accountedfor 11% of 2,569 admissions in 2002, meningitis 1% and malaria 55%. Using thedistrict population, the annual incidence rates for pneumonia and meningitis were420 and 50 cases per 105 children <5 years. However, hospitalized children mostlycame from the part of the district nearest the facility. At the provincial hospital,pneumonia accounted for 22% of 5,179 admissions in 2002, meningitis 3% andmalaria 47%. Using the district population, the annual rates for pneumonia andmeningitis were 1,660 and 200 cases/105. Hospitalized children mainly came fromthe urban area, representing 40% of the district population.Conclusions: A RAT to help MOHs estimate pneumococcal disease burden mayhave to rely on nonstandardized clinical diagnoses. Synthesis of data from en-hanced surveillance sites with varying prevalence of malaria and HIV may helpbridge between vaccine trials and locally-available data to estimate the vaccine-preventable fraction of hospitalized pneumonia.

Community acquired pneumonia (CAP) in childhood – surveillance underes-timates when using ICD10 codes

Fletcher M1, Cartwright K2, Finn A1.1Department of Clinical Sciences, South Bris-tol, University of Bristol, and 2Health Protection Agency, South West Region; UK

Aims: To determine the true incidence of CAP (WHO criteria) presenting at a Re-gional Children’s Hospital, compared with routine ICD10 coding data used for na-tional epidemiological estimations.Methods: CAP in childhood is both common and costly. In the UK, incidenceestimates, used as the basis for cost benefit calculations regarding pneumococcalimmunisation programmes are based on the ICD 10 codes J13, J18, J18.1, J18.8and J18.9 attributed retrospectively.A prospective audit of X-ray diagnosed pneumonia (WHO criteria, developed forepidemiological and vaccine studies) was undertaken, identifying all emergencyadmissions whose chest radiographs (taken within three days of admission) met thecriteria for probable bacterial pneumonia. Additional data collated for each caseincluded course of illness, laboratory investigations and response to therapy.Audit data were then compared with the above ICD10 codes allocated for the sameperiod. Codes were attributed following the child’s discharge from hospital care,by specifically trained coding clerks.Results: These ICD10 codes identified less than 50% of radiological CAP cases.Of the 150 children audited to date, coding data on 50 are included in this abstract.14% of CAP cases were coded J22 (unspecified acute lower respiratory infection)were indistinguishable clinically and radiologically from those coded J189 (pneu-monia unspecified bacteria) and, in some cases J181 (lobar pneumonia, unspeci-fied). In addition, children with empyema were generally correctly coded J869, yetthis category fails to feature in most studies of CAP. Children who had a presentingillness of CAP yet required intensive care tended not to be coded for pneumonia,again potentially resulting in the most severely affected children being excludedfrom surveillance studies.Conclusion: Using selected ICD 10 codes alone can significantly underestimateCAP, and therefore both disease burden and potential value of vaccine programmes.


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EPI-30

Serotypes frequency and Penicillin resistance patterns from S. pneumoniae iso-lated from healthy chindren of Day Care Centers in 12 mexican States .

Gómez Barreto D*, Espinosa de los Monteros LE**, Jiménez V**, Aguilar- ItuarteF*, Tapia R***, Rodríguez Romeo**Infectology Dep..* , Streptococcus Lab. ** from Hospital Infantil de MéxicoFederico Gomez, Secretary of Health***.

Streptococcus pneumoniae is the most important bacterial pathogen commonly re-lated in the upper respiratory tract infections such as pneumonia, sinusitis and otitisin children as well as in adults. It is also considered the principal cause of systemicinfections as bacterial meningitis and bacteriaemia. Penicillin resistance patterns inmany World regions has importantly growth , making therapy management more diffi-cult for such infections. Children less than 5 years old that attend to Day Care Centersare at the highest risk for colonizing from S. pneumoniae and there are several reportsthat the carrier condition is previous to a invasive disease.Materials and metods: This is a transversal , descriptive study to determine serotypesprevalence and penicillin resistant patterns from isolates of S. pneumoniae , from childrenthat attend to Day Care Centers. Every isolate was serotyped and penicillin susceptibilitypattern determined, following guidelines from the NCCLS. Finally, a frequency analysis wasperfomed to obtain isolation percentages, serotypes and susceptibility.Results: In the study, 3144 cultures from nasopharingeal swabs were performed inchildren, both male and female, from 2 to 60 months old. 918 strains were identified andserotyped, representing a 29% of the overall population. Serotypes identified were:19F (23%), 6B12% and non typificable (2%). From all isolates , 54% are serotypes vaccine includedin the 7 valent pneumococcal conjugate vaccine and 18% are serotypes vaccine re-lated, increasing coverage of serotypes included in the 7 valent pneumococcal conju-gate vaccine up to 72%.Penicillin susceptibilities patterns from 918 isolates from S. pneumoniae show only36% penicillin susceptibility , remained 63.9% had diminished penicillin susceptibil-ity. The highest penicillin resistance (>2 mcg/ml) was found in 15% of all isolates.Conclusions: Colonizing S. pneumoniae found in mexican children is similar to thereports in other regions of the World, finding an overall 72% of serotypes included andrelated in the 7 valent pneumococcal conjugate vaccine and represent an acceptableprotection percentage.Pneumococcal penicillin resistant is very high, probably related to a high upper respira-tory tract prevalence in Children that attend to a Day Care Center, therefore is the useof antibiotics.

Serotypes and resistance patterns of Streptococcus pneumoniae from Inva-sive Forms, in the Hospital Infantil de Mexico from 1997 to 2003Gómez Barreto D*, Espinosa de los Monteros LE**, Aldana Rubén*, RodríguezRomeo* *

Dpto. Infectologia* , Laboratorio de Estreptococos ** del Hospital Infantil deMéxico Federico

S. pneumoniae is a pathogen that causes millions of deaths at a global scale, isthe bacteria most commonly related to the upper respiratory tract infections aspneumonia, sinusitis as well as otitis; it is also the most common etiology forbactereaemia and meningitis. In the recent years and increase in the penicillinresistance patterns and other antibiotics had produced changes in strategy ,therapy as well as in prevention like use of a 7 valent pneumococcal conjugatevaccine in childern less than 2 years older.Material and Methods: This is a transversal and descriptive study to determineserotypes prevalence and the penicillin resistance patterns fromStreptococcus pneumoniae isolated in children with pneumococcal invasive dis-ease hospitalizad from 1997 to 2003 in the Hospital Infantil de Mexico (MexicanChildren Hospital).All standards from NCCLS (National Clinical Comitte on Laboratory Standards) forStreptococcus pneumoniae isolations and serotyping were observed. A frequencyanálisis was performed in order to obtain serotypes percentages and susceptibilities.Results: In the sudy , 146 patients with a positive isolation for Streptococcuspneumoniae were included; most prevalent serotypes were 23F (12%), 14 (12%),6 A (10%),19F (7%), 19 A(6%), 9V (5%). Serotypes 1,15,15B,18C,2C and 4 ac-counted 2% each one.Penicillin susceptibility was diminished in 47% from all isolates.Conclusios: From 146 pneumococcal invasive disease forms, 82 (56%) isolates areserotypes included in the 7 valent pneumoccal conjugate vaccine and 29 (20%) areserotypes related for the vaccine, making a total of 111 (76%) isolates of S. pneumoniaeVaccine related. This results shows a potencial protection as high as 76% fromS. pneumoniae.invasive disease forms. Penicillin resistance patterns from S.pneumoniae data report an overall 55 %, such alarming data makes necessary adifferent strategy and therapy approach .


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EPI-32

Pneumococcal serotypes causing severe invasive disease in South Africa

von Gottberg A1, Wasas A1, Rafundisani T1, de Gouveia L1, Madhi SA1, KlugmanKP1,2 and the national surveillance network. 1Respiratory and Meningeal Patho-gens Research Unit (MRC/WITS/NICD), Johannesburg, South Africa; 2Depart-ment of International Health, Emory University, Atlanta, Georgia, USA

Aim: To evaluate serotype distribution of pneumococci causing severe invasivepneumococcal disease (IPD) requiring hospitalisation in South Africa, and review-ing potential vaccine coverage.Methods: All pneumococci isolated from IPD during July 1999 to the end of 2002in laboratories participating in a national surveillance programme in South Africawere reviewed. Repeat isolates from the same patients were excluded. Pneumo-cocci were serotyped using the Quellung method.Results: 792, 2003, 2230 and 2137 episodes of IPD were reported to the centrallaboratory for the three and a half years respectively. Most of these episodes werediagnosed from blood cultures alone, while isolation from CSF constituted approxi-mately 25% each year. Of these episodes, 6696 (93%) had isolates available forfurther testing; five patients had two strains of pneumococci isolated. Serotype 1disease was the most common in all ages in the first 3 years: 171 of 738 viableisolates (23%), 295/1829 (16%), 266/2121 (12%) isolates respectively, while 6A(276/2013, 14%) was the most common serotype in 2002. The increase in the pro-portion of serotype 6A disease was seen in all ages, but was most marked in the lessthan 5-year-olds: 28 of 211 total viable isolates in this age group (13%), 76/562(14%), 98/692 (14%), 146/673 (22%) for each year respectively, P<0.001. The overallcoverage of serotypes contained in the 7-valent conjugate vaccine causing diseasein children under 5 years of age was 54%. If cross-protection within serogroupswas considered, the percentage coverage increased, 77%, 78%, 83%, and 85% foreach year respectively, (P=0.002). The 9-valent vaccine (with cross-protection) cov-ered 1913 (89%) of 2138 cases in children under 5 years overall, compared with1748/2138 (82%) covered by the 7-valent vaccine, P<0.001.Conclusions: Proportion of disease caused by serotype 6A has increased in chil-dren less than 5 years of age.

Streptococcus pneumoniae serotype 1 causing severe invasive disease in SouthAfrica

von Gottberg A1, Wasas A1, Rafundisani T1, de Gouveia L1, Madhi SA1, KlugmanKP1,2 and the national surveillance network. 1Respiratory and Meningeal Patho-gens Research Unit (MRC/WITS/NICD), Johannesburg, South Africa; 2Depart-ment of International Health, Emory University, Atlanta, Georgia, USA

Aim: To describe features of serotype 1 pneumococci causing severe invasive pneu-mococcal disease (IPD) requiring hospitalisation in South Africa.Methods: All pneumococci isolated from IPD during July 1999 to the end of 2002from laboratories participating in a national surveillance programme in South Af-rica were reviewed. Repeat isolates from the same patients were excluded. Pneu-mococci were serotyped using the Quellung method.Results: 792, 2003, 2230 and 2137 episodes of IPD were reported to the centrallaboratory for the three and a half years respectively. Of these episodes, 6696 (93%)had isolates available for further testing; five patients had two strains of pneumo-cocci isolated. The proportion of serotype 1 disease in all ages decreased: 171/738(23%), 295/1829 (16%), 266/2121 (12%) and 224/2013 (11%) respectively foreach year, P<0.001. This reduction was most marked in patients 25 to 44 years old:45 of 182 total viable isolates with known age (25%), 89/458 (19%), 89/579 (15%),and 67/571 (12%) respectively, P<0.001. Overall 748 of 955 (78%) patients withserotype 1 disease were diagnosed from blood culture alone; 197/955 (21%) fromCSF; and 2 and 8 cases from joint fluid or pleural fluid alone respectively. Thenumber of cases of serotype 1 disease roughly followed the seasonality of all IPD,except for a peak of cases in November 1999 (51 of 158 episodes with viableisolates serotyped, 32%) in comparison to November for the subsequent 3 years:24/152 (16%), 18/158 (11%) and 29/168 (17%) respectively, P<0.001.Conclusions: Serotype 1 disease is becoming less common as the paediatricserotypes dominate disease amongst HIV infected young adults. Serotype 1 dis-ease may be occurring as discrete epidemics and the cluster of cases in November1999 needs to be evaluated further.


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EPI-34

Multi-locus sequence typing of S.pneumoniae causing invasive disease in Fin-land

W. P. Hanage1, T. Kaijalainen2, R. Syrjänen2, K. Auranen2, M Leinonen2, P. HelenaMäkelä2, B. G. Spratt1. Imperial College London1, National Public Health Insti-tute, Finland2

Aims: To investigate which serotypes and clones of S. pneumoniae are associatedwith invasive disease in Finland.Methods: 224 isolates of S. pneumoniae from cases of invasive disease in children< 2 years of age in Finland between 1995 and 1999 were serotyped and sequencetypes (STs) were determined by multi-locus sequence typing (MLST).Results: 22 different serotypes and 78 different STs were found. Common serotypeswere 6B, 14, 19F, 23F and 19A (51, 41, 21, 20 and 17 isolates respectively). Com-mon STs were 138, 124, 191, 481, and 156 (28, 20, 14, 12 and 10 isolates respec-tively). The diversity of STs was significantly lower for isolates from invasive dis-ease, compared with a carriage dataset (invasive disease 0.038, CI 0.027-0.048;carriage 0.019, CI 0.014-0.024). Extensive capsular switching is evident in thedataset, including non vaccine types. Among these is serotype 38, which caused 6cases of invasive disease at diverse locations and times. We compared the preva-lence of ST and serotypes among invasive disease with their prevalence in carriageby estimating odds ratios (ORs) for invasive disease, with 95% confidence inter-vals. The ORs of serotypes 14, 18C, 19A, 7F and 6B were significantly greaterthan 1, indicating association with invasive disease. The ORs of 6A and 11A weresignificantly less than 1. Interestingly, the CIs for 6A and 6B do not overlap. Wefound that ST 156, the Spain 9V-3 clone, which in Finland mainly expressed sero-type 14 capsule, is associated with the development of invasive disease (OR= 10.195% CI 1.3 to 79.5). Significant associations with invasive disease were also de-tected for STs 482, 191, 124 and 138, and with carriage for STs 485 and 62.Conclusions: These results demonstrate the invasive phenotype of the serotype 14variant of the Spain9V-3 clone, and differences between members of the sameserogroup in invasive disease potential.

Pneumococcal (Pnc) serotype 1, Haemophilus influenzae type b (Hib) andNeisseria meningitidis group B are most common causes of invasive bacterialinfections (IBI) in children in St. Petersburg, Russia

Harit SM1, Sirkiä K2, Kvetnaya AS1, Kaijalainen T3, Herva E3, Nohynek H3. 1Instituteof Children’s Infectious Diseases (ICID), St. Petersburg (SPb), Russia, 2Hospital Dis-trict of Helsinki/Uusimaa, Finland, 3National Public Health Institute (KTL), Finland

Aims: Role of Pnc serotype 1 in IBI has diminished in many European countriesand USA in past decades. Its role has increased again e.g. in Denmark and Swe-den. As few reports exist on the role of Pnc and other bacteria and their serogroups/types in IBI in Russia, our study attempted to cover this gap in knowledge.Methods: A study on IBI in children (<15 years) was carried out April 1st 2001 toMarch 31st, 2003 in 3 hospitals for children and adolescents in SPb. Childrenincluded were admitted with symptoms of bacterial meningitis, septicaemia, pneu-monia, epiglottitis, arthritis or cellulitis. For bacterial cultures, blood samples weretaken from all, of cerebrospinal fluid (CSF), pleural fluid or joint fluid accordingto clinical manifestations. Samples were cultured considering growth requirementsof S.pneumoniae, H.influenzae and N.meningitidis. Identification and serogroup/type of all isolates were performed first in ICID in SPb and then confirmed inreference laboratory for Pnc and H.influenzae in KTL, Oulu, Finland.Results: S.pneumoniae was isolated from 10 patients, H.influenzae from 9, andN.meningitidis from 31 by culture of blood and/or CSF. Serotype 1 was most com-mon (7/10) of Pnc, followed by 8, 19A and 19F. All H.influenzae isolates wereHib. Group B was most common (23/31, 74%) of N.meningitidis, followed by C(6/31, 19%) and A (2/31, 7%).Conclusions: Serotype 1 was the most common Pnc serotype in IBI in children inSPb, resembling recent situation in Sweden. It has been uncommon in Finland at leastsince 1980’ies, causing in 2002 no IBI in children and only 1% of IBI in adults, de-spite frequent contacts from Finland both to east, Russia, and to west, Sweden. It willbe seen whether a virulent clone of Pnc serotype 1 will spread to and in Finland as itdid recently in Sweden (Henriques-Normark et al. 2001). The dominance ofH.influenzae type b and of N.meningitidis group B could be expected.


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EPI-36

Characteristics of Individual Pneumococcal Serotypes

William P. Hausdorff1, Daniel R. Feikin2, Keith P. Klugman3. GlaxoSmithKlineBiologicals1, King of Prussia, PA; Centers for Disease Control & Prevention2, At-lanta, GA; Emory University3, Atlanta, GA.

Aims: To identify consistent epidemiological differences among individual pneu-mococcal serotypes prior to the introduction of conjugate vaccine into national im-munization programs.Methods: Systematic review and analysis of recent endemic and outbreak diseaseliterature.Results: Certain serotypes appear to predominate within narrow pediatric age win-dows. In neonates and infants <6 mo old, serotypes 1,3,5, & 7F are each oftenprominent; together they can comprise 20-40% of all isolates, vs. 40-50% forserogroups represented in the 7-valent conjugate vaccine (PCV7: 4,6,9,14,18,19,23).PCV7 types generally predominate in 6-24 mo olds, when disease incidence is high-est. In children >2 yrs old, especially >5, serotype 1 often becomes prominentagain, along with 18C, each comprising �15-25% of all blood (serotype 1) or blood& CSF isolates (serotype 18C). In adults 20-65 yrs old, a relatively low incidenceage group, the diversity of serotypes increases, and serotype 1 is more minor. Inadults >65 yrs old, PCV7 types again increase in relative proportion, a pattern alsoseen in HIV+ individuals. PCV7 types, especially 4 & 14, caused several recentoutbreaks in young children in day care centers and in elderly adults in long-termcare facilities in industrialized countries. Serotype 1 was responsible for severalrecent outbreaks in adults in homeless shelters and impoverished communities.Regarding association with specific disease states, some serotypes are consistentlyisolated more frequently from CSF or from blood. In addition, serotype 1 causes25%-63% of “complicated” pneumonias, especially in children >2; serotype 3 alsoappears to be an important cause.Conclusions. Some serotypes are particularly associated with specific age rangesand immune or disease states, and may differ in transmission patterns. These fac-tors should be considered when evaluating the added value of future higher-valentPCV formulations.

Vitamin E and -Carotene Supplementation and Hospital-Treated PneumoniaIncidence in Male Smokers

Hemilä H1, Virtamo J2, Albanes D3, Kaprio J1,2. University of Helsinki, Helsinki1,National Public Health Institute, Helsinki2, Finland. National Cancer Institute,Bethesda, MD, USA3.

Aims: To examine whether vitamin E or b-carotene affects the risk of pneumonia ina controlled trial.Methods: Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study; arandomized, double-blind placebo-controlled trial which examined the effects ofvitamin E, 50 mg/day, and b-carotene, 20 mg/day, on lung cancer using a 2x2 facto-rial design. The trial was conducted among 29,133 males aged 50-69 years, andsmoking at baseline at least five cigarettes per day, and living in southwestern Fin-land. The recruitment took place in 1985-1988 and the intervention lasted up toApril 1993, median 6.1 years. The hypothesis being tested in the present study wasformulated after the trial was closed. The first occurrence of hospital-treated pneu-monia was retrieved from the national hospital discharge register (898 cases).Results: Vitamin E supplementation had no overall effect on the incidence of pneu-monia (relative risk [RR] = 1.00; 95% confidence interval [CI]: 0.88-1.14) nor hadb-carotene supplementation (RR = 0.97; 95% CI: 0.85-1.11). Nevertheless, the ageof smoking initiation was a highly significant modifying factor. Among subjectswho had initiated smoking at a later age (at 21 y ears; n = 7,469 with 196 pneumo-nia cases), vitamin E supplementation decreased the risk of pneumonia (RR = 0.65;95% CI: 0.49-0.86) whereas b-carotene supplementation increased the risk (RR =1.42; 95% CI: 1.07-1.89).Conclusions: Data from this large controlled trial suggest that vitamin E and b-carotene supplementation have no overall effect on the risk of hospital-treated pneu-monia in older male smokers, but our subgroup finding that vitamin E seemed tobenefit subjects who initiated smoking at a later age warrants further investigation.


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EPI-38

The Effect of Pneumococcal Conjugate Vaccine (PCV7) on Colonization andInvasive Pneumococcal Disease (IPD) Among Alaska Native Children: Elimi-nation of a Health Disparity

Hennessy T1, Bulkow L1, Butler JC1, Moore M2,Park S2, Hurlburt D1,ParkinsonA1,Singleton R1, Schuchat A2. Centers for Disease Control and Prevention (CDC),Anchorage1 & Atlanta2, USA

Aims: The rate of IPD for ethnic Alaska Natives (AN) is among the highest in theworld. We used population-based surveillance and 2 colonization studies to assessthe effect of PCV7 vaccine on IPD and nasopharyngeal (NP) carriage among ANafter it was introduced in 2001 for routine use in children.Methods: Alaska laboratories submit S. pneumoniae (SP) isolated from normallysterile sites. NP swabs were obtained from persons of all ages in 8 rural villages(population: 3,900) annually from 1998-2003. We also obtained NP swabs from450 children, 3-59 months old, at 3 urban clinics each winter during 2000-2003. NPswabs were plated directly onto gentamicin blood agar plates and incubated on-siteat 37º C. SP were confirmed by optochin and bile solubility testing; serotyping wasdone using the Quellung reaction.Results: Compared with 1995-2000, annual rates of IPD for PCV7 serotypes amongAN children <2 years in 2001-2002 declined by 90% (306 to 31/100,000, respec-tively) and are now similar to non-Native children (28/100,000 [2001-02]). For thesame time periods, IPD rates for non-PCV7 serotypes were stable for all children<2 years (45 to 47/100,000, respectively). NP carriage of PCV7 serotypes in ruralAN <5 year-olds declined 82% after PCV7 use (55% of SP carriers [1998-2000],10% [2003], p<0.001). This was greater (p<0.001) for those up-to-date (UTD) forPCV7 vaccine (29% of carriers [2001], 3% [2003], p<0.001) than for those notUTD (36% [2001], 25% [2003], p=0.06). Also, in urban AN <5 year-olds, PCV7-type carriage declined in those UTD (42% [2001], 11% [2003], p<0.01) and notUTD (56% [2001], 28% [2003], p=0.03). The proportion of AN 2 year-olds with 3PCV7 doses increased from 36% (Oct 2002) to 86% (Oct 2003).Conclusions: Among Alaska Natives, PCV7 use has eliminated a longstanding healthdisparity of PCV7-serotype IPD through lower PCV7 carriage in vaccinated chil-dren and in those incompletely vaccinated.

Effect of 7-Valent Pneumococcal Conjugate Vaccine (PCV7) on AntimicrobialSusceptibility and Serotype Distribution of Colonizing StreptococcusPneumoniae (SP) in Rural Alaska Children.

Hennessy T, Bruden D, Butler JC, Hurlburt D, Reasonover A, Harker-Jones M,Parkinson A, Arctic Investigations Program/CDC, Anchorage, Alaska.

Aims: We used a community-based colonization study to evaluate SP carriage , SPserotypes, and penicillin (PCN) and co-trimoxazole (COT) nonsusceptibility (NS)after PCV7 was introduced in 2001.Methods:NP swabs were obtained from persons in rural villages (population: 3,900)annually from 1998-2003. Swabs were plated on gentamicin blood agar and incu-bated at 37º C. Serotype (ST) was determined by the Quellung reaction. NS to COT(MIC >1/19 µg/ml) and PCN (MIC >0.064 µg/ml) were determined by agar dilu-tion and E-test, respectively. Up-to-date (UTD) for age was determined using medi-cal records.Results: Overall, 1538 NP swabs were obtained from children <5 years old (66% ofage group).

Compared with 1998-2000, PCV7 serotype carriage in 2002-03 decreased for ST6B, 19F and 23F (p<0.001).Carriage increased for ST 6A (6% to 19%), 10A (1% to7%) and 35B (2% to 9%, p< 0.004,each).Conclusions: In rural Alaska children, PCV7 use has decreased carriage of 3 vac-cine ST and for PCN- and COT-NS strains. Overall, SP carriage has not changeddue to increased colonization with non-vaccine types.


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EPI-40

The impact of active intervention on the spread of penicillin-resistant Strepto-coccus pneumoniae in Swedish day-care centres

Liselotte Högberg1, Birgitta Henriques Normark2, Håkan Ringberg3, Karin Stenqvist4,Hans Fredlund5,

Karl Ekdahl1 and the Swedish EURIS study group. Departments of

Epidemiology1 and Molecular Epidemiology and Biotechnology2, Swedish Insti-tute for Infectious Disease Control, Stockholm; Departments of CommunicableDisease Control Region Skåne3, Västra Götaland Region4 and Örebro county 5,Sweden.

Aims: Policies for handling cases of penicillin-non-susceptible Streptococcuspneumoniae (PNSP) in day care groups vary between different counties in Sweden.In some areas there are no restrictions for these children, while in other regions allchildren in groups where a PNSP index case has been identified are screened, andall carriers are excluded from group day-care. The aim of this study was to evaluatethe effect of this intervention on the PNSP-epidemiology in day care groups.Methods: We followed the incidence in 14 DCC groups with PNSP-spread, by re-peated group screens until no further cases could be identified. All identified carri-ers were excluded in study area A (Skåne county) while they remained in the groupin study area B (Göteborg and Örebro), according to local policies. The interven-tion effect was evaluated by comparing the number of additional cases after thebaseline screen (start of the intervention period) between the two study areas.Results: The relative risk for children in DCCs without active intervention was 6.2(95% CI 1.9 – 20.2). This equals approximately 21 prevented cases in area A duringthe study period. To achieve this effect, 41 children had to be excluded from groupday-care during approximately 171 weeks. Hence one prevented cases equalled ex-clusion of another child for about 8 weeksConclusions: Since the study is based on a limited number of day care centres andchildren, these figures should be regarded as rough estimates. The total cost-benefitof this action has to be put in light of the local situation with regard to the popula-tion prevalence and the distribution of other risk factors.

Surveillance Program of Invasive Streptococcus pneumoniae strains in the prov-ince of Québec, Canada

Jetté L. P. Institut national de santé publique du Québec/Laboratoire de santé publiquedu Québec (LSPQ), Ste-Anne-de-Bellevue, Canada.

Aims : Surveillance of invasive Streptococcus pneumoniae strains was initiated in1996 to study the emergence of resistance and the prevalence of serotypes.Methods : All hospitals send monthly data sheets with information on total numberof cases and susceptibility to penicillin. Invasive strains (1 strain/patient/14 days),sent to LSPQ from sentinel units, are serotyped and tested for antimicrobial suscep-tibility.Results : Between 1996 and June 2003, 8 908 cases were reported for a yearlyincidence rate of 16 cases/100 000 population. Overall, 3 440 strains were ana-lysed. They were isolated from blood (93.4%), CSF (4.4%), and other sterile sites(2.2%). Reported infections were : pneumonia (48.5%), occult bacteremia (33.8%),meningitis (6.1%), otitis/sinusitis (5.4%) and others (6.2%). The majority of infec-tions occurred among people � 65 years (31.5%) and < 5 years (29.4%). Two hun-dred and thirty eight deaths were reported (6.9%). Serotypes 14, 6B, 4, 9V, 23F,18C and 19F accounted for 67% of the strains. Eighty-five percent of serotypesseen in children less than 2 years of age were included in the conjugated 7-valentvaccine. Ninety percent of serotypes seen in people aged two years and over wereincluded in the current 23-valent vaccine. More than 87% of penicillin non suscep-tible strains are included in both vaccines. Mean rates of non susceptibility were :penicillin : 14.9%; ceftriaxone : in non-meningitis cases : 1.1% and meningitiscases : 10.9%; chloramphenicol : 4%; clindamycin : 13%; erythromycin : 14.5%;rifampin : 0.1%; trimethoprim-sulfamethoxazole : 25%; vancomycin : 0% andofloxacin : 2%.Conclusions : 1) S. pneumoniae causes severe infections and mortality in our popu-lation; 2) resistance to antibiotics is increasing; 3) serotypes implicated in invasiveinfections correspond to those found in the conjugated 7-valent or current 23-valentvaccines according to targeted age group.


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EPI-42

Mechanism of epidemic process in pneumococcal infection

Jogolev K.D.1, Zhogolev S.D.2, Ogarkov P.I.2 Immunology Department1, Epidemiol-ogy Department2, Military Medical Academy, St.Petersburg, Russian Federation.

Aims: To study mechanism of epidemic process in pneumococcal infection in mili-tary populations.Methods: Dynamic bacteriological investigations were performed in one of the mili-tary units.Results: In the period of military unit formation the number of pneumococcal carri-ers increases because of recruits “mixing” and overcrowding. The concentration ofpneumococci in the nasopharynx rises. The body immunoresistance abruptly de-creases within the first months of military service as a result of adaptation to serv-ice conditions. Some factors (overcooling, acute respiratory disease, overstrain,hypovitaminosis, stress situations, asynchronia, etc.) can decrease immunoresistance.In the persons weaked due to unfavourable factors pneumococci along or in asso-ciation with various pathogens (viruses, Haemophilus influenza, Chlamydiapneumoniae, Mycoplasma pneumoniae, etc.) cause inflammations of nasopharynx,trachea, bronchi or lung tissue. The level of respiratory tract lesion depends on theintensity of unfavourable factors effect and the body weakness as well as on agentsvirulence. Pneumococcal colonization promotes the penetration of viruses, chlamydiaand other pathogens into epithelial cells of the upper respiratory tract and conse-quently the development of acute respiratory disease. From the other hand thesepathogens contribute to further penetration of pneumococci into the lower respira-tory tract and the development of bronchitis and community-acquired pneumonia(CAP).Conclusions: The causative agents of CAP actively circulate in military populations.This fact does not result in immediate manifestation of the disease. Pneumococcalcolonization is often asymptomatic. CAP in servicemen occurs most frequently asa result autoinfection: pneumococci change their location (from the oropharynxand tracheobronchial tree to lung tissue).

Incidence and outcome of invasive Streptococcus pneumoniae infections in Fin-land, 1995-2002

Klemets P, Ruutu P, Lyytikäinen O, Ollgren J, Nuorti P. National Public HealthInstitute, Helsinki, Finland.

Aims: To determine the age- and sex-specific incidence rates, trends and mortalityassociated with invasive pneumococcal infections (IPI) in Finland during 1995-2002.Methods: All microbiological laboratories performing blood and CSF cultures sub-mitted data on isolation of S.pneumoniae from blood or CSF during 1995-2002.Only the first episode of IPI was included in the analysis. Denominator data forrates were obtained from the Population Statistics Centre, and information on vitalstatus from the Population Information System. The patient’s national identity codewas used for linking databases.Results: We identified a total of 4338 first episodes of IPI. The average annualizedincidence rate was 10.5 per 100 000 population. The age-specific rates per 100 000in age groups <2 (N=375), 2-15 (323), 16-64 (2240), and �65 (1400) years were39.9, 4.5, 8.3 and 22.9, respectively. No trends were observed in the overall or age-and sex-specific rates during the study period. The overall case-fatality proportionsat 7, 28 and 90 days after the first positive culture were 9.1, 13.1 and 16.3 percent,respectively. In patients aged �65 the case-fatality proportions at 7, 28 and 90 dayswere significantly higher in men than in women (18.2 vs. 13.1, 25.1 vs. 18.8 and31.2 vs. 24.4, respectively; p<0.01 for all comparisons). In children aged 0–15 years,the case-fatality proportion at 7 days was 1.3 percent, with no increase over time.Conclusion: In national, population-based surveillance, the overall mortality asso-ciated with IPI was 1.8 times higher 90 days after infection than during the firstweek, suggesting the contribution of pre-existing illness or long term sequelae.Increased long term mortality was most evident in the elderly, among whom menhad significantly higher mortality than women. Further analysis should evaluatethe outcome of IPI controlling for various underlying conditions.


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EPI-44

High prevalence of oxacillin resistant and penicillin susceptible pneumococcalclones in day care centres in Iceland

Kristinsson KG1, Gunnarsdottir Th1, Erlendsdottir H1, Laxdal B2, Gudnason Th2.Deptartment of Microbiology1 and Paediatrics2, Landspitali University Hospital,Reykjavik, Iceland.

Introduction: Testing for oxacillin resistance is the preferred method for screeningpneumococci for non-susceptibility to penicillin. Most oxacillin resistant strainsare also penicillin non-susceptible. High prevalence of penicillin susceptible andoxacillin resistant pneumococci, in healthy children living in the Greater Reykjavikarea, prompted a study on the antimicrobial susceptibility and clonality of thesestrains.Methods: This study was a part of a three-year multicentre European study to re-duce the prevalence of resistance of pneumococci in children (EURIS). Childrenattending day care centres in two communities near Reykjavik were invited to par-ticipate in this three-year prospective study, where nasopharyngeal swabs were takentwice each winter for selective culture of pneumococci. Pneumococci were screenedfor non-susceptibility to penicillin by the oxacillin screening test. All oxacillin re-sistant strains were tested for penicillin susceptibility using the E-test, serotypedand typed by pulsed field gel electrophoresis (PFGE).Results: Culture of 7082 nasopharyngeal swabs taken from 1-6 year old children(mean 4.1 year) yielded 4118 pneumococcal strains (55% carriage rate) of which1119 (27%) were resistant to oxacillin. The penicillin susceptibilities of the oxacil-lin resistant strains were: �0.047 mg/l, 777 strains; 0.064, 95; 0.094, 20; 0.125-0.94, 20; �1, 0. Fifty-nine different clonal types were identified of which the 10most common represented 88% of the strains and the 3 most common 47%. The 3most common clones were of serotypes 6A (n=220), 23F (n=189) and 9V (n=105)and were found in 81-96% of the 27 day care centres.Conclusions: Oxacillin resistant and penicillin susceptible clones are widely dis-tributed in Icelandic day care centres. They have higher penicillin MICs than oxa-cillin susceptible pneumococci and may be precursors of penicillin resistant strains.

Influence of chronic illnesses on incidence of invasive pneumococcal disease inadults

Moe H. Kyaw1, Charles E. Rose Jr.1, Zack Moore2, James Singleton1, Elizabeth R.Zell1, Cynthia G. Whitney1 And Active Bacterial Core Surveillance Team1, 1 CDC, 2

University of North Carolina School of Medicine, Chapel Hill

Aims: To determine the incidence of invasive pneumococcal disease (IPD) in per-sons with chronic illnesses.Methods: We used 1999 and 2000 data from Active Bacterial Core Surveillance(ABCs) and the National Health Interview Survey (NHIS). The number of healthyindividuals and persons with specific chronic and immunocompromising condi-tions who had IPD were obtained from ABCs. Estimates for the U.S. populationwere made by adjusting for race and age using 1999 and 2000 U.S. census data. TheNHIS was used to estimate the number of healthy persons and persons with selectedconditions living in the U.S. We obtained the national estimated figures for personswith HIV/AIDS from CDC and from NHIS.Results: We identified 1716 cases of IPD in healthy adults and 5341 cases in adultswith chronic illnesses. The overall incidence rate ranged from 7.0 cases per 100,000persons in adults 18-34 years to 96.7 per 100,000 in adults >80 years. Comparedwith healthy persons, the incidence of IPD was higher in persons with chronic ill-nesses. Those with immunocompromising conditions such as hematological cancerand HIV/AIDS were at highest risk.Table: Age-specific incidence of IPD (cases per 100,000 persons) by underlying conditions

Conclusions: Persons with chronic illnesses especially those withimmunocompromising conditions, are at higher risk of IPD. Our findings supportthe recommendation for providing pneumococcal vaccination to these at-risk groups.


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Pneumococcal bacteremia among children < 36 months of age (MoA) present-ing with high fever to emergency care facilities of the primary and secondarylevel, in the Metropolitan Region (MR), Chile.

Lagos R1,2, Muñoz A1 , Espinoza M 1 , Levine MM3. 1) Centro para Vacunas enDesarrollo, Chile; 2) Hospital de Niños Roberto del Río; 3) Center for VaccineDevelopment, UMB.

In a pilot study conducted in 1998-1999, we demonstrated that 1.2% of children <36 MoA with high fever (rectal Tº �40ºC) seen in Ped-ERs of the MR and managedas outpatients harbor pneumococcal bacteremia. Subsequently, collection of a bloodculture became a standard practice of ambulatory care for any child < 36 MoA withhigh fever attended in the ER of a government hospital. This was accompanied by a2-fold increase in the incidence of pediatric invasive pneumococcal disease (Ped-IPD) in the MR over that observed when incidence estimates were based only ondetection of hospitalized cases. In addition to hospital ERs, in the MR there areprimary and secondary health care facilities (“SAPUs” and “CRS”, respectively),where children with high fever can seek urgent medical attention. Annually, the 55SAPUs and 6 CRS of the MR attend (roughly) the same number of urgent pediatricvisits as the 8 government hospital Ped-ERs. From July 2003 through January 2003,we studied the prevalence of pneumococcal bacteremia among children < 36 MoAwith high fever presenting to the Quilicura district SAPU and to the Maipœ districtCRS. Recruitment ensued Monday to Friday (except holidays) during hours whenstudy nurses were physically present at the sites. Inclusion criteria included age 0 –35 months; rectal Tº � 40º C at the moment of the visit to the SAPU or the CRS; adecision by the attending physician to manage the child as an outpatient, and in-formed parental consent for performing a hemoculture.Results: 1) During the 7-month study period, 558 children < 36 MoA presented tothe selected facilities with a rectal Tº� 40ºC and were not referred to hospital. 2)All but 4 of the parents of eligible children who were contacted by the study nursesconsented for their child to participate (257/261, 98.5%). 3) S.pneumoniae was iden-tified in 4 of the 257 hemocultures performed on SAPU and CRS outpatients (1.5%).By comparison, during the study period, the Ped-ERs that serve the populations ofMaipœ and Quilicura districts performed 718 “routine” blood culture on outpa-tients < 36 MoA who presented with high fever. S.pneumoniae was identified in 7(1%) of these “routine” blood cultures.Conclusions: The recorded incidence of ambulatory Ped-IPD in the MR would dou-ble if blood cultures were routinely obtained from children with high fever attendedat SAPU and CRS urgent care facilities, in addition to those now collected fromfebrile children seen in Ped-Ers.

Prevalence of pneumococcal bacteremia among children < 36 months of age(MoA) presenting with moderate fever to Pediatric Emergency Rooms (Ped-ER) of the Metropolitan Region (MR), Chile.

Lagos R1,2, Muñoz A1 , Abrego P1 , Levine MM3. 1) Centro para Vacunas enDesarrollo, Chile; 2) Hospital de Niños Roberto del Río; 3) Center for VaccineDevelopment, UMB.

A pilot study conducted in 1998-1999 demonstrated 1.2% prevalence of pneumo-coccal bacteremia among children < 36 MoA with high fever (i.e. rectal Tº > 40º C)seen in Ped-ERs of the MR, and managed as outpatients. Based on this evidence,collection of one blood culture was instituted as a standard practice of ambulatorycare, for any child < 36 MoA presenting to the ER of a government hospital withhigh fever. Subsequent to the introduction of this new standard of ambulatory care,the recorded incidence of pediatric invasive pneumococcal disease (Ped-IPD) in theMR doubled over that observed during preceding years when incidence was basedonly on hospitalized cases. From Sept/01/2002 to Aug/31/2003 we studied the preva-lence of pneumococcal bacteremia among moderately febrile (i.e., rectalTº>39<40ºC) children <36 MoA visiting the same 3 Ped-ERs where the pilot studyhad been conducted. Recruitment ensued Monday to Friday, between 1 and 5 PM;inclusion criteria included age < 36 months; rectal Tº>39º< 40ºC at the moment ofthe ER visit; a decision by the attending physician to manage the child as an outpa-tient, and signed parental informed consent for performing a hemoculture.Results: 1) During the 12-month study, the 3 participating ERs were visited by 13,577children < 36 MoA with Tº in the range of 39ºC to 39.9ºC, and by 3,214 childrenwith higher grade fever. 2) Parents of 837 of the 1,134 moderately febrile childrenwho presented to the ERs during days and hours when recruitment was in progressconsented for their child to participate (73,8%). 3) During matching days and hours,714 children <36 MoA presented with Tº 40ºC, 651 of w hom (91.2%) had a “rou-tine” blood culture performed. 4) Pneumococcal bacteremia was documented among0.7% moderately febrile out-patients, and among 1.2% of those with high fever (6/837 vs 8/651, p>0.05). 5) By multiplying these yields of S.pneumoniae positivehemocultures by the appropriate number of ER visits, we estimated that 97 and 39cases of ambulatory IPD occurred among children with moderate and high gradesof fever (respectively), who visited the 3 ERs during the study period.Conclusions: The recorded incidence of Ped-IPD in the MR would experience afurther 2-fold rise, if blood cultures were routinely collected from young infantsand children presenting to the ER of the government hospitals with moderate fever.


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EPI-48

Incidence density of suspected and radiologically-confirmed community ac-quired pneumonia (CAP) in infants and toddlers of the Metropolitan Region(MR), Chile.

Lagos R1,2, Muñoz A1, Espinoza A3, San Martín O1; Abrego P1; Levine MM4 1)CVD-Chile; 2) Hospital de Niños Roberto del Río; 3) Hospital San Borja Arriarán;4) CVD-University of Maryland, Baltimore.

We assembled a cohort of 520 healthy newborn to participate in a prospective, ob-servational, 24 months long, follow-up study of acute respiratory illnesses (ARI).Inclusions ensued from 09/01/2001 to 08/31/2002, at a rate of 40-50 per month, in18 government primary health care clinics of the MR. ARI are being surveyed bymeans of weekly contacts with the participants, and categorized in the various syn-dromes as per pre-established definitions. Suspected CAP is defined as an ARIepisode that prompts the attending physician to indicate a chest Rx. An investi-gated, suspected-CAP is one whose analogical chest Rx is (are) digitized and inter-preted by 2 or 3 blind, independent readers, using the W.H.O Radiology WorkingGroup procedures and definitions. The endpoints of the digital chest Rx image’sreview process are: a) Consolidation-CAP; b) Non-consolidation-CAP c) No-pneu-monia.Interim results: As of 10/31/2003, 259 participants had experienced 489 episodesof suspected-CAP, distributed as indicated bellow:

Preliminary conclusions: Up to now, almost 30% of the suspected-CAPs episodeswere ruled out by the chest Rx review process, indicating that chest Rx examinationis widely used in Chilean infants and toddlers with ARI. While the clinical rationaleof such standard of pediatric care could be debatable, it creates an ideal setting forunderstanding the spectrum of radiological abnormalities, and for conducting epi-demiological studies on pediatric pneumonias using the chest Rx as the basis forcase definitions.

Nasopharyngeal colonization and antimicrobial susceptibility profile ofStraptococcus pneumoniae and Haemophilus influenzae from the community

Lalitha MK & CAMR Study Group, Christian Medical College and Hospital,Vellore-632 004, India

Background: The nasopharynx of healthy children is often colonized with organisms likeStreptococcus pneumoniae and Haemophilus influenzae; therefore the nasopharynx servesas a reservoir of these pathogens implicated to cause respiratory tract infections and moresevere invasive disease. Studies on prevalence pattern of these pathogens and their sus-ceptibility profile can provide useful indications for more rational therapeutic and pre-ventive strategies.Objective: To determine the prevalence of S.pneumoniae and H.influenzae as nasopha-ryngeal colonizers in healthy school going children attending primary school selectedrandomly from the community and to determine the antimicrobial resistance (AMR) pat-tern of these organisms obtained from the children enrolled.Method: The study was a prospective multicentric study covering five different geographi-cal regions (representing south, north and central) of India. Nasopharyngeal swabs (NP)were obtained from healthy children (6-12 years) attending primary school selected ran-domly from both rural and urban communities with strict inclusion and exclusion crite-ria. NP swabs were transported to the laboratory using the skimmed milk tryptone glu-cose glycecrol (STGG) medium and standard microbiological procedures were used forisolation, identification and antimicrobial susceptibility testing.Results: A total of 11,156 children were enrolled and a high overall colonization rate of52% has been observed among them. S.pneumoniae was the predominant colonizer seen in25% (n=2743) of the subjects. H.influenzae alone accounted for 9% (n=994) of the sub-jects. 18% of children (n=1909) were found to have both S.pneumoniae and H.influenzaeas colonizers. A comparison of the colonization based on the area of residence of the sub-jects enrolled, showed a significantly (p=<0.05) higher colonization of S.pneumoniae andH.influenzae among children from rural settings as compared to that of urban areas. Nocomplete resistance to penicillin was observed among S.pneumoniae isolates (n=4652)with only 4% of isolates exhibiting intermediate resistant. Resistance to cotrimoxazole wasvery high at 85%. Among H.influenzae isolates (n=2903), 11% were resistant to ampicillinand co-trimoxazole resistance was seen in 63% of the isolates.Conclusion: The high rates of colonization seen among healthy children in India indi-cates that a large reservoir of these pathogens exists among healthy school children. Re-sistance to co-trimoxazole is high among S.pneumoniae and H.influenzae limiting theusefulness of this antimicrobial in treatment of children with respiratory tract infections.The AMR pattern as seen in the community will be useful in planning appropriate antibi-otic policy guidelines.


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EPI-50

Acute Otitis Media (AOM) is common among Filipino children < 5 years ofage.

Lintu M1, Heiskanen-Kosma T1, Abucejo-Ladesma E2, Puumalainen T1, SombreroL2, Lupisan S2, Lucero M2, Simoes E3, Nohynek H1 for ARIVAC consortium. 1Na-tional Public Health Institute, Finland; 2Research Institute for Tropical Medicine,Philippines; 3 Imperial College, UK, and University of Colorado

Aims: Epidemiology of AOM is not well documented in many Asian countries. Anongoing effectiveness trial of 11-valent pneumococcal conjugate vaccine in preven-tion of pneumonia in Bohol (Philippines), provides an opportunity to evaluate oc-currence of AOM among Filipino children < 5 years of age.Methods: Children from study area < 5 years of age attending Bohol Regional Hos-pital (BRH) emergency room (ER) Monday to Friday 24 hrs/day and children < 2years attending BRH out-patient department (OPD) during office hours only withsymptoms of cough/difficult breathing and fast breathing (WHO criteria for acutelower respiratory tract infection [ALRI]) were enrolled. In addition, children withonly upper respiratory tract infection (URI) were enrolled one morning and oneafternoon in a week at OPD. The diagnosis of AOM was based on pneumatic otos-copy performed by study physicians. Reflectometry (Ear Checkâ) and tympanometry(Welch Allynâ) were done. No tympanocentheses were made.Results: Total of 1362 children (median age 10.8 months, range 6 weeks-59 months,44% females, 56% males) were enrolled, but 94(7%) cases were excluded due todifficulties in performing otoscopy. AOM was diagnosed in 375 children. In ER,the occurrence of AOM was 27%(140) and at OPD 37% (171) among ALRI pa-tients and 22%(64) among URI patients. AOM was most common in children aged6 to 17 months. It was diagnosed in 25 %(80) of children at 6 weeks -5 months ofage, in 37% (137) at 6-11 months of age, in 32% (89) at 12-17 months of age, in23% (51) at 18- 23 months and in 21% (18) of children at 24-59 months of age. Intotal 31 (2 %) patients had perforation and ear discharge and in 8 cases (26%)Streptococcus pneumoniae was cultured in ear discharge.Conclusions: AOM is a common clinical finding in patients with symptoms of ARIin the Philippines. Pneumococcus was detected in 26% of patients with ear dis-charge and ear drum perforation.

Antibiotic susceptibility and serotypes distribution of invasive and carrier iso-lates of S. pneumoniae from pediatric patients with pneumonia and meningitisin Bangladesh

Mahbubur Rahman1, Shoma S1, Rashid H1, S. Hossain1, AH. Baqui1, R.R. Reinert2, AdnanAl-Lahham2 and R. Breiman1 1ICDDR,B:Centre for Health and Population Research,Dhaka, Bangladesh and 2National Reference Center for streptococci, University Hospi-tal, D-52057 Aauchen, Germany

Aim: Study epidemiology, serotype patterns and antimicrobial resistance of Streptococ-cus pneumoniaeMethods: A prospective study was conducted in three hospitals in Dhaka, Bangladesh tostudy pneumococcal infections in children (<5 years) hospitalized with pneumonia, men-ingitis and septicaemia during 1999-2002. Clinical data were collected, and all patientshad blood cultures. Cerebrospinal fluid (CSF) of meningitis case was analyzed by rou-tine tests. S. pneumoniae antigen was detected by latex agglutination (LA) in culturenegative pyogenic CSF and PCR was performed for lytA in negative samples. Antibioticsusceptibility and serotyping were done by broth dilution test and Quallung reaction.Result: Of 1834 children studied, 73(4%) had pneumococcal infections; 35 (48%) S.pneumoniae were isolated from 1493 (2.34%) pneumonia cases and 38 (52%) S.pneumoniae infections in 293 (13%) meningitis and none from 48 (0%) septicaemiacases. The detection rate was 31.14% (38 of 122) for pyogenic meningitis. Most (81%)infections occurred in infants. The overall case fatality rate was 15%. A nasopharyngealswab was collected from every 7th children enrolled and S. pneumoniae was isolatedfrom 104 (41%) of 255 samples tested. The rates of resistance to penicillin, chloram-phenicol, trimethoprim-sulfamethoxazole, erythromycin, azithromycin, clindamycin,ciprofloxacin and levofloxacin was; 9%, 16%, 64%, 4.5%, 1.5%, 1.5%, 6% and 1.5% forinvasive isolates and 10%, 2.6%, 70%, 4%, 4%, 8.9%, 4.5%, 2.2% for carrier isolates,respectively, none was resistant to amoxicillin, ceftriaxone, moxifloxacin, gatifloxacin,telithromycin, teicoplanin and vancomycin. A multidrug resistant strain was detected in1.5% invasive and 5.5% carrier isolates. The 12 most common serogroups, representing84% of the invasive isolates were 6, 14, 19, 12, 5, 1, 7, 18, 45, 2, 9 and 23 and showed66% correlation with 12 (80%) most common serogroups (6, 19, 14, 23, 9, 7, 13, 15, 21,22, 35 and 37) of carriage strains.Conclusions: S. pneumoniae is a leading cause of life-threatening infections, predomi-nantly in infants. Penicillin and other antimicrbials except trimethoprim-sulfamethoxazolecould be used as empirical therapy. Surveillance of carriage strains for resistance ap-pears useful for therapy. Serogroup results showing new patterns and correlation be-tween invasive and carrier isolates would serve as baseline data for an effective vaccineformulation.


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Network for Surveillance of Pneumococcal disease in the East Africa Region(netSPEAR)

Maranga W.1, English M.2, Scott A.2, Kamau T.3, McDonagh M.4, Oluoch T.2 1.netSPEAR, 2 Wellcome Trust / Kenya Medical Research Institute, 3 Expanded Pro-gramme on Immunization, MOH Kenya, 4. DfID.

Aims: 1. To assess the burden of invasive pneumococcal disease (IPD) in childrenaged 2 months to 5 years in the 7 countries of the WHO East Africa block. 2. Todetermine the serotypes and antimicrobial sensitivity patterns of pneumococci caus-ing IPD in the region. 3. To strengthen sentinel surveillance for invasive Hib diseaseduring introduction of the conjugate Hib vaccine. 4. To facilitate an informed evalu-ation of the case for accelerated introduction of conjugate pneumococcal vaccine inEast Africa. 5. To strengthen communicable disease surveillance and communica-tion between doctors, ministries and other stakeholders.Methods: Through training, motivational visits and conferences, laboratory and clini-cal standardization and consumable support, netSPEAR is developing a network of12 surveillance hospitals in four countries in rural and urban East Africa with plansto expand to all 7 WHO East Africa block countries. It is supported by GAVIspneumoADIP and is integrated into the WHO-AFRO meningitis surveillance net-work. CSF, and in some centres blood samples, are cultured from children meetingpre-defined clinical syndromes and processed with internationally accepted andlocally appropriate laboratory methods. Data are forwarded to netSPEAR in Nai-robi and disseminated to local policy makers, WHO-AFRO, and other internationalstakeholders via email, newsletters, meetings and at www.netspear.org.Results: It is hoped that data generated in the participating countries will enablepolicy makers, development partners and other stakeholders to make informed de-cisions about future preventive interventions. For example, data from one surveil-lance site, Kilifi District Hospital in Kenya, shows culture-positive IPD is an im-portant cause of admission to hospital in Kenya with an estimated incidence of 260/100,00 in children under 2 and 154/100,000 in children under 5 years of age, and acase fatality rate of 30%.Conclusion: Lack of information on the local impact of pneumococcal disease isone factor that may retard implementation of pneumococcal conjugate vaccine. Anetwork of sentinel surveillance may overcome this.

Burden of paediatric invasive pneumococcal disease (IPD) in Europe

McIntosh EDG1, Fletcher MA2, Fritzell B2. Wyeth, Huntercombe Lane South, BerksSL6 0PH, UK1, Wyeth Vaccine Research, Le Wilson II, 80, avenue du Géneral deGaulle, Paris, France.

Aims : To compare reported rates of invasive pneumococcal disease (IPD, meningi-tis or septic bacteremia) among infants and children in European countries.Methods : Wyeth European affiliates submitted the latest available surveillance datafor each country.Results : For infants and children <5 yrs of age

Conclusion: The burden of paediatric IPD in Europe is considerable, even if it re-mains difficult to obtain the actual rates for some countries. The reported incidencevaries by a hundred-fold. Under-reporting and differences in blood culture practicesor reporting methods may distort the true picture. Pneumococcal conjugate vaccinehas the potential to prevent much of this burden of morbidity and mortality.


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Dual Infections in African Children under 5 years of Age with InvasivePneumococccal Disease.

McNally L.M1,2, Jeena, P.M2, Gajee K,2, Sturm A.W2 , Coovadia, H.M2, TomkinsA.M1 and Goldblatt D1. Institute of Child Health, London1 and Nelson R MandelaSchool of Medicine, Durban, South Africa..

Introduction:The pneumococcus is thought to be the most common bacterial patho-gen in paediatric pneumonia. Its role in the morbidity and mortality of HIV infectedadults has been increasingly highlighted. We designed a prospective paediatric pneu-monia study in Durban, South Africa in part to assess the role of the pneumococcus.Methods: Children aged 1 to 59 months admitted to King Edward Hospital, Durbanwith WHO defined (very) severe pneumonia were enrolled. Children were treatedwith high dose benzylpenicillin (200 000 iu / kg / day) and gentamicin (7.5 mg / kg/day). All infants also received high dose co-trimoxazole. Children who failed torespond after 48 hours had a second blood culture (BC) and either a lung aspirate(LA) or non-bronchoscopic bronchoalveolar lavage (NBBAL). All children hadlinked anonymous HIV testing.Results: 362 children were recruited. 218 (60%) responded to initial therapy. Chil-dren who were HIV infected were more likely to fail to respond (78% of non-re-sponders were HIV infected compared to 68% of responders, p=0.002). Overall, 26children [7%] had a positive pneumococcal BC on admission (21 HIV pos, 2 HIVneg) and 8 of these (30%, 7 HIV pos), failed to respond to initial treatment. Two ofthese were too ill to have further testing but the remaining 6 had multiple infectiveagents isolated either from blood culture (1), NBBAL (5) or lung aspirate (1). Noneof the isolated pneumococci had an MIC > 1.0 ug / dl.Conclusions: Streptococcus Pneumoniae was the most commonly isolated organ-ism from blood cultures in children with pneumonia. However, dual pathology waspresent in at least 23 % (6/26) of children admitted with invasive pneumococcaldisease in this study. This may account in part for the significant decrease in inva-sive pneumococcal disease but not pneumonia in HIV infected children reportedfrom the Soweto Pneumococcal Vaccine Study.

PspA families of pneumococcal (Pnc) strains causing acute otitis media (AOM)in children

Melin M1, Hollingshead S2, Briles D2, Kaijalainen T3, Kilpi T1, Käyhty H1. 1NatlPubl Health Inst, Helsinki, Finland; 2Univ of Alabama, Birmingham, Al; 3Natl PublHealth Inst, Oulu, Finland

Background: PspA is a highly variable pneumococcal surface protein. Both family1 and 2 PspAs can confer virulence to pneumococci and inhibit complement depo-sition. Immunisation with PspAs induces protection in animal models against dif-ferent Pnc infections. Information about the PspA families in infections is still sparse.Methods: In the FinOM Cohort Study a middle ear fluid (MEF) sample was takenfrom children under 2 yrs of age in case of AOM. Pnc MEF isolates (N=273) of 109children were PspA-family-typed by whole cell EIA. Confirmation of the PspAtype was done for 10% of the strains by PCR typing.Results: The overall frequencies of PspAs in families 1 and 2 were similar. The twofamilies were evenly distributed among different age groups. PspA typing by PCRconfirmed that a few strains had genes for both family 1 and 2 PspAs. The capsuleserogroups 19 and 6 included strains expressing each PspA family, but strains ofother capsule types showed strong associations with either family 1 or family 2PspA (Table). Differences in the PspA family expressed were observed in consecu-tive AOM isolates of five children. It is possible that genetic events led to thesechanges in PspA expression during infection and carriage.Conclusions: The equal prevalence of PspA families 1 and 2 in AOM suggests thatthe two PspAs would make a good vaccine candidate. Within some serotypes thecapsule type and the PspA family are linked

Table. Percentage of strains (N) in PspA families 1 and 2

NOTE. A significant difference (aP< 0.0001, bP = 0.0124, cP = 0.0005, dP = 0.0067,Chi squared test)


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Trends in Pneumococcal Bacteremia among Adults in Pennsylvania from 2001to 2003.

Metlay J, Fishman N, Mincer T, Edelstein P. VA Medical Center and U. of Pennsyl-vania, Phil., PA.

Aims: For the period 1998 to 2001, vaccination of children with a new pneumococ-cal conjugate vaccine has been reported to reduce the incidence of invasive diseaseamong adults. The aim of this study was to examine trends in adult invasivepneumococal disease from 2001 to 2003.Methods: Since October 2001, we have been conducting population based surveil-lance for bacteremic pneumococcal pneumonia in adults residing in a 5 countyregion in Southeastern Pennsylvania. This region includes a population of 2.9 mil-lion adults, served by 48 acute care hospitals. 44 out of 48 hospitals routinelytransport isolates to our laboratory. Confirmed pneumococcal isolates undergoscreening for resistance to penicillin, erythromycin, tetracycline, and levofloxacinby disk diffusion testing, as specified by NCCLS. Isolates found to be positive inthe disk diffusion screening test are tested with broth micro-dilution testing forconfirmatory purposes. We report annual rates of pneumococcal bacteremia inadults from October 1 through September 30 of the following year. We comparedquarterly levels of susceptibility to each drug using a Mantel-Haenszel Chi Squaretest for trend.Results: A total of 441 isolates were identified during 2001-2002 (population rate =15/100,000). For 2002-2003, a total of 405 isolates were identified (population rate= 14/100,000) (p = 0.22). During the 1st quarter of 2001-2002, the levels of non-susceptibility were 19% penicillin, 12% erythromycin, 7% tetracycline, and 1%levofloxacin. During the 4th quarter of 2002-2003, the levels of non-susceptibilitywere 21% penicillin, 12% erythromycin, 5% tetracycline and 2% levofloxacin (allnon-significant for trend). Ongoing work will examine the distributions of pneu-mococcal serotypes over this time period.Conclusions: We have not observed a decline in the rate of bacteremic pneumococ-cal disease among adults over the two year period from October 2001 through Sep-tember 2003. In addition, levels of drug non-susceptibility have remained constantduring this time period.

Impact of pneumococcal vaccination of children on the risk of bacteremic pneu-mococcal pneumonia among adults in the same household.

Metlay J, Edelstein P, Crossette L, Fishman N.VA Med Center and U of Pennsylva-nia, Phil, PA. USA

Aims: In 2000, a new pneumococcal conjugate vaccine was licensed for use inchildren. Recent surveillance data suggests that rates of invasive disease in adultsmay be declining reflecting a form of herd protection. Our primary aim was todetermine whether vaccination of children protects adults in the same householdfrom bacteremic pneumococcal pneumonia.Methods: We conducted a case-control study nested within a population based sur-veillance study in a 5 county region in Southeastern Pennsylvania. Eligible caseswere all adults with bacteremic pneumococcal pneumonia identified at any of 44participating hospitals. Controls were healthy adults from the 5 county region iden-tified through random digit dialing, with the number of controls selected based onthe number of eligible cases identified each month over the period December 2002through November 2003. Cases and controls were interviewed by telephone andasked to report the number of children under 7 years of age in the household andwhether each had received the pneumococcal conjugate vaccine. We restricted theseanalyses to those subjects who reported living in households with at least one childunder 7 years of age.Results: To date, we have completed interviews with 24 case subjects and 175 con-trol subjects living in households with children. The average age of cases and con-trols was 46 years and 35 years, respectively. 38% of cases reported that at least 1child in the household had received the conjugate vaccine compared to 60% of con-trols (p = .04). After adjusting for age and gender of the subjects, number of chil-dren in the household, and age of the youngest child, cases had a 50% reduced oddsof reporting pneumococcal vaccination of children in the household compared tocontrol subjects (OR = 0.49 95% CI 0.2-1.4).Conclusions: We preliminarily conclude that vaccination of children with the pneu-mococcal conjugate vaccine may reduce the risk of invasive pneumococcal infec-tion among adults in the same household.


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Relative Invasiveness of Pneumococcal (Pnc) Serotypes Among American In-dian Children

Millar EV1, O’Brien KL1, Watt JP1, Bronsdon M2, Reid R1, Parkinson AJ 3, SantoshamM1. 1. Johns Hopkins University, Baltimore, MD; 2. Centers for Disease Controland Prevention (CDC), Atlanta, GA. 3. Arctic Investigations Program, CDC, An-chorage, AK.

Aims: To evaluate the relative invasiveness of Pnc serotypes among Navajo andWhite Mountain Apache (N/WMA) children <5 y.Methods: A group-randomized trial of 7-valent conjugate Pnc vaccine (PnCRM7,Wyeth) was conducted on the N/WMA reservations from 1997-2000. Children re-ceived either PnCRM7 or group C meningococcal conjugate (MnCC, Wyeth). Acarriage study (1998-2000) was nested in the trial with vaccinees swabbed at ap-proximately 7, 12 and 18 mo. old; household members �6 y were also swabbed. Afollow-up carriage study among vaccinees and household children was conductedfrom 2001-02. Calcium alginate nasopharyngeal (NP) swab specimens were storedin STGG media from which Pnc were cultured and typed. Invasive Pnc isolateswere identified through active, population, and laboratory-based surveillance; in-vasive isolates are sent to CDC for serotyping.Results: A total of 2298 NP specimens were collected from N/WMA children <5 y.From 1998-2002, 175 cases of invasive Pnc disease among children <5 y were re-ported; 174 isolates were typed. Serotypes more common among invasive isolatesthan carriage isolates included types 1 [OR: 15.6], 12F [OR: 12.9], 7F [OR: 9.9], 4[OR: 6.1], and 18C [OR: 5.0]. By contrast, serotypes 19F [OR: .59], 23F [OR: .59],NT [OR: .56], 3 [OR: .54] and 6A [OR: .30] were more common among carriageisolates than invasive isolates.Conclusions: Among children <5 y, the invasive potential of Pnc varies consider-ably by serotypes. This finding may have important implications for serotype re-placement carriage and disease in the PnCRM7 vaccine era as serotype switchingmay occur. Assessment of other organism characteristics that relate to invasivenessis warranted.

Impact of a PCV7 Vaccine on Carriage of Nonsusceptible Pneumococci in Ur-ban Alaska Children

Moore M, Park S, Hennessy T, Hyde T, Parks D, Reasonover A, Harker-Jones M,Gove J, Bruden D, Rudolph K, Parkinson A, Butler JC, and Schuchat A, Centers forDisease Control & Prevention, USA

Aim: To determine the impact of routine introduction of 7-valent pneumococcalconjugate vaccine (PCV7) on the community-wide prevalence of cotrimoxazole-(COT-) and penicillin-nonsusceptible (PCN-NS) Streptococcus pneumoniae (SP)among children in urban Anchorage, Alaska.Methods: We obtained nasopharyngeal swabs from 450 children aged 3-59 monthsfrom each of 3 clinics during the winters of 2000-2003 (total N=1800). Specimenswere inoculated on gentamicin blood agar and incubated at 37°C. Nonsusceptibilityto COT (MIC�1/19 �g/ml) and PCN (MIC >0.064 �g/ml) was determined by agardilution and E-test, respectively. Serotypes (ST) were identified by the Quellungreaction. Vaccination status was based on whether subjects were up-to-date for age(UTD).Results: Overall SP carriage ranged between 36 and 44% (P=0.51). Among carri-ers, carriage of PCV7 STs and COT-NS SP declined (table). The prevalence ofPCN-NS PCV7 STs declined by 61% (P<0.0001) while PCN-NS non-PCV7 STsincreased by 80% (P=0.011), with ST 35B representing the greatest increase.

Conclusions: PCV7 appears responsible for a decline in COT-NS and PCV7-typeSP. Although the overall prevalence of PCN-NS SP has remained stable, serotypereplacement with PCN-NS non-PCV7 STs may be


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Rates of penicillin resistant Streptococcus pneumoniae among children corre-late with the biannual epidemic activity of respiratory syncytial virus

Mühlemann K, Büchi W, Gorgievski-Hrisoho M, Aebi C. Departments for Infec-tious Diseases and Pediatrics, University Hospital Bern, and Institute for InfectiousDiseases, University of Bern, Berne, Switzerland; GlaxoSmithKline AG,Münchenbuchsee, Switzerland

Aims: We examined whether trends of pneumococcal antibiotic resistance rates cor-relate with the biannual epidemic activity of respiratory syncytial virus (RSV) ob-served in Switzerland.Methods: Resistance prevalence among pediatric outpatients were analyzed for theperiods 1998 to 1999 and 2001 to March 2003 using an established sentinel surveil-lance network. Resistance trends were compared with reported numbers on RSVinfections and sales of pediatric formulations of antibiotics.Results: Penicillin resistance rates exhibited a two-yearly cycle in phase with thebiannual seasonal RSV epidemics. Resistance rates were higher during the winterseasons 1998/1999 (16.9%), 2000/2001 (13.9%) and 2002/2003 (18.3%) comparedwith winter 1997/1998 (7.6%), 1999/2000 (11.6%), and 2001/2002 (9.5%). Overallpenicillin resistance increased from 11.7% in 1998 to 14.0% in 2002. Antibioticsales showed regular peaks during each winter; sales decreased significantly overthe study period for penicillins (-20.3%), macrolides (-43.9%) and cephalosporines(-44.8%).Conclusion:.This observation suggests a link between pneumococcal resistance ratesand RSV activity, which is independent from antibiotic consumption.

The importance of pneumococcal disease surveillance for assessing local dis-ease burden and the impact of vaccination.

Nelson C and Mhlanga B. World Health Organization, Geneva, Switzerland.(http://www.who.int/vaccines/en/pneumococcus.shtml)

Context: Estimates from the World Health Organization (WHO) suggest that 2.6million children <5y die annually as a result of acute respiratory infection (ARI),mainly pneumonia, making this the leading killer of young children worldwide.Over 1 million of these deaths are attributable toS pneumoniae, i.e. pneumococcus. More than 90% of these deaths occur in develop-ing countries. In spite of the importance of pneumococcal disease, appropriate pneu-mococcal vaccines have yet to be introduced in the developing countries where theyare needed most.

Safe and effective new generation conjugate pneumococcal vaccines are availablein developed countries. Recent funding of the Pneumococcal Accelerated Develop-ment and Introduction Plan (Pneumo-ADIP) will accelerate the development andintroduction of conjugate vaccines that are appropriate for use in developing coun-tries. The Global Alliance for Vaccines and Immunization (GAVI) and the VaccineFund (VF) have made pneumococcal vaccines a priority in their agenda to improvechild survival and this will accelerate the introduction of the newly developed vac-cine.

Recognising the scarcity of local disease burden data and the central importance ofthis information in the process of deciding to introduce a new vaccine, surveillancenetworks are being established in Africa and other regions to facilitate the collectionand dissemination of local evidence that documents disease burden and the impactof vaccination on this burden.

Aims: This presentation discusses WHO’s efforts in sub-Saharan Africa to establishpneumococcal surveillance. Strategies and challenges will be discussed.


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Inventory of drug resistant clones of Streptococcus pneumoniae colonizinghealthy children in Portugal

Nunes S1, Sousa N G 1, Simas C1, Frazão N1, Mato R1, Santos-Sanches I1, DeLencastre H1,2. Instituto de Tecnologia Química e Biológica, Portugal1; TheRockefeller University, New York, USA2.

Aims: To update the inventory of drug resistant pneumococcal clones colonizingthe nasopharyngeal flora of children attending Day-Care Centers (DCCs) in thearea of Lisbon.Methods: Between 2001-2003 (six sampling periods) a total of 4,969 samples werecollected from the nasopharynx of healthy children (6 month to 6 years). S.pneumoniae (Pn) were isolated and tested against a panel of nine antimicrobials.The isolates that were resistant at least one of the antimicrobials (DRPn) wereserotyped and typed by pulsed-field gel electrophoresis (PFGE).Results: A total of 3,539 Pn were isolated and 1,288 (36%) were selected for typing.Twenty-three different serotypes were found: most of the strains (1,018 or 79%)belonged to 5 serotypes (6B, 14, 23F, 19A and 19F); the other 18 serotypes weredistributed among 182 strains; the remaining 88 strains (7%) were non-typeable.Seventy-nine PFGE types were identified: 1,112 or 86% were represented by asfew as 20 PFGE types, while the remainder 176 strains (14%) were quite diverseand were assigned to 59 different PFGE types. Particularly striking was the fre-quency of carriage of four International clones, Colombia23F-26 (n=200), Spain9V-3(n=185), Poland6B-20 (n=135) and Greece6B-22 (n=120) which together (640 strains)accounted for close to 50% of all DRPn recovered. In addition, among the 20 mostfrequently carried clones were included the 8 DRPn clones that represented themajority of the DRPn colonizing children in the Lisbon area DCCs during a previ-ous surveillance study (1996-1998).Conclusions: The results provide a view of the dynamics of pneumococcalpopulations over time in their natural habitat, the nasopharynx of children: a largevariety of clonal types are present and novel clonal types are introduced or emerge.

Effect of conjugate pneumococcal vaccine on invasive pneumococcal disease(IPD) among Navajo.

O’Brien KL1, Weatherholtz R1, Millar E1, Watt J1, Moulton L1, Reid R1, ParkinsonA2, Santosham M1 1. Johns Hopkins University, Baltimore, MD; 2. Arctic Investi-gations Program, CDC, Anchorage, AK.

Aims: We aimed to assess the impact of long term use of conjugate pneumococcalvaccine (PnCRM7) on the epidemiology of IPD among the Navajo population whoare at high risk for IPD. PnCRM7 was used in a randomized efficacy trial amongthose < 2 yo (1997-2000) and then routinely among those < 5 yo.Methods: Active, population, and laboratory based surveillance for IPD was con-ducted defining a case as isolation of S. pneumoniae from a normally sterile site ina Navajo person. Isolates were serotyped using the quellung reaction and chartsabstracted for clinical and demographic information. Annual Indian Health Serv-ice user population statistics (with extrapolations for 2002 and 2003) were used asdenominators for rate calculations. IPD cases from 2001-2003 were compared withthose from 1995-1997.Results: We identified 475 IPD cases in 1995-97 and 285 in 2001-03 (40% reduc-tion). Among those < 5 yo. there was a 64% reduction in cases between the twotime periods (195 vs. 71 cases). Compared to the average number of cases in 1995-97, markedly fewer cases occurred during each of 2001, 2002 and 2003 in children<1, 1-<2, 2-<5 and those 18-<40 years old. The annual rate of vaccine serotype(VT) disease in those < 5 yo. has fallen by 76% between 1995-97 and 2001-03(126/100,000 vs. 29.5/100,000 respectively) although only 55% of disease wascaused by VT strains during the 1995-97 era. The rate of non-vaccine serotype(NVT) disease among those <1 yo. has not changed over this 9 year time period(175/100,000 in 1995-97 vs. 204/100,000 in 2001-03, NS) nor has the rate amongthose <5 yo. (100/100,000 in 1995-97 vs. 79/100,000 in 2001-03, NS).Conclusions: The rate of overall and VT IPD among Navajo children has fallendramatically since PnCRM7 introduction even during a secular trend of decliningproportion of VT IPD. Further analysis of NVT IPD among children and VT IPD inadults is ongoing.


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The cost of treating community-acquired pneumonia (CAP) in children < 2years of age in Bohol, the Philippines, a step towards cost-effectiveness (CE)analysis of an 11-valent pneumococcal conjugate vaccine (11PCV)

Ocampo AF1, Parreño RN1, Nohynek H2, Riley I3, Lucero MG1 for the ARIVACconsortium. 1Research Institute for Tropical Medicine, Philippines, 2National Pub-lic Health Institute, Finland, University of Queensland, Australia3

Aims: To estimate the medical cost, non-medical cost, and indirect caregiver-bornecost per episode of CAP in young children; and to describe the current prescribingpractices of physicians in Bohol in the treatment of CAP.Methods: This study is part of a CE study nested within a phase 3 trial of 11PCVagainst pneumonia in children. Resource utilization data were collected from chil-dren < 2 years of age consulting to a tertiary care government hospital and threeprivate hospitals in Bohol after fulfilling the entry criteria based on the WHO algo-rithm for identifying pneumonia cases. Costs per unit of resource used were com-puted based on the Children’s Vaccine Initiative’s protocol for costing of healthfacilities.Results: 484 cases of CAP were analysed from October 2001 to March 2003. Totaltreatment cost of cases attended at outpatient clinic was US$15 while that of admis-sions ranged from US$48-US$82 and US$80-US$105 in government and privatehospitals, respectively. More than 90% of the total costs were due to medical costswhere room and drug costs were the main drivers. Anti-asthmatic preparations wereprescribed to 61% of children who had no wheeze.Conclusions: There is a high variability in the costs among the hospitals studied,which may be attributed to different physician practices and to the unit costs of theresources utilized. Rationalizing administration of drugs could reduce medical costsand further help families with children experiencing several episodes of pneumoniain a year. This could result in more accurate CE analysis of introduction of PCV insuch settings.

Streptococcus pneumoniae as a main causative agent of pneumonia in service-men of the Russian army

Ogarkov P.I.1, Zhogolev S.D.1, Jogolev K.D.1, Vasilenko A.J.2, Sologub T.S.3, FaustovaM.E.3, Vishnyakova L.A.3 Military Medical Academy1, Pasteur Epidemiological andMicrobiological Research Institute2, Pulmonology Research Institute Pavlov Medi-cal Academy3, St.Petersburg, Russian Federation.

Aims: To determine the etiology of community-acquired pneumonia (CAP) amongservicemen in Russia.Methods: The sputum and serum samples of 378 servicemen with CAP were exam-ined with bacteriological and serological analyses.Results: Etiology was established in 68.3% of the cases. Streptococcus pneumoniaewas appeared to be a main infectious agent. Pneumococcal pneumonia was diag-nosed in 46.6% of all patients examined (or in 68.2% cases with established etiology).Haemophilus influenzae was detected in 41.1 % of all pneumonia cases observed,Chlamydia pneumonia - in 12.4%, Mycoplasma pneumonia – in 4.7%, some otherpathogens (Branhamella catarrhalis, Staphylococcus aureus, Pseudomonasaeruginosa, Moraxella lacunata, etc.) - in 3.5%, viruses (influenza A and B,parainfluenza, adenovirus, rhinovirus) - in 7.8% - both as a single etiological agentand as a mixed infection, most often with S.pneumoniae. Mixed infections accountedfor 24.1% among all patients examined (35.3% in the cases with established etiology).S.pneumoniae was revealed in 74 out of 85 (87.1%) cases with mixed infectioncaused by two pathogens and in all six cases with 3 pathogens. Pneumococcus asso-ciated with H.influenzae was observed most frequently (in 52 out of 91 cases, 57.1%).Associations of pneumococcus with Ch.pneumonia amounted to 19.8% (18 cases);with viruses – 7.7% (7 cases); with M.pneumonia – 5.5% (5 cases), with other bac-teria – 4.4% (4 cases).Conclusions: S.pneumoniae is a main causative agent of pneumonia in servicemenof the Russian army. It is suggested that other pathogens help pneumococcus tomanifest its pathogenic properties, to penetrate into the lower respiratory tract andaffect the lung tissue.


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EPI-66

Association of symptoms and signs with pneumococcal serotype in acute otitismedia

Palmu A1,2, Jokinen J1, Kaijalainen T3, Leinonen M3, Karma P4, Mäkelä PH1, andKilpi T1. National Public Health Institute, Helsinki1, University of Tampere2 andNational Public Health Institute, Department in Oulu3, Helsinki University Hospi-tal4, Finland.

Aims: Clinical symptoms and signs of acute otitis media (AOM) may vary with thepneumococcal serotype causing the disease. Wide-scale pneumococcal vaccinationcould thus change the clinical picture of AOM if the clinical pattern induced byvaccine serotypes was clinically distinct from that caused by non-vaccine serotypes.Methods: Children of the control arm (N=831) in the Finnish Otitis Media VaccineTrial were followed prospectively in eight study clinics from 2 to 24 months of age.If AOM was diagnosed, myringotomy was performed and middle ear fluid was as-pirated for culture of bacterial pathogens. Clinical symptoms and signs of AOMwere routinely recorded on structured case report forms.Results: Serotype distribution among 479 serotype-specific pneumococcal AOMepisodes was similar to that in the previous studies. In 60% of the episodes theisolates were of vaccine serotypes and in 79% of the episodes vaccine or cross-reactive serotypes were isolated. The clinical findings of AOM associated with dif-ferent serotypes and serotype categories were largely similar. Only earache wasmore common in episodes due to vaccine and cross-reactive serotype compared toall other serotypes (42% vs. 29%, respectively, difference 13%; 95% CI 2-24).Conclusions: Introduction of pneumococcal conjugate vaccine and the potentialserotype replacement is unlikely to result in any remarkable alteration in clinicalfindings of AOM attacks in infants.

Evidence for clonal spread of levofloxacin resistance in invasive Streptococcuspneumoniae in the United States

Mathias W. R. Pletz1,2, Lesley McGee1,2, James H. Jorgensen3, Bernard Beall2, Ri-chard R. Facklam2, Cynthia G. Whitney2, Keith P. Klugman1,2 and The Active Bac-terial Core Surveillance Team2. Rollins School of Public Health1, Centers for Dis-ease Control and Prevention2, Atlanta, GA, and University of Texas Health ScienceCenter3, San Antonia, TX, USA

Aims: This study characterized invasive levofloxacin-resistant (MIC 8mg/l) Strep-tococcus pneumoniae isolates obtained from the Active Bacterial Core Surveil-lance from 1998-2002.Methods: Isolates were characterized with respect to capsular serotypes, antimicro-bial susceptibility profiles, pulsed-field gel electrophoresis (PFGE), and multilocussequence typing (MLST). The quinolone resistance-determine regions (QRDRs)of the gyrA, gyrB, parC and parE genes were sequenced and isolates were testedfor efflux mechanism.Results: Fifty out of 15,452 isolates (0.32%) with a levofloxacin MIC�8mg/l weredetected. Within these 50 isolates 17 (34%) serotypes were represented, withserogroup/type 6, 9, 14, 19, and 23 accounting for 32 (64%) of isolates, only three(6%, all serotype 35B) isolates were not covered by the 23 valent vaccine. Eighteen(36%) isolates were co-resistant to penicillin, 22 (44%) to macrolides and 14 (28%)exhibited co-resistance to both. Twenty-five (50%) isolates were resistant to at leastthree drug classes. Of the six fluoroquinolones tested gemifloxacin was the mostactive against these levofloxacin-resistant isolates (MIC

90: gemifloxacin <

garenoxacin < moxifloxacin < gatifloxacin < levofloxacin < ciprofloxacin). Iso-lates within the same PFGE cluster frequently exhibited the same or a very similarmutation pattern in their QRDRs. In eight (16%) isolates there was evidence of anactive efflux mechanism. PFGE grouped 39 (78%) isolates into 11 clusters. MLSTrevealed that five of the international clones as identified by the PneumococcalMolecular Epidemiology Network accounted for 24 (48%) of the strains. TheSpain23F-1 clone was the most frequent (n=8, 16%).Conclusion: Our results suggest that there is considerable evidence for the clonalspread of levofloxacin resistance among invasive Streptococcus pneumoniae iso-lates in the US in contrast to earlier surveillance studies reporting a lack of clonalityin respiratory isolates.


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EPI-68

Genetic Relatedness Between Serotypes of Penicillin NonsusceptibleS. pneumoniae (PNSP), Unrelated to the Conjugate Vaccine (non-VT), andSerotypes Included in the vaccine (VT).

Porat N1, Arguedas A2, Brilla E2, Trefler R1,Dagan R1. 1Ben-Gurion University ofthe Negev, Beer-Sheva, Israel; 2Universidad de Ciencias Medicas, San José CostaRica.

Background: PNSP are confined mainly to a few serogroups. Capsular transforma-tion may serve as a mechanism for spreading antibiotic resistance to new serotypes.Methods: We analyzed 148 S. pneumoniae (Pnc) strains including 45 isolates ex-pressing serotype 11A and 46 isolates expressing serotype 15B/C, isolated during1998-2003 from Israeli children <3 yrs, and 57 isolates expressing serotype 19Fisolated during 1998-2001 from Costa Rican children <7.5 yrs. All isolates werecharacterized by antimicrobial susceptibility and pulsed field gel electorophoresis(PFGE) generated by SmaI and ApaI digestions.Results: PFGE profiles showed that 49/57 (86%) of seroytpe 19F strains, and 19/46(41%) of seroytpe 15B/C strains belonged to the same clone (0-3 band difference),indicating the similarity in the genetic background of the 2 serotypes. The vastmajority of the strains belonging to this clone (80% and 100% for serotype 19F and15B/C respectively) were PNSP. For serotype 11A, the PFGE profiles showed that8/10 (80%) of the PNSP strains belonged to a single clone, which was very similarto the international Spain9V-3 clone (maximum 2 band difference), indicating thatthese 2 clones are closely related.Conclusions: The appearance of PNSP non-VT clones may be due to capsular trans-formation. Capsular transformation of “international clones” expressing VTserotypes (9V/14 and 19F), with non-VT, even though appears to be rare at present,can have implications on the long-term effectiveness of the conjugate vaccine. Onepossible consequence of the phenomenon may be serotype replacement followingmass vaccination, with emergence of important PNSP non-VT clones.

Clinical review as a tool to improve the specificity of diagnosis of lower respira-tory tract infection (LRTI) used as a tertiary endpoint in pneumococcal conju-gate vaccine effectiveness study

Puumalainen T1, Ladesma E2, Heiskanen-Kosma T1, Quiambao B2, Laot T2, Feroldi,E3, Lupisan S2, Simoes E4, Lucero M2 , Nohynek H1, Riley I5, for ARIVAC consor-tium. 1National Public Health Institute (KTL), Finland; 2Research Institute for TropicalMedicine, Philippines, 3Aventis Pasteur, France, 4University of Colorado, USA, 5Uni-versity of Queensland, Australia

Aims: The WHO/IMCI definition for pediatric LRTI (cough and/or difficult breath-ing and fast breathing) is sensitive but not specific screening tool to identify chil-dren in need of medical management. ARIVAC consortium uses this definition asentry point to case ascertainment in effectiveness trial of 11-valent mixed-carrierpneumococcal conjugate vaccine in Bohol, the Philippines. Specificity of diagnosisis more important than sensitivity when measuring the effect of a vaccine againstthe main study endpoint -pneumonia, which is only partly caused by pneumococci.The study team is now reviewing all hospital admissions in order to identify thosepatients who had 1) diseases other than LRTI; 2) diseases predisposing to pneumo-coccal infections; and 3) LRTI further defined in more specific diagnostic catego-ries, in which the proportion of infections caused by pneumococci, and thus thevaccine effectiveness, might differ.Methods: Based on available validated clinical, laboratory and radiological data agroup of pediatric infectious diseases specialists developed standard ICD-10 codeddefinitions for bacterial and viral pneumonia, non-specific LRTI, bronchiolitis andasthma. The method was tested in two pilot reviews prior to currently on-goingreview of all hospital admissions.Results: Up to June 2003, 10,662 infants have received the study vaccine or pla-cebo. In total 1009 hospitalised patients have fulfilled the WHO definition for LRTI.Of those 896 cases had a discharge diagnosis of pneumonia. The results of the re-view of approximately 500 first patients will be available in March 2004.Conclusions: The experience from the pilot reviews suggests that structured expertreview of patient data will improve the specificity of diagnoses and thus facilitatethe understanding on vaccine effect on childhood pneumonia.


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EPI-70

Abstract withdrawn

Epidemiology of invasive pneumococccal disease (IPD) in the pediatric popu-lation of Germany: Nationwide surveillance, 1997-2002

Reinert RR1, Al-Lahham A1, Siedler A2, Toschke M3, Cil MY1, Lütticken R1, vonKries R3. 1Institute of Medical Microbiology and NRCS, Aachen, 2Robert KochInstitute, Berlin, 3Institute for Social Pediatrics, Munich, Germany

Aims: A population based nationwide study was initiated in 1997 to monitor theepidemiology of invasive pneumococcal disease (IPD) in the pediatric population(below16 years of age) of Germany.Methods: All microbiological laboratories and pediatric hospitals were asked inde-pendently to report IPD and to send the isolates to the NRCS for confirmation ofspecies diagnosis by optochin and bile solubility testing and for serotyping by theNeufeld Quellung reaction. Incidence rates were calculated by capture-recapturemethod. MICs were tested by the microdilution method according to NCCLS.Results: During the period January 1997 to December 2002, a total of 2673 caseswere reported either by hospitals or by laboratories. The incidence of IPD amongchildren < 2 years of age was higher than in older age groups and increased from18.2 in 1997 to 21.4 in 2002 per 100,000 children. Predominant serotypes in allcases were: 14 (24.6%), 23F(7.6%), 1 (7.4%), and 18C (7.1%). 57.9% of childrenwith IPD were < 2 years of age (meningitis pts 60.2%) and 80.9% were < 5 years.Coverage rate by the 7-valent pneumococcal conjugate vaccine (7v-PCV) with thecross reactive serotype 6A was 72.1% (age group 6-11 months) and 78.0% (agegroup 1-<2 years). Coverage of the 7v-PCV in the age group 2-<5 years (74.8%)was comparable to the age group 1-<2 years. Coverage of the 11v-PCV (plus 6A)in all cases was 81.0%. The resistance rate (intermediate, resistant) to penicillinwas (5.1%, 0.5%) and to erythromycin (20.2%, 0.1%).Conclusions: Incidence data are on a comparable level with other developed coun-tries. The level of penicillin resistance among pediatric IPD in Germany is one ofthe lowest worldwide. Vaccination of children at high risk is advisable.


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EPI-72

Invasive pneumococcal diseases in age group 0-36 months: results from a per-spective surveillance program in Nothern-eastern Italy.

Romano G1, Poli A1, Tardivo S1, Chiamenti GP2. Department of Medicine and Pub-lic Health-University of Verona1, 2Italian Federation of Family Paediatricians

Aims: The purpose of this study was to provide an estimated incidence of invasivepneumococcal diseases (IPDs) in the age group 0-36 months within the Northern-eastern area of Italy.Methods: The survey, started up on October 2002, involved three Italian Regions ofNorthern-eastern area of our country (Veneto, Friuli Venezia-Giulia and Trentino-Alto Adige). It was randomly selected a sample of 114 family paediatricians (10%of total paediatricians in the area), integrated by a pool of paediatricians to substi-tute any drop-out. Eighty-seven paediatricians, among selected, accepted to be in-volved (76,3%), with a total number of 20,373 observed children < 36 months aged.A protocol was defined to enroll all suspected cases and for their diagnosis, throughidentifying highly selective criteria, specific for age groups and completed by labtests performing as outpatients (C Reactive Protein and test kit for urinary tractinfections exclusion). The criteria for case definitions have been conditioned bypresence or absence of focal infections. In the first situation, the classic symptomsof IPDs (meningitis, osteoarthritis, pneumonia, cellulitis) were considered. If a fo-cal infection was absent, the Yale’s Scale Score was adopted for children aged < 36months suffering from a persistent and/or intermittent fever. In these cases, a nega-tive urinary test, if associated with high values of C Reactive Protein (� 40 �g/mL), would point out need for a blood culture, that has been performed in refer-enced hospital departments.Results: During the first 12 months of survey, 39 suspected cases were reported bysentynel paediatricians, with 12 positive blood cultures. The estimated yearly cu-mulative incidence was equal to 58.90 cases/105 children aged < 36 months (C.I.95%:30.38-102.71). The reported pathologies were, in a frequency order, occultbacteriemias (7), meningitis (2), sepsis (2) e pneumonia (1).Conclusions: The survey showed a significant frequency of IPDs in the age group0-36 months, close to French (59.2 cases/105 children/year in the age group 0-24months) and Spanish data (59.6/105 children/year).

Impact of the Severity of Winter Influenza-like Illnesses (ILIs) and HeptavalentPneumococcal Conjugate Vaccine (PCV7) on Invasive Pneumococcal Infections(IPD) in Children and Adults at a Health System in New York

Rubin L,1, 2 Shafinoori S,1, 2 Ginocchio CC,2 Greenberg AJ,3 Yeoman E,3 Cheddie M.3

Schneider Children’s Hospital, New Hyde Park NY, 2 Dept Laboratory Medicine, 1,

2North Shore-Long Island Jewish Health System, Lake Success, NY, and 3 NassauCounty Department of Health, Mineola, NY.

Aims: Viral upper respiratory ILIs predispose to IPD and annual variation in theseverity of ILIs may influence the rate of IPD. We hypothesized that annual varia-tion in the severity of ILIs may confound interpretation of the impact of PCV7 onrates of IPD in children and adults.Methods: The annual cases of IPD and emergency department (ED) visits of pa-tients in seven hospitals of a health system in New York during the 2 years prior to(7/98-6/00) and 3 years since (7/00-6/03) routine use of PCV7 in young childrenwere tabulated. The annual severity of ILIs was studied using prospective surveysof cases of ILIs at all 13 acute care hospitals and absences from school due to ILIsfrom all schools in Nassau county where most of the system hospitals are located.Results: Sixty-seven percent of the 634 IPD cases occurred during the 6 months ofNov-April that include the winter. Compared with the pre-PCV7 years, there were67 & 72% reductions (P<0.0001) in the rates of IPD episodes (cases/105 ED visits)in children <2 y & 2-4 y of age, respectively and 36 & 28% reductions in the rate ofIPD in adults 18-49 y (P<0.05) & >64 y (P<0.01), respectively, during the PCV7years. During the three PCV7 winters, the mean seasonal episodes of ILI reportedby hospitals (3,455) was 47% lower than during the pre-PCV7 winters (1,842;P<0.0001) & the mean seasonal episodes of ILI at schools was18% lower (meanpre-PCV7, 11,065 vs PCV7 period, 9,076; P<0.0001).Conclusion: In addition to efficacy of PCV7 and herd-type immunity, the milderwinter respiratory viral seasons during the PCV7 period may have contributed to theobserved reduction in rates of IPD.


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EPI-74

The majority of drug-resistant pneumococci isolated from children attendingday-care centers in two cities of Portugal are representatives of several interna-tionally disseminated clones

Sá-Leão R1, Sousa NG1, Nunes S1, Simas C1, Mato R1, Frazão N1, Crisóstomo I2,Santos-Sanches I1,3, de Lencastre H1,2. Instituto de Tecnologia Química e Biológica1,Oeiras, Portugal; The Rockefeller University2, New York, USA; Centro de RecursosMicrobiológicos3 da Universidade Nova de Lisboa, Monte da Caparica, Portugal.

Aims: A total of 1288 drug-resistant Streptococcus pneumoniae (DRPn) were re-covered from nasopharyngeal samples of children attending day care centers inLisbon and Oeiras, Portugal, between 2001-2003. Molecular typing of all strains bypulsed-field gel electrophoresis (PFGE) identified 79 clones. Of those, 28 had fiveor more isolates and accounted for 91% of the drug-resistant strains. The purpose ofthis study was to compare by multilocus sequence typing (MLST) the genetic back-ground of these 28 clones with those of other strains recovered in several countriesand deposited in the MLST database.Methods: Multilocus sequence typing was done on 55 strains representative of 28DRPn clones with more than five isolates.Results: Of the 28 clones defined by PFGE, 13 had sequence types typical of 10PMEN (Pneumococcal Molecular Epidemiology Network) clones. Ten additionalclones had sequence types that matched or differed at a single locus from allelicprofiles available at the MSLT database. The majority of these strains were invasiveand all were from countries distinct from Portugal. The remaining five clones (ac-counting for 42 strains) had unique sequence types not described so far.Conclusions: The majority (at least 88%) of the strains identified in our study be-long to 23 clones which were also identified in other countries and which can bothcolonize and cause disease. The nasopharynx of children attending day care centersis a suitable reservoir to improve our understanding of the pneumococcal popula-tion structure.

Clinical and epidemiological characteristics of children with invasive Pneu-mococcal infection in Manhiça, a rural area of Southern Mozambique.

B.Sigaœque1, A. Roca1,2, I. Mandomando1, S. Sanz1,2, M. Levine3, P.Alonso1,2. HealthResearch Center, Manhiça, Mozambique1, Centre de Salut Internacional (CSI), Hos-pital Clinic, IDIBAPS, Barcelona, Spain2, Center for Vaccine Development, BaltimoreMD, USA3.

Aim: In African children, Pneumococcus is a leading cause of bacteraemia, pneumo-nia and meningitis. High prevalence of HIV and malnutrition are responsible for anincreased incidence of invasive infections by Pneumococcus in such continent com-pared to developed countries. The aim of our study was to describe the clinical presen-tation of the children admitted to Hospital with invasive Pneumococcal disease inManhiça, a rural area of southern Mozambique.Methods: As part of clinical management of children admitted to Hospital, prospectivesurveillance for invasive bacterial infection is ongoing at the Manhiça District Hospital.Hereby, we have analysed clinical samples routinely collected from children admittedto wards during two calendar years (from January 2001 to December 2002).Hemocultures were performed to all admitted children <2 years of age or with axillarytemperature >39ºC. Besides, spinal fluid samples (SFS) were cultured from children <5years of age admitted to hospital with neurological symptoms.Results: During the years 2001 and 2002, more than 12.000 children were admitted tohospital. From them, 10.409 hemocultures were performed. Pneumococcus was thesecond bacteria most commonly found, accounting for 214 isolations in the two yearsperiod. The table below shows the most frequent clinical symptoms associated withPneumococcal invasive infection. Detailed resulsts of the clinical characteristics andoutcomes, age distribution and seasonal patterns of the Pneumococal invasive infec-tion will be presented. Results on the findings of the SFS will be presented also.

-: Not found; +: unfrequently found; ++: sometimes found; +++: frequently found; ++++: always found

Conclusions: Our results show that Streptococcus pneumonia is one of the most im-portant bacterial infections in children and infants in Manhiça. Our data support thenecessity for a vaccine that should be effective since a very young age.


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EPI-76

Streptococcus pneumoniae: Outcome of patients and antimicrobial susceptibil-ity patterns of isolates in South Africa in 2003.

Soma K1, Quan V1, von Gottberg A1, de Gouveia L1, Madhi SA1, Schuchat A2,Klugman KP1, 3 and the national surveillance network. 1RMPRU (MRC/WITS/NICD), Johannesburg, South Africa; 2Centers for Disease Control and 3Departmentof International Health, Emory University, Atlanta, Georgia, USA

Aims: To analyse the outcomes of patients with invasive pneumococcal disease (IPD)in South Africa and to review the susceptibility patterns of the isolates from theseepisodes.Methods: Isolates from IPD were referred to a reference laboratory. Clinical infor-mation was collected from selected centres. Antimicrobial susceptibility testing wasperformed according to NCCLS guidelines.Results: 2378 cases of IPD were reported to the reference laboratory by 1 Decem-ber 2003. The outcome was known for 1069 patients, of these 342 (32%) died inhospital. The case fatality rates (CFR) for different age groups were: <5 years, 111/390 (28%); 5- to 24-years, 39/184 (21%); 25- to 44-years, 125/348 (36%); 45- to64-year-olds, 55/108 (51%); >65 years, 8/25 (32%); P<0.001. HIV serostatus wasknown in 711 patients, with 621 (87%) being infected. The clinical diagnosis waspneumonia in 596/1090 cases (55%) and meningitis in 437/1090 (40%). Of caseswith meningitis, 130/159 (82%) were HIV infected, and with pneumonia, 358/401(89%), P=0.02. The CFR in cases with meningitis was 142/294 (48%), and in pneu-monia 135/578 (23%), P<0.001. HIV infection is associated with mortality in casesof meningitis, (58/123, 47% vs 4/26, 18%, P=0.006). Adults (15 years and older)with meningitis (91/155, 59%) have a higher CFR than children <15 years, (50/136,37%) P<0.001. Penicillin resistance was documented in 186/696 (27%); and wasmore prevalent in children <15 years (120/308, 39%) than in adults (49/290, 17%),P<0.001. Thirteen (2%) isolates (2 from CSF) of 696 tested were intermediatelyresistant to ceftriaxone.Conclusions: The proportion of cases with meningitis is high, and these cases havea high mortality. The presence of resistance to third generation cephalosporins is ofconcern.

Surveillance for radiologically confirmed pneumonia and pneumococcal inva-sive disease among children 0-23 months of age in Manhiça, Mozambique

Soriano-Gabarró M1, Sigauque B2, Mandomando I2, Flannery B1, Feikin D1, CorachanM3, Roca A23, Schuchat A1, Alonso PL23. CDC, Atlanta GA1, Manhiça Health Re-search Center, Manhiça, Mozambique2, Centre de Salut Internacional (CSI), Hospi-tal Clinic, IDIBAPS, Barcelona3

Aim: Since July 2003, a prospective hospital-based surveillance of pneumonia andpneumococcal invasive disease among children 0-23 months of age is currently be-ing implemented in Manhiça, Mozambique, following a generic protocol to esti-mate the burden of pneumonia and pneumococcal disease among young children.The main purpose of the project is to estimate the incidence of hospitalized clinical,radiographic pneumonia and pneumococcal invasive disease, and measure the po-tential impact of new conjugate pneumococcal vaccines in an African country.Methods: The clinical case definition of pneumonia relies on WHO IMCI criteriaand the radiological definition is based on the criteria used in the pneumococcalconjugate vaccine clinical trials (consolidation or alveolar infiltration with or with-out pleural effusion). All severe pneumonia cases are hospitalized. A chest x-ray isrequested within 48 hours of admission and blood cultures are performed on allchildren 0-23 months of age. Cerebrospinal fluid is collected on children with sus-pected meningitis. Chest X-rays are interpreted by a local clinician and a radiolo-gist. Discordant readings are decided by WHO radiologists.Results: The Manhiça District has a population of 130,350; 68,700 of which arepersons in a demographic surveillance area with ongoing population census (9743children less than 5 years of age live in this area). The Manhiça Hospital covers thispopulation. Hib vaccine is not in use. In 2001, 13,783 children were seen at theManhiça Hospital (of which 3570 were hospitalized). Visits and admissions for 2003as well as incidences of clinically defined pneumonia, severe pneumonia andradiologically confirmed pneumonia cases are being assessed. The role of bacteremicpneumonia and pneumococcal invasive disease is also being measured. Data corre-sponding to the 6 first months of surveillance will be presented.


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Properties of novel international drug-resistant pneumococcal clones identi-fied in day-care centers (DCCs) of Lisbon, Portugal

Sousa N G1, Sá-Leão R1, Nunes S1, Simas C1, Frazão N1, Mato R1, Crisóstomo I2,Santos-Sanches I1, de Lencastre H1,2. Instituto de Tecnologia Química e Biológica1,Oeiras, Portugal; The Rockefeller University2, New York, USA

Aims: To describe the properties of 10 drug-resistant pneumococcal (DRPn) cloneswith epidemic behavior, recovered from nasopharyngeal samples of children at-tending DCCs in the area of Lisbon, Portugal, between 2001-2003.Methods:Strains were characterized by antibiogram, serotyping, pulsed-field gel electrophore-sis and multilocus sequence typing (MLST).Results: Ten epidemic clones accounted for 231 DRPn strains (18% from a total of1,284 DRPn), being the majority disseminated in several DCCs. All strains wereresistant to erythromycin often associated with resistance to clindamycin (81.4%)and tetracycline (68.4%). Three clones with serotypes 19A, 19F and non-typeable(NT) had intermediate resistance to penicillin. The remaining seven clones werefully susceptible to this drug, expressing serotypes 6A, 6B (2 clones), 19A, 10, 33F,and NT. All ten clones had sequence types (ST) equal or related to those of otherisolates distributed internationally (mainly in Europe) and deposited in the MLSTdatabase. The majority of the international isolates had been recovered from inva-sive sources.Conclusions: These ten clones are of diverse capsular types and have the potentialto colonize and cause invasive disease. We propose they should be considered forinclusion in the Pneumococcal Molecular Epidemiology Network (PMEN); theserotypes and STs are the following: 6A ST892, 6B ST469, 6B ST176, 19A ST230,19A ST416, 19F ST89, 10 ST 1154, 33F ST717, NT ST344, NT ST180.

Hospital surveillance underestimates the incidence of bacterial meningitis inchildren in South Asia

Steinhoff MC, Minz S, Balraj V, Lalitha MK, Murali N, Cherian T, Joseph A. CMCHospital, Vellore, India; and Johns Hopkins University, USA.

Aims: To assess of the completeness of hospital surveillance for childhood menin-gitis in South Asia, home to 167 million children.Methods: Prospective surveillance in part of a single district in India, with 56,153children under 5 years of age during 1998 and 1999. Surveys showed 3 of 33 hospi-tals and one of 88 medical practitioners did lumbar puncture (LP) in children. Men-ingitis cases were identified in 3 hospitals by daily review of ward and laboratoryrecords. A survey of all childhood deaths was done simultaneously, and hospitalroutines were evaluated.Results: We found 97 cases of possible meningitis (> 10 WBC ml in CSF), [inci-dence/100,000 children per year = 86]. 34 of these cases were probable bacterialmeningitis (> 100 WBC/ml CSF), and 16 had laboratory bacterial isolates [incid =14]. There were 6 deaths in hospitalized children with possible meningitis. Thesurvey of childhood deaths showed 8 deaths with meningitis/encephalitis symp-toms [incid = 40] but none had a LP. The rate of possible meningitis cases/100childhood admissions per year ranged from 0.1 to 3.5 in the 3 surveyed hospitals.The laboratory-proven bacterial meningitis cases ranged from 3% to 25% of allpossible meningitis cases in hospitalConclusions: Hospital surveillance in settings with variable access to medical care,and differing diagnostic routines for LP will underestimate childhood meningitis.Our evaluation of deaths outside hospitals, and admissions without LP suggest hos-pital surveillance alone underestimated bacterial meningitis incidence by approxi-mately 50% in this region. These data are similar to observations on missed menin-gitis cases reported from London in the 1990’s. Surveillance studies for bacterialmeningitis in South Asia should included verbal autopsy for all deaths out of hospi-tal, and measure local variation in LP practice, including rates of LP in populationsand in individual hospitals [supported by V&B, W.H.O]


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EPI-80

Pneumococcal serotypes/serogroups causing invasive disease in children of SouthAsia: limited data reveals substantial variation between countries.

Steinhoff MC, Ganesan A. Bloomberg School of Public Health, Johns HopkinsUniversity, USA.

Aims: To assess the distribution of pneumococcal serotypes/serogroups [STG] pub-lished from the 8 countries of South Asia, which have 167 million [27%] of theworld’s children. These data are needed to estimate potential pneumococcal vaccineeffectiveness.Methods: We reviewed publications in English after a systematic search throughPubMed and other sources to find reports of pneumococcal STG in childhood inva-sive disease in South Asia. From 1976-2002 we found 10 reports with 1105 isolatesfrom 11 hospital centers in 3 countries.Results: Bangladesh had 539 isolates from 1 hospital, Pakistan 168 from one re-gion, and India 396 from 8 cities. The rate of isolates per million children varied 10fold among these 3 countries. There are apparently no serotype data available fromAfghanistan, Bhutan, Maldives, Nepal and Sri Lanka. There was little similarity inthe rank ordering of common serotypes among the 3 countries. For example, the top3 serotypes (33 to 53% of the individual country isolates) are unique for each coun-try. Using these published data, the proportion of invasive disease potentially pre-ventable with 7 valent/11 valent conjugate vaccines using these data is 53/55% forPakistan, 27/51% for Bangladesh, 27/79% for India. Data on STG distribution fromadult invasive disease were available from India only.Conclusions: Of all global regions, S. Asia has the lowest rate of published serotypedpneumococcal isolates/million residents. These limited published data may not beadequate to assess potential vaccine effectiveness in these neighboring countries,because of variations in sampling of populations, and in diagnostic routines forobtaining blood culture and lumbar puncture. Expanded surveillance of pneumo-coccal disease among children and adults in the South Asian region is needed todevelop national vaccine and antibiotic policies.

Invasive pneumococcal disease in Indian children: Distribution of serotypes/serogroups in 6 cities 1993-2003.

Thomas K, Lalitha MK, Steinhoff MC, for the IBIS project group, Indian ClinicalEpidemiology Network, INCLEN, India and USA.

Aims: Because serotype distribution of pneumococcal disease varies over time,across geography, and by age group, we carried out prospective hospital surveil-lance among Indian children with invasive pneumococcal disease, to determine se-rotype distribution relative to vaccine serotypesMethods: Children with clinical or laboratory criteria for meningitis, pneumoniaand other invasive infections were enrolled at hospitals in Delhi, Lucknow, Mumbai,Nagpur, Thiruvananthapurum and Vellore from 1993-2003. Standard serotyping/grouping of isolates was done with antisera obtained from Statens Seruminstitut(Denmark).Results: These 6 teaching hospitals have a total of 12,000 beds, and admit 245,000both local and referred patients annually. 1,526 clinical specimens were obtainedfrom children and we present preliminary data on 184 characterized isolates. Therewas only 1 isolate of serogroup 9 (7 valent) in this broad sample of Indian childrenand no isolates of serogroup 3 (11 valent). Serotypes among CSF isolates werestatistically different from blood isolates. Potential effectiveness of the current 7valent/proposed 11 valent conjugate vaccines is 35%/77% in children under 5 monthsof age; 57%/67% in children 6 to 11 months, and 52%/72% in children 12 to 59months, respectively. The proportion of serotypes covered by the 7 valent vaccine issignificantly lower in the 0 to 5 month age group, compared to the older childrenConclusions: The distribution of serotypes in this multicenter study had substantialvariation by age group and by anatomic site. However, there was little variation overthe 10 year period of potential overall effectiveness against invasive disease by the 7valent vaccine serotypes. Continuing surveillance is needed for vaccine and antibi-otic policy. Inclusion of local pneumococcal serotypes in proposed vaccines mayimprove protection of the 116 million Indian children, who are 19% of the world’schildren. [Supported by INCLEN, and USAID]


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Epidemiologic surveillance of pneumococcal disease in children from twomonths to two years of age in Cordoba city, Argentina; between 1999-2002.

M. Tregnaghi1, A. Ceballos1, R. Rüttimann1, J. Ussher1, P. Peeters2; 1Hospital InfantilMunicipal, Cordoba, ARGENTINA, 2GlaxoSmithKline Biologicals, Rixensart, BEL-GIUM.

Aim: This study was designed to investigate the incidence and impact of invasive pneu-mococcal disease in children from two months to 2 years of age in Cordoba city.Methods:This was a descriptive, 3-years prospective study. Results from these 3 yearsof survillance (Dec. 1999 to Dec. 2002) are reported. Fourty-two percent (42%) of thepediatric population in Cordoba city is covered by the public health system in twopediatric hospitals and 80 primary health care centers. All cases of invasive pneumo-coccal disease and obvious pneumonia meeting the following inclusion criteria wereincluded: children from 2- 24 months of age with residence in Cordoba city presentingaxillary temperature � 39ºC or clinically suspected pneumonia, meningitis, peritoni-tis, pericarditis, arthritis, or sepsis. Invasive pneumococcal disease and obvious pneu-monia were defined as S. Pneumoniae isolated from normally sterile body fluid orconsolidation >2.5 cm in chest X-ray respectively. Results:There were a mean of 28,737 children per year under survillance. During thethree years, a mean of 7,301 (25.4%) children from the study cohort fulfilled the inclu-sion criteria. There were 183 (0.83%) documented cases of invasive pneumococcaldisease and 2088 (9.53%) documented cases of obvious pneumonia. Invasive pneumo-coccal disease mean annual rate was 212.27/100,000, and 2421.96/100.000/year forobvious pneumonia. Stratified by age, 114.85/100,000, 242.47/100,000, 253.95/100,000and 208.01/100,000 strains of S. pneumoniae were isolated from children 2-5, 6-11,12-17 and 18-24 months old respectively. The three main invasive pneumococcal dis-ease diagnosed were pneumonia with S. pneumoniae isolation, occult bacteraemia andmeningitis which mean annual incidence were 99.77/100,000, 91.62/100,000 and 8.10/100,000 respectively . The 91.1% of the isolated serotypes in the cohort under surveil-lance is covered by the 11 serotypes included in the GSK´s conjugated pneumococcalvaccine under development (1,3,4,5,6B,7F,9V,14,18C,19F,23F). Two children died be-cause of meningitis (overall mortality 1.09%).Conclusion: We report a high incidence of invasive pneumococcal disease and obviouspneumonia in children less than 2 years of age in Cordoba city. The high burden ofpneumococcal and respiratory diseases in this age group can potentially be reduced byimmunizing young children with multivalent pneumococcal conjugate vaccines.

Surveillance of Streptococcus pneumonia infection in Arkhangelsk, Russia

Tulisov A, Buzinov R., Gordienko T., Grishina L.State Sanitary and Epidemiological Surveillance Centre in Arkhangelsk region,Arkhangelsk, Russia.

Aims: The purpose of the surveillance was to evaluate the burden of Streptococcuspneumonia infection and to define main groups at risk.Methods: Data of routine epidemiological surveillance of laboratory confirmedbacterial meningitis cases in Arkhangelsk city in 2000-2002 were used. Cases ofmeningitis cases caused by Streptococcus pneumonia from 1995 to 2002 were ana-lysed.Results: Forty six bacterial meningitis cases were revealed in 2000-2002 inArkhangelsk. In total 32,6% of all meningitis cases were caused by Streptococcuspneumonia, 43,5% of cases were caused by Neisseria meningitides and in 23,9% ofcases pathogen was not identified.Overall 31 cases of bacterial meningitis cased by Streptococcus pneumonia wereregistered in Arkhangelsk in 1998-2002. Incidence rate ranged from 0,5 per 100 000population in 1997 to 2,0 per 100 000 population in 2001. 20 cases (64,5%) oc-curred among adults 20 years of age and older, 3 cases (9,7%) occurred amongchildren at the age from 3 to 6 years and the same number of cases were registeredat the age from 7 to 14 years old, 2 cases (6,5%) occurred among children at the ageunder 1 year and 2 cases (6,5%) were registered at the age from 1 to 2 years. Only1 case (3,2%) was revealed at the age from 15 to 19 years old.Conclusions: The results of the surveillance showed the importance the problem ofmeningitis caused by Streptococcus pneumonia in Arkhangelsk. All age groups areaffected; adults over 20 appeared to be the main risk group. It is necessary to spreadthe surveillance system for Streptococcus pneumonia meningitis all over Arkhangelskregion and to establish the system for pneumonia caused by Streptococcus pneumo-nia surveillance. Laboratory diagnostics should be improved to identify the patho-gens, which causes meningitis in cases of ”unknown etiology”.


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EPI-84

Serotype distribution and antibiotic resistance of invasive and non-invasive Strep-tococcus pneumoniae in a rural area of southern Mozambique

Valles Casanova FX.1,2, Flannery B.3, Mandomando I.1, Sigauque B. 1, Roca A. 2, LevineM. 4, Feikin D3, Schuchat A. 3, Soriano-Gabarro M. 3, Alonso P.2

Manhiça Health Research Center, Manhiça, Mozambique1, Centre de Salut Internacional(CSI), Hospital Clinic, IDIBAPS, Barcelona, Spain2, Centers for Disease Control andPrevention, Atlanta GA3, Center for Vaccine Development, Baltimore MD, USA4.

Aim: to describe the serotype distribution and antibiotic resistance pattern of invasiveand non-invasive pneumococcal isolates from children in this area.Methods: we carried out a cross sectional nasopharyngeal carriage survey among chil-dren <5 years of age (median age= 1 year) attending to the outpatient department of theManhiça District Hospital, where prospective surveillance for invasive pneumococcaldisease is ongoing. Invasive pneumococci were isolated from blood and cerebrospinalfluid of children <15 years of age (median age= 2 year ) admitted to hospital betweenAugust 2002 and July 2003. Antibiotic susceptibilities were determined by disk diffu-sion and E-test and isolates were serotyped using the Quellung reaction. Proportionswere compared using the X2 test.Results: the prevalence of nasopharyngeal colonization was 87% among children <5years of age.Among 198 non-invasive isolates serotyped the five most commonly car-ried-serotypes were 19F ( 26%), 19A (8%), 23F ( 7%), 6A ( 7%) and 6B (6%). Among101/161 invasive isolates serotyped the most common serotypes were 1 (42%), 5(9%), 14 (7%), 6A (5%) and 23F (5%). Serotypes included in the 7-valent conjugatevaccine accounted for 52% of the non-invasive isolates but only 19% of the invasiveisolates; adding serotypes 1 and 5 increased the coverage of invasive isolates to 74%.Resistance to cotrimoxazole was prevalent: 60% of non-invasive and 42% of invasiveisolates were cotrimoxazole-nonsusceptible. Although 52% of non-invasive isolates werenonsusceptible to penicillin, only 18% of invasive isolates were nonsusceptible; nonewere fully resistant. Resistance to erythromycin and chloramphenicol was uncommon(2% each).Conclussions: we found a very high prevalencee of nasopharyngeal colonization amongchildren <5 years but a very different pattern of antibiotic resistance and serotype dis-tribution between carriage and invasive isolates. Due to the high proportion of invasivedisease caused by serotypes 1 and 5, the 9-valent conjugate vaccine formulation wouldprovide much greater coverage than the 7-valent vaccine in this population.

Radiographic Surveillance for Community Acquired Pneumonia

Watt JP, O’Brien KL, Whitney CG, Abboud-Donaldson R, Reid R, Ferro S, SantoshamM. Johns Hopkins University, Baltimore MD, USA; CDC, Atlanta, GA, USA; AventisPasteur Ltd., Toronto, Canada.

Aims: Where chest radiographs (CXRs) are routinely taken, radiographic surveil-lance could be an efficient way to identify pneumonia cases for research. This studyaimed to develop methods for radiographic surveillance in White Mountain Apacheadults, a population at high risk for pneumococcal disease.Methods: A retrospective review of CXR records was carried out at the primaryhospital serving the population. The report for each CXR taken of a person > 40years was reviewed to identify possible pulmonary abnormalities. Medical recordswere then screened, and episodes with at least one sign or symptom consistent withpneumonia and without nosocomial exposure were included. A standardized read-ing of acute, baseline and follow-up CXRs was done by a radiologist blinded toclinical information.Results: Of 1,634 CXRs taken, 1,600 (97.9%) were evaluated. The clinical CXRreport showed no pulmonary abnormalities in 1,272 (79.5%) and another 137 metexclusion criteria. Of 191 remaining episodes, 108 (56.5%) had a clinical pneumo-nia diagnosis and 136 (71.2%) had a clinical CXR reading consistent with pneumo-nia. There was often disagreement between clinical diagnosis, clinical CXR read-ing and standardized CXR reading. Of episodes with a pneumonia diagnosis, 94(87.0%) had a clinical reading consistent with pneumonia. The standardized read-ing from 92 episodes with a pneumonia diagnosis classified 36 (39.1%) as acuteinfiltrate, 15 (16.3%) as chronic changes, and 41 (44.6%) as no infiltrate. Thestandardized reading from 119 episodes with a clinical reading consistent with pneu-monia classified 49 (41.2%) as acute infiltrate, 16 (13.4%) as chronic changes, and54 (45.4%) as no infiltrate.Conclusions: We developed a method of radiographic surveillance for pneumonia.Standardized reading of CXRs often disagreed with clinical diagnosis or clinicalreading. Reproducible case definitions for adult pneumonia are needed for epide-miological research and clinical trials. The role of standardized CXR reading insuch case definitions should be further evaluated.


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EPI-86

Epidemiology of Pneumonia among White Mountain Apache Adults

Watt JP, O’Brien KL, Whitney CG, Abboud-Donaldson R, Reid R, Ferro S,Santosham M. Johns Hopkins University, Baltimore MD, USA; CDC, Atlanta,GA, USA; Aventis Pasteur Ltd., Toronto, Canada.

Aims: Few population-based studies have evaluated the epidemiology of commu-nity-acquired pneumonia (CAP). This study aimed to determine the incidence andclinical characteristics of CAP in a population at high risk for pneumococcal dis-ease. These data will be useful for the design of future vaccine trials.Methods: Possible cases were identified by retrospective review of radiology recordsat the primary hospital serving this population. Persons � 40 years of age withabnormal chest X-rays (CXR) were selected. Clinical CAP was defined as epi-sodes meeting the clinical case definition and having a diagnosis of pneumonia.Radiographic CAP was defined as episodes meeting the clinical case definitionwith evidence of an acute infiltrate on standardized CXR evaluation that was blindedto clinical information.Results: The screening procedure identified 191 episodes of possible CAP in 3,301person-years. Of these, 100 (30/1,000 person-years) were clinical CAP. CXR re-view was completed for 89.0% of episodes meeting the clinical definition. Of these,57 (17/1,000 person-years) fulfilled radiographic criteria. The incidence of radio-graphic CAP was higher for persons � 65 years compared with those 40-64 years(35 vs. 14/1,000 person-years, respectively). The incidence was similar for malesand females (14 vs. 20/1,000 person-years, respectively). In the radiographic CAPgroup there were 30 (52%) hospitalisations and 2 (4%) deaths. Blood and sputumculture were obtained from 27 (1 pneumococcal isolate) and 19 (7 pneumococcalisolates) radiographic CAP episodes, respectively. Of persons with radiographicCAP, 32 (56%) had received pneumococcal polysaccharide vaccine from 0-21 (me-dian 0.6) years prior to the episode.Conclusions: CAP is approximately 2-3 times more common in this populationthan in the general US population. The incidence is particularly high among per-sons 65 years and older. The proportion of CAP due to pneumococcus is not known.Additional studies are underway to determine the burden of pneumococcal CAP.

Serotypes and molecular clones responsible for invasive pneumococcal diseaseand discharging ears in Metropolitan NSW, Australia - Implications for theeffectiveness of a 7 valent conjugate vaccine program in Australia.

Watson M1, Brett M1, Warren S1, Brown M1, Stewart MG1 for the NSW Pneumo-coccal Network, The NSW Pneumococcal Reference Laboratory, The Children’sHospital at Westmead, Sydney, Australia1.

Aims: The purpose of this study was to examine the pneumococcal serotypes andmolecular clones responsible for penicillin resistance in invasive pneumococcaldisease and discharging ears in children less than 15 years of age in metropolitanNew South Wales, Australia.Methods: All microbiology laboratories in metro NSW prospectively referred iso-lates of Streptococcus pneumoniae from sterile body sites and discharging earsbetween July 1st 2000 and June 30th 2003 to the NSW State Reference Laboratory.Isolates identified routinely as having reduced susceptibility to penicillin were con-firmed using NCCLS disc diffusion and E test. A representative sample of penicil-lin resistant isolates were typed using multi-locus sequence typing (MLST) and thecorresponding Box PCR patterns determined. The majority of penicillin resistantisolates were then screened using Box PCR and those isolates with atypical BoxPCR patterns were also subjected to MLST.Results: 607 of 689 ear isolates and 708 of 736 invasive isolates were available forserotyping. Serotype 19F accounted for 34.4% of all ear isolates and 68.8% ofpenicillin resistant ear isolates and 13.3% of all invasive isolates and 30.1% ofpenicillin resistant invasive isolates. Multiple variants of the Taiwanese 19F clonepredominated including ST 320 and ST 352. Serotype 14 accounted for 35% of allinvasive isolates and 19.6% of penicillin resistant invasive isolates but only 9.4% ofall ear isolates and 7% of penicillin resistant ear isolates. The predominant penicil-lin resistant molecular clone of serotype 14 was ST 156.Conclusions: The predominance of serotype 19F as a cause of penicillin resistantdischarging ear isolates is important due to the reduced efficacy of the 7 valentconjugate vaccine for this serotype in middle ear disease and the potential to selectout these resistant clones with introduction of the vaccine.


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EPI-88

The epidemiology of community-acquired pneumonia and its different formsin Germany from 1996 to 2001

JAI Weigl1,2, W Puppe1, HJ. Schmitt2, RR Reinert3 Univ. Children’s Hosp. Kiel1,Mainz2, National Reference Center Aachen3, Germany

Aims: To generate detailed data on CAP in regard to incidence and pathogen-spe-cific fractions, esp. related to S. pneumoniae.Method: Several forms of epidemiological and molecular-biological studies wereperformed to elucidate the epidemiology of CAP on different levels. RSV and in-fluenza serve as scale for comparison of the attributable burden of CAP. Fractionsdue to S. pneumoniae are estimated by serological data from Finland and VE datafrom the Kaiser Permanente data. Serotype-specific data for pleural empyema gen-erated by ESPED and the German Reference Center are used to characterize the tipof the iceberg of CAP.Results: Retrospective cohort studies from 1999-2001 with the public health serv-ice in Schleswig-Holstein revealed the baseline cumulative incidence per year of allforms of CAP in preschool children as 1800/105 for under-1 year and 1500/105

under-5 years of age. A cross-sectional study for hospitalized (severe) CAP from1996 to 2000 in the area of Kiel found an incidence of 1113/105 under-1 year, 684/105 under-5 years and 300/105 under-16 years of age; i.e. 60% of under-1 year and45% under-5 years with CAP had to be hospitalized; in under-1 and under-5 yearold children, 34% and 15% of severe CAP were RSV-positive, 1% and 2% wereinfluenza-positive and 4% and 6% would have been due to the serotypes in the7PNC, respectively. The incidence of parapneumonic effusion was 16/105, highestin the second year of life (27/105); 6 of 13 isolates would have been covered by thepanel of the 7PNC (11 of 13 by 11PNC). Extrapolated to Germany, 341 hospital-ized and 214 ambulatory cases of CAP in children under 1 year and 1,620 hospital-ized and 1,945 ambulatory CAP in under 5 years old would be preventable per year.Conclusions: The incidence and the fraction of children hospitalized decline byage. RSV is by far a more important player than influenza. The impact of S.pneumoniae lies in-between. The 11PNC would be desirable for Germany.

Pneumococcal meningitis in North Yorkshire, England 1997 – 2002

Weightman NC, Sajith J. Friarage Hospital, Northallerton, UK

Aims: To assess the incidence, features and outcomes of pneumococcal meningitisin North Yorkshire, England.Methods: All microbiologically confirmed cases of pneumococcal meningitis forthe area were ascertained from Public Health and Microbiology Laboratory recordsfor the 6 years 1997 – 2002 inclusive, and the case notes, where available, werereviewed.Results: A total of 50 cases of microbiologically confirmed pneumococcal menin-gitis occurred during the study period, giving an overall incidence of 1.1 per 100,000population per year. The incidence was markedly seasonal, 76% of cases presentingin the winter months (October to March). The male:female ratio was exactly 1:1.Patients’ ages ranged from 1 month to 89 years (mean 31.7 years); the age groupwith the highest incidence (22 per 100,000 population per year) was children under1 year old. The average time from admission to antibiotic administration was 3.7hours, (3.5 hours in survivors, 4.4 hours in non-survivors). Where blood cultureswere taken, these were positive in 73% of cases, and where cerebrospinal fluid sam-ples were taken these were positive in 79% of cases. The overall mortality was 28%.Survivors were followed up for an average of 21.6 months, and of these 28% suf-fered long-term sequelae, the majority of a chronic neurological nature. Glasgowcoma scores on admission for patients who died, were left with long term sequelae,or made full recoveries, were 8.2, 10.5 and 11.3 respectively. There were no cases ofpenicillin resistant pneumococci isolated during this study.Conclusions: Pneumococcal meningitis is more common in North Yorkshire thanreported for England and Wales generally, but this could be the result of better caseascertainment locally. It remains a rare disease, but carries a high mortality and therisk of long term sequelae. Low Glasgow coma scores on admission are associatedwith poor outcome.


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EPI-89

EPI-90

Rapid pneumococcal serotyping with monoclonal antibodies and a flowcytometer

Yu J, Lin J, Nahm MH Department of Pathology, University of Alabama at Bir-mingham, USA

Aim: To develop a rapid pneumococcal serotyping assay useful for studying 7-valentvaccines.Method: We have developed a set of monoclonal antibodies and a quantitativemultiplexed-assay for pneumococcal capsular polysaccharide(PS). The monoclonalantibodies were produced by immunizing mice with capsular PS-protein conju-gates. We have produced monoclonal antibodies specific for capsular PS serotypes4, 6B, 9V, 14, 18C, 19F, or 23F. The assay uses cytometric bead-array technologyand is based on our previous work (Park et al., CDLI 2000). This assay uses 15different beads distinguishable by a flow cytometer and each bead is coated with adifferent PS. A mixture of PS-coated bead is incubated with pneumococcal lysateand a mixture of monoclonal antibodies. Bead-bound monoclonal antibodies arevisualized with fluorescein-conjugated anti-mouse Ig antibody. Pneumococcal lysatereduces fluorescence of the beads coated with matching serotype only.Result: Assay is sensitive. 50% reduction in fluorescence is achieved with pneumo-coccal lysate after 50 fold dilution for 9V and 250 fold dilutions for all other serotypes.100 ng/ml of PS reduces fluorescence of the matching bead by about 50%. Specificityof the assay was established with lysate of pneumococci of all the known (90)serotypes. Also, the assay should identify all members in each serotype since theassay correctly identified all (7) different isolates belonging to each assay serotype.The assay is fast: it can be used to serotype several hundred isolates per day.Future work: To be able to evaluate cross-protection and 11-valent vaccines, we arecurrently producing monoclonal antibodies to serotypes 1, 3, 5, 6A, 7F and 19A.This pneumococcal serotyping assay should be useful to determine whether a pneu-mococcal isolate expresses a serotype included in a pneumococcal conjugate vac-cine. This capability will be useful to monitor serotype shifting and vaccine effi-cacy.

Utility of respiratory rates in screening for radiological pneumonia in children

Zaman SMA for The Gambian Pneumococcal Vaccine Trial Group Medical Re-search Council Laboratories, The Gambia

Aims: To assess the sensitivity of respiratory rates in a trial to maximize the detec-tion of radiological pneumonia as defined by WHO Radiology Working Group.Methods: During the surveillance for identification of pneumonia as a part of theongoing Pneumococcal Vaccine Trial in Upper and Central River Divisions of TheGambia, 3605 children 2-29 months of age presenting with cough or symptomspossibly indicating difficult breathing for less than two weeks at outpatients, orhospitalized with other signs of possible invasive pneumococcal disease, werescreened by counting the respiratory rate (RR) and xrayed. Six hundred and sixtyradiological pneumonias were identified between February 2002 to October 2003in two health facilities and their trekking clinics. We calculated the sensitivity ofWHO defined respiratory rate (RR) cut-offs for ALRI for diagnosing radiologicalpneumonia.Results: For infants, the overall sensitivity of RR cut-off of �50/minute was 94%(95% confidence interval (CI) 92-98%; n=2276). The overall sensitivity could beincreased to 99% (95% CI 98-100%) by lowering the RR cut-off to �40/minute,this would lower the specificity from 6% to 2%. The lowest sensitivity was in se-verely wasted (83%, 95% CI 34-99%; n=26) and afebrile infants (91%, 95% CI 82-96%; n=680), which could be raised to 100% and 99% respectively by lowering theRR cut-off to �40/minute. For children 12-30 months of age, the overall sensitivityof RR cut-off of �40/minute was 96% (95% CI 94-98%; n=2679). The sensitivitycould be increased to 99% (95% CI 98-100%) by lowering the RR cut-off to �30/minute, lowering the specificity from 2% to 1%. The sensitivity in severely wasted(92%, 95% CI 77-99%; n=96) or afebrile (88%, 95% CI 78-94%; n=721) childrencould be raised to 100% and 97% by lowering the RR cut-off to �30/minute.Conclusions: The WHO defined RR cut-offs for diagnosing ALRI are sensitivetools for identification of radiological pneumonia. Lowering RR cut-offs by 10/minute in severely malnourished and afebrile children would increase the detectionof radiological pneumonia. Further studies are required to assess the cost and valueof lowering RR cut-off for screening for radiological pneumonia in communitystudies of pneumonia burden or vaccine trials.


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EPI-91

Comparative study on serotype distribution and antimicrobial resistance pat-terns in Streptococcus pneumoniae isolates from hospitalized pediatric patientswith respiratory infections in urban and rural area, China

Zhao GM1, Black S,2, Shinefield H,2. School of Public Health, Fudan University,Shanghai, China1, The Kaiser Permanente Vaccine Study Center, Oakland, USA2.

Aims: Surveillance for pneumococcal respiratory illness was conducted in Childrenhospitalised in urban and rural area respectively from August 2000 to April 2003.Methods: Sputum cultures were obtained from pediatric patients <3 years old ad-mitted with pneumonia or respiratory distress by tracheal aspirate. All pneumococ-cal isolates were serotyped and tested for antibiotic susceptibility. In addition clini-cal information on the patients including prior antibiotic history was abstracted.Streptococcus pneumoniae tracheal isolations were attempted in a total of 1013 and999 pediatric patients hospitalized in the urban and rural areas respectively duringthis period. Among these samples, 112 and 42 specimens were S. pneumoniae posi-tive respectively. All the positive isolates underwent serotyping and antibiotic sus-ceptibility testing.Results: More than 80% of all patients had received antibiotic treatment beforeadmission to the hospital in both areas. Five serotypes (19F, 23F, 6A, 14, 6B) of S.pneumonia accounted for 81% (91/112) and 62% (26/42) in urban and rural arearespectively. Other serotypes accounted for 12% (13/112) and 33% (14/42), 7% (8/112) and 5% (2/42) of isolates cound not be typed by quelling test. In these twoareas, moderate and high level penicillin resistance (MIC, 0.1-1.0 µg/mL) werefound in 58% and 57% of isolates respectively. The percentage resistant to otherantibiotics were different in urban and rural areas. Among the isolates that were notsusceptible to penicillin, serotype 19F was the most common.Conclusions: S. pneunomiae is a common cause of respiratory illness requiringhospitalisation in young children in China, with antibiotic resistance increasinglycommon. Five serotypes account for most disease.


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Strengths and limitations of bacterial specific techniques

*Goldblatt, D

Institute of Child HealthImmunobiology Unit30 Guilford StreetLondon WC1N 1EHUNITED KINGDOM

[email protected]

Use of radiology and adjunct non-specific techniques to diagnose bacterial pneu-monia

*Cherian, T

World Health OrganizationInitiative for Vaccine Research20 Ave. AppiaCH-1211 Geneva [email protected]


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Lung tap after all?

*Peltola, H

Helsinki University Central HospitalHospital for Children and AdolescentsP.O.Box 281FI-00029 [email protected]

Vaccine probe to determine the burden of pneumoccoal pneumonia

*Scott, A

Wellcome Trust/Kenya Medical Research InstituteP.O.Box [email protected]


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Role of procalcitonin (PCT) and C-reactive protein (CRP) in augmenting effi-cacy estimates of a 9-valent pneumococcal conjugate vaccine (PncCV) againstradiologically diagnosed pneumonia.

Ramjee Heera J1, Madhi SA1, Klugman KP1,2. 1Respiratory and Meningeal Patho-gens Research Unit, South Africa; 2 Rollins School of Public Health, Emory Uni-versity, USA.

Aims: Determine whether PCT and CRP may improve the specificity of definitionof radiologically confirmed pneumococcal pneumonia.Methods: Part 1: PCT was measured in HIV-infected and HIV-uninfected childrenwith bacteraemic pneumococcal pneumonia from a historical cohort for whom se-rum samples, obtained within 24 hours of admission, were available. Part 2: PCTand CRP were measured in 322 sera from HIV-uninfected children randomized in aPncCV trial, with radiologically confirmed pneumonia based on WHO criteria.Results: Part 1: The median values of PCT (ng/ml) among 27 HIV-uninfected and85 HIV-infected children were 10.2(range:0.1-181.3) and 2.9(range:0.1-247.8) re-spectively, with the overall median 4 (range:0.1-247.8). Part 2: The vaccine effi-cacy of CXR confirmed pneumonia for those children in whom serum sampleswere available was 21.1% (95%C.I. 1.8-36.7). The point estimate of vaccine effi-cacy when interpreted together with PCT was 20% (95%C.I.-6.5-39.9) when PCTwas <1.0 ng/ml; 30% (95%C.I. –38.5-64.6) if PCT was 1.0-1.9; 26.1% (95%C.I.-38.4-60.5) if PCT was 2-4.9 ng/ml and 44.8% (95%C.I.5.9-67.6) if PCT levels were�5.0 ng/ml. Vaccine efficacy calculations for alveolar consolidation interpretedtogether with CRP were 21.9% (95%C.I. –5.6-42.2) for CRP levels �40mg/l; 31.0%(95%C.I. 0.8-52.0) for CRP levels �80 mg/l and 37.8% (95%C.I.3.9-59.7) for CRPlevels �120mg/l . The highest point estimate was obtained when interpretingradiologically confirmed pneumonia combined with CRP levels of �80mg/l andPCT levels of �5ng/ml; for which the vaccine efficacy was calculated to be 60.0%(95%C.I.21.9-79.5).Conclusion: The specificity of a radiological diagnosis of pneumococcal pneumo-nia is improved when interpreted together with CRP and procalcitonin levels inHIV-uninfected children.

Role of Conventional Qualitative versus Real-Time Quantitative PCR to Diag-nose Community-acquired Streptococcus pneumoniae (SP) Pneumonia in Hos-pitalized Immunocompetent Children.

Michelow I.C.1, Saukkoriipi A.2, Olsen K.1, Lozano J.1, McCracken G.H.1, LeinonenM.2. 1University Of Texas Southwestern Medical Center, Dallas, TX, U.S.A; 2 Na-tional Public Health Institute, Oulu, Finland.

Aims: Novel rapid and accurate tests are needed to diagnose the causes of pneumo-nia in children. We compared conventional single-step and real-time PCR tests todetect SP in blood and pleural fluid (PF) samples from a well-characterized studypopulation.Methods: 154 hospitalized children with radiologically confirmed acute pneumo-nia were studied. Their clinical, lab., and pneumolysin-based conventional PCR datahave been published. A real-time PCR assay that utilizes fluorescent detection wasused in a blinded fashion to target the pneumolysin gene in whole blood (WB),serum, buffy coat, and PF samples if available. Their clinical, lab.and radiologiccharacteristics were collated prospectively.Results: Sensitivity of both PCR techniques among 8 children with SP bacteremiawas 100%. Among 6 children with SP empyema, sensitivities for qual. and quant.PCR were 100% and 67%, resp. Overall, 68 patients (44%) were diagnosed with SPpneumonia based on conventional PCR results. Among these children, the overallsensitivity of the quant. PCR tests was 73%. Specifically, when concordant sampleswere compared, sensitivities ranged from 55% (WB) to 77% (serum). Pos and negpleural fluid PCR results were similar for qual. vs quant. methods (86 vs 89%, and14% vs 11%, resp., P=0.38) Significant correlations existed between results of eachPCR method and various markers of inflammation (procalcitonin, WBC, pleuraleffusion) or duration of hospitalization. Band-form neutrophil percentage corre-lated with SP genome counts (P=0.009). Nasopharyngeal SP colonization did notinfluence PCR results.Conclusions: The results demonstrate strong agreement between conventional andreal-time pneumolysin-based PCR assays to diagnose severe SP pneumonia as de-fined by positive blood or PF cultures in hospitalized children. PCR positivity andSP DNA counts correlated with selected markers of inflammation and disease se-verity.

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Serotype-specific PCR for detection of Streptococcus pneumoniae

Adegbola R1,2, Farley J2, Fletcher L2,4, Xie X2, Zagursky R2,3. Medical ResearchCouncil Laboratories, The Gambia1, Wyeth Vaccines, W. Henrietta2 and Pearl River3,University of Rochester Medical Centre, Rochester4, NY, USA.

Aim: The objective of this study was to develop a sensitive and rapid PCR assay fordetection of nine pneumococcal serotypes commonly found in The Gambia. Methods: Pneumococcal strains used were of serotypes 1, 4, 5, 6B, 9V, 14, 18C,19F and 23F. Serotypes were identified by Quellung reaction. PCR oligonucleotideprimers were designed based on published CPS gene DNA sequences and our ownsequencing of serotype 5. Alignment of the capsule biosynthesis DNA region of allnine serotypes revealed two class-specific sequences for PCR primer design fordetection of serotypes 1, 5, 14, 18C and 19F (Class I) and serotypes 4, 6B, 9V and23F (Class II). Small sequence variations were used to make another set of PCRprimers with specific homology to desired serotypes. In the first step of this assay,PCR reaction mixture consisted of 45 �l of ABGene ReddyMix PCR master mixand 2.5 �l of each 20�M classing primer. A bacterial colony or frozen bacterialcells were scraped with a pipette tip and swirled into 10 �l volume of the reactionmixture in a reaction tube. Samples were heated in a thermocycler to 960C for 3 minhot start and 35 cycles of denaturation, annealing, extension and extra-extension.PCR reactions were analysed by agarose gel electrophoresis, stained with ethidiumbromide and visualized on a KODAK Image Station. In the second step, a two-stepannealing PCR protocol was used to identify five individual serotypes from cul-tures identified as Class I using Class I identification primers. Similarly, a three-step annealing protocol was used to identify four individual serotypes from culturesidentified as Class II serotypes using Class II primers.Results: The new PCR assay detected the desired nine pneumococcal serotypes andshowed no cross-reactivity with non pneumococcal bacterial samples.Conclusions: A two-step PCR assay was successfully developed for serotype-spe-cific detection of the nine-valent pneumococcal conjugate vaccine now in use in theGambian efficacy trial. Further work to evaluate sensitivity and specificity of theassay for direct detection of pneumococcal serotypes in nasopharyngeal specimensis underway in The Gambia.

Pilot study of the use of procalcitonin to predict bacterial pneumonia in theGambia

Cutts FT for the Gambian Pneumococcal Vaccine Trial Group, Medical ResearchCouncil, The Gambia

Aims: To determine whether procalcitonin (PCT) can be used as a marker of acutebacterial infection in Gambian children with a radiological diagnosis of pneumo-nia.Methods: Serum samples were obtained from a random sample of children withculture-confirmed invasive pneumococcal infection, those with other bacteraemias,those with non-bacteraemic radiological pneumonia, and those with non-specificfever. Laboratory assays were blinded to clinical status of the child. Procalcitoninwas measured using the BRAMs kit and results expressed in ng/ml.Results: The median PCT was 0.7ng/ml (range 0.1-380.7), and 34% of children hadlevels of at least 2ng/ml. Raised PCT levels (>=2ng/ml) were more common amongchildren with radiological pneumonia (42% of 45) than those without (29% of 97)(p=0.2). Using a cut-off of 0.5, the difference was smaller (63% vs 55% respec-tively). Among children with radiological pneumonia, raised PCT levels were slightlymore common among those with bacteraemia (45% of 22) than those without (39%of 23). Among 6 children with a positive bacterial culture from lung-aspirate, how-ever, only one had a level >=2ng/ml while 4 had levels below 0.5. On logisticregression, controlling for presence of bacteraemia and malaria, the adjusted oddsratio for a PCT level of >=2ng/ml among children with radiological pneumonia was1.8 (95% CI, 0.8, 4.0; p=0.1). The adjusted ORs were 1.5 (95% CI, 0.6, 3.6) formalaria and 1.33 (95% CI, 0.6. 2.7) for bacteraemia. There was no evidence ofinteraction between xray consolidation and either malaria or bacteraemia. ReceiverOperating Curves showed that a cut-off of 2ng/ml had only 50% sensitivity and64% specificity for radiological pneumonia. To increase specificity to over 80%, acut-off of 20 or higher would be needed but this would have less than 25% sensitiv-ity. Restricting analyses to bacteraemic and/or non-parasitaemic children with ra-diological pneumonia had little effect on these estimates.Conclusions: Raised procalcitonin correlated poorly with bacteraemic pneumoniaand does not appear helpful in the Gambian context to differentiate bacterial fromother pneumonias.

DGN-11

DGN-12


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Evaluation of the Denka Seiken (Tokyo, Japan) slide agglutination test kit as arapid routine pneumococcal serogrouping/typing method

de Gouveia L1, von Gottberg A1, Wasas A1, Klugman KP1,2. 1Respiratory and Me-ningeal Pathogens Research Unit (MRC/WITS/NICD), Johannesburg, South Af-rica; 2Department of International Health, Emory University, Atlanta, Georgia, USA

Aim: We evaluated the ability of the above slide agglutination test kit to accuratelyidentify Streptococcus pneumoniae serogroups/types in comparison to the Quellungtest.Methods: 115 invasive paediatric isolates from one hospital in Johannesburg col-lected from 2000 to 2001 and 40 clinical strains received during March 2003 as partof a national surveillance programme for pneumococcal invasive disease in SouthAfrica were serotyped in a blinded fashion by one operator using the Denka Seikenkit acccording to manufacturer’s instructions. Methodology was slightly altered af-ter the first ten strains by making a turbid suspension of the isolate in saline.Results: Of the 115 strains tested, 96 (83%) were correctly serogrouped/typed ini-tially (19 strains were either incorrectly grouped, or gave non-specific/no reactions).After unblinding, 12 were correctly identified and 7 remained non-groupable. Ofthe second set of isolates, 35/40 (88%) correctly matched the Quellung reaction.After unblinding 1 was correctly identified and 4 remained non-groupable. Nine of155 (6%) strains remained ungroupable/untypeable showing non-specific cross-re-actions in 2 or more monovalents, or no reaction. The 155 strains tested belonged to22 different serogroups/types (most common: 6, 19, 23, 14).Conclusions: Some problems were: very weak/no agglutination with serogroups 10and 22, weak agglutination of mucoid serotype 3 strains with polyvalent 1 and noavailability of antisera for the detection of serotype 13. Once unblinded, agglutina-tion was easier to interpret in the monovalent antisera. This kit allowed rapid, cost-effective, easily interpretable serogrouping of 131/155 (84.5%) of pneumococcalstrains initially, and 144/153 (94%) after unblinding (excluding the 2 strains of se-rotype 13).

Streptococcus pneumoniae in sputum samples, comparison of the diagnosingmethods and the antimicrobial sensitivity

Kurti A1,2, Mulliqi Gj1,2, Raka L1,2, Jaka A1,2, Jakupi Xh1,2, Begolli L1,2, Berisha L1,2.National Institute of Public Health of Kosova, Prishtinë1, University of Prishtina,Medical School2, Prishtinë, Kosovë

Aims: Determination of the frequency of isolation of Streptococcus pneumoniae insputum samples, comparison of the diagnosing methods and analysis of the antimi-crobial sensitivity.Methods: Isolation and identification of Streptococcus Pneumoniae has been car-ried out initially with standard methods (culture, Gram staining). Suspicious colo-nies have been tested with optochin (ethyl hydrocupreine), slidex Pneumo-Kit andrapid ID 32 STREP. Sensitivity has been tested on the Miller-Hinton agar throughdiffusion method using 1µg oxacillin and 15 µg erythromycin discs. Strains of Strep-tococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATTC 2921 have beenused for quality control.Results: Out of 1819 sputum samples of hospitalised patients analysed within thetwo-year timeframe (2001-2002), Streptococcus pneumoniae has been isolated andidentified in 235 samples or 12.91%. Leukocytes and Gram positive diplococcihave prevailed in the Gram stained direct slides of 235 positive samples. Male-female ratio has been 1.6:1 (145:90). In 24 samples (10%), where optochin test wasnegative while Pneumo-Kit was positive, a confirmation using rapid ID 32 STREPhas been performed. Resistance in oxacillin has appeared in only one case while inthree cases erythromycin has emerged as intermediary.Conclusions: The slidex Pneumo-Kit has shown 100% specificity in the sputumsamples, while the specificity of the optochin was 90%. In the absence of moresophisticated assays, additional testing with rapid ID32 STREP was sufficient forthe optochin negative cases. Furthermore, penicillin remains the first option drugfor our positive samples.

DGN-13

DGN-14


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DGN-15

DGN-16

Diagnostic value of microscopic examination of gram-stained sputum (GSS)and sputum culture (SC) in patients with bacteremic pneumococcal pneumo-nia due to Streptococcus pneumoniae (Spn)

D. Musher,1,2 R. Montoya,1 A. Wanahita.1 VAMC,1 Baylor College of Medicine,2

Houston, TX

Background, Aims: Clinicians continue to question the usefulness of microscopicexamination of GSS and SC in diagnosing pneumonia, based on the reportedly lowsensitivity of these techniques. Our aim was to study the sensitivity of GSS and SCin patients with proven Spn pneumonia.

Methods: This was a retrospective study of 105 patients with bacteremic Spn pneu-monia during 6 years.

Results: GSS showed gram positive cocci in pairs and chains, and SC yielded Spn in31% and 44% of all cases of Spn pneumonia, respectively, appearing to suggestvery low sensitivity. But, sputum was never submitted to the laboratory in 31 casesand, in 16 others, the sample was inadequate. Excluding these cases, GSS and SCsensitivities were positive in 57% and 79%, respectively. If patients who receivedantibiotics for >24 hr were excluded, GSS showed Spn in 63%, and SC documentedtheir presence in 86%. Sensitivity rose in inverse proportion to the duration ofantibiotic therapy (p<.05); in patients who provided a sputum before antibioticswere given, the sensitivities of GSS and SC were 80% and 93%, respectively.

Conclusions: GSS within 18 hr of antibiotic administration and SC within 24 hryielded the correct diagnosis in >80% cases of Spn pneumonia. Analyzing data inthis fashion emphasizes the importance of obtaining specimens promptly for micro-biological study and helps explain discrepancies in earlier reports on the usefulnessof these techniques. Our failure to identify Spn in sputum from >50% of cases ofproven pneumococcal pneumonia supports the notion that the true incidence of Spnas the cause of community-acquired pneumonia is substantially greater than hasbeen suggested by some recent reports.

Nasopharyngeal carriage and pneumococcal urine antigen test in healthy eld-erly subjects

Palmu A1,2, Kaijalainen T3, Leinonen M3, Mäkelä PH1, and Kilpi T1. National PublicHealth Institute, Helsinki1, University of Tampere2 and National Public Health Insti-tute, Department in Oulu3, Finland.

Aims: Streptococcus pneumoniae (Pnc) is a common mucosal resident in nasophar-ynx, especially in young children, even when healthy. However, the extent of Pnccarriage in healthy elderly living at home is largely unknown. Estimates rangingfrom 8 to 35% in special populations or risk groups have been presented.Methods: Healthy elderly subjects at least 65 years old were recruited in a vaccineimmunogenicity study and nasopharyngeal swab (NPS) was taken to assess the car-riage of Pnc and Haemophilus influenzae (Hi). After sampling the NPS was placedin STGG transport medium and frozen for later analysis. The presence of Pnc wasanalysed by direct culture and after enrichment by culture and the quellung method.Additionally, a urine sample was taken at the same time and analysed on-site toassess the effect of the carriage on urinary antigen test (Binax NOW).Results: Of the 160 NPS samples analysed 6 (4%, 95% confidence interval 1-7%)were found positive for Pnc. Of these 6 samples, 4 were positive in both cultures, 1in the direct culture only and 1 in the culture after enrichment only. In only onesample the quellung method was positive (being positive in both culture methods,too). The urine antigen tests were all negative for the 160 subjects.Conclusions: The pneumococcal carriage prevalence is low in healthy elderly sub-jects. The negative urine test results in the healthy elderly, also in the small numberof those with pneumococcal carriage, suggest that the urine antigen test has thepotential of being highly specific in detecting Pnc disease.


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DGN-17

DGN-18

Use of a rapid immunochromatographic assay for Streptococcus pneumoniaeantigen (Binax NOW) on specimens additional to urine from patients present-ing with community-acquired pneumonia

Rafundisani T1, von Gottberg A1, Musabeyezu E2, Perovic O3, Feldman C2, KlugmanKP1,4. 1Respiratory and Meningeal Pathogens Research Unit (MRC/WITS/NICD), 2

Department of Medicine and 3Department of Clinical Microbiology and InfectiousDiseases, University of the Witwatersrand, Johannesburg, South Africa; 4Depart-ment of International Health, Emory University, Atlanta, Georgia, USA

Aim: Comparison of pneumococcal antigen detection in urine, nasopharyngeal (NPS)and oropharyngeal (OPS) swabs collected on admission from patients with commu-nity-acquired pneumonia (CAP) with isolation of pneumococci from sputum, blood,NPS and OPS cultures.Methods: 16 adult patients admitted with CAP were recruited. All specimens, ex-cluding urine, were cultured for Streptococcus pneumoniae according to standardpractices. Urine, NPS and OPS specimens were tested for pneumococcal antigen(Binax NOW, Binax, Portland, ME). Pneumococci were serotyped using the Quellungmethod.Results: Five of the 16 patients had pneumococcal bacteraemia, and all were alsopositive for urinary antigen and had positive cultures from NPS and OPS. All strains(except one from OPS) correlated phenotypically with the invasive strains. Of allthe pharyngeal swabs (NPS and OPS) tested for antigen in these bacteraemic pa-tients, only one was OPS Binax negative. Three patients with bacteraemia also hadpneumococci isolated from sputum. A further 7 of 11 patients with non-bacteraemicpneumonia were also colonised (total colonisation 12/16, 75%). Four of the 12 colo-nised patients (33%) were colonised with serotype 1 pneumococci. Only one pa-tient positive for pneumococcus on antigen detection and culture of OPS and NPS,was negative on urinary antigen detection.Conclusions: Carriage can be determined by performing antigen testing on NPS orOPS, however the urinary antigen test results may correlate more closely with truelower respiratory tract disease.

Use of multiplex PCR for the detection of pneumococcal multiple carriage

Ramirez, M.1,2, Brito, D. A.1, de Lencastre, H.1,3, 1ITQB/UNL, Oeiras, Portugal,2Faculdade de Medicina, Lisbon, Portugal, 3The Rockefeller University, New York,USA

Aims: We developed a multiplex PCR-based method for the detection ofepidemiologically important pneumococcal capsular types without using immuno-logical techniques (J. Clin. Microbiol., 2003, 41: 2378). This method was expandedand modified to detect and characterize multiple serotypes in heterogeneous sam-ples.Methods: All the primers previously described were combined into three multiplexreactions. Boiled McFarland 5 suspensions of nasopharyngeal flora grown over-night on blood agar plates were used as template (Total Bacterial Growth, TBG).Sensitivity was determined by analyzing the possible combinations of distinctserotypes in a mixture. A set of 296 TBGs with corresponding isolated colonies(IC), from asymptomatic children, constituted the pilot study.Results: The sensitivity of the method was 1/100 to 1/1000. To detect the minorpopulation using conventional serotyping (p=0.95), one would have to type 300 to3000 IC, respectively. The IC and TBG results were 100% concordant and at least64 TBGs had � 2 serotypes. The distribution of the eight prevalent types was differ-ent in the IC from the analysis of TBGs when � 1 serotype was detected.Conclusions: Our method covers the majority of serotypes colonizing children, isfast, cheap, and deployable in large scale, and allows multiple carriage detectionwithout the need to isolate different colonies, thus constituting a suitable techniqueto better characterize the nasopharyngeal pneumococcal population before and af-ter vaccination.


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DGN-19

DGN-20

Procalcitonin not better than C-reactive protein in identifying severe pneumo-coccal infections

Rautio L1, Puumalainen T1, Vuori-Holopainen E2, Ollgren J1, Lucero M3, Peltola H2,Nohynek H1 ARIVAC consortium; 1National Public Health Institute, 2University ofHelsinki, Finland; 3Research Institute for Tropical Medicine, the Philippines

Background: Serum concentrations of procalcitonin (PCT), the precursor of thehormone calcitonin, increase in severe bacterial infections. Some studies suggestPCT to be better than C-reactive protein (CRP) as an acute phase marker of invasivebacterial infections in childhood.Aims: To compare PCT with CRP and evaluate if PCT is more sensitive than CRP indistinguishing severe childhood bacterial, especially pneumococcal (Pnc) infectionsfrom other infections.Methods: 121 children (103 from the Philippines, age 0,8-22 months and 18 fromFinland, age 0,8-12 years, median 23 months) were all hospitalised due to symp-toms of severe infection (meningitis, sepsis, pneumonia). The Finnish children hadculture (transthoracic needle aspirate) proven Pnc alveolar pneumonia; 2 had alsoPnc in blood culture. Of the Filipino children, altogether 52 had alveolar pneumonia(including 6 sepsis (3 Pnc) and 4 meningitis, (2 Pnc)). In addition to these pneumo-nia patients, 6 had bacterial sepsis (1 Pnc) and 5 meningitis (1 Pnc).Results: PCT correlated significantly with culture proven bacterial infections andespecially Pnc infections. PCT concentration profile was very similar to that ofCRP in terms of the severity of infection. In ROC analysis, culture-proven Pnc in-fections had the best cut-off values at 1,45 ng/ml for PCT (sensitivity of 91,3%,specificity of 72,6%) and 99,5 mg/l for CRP (95,7% and 75,8%, respectively). Thearea under the curve was 0,830 for PCT and 0,856 for CRP, which both are consid-ered excellent discrimination. The odds ratio for the Pnc-positive vs. culture nega-tive cases to have PCT>1,45 ng/ml was 36 (95%CI 7,7-165,5) and CRP>99,5 mg/lwas 95 (95%CI 11,9-756,8).Conclusions: Both PCT and CRP distinguish severe culture proven Pnc infectionswell but PCT does not add to the value of CRP. Instead, at present, CRP is mucheasier and cheaper (PCT measurement is >10 times more expensive) to measuremaking it considerably more cost-effective than PCT.

Eradication of Streptococcus pneumoniae from middle ear fluid

Ruohola A1, Meurman O2, Ruuskanen O1. Department of Pediatrics1 and Microbi-ology2, Turku University Hospital, Turku, Finland.

Aims: To determine the eradication of Streptococcus pneumoniae (Pnc) from mid-dle ear fluid (MEF) and the duration of otorrhea when Pnc occurs as a single patho-gen or as a co-pathogen (with either Haemophilus influenzae, Moraxella catarrhalis,Staphylococcus aureus, or Streptococcus pyogenes) in effectively treated and non-treated patients.Methods: 64 children, aged < 7y, with acute (< 48h) tube otorrhea and concomitantrespiratory infection. The duration of otorrhea was determined by suctioning MEFdaily through the tympanostomy tube. Bacterial culture of MEF was done beforeand daily during treatment that was amoxicillin-clavulanate or placebo according todouble-blind randomization. Mann-Whitney U test was used to test the differencesbetween medians.Results: In the amoxicillin-clavulanate group, the mean/median duration of otorrheawas 2,1/2,0 days whenPnc occurred as a single pathogen and 4,9/5,0 days when Pnc was a co-pathogen(p=0,014). Duration to eradication of Pnc was 1,0/1,0 vs. 1,7/1,0 when Pnc oc-curred as a single vs. co-pathogen (n.s.). In the placebo group, the mean/medianduration of otorrhea was 5,3/6,0 days when Pnc occurred as a single pathogen and8,0/8,0 days when Pnc was a co-pathogen (p=0,082). Duration to eradication of Pncwas 4,3/4,5 vs. 6,4/8,0 when Pnc occurred as a single vs. co-pathogen (n.s.).Conclusions: During amoxicillin-clavulanate treatment sensitive Pnc is eradicatedrapidly from MEF. Without treatment the presence of other pathogens may prolongthe eradication of Pnc. The duration of otorrhea is shorter when Pnc occurs as asingle than as a co-pathogen.


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DGN-21

DGN-22

Comparative serotyping of Streptococcus pneumoniae using Denka Seiken(Tokyo, Japan) slide agglutination kit and a locally made anti-serum coatedlatex agglutination reagent.

Saaka M, Secka O, Corrah T, Adegbola R Medical Research Council Laboratories,The Gambia

Aim: Quellung reaction is regarded as the gold standard for serotyping of pneumococ-cal isolates but the method is costly and cumbersome to perform. We aimed to comparethe usefulness of a commercially available agglutination kit against our locally pre-pared antiserum coated latex agglutination (LA) reagent for serotyping of pneumo-cocci.Methods: One hundred consecutive isolates obtained from normally sterile sites frompatients with pneumococcal disease at the Medical Research Council hospital in TheGambia (May, 2001 to September, 2002) were used for the study and S. pneumoniae(ATCC 49619) was included for quality control. Following isolation from clinical speci-mens, colonies of each bacterial culture were suspended in normal saline to Marcfarlandstandard turbidity number 0.5. Equal volumes (50µl each) of locally prepared antise-rum-coated latex suspension and bacterial suspension were tested by agglutination toidentify associated main groups (A-I) and specific factor types within two minutes ofrocking. Cultures were suspended in vials containing 15% glycerol broth and stored at-700C until required for further testing. All strains were sub cultured onto ColumbiaAgar with 5% sheep blood, incubated overnight (18-24 hours) at 350C in 5% CO2 andserotyped in a blinded fashion by one technician who had no knowledge of the LAresults using the Denka Seiken Kit following manufacturer’s instructions.Results: Denka Seiken kit and our LA reagent were used successfully to separate all100 isolates into 19 serogroups/types. Serotype 1 was most frequent (23%), followedby serogroup 6 (17%) with serogroups 8 and 29 being the least common. LA reagentwas used to factor-type serogroups 6 and 23 into 6A or 6B and 23A or 23F respec-tively.Conclusions: In this series of 100 invasive pneumococcal isolates from The Gambia,Denka Seiken latex agglutination kit and a locally made LA reagent gave similarserotyping results. The tests were rapid and easy to interpret. Denka Seiken test wasfaster and easier to interpret but cannot be used for factor typing and is probably not ascost effective as our locally prepared reagent.Acknowledgements: Denka Seiken and Anne A von Gottberg

Real-time PCR and melting curve analysis may be helpful in distinguishingpneumococci from other a-hemolytic streptococci

Saukkoriipi A, Kaijalainen T, Herva E, Leinonen M. National Public Health Insti-tute (KTL), Oulu, Finland.

Aims: The purpose of this study was to find out if pneumolysin (ply) PCR positivea-hemolytic streptococci really contain the ply gene as detected by using real-timePCR. The aim was also to study if the fragment of the ply gene amplified in PCR isidentical to that found in typical pneumococci (Pnc).Methods: 11 �-hemolytic streptococcal strains not fulfilling all conventional diag-nostic criteria for Pnc were analyzed by real-time PCR based on amplification of a206-bp fragment of the ply gene. PCR was carried out once using a pair of se-quence-specific hybridization probes for detection of PCR products and once using� double-stranded DNA binding dye. In the latter analysis, melting curves of thePCR products were studied. The PCR products were sequenced and results werecompared to ply gene (GenBank X52474).Results: A PCR product was detected with the hybridization probes in all 11 strains(no products in negative controls). However, all but one the amplification curvesremained very low compared to the pneumococcal standard. A slightly differentproduct melting temperature was seen for these 10 strains compared to standardsanalyzed in the same run. When the amplified fragments of these strains weresequenced, all were found to closely resemble the ply sequence except that they allcontained two mutations (G→A and C→T) at the binding site of one of the probes.Four strains also had a mutation (C→T) at the binding site of the other probe. Thestrain with a normal amplification curve neither had a differing product meltingtemperature nor contained mutations in the 206-bp fragment as compared to thepublished sequence.Conclusions: Non-pneumococcal a-hemolytic streptococci harboring pneumococ-cal genes may cause false positive results in PCR assays targeted to these genes.Real-time PCR and melting curve analysis of PCR products may help to distinguishPnc from other a-hemolytic streptococci.


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DGN-23

DGN-24

Combining diagnostic techniques to estimate the proportion of adult pneumo-nia in Kenya caused by Streptococcus pneumoniae

Scott J A G. Wellcome Trust / Kenya Medical Research Institute, Kilifi, Kenya

Aims: To estimate the proportion of cases of pneumonia caused by pneumococci inKenya and determine the optimum combination of diagnostic tests to broaden thisevidence to other under resourced settings.Methods: The study integrates results of validation studies of four diagnostic tech-niques with original data from an aetiology study. Cases were 281 inpatients withacute community-acquired X-ray confirmed pneumonia presenting to two hospitalsin Kenya. Controls were 254 patients, in three samples, presenting to the same hos-pitals but without pneumonia, meningitis or septicaemia, and 47 healthy volunteers.Sensitivity, diagnostic yield, and specificity were estimated for the diagnostic testsalone and in combination. Sensitivity of individual tests or combinations was esti-mated in 74 patients with blood or lung aspirate culture evidence of S. pneumoniae.Individual test specificities were estimated in the sick control groups and thespecificity of combinations of tests was modelled.Results: Sensitivity of a single blood or lung aspirate culture alone was 0.69. Sensi-tivity of capsular antigen detection in urine, enzyme immunoassay for pneumolysinimmune complexes, ELISA for IgG antibodies to Pneumococcal surface adhesin A(PsaA), and PCR of lung aspirates for psaA, the gene encoding PsaA, had sensitivi-ties of 0.49, 0.19, 0.96, and 0.69, respectively, and specificities �0.98. Results wereindependent of HIV. Three simple tests, (blood culture, capsular antigen detection,and PsaA ELISA) defined 62% of pneumonia episodes as pneumococcal with acombined specificity �0.96.Conclusions: Streptococcus pneumoniae causes nearly two thirds of the episodes ofadult pneumonia in Kenya. A combination of three uncomplicated techniques couldbe used to estimate the hospital burden of pneumococcal pneumonia more widelyin the developing world.

Unmasking the killer: The use of molecular methods to investigate culture nega-tive pneumonia cases.

C.L. Sheppard1, T.G.Harrison1, R.C.George1, J.E. Salmon2, M. Lyons2 1. R.S.I.L.,HPA Central Public Health Laboratory, London UK. 2. NPHS Wales

Introduction: In the first half of 2003 a number of cases of initially unexplainedpneumonia in individuals less than fifty years of age were noticed in a small town inthe UK and thought possibly epidemiologically linked. This included six affectedindividuals within two family groups. A further five additional cases aged less thanfifty years were identified in the surrounding locality. Initial routine testing by cul-ture was inconclusive however pneumococcal infection was suspected and variousmolecular methods for non-culture detection and characterisation of pneumococciwere used to investigate the putative cluster.Materials and methods: Sample remnants from 10 patients were obtained. Pneumo-coccal PCR was applied to all tissue, blood and serum samples and, when positive,non-culture MLST was attempted. Urine samples were tested with the Binax NOW¨

pneumococcal antigen test and where positive a serotype-specific ELISA test wasused to determine serotype.Results: Two lung tissue samples from the patients who died were positive by PCRand yielded differing MLST sequence types (ST237 and ST235). Urine obtainedfrom one of these deceased patients was also positive for pneumococcal antigen ofserotype 1 as was a urine sample from a second case in the same household. Afurther 3 patients who were not part of the family clusters were urine antigen posi-tive, two for serotype 1 and one for serotype 4. For two of these cases serum PCRwas also positive but MLST was not possible on these samples.Conclusions: Using the non-culture techniques, useful information was gained aboutthe cluster of cases of unexplained pneumonia. In addition to confirming the aetiol-ogy of the disease in six of the cases, valuable epidemiological data was obtainedthat showed that there was not a single outbreak but that there may have been asmaller cluster of related cases within a high background of pneumococcal pneu-monia.


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DGN-25

DGN-26

Characterization of a new pneumococcal antigen for absorption of serum sam-ples

Skovsted I. C., Kerrn M. B. and Konradsen H. B. Statens Serum Institut, Denmark

Aims: Pneumococci contains a common antigen, which has not been characterizedbefore. Antibodies against this antigen interferes with the pneumococcal ELISA(quantitation of human antibodies towards pneumococcal capsular polysaccharides).The new antigen is a cell-wall polysaccharide (C-Ps2) and is a contaminant of com-mercially available pneumococcal capsular polysaccharides.The aim of the study was to investigate which serotypes contain the antigen in largequanteties and to find the optimal absorption of antibodies against it in serum sam-ples.Methods: An ELISA test was set-up to measure the amount of absorbed unspecificantibody. Ten serum samples was examined after absorption with C-Ps (SSI) orcapsular polysaccharides from serotypes 9A (SSI), 9V (ATCC) and 22F (ATCC),F-antigen (SSI) and a combination (1:1) of C-Ps with 9A, 9V, 22F and F-antigen,respectively.Results: The result showed that serum absorbed with a combination of C-Ps/9V, C-Ps/9A, C-Ps/22F and C-Ps/F-antigen had 42%, 41%, 30% and 5% lower amountsof unspecific antibodies than when C-Ps was used alone.Conclusions: We conclude that before preforming a pneumococcal ELISA serumabsorption with a combination (1:1) of C-Ps/9V or C-Ps/9A is recommended. (Cur-rently SSI is working on making rough strains of serotypes 9V and 22F to purifieand isolate the new C-Ps. We are planning to publish our findings at ISPPD-4.)

Clinical features in pneumococcal meningitides with children.

Skripchenko N.V., Vilnitz A.A., Ivanova M.V., the Research Institute of Children¢sInfections, St.-Petersburg, Russia

Aim: a study of peculiarities in the clinical course of pneumococcal meningitides(PM) with children.Methods: 25 children with PM including children younger then 1 year (32%), chil-dren of 2-4 years of age (16%) and children older then 5 years (52%), have beenadmitted and studied at our Institute in 2000-2002.Results: the most acute onset of the disease was seen in 84% of the cases; it wascharacterized by high fever 39,4±0,3C in 96% of cases; in 56% of the patients re-peated vomiting was observed. Obtundation, letargia and coma were seen in 75% ofthe cases. During the first day of the disease scarce small-spot and hemorragic rashappeared on the trunk and limbs in 40% of the patients. Nuchal rigidity was foundin 92%, positive Kernig sign in 72%, while Brudzinski sign in 28% of all the cases.Focal neurological symptoms in the acute phase of PM were seen in 16% of thepatients. In the acute phase prevailed the following complications: cerebral edema(46%), inadequate antidiurethic hormone secretion syndrom (38%), hydrocephalicsyndrome (24%). From the very PM onset 2 patients (8%) had acute hearing loss.PM outcomes included: absolutely recovered without any neurogic deficit 60% ofall the cases, PM residua 40% including occlusive hydrocephalia 4%, intracranialhypertension 12%, severe hearing loss 8%, and astenic state 16%; no fatal caseswere observed.Conclusions: pneumococcal meningitis is a severe form of pneumococcal infectionhaving the most complicated course and invalidating sequel in children, which re-quires prevention measures such as specific vaccination.


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DGN-27

DGN-28

The effect of medias on pneumococcal growth

Slotved H-C, Kerrn M.B., Serum Production, Department of Bacteriology, Mycol-ogy and Parasitology, Statens Serum Institut, Copenhagen, Denmark.

Aims: The aim was to test Todd-Hewith broths (TH broths) for the ability to propa-gate pneumococci and thereafter to evaluate the serotyping result obtained by thePneumotest-Latex kit (SSI).Methods: TH broths from four different producers (Oxoid, Sigma, Difco and SSI)were tested and compared with serum broth (SSI). Eight pneumococcal strains (dif-ferent serotypes) were inoculated into the broth with start inoculums of 101, 103 and106 CFU/mL. After incubation overnight viable bacterial counts and visible growthwere recorded. All pneumococci were serotyped with the Pneumotest-Latex kit.Results: After incubation, the bacterial counts in all TH broths were on averagebetween 106 - 107 CFU/mL, while serum broth showed an average growth of 108

CFU/mL depending on serotype and initial inoculum. The TH broths from Oxoidand Sigma showed on average the highest growth followed by Difco and TH brothfrom SSI. In general, serum broth showed a more pronounced visual growth thaneach of the four TH broths. Serotyping with serum broth resulted in positive andcorrect latex typing results for all serotypes and initial inoculum, while the resultswith the TH broths showed some false negative results depending on inoculum andserotype.Conclusions: There is a difference between the performances of TH broths fromdifferent producers. Only serum broth showed stable growth for all serotypes tested.The broth used to culture pneumococci has an effect on the latex serotyping results,which are more pronounced with TH broths than with serum broth.

Can serology for pneumococcal serotypes be used for establishment of pneu-mococcal pneumonia?

StrŒlin K1, Kaltoft MS2, Holmberg H1, Olcén P3, and Kondradsen HB2. Depatmentof Infectious Diseases, Örebro University Hospital, Örebro, Sweden1, Streptococ-cus Unit, Statens Serum Institut, Copenhagen, Denmark2, Department of ClinicalMicrobiology, Örebro University Hospital, Örebro, Sweden3.

Aims: A serotype specific ELISA was tested for establishment of pneumococcalaetiology in pneumonia.Methods: In 70 adults with pneumonia, culture positive for Streptococcus pneumoniaein blood (n=19) or sputum/nasopharynx, and 5 healthy adult pneumococcal carri-ers, the pneumococcal strains were serotyped. In all subjects, an ELISA was per-formed, in which serotype specific pneumococcal antibodies were measured in pairedsera against the patient’s own serotype and 6 standardised serotypes (1, 4, 7F, 14,18C, and 19F), one serotype per reaction. A geometric mean (GM) was calculatedfor the 6 serotypes, and a 2.5-fold antibody increase to the patient’ s own serotypeor in GM was regarded as significant.Results: Among 19 blood culture positive and 51 blood culture negative pneumoniapatients, the serology was significant both to the own serotype and in GM in 6 and8 patients, respectively, to the own serotype alone in 1 and 10, respectively, and inGM alone in 1 and 1, respectively. Using positive blood culture or significant serol-ogy to the own serotype as expanded gold standard, the sensitivity and specificityfor use of the 6 standardised serotypes were 42% (15/37) and 97% (33/34), respec-tively. Of 28 patients with culture serotypes included in the 6 standardised types and42 with other serotypes, 8 (29%) and 8 (19%) patients, respectively, were GM posi-tive (P = 0.35). Of the latter 8, 7 were positive with blood culture or serology to theown serotype. In the 5 controls, none had significant serology to the own serotypeor in GM.Conclusions: As serology to the own serotype was significant in 18 of 51 bloodculture negative patients, it could be a useful tool in epidemiological studies forestablishment of pneumococcal pneumonia. Serology against 6 standardisedserotypes may also be used, since it showed a high specificity and was positive in 9blood culture negative patients. Such a serology would not require isolation andserotyping of strains.


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Clinical vs Research Reading of Radiographs in Evaluation of Pneumonia Vac-cine Effectiveness.

S Black, H Shinefield, J Hansen, B Fireman (Kaiser Permanente Vaccine StudyCenter, Oakland, USA); T Cherian (WHO, Geneva) and Jane Benson ( Johns HopkinsUniversity, Baltimore)

Aim: To compare utility of the treating radiologist reading of radiographs withresearch readings obtaining utilizing WHO guidelines and cross trained readers.Methods: 7-valent pneumococcal conjugate was evaluated in a RCT including 37868infants. Effectiveness against pneumonia was evaluated using the original treatingradiologist reading and was 17.7% (95% CI=4.8-28.9%) in intent-to-treat (ITT) and20.5% (95%CI= 4.4-34%) in per protocol (PP) for pneumonia with a positive film.The 2841 sets of radiographs from this trial were all scanned and blindly read bytwo WHO cross-trained readers. Readings non-concordant as positive (consolida-tion or pleural effusion) were read by a two radiologist panel as were quality controlsets.Results: 250/2841 (8.8%) of films were read as positive by both readers. An addi-tional 129 were read as positive by reader A only and 142 by reader B only for atotal of 521 read positive by one or more reviewers. The concordance rate betweenthe two reviewers was 250/521= 48%. Of the 271 discordant films, 45/129 (34.9%)of reader A and 66/142 (46.5%) for reader B were finalized as positive by theadjudicating panel. Overall 361 films were finalized as positive (12.7%). With these361 images as the “gold standard”, the sensitivity and specificity of reader A were82% and 97% and 88% and 97% for reader B, respectively. Kappa for the two read-ers was 0.59. Of the 25 control films read as positive by both A and B, 80% werealso read as positive by the panel and all 25 control negative films were read asnegative by the panel. Using these results, the efficacy against first episode of x-rayconfirmed pneumonia adjusting for age, gender and year of vaccination, ITT = 25.5%(95%CI=6.5-40.7%, p=0.011) and PP = 30.3% (95% CI= 10.7-45.7%, p=0.0043)Conclusion: Research reading of x-rays using WHO criteria increased point esti-mates of vaccine efficacy presumably through improved specificity. However, theseresults were not significantly different from the original analysis.


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Pneumococcal Accelerated Development and Introduction Plan Mission andObjectives

Levine, O. GAVI’s Pneumococcal ADIP

Pneumococcal disease is a major killer of children in developing countries. Effec-tive vaccines exist to prevent it and yet, experience with other similar vaccines showsthat it can take >15 years to assure that the vaccine reaches even 10% of the childrenin the world’s poorest countries. The Pneumococcal Vaccines Accelerated Develop-ment and Introduction Plan (PneumoADIP) is an independent group located at JohnsHopkins Bloomberg School of Public Health and funded by the Global Alliance forVaccines and Immunizations (GAVI). The ADIP’s mission is to improve child healthby accelerating the evaluation of and access to new life-saving pneumococcal vaccinesfor the world’s poorest children. PneumoADIP aims to shorten the time lag be-tween the use of a new vaccine in rich countries and its use in poor countries byworking to achieve a sustainable, affordable supply of quality vaccines by reducingthe uncertainty of demand for the vaccine in developing countries. ThePneumoADIP’s strategy will be reviewed in detail, including an assessment of thevalue of accelerated pneumococcal vaccination in developing countries.

Current perceptions and attitudes regarding ARI prevention in four develop-ing countries

Kvist, H. GAVI’s Pneumococcal ADIP

Background: Communicating the value of pneumococcal vaccination is one of the3 main areas of the PneumoADIP strategy. In order to communicate effectively, thePneumoADIP is undertaking a systematic assessment of the current perceptionsand attitudes and specifically the priority assigned to prevention of ARI in child-hood. The work was conducted by Edelman, Inc. and supported by the CHANGEProject at AED with funding from USAID and technical support from thePneumoADIP.Study objectives and method: The perception and attitude research was executed infour developing countries with different economic and health challenges. The coun-tries visited were: India, Thailand, Senegal and MalawiInterviewees included senior Government officials, Secretaries of Health, heads ofEPI programs and NGO’s, representatives from UNICEF and WHO and opinionleaders in vaccine research and pediatrics.The aim of the study was to assess current national health care priorities and todetermine the level of awareness of ARI as a child health problem and the prioritycurrently assigned to preventing itFindings: ARI is not on the list of healthcare priorities in any of the visited countriesand the unprompted awareness of pneumonia as a major cause of mortality andmorbidity is low. Interviewees were not aware of data on ARI burden and the causesof ARI. However, when prompted many of the interviewees recognized ARI/pneu-monia as a leading cause of childhood illness and death.Conclusion: These findings indicate a need for improving awareness of existingdata on ARI in developing countries and supporting efforts to collect additionaldata where they are not available. A major effort is needed to demonstrate to deci-sion-makers that ARI is preventable and that they can access solutions to this im-portant child health problem.


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Innovative Options for Financing and Supply of Pneumococcal Vaccines forDeveloping Countries

Accelerated introduction of new, life-saving pneumococcal vaccines into develop-ing countries will require an adequate, affordable, sustainable supply of vaccinesand the financing to procure it. Recently, financing for accelerating and sustainingvaccines into developing countries has received an unprecedented amount of atten-tion and energy. The existence of the Vaccine Fund, a fund of >$1 billion for sup-porting immunizations in the 75 lowest income countries of the world, is the bestknown of these innovations. Other innovative approaches for assuring increasedfinancing and/or making more effective use of financing are also being exploredand implemented. These include securitization of Vaccine Fund financing, “buy-downs” of World Bank loans, the Vaccine Fund, guaranteed purchase contracts, andthe use of ‘put’ options. On the financing side, the PneumoADIP is working withthe groups in this area, including donors and vaccine manufacturers, to determinewhether and how these potential innovative options and mechanisms might be ap-plied to pneumococcal vaccines and the impact of using these on accelerating vac-cine use and pneumococcal disease prevention in the world’s poorest countries.

Introduction to PneumoADIP surveillance and research projects

Deloria-Knoll M. Johns Hopkins Bloomberg School of Public Health, Baltimore,Maryland, U.S.A.

Aims: A major priority of the PneumoADIP is to establish the value of pneumococ-cal conjugate vaccine by providing essential information on disease burden andvaccine impact in targeted countries to enable local decision makers, GAVI, and itspartners to prioritize its introduction.Methods: Information essential for prioritizing vaccine introduction includes: (1)local or regional data on proven pneumococcal disease (pneumonia, meningitis,sepsis), serotype distribution, and mortality; (2) data on cost-effectiveness, antibi-otic resistance, or otitis media; and (3) documentation of the impact of vaccineintroduction. Working with Scientific Advisors, the PneumoADIP synthesizes ex-isting data, and identifies and communicates key research gaps to be filled by ADIPand non-ADIP activities. Research investments will be strategically targeted tomaximize impact on policy decisions while recognizing the limited scope of theADIP budget and timeline. Communication with local decision-makers will ensurethat research addresses health outcomes of interest.Results: PneumoADIP has already funded (1) surveillance networks in several coun-tries in Africa and Asia to identify laboratory-confirmed invasive S. pneumoniaedisease, (2) immunogenicity and safety trials to assess the value of alternative vac-cination regimens, (3) economic analyses to assess cost-effectiveness of vaccina-tion and cost of illness, and (4) ancillary studies nested in existing large-scale vac-cine trials to improve our understanding of the vaccine’s efficacy against pneumo-nia. A meeting of Scientific Advisors contributed to the identification andprioritization of future ADIP activities.Conclusions: The PneumoADIP is an effort to strategically use its resources to ad-dress issues important in evaluating the introduction of a new pneumococcal vac-cine and to identify and influence research priorities of other agencies supportingresearch. If successful, the PneumoADIP could significantly accelerate access toaffordable, life-saving pneumococcal vaccines for developing countries.


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Health economic impact of pneumococcal disease and its prevention

Sinha A1, 2, Lee G3, Lieu TA3. Brigham and Women’s Hospital1; Depts. of Medicine2

and Ambulatory Care and Prevention3, Harvard Medical School and Harvard Pil-grim Health Care, Boston, USA

Vaccination of children in developing countries against pneumococcal infection hasgreat potential to save lives and reduce disability. Pneumococcus is the leadingbacterial source of acute respiratory infections, which are a major cause of childmortality. An updated economic analysis is needed to inform the overall investmentcase for vaccine purchase and to identify the optimal areas for investment in currentdata collection and vaccine trials. We are conducting a cost-effectiveness analysis toassist decisions about investment in pneumococcal vaccine purchase and to helpinform priorities for the Global Alliance for Vaccines & Immunization’s (GAVI’s)Pneumococcal Vaccine Accelerated Development and Introduction Plan (ADIP).The decision analytic model evaluates three alternative strategies: No intervention,accelerated uptake with vaccine funding, and accelerated uptake without vaccinefunding. The analysis is being conducted from a societal perspective and with asummary outcome measure of US$ per disability-adjusted life-year saved. Theanalysis addresses: 1) the cost-effectiveness of pneumocccal vaccination for low-income countries; 2) the cost-effectiveness of pneumococcal vaccination under al-ternative scenarios, e.g., for selected regions, for middle-income countries, and foralternative vaccine regimens; 3) identification of the key factors that drive the healthand economic impact of pneumococcal vaccination via sensitivity analyses. Thisanalysis hopes to provide systematic projections of the health and economic impactof pneumococcal vaccination of children in developing countries. It will yield in-formation toward the optimal choices for current vaccine funding decisions, as wellas identify the most fruitful areas for further information gathering.

Progress of network for surveillance of pneumococcal disease in East Africaregion (netSPEAR) project.

Maranga W.1, English M.2, Scott A.2, Kamau T.3, McDonagh M.4, Oluoch T.2 1.netSPEAR, 2 Wellcome Trust / Kenya Medical Research Institute, 3 Expanded Pro-gramme on Immunization, MOH Kenya, 4. DfID

Aims: 1. To assess the burden of invasive pneumococcal disease (IPD) in childrenaged 2 months to 5 years in the 7 countries of the WHO East Africa block. 2. Todetermine the serotypes and antimicrobial sensitivity patterns of pneumococci caus-ing IPD in the region. 3. To strengthen sentinel surveillance for invasive Hib diseaseduring introduction of the conjugate Hib vaccine. 4. To facilitate an informed evalu-ation of the case for accelerated introduction of conjugate pneumococcal vaccine inEast Africa. 5. To strengthen communicable disease surveillance and communica-tion between doctors, ministries and other stakeholders.Methods: Through training, motivational visits and conferences, laboratory and clini-cal standardization and consumable support, netSPEAR is developing a network of12 surveillance hospitals in four countries in rural and urban East Africa with plansto expand to all 7 WHO East Africa block countries. It is supported by GAVIspneumoADIP and is integrated into the WHO-AFRO meningitis surveillance net-work. CSF, and in some centres blood samples, are cultured from children meetingpre-defined clinical syndromes and processed with internationally accepted andlocally appropriate laboratory methods. Data are forwarded to netSPEAR in Nai-robi and disseminated to local policy makers, WHO-AFRO, and other internationalstakeholders via email, newsletters, meetings and at www.netspear.org.Results: Initial foundation conference was held in Nairobi Kenya in November 2003.SOPS, algorithms, hospital performance indicators and hospital sites assessmentchecklist developed. The data management software development is at an advancedstage and proposed initial sites assessment visits are currently ongoing. Existingdata from one surveillance site, Kilifi District Hospital in Kenya, shows culture-positive IPD is an important cause of admission to hospital in Kenya with an esti-mated incidence of 260/100,00 in children under 2 and 154/100,000 in childrenunder 5 years of age, and a case fatality rate of 30%.


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Gambia PVT

Cutts, FT Medical Research Council, The Gambia

Aims: To estimate the health and economic impact of the 9-valent pneumococcalconjugate vaccine.Methods, results, conclusions: The PneumoADIP is currently sponsoring 5 activi-ties nested within the larger phase 3 trial of pneumococcal conjugate vaccine. Ac-tivity 1: Further validation of x-ray classification of pneumonia. Activity 2: An analy-sis of the use of serologic markers of acute inflammatory responses and nasopha-ryngeal colonization for the improved diagnosis of likely bacterial pneumonia (incoordination with the ARIVAC vaccine trial in the Philippines). Activity 3: An analy-sis of the effect of 9-valent pneumococcal conjugate vaccination of infants on ac-quisition and transmission of S. pneumoniae. Activity 4: An analysis of the cost-effectiveness of pneumococcal conjugate vaccination in the Gambia. Activity 5: Aspatial analysis, using geographic information systems, of the relationship betweenaccess to care and pneumococcal disease burden. An update on the status of allactivities will be discussed and a brief overview of the results will be shared.


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DISCUSSION AND CLOSING REMARKS


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Pneumococcal vaccines and ecology of pneumococci

*Lipsitch, M

Harvard School of Public HealthDept. of Epidemiology677 Huntington AvenueBoston, MA 02115UNITED [email protected]

Pneumococcal carriage and clinical disease

*O’Brien, K

Johns Hopkins Bloomberg School of Public Health621 N. Washington St.Baltimore, MD 21205UNITED [email protected]


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Adherence Interactions among Competing Pneumococcal (Pnc) Vaccine (VS)and Non-vaccine Serotypes (NVS) in an in vitro tissue culture assay.

Rajam G, Romero-Steiner S, Jackson D, Whitney CG, Carlone GM. CDC, Atlanta,Georgia, USA 30333.

Aims: Marked reduction in Pnc invasive disease caused by VS was reported sincethe introduction of the conjugate vaccine in 2000.The serotypes responsible for theremaining Pnc disease may include both VS and NVS. To predict potential replace-ment serotypes, we determined the capacity of NVS strains to out compete VS strainsfor attachment in an in vitro adherence model using nasopharyngeal epithelial cells.Methods: Detroit 562 cells were co-infected with a VS and a NVS strain to yield~100 CFU/well per strain. A representative strain from VS 9V, 19F and 23F wastested in triplicate against 12 NVS strains (6A, 7F, 8, 9N, 10A, 11A, 12F, 15C, 16F,31, 33F, and 35B). The adherence capacity (CFU/well) for an individual isolate wasdetermined without antibiotic selection; however, an antibiotic resistance marker inVS was used for selection during co-infection. Percent reduction or increase in ad-herence of either VS or NVS was determined by comparing the adherence in thepresence or absence of antibiotic of the co-mixed isolates.Results: While there was a significant (P<0.05) reduction in adherence of NVS(mean reduction = -47%), an increase in adherence (mean increase = +27%) of VSwas found under competition conditions. Of the VS strains, 9V-N4707 and 19F-N6133 out-competed all 12 NVS strains, with up to 1.6 fold rise in adherence undercompetition conditions. In contrast, 23F-N3476 was out competed by 15C-4476,31-2247, and 35B-1766 with up to 2.2 fold rise in NVS adherence. Competitionwith VS did not markedly reduce (<25% of adherence) the adherence capacity ofcertain NVS (7F and 8 against 9V; 16F, 33F, 9N and 10A against 19F, and 31, 35Band 15C against 23F).Conclusions: VS may have a greater potential to adhere to nasopharyngeal cellsthan NVS. However, certain NVS strains have exhibited the potential to out-com-pete VS for cell attachment. This in vitro tissue culture model is useful for estimat-ing the carriage replacement potential of NVS.

Capsular switching was detected in approximately 1% of pneumococci carriedby children.

Kaltoft MS, Konradsen HB. WHO Collaborating Centre for Reference and Researchon Pneumococci, the Streptococcus Unit, Statens Serum Institut, Copenhagen, Den-mark.

Aims: Our purpose was to determine the frequency of capsular switching, i.e. hori-zontal exchange of capsular genes between pneumococcal isolates carried simulta-neously in nasopharynx, in colonized children.Methods: A study was conducted to determine the prevalence of nasopharyngealcarriage of pneumococci among children attending Danish day care centres (DCCs).The number of pneumococcal strains carried was found by use of direct serotypingof an enrichment broth culture. 1.5 ml serum broth was used as a transport mediumand as an enrichment broth. Specimen collection, species identification andserotyping of the isolates were performed according to standard procedures. Allisolates were genotyped by Pulsed Field Gel Electrophoresis (PFGE) in a CHEF-DR III, following restriction digestion with Smal. PFGE profiles were analysed byvisual inspection. Interpretation of PFGE pattern interrelationships was performedaccording to the criteria of Tenover and coworkers.Results: On average 38% (505) of the children (age 46 ± 18 months) in 27 day carecentres participated. 283 (56 %) were colonized by pneumococci. In 26 (9.2 %)carriers we found 2 distinct serotypes of pneumococci. In three (11.5 %) pairs theisolates had identical PFGE type. In all three pairs one of the serotypes had a PFGEtype, which were prevalent for this particular serotype. For the other serotypes inthe pairs this was the only occasion where we detected a serotype with this PFGEtype in the study. For two of the cases the “donor” serotype was found among theother isolates in the DCC.Conclusions: Two different serotypes were detected in 9.2 % of the carriers and11.5 % of these had identical PFGE types. The explanation of this finding could becapsular switching. If so, this means that, in approximately 1% of carriage in thisstudy capsular switching had taken place.


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Nasopharyngeal carriage of multiple Streptococcus pneumoniae isolates in asemi-closed community in the Kilimanjaro region of northern Tanzania.

Oriyo N, Charalambous BM, Sinclair A, and Gillespie SHMedical Microbiology, Royal Free & University College medical School, LondonNW3 2PF

Aim: To test for the possible carriage of multiple S. pneumoniae isolates as definedby serotype and antibiotic susceptibility.Methods: Healthy children under 7 years of age were recruited and naso- and oro-pharyngeal swabs taken. Either 4 or 8-10 pneumococcal isolates from each positiveplate were serotyped by slide agglutination with typing reagents from Statens Se-rum Institute and antibiotic susceptibilities were performed by E-test (NCCLS) fol-lowing standard procedures.Results: At the time of sampling the pneumococcal carriage rate in this communitywas 56%. When 4 isolates were picked 80% (20/25) of the samples were found tocontain multiple serotypes; 56% (14/25) had two different serotypes, and 24% (6/25) contained 3 different serotypes. When 8-10 isolates were picked 92% had mul-tiple serotype colonisations, of which 58% (7/12) had 2 serotypes, 17% (2/12) had3 serotypes, 8% (1/12) had 4 serotypes, and 8% had 5 serotypes. These percentagescould be higher as the data is limited to the serotypes present in the 23-valent polysac-charide vaccine, and the non-vaccine and the non-typable isolates were groupedtogether. Co-existence of antibiotic sensitive and non-susceptible isolates was alsoobserved. Combining the data from both the 4 and 8-10 picks 35% (12/34) hadmultiple sensitivities to penicillin, 27% (9/34) to cotrimoxazole, 9% (3/34) to tetra-cycline and 3% (1/34) to erythromycin.Conclusion: Data from this pilot study indicate that in this semi-closed rural com-munity where more than half the children sampled were carriers the levels of multi-ple colonisation by different serotypes is relatively high. The co-existence of bothdrug sensitive and non-susceptible organisms also appears to be a relatively com-mon event.

Pneumococcal Carriage Is Higher In Ugandan Adults Infected With HIV ThanIn The Uninfected, Shows Seasonal Variation And Is Directly Associated WithPneumococcal Disease Rates.

French N1, Watera C2, Moi K2, Whitworth J2,3, Gilks C4. 1 Liverpool School ofTropical medicine, UK, 2 MRC programme on AIDS, Uganda, London School ofTropical medicine, UK, Imperial College of Science and Technology, UK.

Aims: HIV-infected adults are at increased risk of pneumococcal disease. We wishedto establish whether they are at increased risk of carriage and the relationship ofcarriage to disease events.Methods: A cohort of HIV-infected adults attending an out-patient care clinic hasbeen followed up since 1995. Pernasal sampling of participants has been under-taken at regular intervals as well as comprehensive surveillance for pneumococcaldisease. An HIV-uninfected control group of similar age and social backgroundwho attended the clinic for HIV testing were also sampled. Pernasal swabs weretaken into Amie’s transport media and plated on to blood agar within 3 hours ofcollectionResults: Swabs were collected from 2604 HIV-infected adults, pneumococci wererecovered from 670 (25.7% CI 23.3 – 28.1), and from 631 HIV-uninfected adults,113 (17.9% CI 14.9-20.9) grew pneumococci (x2 infected vs uninfected = 16.94,P<0.001). Carriage peaked in the cooler drier months of June-August after the rainswhen humidity remains high. The carriage rate had a direct temporal associationwith disease events, with disease peaking in June-August, peak disease rate of IPD34.5/1000 person years.Conclusion: Pneumococcal carriage is significantly elevated in HIV-infected Afri-can adults compared to the uninfected. This suggests there is a defect in mucosalimmunity, which allows longer duration of carriage of a single serotype and/or greaterdensity of carriage and/or greater numbers of serotypes to be carried. Investigationsof mucosal immunity and carriage dynamics are planned. In addition, in regions ofhigh HIV prevalence in Africa, the ability of conjugate pneumococcal vaccines givento children to produce herd immunity may be affected by a large reservoir of pneu-mococcal carriage in adults.


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Natural competition between Streptococcus pneumoniae and Staphylococcusaureus during colonisation in healthy children

D. Bogaert1, A. van Belkum2, M. Sluijter1, A. Luijendijk2, R. de Groot1, H.C. Rümke3,H.A. Verbrugh2, and P.W.M. Hermans1. 1Department of Paediatrics, 2Department ofMedical Microbiology and Infectious Diseases, 3Vaxinostics, Erasmus MC, Rotter-dam, The Netherlands

Background: Recently, a large randomised double-blind trial with a 7-valent pneu-mococcal-conjugate vaccine was conducted in children suffering from recurrentacute otitis media. A complete shift in pneumococcal colonisation towards non-vaccine serotypes was observed. In addition, an increase in Staphylococcus aureus-related acute otitis media was found after vaccination (Veenhoven, 2003. Lan-cet:361;2189-95).Methods: We investigated the prevalence and determinants of nasopharyngeal car-riage of S. pneumoniae and S. aureus in 3193 healthy children 1-19 years of age. Inaddition, we performed serotyping of all pneumococcal isolates. Finally, we inves-tigated the correlation between S. aureus and S. pneumoniae carriage.Results. Determinants of nasopharyngeal carriage of S. pneumoniae (19%) wereage (peak incidence: 3 years) and regular day-care visits (OR: 2.14, 95% CI: 1.44-3.18). Risk-factors for S. aureus carriage (36%) were age (peak incidence: 10 years),male sex (OR: 1.46, 95% CI: 1.25-1.70), large families (� 5 members, OR: 1.17,95% CI: 1.00-1.37) and passive smoking (OR: 1.22, 95% CI: 1.04-1.42), whereasactive smoking was inversely related to S. aureus carriage (OR: 0.75, 95% CI: 0.53-1.04). Pneumococcal serotype analysis showed 42% vaccine type and 58% non-vaccine type pneumococci. Multivariate regression analysis showed a negative cor-relation for co-colonisation of S. aureus and vaccine-type pneumococci (OR: 0.68,95% CI: 0.48-0.94), but not for S. aureus and non-vaccine serotypes.Conclusion: These observations suggest the presence of a natural competitive bal-ance between vaccine-type pneumococci and S. aureus during colonisation, whichmay explain the increase in S. aureus-related otitis media events found after vacci-nation.

Molecular dynamics of pneumococcal colonization in response to pneumococ-cal conjugate vaccination in children with recurrent acute otitis media

Debby Bogaert1, Reinier H Veenhoven2, Marcel Sluijter1, Wim JW Wannet3, Ger TRijkers4, Wil HF Goessens5, Anne G. Schilder6, Lieke EAM Sanders4, Ronald deGroot1, and Peter WM Hermans1

1Department of Pediatrics, 5Department of Medical Microbiology and InfectiousDiseases, Erasmus MC-Sophia, Rotterdam, 2Department of Pediatrics, SpaarneHospital Haarlem, 3National Institute for Public Health and the Environment,Bilthoven, 4Department of Immunology, 6Department of Otorhinolaryngology, Uni-versity Medical Center, Utrecht, The Netherlands

Introduction: A randomized double-blind trial with a 7-valent pneumococcal conju-gate vaccine was conducted in The Netherlands among 383 children, aged 1-7 years,with a history of recurrent acute otitis media. No effect of vaccination was found onthe pneumococcal colonization rate. However, a shift in serotype distribution wasclearly observed (Veenhoven, 2003. Lancet: 361:2189-95).Methods: We investigated the molecular epidemiology of 921 pneumococcal iso-lates retrieved from both the pneumococcal vaccine (PV) and control vaccine (CV)group during the vaccination study.Results: Within individuals a high turnover rate of pneumococcal genotypes wasobserved, which was unaffected by vaccination. Comparison of the genetic struc-ture before and after completion of the vaccination scheme revealed, despite a shiftin serotypes, genetic homology between 70% of the pneumococcal populations.The remaining isolates (30%) were equally observed in the PV and the CV group.In addition, the degree of genetic clustering was unaffected by vaccination. How-ever, within the population genetic structure, non-vaccine serotype clusters with theserotypes 11, 15 and 23B became predominant over vaccine-type clusters as a resultof vaccination. Finally, overall pneumococcal resistance was low (14%), and, albeitnot significant, a reduction in pneumococcal resistance as a result of pneumococcalvaccination was observed.


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Risk factors for carriage of Streptococcus pneumoniae in children attendinginternational schools in Hong Kong

Boost MV, O’Donoghue MM, Dooley JS, School of Nursing, Hong Kong Polytech-nic University, HK SAR.

Aims: Carriage rates of S. pneumoniae in primary school age children are low inHong Kong and have been suugested to be related to socio-economic status, ethnic-ity or climate. This study investigated associations between ethnicity, length of stayin Hong Kong and familial factors, and carriage of S. pneumoniae.Methods: In a cross-sectional survey of children attending the first year of 13 inter-national primary schools, swabs were collected and cultured for S. pneumoniae andantibiotic susceptibility testing was performed on all isolates. Parents of participat-ing children completed a questionnaire giving details of age, sex, number of sib-lings, kindergarten attendance, ethnicity, housing type, and length of stay in HK.Results: 69% of children, average age of 66 months, were sampled. 23 (4.5%) car-ried S. pneumoniae but only two strains were resistant to penicillin, as well as totetracycline and erythromycin. Seven strains showed resistance to either tetracy-cline (4) or erythromycin (2) or both (1). No ciprofloxacin resistance was observed.Carriage rates varied between schools (p = 0.002) but there was no associationbetween the age of the child and carriage in the age range sampled. Travel abroad,especially to Europe, appeared to increase risk of carriage, but did not reach signifi-cance. Hospitalisation, visits to the doctor and recent antibiotic use did not signifi-cantly increase carriage risk. Day care use (94%) was not significantly associatedwith carriage. Carriage was somewhat higher in those with Chinese ethnicity, butdid not reach significance. Carriage was higher in children of any ethnicity, whohad not lived in HK for 4 years (p = 0.04).Conclusions: Primary school children of non-Chinese ethnicity have a low rate ofcarriage of S. pneumoniae, similar to that of locally-born ethnic Chinese, if theyhave lived in HK for at least 4 years. This suggests that local conditions, rather thangenetic differences, favour a low rate of carriage. Climate, early use of day care andhigh living standards may result in low carriage of S. pneumoniae in this area.

Nasopharyngeal carriage and antimicrobial resistance of pneumococcus in Bra-zilian adolescents.

Cardozo DM1,3, Nascimento-Carvalho C1, Brandão MA2, Azevedo GMS2, SouzaFR1, Silva NMS1. Federal University of Bahia1, LACEN-BA2, FAPESB3, Brazil.

Introduction: Respiratory infections are a major cause of morbidity and mortalityamong adolescents. Pneumococcus colonizes the nasopharynx where it replicatesand from which it gains access to parts of the body that are normally free of infec-tion, mainly the lungs. There has been an increasing number of reports of antimi-crobial resistance among pneumococcal isolates from children or adults.Aims: To determine the frequency of nasopharyngeal carriage, to record the level ofantimicrobial resistance, and to identify risk factors for pneumococcal infectionamong adolescents.Methods: This is a cross-sectional study of adolescents randomly recruited frompublic schools located in every District of Salvador, Northeast Brazil, from Novem-ber 2002 to July 2003. Demographic, clinical and epidemiological data were col-lected. Nasopharyngeal samples were inoculated onto sheep agar with gentamicinand pneumococcus strains were identified by using bile solubility and optochindisc. Disc diffusion was used for surveillance of antimicrobial resistance.Results: Of 1,013 adolescents 53.3% were females and the median age was 15 years(mean 14.6+2.3). Pneumococcus was recovered from 8.3% and pneumococcal colo-nization was associated with having a smoker in the household (11.5vs6.6, p<0.05),having an upper respiratory infection(URI) during recruitment (15.0%vs5.9%,p< 0.05) and having a history of asthma (18.9%vs7.9%, p<0.05). Resistance wasdetected to tetracycline (20.2%), erythromycin (4.8%), trimethoprim-sulfa-methoxazole (38.1%) and penicillin (21.1%). All isolates were susceptible to chlo-ramphenicol, clindamycin, rifampin and vancomycin.Conclusions: Pneumococcal nasopharyngeal carriage was detected in 8.3% of ado-lescents and penicillin resistance was described in 21.1% of the strains. Having asmoker in the household, current URI or a history of asthma may be risk factors forpneumococcal colonization in adolescents.


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Serotype, molecular type and antimicrobial susceptibility of pneumococcal car-riage strains in Aboriginal and non-Aboriginal children in arid Western Aus-tralia

Carville KS1,2, Lehmann D1, Bowman J3, Riley TV3,4, Murphy D5, Elsbury D1, StokesA1, Finucane J1, Monck R1 for the Kalgoorlie Otitis Media Research Project. TelethonInstitute for Child Health Research, Perth1, National Centre for Epidemiology andPopulation Health, Australian National University2, Division of Microbiology &Infectious Diseases, PathCentre, Perth3, Department of Microbiology, University ofWestern Australia4, Public Health Bacteriology Laboratory, Brisbane, Australia.

Aims: To examine serotype, molecular type and antimicrobial susceptibility pat-terns of pneumococci (Pnc) isolated from the upper respiratory tract (URT) of youngAboriginal and non-Aboriginal children.Methods: 199 Pnc isolates (97 from non-Aboriginal and 102 from Aboriginal chil-dren) from 99 children were examined. Serotyping,PFGE and antimicrobial susceptibility testing was performed on all isolates.Results: The most frequently carried Pnc serotypes were 6B (20%), 19F (10%),19A (9.5%), 11A (8%), 6A (7%), and 16F (6%). Serotype 6B was isolated lessoften from Aboriginal than non-Aboriginal children (p=0.01) whereas Aboriginalchildren more frequently carried non-vaccine type 16F (p<0.01). The most geneti-cally diverse serotypes were 6A, 14, 19F, 23F, 15B, 19A and 9V. Eleven Pnc, all ofwhich were serotype 6B and the same molecular type and isolated only from non-Aboriginal children, were resistant to chloramphenicol, penicillin, cotrimoxazole,tetracycline and erythromycin. One other isolate (serotype 23F) was also multi-resistant but of a different molecular type.Conclusions: Aboriginal children in this cohort are less likely than non-Aboriginalchildren to carry vaccine types of pneumococci, such as 6B. The extent of geneticdiversity of carriage strains varies between serotypes. A multi-resistant clone, pri-marily belonging to serotype 6B, carried by non-Aboriginal children has been iden-tified and will be characterised further.

Intranasal (IN) exposure to Streptococcus pneumoniae (SP) and protectionagainst IN colonization: impact of capsular serotype (ST) on colonization andon homologous and heterologous protection

Trzci ski K1, Thompson CM1, Malley R2 and Lipsitch M1. Harvard School of PublicHealth1, Children’s Hospital, Harvard Medical School2, Boston, Massachusetts

Aims: Using otherwise isogenic strains that differ only in ST, to test the hypothesesthat: (a) ST modulates the success of IN colonization; (b) homologous (same ST)exposure provides greater protection against a given challenge than does heterolo-gous exposure; (c) ST affects the degree of heterologous protection conferred by INexposure; and to establish correlates of protection against IN carriage.Methods: We previously created variants of strain TIGR4 that differed only at thecps locus conferring ST of 6B, 7F or 14. Groups of 20 C57BL/6 mice were exposedIN on 3 consecutive weeks to 6B, 7F, 14 or saline and cured of colonization 1 weeklater with rifampin. Two weeks later, they were bled and sampled for saliva, chal-lenged IN with ~ 10^6 cfu of 6B, 7F, or 14, and assessed for colonization 1 weeklater by retrograde tracheal wash. All combinations of exposure (3 ST+saline) andchallenge (3 ST) were assessed.Results: There was no difference in the degree of protection between mice that hadprior homologous exposure (probability of colonization reduced by median 57%,range 24-93%) and those with heterologous exposure (median reduction 54%, range24-84%). ST 14 was the most efficient colonizer (100% of naive animals colo-nized, vs. 70% and 74% for 6B and 7F, p<0.05). 14 also induced the strongestprotection vs. heterologous challenge, and protection against ST 14 challenge wasthe least effective. In a subset of 49 SP exposed animals for which ELISA wasperformed, PsaA and PspA IgG concentrations were associated with reduced risk ofcolonization (p<.005). Salivary IgA showed a similar trend (non-significant).Conclusions: In this model IN exposure to a strain of heterologous ST can induceprotection as good as or better than exposure to a homologous ST strain. ST canaffect the success of IN colonization, the ability to stimulate immune responses thatare not ST-specific, and the susceptibility to such antibodies. Work is ongoing toestablish additional humoral and mucosal correlates of protection in this model.


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Pneumococcal carriage in healthy Norwegian children attending day carecenters

Pedersen MK1, Sørdal JO2, Aaberge IS1, Høiby EA1, Frøholm LO1, Caugant DA1.Norwegian Institute of Public Health1, Linderud legesenter2, Oslo, Norway

The aim of this study was to determine the rate of nasopharygeal carriage, serotypedistribution and antimicrobial susceptibility of S. pneumoniaein healthy Norwegian children.Background: In April 2003 two cases of pneumococcal meningitis occurred in oneday care center in Oslo. Both children suffered a serotype 14 infection. One monthafter the episodes, this study was undertaken among healthy children from the af-fected day care center and from 3 other day care centers in Oslo.Methods: Nose and throat samples from 94 children, age 1 – 6 years, were col-lected in the period from May to June 2003. The samples were screened for S.pneumoniae by standard laboratory methods. All isolated pneumococcal strainswere serotyped using antisera from Statens seruminstitutt, (Copenhagen, Denmark).MIC testing with penicillin, cefotaxime, ceftriaxone, ciprofloxacin, erythromycin,doxycycline, clindamycin and chloramphenicol were performed by the Etest method,(AB Biodisk, Solna, Sweden).Results: The total carriage rate was 45 % (43 of 94). The carriage rate varied amongthe day care centers from 23% to 75%; the carriage rate in the center where the twocases had occurred was intermediate (43 %). Carriage was most frequent amongthe children 2 years of age (70 %). Eleven serotypes were represented among the43 isolates. The most frequent ones were serotypes 6A, 23F, 6B and 19F; 5 strainswere non-typable. No serotype 14 isolate was found among the healthy carriers.None of the strains were resistant to any of the drug tested. However, two strainsshowed reduced susceptibility to penicillin( MIC 0,125), one to ceftriaxone (MIC1,5), one to chloramphenicol (MIC 4), and four to ciprofloxacin (MIC 2).Conclusion: Our analyses showed a relatively high rate of pneumococcal carriageamong healthy Norwegian children attending day care centers. The children harboreda variety of serotypes. None of the strains were resistant to any of the drug tested.

Importance of Streptococcus pneumoniae isolation carriers and pneumococ-cal invasive disease in pediatric population

Gómez-Barreto D*, Martínez C, Espinosa de los Monteros Luz Elena**.Dpto. Infectologia* , Labaoratorio de Bacteriología ** del Hospital Infantil deMéxico(Infectious Diseases Department, Bacteriology Department** Mexican ChildrenHospital)

The relevance between the carrier condition and the pneumococcal invasive dis-ease of Streptococcus pneumoniae is not clear. There are several studies not con-clusive , however in México it has not even yet documented in the past.Objective: Establish relevance of the relationship among a carrier condition andpneumococcal invasive disease.Materials and Methods: A transversal study and a comparative análisis wasperformed among proportion of Streptococcus pneumoniae serotypes and peni-cillin susceptibilities from 918 children and 146 pneumococcal invasive diseaserelated patients, purpose was to establish differences or associations among thetwo groups.Results: Statistically significant differences were found (P<0.05) among sero-type carriers and pneumococcal invasive disease, with a higher proportion forserotype 14, 4 and 9V in the pneumococcal invasive disease and also 19F and 6Afor the carriers. An age – related negative association was also observed for thecolonization of Streptococcus pneumoniae with a diminished susceptibility forpenicillin.Conclusions: This study provides information from Streptococcus pneumoniaeepidemiology as a colonizer and producer of pneumococcal invasive disease andprovided new data regarding frequency of certain serotypes not observed before.


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Streptococcus pneumoniae serotype 23F cultivation under aerobic and anaero-bic atmospheres: differences on cell associated capsular polysaccharide

Gonçalves,VM1, Carneiro, S2, Giordano, RC3, Tanizaki, MM1. 1 Centro deBiotecnologia and 2 Lab. Biologia Celular, Instituto Butantan, Brazil. 3 DeptoEngenharia Química, UFSCar, Brazil.

Streptococcus pneumoniae is capable of a marked intrastrain variation betweenopaque and transparent phenotypes. Opaque variants present greater amount ofcapsular polysaccharide (PSC) and they are more virulent in models of systemicinfection, while transparent variants show increased adherence to epithelial cells innasopharyngeal carriage models.Two different atmospheres were employed to cultivate S. pneumoniae in 5L reactor(Bioflo2000, New Brunswick) using the medium and culture conditions previouslyestablished [Gonçalves et al 2002]: i) nitrogen-sparged only and ii) nitrogen spargingfollowed by air when the growth reached the stationary phase. In the second case,when nitrogen was replaced by air the amount of capsular polysaccharide (PSC) inthe medium increased from 300 mg/L to 400 mg/L, whereas tight cell bound PSCdecreased from 400 mg/L to 150 mg/L. These differences seem not be related tocellular lysis, since CFU profile was very similar in both cultures. Immunoelectronmicroscopy of bacteria from cultivation in nitrogen and in air clearly showed differ-ences on the quantity of cell associated PSC: the amount of tight cell bound PSCwas higher in bacteria growing under nitrogen than in those growing under air. Thedifferences shown in immunoelectron microscopy could suggest the occurrence ofan intrastrain variation as consequence of changing the atmosphere, thus the oxy-gen could play a role on the modulation of the phase variation.

Pneumococcal carriage during the first year of life among children in Savar,Bangladesh

Granat S1, Zakaria MM2, Piirainen L2, Herva E3, Ollgren J3, Auranen K4, MäkeläPH5

National Public Health Institute (KTL), Helsinki1 and Oulu3, Finland, GVRL2, Savar,Bangladesh

Aims: To describe the pattern of pneumococcal carriage during the first year of lifein Savar, Bangladesh.Background: To provide more data on pneumococcal carriage in South-East Asiawe performed a longitudinal study of Bangladeshi families. The carriage preva-lence and acquisition data have been presented previously (Poster, ISPPD-3 Alaska);pneumococcal acquisition was fast (50% of children had encountered pneumococciat least once by the age of 8 weeks) and the point prevalence stayed around 50%from the age of approximately 16 weeks onwards.Methods: From 96 families with a newborn child, nasopharyngeal swabs were col-lected from the newborn every 2 weeks until 4 months of age and subsequenlyevery 4 weeks until the end of follow up. Pneumococci were isolated and serotypedas described earlier. A pneumococcal carriage episode was defined as the time pe-riod between two samples of the same serogroup, with at most one negative samplein between, plus 2 weeks to account for the sampling frequency. The mean durationof carriage episodes was estimated by applying an exponential model.Results: During the first year of life a total of 360 pneumococcal carriage episodeswere observed among the 96 children enrolled (median 4 episodes/child). The numberof different serogroups encountered per child varied between 1 and 7, the medianwas 3. Thus, the same child had seldom more than one carriage episode of the sameserogroup/type. Carriage episodes were mostly short, with only one positive samplein most episodes. The estimated mean duration for all carriage episodes was 46days while the two most common serogroups 6 (55 episodes) and 19 (50 episodes)were carried clearly longer, 63 days both.Conclusions: Children in a semi-rural area of Bangladesh are extensively exposedto pneumococci and experience several short episodes of carriage by differentserogroups.


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A Model to Study the Impact of Pneumococcal Conjugate Vaccine on Asymp-tomatic Nasopharyngeal (NP) Carriage of S. pneumoniae (SP) in Calgary,Canada.

Kellner JD, MacDonald J, Church DL, Scheifele D, Tyrrell G. CASPER (CalgaryArea S. pneumoniae Epidemiology Research), Calgary, Canada.

Aim: Establish a project to evaluate 3-year trend in NP carriage of SP (vaccine-serotype (VT), VT-related (VTR) and non-VT (NVT)) in children after introductionof routine 7-valent pneumococcal conjugate vaccine (PCV7) immunization pro-gram in the province of Alberta, Canada.Methods: Routine PCV7 immunization began in Alberta for all children born afterJune 30, 2002 and for older children with high-risk conditions, but with no catch-upprogram. Calgary (pop ~1,000,000) is a city in Alberta. We will conduct 6 twice-yearly surveys of NP carriage in children aged 12 mos, 18 mos and 4.5 years attend-ing community health centres (CHCs) for immunizations. Swabs are plated within6 hrs and SP is identified using standard methods. Isolates are serotyped and antibi-otic susceptibilities are determined.Results: The first survey took place in June & July 2003. Children who had alreadyreceived PCV7 were excluded from this initial survey. A total of 485 children par-ticipated and 108 (22%) carried SP. The carriage rate of children cared for at daycarecentres (42%) was higher than children attending dayhomes (31%) or cared for athome (20%, P=0.005). Children with siblings carried SP more often than childrenwithout siblings (26% vs, 16%, P=0.02). Carriage in 12 and 18 month olds (25%)trended higher than 4.5 year olds (18%, P=0.07). Isolates were serotyped: 68.8%were VT, 8.3% were VTR and 22.9% were NVT. 5% of isolates were penicillin non-susceptible, 10% were erythromycin non-susceptible and 22% were TMP/SMX non-susceptible. The second survey took place in Dec 2003 & Jan 2004 (results pend-ing).Conclusions: These initial results provide baseline data obtained soon after the startof routine PCV7 immunization program, from children who have not received PCV7.The SP carriage rate and serotypes carried will be influenced not only by vaccina-tion status but by factors such as daytime care status and age.

Pneumococcal carriage in older children and adults in a high risk population.

Mackenzie G1, Leach A1, Morris P1 and Carapetis J2. Menzies School of HealthResearch1, Darwin, Australia. Centre for International Child Health, University ofMelbourne2, Dept. of Paediatrics, Australia.

Aims: To determine pneumococcal (Spn) nasopharyngeal carriage rates and factorsinfluencing carriage in older children and adults at high risk of pneumococcal dis-ease.Methods: In 2002, Four Australian Aboriginal communities participated in this sur-vey. Nasopharyngeal swabs were performed for Aboriginal adults and childrengreater than three years of age. Swabs were processed using standard microbiologi-cal techniques for culture, identification and serotyping of pneumococci. Informa-tion regarding demographics and possible risk factors for carriage was recorded.Results: 537 swabs were performed. After analysis of 33%(176/537) of the data,the overall Spn carriage rate was 52% (91/176). The Haemophilus influenzae car-riage rate was 45% (79/176). Spn carriage in males and females was 57% (48/84)and 47% (43/91) respectively. The Spn carriage rate in those less than 5, 5 to 16,and greater than 16 years of age was 84% (16/19), 71% (32/45) and 38% (43/112)respectively.Twenty-four different Spn serotypes were isolated. Predominant serotypes were:19A, 6A, 19F and 7. Spn serotypes were categorised in relation to the 7-valentpneumococcal conjugate vaccine (7PCV). Carriage rates of vaccine types (VT),vaccine related types (VR) and non-vaccine types (NV) were 10% (17/176), 13%(22/176) and 30% (52/176) respectively.For those less than 5, 5 to 16 and greater than 16 years of age, predominant Spnserotypes were VR types (10/16), NV types (20/32) and NV types (29/43) respec-tively.Conclusions: Preliminary results suggest high nasopharyngeal Spn carriage rates inolder children and adults in this population. Spn serotypes were diverse and pre-dominantly unrelated to the 7PCV. Complete data will be presented including indi-vidual and family structure risk factors for Spn carriage. This data will provide abaseline for evaluation of the impact of infant 7PCV on Spn carriage in unimmunisedcontacts.


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Nasopharyngeal Carriage of Pneumococci in Children Admitted with SeverePneumonia.

McNally L.M1,2, Jeena, P.M2, Gajee K,2, Sturm A.W2 , Coovadia, H.M2, TomkinsA.M1 and Goldblatt D1. Institute of Child Health, London1 and Nelson R MandelaSchool of Medicine, Durban, South Africa..

Aim: To determine whether HIV status, age and antibiotic use impact on the car-riage of pneumococci and other organisms in children admitted with severe pneu-monia in an HIV endemic area and to compare rates of isolation of pneumococciusing nasopharyngeal versus oropharyngeal swabs in this population.Methods: Children aged 1 to 59 months admitted to King Edward Hospital, Durbanwith WHO defined (very) severe pneumonia were included. All children had na-sopharyngeal and oropharyngeal swabs taken before anti-microbials were com-menced. All children also had linked anonymous HIV testing.Results: 362 children were enrolled in the study (median: 4.9 months) of whom 185(51%) were colonised with pneumococci on admission. 134 children had pneumo-cocci isolated exclusively from nasopharyngeal swabs, 6 from oropharyngeal swabsand 45 from both (P<0.001). Colonised children were significantly older than theiruncolonised peers (mean :11.9 vs 9.2 months; p=0.03), although colonisation oc-curred early in this cohort (peak 2.7 months). There was no statistically significantdifference in the HIV rates of colonised (68%) and uncolonised (71%) childrenoverall, although for the HIV infected cohort, a significant trend was seen for colo-nisation increasing with age (X2 for trend: p<0.01). There was no difference in theadmission urinary antimicrobial activity between colonised (38%) and uncolonised(41%) children nor in the response rates to pneumonia treatment (63% vs 57%respectively).Conclusions: Nasopharyngeal swabs are significantly better than oropharngeal swabsat determining pneumococcal carriage in both HIV infected and uninfected chil-dren under 5 years old. HIV infected children had increased rates of pneumococcalcarriage with age but this was not seen in HIV uninfected children. In both infectedand uninfected children, the presence of pneumococci in the nasopharynx on ad-mission had no impact on their subsequent response to pneumonia therapy.

Epidemiology of Pneumococcal (Pnc) Carriage among American Indian Chil-dren

Millar EV1, O’Brien KL1, Watt JP1, Bronsdon M2, Reid R1, Santosham M1. 1. JohnsHopkins University, Baltimore, MD; 2. Centers for Disease Control and Prevention(CDC), Atlanta, GA.

Aims: To describe Pnc carriage among Navajo and White Mountain Apache (N/WMA) children.Methods: A group-randomized trial of 7-valent conjugate pneumococcal vaccine(PnCRM7, Wyeth) was conducted on the N/WMA reservations. A carriage studywas nested in the trial to evaluate the impact of PnCRM7 on Pnc carriage amongvaccinees and their household siblings. Depending on the community of residence,study children received either PnCRM7 or control vaccine, a group C meningococ-cal conjugate (MnCC, Wyeth). Vaccinees were swabbed at approximately 7, 12 and18 months of age; household members �6 y were also swabbed at these visits.Nasopharyngeal specimens were collected with calcium alginate swabs, placed inSTGG transport media and sent to the CDC for Pnc identification and serotyping.We analyzed data among children living in MnCC-randomized communities to de-scribe the epidemiology of Pnc carriage among American Indian children <6 y ofage. Generalized estimating equations (GEE) were used to control for householdclustering and repeated measures of study children.Results: We enrolled 412 children from MnCC-randomized communities; 1068 speci-mens were obtained, an average of 2.6 (range, 1-3) specimens per child. Pnc wasisolated at least once from 381 (91%) children. Sixty-seven percent of specimenswere positive for Pnc. Age <2 y [OR: 2.2], male sex [OR: 1.5], daycare attendance[OR: 1.9], and having a sibling colonized with Pnc [OR: 4.3] were associated withan increased risk of carriage. Thirty-six percent of Pnc isolates belonged to serotypesin PnCRM7.Conclusions: The high carriage prevalence among N/WMA children reflects anintense exposure to the pneumococcus. The lack of modifiable risk factors high-lights the importance of vaccination for prevention of carriage and disease by vac-cine serotypes. Frequent carriage of serotypes not in PnCRM7 may have importantimplications for prevention of pneumococcal disease in the future.


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Risk factors for Streptococcus pneumoniae carriage in healthy children attend-ing day-care centers (DCCs) in Lisbon and Oeiras, Portugal

Simas C1, Frazão N1, Nunes S1, Sousa NG1, Mato R1, Saldanha J1, Brito-Avô A1,Santos-Sanches I1 and H de Lencastre1,2. Instituto de Tecnologia Química e Biológica,Portugal1; The Rockefeller University, New York, USA2.

Aims: To assess which demographic and clinical factors are associated with S.pneumoniae (Pn) carriage among healthy children attending 14 DCCs in Lisbonand Oeiras, Portugal.Methods: Between 2001 and 2003, 4,969 nasopharyngeal swabs were obtained fromchildren under 7 years old attending DCCs. A total of 3539 Pn strains were isolatedand determined the antibiotypes. Questionnaires containing demographic and clini-cal information were collected at sampling and analyzed.Results: The overall carriage rate of Pn was very high (71%). Out of 3539 Pn strainsisolated, 795 (22%) were resistant to penicillin (PRPn) and 863 (24%) were resist-ant to erythromycin (ERPn). Young age (less than 3 years old) (p=0.000) was foundto be a risk factor for Pn and PRPn carriage. Exposure to cigarette smoke (p=0.023)was found to be a risk factor for Pn carriage. Antibiotic consumption at sampling or1 month before sampling (p�0.008) are risk factors for both PRPn and ERPn car-riage. Previous otitis media episodes (p=0.000) is a risk factor for PRPn carriagewhile throat infections are risk factors not only to PRPn (p�0.005) but also to Pn(p�0.005) and ERPn (p�0.005) carriage. Asthma/bronchitis or pneumonia(p�0.005) were found to be risk factors for PRPn carriage. Previous hospitalizationand child gender were not found to be associated to Pn carriage.Conclusions: When compared with other developed countries the overall carriagerate of Pn was very high which is a major problem since it could lead to higherincidence of pneumococcal disease. As in previous studies, in our setting, youngage, otitis, throat infections, asthma/bronchitis, pneumonia and antibiotic uptakeare risk factors for carriage of antibiotic resistant Pn. Risk factors identification isof great importance in order to apply correct measures to avert colonization anddisease.

Molecular epidemiology of pneumococcal colonization in healthy dutch childen

M. Sluijter1, D. Bogaert1, N. Lemmens-den Toom2, WHF. Goessens2, R. de Groot1,and PWM. Hermans1

1Department of Pediatrics, Erasmus MC-Sophia, Rotterdam, The Netherlands2Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rot-terdam, The Netherlands

Introduction: Streptococcus pneumoniae is a common cause of invasive diseasessuch as sepsis and meningitis in young children. Colonization with this species iscommon, particularly in young children. The distribution of pneumococcal serotypesis age-related, with a shift from vaccine – to non-vaccine serotypes after the age ofthree years.Methods: We characterized the genetic background and resistance profiles of 578pneumococcal isolates from healthy Dutch children aged 1-19 years by means ofRFEL genotyping and susceptibility testing.Results: In total, 337 genotypes were observed, which consisted of 153 unique geno-types and 184 genotypes, which were shared by two or more strains. The latter184genotypes, representing 425 isolates, comprised 92 genetic clusters. In contrast tothe observed age-related serotype distribution, the genetic background of the strainswas not age-related. Comparison of our population to the PMEN database revealedthe presence of 5 international clones, i.e. Spain9V-3 (10 isolates), England 14-9 (4isolates), Tennesee23F-4 (2 isolates), CSR14-10 (1 isolate) and Sweden15A-25 (1 iso-late). In total, 19% of all strains showed resistance to one or more antibiotics. Re-sistance to cotrimoxazole, tetracycline, erythromycin and penicillin was found in13.9%, 6.4%, 5.0% and 3.4%, respectively. Multidrug resistance was found in 2.4%of all strains.Conclusion: Pneumococcal colonization isolates from healthy Dutch children rep-resent a heterogeneous, mostly susceptible genetic population with a high tendencyto spread horizontally and without an age-related distribution.


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Measuring the burden of nasopharyngeal colonization in two populations atmedium to high risk of persistent severe otitis media: the BLOOM (BacterialLoad On Otitis Media) project.

Smith-Vaughan H, Byun R, Harrington B, Jacups S, Nadkarni MA, Jacques NA,Hunter N, Leach AJ, Morris PS. Menzies School of Health Research, Darwin, andInstitute of Dental Research, Westmead Millennium Institute and Westmead Centrefor Oral Health, Sydney, Australia.

Background: Rates of severe otitis media (acute otitis media with perforation,AOMwiP, or chronic suppurative otitis media, CSOM) are less than 1% in childrenattending Darwin child care centres compared with an average of 25% for Aborigi-nal children in remote communities. Natural cure is rare and standard antibioticregimens fail in almost all Aboriginal children with OM.Aim: To test the hypothesis that the high prevalence of CSOM or AOMwiP in Aus-tralian Aboriginal children is related to density and diversity of nasopharyngealbacterial load.Methods: Nasopharyngeal bacterial load was estimated in children aged 18-36 monthsfrom two populations; Aboriginal children living in remote communities and non-Aboriginal children attending urban child care centres. 100 nasopharyngeal swabsfrom independent children were randomly selected and staff blinded to the popula-tion group. Using quantitative and semi-quantitative colony counts, the density ofpneumococcus, Haemophilus influenzae, Moraxella catarrhalis and total bacterialload were estimated. Real-time PCR technology is being employed to support thesefindings.Results: Analysis of colony counts and clinical diagnosis of worse ear revealed asignificant association between the nasopharyngeal load of the three organisms andear state. Real-time PCR data will be included, and analysis by population groupwill be presented.Conclusions: Preliminary analyses demonstrate that the nasopharyngeal load ofpneumococcus, H. influenzae and M. catarrhalis are significantly associated withseverity of otitis media in these populations. Future studies will assess the impactof high bacterial load on efficacy of standard antibiotic treatment regimens andcurrent and novel pneumococcal conjugate vaccine schedules.

Direct Competitive Interactions Between Streptococcus pneumoniae (SP) strains

Thompson CM1, Malley R2 and Lipsitch M1. Harvard School of Public Health1,Children’s Hospital, Harvard Medical School2, Boston, Massachusetts

Backgroud/Aims: Population effects of serotype(ST)-specific vaccines will dependon the frequency, strength, and nature of competition between SP strains of differ-ent ST. We tested the following hypotheses: (a) naturally occurring SP strains in-hibit each other’s ability to colonize mice intranasally (IN); (b) the second strain tocolonize a mouse IN is at a disadvantage to the first; (c) in a pair of strains, if Ainhibits B then B will not inhibit A; (d) inhibition is transitive: if A inhibits B, and Binhibits C, then A inhibits C.Methods: Using 7 strains from the CDC’s ABCs collection, ST 23F, 14(2), 6B(2),7F, 10A, we performed 28 pairwise competition experiments in which a resident Rstrain or saline (S) was inoculated IN into a mouse on day 0, followed by a challengeC strain or S on day 4, and IN colonization with both was assessed on day 11 byretrograde tracheal wash. Inhibition was assessed as a significant reduction in colo-nization (cfu/nasal wash) by R or C in mice receiving both, relative to mice receiv-ing that strain plus S.Results: In 11/28 cases, the R strain was inhibited by the C strain. C was inhibitedby R in 7/28 cases (difference not significant). Of the 14 strain pairs tested, neitherstrain had a detectable effect on the other in 3 cases; both inhibited each other in 2cases, and inhibition was unidirectional (A inhibited B but B had no effect on A) in9 cases. In these 9, the inhibiting strain had an effect whether it was R or C in 4cases. One nontransitive interaction was observed, in which a 23F inhibited a 6B,which inhibited a 14, which inhibited the original 23F. A diagram of all interactionswill be presented.Conclusions: Measurable inhibitory interactions between colonizing strains are com-mon, and are approximately equally common regardless of the order of exposure tothe strains. Mutual inhibition between two strains was rare but did occur. Onenontransitive interaction was observed; such interactions, if common, may facili-tate diversity in pneumococci.


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Involvement of glycosaminoglycans in pneumococcal adherence to mucosal epi-thelial cells?

Tonnaer E.L.G.M.1, van Kuppevelt T.H.1, Rijkers G.T.2, Curfs J.H.A.J1. UniversityMedical Center Nijmegen 1, University Medical Center Utrecht2, the Netherlands.

Aims: The purpose of this study was to investigate the possible involvement ofglycosaminoglycans in the adhesion of pneumococci. Adhesion is considered to bethe first and therefore crucial event in the pneumococcal colonization of the na-sopharyngeal mucosa. Glycosaminoglycans are found in most mammalian cells andtissues as well as in lower vertebrates and invertebrates. Glycosaminoglycans arenegatively charged, linear, repeating disaccharides. They play a role in a wide vari-ety of biological phenomena. In particular, they have been shown to mediate adher-ence of different pathogenic microorganisms to various epithelia.Methods: In vitro, adherence of FITC-labeled pneumococci to human mucosal epi-thelial cells with and without potential inhibitors, was studied (fluorescencemicroscopy). Electronmicroscopical techniques (immuno-EM and cupromeronic-blue staining) were used to compare the nasopharyngeal mucosae of control ratsand those of rats inoculated with pneumococci.Results: Cupromeronic-blue staining clearly showed the presence ofglycosaminoglycans on the sites of contact between pneumococci and mucosal epi-thelial cells of the rat. In addition, also current in vitro experiments with FITC-labeled bacteria indicate that glycosaminoglycans are involved in pneumococcaladherence to human mucosal epithelial cells. Experiments aimed at a further char-acterization of the specific types of glycosaminoglycans are being conducted.Conclusions: Preliminary results indicate a role for glycosaminoglycans in pneu-mococcal adherence to mucosal epithelial cells. The results of ongoing experimentsto obtain detailed information on the types of glysoaminoglycans involved will bediscussed. These results may have implications for the development of new thera-peutic strategies.This study was supported by a grant from the NWO Medical Research Organization(grant 904-61-092)

Comparison of oropharyngeal with nasopharyngeal swabs in detecting car-riage of Streptococcus pneumoniae in HIV-infected mineworkers in South Af-rica.

Wasas A1, von Gottberg A1, Charalambous S2, Grant A2,3, Moloi V2, Magadla B2,Churchyard G2, Klugman KP1,4. 1Respiratory and Meningeal Pathogens ResearchUnit (MRC/WITS/NICD) Johannesburg, South Africa; 2Aurum Health Research,Orkney, South Africa; 3London School of Hygiene & Tropical Medicine, UK; 4De-partment of International Health, Emory University, Atlanta, Georgia, USA

Aim: To compare the yield of oropharyngeal with nasopharyngeal swabs in detect-ing carriage of Streptococcus pneumoniae in HIV-infected miners.Methods: Mineworkers attending two HIV clinics between May 2002 and June 2003were recruited. Nasopharyngeal (NPS) and oropharyngeal swabs (OPS) were col-lected from participants, placed into skim milk-tryptone-glucose-glycerin medium(STGG) and subcultured on selective media to detect pneumococcal growth. Pneu-mococci were serotyped using the Quellung method, and susceptibility testing wasperformed according to NCCLS specifications.Results: 856 mineworkers (80% living in hostels) were enrolled. The mean age was42 years (range 25-59); all but two participants were male. The mean CD4+ cellcount was 333 cells/dl. 294 (34%) patients were on trimethoprim-sulphamethoxazole(TMP-SMX) preventive therapy. Both NPS and OPS were obtained from 855 par-ticipants. Pneumococcal carriage was confirmed in 75/855 (8.8%) miners. In 8/75(11%) cases both NPS and OPS yielded pneumococci; while carriage was deter-mined in 53/855 by NPS and 30/855 by OPS, Mc Nemars test, P < 0.01. Carriagewas highest in the winter months, June to August. One miner was colonised withtwo pneumococcal strains. The most prevalent serotypes were 19F (8/76, 10%); and23F, 6A and 6B each representing 7/76, 9%. Eleven (14%) isolates wereintermediately resistant to penicillin. Overall prevalence of TMP-SMX resistancewas documented in 29 (38%).Conclusions: Performing NPS alone would have missed 22/75 (29%) of pneumo-coccal carriers.


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Pneumococcal carriage in military populations

Zhogolev S.D.1, Ogarkov P.I.1, Jogolev K.D.2 Epidemiology Department1, Immunol-ogy Department2,Military Medical Academy, St.Petersburg, Russian Federation

Aims: To investigate the pneumococcus circulation in military populations.Methods: 307 servicemen were observed with bacteriological analyses during 6-9months.Results: 45% of this population became carriers of Pneumococcus. In 69.6% ofthem the carriage was “transitory”, in 28.3% it was “short-term” within 2 monthsand in 2.1% it was “chronic” (within 6-9 months). Untyped strains of pneumococciisolated from carriers prevailed and amounted to 77.6%. Typed strains (33) of Strep-tococcus pneumoniae belonged to 18 various serotypes: 3(2 strains), 4, 6(2), 7(2),8(2), 9(1), 10(1), 11(2), 15(2), 18(4),19(3), 22(1), 23(1), 24(1), 25(1), 26(1), 29(4),38(1). In December-January (1-2 months after the military unit formation) the phaseof epidemic transformation was followed by the phase of epidemic spreading ofPneumococcus. At this period the number of pneumococcal carriers was the largestand accounted for 45%. The mean geometric concentration (MGC) of pneumococ-cus in the carriers (5.9 lg/ml) and number of typed cultures were maximal at thisphase. In March-April (the phase of transformation reservation) and in June (thephase of reservation) the pneumococcal carriers accounted for 19.7% (2.3 timesdecrease). MGC of pneumococci became lower (4.9-5.2 lg/ml). The number of typedforms of circulating pneumococci decreased 2.2-3.9 times. The phase of epidemicspreading of pathogen coincided with a maximal morbidity of acute respiratorydisease (ARD), acute bronchitis and pneumonia. Pneumococcus appeared to be afrequent causative agent not only in pneumonia but in other acute respiratory infec-tions as alone and in association with other pathogens (viruses, Chlamydia, etc.).Conclusions: Serotypes of Pneumococcus circulating in military populations inRussia were detected. The main characteristics of epidemic process phases in pneu-mococcal infection were demonstrated.


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Ecology of antibiotic resistant pneumococci and antibiotic use

*Dagan, R

Ben-Gurion UniversitySoroka Medical CenterPediatric Infectious Diseases Unit, POB 151Beer Sheva, [email protected]

Molecular mechanisms and antimicrobial resistance in the pneumococcus

*Hakenbeck, R

University of KaiserslauternPaul Ehrlich Str. 23D-67663 [email protected]


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Incremental increases in fitness cost with successive acquisition of low-affinityPBPs in Streptococcus pneumoniae evaluated by competition in intranasal colo-nization of infant rats

Trzcinski K1, Thompson CM1, Dowson CG2, Gilbey A2, Lipsitch M1, Harvard Schoolof Public Health, Boston, Massachusetts,1 University of Warwick, Coventry, UnitedKingdom2

Aims: To evaluate the impact of resistant alleles of penicillin-binding proteins (PBPs)on the ability of newly emerged penicillin-resistant (PEN-R) strains to compete forupper respiratory tract (URT) colonization with their penicillin-susceptible (PEN-S) ancestors.Methods: PEN-S serotype 6B and 9V strains were transformed into derivatives ex-pressing resistant forms of PBPs (2x; 2x and 2b; or 2x, 2b, and 1a) with PCR ampli-fied fragments of pbp genes of same serotype PEN-R strains. In addition, strainD39 was transformed into PEN-R derivatives with plasmid-cloned pbps from a highlyresistant isolate 159. Penicillin and cefotaxime MICs were elevated in transformantsas expected. In competition model groups of 8 to 13, 3- to 4-day old infant rats wereinoculated intranasally with a mix of 1.3-2.2x10^6 colony forming units (CFU) ofPEN-R resistant strain and its PEN-S ancestor (ratio R:S ~ 1:10). For 5 consecutivedays nasal washes were collected from all animals to asses the ratio of PEN-R:PEN-S cells colonizing the URT.Results: After five days 0.4log10 (+SE=0.3, p>0.05) decline of the PEN-R:PEN-Sratio was noted for the derivative carrying low affinity form of pbp2x, and 1.5log10(+0.4), and 2.2log10 (+0.4) declines were observed for 2x,2b and 2x,2b,1atransformants respectively for serotype 6B isolates. A decline of 1.4log10 (+0.2)was observed for the 2x transformant of the serotype 9V strain at day 5, whereas its2x,2b and 2x,2b,1a transformants disappeared from all animals by day 4 (a declineof at least 2.6log10). Declines of 0.6log10 CFU (+0.4, p>0.05), and a significant2.0log10 (+0.6) were observed for 2x and 2x,1a,2b transformants of D39 on day 5.All declines had p<0.02 by 2-tailed t test except where noted.Conclusions: The cost of penicillin-resistance acquisition for the S. pneumoniaestrain competing with its susceptible ancestor to colonize URT increases with thenumber of resistant pbp alleles acquired.

Global developments in the clonality of the resistant pneumococcus

*McGee, L

Emory University - RSPH1518 Clifton Rd - L22Atlanta GA 30322UNITED [email protected]


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The effect sub-inhibitory concentrations of ciprofloxacin on mutation rate ofStreptococcus pneumoniae.

Stephen H. Gillespie Lorraine Simara Anne L. Dickens

Aim: Fluoroquinolones with enhanced activity to Gram positive organisms havebeen incorporated into several international guidelines for the therapy of acute com-munity acquired pneumonia. We therefore aimed to study the effect of sub-inhibi-tory concentrations of ciprofloxacin on the rate at which resistance mutations emergedin vitro.Method: Mutation rates were determined for S. pneumoniae by a median mutationfluctuation assay methodology and mutation rates calculated using Drake’s formula.Within each experiment a minimum of five broths were inoculated.Results: Incubation of S. pneumoniae in the presence of half MIC ciprofloxacinincreased the rate of mutation by approximately two orders of magnitude. At aquarter MIC the increase in mutation rate was approximately 10 fold and at oneeighth MIC 2-3-fold.Conclusions: These data suggest that sub-inhibitory concentrations offluoroquinolones may increase the rate at which resistance mutations emerge. Theseresults will be discussed in the light of the pharmacokinetics of fluoroquinolones.


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Multilocus sequence typing of macrolide resistant S. pneumoniae isolates re-covered from eleven European centers

Al-Lahham A1, Schmitz FJ2, Ringelstein A1, Lütticken R1, Cil MY1, Reinert RR1.NRCS and Institute of Med. Microbiology in Aachen1, Institute of Microbiologyand Hygiene in Minden2, Germany

Aims: Macrolide resistance among isolates of S. pneumoniae is a problem of in-creasing concern worldwide. The aim of this study is to characterize the erythro-mycin resistant pneumococcal strains (erythromycin > 0.5 mg/L) with multilocussequence typing (MLST).Methods: 84 clinical strains of erythromycin resistant S. pneumoniae (ERSP) werecollected from 11 university medical centers in 1997. MICs to penicillin G (PEN),cefotaxime (CETA), amoxicillin (AMOX), clindamycin (CLI), quinupristin-dalfopristin (Q-D), vancomycin (VAN), teicoplanin (TEI), ciprofloxacin (CIP),roxithromycin (ROXI), clarithromycin (CLA), spiramycin (SPI), azithromycin (AZI)and telithromycin (TELI) were determined by the microdilution method accordingto NCCLS. Strains were serotyped by Neufeld Quellung reaction. Macrolide re-sistance genes (erm(B) and mef(A)) were analyzed by PCR.. MLST was performedaccording to standard methods.Results: The resistance rate of the ERSP to other antibiotics was as follows: PEN4.8%, CETA 4.8%, AMOX 9.5%, CLI 77.4%, ROXI 95.2%, CLA 90.5%, SPI 89.3%,AZI 90.5%. All strains were sensitive for TELI, CIP, TEI, VAN, and Q-D. Serotypes14 (31%), 23F (24%) and 6B (17.9%) were the most prevalent. The percentage ofERSP strains with mef(A) was 19.1% and with erm(B) 78.8%. Predominant se-quence types were: ST 81 (26.8%), ST 143 (12.2%), ST 156 (6.1%) and ST 658(6.1%). All strains of ST 81 originated from France and Spain. Isolates of ST 143originated only from France. Four isolates (4.9%) possessed new allele combina-tions.Conclusions: The erm(B) genotype is the predominant mechanism among ERSPstrains in Europe. Sequence types 81 and 143 were predominant. MLST is a usefulmethod for analyzing clonal relatedness.

A multicenter surveillance of resistance in S. pneumoniae and S. pyogenes strainsand development of a LightCycler PCR method for rapid detection of mac-rolide resistance

Al-Lahham A, Franken C, Neuberger N, Cil MY, Lütticken R, Reinert RR. Instituteof Medical Microbiology and National Reference Center for Streptococci, Aachen,Germany

Aims: RTIs caused by S. pneumoniae and S. pyogenes are serious health problemsworldwide. In a multicenter study covering 10 clinical laboratories, a total of 241 S.pneumoniae and 236 S. pyogenes strains were collected from children with com-munity acquired infections in 2002-2003. In this study a new RT-PCR protocol wasdeveloped for rapid detection of macrolide resistant genotypes in both organisms.Methods: MICs were determined according to NCCLS by microbroth dilutionmethod; macrolide resistance phenotypes of both pathogens were determined bydouble disk diffusion test. The Neufeld Quellung reaction method was used to se-rotype S. pneumoniae isolates and the emm typing method was used to type S.pyogenes. RT PCR was used to detect macrolide resistant genotypes of both organ-isms.Results: Resistance rates of S. pyogenes isolates were as follows (I and R): penicil-lin 0%, erythromycin A 14%, clindamycin 0%, and gatifloxacin 0%. S. pneumoniaeisolates showed the following resistance rates (I and R): penicillin 5%, cefotaxime1.7%, amoxicillin 0.8%, erythromycin 19.9%, clindamycin 7.5% and gatifloxacin0%. Telithromycin was 100% active against all S. pneumoniae (MIC

90 = 0.125 mg/

L) and S. pyogenes (MIC90

, 0.5 mg/L). 30 of 48 macrolide-resistant pneumococcalstrains (62.5%) were M phenotypes possessing the mef(A) genotype and 18 (37.5%)were cMLS

B possessing the erm(B) genotype. 33 isolates of S. pyogenes were mac-

rolide resistant, among which 31 (93.9%) were M phenotypes. All macrolide re-sistant strains showed products using the RT-PCR compared to the conventionalPCR method.Conclusions: Macrolide resistance is an increasing problem in Germany.Telithromycin was highly active against both organisms. RT-PCR assay al-lows a rapid detection of macrolide resistant genotypes in both S. pneumoniaeand S. pyogenes.


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Distribution of subclasses mefA and mefE genes among macrolide resistance(M phenotype) in Streptococcus pneumoniae, viridans group streptococci andStreptococcus pyogenes clinical isolates.

Ardanuy C1, Liñares J1, Tubau F1, Domínguez MA1, Pallarés R2, Martín R1. TheSpanish Pneumococcal Infection Study Network G03/103. 1Servicios deMicrobiología. 2Enfermedades Infecciosas. Hospital Universitari de Bellvitge, Bar-celona, Spain.

Aims: In Spain the majority of macrolide resistant S. pneumoniae strains harborermB gene, whereas mefA/E genes are the most frequent among macrolide resistantviridans group Streptococcus (VGS) and S. pyogenes strains. The aim of this studywas to know the distribution of subclasses mefA and mefE genes among clinicalstreptococcal isolates with M phenotype.Methods: Detection of mefA/E genes was performed by PCR in 107 M phenotypeerythromycin resistant strains (36 S. pneumoniae, 47 VGS and 24 S. pyogenes)isolated from adult patients during the 1998-2003 period. Amplicons were digestedwith BamHI to differentiate between subclasses mefA and mefE genes. Serotypesand PFGE (SmaI) profiles of S. pneumoniae isolates were studied.Results: All 107 isolates had a positive PCR for mefA/E genes. The distributions ofsubclasses mefA and mefE were as follows: 32 S. pneumoniae strains had mefE and4 pneumococci had mefA, 46 isolates of VGS had mefE and 1 had mefA, whereas allS. pyogenes isolates had mefA. The serotype distribution of S. pneumoniae was asfollows: serotype 14 (39%), 23F (6%), 23A (3%), 6A (3%), 19(3%) and non-typable(47%). Twenty-seven different PFGE patterns were found among 36 S. pneumoniaeisolates studied. Among serotype 14 pneumococcal strains 5 PFGE patterns werefound: 7 isolates belonged to Spain9V-3-14 clone, 4 isolates to England14-9 cloneand 3 isolates were genetically unrelated.Conclusion: The majority of pneumococci and VGS had mefE, whereas all S.pyogenes had mefA. The most frequent serotype found among pneumococci withM phenotype was serotype 14 and 78% of strains of this serotype belonged to 2international clones (Spain9V-3-14 carrying mefE and England14-9 harboring mefA).

Carriage and antibiotic resistance of Streptococcus pneumoniae in childrenattending pre-school in Guangzhou, China

Boost MV, O’Donoghue MM, Dooley JS, School of Nursing, Hong Kong Poly-technic University, HK SAR

Aims: Limited studies on carriage rates of S. pneumoniae have been performed andnone published for Southern China. As levels of carriage in primary school chil-dren observed in Hong Kong are low, investigation of levels in a neighboring areaof China could help to understand these low levels.Methods: Four hundred and forty children attending two private pre-schools inGuangzhou were sampled for carriage of S. pneumoniae. Antibiotic susceptibilitytesting was performed. Parents completed a questionnaire giving details of age,sex, number of siblings, housing type, hospitalisation and antibiotic use.Results: A carriage rate of 2.7% was found overall. 83% of strains were resistant topenicillin, tetracycline and erythromycin. 80% of penicillin resistant strains werealso resistant to chloramphenicol. Age and sex were not associated with carriage.Few children had a sibling except in the case of twins. No association was foundbetween recent hospitalization or antibiotic use and carriage of S. pneumoniae.Knowledge of antibiotic use was low but these drugs are readily available over-the-counter.Conclusions: The carriage rate found conf irms the low rate of carriage ofS. pneumoniae in primary school children in Southern China observed inHong Kong. High use of day care at an early age may allow immunity tomany strains to develop at a younger age than seen elsewhere and thus byprimary school age carriage is rare. Opportunities for introduction of newstrains may be reduced in this community due to the very low numbers ofyounger siblings who may act as a reservoir of strains. Easy availabilityand high use of antibiotics may favour the survival of resistant strains.


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High levels of antibiotic resistance in strains of S. pneumoniae carried persist-ently in kindergarten children.

Boost MV, Lo C, O’ Donoghue MM, School of Nursing, Hong Kong PolytechnicUniversity, HK SAR

Aim: Most strains of S. pneumoniae are carried by young children for relativelyshort periods of time. In a longitudinal study of carriage of S. pneumoniae in kin-dergarten children, a few children were colonized throughout the six month sam-pling period. The antibiotic susceptibilities and clonal relationships of strains car-ried persistently by young children were investigated.Methods: Nasopharyngeal swabs were collected on three occasions over a six monthperiod from 780 children attending 5 kindergartens and cultured for S. pneumoniae.Antibiotic susceptibility testing was performed and pulsed field gel electrophoresiscarried out on stains with similar susceptibility patterns isolated from the samechild on subsequent samplings.Results: Carriage rates fell from 19.7% on the first sampling to 10.7% on the thirdbut penicillin resistance in isolates rose from 47% to 65.7%. Penicillin resistance instrains isolated on two occasions from the same individual was higher (63%) andreached 87.5% in individuals colonized on all three occasions. Pulsed field gel elec-trophoresis revealed that of eight children colonized on all three occasions, fourcarried the same strain throughout the sampling period.Conclusions: Antibiotic resistant strains seem able to remain in circulation for longerperiods in the kindergartens, probably due to antibiotic selective pressure. Certainantibiotic resistant strains are able to persist in some children. This may be due tolow immunogeniecity of these strains or failure in some children to develop immu-nity to certain strains.

Detection of penicillin and vancomycin tolerance in clinical isolates of Strepto-coccus pneumoniae in Hong Kong

Boost MV, Ko WM, O’Donoghue MM, School of Nursing, Hong Kong PolytechnicUniversity, HK SAR.

Aims: Antibiotic tolerance allows bacteria to survive in the presence of antibioticconcentrations above the determined minimum inhibitory concentration (MIC).Although the MIC is unchanged, tolerant bacteria are not lysed or killed in thepresence of effective antibiotic therapy. As antibiotic therapy cannot eradicate theinfective agent, the infection can relapse once the antibiotic therapy is removed.Antibiotic tolerant bacteria cannot be detected using conventional susceptibility testsas they appear to be sensitive. Penicillin tolerance in S. pneumoniae was first recog-nized in 1985, but vancomycin tolerance only in 1999. Surveys have shown thepresence of vancomycin tolerant strains in isolates from USA, Sweden and Italy.Methods: Fifty clinical isolates of S. pneumoniae were investigated for tolerance topenicillin and vancomycin. The MIC for both drugs was determined by broth dilu-tion. Logarithmic growth phase cultures were tested by a cell-lysis assay in the pres-ence of 10 times the MIC of the antibiotics. Those strains which showed resistanceto lysis, showing less than a 30% reduction in OD, were suspected to be tolerant andwere further characterized by means of a time-kill assay, in which viable countswere performed hourly for four hours following addition of antibiotic.Results: Five strains (10%) appeared penicillin tolerant by the cell lysis assay. Oneof these strains was also tolerant to vancomycin. Time kill assay confirmed toler-ance with less than 2 log decrease in viability within four hours exposure to 10times the MIC level of the antibiotic under test.Conclusion: Penicillin tolerance was detected in 10% and vancomycin tolerance in2% of clinical isolates studied. This is the first report of vancomycin tolerance inHong Kong. As vancomycin tolerant strains appear susceptible by routine methods,laboratory detection of these strains is necessary to avoid treatment failure.


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Emergence of fluoroquinolone resistance among international penicillin-resist-ant Streptococcus pneumoniae clones in Spain

A. G. de la Campa1, L. Balsalobre1, C. Ardanuy2, A. Fenoll1, E. Pérez-Trallero3, J.Liñares2, The Spanish Pneumococcal Infection Study Network. Centro Nacional deMicrobiología, Instituto de Salud Carlos III, Majadahonda, Madrid1, Hospital deBellvitge, Barcelona2, Hospital Donostia, San Sebastian3, Spain.

Aims: To investigate the epidemiology of fluoroquinolone resistant pneumococciand the actual incidence of recombinant resistant strains originated by interspecifichorizontal transfer.Methods: Strains (one isolate per patient) isolated during 2002 in Spain were iden-tified by standard methods. MICs to 5 fluoroquinolones and other antimicrobialagents were determined by microdilution as recommended by the NCCLS.Ciprofloxacin-resistant (CipR) strains were characterized by PFGE and sequencingof the quinolone-resistance determining regions (QRDRs) of parC, parE and gyrA.Results: A low frequency (2.6%, 75 of 2,884) of CipR strains was found. The 14low-level (MIC 4-8 �g/mL) CipR strains had single parC resistance mutationswhereas the 61 high-level (MICs >8 �g/mL) CipR strains, had either 2 (parE orparC + gyrA) or 3 mutations (parC + parE + gyrA or parC + gyrA). Although 37different PFGE patterns were observed, 48 strains belonged to 9 PFGE types repre-senting prevalent pneumococcal clones, with 30 strains belonging to 4 multi-drugresistant clones: Spain23F-1, Spain6B-2, Spain9V-3 and Spain14-5. Strains have acquiredresistance either by point mutation (70 of 75 strains) or by recombination with viridansstreptococci (5 strains).Conclusions: The majority (68%) of the CipR strains were penicillin-resistant and37% were multi-drug resistant. CipR isolates are being selected from existing clones.These clones, now CipR, could be disseminated, and also the recombinant CipRisolates belonging to international clones. If fluoroquinolones are widely used, re-sistance in S. pneumoniae may appear to be an important problem in the near future.

Pneumococcus pneumonia: a prospective multicentre study of young childrenin Latin America

Research Group of the Collaborative multicentre study on Acute RespiratoryInfections and Bacterial Resistance (CARIBE)*Study funded by: PAHO and WHO

Background: Streptococcus pneumoniae is presumed to be the primary bacterialcause of community acquired pneumonia in children, but limited information isavailable regarding the characteristics of the disease in Latin AmericaMethods: The study was a multicentre prospective observational investigation. Chil-dren aged 3 to 59 months hospitalized due to pneumonia in 13 centers in Argentina,Brazil, Dominican Republic, and Peru were included. Blood culture was performedin all children and pleural fluid culture was done when clinically indicated. Thepatients were treated with ampicillin (150mg/kg) or penicillin (200.000U/kg) andfollowed-up until discharge.Results: We selected 2512 children from Argentina (750), Brazil (926), Peru (228),and Dominican Republic (572).Pneumococcus was isolated from blood and/or pleuralfluid of 292 (12%) children, with 48% of these strains being penicillin susceptible,24% with intermediate resistance, and 21% fully resistant. We were able to serotype257 strains. The major serotypes identified included 14 (51%), 1 (10%), 6B (9%), 5(7%) and 6A (4%).Therapeutic success ,using penicillin, was achieved in 199 (70%)of the children with pneumococcal pneumonia. The results of the multiple regres-sion analysis showed that there was no influence of penicillin susceptibility uponthe treatment failure.

CARIBE Research Group: Agosti M.R.; Benguigui Y.; Berezin E.W.; BrandileoneM.C.C.; Camargos P.; Cardoso M.R.A.; Cociglio R.; Coradin H.; Diaz M.E.R.;Feris J.M.; Ferrero F.; Grenon S.; Maggi R.S.;March M.F.B.P.; Martearena C.R.;Nascimento-Carvalho C.M.C.; Pascua C.F.; Regueira M.; Ruvinsky R.; Sanchez J.;Sant’anna C.C.; Souza L.S.F.; Tagliaferri P.; Urbano C.


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The carriage of penicillin non-susceptible Streptococcus pneumoniae in a re-mote village in Northern Tanzania: an analysis by serogroup and minimuminhibitory concentration (MIC).

Charalambous, B.M.1, Batt, S.L.1, Sam, N.E.2, & Gillespie, S.H.1*.1Department of Medical Microbiology, University College London, Royal FreeCampus, Rowland Hill St, London NW3 2PF. 2Clinical Laboratory, KilimanjaroChristian Medical College, Moshi PO Box 3010, Tanzania

Aims: To test the hypothesis that the likelihood of antimicrobial non-susceptibilityin S. pneumoniae is associated with the carriage rate.Method: Susceptibility testing was performed on Streptococcus pneumoniae iso-lated from healthy children in a remote urban village in the Rombo district of NorthernTanzania. Antibiotic sensitivity and serogroup were determined using standardprotocols and the �2 test was used to compare the proportion of resistant organismsin each serogroup to the proportion of resistant isolates in the whole population oforganisms. Regression analysis was performed to determine the covariance be-tween the number of resistant isolates and penicillin minimum inhibitory concen-tration (MIC) with carriage levels.Results: Regression analysis suggests that the number of antibiotic resistant iso-lates increases with the total number of isolates in each serogroup carried (r2 = 0.78;p<0.0001). Serotype 19 appears to have a higher proportion of resistant isolatesthan the overall population. A significant correlation was observed between themean penicillin MIC of each serogroup and the number of isolates in each serogroup(Pearson correlation = 0.21; p = 0.0002) suggesting that the MIC increases with theprevalence of the serogroup being carried.Conclusions: These data support the hypothesis that it is the carriage rate that is themajor risk factor for resistance emerging in S. pneumoniae.

Streptococcus pneumoniae carrying the resistance determinants mef(E) andtet(M): Characterization of mega and Tn916-like genetic elements and theirlinkage

Del Grosso M 1, Scotto d’Abusco A 1†, Iannelli F 2, Pozzi G 2, Pantosti A 1. IstitutoSuperiore di Sanità, Rome,1 and University of Siena, Siena,2 Italy

Aims: The aim of this work was to examine the association of the macrolide andtetracycline resistance genes in S. pneumoniae clinical strains carrying mef(E) andtet(M).Methods: Seven Italian clinical isolates of S. pneumoniae carrying mef(E) and tet(M)were studied. Three of these carried also erm(B). Proximity between mef(E) andtet(M) was investigated by extended PCRs with four combinations of primers pairs.The structure of the mega element, containing mef(E), and that of Tn916-like ele-ment, containing tet(M), were mapped by a series of PCR reactions. Conjugationexperiments were carried out using an unencapsulated transformation-defective re-cipient strain. Transformation experiments were carried out in presence of compe-tence-stimulating peptide.Results: In three strains, a novel element designed Tn2009 was found. This consistsin a composite Tn916-like element in which mega is integrated in ORF6 of Tn916.Tn2009 could not be transferred by conjugation to a recipient pneumococcal strain,but was transferred by transformation. The three strains carrying erm(B) in additionto mef(E) and tet(M) did not carry Tn2009, but an element similar to the knowncomposite element Tn3872, including erm(B) (Tn917) integrated in Tn916, whilemega was inserted in a different site. In one strain the linkage between mega andTn916 was not demonstrated.Conclusions: The study of the association of the resistance genes mef(E) and tet(M)in S.pneumoniae demonstrated that these determinants can be associated. In par-ticular, a novel element designed Tn2009 was identified in which mef(E) and tet(M)genes are associated in the same transposon. Although Tn2009 could not be trans-ferred by conjugation, it could be disseminated by transformation to other pneumo-coccal isolates, representing a further contribute to the spread of both macrolideand tetracycline resistance.


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International context of Portuguese erythromycin resistant pneumococcal clonesfrom serotype 14 of invasive origin.

Dias R1, Caniça M1. Antibiotic Resistance Unit, National Institute of Health Dr.Ricardo Jorge1, Lisbon, Portugal

Aims: Resistance to macrolide in Streptococcus pneumoniae (Sp) is of major con-cern in the last few years in Portugal. A molecular study was used to describe thegenetic background of these Sp clones, allowing the evaluation of spread and clonalexpansion on resistant serotype 14 (the predominant serotype in Portugal).Methods: 614 consecutive isolates were collected in the Antibiotic Resistance Unitin NIH, from blood, CSF and pleural liquid, between 1999 and 2002 (first semes-ter), in the scope of a multicenter study with the participation of 26 hospitals. MICsto 10 antibiotics were determined by agar dilution method. Serotype was performedby Dot-Blot and Quellung reaction. Genotyping of macrolide resistant strains fromserotype 14 (n=25) was performed by PFGE and MLST. BURST analysis was doneto infer evolutionary relationships.Results: The resistance to erythromycin (Ery), from 1999 to 2002, was 12.7%, 11.7%,16.8%, 13.1%, respectively. Among Ery resistant Sp from serotype 14 were found 4Sequence Types (ST), which clustered in two clonal complexes (CC). CC-1 includedST156, ST143 and ST1042. CC-2 included ST15 and ST9. ST156 and ST15 iso-lates showed the greatest variability of PFGE profiles, and are putative primaryfounders from CC1 and CC2, respectively. Clone ST156 is a Spain 9V-3-14 clonewhich acquired resistance to macrolides. ST1042 is a SLV variant from ST156.ST143 was the major ST from this study (n=8) and is a DLV from ST156. ST9clone is an England14-9 clone and a putative sub-group founder from CC2.Conclusions: Our results suggest that emergence of macrolide resistance amonginvasive Sp in Portugal are due to the presence of internationally disseminated clones.Considering the selective pressure caused by antimicrobial therapy, monitoring clonaldissemination of Sp antimicrobial resistant is of high concern in Portugal.

Antimicrobial susceptibility and serotype distribution of invasive pneumococciisolated in Portuguese children

Dias R1, Caniça M1. Antibiotic Resistance Unit, National Institute of Health Dr.Ricardo Jorge1, Lisbon, Portugal

Aims: Streptococcus pneumoniae (Sp) is one of the most important bacteria causinginvasive disease (ID) in Portugal. We evaluated the serotype-antimicrobial suscepti-bility relationship in invasive disease caused by Sp in pediatric age in Portugal.Methods: 171 consecutive isolates were colleted in the Antibiotic Resistance Unitin National Institute of Heath, from blood, CSF and pleural liquid, during threeyears, from 18 hospitals. MICs to penicillin (Pen), cefotaxime (Ctx), ceftriaxone(Ctr), tetracycline (Tet), erythromycin (Ery), clindamycin (Cli), chloramphenicol(Cm), ofloxacin (Ofl) and ciprofloxacin (Cip) were determined by agar dilutionmethod (NCCLS). Serotype was performed by Dot-Blot and Quellung reaction.Results: MICs90 (mg/L) ranged as follow: 0.5-1 to Pen, 0.25-1 to Ctx, 0.5-1 to Ctr,8-32 to Tet, 8-32 to Ery, 32-64 to Cli, 4 to Cm, 2 to Ofl and 1-2 to Cip. Serotypes 14,1, 23F, 6B, 7F, 19F, 9V, 3, and 6A (descending order) represented 80% of the iso-lates causing ID in children by Sp. Invasive disease in children from age group < 2years was due to a higher number of serotypes than in age group 3-5 years. Serotypes14, 1, 6B and 7F were more frequent in blood and serotypes 23F, 19F and 6A inCSF. Serotype 14 was prevalent among Pen (60%) resistant strains. Serotype 6Bwas prevalent among Ery (30%) and Tet (43%) resistant strains.Conclusions: In this study we showed that serotypes from 7-valent pneumococcalvaccine represented 59% and 69% in the age groups < 2 years and 3-5 years, respec-tively. The results also suggest that vaccine serotypes and vaccine serotypes plusvaccine-related serotypes represented 56% and 64%, respectively. Antimicrobialresistance could be reduced by the use of a vaccine against invasive pneumococcaldisease.


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The membrane associated serine/ threonine kinase of Streptococcuspneumoniae:Percetion and response to cell-wall stress and alkaline pH ; Posi-tive control on competence and virulence

Jose Echenique*, Linda Novakova**, Aras Kadioglu***, Susana Romao*,PavelBranny**, Peter Andrew***, Marie-Claude Trombe* ; *Université Paul SabatierIFR 31 CHU Rangueil, 31403Toulouse France ; **Institute of Microbiology, CzechAcademy of Sciences, CZ-142 20 Pragu 4, Czech Republic ;***University of Leices-ter, Leicester LE1 9HN UK

Aims: Functional and physiological characterization of the serine/threonine kinase ofS. pneumoniaeMethods: Biochemical characterization of wild type and mutated the recombinant pro-teins Expression and localization in S. pneumoniae by activity tests andimmunodetection. Physiological implication of the kinase in comCDE expression, an-tibiotic susceptibility, autolysis, competence and virulence by mutation analysis andgenetic dissection using Northern blots, transformation, autolysis, resistance tests andvirulence studiesResults: Streptococcus pneumoniae genone contains a single gene stkP (EMBL acces-sion N°AF285441), encoding a serine/threonine kinase carying the PASTA signature.The recombinant protein shows indeed threonine, serine autokinase activity and presentsthe biochemical properties of eukaryotic serine/threonine kinases. In S. Pneumoniaethe protein is associated to the membrane-cell wall fraction. Insertion mutation in stkPincreases the sensitivity of the bacteria to the cell-wall directed inhibitors penicillin Gand vancomycin culminating to LytA-dependent autolysis. This suggests that functionnalStkP is involved in the response to cell-wall defects induced during growth in the pres-ence of low concentrations of cell-wall inhibitors. Functional StkP was also found to beinvolved in the protection against Lyt-A dependent autolysis during exponential growthat alkaline pH (pH 8). Analysis of “population phenotypes” revealed the crucial role ofStkP in the positive regulation of the central competence operon comCDE and also inlung colonisation and blood stream-invasion in mouse.Conclusions : It is proposed that StkP with its PASTA signature allows to respond tocell-wall-stress culminating in Lyt-A dependent autolysis, induced by low concentra-tions of penicillinG and vancomycin and also during exponential growth at alkalinepH. Competence trigger and virulence expression might belong to the pneumococcaladaptative strategy to these stress.These data are from manuscripts JB 01536-03 and IAI 01646-03 under review.

Genetic characterization of rifampicin-resistant Streptococcus pneumoniae iso-lated in Spain (1990-2003)

MJ. Ferrándiz1, C. Ardanuy2, J. Liñares2, E. Pérez-Trallero3, E. Cercenado 4, A.Fleites5, A. G. de la Campa1, The Spanish Pneumococcal Infection Study Network.CNM, Instituto de Salud Carlos III, Majadahonda, Madrid1, Hospital de Bellvitge,Barcelona2, Hospital Donostia, San Sebastian3, Hospital Gregorio Marañón, Ma-drid4, Hospital Central de Asturias, Oviedo, Spain.

Aims: To deepen in the epidemiology of rifampicin-resistant (Rif-R) pneumococciand the molecular mechanisms involved in this phenotype.Methods: 66 Rif-R strains were characterized by PFGE and sequencing of two rpoBregions: residues L37-V175 (cluster N) and T461-E680 (clusters I, II and III). Trans-formation of a Rif-S strain with DNA fragments containing Rif-R mutations wereperformed.Results: A 73% of the Rif-R strains was penicillin-resistant and a 53% penicillin-and erythromycin-resistant. Although 36 different PFGE patterns were observed,34 strains belonged to 4 PFGE types representing prevalent pneumococcal clones,with 25 strains belonging to 3 multi-drug resistant clones: Spain23F-1, Spain6B-2 andSpain9V-3. Strains have acquired resistance either by point mutation or by recombi-nation with viridans streptococci (7 of 66 strains). Rif-R isolates had single or dou-ble changes in RpoB at cluster N (Q150), cluster I (S481, S482, S485, Q486, D489,S495, H499, R501, L506), cluster II (P537), or downstream cluster II (N547). Trans-formation experiments allowed to correlate Rif MIC and rpoB mutation in approxi-mately half of the clinical isolates, whereas the other part showed greater MICs thanthose expected according to their rpoB mutation.Conclusions: The majority of the Rif-R strains are being selected from pre-existingmultidrug-resistant international clones. Genetic experiments show that, althoughrpoB mutations are involved in Rif-resistance, additional mutations, in regions notsequenced in this study, could be involved in Rif-resistance.


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The mutability of Streptococcus pneumoniae with different mutations in gyrA,parC and parE

Stephen H. Gillespie Lorraine Simara Anne L. Dickens

Aim: There is increasing concern about the increase in the prevalence of resistanceto fluoroquinolones among clinical isolates of Streptococcus pneumoniae. We havepreviously shown that Streptococcus pneumoniae with mutations in parC and gyrAacquire a second mutation at a higher rate we therefore sought to determine whetherthis hypermutability was limited to the genes of the topoisomerase/gyrase system.Method: We studied the rate at which different gyrA and parC mutant strains mu-tated to rifampicin resistance. Mutation rates were calculated using the Drake for-mula. A total of three single mutants, four double mutants and one triple mutantwere tested.Results: There was no significant difference in the rate at which mutations to ri-fampicin resistance emerged (range 1.5 - 4.0x10-8 mutations per cell division).Conclusion: From this we conclude that mutation in the topoisomerase IV and gyrasegenes does not produce general hypermutability.

Increasing beta-lactam resistance in invasive pneumococcal isolates causingdisease in South Africa from 1999 to 2002.

von Gottberg A1, de Gouveia L1, Wasas A1, Rafundisani T1, Madhi SA1, KlugmanKP1,2 and the national surveillance network. 1Respiratory and Meningeal PathogensResearch Unit (MRC/WITS/NICD), Johannesburg, South Africa; 2Department ofInternational Health, Emory University, Atlanta, Georgia, USA

Aim: To measure resistance to beta-lactam antibiotics in pneumococcal isolates caus-ing invasive pneumococcal disease (IPD) requiring hospitalisation.Methods: All pneumococci isolated from IPD during July 1999 to the end of 2002in laboratories participating in a national surveillance programme in South Africawere reviewed. Repeat isolates from the same patients were excluded. Susceptibil-ity testing was performed according to NCCLS specifications.Results: 792, 2003, 2230 and 2137 episodes of IPD were reported for the three anda half years respectively. Most of these episodes were diagnosed from blood cul-tures alone, while isolation from CSF constituted 1790/7162 (25%). Most episodesoccurred in the less than 1-year-old age group (1706/5937, 29%) and in the 25- to44-year-old adults (1915/5937, 32%), with similar proportions for each year. Ofthese episodes, 6696 (93%) isolates were available for further testing. Intermediateresistance to penicillin increased over these years: 136/738 (18%), 301/1828 (16%),412/2120 (19%) and 456/2010 (23%) respectively, P<0.001. The number of isolatesdemonstrating high-level resistance to penicillin (� 2 mg/L) also increased: 0, 1, 1and 16 respectively. Four isolates (2 from CSF) in the last 2 years demonstratedMICs of 1 mg/L to ceftriaxone. The proportion of penicillin-resistant isolates washighest in the less than 5-year-old age group, and this increased over the years: 58/211 (27%), 136/562 (24%), 204/691 (30%), 239/673 (36%), P<0.001. In patients15 years or older, the overall proportion of resistant isolates was 379/2942 (13%).Conclusions: Beta-lactam antibiotic resistance in pneumococci isolated from pa-tients with invasive disease is increasing and needs to be monitored carefully.


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Evolution of higher MIC levels against penicillin in an international serotype19F clone possibly by uptake of PBP genes from non-encapsulated pneumo-cocci

Hauser C, Aebi S, Mühlemann K, Institute for Infectious Diseases, University ofBern, Switzerland

Aims: To analyze the nature of low-level resistance in an international Streptococ-cus pneumoniae serotype 19F clone, which became epidemic in Switzerland during1998/99.Methods:108 oxacillin non-susceptible (disk diameter <20mm) nasopharyngeal se-rotype 19F isolates from children with respiratory tract infections were typed bypulsed-field gel-electrophoresis (PFGE) and selected isolates from PFGE clusterswere subjected to multilocus sequence typing (MLST). MICs against penicillin weredetermined by E-test. Penicillin binding protein (PBP) genes 1a, 2b and 2x wereanalyzed for selected isolates by restriction length polymorphism (RFLP) andsequencing.Results: 70 (64.8%) of the 108 isolates belonged to the PFGE clone H (Sáo-Leão etal. J Infect Dis 2002) and included MLST ST 177 and 179. MIC values of isolates inPFGE clone H ranged between 0.023 and 1.0 mg/L. An MIC �0.06 mg/L wasfound in 36 (51.4%) isolates and the highest MIC was 1 mg/L. By RFLP and se-quence analysis isolates with penicillin MICs <0.094 mg/L showed identical muta-tions in pbp 2x. But 2 isolates with an MIC of 0.094 mg/L and 1 mg/L, respectively,differed from this pattern. Both had a pbp2x with high homology to the pbp2x fromnon-encapsulated pneumococci isolated in Switzerland. Additional mutations werefound in the pbp1a genes of both strains and in pbp2b of the strain with a MIC of 1mg/L.Conclusion: Low-level penicillin resistance in an international S. pneumoniae sero-type 19F clone was characterized by mutations in pbp2x, which correlated with anoxacillin disk test diameter <20mm. In the course of their evolution to higher MICvalues two isolates in this clone acquired pbp2x fragments which were also found innon-encapsulated pneumococci. Non-encapsulated S. pneumoniae may serve as areservoir of resistance-mediating PBP genes.

Tracking Longitudinally Quinolone-Resistance in Streptococcus pneumoniaein Hong Kong.

Ip M, Lyon DJ, Tsang L, Cheng AFB. Dept of Microbiology, Chinese University ofHong Kong, Prince of Wales Hospital, Shatin, Hong Kong.

Aims: Previous studies from Hong Kong reported high prevalence of quinoloneresistance in Streptococcus pneumoniae (SPNE). We tracked longitudinally the preva-lence of quinolone resistance in S. pneumoniae in a Hong Kong teaching hospitalfor the periods of 1994 to 2003. The QRDR of the gyrA and B, parC and E geneswere also examined by polymerase chain reaction – single-stranded conformationalpolymorphism (PCR-SSCP) and sequence analysis.Methods: 1363 non-duplicate SPNE isolated in 1994 – Sept, 2003 at Prince of WalesHospital, a 1350 bed teaching hospital in Hong Kong were studied. The MICs topenicillin, cefotaxime, erythromycin, ciprofloxacin, levofloxacin, gatifloxacin andmoxifloxacin were determined (NCCLS). SPNE with ciprofloxacin MIC �4.0 ug/ml were serotyped using Pneumotest antisera, examined by pulsed-field gel elec-trophoresis and multilocus-sequence typing (MLST). Mutations at the QRDR ofthe gyrA and B, parC and E genes were also characterized and examined by PCR-SSCP and sequence analysis.Results: The overall rate of resistance to levofloxacin (MIC �4.0 ug/ml) was 2.1%(ranged 1.6% in 1994-1999, to 2.8% in 2000). The rates for the year 2001, 2002,2003 till Sept, were 2.5%, 2.1% and 2.5% respectively. Serotyping and moleculartyping of these isolates indicated the majority belonged, but not confined, to peni-cillin-nonsusceptible SPNE of the Spanish 23F, 19F and 6B clones. Characteristicmutations that occurred included Ser→Phe at position 81 for gyrA gene, Ser→Pheat position 79, Ile→Leu at 81, Asp→Asn at 83, Asn→Asp at 91, Lys→Asn at 137for parC gene, and Asp→Asn at 435, Ile→Val at 460 for parE gene.Conclusions: Resistance rates to levofloxacin in SPNE have remained stable at aHong Kong teaching hospital in the last decade.


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Antibiotic susceptibility and vaccine coverage of nasopharyngeal (NP) andoropharyngeal (OP) Streptococcus pneumoniae (SP) in daycare (DCC) attendees,St. Petersburg, Russia

Katz A1, Timchenko VN2, Leibovitz E1, Greenberg D1, Porat N1, Peled N1, DaganR1, Ossipov IB2. Ben-Gurion Univ, Beer-Sheva, Israel1 and Acad of Pediatr, St.Petersburg, Russia2

Aims: To study serotypes and antibiotic susceptibility of NP and OPSP in healthyDCC attendees in St. Petersburg.Methods: NP and OP SP was investigated in June 2003 in 125 children aged 16-70m. Susceptibility to penicillin (Pen) and ceftriaxone (CRO) was determined by E-test and to other 5 antibiotics by disk diffusion.Results: 83 SP were isolated in 75/125 (60%) children, of which 36 (48%) NP, 12(16%) OP and 27 (36%) both (19/27 [70%] identical). 100% and 98% isolates weresusceptible to CRO and Pen, respect. Of 81 isolates, 51 (61%), 27 (33%), 16 (19%),14 (17%) and 5 (6%) were nonsusceptible to TMP/SMX, tetracycline, clindamycin,erythromycin (ERY) and chloramphenicol, respect. 17/83 (21%) isolates weremultidrug resistant (� 3 antibiotic classes, MDR). The most frequent serotypeswere 6B (18%), 6A (12%), 23F (10%) and 3, 15B/C and 19F (8% each). 45% wereof serotypes included in the 7-valent conjugate pneumococcal vaccine (PCV); 16%were of related serotypes; 69% were of serotypes included in the 11-valent PCV.Serogroup 15 was found in 12/83 (15%) cases. 65%, 57%, 32% and 27% of 7-valent PCV serotypes were resistant to TMP/SMX, tetracycline, clindamycin andERY, respect. The respective figures for 6A and 19A were 92%, 31%, 31% and 23%and for non 7-valent PCV types 42%, 6%, 0% and 3%. The respective figures forMDR were 100%, 94%, 71% and 76%. 76% of all MDR isolates were covered by 7-valent PCV.Conclusions: 1) While Pen resistance rate was low, high resistance rates to TMP/SMX and tetracycline were found; 2) Resistance rate to ERY was higher than re-ported in other Russian cities; 3) PCV coverage of SP serotypes was modest, but thevaccine may potentially reduce MDR-SP.

Antimicrobial susceptibility in pneumococci and other respiratory bacterialpathogens among children in Greenland: little antibiotic resistance in spite ofhigh antibiotic use

Koch, A1, Tvede, M2, Høgh Andersen, L1, Hjuler, T1, Melbye, M1. Statens SerumInstitut1, Copenhagen, Denmark. Rigshospitalet University Hospital2, Copenhagen,Denmark

Background: In Alaskan natives the prevalence of penicillin-resistant S. pneumoniaeisolates is high. In Greenland respiratory tract infections in children are frequent,and antibiotic use is high. Still, local data on antimicrobial sensitivity hardly exist.Methods: A population-based cohort of children aged 0-4 years was followed fortwo years in Sisimiut, Greenland. Throat swabs were taken when symptoms of res-piratory tract infection presented and every half-year without symptoms. In case ofacute purulent ear discharge a swab was taken from the middle ear. Antibiotic sus-ceptibility was determined by the disk diffusion method with Danish Blood Agarand Rosco New Sensitabs. Antimicrobial susceptibility was defined as susceptible,intermediate, or resistant.Result: Overall, 1624 swabs from 376 children (81% of cohort) were cultured and2560 isolates identified. All S. pneumoniae (n=153) isolates were susceptible toPenicillin, Ampicillin, Erythromycin and other drugs tested. Only 1 of all 270 S.pneumoniae isolates was intermediately susceptible to Penicillin. Of 237 H.influenzae isolates 92% were susceptible to Ampicillin, and 94% susceptible toErythromycin. All Ampicillin resistant strains were �-lactamase producing. Of 228�-haemolytic streptococcal isolates 98% were susceptible to Erythromicin. Of 35Moraxella spp. isolates 49% were susceptible to Ampicillin and 51% resistant, while100% were susceptible to Erythromycin. There was no Meticillin resistant S. aureus(MRSA). The distribution was unchanged if only throat swabs were analysed.Conclusions: Antibiotic resistance is low in Greenland and similar to that in Scandi-navia. Penicillin resistance is less in Greenland than in Alaskan natives. Surveil-lance of antimi-crobial susceptibility in Greenland is warranted. In 2000 Greenlandjoined the International Circumpolar Surveillance of infectious diseases. (MRSA). The distribution was unchanged if only throat swabs were analysed.


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Comparative in vitro activity of telithromycin and seven other antimicrobialsagainst 200 well-characterized erythromycin-resistant (Ery-R) and 100 -sus-ceptible Streptococcus pneumoniae strains

Liñares J1, Ardanuy C1, Fenoll A2, Tubau F1, Perez E1, Dominguez MA1, PallarésR3, Martín R1, The Spanish Pneumococcal Infection Study Network G03/103.1Servicios de Microbiología y 3Enfermedades Infecciosas, Hospital de Bellvitge,Barcelona, 2CNM Instituto de Salud Carlos III, Madrid, Spain.

Aim: To compare the activity of telithromycin and 7 other antimicrobials against200 Ery-R pneumococci (100 ermB+ and 100 mefA/E+), and 100 Ery-S strainsisolated from adult patients.Methods: The antibiotic susceptibilities of 200 Ery-R and 100 Ery-S pneumococciwere studied. The MICs of 8 antimicrobials were performed by microdilution. Thein vitro activity of telithromycin was studied with and without 10µg/ml of reser-pine. Macrolide resistance genes were detected by PCR.Results. The MIC

90 (µg/ml) and frequency of resistance among Ery-S isolates,

mefA/E+ isolates and ermB+ isolates, were as follows: penicillin 2 (33%), 2 (58%),2 (85%), cefotaxime 1 (15%), 1 (26%), 1 (49%), erythromycin 0.12 (0%), 16 (100%),>128 (100%); clindamycin 0.12 (0%), 0.12 (0%), >128 (100%); tetracycline 0.5(8%), 32 (18%), 64 (93%); chloramphenicol 4 (6%), 4 (3%), 16 (59%);cotrimoxazole 4/76 (40%), >4/76 (65%), >4/76 (68%). The range, MIC

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telithromycin were as follows:Ery-S (<0.01 , <0.01, <0.01), Ery-R mefA/E+ ( 0.03-1, 0.25, 0.5) and Ery-R ermB+(<0.01-1,<0.01, 0.25). The majority (85%) of ermB+ isolates were inhibited by0.06µg/ml of telithromycin , whereas only 10% of mefA/E+ were inhibited at thisconcentration. When the MICs of telithromycin were supplemented with 10µg/mlof reserpine 97 % of ermB+ and 68% of mefA/E+ isolates were inhibited by 0.06µg/ml. The MICs of telithromycin for the majority of Ery-R decreased 16-fold inpresence of reserpine.Conclusions: Telithromycin was the most active antimicrobial studied against Ery-S and Ery-R pneumococci. The decrease of MICs of telithromycin with reserpineagainst Ery-R isolates might suggest the presence of an efflux mechanism.

Efficacy of a 9-valent pneumococcal conjugate vaccine in preventing drug-resistant invasive pneumococcal disease

Madhi SA1, , Huebner RE1, Mbelle N1 , Klugman KP1,2 for the Pneumococcal Vac-cine Trialist Group.1Respiratory and Meningeal Pathogens Research Unit, Johannesburg, South Af-rica. 2 Rollins School of Public Healt, Emory University, USA

Aim: To determine the effficacy of a PncCV in preventing drug-resistant invasivepneumococcal disease (IPD) among children. Methods: 39 836 children, including ~2 510 of whom were HIV-1 infected wereenrolled in a phase 3 trial evaluating the PncCV in South Africa. The antibioticsusceptibility profile of the pneumococcal isolates obtained from normally sterile-body sites in children hospitalized for IPD was screened by the disk-diffusionmethod. Isolates that expressed reduced antibiotic susceptibility by this methodwere further evaluated by for antibiotic susceptibility using the E-test or the brothmicrodilution method for minimal inhibitory concentrations. The NCLLS break-points were used in characterising antimicrobial susceptibility profiles. The studywas started in March 1998 and the outcomes reported include surveillance untilJanuary 2003. Results: Overall, the vaccine reduced IPD due to penicillin-resistantpneumococci by 52.2% (95%C.I. 4.9-76.0). Similar reductions were seen in IPDcaused by strains resistant to erythromycin (70%; 95%C.I. 25.3-88.0), clindamycin(72.2%; 95%C.I. 25.2-89.7); tetracycline (62.5%; 95%C.I. 4.2-85.3) andtrimethoprim-sulfamethoxazole (52.5%; 95%C.I. 18.1-72.5).Conclusion: The PncCV serotypes represent most of the serogroups that are asso-ciated with drug-resistance (including serotypes 6B, 9V, 14, 19F and 23F). Thevaccine has had a significant impact on preventing drug-resistant related IPD, how-ever, the potential for drug-resistant pneumococci to appear in non vaccine serotypesrequires vigilance.


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Molecular aspects of Macrolide-Resistance in Streptococcus pneumoniae Iso-lates at the Far East of Russia

Martynova A.V. Epidemiology Department, State Vladivostok Medical UniversityVladivostok, Russia

Backgrounds: Macrolide resistance has been reported to be high among pneumo-cocci in Asian countries, but there is no information on macrolide resistance ofStreptococcus pneumoniae at the Far East of Russia.Aims: To further define molecular mechanisms of macrolides resistance in pneu-mococci strains at the territory of the Far East of Russia.Methods: MICs of penicillin, erythromycin, clarithromycin, and clindamycin weredetermined by the agar dilution method. PCRs were performed with appropriateprimers.Results: A total of 35,82% (48 of 134 strains) of the S. pneumoniae strains wereresistant to erythromycin with a MIC of 1,0 g/ml. Of these, 31,25% (15of 48)showed an MLSB phenotype with erythromycin and clindamycin ; 66,6% (32 of48) showed resistance to erythromycin alone (M phenotype), with a MIC50 andMIC90 of 8,0 g/ ml. One isolate was postive with both ermB and mefE primers. Ofthe isolates expressing the MLSB phenotype, only the ermB gene was detected in86,63% (13 strains of 15) of the isolates by PCR. Two isolates were repeatedlynegative on testing for ermB but were positive for ermA gene. All the isolates ex-pressing the M phenotype were positive for the mefE gene by PCR. The majority ofthe M-phenotype strains (84,37%, or 27 of 32) had constitutive resistance (cMLphenotype); only 15,625% of these strains had inducible resistance (iML pheno-type). Before 2000, it was recorded that among the erythromycinresistant S.pneumoniae isolates, the majority (78%) had an ML phenotype and 22% had an Mphenotype. All S. pneumoniae isolates exhibiting a cML or iML phenotype harboredthe ermB gene.Conclusion: This study indicated a high percentage of erythromycin resistanceamong clinical isolates of S. pneumoniae in the Far East of Russia. It requires amore careful approach to diagnostics of macrolide resistance in pneumococci inthe clinical microbiology laboratory, particularly in areas with high rates of it.

Molecular Characterization of Macrolide-Resistant Nasopharyngeal CarriageIsolates of Streptococcus pneumoniae from Tennessee Children Less than OneYear of Age

Charles A. Mayfield, 1 Crystal N. Johnson, 1 2 Stephen A. Moser, 1 Kathryn M.Edwards, 2 William H. Benjamin, Jr., 1 Susan K. Hollingshead, 1 Ken B. Waites 1

University of Alabama at Birmingham, 1 Birmingham, AL and Vanderbilt Univer-sity, Nashville, TN, USA. 2

Aims: We evaluated non-duplicate nasopharyngeal carriage isolates of Streptococ-cus pneumoniae obtained from 35 children < year of age in Nashville, TN toassess molecular epidemiology of macrolide resistance.Methods: MICs were determined for erythromycin, penicillin, and clindamycin bymicrobroth dilution. Polymerase chain reaction was used to detect mef(A) and erm(B)to assess mechanisms of macrolide resistance. Pulsed field gel electrophoresis(PFGE) was performed to assess genetic relatedness of isolates to one another andto 16 international clones.Results: 32 of 35 (91.4%) macrolide-resistant isolates were intermediate or fullyresistant to penicillin and 16 (45.7%) were resistant to penicillin and clindamycin.15 isolates contained mef(A), 18 contained erm(B), and 2 contained both. Capsularserogroups were 6 (14 isolates), 19 (8 isolates), 14 (5 isolates), 9 (3 isolates), 23 (2isolates), with 3 non-typeable. PFGE showed 20 (57.1%) isolates were unique fromone another and from the international clones. The remaining 15 could be classi-fied into 7 clusters, each containing 2 to 3 isolates that possessed �90% similarityto one another. Two isolates, both containing mef(A) and erm(B), had 80% similar-ity to Taiwan19F-14 and 1 isolate had 83% similarity to Spain6B-2. No other interna-tional clones appeared related to any of these isolates.Conclusions: Young Tennessee children can be colonized with macrolide-resistantS. pneumoniae, some of which possess high-level resistance mediated by erm(B),and are concomitantly resistant to drugs in other classes. Over half of the childrenharbored pneumococci that were related to at least one other strain in the studypopulation. Among 7 different clusters of related organisms, only 1 was related toan international clone while the others probably reflected clones endemic to thegeographic region


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Molecular characterization of very high-level penicillin resistant S.pneumoniaeisolates

McGee L1, Beall B2, Schrag S2, Whitney C2, Klugman K1. Rollins School of PublicHealth, Emory University, Atlanta GA, USA States1, Centers for Diseases Controland Prevention, Atlanta GA, USA

Aim: To characterize the molecular relatedness of invasive S. pneumoniae isolateswith penicillin minimum inhibitory concentrations (MICs) of 8 µg/ml.Methods: Seventy-nine pneumococcal isolates with penicillin MICs of 8 µg/mlwere identified amongst invasive surveillance isolates collected through CDC’sActive Bacterial Core Surveillance in the United States during 2000 and 2001.Strains were characterized by antibiotic susceptibility patterns, serotyping, PFGE,MLST and PBP restriction analysis to determine the clonality of isolates and tocharacterize their pbp genes.Results: All very high-level (MIC 8 µg/ml) penicillin-resistant strains were alsoresistant to other b-lactam antibiotics. Seventeen (21.5%) isolates had amoxicillinMICs of 16 µg/ml and 76% (n=60) showed cefotaxime MIC s 8 µg/ml. The ma-jority of these isolates were resistant to other drug classes. Serotypes 23F (n=32)and 14 (n=35) accounted for 85% of these strains. PFGE and MLST analysesshowed the isolates to be highly clonal with 90% of isolates belonging to 5 of theinternational clones described by the PMEN. The Tennessee23F-4 (n=31) and Eng-land14-9 (n=28) accounted for the largest proportions (74.7%) of isolates. PBP 2X,2B and 1A restriction analyses of the 79 isolates showed 11, 14 and 5 patternsrespectively. Isolates within each PFGE/MLST cluster showed similar RFLP re-striction patterns of their pbp 1A, 2B and 2X genes.Conclusions: The clonality and association of a very high-level penicillin resist-ance with multiple drug resistance requires further monitoring and highlights theneed for novel agents active against the increasingly resistant pneumococcus.

Clinical patterns and microbiology of pneumococcus associated with invasivedisease in Salvador.

Nascimento-Carvalho C1, Moreno-Carvalho O2, Alves NN1, Caldas RM1, BarberinoMG3, Duarte J3, Brandão MA4, Guerra ML5, Brandileone MC5, Di Fabio JL6. UFBA1,FSJ2, HA3, LACEN4, IAL5, PAHO6.

Aims: To describe the types of organisms and the clinical aspects of the disease inpatients from whom pneumococcal strains were isolated from normally sterile bodyfluids.Methods: From Nov 1997, a 6-year laboratory-based survey was conducted at 3clinics in Salvador, Brazil. Isolates were documented to be pneumococcus. Peni-cillin resistance was screened by disc and non-susceptible strains had MIC deter-mined. Disc diffusion or MIC tests were used to evaluate resistance to otherantimicrobials. Serotyping was done. Clinical data were collected on a standardquestionnaire.Results: 140 strains were isolated from 138 patients; 2 patients with meningitis hadpneumococcus isolated from both blood and CSF. The median age was 3yrs, (mean13.4+22.4yrs, range 1 mo-86.5yrs) with 79.7% of all isolates being recovered frompatients<20 years of age. 54.3% were males. Strains were recovered in blood(85.0%),CSF(10.7%), other fluids(4.3%). The clinical diagnosis of bacteremic patients werepneumonia(71.4%), fever without localizing signs(11.8%), upper respiratory in-fections(8.4%), meningitis(3.4%), others(5.0%). Bacteremia was age-related(10.3+19.7 vs 32.6+28.4yrs, p<0.05, mean difference 95%IC 8.2-36.3). The pres-entation of patients from whom strains were isolated in fluids other than bloodwere meningitis(71.4%), pneumonia(14.2%), others(14.4%). Of the 89 isolates forwhich antimicrobial resistance and serotyping tests were performed, resistance wasdetected to penicillin(19.1%), trimethoprim-sulfa(62.9%), tetracycline(21.3%),ofloxacin(4.9%), erythromycin(4.5%), clindamycin(2.2%). All isolates were sus-ceptible to chloramphenicol and vancomycin. 19 different serotypes were identi-fied. Penicillin-resistant strains belonged to serotypes 14(58.8%), 6B(23.5%),19F(11.8%), and 23F(5.9%).


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Clustering of penicillin resistance in Streptococcus pneumoniae (Spn) in ruralPhilippines, 1994 - 2000.

Nissinen A1, Sombrero L2, Esparar G2, Lupisan S2, Lindgren M3, Herva E4, RuutuP5, ARIVAC consortium. Central Hospital of Central Finland, Jyväskylä, Finland1;Research Institute for Tropical Medicine, Manila2; Philippines, National PublicHealth Institute (KTL), Turku, Finland3; KTL, Oulu, 4; KTL, Helsinki5.

Aims: We investigated penicillin resistance in Spn isolated in nasopharynx (carrierisolates, NPA), blood, or cerebrospinal fluid (CSF) (invasive isolates) in a prospec-tive study of hospitalised infants with signs or symptoms of pneumonia, sepsis, ormeningitis in the rural province of Bohol, the Philippines.Methods: NPA samples were cultured on selective blood agar plates, blood andCSF samples on blood agar plates which were incubated in candle jars at 36 oC ad.twice over-night. Spn suspect colonies were identified by optochin test and serotyped.Susceptibility to oxacillin, tetracycline, chloramphenicol, erythromycin andtrimethoprim-sulfa was tested according to the National Committee for ClinicalLaboratory standards. All oxacillin-resistant isolates were tested for minimal in-hibitory concentration of penicillin by the E-test. In addition, isolates with identi-cal resistance profiles and serotype were analysed by pulse-field gel electrophore-sis (PFGE) to assess their similarity in detail.Results: Among the 1206 NPA isolates and 20 invasive isolates, altogether 20 andtwo, respectively, penicillin-non-susceptible strains (PNSP) were found. Six of theNPA isolates and both invasive isolates were of serotype 14. Of the remaining NPAisolates, 5 were not serotyped, two were of serotype 23F, two of serogroup D, one ofserogroup H, and one each of serotypes 4, 5, 33, and 34. All of the serotype 14isolates had decreased susceptibility to penicillin and were resistant to chloram-phenicol, tetracycline, and trimethoprim-sulfa. Among them, two clusters were iden-tified by PFGE.Conclusions: PNSP are rare in rural Philippines. However, two multi-resistant se-rotype 14 clusters have been identified both among carriage and invasive Spn iso-lates.

Major serotypes among penicillin non-susceptible pneumococci isolated fromnon-invasive disease in Portugal

Pissarra C1, Dias R1, Caniça M1. Antibiotic Resistance Unit, National Institute ofHealth Dr. Ricardo Jorge1, Lisbon, Portugal

Aims: The purpose of this study was to evaluate serotype-antibiotype correlationamong penicillin non-susceptible (PNS) isolates from non-invasive pneumococcaldisease in Portugal.Methods: We studied 445 non-invasive Streptococcus pneumoniae (Sp), with lowor high resistance to penicillin (Pen) (MIC>=0.1mg/L), isolated from different speci-mens in 16 Portuguese hospitals, and collected in the Antibiotic Resistance Unit inNational Institute of Health, between 1994 and 2000. MICs to penicillin, cefotaxime(Ctx), ceftriaxone (Ctr), tetracycline (Tet), erythromycin (Ery), chloramphenicol(Cm), clindamycin (Cli) and ofloxacin (Ofl) were determined by agar dilution method(NCCLS). Serotype was performed by Dot-Blot and Quellung reaction.Results: Serotypes 23F, 9V, 14, 15A and 19F represented 80% among PNS isolates.Among the most predominant antibiotypes: 29% were PNS alone (80% of whichwere from serotypes 14, 9V, 23F and 6A); 15% were PenCtxCtr (95% from serotypes9V and 14); 12% were PenTetCmCtxCtr (96% from serotype 23F); and 7% werePenEryCliTet (57% from serotypes 15A, 6B, 19F). All PNS isolates from serotype15A were also Ery resistant, representing 5% from all studied strains. Overall weredetected 25 different multidrugresistant phenotypes.Conclusions: In this study we showed that penicillin resistance andmultidrugresistance of non-invasive S. pneumoniae strains were mostly associatedwith serotype 23F. Serotypes 15A and 6A, found among the sample (5% and 3%,respectively) are not included in the 7-valent pneumococcal conjugate vaccine. Ourwork highlights the importance of monitoring the serotype and antimicrobial sus-ceptibility of isolates from patients with non-invasive pneumococcal disease in Por-tugal.


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Analysis of clinical isolates of S. pneumoniae with reduced susceptibility toamoxicillin

du Plessis M 1, Bingen E 2, Klugman KP 1,3. National Health Laboratory Service,South Africa 1; Laboratoire de Microbiologie, Paris, France 2; Department of Inter-national Health, Emory University, USA 1,3.

Aims: To establish the mechanism/s of resistance to penicillin and amoxicillin inpneumococcal isolates originating from France. These isolates also exhibit an unu-sual phenotype in that their minimum inhibitory concentrations (MIC) foramoxicillin exceed that of penicillin.Methods: All five pbp genes and the mur gene of 9 isolates (Amox MIC range, 1-16 �g/ml) were examined for mutations. Transformation experiments were carriedout using cloned pbp and mur genes as well as genomic DNA from isolate 72521(pen and amox MICs, 2 and 4 �g/ml, respectively). Penicillin-susceptible strain R6was used as the recipient in all transformation experiments. In addition, Hungarianisolate 3191 (pen and amox MICs, 16 and 8 �g/ml, respectively) exhibiting theusual phenotype of penicillin/amoxicillin MICs, was included in the study. The pbpand mur genes from this isolate were compared to those of the French isolates andwere also used in transformation experiments.Results: Transformation of R672521/2x/2b/1a mutants with genomic DNA72521 resulted in2/14 transformants with an altered mur gene, however, the transformants with un-altered mur genes also exhibited increased MICs equivalent to that of the donorstrain. The mur72521 gene, when transformed on its own, did not have any effect onresistance. Transformation of R672521/2x/2b/1a with mur3191 resulted in mutants withunaltered mur genes, however, transformation with genomic DNA3191 showed thatin 3/6 transformants, pbp1a72521 was replaced by pbp1a3191. Although alltransformants exhibited increased MICs, only 2 transformants harboured an al-tered mur gene. The significance of pbp1a is also highlighted in transformationstudies whereby combinations of pbp genes from both isolates were used. A 754-bp fragment of pbp2b72521, which excludes the KTG motif and a portion of the SSNmotif, was able to transform R672521/2x to increased levels of resistance.

Antimicrobial resistance in invasive vs. non-invasive Streptococcus pneumoniaestrains in Finland 2002

Rantala M1, Huovinen P1, Jalava J1, and the FiRe Group2. 1National Public HealthInstitute, Turku, Finland; 2 The Finnish Study Group of Antimicrobial Resistance

Aims: The objective of this study was to monitor antimicrobial resistance and todetect macrolide resistance genes in invasive (n=129) and non-invasive (n=878) S.pneumoniae strains in Finland in 2002.Methods: Bacterial strains were obtained from 24 FiRe laboratories. The suscepti-bility to antimicrobials was tested by agar plate dilution technique. The presence ofmacrolide resistance genes, mef(A/E), erm(B) erm(TR), was studied by multiplex-PCR from strains with erythromycin MIC�0.25 mg/l.Results: Invasive S. pneumoniae strains showed more frequently reduced suscepti-bility to telithromycin (MIC�1 mg/l) than non-invasive strains (12% vs. 6%,p<0.05). Invasive pneumococci had less I+R strains to penicillin (MIC�0.125 mg/l) than invasive strains (5% vs. 13%, p=0.005). In both groups 20% of strains wereresistant to erythromycin (MIC�0.5 mg/l) and 10% to clindamycin (MIC�0.5 mg/l). Macrolide resistance genes was tested from 199 non-invasive pneumococci: 46%(n=92) carried mef(A/E), 39% had (n=78) erm(B), 2% (n=4) had both, one strainharbored erm(TR) and 23 were negative. Of 26 invasive strains 54% (n=14) hadmef(A/E), 42% (n=11) had erm(B), and one was negative. Resistance to moxifloxacin(MIC�2 mg/l) was observed only in non-invasive strains (1.6%). Only a few strainshad reduced susceptibility to meropenem (MIC�0.5 mg/l): in invasive 0.02% vs.non-invasive strains 0.3%.Conclusions: Penicillin resistance in invasive pneumococci seems to have remainedon the same level in Finland as it was 1999-2000. The difference in penicillin re-sistance in invasive vs. non-invasive strains may be explained by larger proportionof intermediate strains among non-invasive pneumococci. Resistance to macrolidesis increasing. Difference in susceptibility to telithromycin among invasive vs. non-invasive pneumococci might be due to spread of special clone(s) and is worth offurther investigation.


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Characterization of Macrolide Resistant Streptococcus pneumoniae IsolatesFrom Alaska: 1986-2002.

K. Rudolph, A. Reasonover, M. Harker-Jones, D. Hurlburt, A. Parkinson, T.Hennessy, J. Butler. Arctic Investigations Program, Anchorage, AK.

Aims: Resistance of S. pneumoniae (Sp) to macrolides is increasing in many partsof the world and is generally mediated by ribosomal modification or active drugefflux. The objectives of this study were to determine the prevalence of macrolideresistance and to characterize macrolide-resistant determinants among invasive pneu-mococcal isolates recovered in Alaska.Methods: 2062 invasive pneumococcal isolates were collected as part of statewidesurveillance from 1986 – 2002. Serotyping was done by Quellung reaction. Sus-ceptibility testing was performed by microbroth dilution according to NCCLS guide-lines. Macrolide resistant isolates were screened for erm(B) and mef(E) by PCR.PFGE was performed with SmaI enzyme.Results: Overall macrolide resistance was 8.3%, increasing from a low of 0.8% in1988 to 18.4% in 1997 with a decline to 8% in 2002. Ninety-two percent of eryth-romycin resistant isolates were multidrug resistant (2 or more antimicrobial classes);63% and 88% were nonsusceptible to penicillin and trimethoprim/sulfamethoxazole,respectively. Predominant serotypes of erythromycin resistant strains were 6B (34%),9V (12%), and 14 (30%). Eighty percent (n=137) carried mef(E) and 9.3% (n=16)erm(B). Two isolates carried both mef(E) and erm(B), and 16 isolates were nega-tive for both genes. The prevalence of mef(E) positive isolates steadily increased,peaking at 17.5% of all isolates in 2000. Isolates harboring erm(B) were not seenuntil 1992, but accounted for <4% of isolates each year. Erythromycin MIC’s formef(E)+ isolates ranged from 0.5 µg/ml – 64 µg/ml, which included 4 (2.9%) iso-lates with MICs of 64 µg/ml; for erm(B)+ isolates, MIC’s ranged from 4 µg/ml – 64µg/ml. The mef(E) containing isolates comprised 6 major PFGE clones and in-cluded isolates of serotypes 6B, 9V, 14 and 19F.Conclusions: The predominant mechanism of macrolide resistance among inva-sive Sp isolates in Alaska is active efflux, and increasing rates of resistance are dueto a steady increase in the presence of mef(E).

Capsular switch of a serotype 9V clone explain the rapid increaseof“Streptococcus pneumoniae isolates of serotype 14 with reduced susceptibil-ity to penicillin in Sweden .

Sjöström K, Vallhagen J, Morfeldt E, Fernebro J, Normark S, Henriques NormarkB. Swedish Institute for Infectious Disease Control, Solna, and Microbiology andTumorbiology Center, Karolinska Institutet, Sweden.

In Sweden the frequency of pneumococci with reduced susceptibility to penicillinremains low, though with regional differences. One clone of serotype 9V, whichhas also been recognized internationally, has dominated, but lately we have ob-served an increase of serotype 14 isolates with a reduced susceptibility to penicil-lin.

Aims: To investigate whether the increase of serotype 14 was due to an increase ofa specific clone and if a capsular switch had occurred between serotype 14 and 9Visolates and in that case why type 9V is involved in horizontal gene transfer events.Methods: All pneumococci with reduced susceptibility to penicillin in Sweden aresent to our laboratory at the Swedish Institute for Infectious Disease Control since1998. During 1999-2002, 320 clinical isolates of serotype 14 with reduced suscep-tibility to penicillin were collected, predominantly from the nasopharynx, and fromchildren up to 6 years. These isolates were characterized by antibiotic susceptibil-ity testing and by using pulsed-field gel electrophoresis (PFGE) and multilocussequence typing (MLST). Also, 9V isolates were characterized using the samemethods. In addition genes involved in DNA maintenance, i.e. dinP, were sequenced.Results: The major part of the serotype 14 isolates belonged to the same clone.Isolates belonging to the predominating clone of type 9V in Sweden showed thesame PFGE pattern and sequence type (ST) as the type 14 clone. The 9V clone wasfound to carry a partial deletion of dinP, encoding error prone DNA polymerase IV,resulting in a truncated product. The same dinP deletion was also found in isolatesof the type 14/9V switch clone as well as in isolates belonging to other type 9Vclones. Isolates belonging to other serotypes had a wildtype dinP gene.


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Selection of penicillin-susceptible pneumococcal strains following transforma-tion of a penicillin-resistant strain with a penicillin susceptibility gene

Smith AM, Klugman KP. Respiratory and Meningeal Pathogens Research Unit,National Institute for Communicable Diseases, Johannesburg, South Africa.

We report a technique useful for transformation experiments involving bacteria,such as the pneumococcus, which are naturally competent for DNA transforma-tion. It allows the selection of antibiotic-susceptible transformants following thetransformation of a resistant strain with an antibiotic susceptibility gene. We showthe effectiveness of this technique through the selection of penicillin-susceptibletransformants following the transformation of a penicillin-resistant pneumococcalstrain with a penicillin-susceptibility gene. Our transformation recipient was peni-cillin-resistant strain R62X/2B/1A/mur with a penicillin MIC of 16 µg/ml. The super-script lettering (2X/2B/1A/mur) indicates that the strain has altered penicillin-bindingproteins (PBPs), PBP 2X, PBP 2B, and PBP 1A; as well as altered murM. Thesealtered proteins collectively confer the high-level penicillin resistance. Our aimwas to transform strain R62X/2B/1A/mur to penicillin-susceptibility using an unalteredpbp2X gene. To select for this penicillin-susceptible phenotype, we created a trans-forming DNA fragment, which contained an unaltered pbp2X gene and an erythro-mycin resistance gene cassette inserted into a neighbouring non-essential gene,yllC. Penicillin-susceptible transformants could therefore be selected by virtue oftheir simultaneous transformation to erythromycin resistance. Analysis of erythro-mycin-resistant transformants revealed penicillin-susceptibility (MIC, 0.03 µg/ml)through the introduction of an unaltered pbp2X gene, thereby creating strain R62B/

1A/mur. Our results demonstrate the essential role that altered PBP 2X plays in thedevelopment of penicillin-resistance in the pneumococcus. With an unaltered PBP2X in place, other penicillin resistance determinants (altered PBP 2B, altered PBP1A, and altered murM) cannot exert their effect, such that penicillin-susceptibilitywill exist in the pneumococcus.

Distribution of PMEN clones in Scottish pneumococcal isolates analysed byMLST.

A Smith1, J Jefferies2, S C Clarke3, C Dowson4, T J Mitchell2 & G Edwards3. 1Infec-tion Research Group, Glasgow Dental Hospital; 2Division of Infection and Immu-nity, University of Glasgow; 3SMPRL, Stobhill Hospital, Glasgow; 4Dept. of Bio-logical Sciences, University of Warwick.

Aims: To compare 250 Scottish pneumococcal isolates analysed by multilocus se-quence typing (MLST) to the globally significant antimicrobial resistant clonesidentified by the Pneumococcal Molecular Epidemiology Network (PMEN clones).Methods: MLST of pneumococcal isolates was performed as previously published(Jefferies et al., Mol. Biotech. 2003; 24: 303-307). Antimicrobial susceptibilitywas determined by Etest methodology.Results: The 250 isolates contained over 30 different serogroups. 91 Sequence types(ST’s) were assigned to the Scottish isolates, of which 41 were new to the MLSTdatabase. Three of the 25 STs of the PMEN clones were present in the Scottishcollection, these were ST156, (7 isolates) ST9 (8 isolates) and ST377 (1 isolate).With the exception of one Scottish ST156 isolate (erythromycin MIC 2.0mg/L) allScottish isolates related to the PMEN clones had lower penicillin and erythromycinvalues than those described for the corresponding type strain. Although the numberof PMEN STs in our collection is low, there are a number of single (SLV) anddouble locus variants (DLV) of these STs present. The highest level of resistance topenicillin and erythromycin were found in two serotype 19 (ST 320) isolates. ThisST is not one of the previously recognised PMEN clones. Our collection includedan erythromycin resistant (>256mg/l) serotype 6 isolate with a new ST (814).Conclusions: Several Scottish pneumococcal isolates had identical ST’s to some ofthe PMEN clones but reduced levels of susceptibility compared to the type strains.These results provide a snapshot of resistance levels and the genotypic backgroundof Scottish pneumococcal isolates in comparison with the PMEN clones.


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Mutational analysis of the vex123-vncRS locus in S. pneumoniae T4

Sublett J, Haas W, and Tuomanen E. St. Jude Children’s Research Hospital, Mem-phis, TN, USA

Aims: Determine the role of the vex123-vncRS locus in vancomycin-stress response,tolerance, and autolysis.Background: The locus, encoding an ABC transport system (Vex123), a proposed ex-tra-cellular signaling peptide (Pep27), and a two-component system (VncRS), wasimplicated in the regulation of cell death via autolysis and vancomycin-tolerance. Re-cent findings have challenged this model, resulting in a re-evaluation of previous find-ings.Methods: The genes for vex3, pep27, vncR, vncS, lytA (autolysin, positive control), andSP2155 (IgA1 protease, negative control) were individually replaced with the ermBresistance cassette via site-specific recombination mutagenesis. The response of thesemutants to vancomycin challenge in mid logarithmic growth phase was determined byfollowing changes in optical density (OD

620) and colony forming units over a period of

four hours after addition of the antibiotic. For microarray experiments, RNA was iso-lated from mutant strains and compared to the T4 wild-type strain using whole-ge-nome cDNA microarrays as described by the manufacturer (Pathogen FunctionalGenomics Resource Center, Institute for Genomic Research (TIGR), Rockville, MD).Results: A strain that lacks the autolysin LytA did not undergo autolysis after additionof the antibiotic and was therefore considered vancomycin-tolerant. Strains with muta-tions in the genes for Pep27, VncR, VncS, or the IgA1 protease control did not showany reduced rate of lysis when compared to the wild-type strain. In contrast, mutationsin the vex3 gene did yield a strain with a reduced rate of autolysis, which fulfilled thedefinitions for tolerance. All strains tested showed a decrease in the rate of vancomy-cin-induced lysis when the strains were grown in the presence of erythromycin.Microarray analyses revealed that only very few genes are differentially regulated inthe absence of vncR or vncS, suggesting that the two genes might activate, rather thanrepress, transcription. Microarray data also support the hypothesis that transcription ofvex123 is under regulatory control of VncRS.Conclusions: 1) The vex123-vncRS locus’ main function in S. pneumoniae is not theregulation of autolysis in response to vancomycin-treatment, although a mutation invex3 does reduce the rate of autolysis. 2) Erythromycin does make S. pneumoniaemore tolerant towards vancomycin, which could explain why previous reports sug-gested a role for vncRS in tolerance. 3) The VncRS two component regulatory systemseems to control expression of vex123 in response to an unknown stimulus.

Nasopharyngial carriage and antimicrobial resistance of Streptococcuspneumoniae in healthy children in Estonia during two study periods.

Tamm E,1 Lutsar 1, Naaber P2, Kõljalg S2, Maimets M3. Dept. of Pediatrics TartuUniversity1, Dept. of Clinical Microbiology United Laboratories2, Dept. of Infec-tious Diseases Tartu University3, Estonia.

Aims: To determine rates of nasopharyngeal (NP) carriage and antimicrobial re-sistance of S.pneumoniae in healthy children in Estonia and to find out if the resist-ance is emerging.Methods: In winter 1999/2000 (1-period) and 2003 (2-period) NP swabs collectedfrom healthy 2 –7 year old children in 29 day care centers of Tartu, Tallinn andJõhvi were assessed for presence of S.pneumonie, it’s antibiotic susceptibility andserotypes.Results: A total of 396 and 289 children (mean age 4 years) were studied in 1- and2-period respectively.The NP colonisation rate remained unchanged, being 46% inthe first and and 40% in the second period. Between these two periods penicillinnonsusceptibility rate of S.pneumoniae decreased from 9% to 1% ( p<0.05). Allpenicillin nonsusceptible strains were categorised intermediately resistant. A non-significant increase from 4% to 8.5% was seen in erythromycin resistance. All 10erythromycin resistant strains isolated in 2003 showed high resistance ( MIC 8 - >256 �g/ml ), of which 5 were clindamycin resistant. A total of 71% of isolates in1999/2000 and 61% in 2003 were resistant to trimethoprim-sulfametoxazole. Ofall isolates 3% in 1999/2000 and 6% in 2003 were considered multiresistant. Inboth periods the most commonly colonising serotypes were in order of frequency19, 23, 6, 14 and 15. Of 261 serotyped strains 238 ( 91%) belonged to the 21vaccine-related types. 40% of penicillin nonsusceptible strains in 1999/2000 and40% of erythromycin nonsusceptible strains in 2003 were nontypable (rough).Conclusions: The pneumococcal resistance rate of nasopharyngial isolates to peni-cillin and erythromycin in Estonia is minimal and has not increased from 1999 to2003.


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Antibiotic resistance development in Streptococcus pneumomoniae: is there arole for mutation frequency?

Vallhagen J, Morfeldt E, Blomberg C, Henriques Normark, B. Swedish institute forinfectious disease control, Solna, and Microbiology and Tumorbiology Center,Karolinska Institutet, Sweden Pneumococci acquire penicillin resistance by hori-zontal gene transfer form other streptococcal species. If there is a fitness cost foracquiring resistance and whether compensatory mutations occur is not determinedyet. Mutation frequencies have been shown to be important for resistance develop-ment in other bacteria.

Aim: To see whether there is a correlation between antibiotic resistance develop-ment and mutation frequency in pneumococci.Method: The mutation frequency to rifampicin was measured in 254 clinical pneu-mococcal isolates; 155 penicillin susceptible, 51 penicillin non-susceptible and 48multi-drug resistant. The isolates were selected to be of different serotypes, themajority collected in Sweden but a few were international isolates causing invasivedisease or carriage. In addition, mutation frequencies to streptomycin is being meas-ured in some of these isolates. Also, the types of mutations that gives resistance tothe two antibiotics are examined by sequencing the rpoB- and rpsLgenes.Results: There was a tendency that penicillin susceptible isolates had lower muta-tion frequencies to rifampicin than penicillin non-susceptible and/or multi-drugresistant isolates. Analysis of the mutation frequency to streptomycin is ongoing toverify these results.Conclusion: Mutation frequency does, as it appears, play a role in antibiotic resist-ance development since it seems that clinical isolates that show antibiotic resist-ance has higher mutation frequencies to rifampicin. Further experiments are, how-ever, needed to confirm this.

Risk Factors For Antibiotic Resistance in Infections due to Streptococcuspneumoniae

Vanderkooi OG, Powis JE, Low DE, Green K, Pong-Porter S, Plevneshi A, ShigayevaA, McGeer A and the Toronto Invasive Bacterial Disease Network. Mount SinaiHospital, Ontario, Canada.

Aims: To identify those factors associated with a risk of resistance amongst pneu-mococci from patients with invasive disease. Methods: The Toronto Invasive Bac-terial Disease Network has been conducting prospective surveillance for invasivebacterial diseases in the Toronto and Peel Region (population 3.4M people) since1995. All isolates are collected, and susceptibility testing done according to NCCLSguidelines. Study nurses review charts, and interview patients and physicians. Riskfactors for resistance included age, underlying illness, vaccination status, socio-economic status, and prior antibiotic use. Results: A total of 3543 invasive S.pneumoniae isolates were collected from 1995 to 2002. The mean age of the pa-tients was 49.8 (± 30.7) years with 45.9% females. In univariate analysis, age, un-derlying illness and prior antibiotic use were associated with resistance. Inmultivariate modelling, underlying illness was not associated with antibiotic resist-ance, except that diabetes mellitus (DM) was associated with fluoroquinolone (FQ)resistance (OR=2.9, p=.01). Antibiotic use in the previous 3 months was signifi-cantly associated with resistance in both univariate and multivariate analysis. Re-sistance to any antibiotic class was associated with age <18yrs, OR 1.7 (p<.0001)and 1.4 (p=.0015) respectively. Risk factors for penicillin resistance were age <18yrs (OR 1.8, p<.0001) and antibiotic use (OR 1.9, p<.001). Only antibiotic usewas significant for ceftriaxone resistance (OR 1.7 p=0.008) while DM (2.9, p=.01)and antibiotic use (OR 2.2, p=0.04) were significant for FQ resistance. Risk factorsfor trimethoprim-sulfamethoxazole resistance were age < 18 yrs (OR 1.5, p<.001)and antibiotic use (1.7, p<.0001 with similar results seen for erythromycin (OR 1.8(p<.001) and 1.7 (p<.001) respectively). Conclusions: The most important risk fac-tor for antibiotic resistance in invasive pneumococcal infections is prior antibioticuse. Children are more likely to be infected with an antibiotic resistant strain. Un-derlying illness is generally not a risk factor for resistance.


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Models and data to distinguish mechanisms for the acquisition of pneumococ-cal immunity

*Lainé, C

Harvard School of Public HealthEpidemiology Dept. / 677 Huntington AvenueBoston, MA 02115UNITED [email protected]

What is the reproduction number for pneumococcal infection, and does it matter

*Farrington, P

The Open UniversityDept. of StatisticsWalton HallMilton Keynes MK7 6AAUNITED [email protected]


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MOD-04

Estimating the transmission parameters of pneumococcal carriage in house-holds

Melegaro A1, 2, Gay N1, Medley G2. 1Modelling and Economics Unit, CDSC, HealthProtection Agency, London, 2University of Warwick

Aims: To investigate Streptococcus pneumoniae transmission dynamics and to pro-vide estimates of pneumococcal (Pnc) duration of carriage and acquisition ratesfrom the community and from infected individuals within the household.Method: Data from a longitudinal study on Pnc carriage in UK families was used.Ten consecutive swabs were taken at 4-week intervals from all members of 121households and Pnc isolates were serotyped using standard methods. A density de-pendent transmission model was fitted to derive maximum likelihood estimates ofthe duration of carriage and acquisition rates from the community and from in-fected individuals within the household. Parameter values were estimated for allserotypes together, and for the most common individual serotypes for children (<5years) and adults (5+ years).Results: The duration of carriage is longer in children less than 5 years of age thanin older family members (51 vs. 19 days). Children are 3 to 4 times more likely thanadults to acquire Pnc infection from the community. Transmission rates within thehousehold suggest that adults are more infectious but less susceptible than children.Transmission within the household is most important in large families. The propor-tion of household acquired infection ranges from 29-46% in households of threepersons to 38-50% in larger households. Evidence of density dependent within-household transmission is found, although the strength of this relationship is notwell-defined.Conclusions: Household-acquired infection makes a major contribution to Pnc trans-mission in families both for children and adults. Age-specific differences in theduration of carriage have been observed with children carrying Pnc for longer. Dif-ferences in transmissibility and duration of carriage between serotypes may haveimplications for the effectiveness of vaccination programs.

A Bayesian approach to investigate S. pneumoniae carriage in a follow-up ofchildren in schools

Cauchemez S1, Temime L1, Guillemot D2, Varon E3, Boëlle PY14. INSERM U4441,Institut Pasteur2, Centre de Référence du Pneumocoque3, Assistance Publique -Hôpitaux de Paris4, Paris, France.

Aims: Estimates of the acquisition rate and average duration of carriage of S.pneumoniae (Pnc) are critical in mathematical models trying to predict vaccineimpact and trends in antibiotic resistance.Methods: We estimated these parameters using data from a 5 months longitudinalfollow-up of 2819 children in 50 schools in France. 7377 samples were available forserotyping and penicillin resistance determination, antibiotic (ATB) exposure wasalso documented. For each child, the data were augmented to describe the detailedtime course of Pnc carriage. The hierarchical Bayesian framework ensured consist-ence between observation and augmented data; described the dynamics of Pnc ac-quisition and clearance; grouped serotypes into clusters according to their charac-teristics; defined the prior distribution of the parameters. Markov Chain Monte Carlosampling was used to sample from the posterior distribution of the parameters.Results: Application to the real data yielded a person to person transmission risk of0.00046 day-1 (95% Credible Interval, CI [0.00030,0.00052]) in a school of size100 and an average duration of carriage of 22 days (CI [18,26]). The observedserotypes were split into two clusters according to high/low prevalence, but no clus-tering was detected for the durations of carriage. In a simulation study, we foundthat the epidemiological parameters were accurately estimated in this model andthe number of serotype clusters reflected the simulation scenarios. In particular, themethod succeeded detecting short difference in durations of carriage (of the orderof a week).Conclusions: There was no evidence that the different strains of Pnc had differentdurations of carriage, albeit the method could detect differences of the order of theweek. While the duration of carriage was shorter than previously reported, it shouldbe noted that neither ATB exposure nor resistance were taken into account. Furtherdevelopments are under way to incorporate these data.


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Evaluating PnC-7 herd immunity impact in German adults

Claes C1, Reinert R2, Schulenburg JM1

1 Institute for Insurance Economics, University of Hanover, Germany2 National Reference Centre for Streptococci, University Hospital Aachen, Germany

Background: In Germany the conjugate vaccine PnC-7 is only recommended forchildren with increased health risks although a general vaccination would redeem51.1 % of vaccination costs due to avoidable direct health care expenditures and100 % from societal view including indirect costs. Additional US-american studiesshowed evidence for a reduced rate of pneumococcal diseases in unvaccinated adults(herd immunity).Methods: In a Markov model the herd immunity effect was transferred to Germancircumstances. Adjacent herd immunity further key data were essential: incidencesof invasive pneumococcal diseases (IPD) and pneumococcal pneumonia, fatalityrates, age specific population structure and health care expenditures for treatmentof pneumococcal infections.Results: The herd immunity of PnC-7 is able to avoid 59,259 pneumococcal infec-tions (IPD and pneumococcal pneumonias) in German adults every year, 2,688deaths, and direct health care costs about 54.6 million EURO. The latter refinancesvaccination costs with a further share of 27.3 %. The sensitivity analysis showedthat the estimates were conservative. In comparision to presence the ageing Ger-man population will effect more avoidable infections, deaths and health care expen-ditures during the next two decades although the German population will decline.Conclusions: A general vaccination recommendation will lead to a net vaccinationcost burden of merely 21.6 %. Additionally the risk group of senior citizens withunsatisfying polysaccharid vaccination compliance can be protected within someextent.

Cost-effectiveness analysis of pneumococcal conjugate vaccine in children

Melegaro A1, 2, Edmunds WJ1. 1Modelling and Economics Unit, CDSC, Health Pro-tection Agency, London, 2University of Warwick

Aim: To establish whether universal vaccination of infants with the pneumococcalconjugate vaccine is likely to be cost-effective from the perspective of the healthcare provider (NHS).Mehtod: Two cohorts – one vaccinated and one unvaccinated - were followed overtheir lifetime, and the expected net costs and benefits (measured in terms of life-years and Quality Adjusted Life Years gained) were compared in the two cohorts.The impact of long term effects of the vaccine, such as herd immunity and serotypereplacement, were investigated and their relative importance was assessed by per-forming univariate sensitivity analysis and multivariate Monte Carlo simulations.Results: Under base-case assumptions (no herd immunity and no serotype replace-ment) the programme is not expected to be cost-effective from the NHS perspectiveat the current price of the vaccine. A reduction of the cost of the vaccine to half ofits current level could bring the cost per QALYs gained within normally acceptableranges. If the burden of disease is significantly underestimated by current surveil-lance systems, then the cost per QALY gained approaches acceptable levels at thecurrent vaccine price. The introduction of herd immunity effects substantially re-duces the burden of pneumonia among the elderly and significantly improves thecost per life year and QALY gained. However, serotype replacement has been shownto occur both in carriage and disease and may counterbalance the advantages ofherd immunity.Conclusions: The use of pneumococcal conjugate vaccine in infants is not justifiedeconomically at current vaccine prices. However, this conclusion is sensitive toassumptions regarding the current burden of pneumococcal disease and the futureimpact that vaccination will have in the unvaccinated and on the future serotypedistribution.

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A simulation model for pneumococcal carriage

Robertson C 1,2, Bramley JC1. Scottish Centre for Infection and EnvironmentalHealth1, University of Strathclyde2, Glasgow, Scotland, UK.

Aims: To investigate the feasibility of constructing a simulation model for the car-riage of pneumococci in the community.Methods: The model takes account of population dynamics and is based upon house-holds, with individuals as the unit of transmission. Parameters for population agestructure, birth and death rates, and household composition use national statisticsfor Scotland. Four other grouping structures were created to represent neighbour-hoods, wider family, work and school environments. Households are merged toaccount for drift to single-person units. Five arbitrary pneumococcal serotypeswere considered for transmission, which was assumed to be related to age, serotypeand immunity, and to take place only if there was sufficient contact between indi-viduals. A matrix of transmission was constructed. The duration of carriage couldbe varied by serotype and multiple carriage was possible. Input parameters werebased on published data wherever possible. The basic unit of time for simulationwas one week and simulations were run for 10-20 years.Results: A stable population model was constructed. Results obtained for indi-vidual-to-individual transmission were computationally very slow and, therefore, a‘contact score’ was developed to reflect the weighted total number of carriers anindividual comes into contact with.Conclusions: Construction of a simulation model for pneumococcal carriage is fea-sible. Further work is required to clarify some pneumococcal parameters and in-vestigate sensitivity. The model can easily be extended to include invasive, non-invasive disease and mortality. Vaccination effects against specific serotypes couldalso be incorporated, to study the potential effects of serotype replacement. Themethodology could also be used as a basis for exploring the dynamics of otherrespiratory infections.

Impact of conjugate vaccination on the incidence of meningitis due to PRP inhigh vs. low antibiotic exposure environments.

Temime L1, Boëlle PY1,2, Guillemot D3. INSERM U4441, Université Pierre et MarieCurie2, Institut Pasteur3, France.

Aims: The frequency of meningitis due to penicillin G-resistantS. pneumoniae (PRP) has increased in recent years, making treatment failure morelikely; it is currently expected that pneumococcal conjugate vaccines could curbthis trend.Methods: We investigated this issue using a mathematical model describing trans-mission of pneumococcal strains and selection of pneumococcal resistance to peni-cillin in the community, with two sets of parameters chosen to reflect the widelydifferent current situation regarding resistance and antibiotic exposure in the USAand in France. Parameters describing the natural history of colonization of S.pneumoniae, the frequency of contacts in the population and the characteristics ofantibiotic consumption were estimated from the literature for the two countries.Results: Our main finding is that while widespread vaccination may curb the trendin resistance selection, the level of antibiotic exposure in the community stronglyhinders this effect. Indeed, in relatively low antibiotic exposure environments suchas the USA, large scale vaccination allows to prevent a large part of PRP meningitiscases. But in high antibiotic exposure environments such as France, vaccinationalone does not lead to a comparable reduction in PRP meningitis incidence unless itis associated with a global reduction in antibiotic prescriptions to children.Conclusions: This study suggests that antibiotic exposure reduction will remain ofprimary importance for the control of PRP meningitis despite wide scale use ofpneumococcal conjugate vaccines. This is especially important in Europe, whereantibiotic consumption rates may vary up to fourfold with southern European coun-tries the biggest consumers in the western world.

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The influence of competition and vaccination on the coexistence of two pneu-mococcal serotypes

Zhang Y1, Auranen K2,3, Eichner M1. Dept. for Medical Biometry, University ofTübingen, Germany1; National Public Health Institute (KTL), Finland2; Dept. ofMathematics and Statistics, University of Helsinki3, Finland.

Aim: To study by means of mathematical modelling and computer simulations theinfluence of competition and vaccination on the coexistence of two pneumococcalserotypes.Methods: We establish a deterministic system of differential equations which repre-sent two modified versions of the SIRS model considering direct and indirect com-petition. In the case of direct competition, the serotypes are assumed to influenceeach other only through simultaneous colonization, whereas in the case of indirectcompetition, colonization by one serotype is affected by previous colonization ofthe other serotype through cross-reactive immunity. The two models are studiedwith three different effects of competition which may either reduce (i) the suscepti-bility, (ii) duration of carriage, or (iii) force of infection, caused by super-colonizedindividuals (under direct competition) or by individuals that are immune against theother serotype (under indirect competition). We study the potential for serotypereplacement under vaccination with a mono-valent vaccine.Results: All three effects of competition influence the coexistence of the two serotypessimilarly. Both serotypes can only coexist if competition is moderate. Vaccinationagainst a target serotype influences the prevalence of the target serotype in differentways, dependent on the mode of competition: serotype replacement can only occurunder direct but not under indirect competition. Under both modes, competitionalways reduces the critical vaccination coverage.Conclusions: Mathematical models and computer simulations show that the possi-bility of serotype replacement strongly depends on the mode of competition. It is,therefore, important to investigate experimentally whether competition operatesdirectly or indirectly.

MOD-15

A mathematical model to compare the impact of a conjugated vs polysaccharidepneumococcal vaccine for elderly persons

Verstraeten T, Hausdorff W, Poolman J. GlaxoSmithKline Biologicals, Rixensart, Bel-gium

Background: A recent comparison (Fry 2002) of the potential benefits of conjugatedand polysaccharide (PS) pneumococcal vaccines suggested that conjugated vaccinesrequired longer duration of protection in order to have similar impact on invasive pneu-mococcal disease (IPD) among the elderly. There is evidence, however, that conju-gated vaccines also improve the proportion of elderly responding to vaccination.Objective: To evaluate how the impact on IPD among elderly would differ between aconjugated vaccine with less valences, but better response rate (defined as the percent-age of individuals showing a “protective” immune response over a defined threshold),and a PS pneumococcal vaccine.Methods: We assumed the response rate and clinical protection against IPD offered bythe PS vaccine to be 60% for each serotype. We then modelled the impact of a hypo-thetical conjugated vaccine on each individual serotype by assuming various increasesin response rate. To estimate the overall impact of each vaccine, we weighted the roleof each serotype on the available epidemiological data.Results: A conjugated pneumococcal vaccine can have an equivalent impact on IPD ascompared to the PS vaccine with relatively minor increases in response rates. Thiscould be achieved, for example, by an 11-valent vaccine that would increase the re-sponse rate for the three most prevalent serotypes by 25% in Europe or 29% in the US.It could equally be achieved by an 11-valent vaccine that would improve the responserate for all serotypes by 14% in Europe or 17% in the US. Any cross-protective effector increase in the number of serotypes included would lead to a superior profile forsuch a conjugated vaccine.Conclusion: Conjugated pneumococcal vaccines that increase the response rates overa defined threshold for some or all serotypes can have an equivalent population-levelimpact on IPD as the PS vaccines, without having to rely on longer duration of protec-tion. Assuming that clinically relevant thresholds for the immune response can be iden-tified, this model can be applied to assess the potential of vaccines in development.

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GEN-01

GEN-02

Comparative genomics of pneumococci

*Tettelin, H

The Institute for Genomic Research9712 Medical Center DriveRockville, MD 20850UNITED [email protected]

Structure and evolution of the cps (capsular polysaccharide) region

*Bentley, S

Wellcome Trust Sanger InstituteHinxtonCambridge CB10 1SAUNITED [email protected]


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GEN-04

Gene alterations occurring in Streptococcus pneumoniae as a result of biofilmgrowth

Ogburn, E., Dowson, C. University of Warwick, UK., Mitchell, T. University ofGlasgow. Gillespie, S. Royal Free Hospital, London.

Aims: Asymptomatic carriage of S. pneumoniae as a biofilm in the nasopharynx iswidespread and serves as the main reservoir in causing infections. One of the ma-jor virulence determinants is the capsular polysaccharide with the effects of cap-sule loss being well documented. This work determines the genetic events that leadto capsule loss in S. pneumoniae type 3 laboratory and clinical strains, which, inturn, may lead to alterations elsewhere in the pneumococcal genome contributingto the evolution of this bacterium.Methods: Using the sorbarod biofilm system to grow S. pneumoniae type 3 leadsto a high frequency of capsule loss. PCR amplification was used to identify altera-tions within the capsule loci of biofilm and clinical acapsular variants. Furtheranalysis of the entire genome of one acapsular sorbarod variant with DNAMicroarray technology was then performed. PCR amplification of any genes de-termined to be absent reiterated the microarray result.Results: Random deletions and duplications were found within the capsule locilaboratory strains ranging from 11-239bp, larger deletions were discovered in theclinical type 3 strains. DNA Microarray results showed a large number of genesfound in the wildtype strain were deemed “not present” in the sorbarod variantwith PCR amplification proving this to be a true result.Conclusions: Capsule loss in S. pneumoniae type 3 occurs due to random duplica-tions and deletions in laboratory strains with deletions also present in clinical strains.Not only does capsule loss affect the capsule loci, many genes throughout the ge-nome appear to be altered.

Pneumococcal vaccine development including genomically derived proteins

*Poolman, J

Research and DevelopmentGlaxo Smith Kline BiologicalsRue de l’Institut 89B-1330 [email protected]


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GEN-05

Tissue-specific regulation of iron uptake and oxidative stress responses in Strep-tococcus pneumoniae by two-component signal transduction

Andrew T. Ulijasz1, David R. Andes2, Jeremy D. Glasner3, and Bernard Weisblum1*Departments of Pharmacology1, Medicine2, Veterinary Medicine3 University ofWisconsin, Madison, WI USA

We have constructed a deletion mutant in Streptococcus pneumoniae of an orphantwo-component response regulator needed for virulence, named RR489 by Throupet al. [Mol Microbiol. 35:566 (2000)]. In the present studies rr489 was found to berequired for lung but not hip infection in a murine model, which suggests a tissue-specific role. High density DNA microarrays were used to measure mRNA abun-dance of the �rr489 mutant and a tetracycline inducible rr489 strain relative to wildtype R800 S. pneumoniae. Genes which exhibited the greatest differential expres-sion in mRNA abundance were piuB and piuA which are involved in Fe-hemin trans-port and were repressed by rr489. Conversely, two putative H2O2 resistance orthologsDpr (Fe storage) and AdhE (Fe alcohol dehydrogenase) were upregulated. H2O2killing and hemin-streptonigrin susceptibility assays were performed and correlatedwith array data. Addition of 40 mM H2O2 to �rr489 mutant and wild-type cellsresulted in half-lives of 35 and 45 minutes, respectively, indicating a lower toler-ance in the �rr489 mutant to H2O2. Streptonigrin, an antibiotic which requires ironfor activity, showed a 43-fold reduction in viable colonies after 60 minutes in �rr489compared to wild-type R800 S. pneumoniae. By using recombinant GST-RR489, afootprint was found within the promoter region of piuB, an Fe-permease subunitand the first of 4 genes in the piu operon described by Brown et al. [Mol Microbiol.40:572 (2001)]. Our results indicated three distinct binding sites within the piuBpromoter and revealed a consensus sequence similar to that of CovR, a S. pyogenesglobal response regulator with which RR489 shares its closest similarity. Theseobservations suggest that RR489 is a unique two-component response regulatorwhich functions as a possible Fur-IdeR-DtxR-like functional ortholog in S.pneumoniae.


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GEN-12

Phylogenetic analysis of a aliB homologue found in the capsule region of non-encapsulated Streptococcus pneumoniae

Patrick Bättig1, Lucy J. Hathaway1, Kathrin Mühlemann1. 1Institute for InfectiousDiseases, University of Bern, Switzerland.

Aims: To analyse the phylogenetic relationships of a recently described aliB homo-logue present in the capsule region of non-encapsulated pneumococci.Methods: BLAST search at the NCBI (http://www.ncbi.nlm,nih.gov/BLAST/) andTIGR microbial database (http://www.tigr.org/tdb/mdb/mdbinprogress.html).Phylogenetic studies were done with the Lasergene software (DNASTAR Inc, Madi-son, WI, USA).Results: BLAST analysis revealed that the aliB-like ORF2 (Hathaway et al. submit-ted) was related to:a) an open reading frame (ORF) in the capsule region of the Streptococcus mitis

genome (93% nucleotide homology over 1889 bp).b) spr0311, a gene encoding for a hypothetical protein in the capsule region of S.

pneumoniae R6 (92% homology over 163 bp at the 5’ end of aliB-like ORF2)genome. Spr0311 is present in encapsulated pneumococci of various serotypes(1, 2, 3, 4, 6B, 8, 14, 18C, 19A, 19F, 23F, 33F and 37), is located downstream ofdexB and upstream of a putative transposase, and is remarkably conserved be-tween serotypes.

c) a short region (32 bp) after the putative transposase in the capsule operon of S.pneumoniae R6, which matches a region at the 3’ end of aliB-like ORF2. Ahomologue of this region is present in about half of the encapsulated strainscarrying the spr0311 homologue.

Conclusions: The aliB-like ORF2 carried by some non-encapsulated pneumococciis related to genes in the capsule region of S. mitis and encapsulated pneumococci.Recombination events between encapsulated strains and S. mitis may be a mecha-nism of capsule loss in S. pneumoniae.

Genomic organization and molecular analysis of the inducible prophage EJ-1,a mosaic myovirus from an atypical pneumococcus

García E, López R, Romero P. Centro de Investigaciones Biológicas, CSIC, Spain.

Aims: The purpose of this work is to analyze the genomic organization of the induc-ible bacteriophage EJ-1 from S. pneumoniae. This is the first report on the genomeof a myovirus infecting a low G+C content Gram-positive (LGP) bacterium.Methods: EJ-1 was induced with mitomycin C from an atypical pneumococcus (strain101) that was isolated in 1987 in a Spanish hospital from the blood of a patientsuffering from pneumonia. This strain contains two lytic amidase genes, i.e., onefrom the host (lytA

101) and another from the phage (ejl). Sequencing of the phage

genome was carried out using standard techniques.Results: The genome of the EJ-1 prophage (42,935 bp) is organized in 73 openreading frames (ORFs) and in, at least, five major clusters. Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible func-tions to 52 ORFs. The predicted proteins coded for the EJ-1 genome revealed simi-larities in the lysogeny, DNA replication, regulation, packaging, and head morpho-genesis protein clusters with those from several siphoviruses infecting lactic acidbacteria. However, the proteins encoded by genes orf53 to orf64, corresponding toputative tail proteins of the virion, were very similar to those of the defective Bacil-lus subtilis myovirus PBSX with the notable exception of the gene product of orf57(the tape measure tail protein) that was similar to proteins from phages infectingGram-negative bacteria.Conclusions: An abundant presence of lytic and/or temperate phages has been pro-posed to exist in the pneumococcus although a precise role of phage genes in andhuman disease remains to be established. The genomic exploration of the firstmyovirus (EJ-1) infecting LGP bacteria, a bacterial group embraces important patho-gens (as S. pneumoniae or S. pyogenes) as well as microorganisms of remarkableindustrial importance (as S. thermophilus, lactococci, or lactobacilli), represents animportant step in this direction.


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GEN-14

Vancomycin stress-response in sensitive and tolerant strains of S. pneumoniae

Haas W, Kaushal D, and Tuomanen E. St. Jude Children’s Research Hospital, Mem-phis, TN, USA

Aims: Characterization of the changes in gene expression after treatment with vanco-mycin in two strains of S. pneumoniae that are sensitive (strain T4) or tolerant (strainTupelo) to the antibiotic. Background: Strains of S. pneumoniae have developed resist-ance to most commonly used antibiotics with the exception of vancomycin. Vancomy-cin-tolerant strains do not grow or undergo autolysis in the presence of the antibiotic asresistant or sensitive strains would do. Tolerance can lead to treatment failure and mightlead to the emergence of resistant strains. Methods: RNA was isolated from T4 or Tupelo cultures that were treated for 10 or 20min with vancomycin, and from untreated control cultures. Whole genome cDNAmicroarray analysis was performed as described by the supplier of the arrays (PFGRC,Institute for Genomic Research (TIGR)). Results: Both strains responded to the vancomycin challenge by increasing the ex-pression of heat shock proteins and chaperonins, transcriptional regulators, cholinebinding proteins CbpG and CbpF, and two ABC transport systems. Among the down-regulated genes were RNA polymerase and enzymes involved in spermidine and xan-thine metabolism. Genes involved in aminosugar metabolism were differentially regu-lated in both strains as well, suggesting a shift from peptidoglycan synthesis toaminosugar degradation. Alcohol-dehydrogenases, generally involved in fermentationprocesses, were up-regulated in T4 and down-regulated in strain Tupelo. Among thegenes that were differentially expressed in T4, but not in strain Tupelo, were the CiaRHregulon (shown to be involved in penicillin resistance and competence), the proteaseClpE, hemolysin, and neuraminidase B (increase in transcription in response to vanco-mycin), several glucosyltransferases and capsule specific genes, ABC transporters,ribosomal proteins, and genes involved in DNA replication (decrease). Genes that weredifferentially regulated in strain Tupelo, but not in T4, were the ATP synthase operon(increase), the tryptophan, purine and sodium ATP synthase operons, and the catabolitecontrol protein ccpA (decrease). Conclusions: Vancomycin-challenge induces a strain-specific stress response in thesensitive strain T4 and the tolerant strain Tupelo. Despite these differences, certaingenes are differentially expressed in both strains, suggesting a common triggeringmechanism. Further work is required to establish a regulatory interaction map for S.pneumoniae and to determine the cause of tolerance in strain Tupelo.

Homologues of aliB are found in the capsule region of non-encapsulated Strep-tococcus pneumoniae

Hathaway L.J., Stutzmann Meier P., Bättig P., Aebi S, Mühlemann K. Institute forInfectious Diseases, University of Bern, Switzerland.

Aims: To study the epidemiology of non-encapsulated pneumococci in Switzerlandand to characterize their capsule locus.Methods: Pneumococci were isolated from 1980 nasopharyngeal and 215 bloodsamples, collected as part of two nationwide survey studies between 1998 and 2002.27 non-encapsulated isolates were identified by the Quellung reaction and charac-terized by bile solubility, antibiotic susceptibility and multilocus sequence typing(MLST). PCR of the capsule locus was performed and the PCR products sequencedfor four isolates. The capsule regions were also analyzed by Southern blot. Expres-sion of putative open reading frames located with the capsule operon was assessedby RT-PCR and Northern blot and in vitro translation was performed.Results: MLST and capsule region analysis divided the non-encapsulated pneumo-cocci into two groups: Group I was closely related to encapsulated strains whereasgroup II, which had a clonal population structure, lacked capsule genes but insteadcarried a homologue of the oligopeptide transporter aliB, named here as aliB-likeORF 2. Also, some non-encapsulated strains had an additional homologue, namedaliB-like ORF 1. AliB-like ORF 1 was transcribed and could be translated in vitro.Conclusions: In some strains, loss of capsule expression was associated with loss ofcapsule genes and importation of one or two aliB homologues into the region of thecapsule locus.


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GEN-16

CodY-transcriptional regulation in Streptococcus pneumoniae

W.T. Hendriksen1, A. de Jong2, P.V. Adrian1, R. de Groot1, O.P. Kuipers2, and P.W.M.Hermans1

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1 Department of Pediatrics, Erasmus Medical Center, Rotterdam, the Netherlands.2 Department of Molecular Genetics, University of Groningen, the Netherlands.

Aim: CodY is a pleiotropic DNA-binding repressor, which is known to play a regu-latory role in amino acid metabolism in Bacillus subtilis and Lactococcus lactis. Inthis study, we investigated the regulation of gene transcription by the CodY homo-logue in Streptococcus pneumoniae in order to predict its role in pathogenesis.Methods: The gene encoding CodY was inactivated in strain D39 (serotype 2). Thein vitro growth characteristics of D39�codY in THY-medium were compared withthe parental strain by monitoring the optical density in time. Total RNA was isolatedat various time points during growth of both wildtype and mutant strain. The RNAof both strains was reverse transcribed, fluorescently labeled using Cy3/Cy5, andapplied to microarray slides to identify the transcriptome profiles.Results: D39�codY displayed a significant reduction in growth when cultured inTHY-broth at 37°C in a static flask culture, which suggests that the codY mutationhas severe effects on the global transcription. Several genes were upregulated by thedisruption of codY. These genes comprised members involved in nitrogen/aminoacid metabolism, metal uptake, competence energy transduction and fatty acid me-tabolism.Conclusions: In S. pneumoniae CodY regulates not only genes involved in peptideuptake, e.g. the ami-operon, carA, thd1 and brnQ, which are up-regulated in the�codY mutant, but also other operons involved in thiamine synthesis (thi-operon),and acetoacetate synthesis (ilv-operon). Since pleiotropic regulators are usually in-volved in many different cellular and physiological processes, the relevance of CodYin pathogenesis will be discussed.

Transcriptome analysis of Streptococcus pneumoniae TCS mutant �rr09

Hendriksen, W.T.1, C.E. Blue2, P.V. Adrian1, R. de Groot1, T.J. Mitchell2, and P.W.M.Hermans1.Erasmus MC1, Rotterdam, The Netherlands; University of Glasgow2, UK.

Aim: Two component signal transduction systems (TCSs) play an important role inthe transcriptional adaptation of bacteria to the environment and environmentalchanges. Recent studies have demonstrated that TCS09 of Streptococcus pneumoniaeis virulence-associated, since mutants lacking this system are reduced virulent inmurine animal models (Blue et al., 2003, Infection and Immunity, 71(8):4405-13).In the present study, we investigated the transcriptional changes in pneumococcalmutants lacking the response regulator of TCS09 (�rr09) by microarray analysis.Methods: The gene encoding rr09 was inactivated in the pneumococcal strains D39and TIGR4. The in vitro growth characteristics of D39�rr09 and TIGR4�rr09 inTHY-medium were compared with the parental strains by monitoring the opticaldensity in time. RNA was isolated at two time points during growth, reversely tran-scribed, fluorescently labeled using Cy3/Cy5, and applied to genomic amplicon-based microarray slides.Results: TIGR4�rr09 and D39�rr09 showed no changes in in vitro growth charac-teristics when cultured in THY-broth at 37°C in a static flask culture. However, thetranscriptional pattern of TIGR4�rr09 displayed clear differences compared to theparental wild type strain. Preliminary results showed that several genes involved inamino acid-, sugar uptake-, and carbohydrate metabolism were down regulated inthe TIGR4�rr09. In addition, certain genes involved in iron metabolism and somecarbohydrate metabolic genes were up regulated in the TIGR4�rr09. Transcrip-tional analysis of D39�rr09 is currently in progress.Conclusion: The preliminary microarray data suggest a link between TCS09 andseveral metabolic pathways in S. pneumoniae. In addition, the relevance of TCS09in pneumococcal virulence suggests an important role of these pathways in vivo.


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GEN-18

Implications for Vaccine Development Based on Pneumococcal PopulationStructure

Huot H, Bouchet V, Leroy M, Pelton S, Goldstein R. Division of Pediatric Infec-tious Diseases, Boston University Medical Center, Boston, MA USA.

Aim: Phylogenetic characterization of a comprehensive collection of S. pneumoniae(Sp).Collection: 1100 isolates involving diverse categories of disease: invasive (25%),upper respiratory, ie otitis media (19%), lower respiratory (6%); carriage (50%),the reservoir of disease causing isolates; and a set of Sp horse isolates. This collec-tion is also geographically diverse (15 countries), and encompasses 52 serotypes(ST) and non-typable isolates.Results: Characterization of the population structure revealed an absence of clus-tering of isolates by clinical syndrome. As well, carriage isolates spanned the ge-netic diversity of the population. This implies that eradication of disease-causingSp would likely require eradication of carriage isolates. Also found is what we referto as “fragmented clonality”: while some ST-based dendrogram clustering is ob-served, multiple clusters of a particular ST are seen within a unique geographiclocation. This has potentially significant implications regarding future efficacy of acapsule-based vaccine. Unlike the case of‘H. influenzae where the Hib vaccine elimi-nated most invasive disease, our study suggests that eradication of Sp of particularSTs may not have as large an impact, as (i) specific STs were not found to beassociated with specific forms of disease, and (ii) vaccine and non-vaccine STswere often found to be genetically closely related, suggesting that any capsule typecould become associated by horizontal transfer with any genetic backbone, allow-ing for emergence of new potentially pathogenic combinations. Similarly, absenceof clustering by geography suggests that any location is seemingly susceptible toentry by isolates of any genetic backbone and ST.Conclusion: To overcome limitations associated with Sp capsule-based vaccines,exploitation of a robust dendrogram depicting population-based diversity of a spe-cies such as that described here will be useful in evaluating non-capsular vaccinecandidates both present and conserved across all members of the species.

Molecular Ecology of pneumococcal virulence genes.

J. M. C. JEFFERIES1, S. C. CLARKE1, 2, A. SMITH3, C. DOWSON4 AND T. J.MITCHELL11.Division of Infection & Immunity, University of Glasgow, GlasgowUK.2.Scottish meningococcus and pneumococcus Reference Laboratory, GlasgowUK.3.Infection Research Group, University of Glasgow, Glasgow, UK.4.Dept. OfBiological Science, University of Warwick, Coventry, UK.

Aims: To understand the relationships between pneumococcal disease isolates andmap virulence gene sequence data from pneumococci and related viridans strepto-cocci onto this in order to understand the distribution of these allelic variants andthe capacity for horizontal transfer. Due to the inherent variability of the pneumo-coccal genome it is fundamental to discover which aspects of the genome providestable targets for therapeutic interventions.Methods: Multilocus sequence typing (MLST) has been used to build a phylogeneticframework based on housekeeping genes of 250 pneumococcal Scottish diseaseisolates provided by the Scottish Meningococcus and Pneumococcus ReferenceLaboratory (SMPRL). PCR and sequencing of ply, hylA and nanA genes was thencarried out and the sequences mapped onto the MLST data. Related oral strepto-cocci were also screened by PCR for the presence of the three genes has revealedthe potential for horizontal recombination.Results: Pneumococcal serogroups/types can be made up from a number of geneti-cally distinct clones or sequence types. Distinct Sequence Types (STs) can containisolates of more than one serogroup. Relatively low numbers of viridans strepto-cocci contain homologues of pneumococcal virulence genes and allelic differencesbetween these two populations of genes exist.Conclusions: There is evidence for horizontal transfer of genes between pneumo-cocci and the closely related viridans streptococci. Hyaluronidase and neuramini-dase are less well conserved than pneumolysin, this may be attributed to evolution-ary pressure from the host immune system due to their location on the cell surface.Further work to determine differences in activity between various alleles of thesevirulence proteins is being undertaken in our laboratory.


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Typical pneumococcal lytA alleles in Streptococcus mitis: Evidence for bacteri-ophage-mediated gene transfer

López R,., García E, Romero P. Centro de Investigaciones Biológicas, CSIC, Ma-drid, Spain

Aims: Streptococcus mitis, a pneumococcal-relative, is a leading cause of infectiveendocarditis. Pneumococcal LytA amidase appears to contribute to higher morbidityand mortality during pneumococcal infection. We report here the isolation of two S.mitis phages that code for lytic enzymes homologous to LytA and provide results onthe basic role of phages in the spread of genes implied in virulence.Methods: Phages were isolated from the lysogenic S. mitis strains B6 and HER treatedwith mitomycin C. DNA was prepared by treatment of purified phages with SDS andproteinase K. lytA

B6 and lytA

HER isolated from B6 and HER, repectively, were cloned

and expressed in Escherichia coli DH-5�.Results: Two new temperate phages exhibiting a Myoviridae (�B6) and a Siphoviridae(�HER) morphology have been isolated from strains B6 and HER,. respectively. ThelytA

HER and lytA

B6 genes were very similar (87% nt identity), and have identical size

(957 bp). lytAHER

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are homologous to the lytA gene characteristic of S.pneumoniae and of those lytic genes described in pneumococcal temperate phages.Most interestingly, an inactive E-form of the LytA

HER and LytA

B6 amidases are the

primary products. These inactive forms can be converted to the full biochemical activeC-form in the presence of choline but, in remarkable contrast with the case of thepneumococcal amidase, the C-form can be “re-converted” to the E-form by dialysis. Adendrogram of genetic relationships between the 40 lytA-like alleles sequences reportedso far suggests that typical and atypical lytA alleles form two independent clades.Conclusions: i) the genome of some S. mitis possesses typical pneumococcal lytA-likegenes; ii) phage-coded lytic enzymes of S. mitis exhibit the peculiar characteristic of“conversion”, a property that appeared to be exclusive of the major pneumococcalautolysin; iii) our findings provide strong support to previous proposal on the basicrole of pneumococcal-related phages in the spread of genes implied in virulence.Conclusions: i) the genome of some S. mitis possesses typical pneumococcal lytA-likegenes; ii) phage-coded lytic enzymes of S. mitis exhibit the peculiar characteristic of“conversion”, a property that appeared to be exclusive of the major pneumococcalautolysin; iii) our findings provide strong support to previous proposal on the basicrole of pneumococcal-related phages in the spread of genes implied in virulence be-tween species living in common biological habitats.

Generation of capsule diversity in Streptococcus pneumoniae

van Selm S1, Kolkman MAB2, van der Zeijst BAM3, van Putten JPM4. Erasmus MC- University Medical Center Rotterdam1, Genencor International, B.V., Leiden2,Netherlands Vaccine Institute, Bilthoven3, Utrecht University4, The Netherlands.

Aims: In this project, we studied the molecular basis of the diversity in capsularpolysaccharide (CPS) structures among pneumococcal serotypes via comparisonof the capsule genes of related serotypes.Methods: Relevant capsular polysaccharide biosynthesis (cps) locus of differentpneumococcal serotypes were PCR amplified, sequenced and genetically analysed.Emphasis was placed on genes encoding glycosyltransferases, O-acetyltransferasesand CPS repeating unit polymerases. The role of horizontal gene transfer in theevolution of pneumococcal capsular serotypes was investigated via directed func-tional gene replacement of a putative CPS repeating unit polymerase.Results: The variable presence of an O-acetyl group in the CPS of serotypes 15Band 15C correlated with the length of a short tandem TA-repeat. The reversibleswitching between these two serotypes may well be explained by a slipped strandmispairing based variation in the length of the short tandem repeat sequence. Forseveral other serotypes that vary in O-acetylation, identical putative O-acetyltransferase genes could be found, indicating that the cause of the variable O-acetylation and thus of capsule diversity may reside outside the cps loci. Functionalreplacement of a CPS repeating unit polymerase of serotype 15B with the corre-sponding gene of serotype 15H caused a switch in capsular serotype. Single basedeletions or larger deletions within cps genes were identified that relate to the di-versity in capsular serotypes. Indications of horizontal gene transfer were found inthe presence of remnants of rhamnose biosynthesis genes.Conclusions: Our data indicate that deletions, slipped strand mispairing, factorsoutside the cps locus and horizontal gene transfer contribute to the generation ofcapsule diversity.


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Molecular mechanisms of invasive pneumococcal disease

*Weber, J

Humboldt University, Dept. of Neurology, ChariteSchumannstr. 20/21D-10117 [email protected]

Contribution of pneumococcal proteins to pathogenesis

*Mitchell, T

Univerisity of GlasgowDiv. of Infection and ImmunityJoseph Black BuildingG12 8QQ GlasgowUNITED [email protected]


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Regulation of capsule expression

*Yother, J

University of Alabama at Birmingham845 19th St. S.Birmingham, AL 35294UNITED [email protected]

Pneumococci and TLRs: The double-edged sword of immunity

*Malley, R

Channing LaboratoryBrigham and Women’s HospitalBostonMA 02115UNITED [email protected]


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Reduction in Nasopharyngeal Carriage of Streptococcus pneumoniae due toHaemophilus influenzae in Murine Model of Co-colonization

E. Lysenko and J.N.Weiser. Department of Microbiology, University of Pennsylva-nia, Philadelphia, PA

Aims: An animal model was developed to study the effect of bacterial interactionduring co-colonization with H.influenzae and S.pneumoniae.Methods: SCID mice were given intranasally either a type b H.influenzae strain, atype 23F S.pneumoniae clinical isolate, or both. Nasal washes at 24 or 72 h post-inoculation were used to determine levels of colonization by quantitative cultureand levels of MIP2 as a marker of pro-inflammatory innate immunity on the mu-cosal surface.Results: Both bacteria were able to efficiently colonize the nasopharynx of SCIDmice at similar levels. Levels of H.influenzae were not affected by the presence ofS. pneumoniae. In contrast, colonization by S. pneumoniae was significantly re-duced at 24 and 72 h when given together with H.influenzae, as well as followingpre-colonization with S.pneumoniae prior to infection with H.influenzae. Infectionwith a mixture of heat killed H.influenzae and live S.pneumoniae did not reducepneumococcal colonization. By 72 h, MIP2 concentrations in nasal washes of micecolonized with either species alone were significantly lower than in the mixed in-fection. However, no direct correlation between colonization efficiency and levelsof MIP2 associated pro-inflammatory responses was found.Conclusions: In a SCID mouse model the presence of live H.influenzae during co-infection caused up to a 100-fold decline in level of pneumococcal colonization.Since this effect was seen in SCID animals, it is not due to adaptive immunity. Thelack of correlation between levels of bacterial colonization and concentrations ofMIP2 also suggests that this effect is unlikely to result from pro-inflammatory re-sponses. Therefore, the reduction in pneumococcal colonization appears to be dueto bacterial-bacterial competition. Our findings have implication for preventativestrategies that would alter colonization dynamics.


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Role of Choline Binding Protein G (CbpG) in pneumococcal infection

Antikainen J1*, Mann B2*, Orihuela C2*, Gao G2, Korhonen TK1, Tuomanen E2

Division of General Microbiology, University of Helsinki, Finland1, Department ofInfectious Diseases, St. Jude Children’s research hospital, Memphis, USA2 *con-tributed equally to this work

Aims: Functional characterization of CbpG, a putative serine protease, important invirulence of S. pneumoniae.Methods: cbpG and cbpG mutant lacking the choline binding domain were con-structed from S. pneumoniae TIGR4 strain and unencapsulated TIGR4R strain byinsertion duplication mutagenesis. CbpG was also expressed on the surface of Lacto-bacillus casei to characterize proteolytic function. Lung epithelial, nasopharynxand rat brain endothelial cell lines were used in adhesion assays and female Balb/cJmice to study in vivo colonization and causing of the invasive disease.Results: cbpG sequences from clinical isolates revealed two forms of the gene. 25%of strains from healthy carriers, pneumonia, and meningitis cases expressed a CbpGprotein that lacked the choline binding domain as a result of a conserved slip-strandmutation in the sequence, otitis media strains almost never showed this variation.All mutants were attenuated in their ability to bind epithelia but had no loss ofbinding to endothelia. In mouse model studies, CbpG deficient mutant strain wasattenuated in its ability to initially colonize the nasopharynx and was dramaticallyattenuated in its ability to survive or replicate in the blood stream or enter to CSF incontrast to the wild type. Only mice infected with the wild type strain developedpneumonia. L. casei expressing CbpG degraded both casein and fibronectin and thesame was seen with the choline extractions of the wild type streptococci strains.Conclusions: We have shown that choline binding protein G functions as an adhesin,an invasin and has proteolytic activity.

The functional implications of allelic variation in secretory-component bind-ing domains of pneumococcal choline-binding protein A (cbpA)

Balakrishnan I1, Charalambous BM1, Gillespie SH1. Department of Medical Micro-biology, Royal Free Campus, Royal Free and University College Medical School,University College London, UK.

Aims: To study the functional implications of allelic variation in secretory-compo-nent binding domains (SCBD) of pneumococcal choline-binding protein A (cbpA)Methods: Allelic variation in pneumococcal SCBD amongst blood culture isolatesof a defined patient group was identified and characterised by PCR amplificationand sequencing. Real-time binding kinetic analysis using a resonant mirror biosen-sor provided evidence of variation in binding kinetics between isolates with differ-ent SCBD sequences and secretory IgA (sIgA). However, these data are affected byvariable expression levels of cbpA and other molecules which may interact withsIgA. To enable molecular studies of the functional implications of our findings,gene sequences encoding different allelic variants of the second of the two repeatsecretory-component binding domains (R2-SCBD) of cbpA were cloned and ex-pressed in E.coli. Binding studies were then conducted on the recombinant peptides.Results: Competition ELISA studies indicate variation in the binding interactionsbetween allelic variants of R2-SCBD and sIgA. Confirmatory studies usingchaotropic ELISA and resonant mirror biosensor analysis are currently in progress.Conclusions: Our studies indicate that allelic variation in SCBD of pneumococcalcbpA affects its binding characteristics with sIgA. The implications of these find-ings on pneumococcal adhesive and invasive capabilities require further study.


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Multifunctional activity of glycolytic proteins of Streptococcus pneumoniae

1Bergmann S., 2Rohde M., 2Bracht D., and 1Hammerschmidt S. 1Research Centerfor Infectious Diseases, University of Würzburg, Germany. 2German Research Centerfor Biotechnology (GBF), Microbial Pathogenicity, Braunschweig, Germany

Aims: Identification of plasmin(ogen)-binding proteins of Streptococcus pneumoniaeand characterisation of specific binding sites.Methods: Plasminogen overlay assays of pneumococcal cell wall proteins identi-fied the �-enolase (Eno) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)as plasminogen-binding proteins. Subcellular localisation of both glycolytic en-zymes was carried out by immunoelectron microscopy and 2-D-gel electrophore-sis. In order to detect putative plasminogen-binding sites, blot overlays were per-formed with membranes containing overlapping synthetic peptides of the enolaseprotein sequence. Amino acid substitutions in the internal binding site of Eno anddeletions of C-terminal lysines confirmed identified binding sites. Using surfaceplasmon resonance (SPR) technique the binding dynamics were determined. Withrespect to the �-enolase, the impact of plasminogen-binding motifs was investi-gated by intranasal infection of mice with pneumococcal eno mutants.Results: The enolase and the GAPDH of S. pneumoniae were identified asplasmin(ogen)-binding proteins. Both glycolytic enzymes are displayed on the pneu-mococcal cell surface. SPR studies and spot membrane analysis provided evidencethat an internal binding site in Eno is substantial for the interaction with plasmino-gen.Conclusions: The glycolytic enzymes such as the �-enolase and GAPDH have anessential role for pneumococcal viability and most likely also an important func-tion in pneumococcal pathogenesis which is displayed on the bacterial cell surface.

Integrity of the fourth domain of pneumolysin: essential for maintenance ofpneumococcal virulence?

Bortoni-Rodriguez ME, Hirst RA, Kadioglu A, Munkundan S, O’Callaghan C,Andrew PW. Dpt. of Infection Immunity & Inflamation. Univeristy of Leicester,U.K.

Aims: The possibility that an important contribution to pneumococcal virulencewas provided by domains 1-3 of pneumolysin (ply) and independent of haemolyticactivity or complement activation was proposed by Baba et al., 2002. This hypoth-esis was tested by comparing the ex-vivo and in vivo growth characteristics andvirulence levels of a pneumococcal pneumolysin mutant (�4) with a wild type S.pneumoniae D39 (wt).Methods: The 3’end of the‘ply gene for pneumolysin on the chromosome of‘S.pneumoniae D39 was interrupted by the insertion of a spectinomycin gene. Thisproduced a mutant (�4) whose ply protein lacks the expression of the last 34 aminoacids of the fourth domain (d4). The construction was confirmed by PCR. Assayswere done to compare the haemolytic activity of �4 with that of the wt. Tests inciliated ependymal cells from rats were done with total bacterial protein. IV infec-tions in mice were done with wt and �4 passaged bacteria, blood was collected atdifferent time points of infection by tail bleeding, and viable counts were done.Survival time of the mice was as well recorded.Results: The mutant �4 presented no haemolytic activity in contrast to significantlyhigh levels of activity with wt. �4 was still capable of stopping the beating of ciliain ependymal cells, but there was a significant delay compared to the wt (p<0.05).A significant difference in growth between 24-48 hours following IV infection inmice was observed between the two strains of bacteria (p<0.05). The mortality formice infected with wt S. pneumoniae was of 85%, but only 25% for �4.Conclusions: The requirement for d4 varied according to the array. Integrity of d4was necessary for virulence following systemic infection and for haemolysis whereasactivity against ependyma was dependent on d4.


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Presence of zinc metalloproteinases in clinical isolates of Streptococcuspneumoniae

Camilli R1, Del Grosso M1, Memmi G2, Pozzi G2, Pantosti A1, Oggioni M R2. IstitutoSuperiore di Sanità, Rome1, University of Siena2, Italy.

Aims: The purpose of this work was to investigate on the presence and the distribu-tion of the zinc metalloproteinases classes ZmpB, ZmpC and Iga, in invasive iso-lates of Streptococcus pneumoniae.Methods: A total of 140 strains collected from cases of bacteremia or meningitiswere studied. The collection included the most prevalent serotypes isolated during alarge nationwide study in Italy in the years 1999-2000. The presence of eachmetalloproteinase gene was detected by PCR using a couple of primers, designedon the conserved regions surrounding the respective gene, to amplify a fragment ofapproximately 6 kb for zmpB and 8 kb for zmpC and iga, respectively. For the igaclass, the presence of iga2 was investigated with a second PCR using a differentdownstream primer, designed inside the iga2 gene.Results: All the strains analysed, with a few exceptions, carried zmpB; converselyonly 14 % of strains carried zmpC. Interestingly, all the strains belonging to sero-type 8 harboured this gene. Iga gene class was present in the majority (75%) of thestrains analysed. In particular, approximately 40% of the strains carried only igaand 35 % the association iga/iga2. Iga alone was more commonly found in serotypes3 and 6A/6B isolates, while the association iga/iga2 was more frequent in serotypes7F and 14 isolates. No association was found between presence of a metalloproteaseand type of invasive disease or age of patient.Conclusions: Our study showed that zmpB, iga and, to a lesser extent, zmpC arecommon among invasive isolates of S.pneumoniae. These data support the hypoth-esis that the zinc metalloproteinases contribute to the virulence of the pneumococ-cus.

Pneumolysin induces CXCL8 production in human upper respiratory tract epi-thelial cells.

Dogan S1, Zhang Q1, Murdoch C2, Mitchell T3 and Finn A1. Department of ClinicalSciences South Bristol, University of Bristol, Bristol, UK1, Divison of Genomic Medi-cine, University of Sheffield Medical School, Sheffield, UK2, Division of Infection &Immunity, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow,UK3.

Aims: In murine studies, pneumolysin (Ply), a cytolytic toxin of Streptococcuspneumoniae, has been shown to be an important virulence factor. Used as vaccine itcan prevent infection and/or carriage. CXCL8 is a potent inflammatory mediator andimmune modulator. We investigated the effect of Ply on CXCL8 production in thehuman upper respiratory tract epithelial cell line (Detroit 562).Methods: Concentrated culture supernatants (CCS) from a type 2 strain of pneumo-coccus (D39)(Ply+) and CCS from a daughter mutant Ply-negative strain (Ply-),recombinant Ply and mutant recombinant Ply toxoid F433 (which lacks cytolytic activ-ity) were incubated with Detroit cells and CXCL8 release measured by ELISA. Pres-ence/absence of Ply in the CCSs was confirmed by Western immunoblotting. The roleof calcium was investigated by using calcium-free media and by using the intracellularcalcium chelator BAPTA-AM.Results: Secretion of CXCL8 was induced in Ply+ CSS-stimulated cells in a dose de-pendent fashion, while it was reduced to 40% following stimulation by equivalent con-centrations of Ply- CCS (p<0.05). Pre-incubation of CCS with proteinase K and boil-ing both abolished CXCL8 production, suggesting that the activity is due to protein.Cell pre-treatment with BAPTA-AM reduced CXCL8 release to 60%. Use of calciumfree media abolished CXCL8 release but only when Ply was used at concentrations >0.6ug/ml.Stimulation with both rPly and rPly toxoid F433 induced dose dependent CXCL8 re-lease but this was reduced to 24% by F433 compared to rPly (at protein concentrationsof 0.25ug/ml).Conclusions: Ply induces CXCL8 release in human upper respiratory epithelial cells.Signalling pathways involve calcium. Some, but not all induction is due to cytolyticeffects of the toxin. These data may have important implications for the use of Ply as amucosal vaccine.


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SpsA (CbpA) of Streptococcus pneumoniae binds to human specific motifs indomains 3 and 4 of polymeric Ig receptor

Christine Elm1, Ranveig Braathen2, Finn-Eirik Johansen2, Sven Hammerschmidt1.1Research Center for Infectious Diseases, University of Würzburg, Germany. 2Insti-tute of Pathology, Laboratory for Immunohistochemistry and Immunopathology,Rikshospitalet University Hospital, Oslo, Norway.

Aims: Identification of the minimal SpsA (CbpA) binding sites in polymeric Igreceptor (pIgR) expressed by epithelial cells and elucidation of the impact of SpsA-pIgR interaction on pneumococcal invasion.Methods: Human-mouse chimeric molecules were constructed by PCR and clonedin pcDNA3.1 his6 for expression. The ectodomain sequence of pIgR was dividedinto 200 overlapping peptides and peptides were synthesized as array of spots on anaminopegylated cellulose membrane. Amino acid substitutions were introduced inidentified binding sites and binding to SpsA was assayed in an ELISA. The impactof putative binding motifs for pneumococcal adherence and invasion was tested inblocking experiments using chimeric molecules and synthetic peptides. Internali-zation of pneumococci or SpsA coated latex beads in pIgR expressing cells or cellsexpressing mutated pIgR was analysed by confocal laser scanning microscopy andelectron microscopy. The transwell system was used to analyse changes of the hostcell morphology upon infection.Results: Binding sites in the third and fourth Ig-like domain of pIgR mediate bind-ing of SpsA. Amino acid substitutions identified critical amino acids of the bindingmotifs which are unique to the human pIgR. Electron microscopy revealed thechanges of the host cell morphology during pneumococcal transmigration medi-ated by the SpsA-pIgR interaction.Conclusions: The SpsA-pIgR interaction is species specific because critical aminoacids for this interaction are unique to the human pIgR. This interaction provides anefficient mechanism for pneumococcal transmigration.

The Effects of Pneumococcal Hyaluronidase on Human Ciliated Epithelium invitro.

Charles Feldman1, Ronald Anderson2, Timothy Mitchell3. Division of Pulmonology1,Department of Medicine, University of the Witwatersrand, Johannesburg, SouthAfrica, MRC Unit for Inflammation and Immunity2, University of Pretoria, Preto-ria, South Africa, Division of Infection and Immunity3, University of Glasgow, Glas-gow, Scotland.

Aim: The aim of this study was to investigate the effects of recombinant hyaluroni-dase (HYL), one of the protein toxins produced by the pneumococcus, on humanciliated epithelium in vitro. These investigations were conducted in the absence andpresence of pneumolysin (PL), another protein virulence factor of the pneumococ-cus that is known to effect ciliated epithelium.Methods: Effects of HYL on human ciliated epithelium were studied using nasalbrushings obtained from the inferior nasal turbinate of healthy volunteers. Ciliarybeat frequency (CBF) was measured using a phototransistor technique and damageto the structural integrity of the epithelium (ED) was measured using a visual scor-ing index.Results: In concentrations between 0.1�g/ml and 10�g/ml HYL alone had no ef-fects on CBF and did not cause ED. On its own PL at concentrations of 50�g/mland 100�g/ml effected both ciliary function and caused ED (CBF of control epithe-lial strips was 11.6 Hz, no ED; CBF of strips incubated in PL 50�g/ml was 9.7 Hz,22% ED; CBF of epithelial strips incubated in PL 100�g/ml was 8.6 Hz, 34% ED).These effects of pneumolysin on the epithelium were significantly enhanced bypre-incubation of the epithelium with HYL (final concentration 10�g/ml) for 30minutes at 37oC (CBF of control epithelial strips was 11.6 Hz, no ED; CBF ofepithelial strips pre-incubated with HYL followed by PL 50�g/ml was 8.3 Hz, 34%ED; CBF of epithelial strips pre-incubated with HYL followed by PL 100�g/mlwas 7.7 Hz, 40% ED).Conclusions: Although hyaluronidase does not have any direct toxic effects on hu-man ciliated epithelium in vitro, it does enhance the injurious effects mediated bypneumolysin.


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Conserved, non-essential genes that may be associated with virulence

Fernebro J1, 2, Wolf-Watz3, Normark S2, and Henriques Normark B1, 2. 1Dep of Bac-teriology, Smittskyddsinstitutet, Solna, Sweden, 2Microbiology and Tumor biologyCenter, Solna, Sweden, 3Department of Molecular Biology, Umeå University, Umeå,Sweden.

Aims: Several conserved genes have been identified in Yersinia pestis that havehomologues in a number of other human-specific pathogens, e.g. Streptococcuspneumoniae, Neisseria gonorrhoeae and Helicobacter pylori. When mice were in-fected with Yersinia pseudotuberculosis in-frame deletion mutants of these genes,five genes were found to affect virulence. The functions of these virulence-associ-ated genes (vags) are unknown. The aim of this project is to find out if these genesare also non-essential in Streptococcus pneumoniae and if they are associated withvirulence in this pathogen.Methods: The five vags were knocked-out in the sequenced TIGR4 strain usingPCR ligation mutagenesis. The mutants are being tested for virulence in a mousemodel. An intranasal route of infection is used.Results: All of the vag genes were successfully knocked-out. None of these dele-tions seem to have any effect on the in vitro growth rate. Results on how the dele-tions of pneumococcal vag genes affect virulence will be presented.Conclusions: Even though the five vag genes are conserved among a large numberof pathogens, they are non-essential in Streptococcus pneumoniae. Virulence stud-ies will tell whether or not virulence-associated genes in a Gram-negative organismlike Yersinia pseudotuberculosis are also required for virulence of a Gram-positiveorganism like Streptococcus pneumoniae, with a completely different mode of caus-ing infectious disease.

Docosahexaenoic Acid Antagonizes Pneumolysin-Mediated, Calcium-Depend-ent Activation of NFkB and Synthesis of Interleukin-8 in Human Neutrophils

Heidi Fickl,1 Riana Cockeran,1 Helen C. Steel,1 Charles Feldman,2 Timothy J.Mitchell,3 Ronald Anderson1. 1Medical Research Council Unit for Inflammationand Immunity, Department of Immunology, Faculty of Health Sciences, Universityof Pretoria, Pretoria, South Africa, 2Division of Pulmonology, Department of Medi-cine, University of the Witwatersrand, Johannesburg, South Africa; 3Division ofInfection and Immunity, University of Glasgow, Glasgow, Scotland.

Aim: The objective of this study was to investigate the relationship betweenpneumolysin (8.37 and 41.75 ng/mL)-mediated influx of Ca2+ into neutrophils, ac-tivation of NFkB and synthesis of interleukin-8 (IL-8), as well as possible antago-nism of the toxin by docosahexaenoic acid (DHA, 5 and 10 �g/mL).Methods: Activation and translocation of NFkB were assayed using a radiometricelectrophoretic mobility shift assay, while IL-8 was measured by an ELISA proce-dure.Results: Exposure of neutrophils to pneumolysin was accompanied by activation ofNFkB and synthesis of IL-8, both of which were attenuated by depletion of Ca2+

from the cell-suspending medium, or by pretreatment of the cells with DHA.Conclusions: These observations demonstrate that pneumolysin-mediated activa-tion of NFkB and synthesis of IL-8 are secondary to influx of Ca2+ into neutrophils,and that these potentially harmful proinflammatory activities of the toxin are an-tagonized by DHA.


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The rlrA pathogenicity islet - function and distribution in Streptococcuspneumoniae

Hemsley C1, Hava D L2, Brueggemann A3, Crook D3, Camilli A2, Holden D H1.Department of Infectious Diseases, Centre for Molecular Microbiology and Infec-tion, Imperial College School of Medicine, London, England1, Department of Mo-lecular Biology and Microbiology, Tufts University School of Medicine, Boston,USA2, Department of Microbiology, University of Oxford, Oxford, England.

Aims: The rlrA pathogenicity islet contains virulence genes for serotype 4 Strepto-coccus pneumoniae but is absent from two of the other sequenced pneumococcalgenomes. S. pneumoniae is a naturally transformable organism and recombinationevents are thought to occur in vivo. This and the fact that the islet is flanked byIS1167 insertion sequences allowing integration of the element into the genome tohave occurred by homologous recombination or a transposition event supports theidea that it was/is a mobile element. The aim of this work was to investigate thefunction rlrA pathogenicity islet in different Streptococcus pneumoniae strains andits distribution in clinical isolates to assess if there was a correlation between isletpresence and serotype and/or disease state.Methods: Mutants with deletions in islet genes were assessed for virulence and invitro binding to lung epithelial cells (A549 cell line). The islet was transferred intoan islet naïve strain, D39, and the resultant strain was compared to wildtype forvirulence in murine models and in vitro binding. The distribution of the islet inclinical strains of different serotypes and from carriage and invasive disease wasstudied by dot/blot and pcr.Results: The islet encodes for factors required for binding to A549 cells in vitro.Addition of the islet to D39 led to both increased binding in vitro and increasedvirulence as assessed in murine models of disease. The islet was not evenly distrib-uted amongst pneumococcal serotypes in clinical strains.Conclusions: The rlrA islet encodes for putative adhesins and contributes to viru-lence in murine models. Its uneven distribution amongst clinical strains supportsthe notion that it was /is a mobile element.

The virulence activity of mutant Streptococcus pneumoniae deficient in eitherpneumolysin or autolysin is reduced in an adult rat model of meningitis.

Hirst RA, Patel B, O’Callaghan C and Andrew PW. Department of Infection, Im-munity and Inflammation, University of Leicester, Leicester, LE2 7LX, UK.

Aims: To compare the effect of pneumolysin (�ply) and autolysin (Lyt-A-) negativemutant S. pneumoniae with wild type (D39) S. pneumoniae on the pathogenesis ofpneumococcal meningitis.Methods: Male wistar rats were administered 104colony forming units (cfu) S.pneumoniae: D39 wild type, �ply and Lyt-A- into their cerebrospinal fluid (CSF)via a cisternal catheter. The controls were cisternally injected with sterile phosphatebuffered saline (PBS). The infected rats were assessed for clinical symptoms up tothe lethargic stage of the disease. If no disease was observed 72 hours after infec-tion the rats were euthanased. CSF was collected via the catheter in order to deter-mine the numbers of leucocytes and bacteria. The brains were removed and vibrotomesections (250�m) taken through the 4th and lateral ventricle in order to measure thefunctional integrity of the ependymal cilia by high-speed video analysis of the cili-ary beat frequency (CBF).Results: The average time to the lethargic stage of meningitis in rats infected withD39 pneumococci was 26 hours, this was in contrast to the rats infected with �plyand Lyt-A-, which either had lethargic end points later than 26 hours or did notdevelop meningitis symptoms. All D39 infected rats had bacteria in the CSF, whereas40% of the rats infected with �ply or Lyt-A- had CSF which was clear of bacteria.Bacterial and leucocyte numbers in the CSF were higher in the D39 infected ratswhen compared with �ply and Lyt-A- infected rats. Ependymal CBF was signifi-cantly inhibited in the D39 infected rats around the floor of both ventricles, how-ever, there was little or no effect on the CBF in the rats injected with PBS or in-fected with �ply or Lyt-A- at 26 hours.Conclusions: We have shown that without either pneumolysin or autolysin, S.pneumoniae is less virulent in causing meningitis when compared to the D39 wildtype strain. The CBF measurements correlate well with the disease symptoms andare a good indicator of the severity of meningitis.


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The control of virulence by the two-component system CiaR/H is mediated viaHtrA, a major virulence factor of Streptococcus pneumoniae

IBRAHIM Y.M., KERR A.R., McCluskey J. & MITCHELL T.J. Division of Infec-tion and Immunity, Institute of Biomedical and Life Sciences, University of Glas-gow, Glasgow G12 8QQ, UK.

Aims: The purpose of this study is to investigate the contribution of the High Tem-perature Requirement A (HtrA) heat-shock protein to the CiaR/H phenotype inmediating the pathogenesis of pneumococcal infection.Methods: We constructed ciaR and htrA mutations in strain D39 (type2) and usedthe provided ciaR mutant in strain 0100933 (type3). HtrA was expressed in pAL2plasmid under the regulation of the pneumococcal ami promoter. We compared thegrowth at different temperatures and the sensitivity to hydrogen peroxide of D39mutants to those of the wild type. Western blotting was used to measure the levelsof HtrA expression in the D39 wild type and mutants. We also used our murinemodels of infection to study the role of CiaR and HtrA in the virulence of thepneumococcus and the effect of complementation with HtrA.Results: The phenotypes of D39ciaR and D39htrA mutants were very similar. Thegrowth of both was slower than that of D39 wild type at 40°C, both showed reducedrate of autolysis after stationery phase of growth, and both were more sensitive toperoxide compared to the wild type D39. The two mutants have reduced virulencein mouse models. The ciaR mutant of strain 0100933 was also much reduced in itsoverall virulence. The mutant organism grew very poorly in the lung and only in-vaded the blood stream to very low levels. Competition experiments showed thatthe mutant was much less able to colonise the lungs and also less able to growsystemically. Western blotting indicated that D39ciaR expressed less HtrA than wildtype D39. When HtrA expression was restored to wild-type levels in D39ciaR thestrain was again fully virulent in mice. The growth phenotype and sensitivity toperoxide of D39ciaR were also reverted by expression of HtrA.Conclusion: Our results confirm that the expression of HtrA is regulated by theCiaR/H two-component system and reduced HtrA expression can explain the re-duced virulence of CiaR/H mutants.

The Ami-AliA/B permease of Streptococcus pneumoniae is involved in nasopha-ryngeal colonisation but not in invasive disease

A.R. Kerr1, P.V. Adrian2, S. Estevão2, R. de Groot2, G. Alloing3, J.-P. Claverys3, T.J.Mitchell1, and P.W.M. Hermans2

1Division of Infection and Immunity, University of Glasgow, UK; 2Department ofPediatrics, Erasmus MC-Sophia, Rotterdam, The Netherlands; 3Laboratory of Micro-biology and Molecular Genetics, CNRS-Paul Sabatier University, Toulouse, France

Aims: The Ami-AliA/B oligopeptide permease is an ABC transporter found in Strep-tococcus pneumoniae, which is involved in nutrient uptake. We have investigatedthe role of Ami-AliA/B using experimental animal models.Methods: A series of mutants in aliA, aliB, and amiA either alone or in combinationas double or triple mutations were utilised in murine models of pneumococcal colo-nisation and invasive disease.Results: Inoculation of the nasopharynx with a mixture of the triple mutant (Tr-)and wild type bacteria (D39) resulted in significantly lower numbers of Tr- colonis-ing the nasopharynx. Utilisation of a mixture of the individual mutants and wildtype pneumococci revealed that amiA, aliA and aliB were all required for success-ful colonisation of the nasopharynx. Animals with invasive disease caused by thesemutants had similar survival times, bacterial loads and inflammatory cytokine pro-duction as those infected with wild type pneumococci.Conclusion: Our results show that although the Ami-AliA/B complex is not re-quired for virulence during pneumococcal pneumonia it does play a role in coloni-sation of the nasopharynx.


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Evidence For Pneumococcal Biofilm in Children with Otitis Media with Effu-sion.

Langlands J1, Richmond P1, Fillion P2, Keil T1, Vijayasekaran S1 Thornton R1, CoatesH1, Princess Margaret Hospital for Children1, Pathcentre, Sir Charles Gairdner Hos-pital2, Perth, Western Australia.

Background:. In otitis media with effusion (OME) at least 40-60% of effusions aresterile, however metabolically active bacteria can be detected using polymerase chainreaction.(PCR). Oral antibiotics have poor efficacy in the treatment of OME. Theseobservations may be explained by the presence of biofilm, which is a strategy useduniversally by bacteria for survival and has been demonstrated in chronic human in-fections such as cystic fibrosis and endocarditis. Biofilm renders bacteria resistant toantibiotics and immune attack and has been demonstrated in animal models of OM.Aims: The purpose of this study was to investigate the presence of biofilm in chil-dren with OME using electron microscopy (EM), bacterial culture and bacterialPCR.Methods: 2mm Biopsies of middle ear mucosa from children with OME undergo-ing insertion of ventilation tubes were examined using EM for evidence of bacterialbiofilm. Middle-ear effusion fluid (MEF) was sent for standard culture andmicroscopy (M/C/S) and PCR for pneumococcal pneumolysin.Results: To date, we have collected 16 mucosal samples and 12 samples of MEFfrom children with OME. MEF culture grew Haemophilus influenzae in 3 of the 12samples, but pneumococcus and other middle ear pathogens have not been isolated.Of the remaining 9 samples, 3 (33%) were positive for pneumolysin PCR; and 2 ofthese had cocci bacteria identified in epithelial cells or epithelial stroma on EM. Wehave been unable to demonstrate the presence of pneumococcal biofilm in thesesamples. This may relate to the small amount of mucosa biospied or processingtechniques, which are under review.Conclusions: This provides further indirect evidence of a role for pneumococcalinfection in the pathogenesis of OME. Whether this is due to the presence of pneu-mococcal biofilm or due to sequestration of pneumococci in subepithelial cellsremains to be elucidated.

Solution structure of Choline binding protein A, a key adhesin of Streptococcuspneumoniae

Mann E, Luo R, Heath R, Kriwacki R, Tuomanen E. Department of InfectiousDiseases St. Jude Children’s Research Hospital, Memphis, Tennessee.

The surface of Streptococcus pneumoniae is decorated with 13 known choline bind-ing proteins (Cbp’s) noncovalently bound to phosphorylcholine residues on the cellwall. Choline binding protein A (CbpA), the most abundant of these proteins, is aknown adhesin that binds to human receptors such as polymeric Ig receptor trigger-ing transcytosis. The N-terminus of CbpA contains two 77% identical, �-helicaldomains termed R1 and R2. Using extensive NMR studies, we solved the structureof a coiled-coil region of CbpA (R2) and determined that the R1 and R2 domainsfunction independently in adherence. Multiple repeats of the leucine zipper motif inR2 create an anti-parallel three-helix bundle with a highly charged solvent exposedsurface. The RNYPT motif in CbpA binds secretory component (Hammerschmidt,2000) and is present in both the R1 and R2 domains of the Tigr4 CbpA. The motifis exposed at the end of a helical loop. Mutation of this SC binding site signifi-cantly decreased adhesion and invasion of nasopharyngeal epithelial cells by 45%.Although SC bound both R1 and R2 domains, R2 was the dominant binding site inthe Tigr4 strain. We determined that a helical structure at the N-terminus, DomainA, although not a binding site, is essential for proper folding and dimerization ofthe protein. Disrupting the helical structure of Domain A by site-directed mutagen-esis decreased the adherence of the bacteria to Detroit cells by 70%. Combiningstructural and biological data, we conclude CbpA dimerizes at both the cholinebinding domain and the A Domain, forming a potential interlocking “network” ofthe CbpA on the surface of the pneumococcus.


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Contribution of Neuraminidase in Colonisation by Streptococcus pneumoniae

Manco S1, Kadioglu A1, Paton JC2, Andrew PW1.. Dpt. Infection, Immunity & In-flammation, University Leicester1,U.K. School Molecular & Biomedical Science,University Adelaide2, Australia

Aims: Streptococcus pneumoniae wild-type and its isogenic neuraminidase-nega-tive mutants NanA- and �NanB were used to determine the role of theneuraminidases NanA and NanB in colonisation of the upper and lower respiratorytract.Methods: Bacterial strains: Wild-type Streptococcus pneumoniae serotype 2 strainD39 (WT) was used; also its isogenic neuraminidase-deficient mutants NanA- (con-structed by insertion mutation) and �NanB (constructed by deletion mutation).Model of colonisation: Female MF1 outbred mice that were 6-9 weeks old andweighing 30-35g were lightly anaesthetised with 2.5% (v/v) fluothane over oxygen(1.5 l/min) and 50�l of PBS containing 1x106 CFU of S. pneumoniae was admin-istered into the nostrils. The inoculum was confirmed by plating on blood agarplates following infection. At pre-selected times post-infection blood was collectedby cardiac puncture and the growth of pneumococci in homogenised nasopharynx,trachea and lungs and in blood was determined by viable counting.Results: Mice challenged with WT were moribund by 24h post-infection. In con-trast, none of the mice infected with NanA- or �NanB showed signs of illnessthrough the 2 and 4 days of the experiment, respectively. Wild type pneumococciand �NanB colonised the respiratory tract to a similar extent during the early hoursof infection while NanA- was unable to do so. Neither NanA- nor �NanB was de-tected in the blood at any time, in contrast to the WT.Conclusions: The lack of neuraminidase alters the capability of S. pneumoniae tocolonise the upper and lower respiratory tract and to cause bacteraemia. Neurami-nidase may be required for the development of septicaemia.

Levels of nitric oxide regulate microbial killing and mechanisms of macrophagecell death during pneumococcal infection.

Marriott HM1, Read RC1, Mitchell TJ2, Whyte MKB1, Dockrell DH1. Division ofGenomic Medicine, Sheffield University1 and Division of Infection and Immunity,University of Glasgow2, United Kingdom.

Aims: Nitric oxide (NO) contributes to macrophage host defense and is also an im-portant regulator of the inflammatory response. We have previously demonstratedthat killing of pneumococci is associated with macrophage apoptosis and have in-vestigated the role of NO.Methods: Human monocyte derived macrophages (MDM) were mock infected orinfected with type 1 (Spn), type 2 (PLY+) or pneumolysin deficient pneumococci(PLY-). NO production was measured by DAF-FM, by detection of extracellularnitrite and by detection of nitrotyrosine. Western blots detected levels of inducibleNO synthase (iNOS) and the anti-apoptotic protein Mcl-1. Apoptosis was deter-mined by DAPI or TUNEL staining, mitochondrial membrane permeabilization(MMP) by JC-1 staining and cytochrome c translocation, caspase activation by FITC-VAD and necrosis by PI staining. iNOS was inhibited by 1400W.Results: Spn resulted in increased NO production which was adjacent tophagolysosomes and intracellular pneumococci. Intracellular killing of Spn wasdecreased by iNOS inhibition (1.4x105 (7.8x104-1.7x105) cfu vs. 8.8x104 (4.0x104-1.4x105) cfu, p<0.05 at 6h after infection. Enhanced MDM apoptosis induction bySpn was decreased in the presence of 1400W 7 (5-10)% vs. 19 (18-20)%, p<0.05.Spn infection induced MMP and decreased Mcl-1 in MDM and these were reversedby 1400W. iNOS inhibition inhibited Spn-associated caspase activation and switchedcell death from apoptosis to necrosis. Pneumolysin contributed to NO productionand apoptosis induction: PLY+ 21 (14-28)% vs. PLY- 8 (5-9)% apoptosis, p<0.05.Conclusions: These results suggest NO contributes to killing of internalised pneu-mococci and influences the program of cell death by modulation of Mcl-1 expres-sion and induction of MMP. NO may explain links between microbial killing andhost-mediated macrophage apoptosis.


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Susceptibility to Streptococcus pneumoniae infection after influenza infectionis not increased in the upper respiratory tract

McNamee L, King Q, Harmsen A. Veterinary Molecular Biology Department,Montana State University, Bozeman, MT, United States of America.

Aims: The purpose of this study was to determine if susceptibility to Streptococcuspneumoniae (SP) infection after influenza infection is increased in the upper respi-ratory tract (URT). It is well documented that susceptibility is increased in the lowerrespiratory tract (LRT).Methods: To test our hypothesis that susceptibility to SP infection after influenza isincreased in the URT, C57BL/6 mice were infected intranasally with 1 x 107 colonyforming units (CFUs) SP type 4 for 2, 12 or 24 hours at varying timepoints (0-9days) after a primary PR8 influenza infection (300 PFU/nare). Control mice wereeither uninfected, influenza-infected only, or SP-infected only. To measure suscep-tibility independent of influenza-induced changes in neutrophil function, we usedmice depleted of neutrophils with RB6-8C5 antibody and CXCR2 knockout micethat were infected intranasally with influenza (300 PFU/nare) for 6 days followedby infection with 107 CFUs SP type 4 for 12 hours. Bronchial alveolar lavage fluidswere used for differentials to confirm neutrophil depletion. Control mice includedwild-type C57BL/6 mice and mice that were not influenza-infected. SP CFUs inthe nasal mucosa (URT) and lungs (LRT) were enumerated for all mice.Results: In the LRT, as expected, prior influenza infection significantly increasedsusceptibility to SP infection compared to mice that were infected with SP only,with the peak of susceptibility at 6 days after influenza infection. Surprisingly, inthe URT, we found that prior influenza infection did not increase susceptibility toSP infection, and this effect was not dependent on neutrophil function.Conclusions: These results suggest that the immune response in the URT to co-infection with influenza virus and SP type 4 varies from that found in the LRT.

PspA Protects Pneumococci from Killing by Apolactoferrin and Antibody toPspA Enhances Killing of Pneumococci by Apolactoferrin.

Mirza S1, Hollingshead SK1, Benjamin WH Jr1,2, Briles DE1,1Department of Micro-biology and 2Department of Pathology, University of Alabama at Birmingham, Bir-mingham, AL, USA.

Background: Lactoferrin is an important component of innate immunity, workingthrough its sequestration of iron, its bactericidal activity, and its immune modula-tion. Apolactoferrin (ALF) is the iron-depleted form of lactoferrin and is bacteri-cidal against pneumococci and several other several species of bacteria.Results and Methods: We confirmed the killing of pneumococci by lactoferrin andobserved that pneumococci were also killed by lactoferricin (LFN), an 11-aminoacid fragment from the N-terminus of human lactoferrin. Strains of Streptococcuspneumoniae varied in their susceptibility to ALF. PspA, a pneumococcal proteinthat attaches to the bacterial surface by binding choline, is known to bind lactoferrinto the pneumococcal surface. Using PspA– mutants of 4 different strains of pneu-mococci we observed that PspA greatly reduces the ability of pneumococci to bekilled by ALF. Mutations in two other choline-binding proteins (PspC and PcpA)did not affect killing by ALF. The soluble recombinant N-terminal half of PspAcould prevent killing of pneumococci by ALF and LFN, thus demonstrating thatPspA did not have to be attached to the bacterial surface to inhibit killing. An 11amino acid fragment of PspA was also able to reduce the killing by LFN. Antibodyto PspA enhanced killing by lactoferrin. These findings suggested that binding ofALF to PspA probably blocks the active site(s) of ALF that are responsible forkilling.Conclusions: From these data it seems likely that a major virulence effect of PspA,especially on mucosal surfaces where concentrations of apolactoferrin are high, isto protect against killing by apolactoferrin. Moreover, the ability of antibody toPspA to protect against nasal colonization may be due in part to the ability of theseantibodies to enhance killing of pneumococci by apolactoferrin.


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Genes encoding Toll-like receptor 1 (TLR1) and TLR6 are induced by Streptococ-cus pneumoniae

Moore LJ, Pridmore AC, Dower SK and Read RC. Division of Genomic Medicine,University of Sheffield Medical School, United Kingdom

Aims: TLR2 forms natural heterodimers with TLR1 or TLR6 to increase the specificityof the host innate response to pathogens. TLR2 and TLR6 dimerisation enhances TLR2response to peptidoglycan (PGN) whereas TLR2 and TLR1 coexpression increaseslipoteichoic acid responsiveness. We have shown that low concentration penicillin en-hances proinflammatory responses to Streptococcus pneumoniae mediated via TLR2(Moore et al 2003, JID, 188:1040-8). We measured induction of TLR1 and TLR6 mRNAin primary human cells in response to PGN, and penicillin-treated and -untreatedS.pneumoniae. Methods: Peptidoglycan from Staphylococcus aureus at a final con-centration of 1�g/mL was prepared as a positive TLR2 control. D39 S.pneumoniae at5x106 were treated in MIC ratios of penicillin for 6h during lag growth or early loggrowth. HeLa cells were transfected with expression vectors for TLR2 and TLR1 orTLR6 and CD14 or empty vector control. TLR2-mediated signalling was measured byco-transfection with reporter vectors: pIL8-pluc, a human IL8 promoter linked to afirefly luciferase gene and ptkrLUC, a low level constitutive control tagged to a renillaluciferase gene. IL8 promoter activity was determined by normalisation of values forfirefly with renilla luciferase and empty vector control. Messenger RNA for TLR1 andTLR6 were determined by real-time PCR after culture of human monocyte-derivedmacrophages (MDMs) with peptidoglycan, penicillin treated and -untreatedS.pneumoniae or medium only control. Results: Over-expression of TLR6 in transfectedHeLa cells enhanced the TLR2 response to peptidoglycan and S.pneumoniae. TLR1transfection of HeLa cells did not affect TLR2 activation in response to PGN, but didenhance TLR2 response to S.pneumoniae. In MDMs, TLR6 and TLR1 mRNA wasinduced by live bacteria and/or PGN when compared to medium only controls (P 0.024,n=6). Treatment of lag phase S.pneumoniae with sub-MIC (but not MIC) penicillinenhanced TLR6 and TLR1 mRNA induction further when compared to untreated bac-teria (TLR1; P 0.037, TLR6; P 0.021, n=6). Conclusions: Co-expression of TLR6,TLR1 and TLR2 refine the proinflammatory response to S.pneumoniae and its cellwall constituents. The pneumococcus induces gene expression of TLR1 and TLR6,particularly during release of cell wall fragments by low penicillin concentrations.

Infection of A549 cells with respiratory syncytial virus increases the adherenceof Streptococcus pneumoniae

Mühlemann K, Hathaway L, Mumprecht V, Aebi S. Institute for Infectious Dis-eases, University of Bern, Switzerland.

Aims: Epidemiological data suggest an interaction between respiratory syncytialvirus (RSV) andS. pneumoniae in the human respiratory tract (Mühlemann K et al., submitted). Weevaluated, whether RSV infection affected pneumococcal adherence to human lungepithelial cells.Methods: Confluent monolayers of A549 cells were infected with the RSV longstrain (A2) for 48h and then exposed to 5x10E6 colony forming units of S.pneumoniae laboratory strain D39 or its non-encapsulated mutant R6. Bacterialadherence was measured by plating serial dilutions of trypsinized A549 cells forcolony counts on blood agar plates.Results: Pneumococcal adherence rates to RSV non-infected A549 cells were 0.07%(±0.04%) for strain D39 and 9.26% (±2.78%) for strain R6. RSV infection of A549cells increased adherence rates by 176% for D39 and 251% for R6.Conclusions: RSV infection mediates enhanced adherence of encapsulated and non-encapsulated S. pneumoniae to human alveolar epithelial cells. The molecularmechanism(s) involved are being studied. This result complements epidemiologi-cal observations suggesting a direct interaction between RSV and S. pneumoniae.


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Pneumococcal zinc metalloproteinase ZmpC cleaves human matrixmetalloproteinase 9 and is a virulence factor in experimental pneumonia

Oggioni MR, Memmi G, Maggi T, Chiavolini D, Iannelli F, Pozzi G. LAMMB,Dipartimento di Biologia Molecolare, Università di Siena, Siena, Italy.

Aims: Identification of novel substrates for the pneumococcal zinc metalloproteinasesIga, ZmpC and ZmpB and analyse their contribution to virulenceMethods: Bioinformatic tools were used for the identification of novel zincmetalloproteinases substrates, and biochemical assays were used to analyse cleav-age of human matrix metalloproteinase 9 (MMP-9). Pneumococcal mutants wereconstructed and assayed in animal models of infection.Results: As previously described, the protease activity of Iga was confirmed to bespecifically linked to the zinc metalloproteinase IgA1-protease (annotated asSP1154). The zinc metalloproteinase ZmpC of Streptococcus pneumoniae, anno-tated in the type 4 genome strain as SP0071, was proven to cleave human MMP-9.MMP-9 is a protease cleaving extracellular matrix gelatin and collagen and it is, asmost proteases, activated by proteolytic cleavage. This novel substrate was found bydatabase searches using the consensus pattern of the cleavage site of the other strep-tococcal zinc metalloproteinase characterised. Combined analysis for zmpC pres-ence and MMP-9 cleavage activity in several pneumococcal strains confirmed cor-relation of ZmpC presence to MMP-9 cleavage activity. In a murine pneumoniamodel, infection with a ZmpC-deficient mutant strongly reduced mortality by 75%compared to wild type strains. The contribution of the other two zincmetalloproteinases IgA and ZmpB was also assayed in the same animal model.Conclusions: Our data suggest that all three pneumococcal zinc metalloproteinasesare involved in pneumococcal virulence, possibly at different stages of the infec-tious process.

Microarray Analysis of Streptococcus pneumoniae Gene Expression DuringInvasive Disease

Carlos J. Orihuela1, Jana Radin2, Geli Gao1, Jack Sublett1, Deepak Kashal3 and ElaineI. Tuomanen1. 1Department of Infectious Diseases, St. Jude Children’s ResearchHospital, Memphis, Tennessee. 2Department of Molecular Sciences, University ofTennessee Health Science Center, Memphis, Tennessee. 3Hartwell Center forBioinformatics and Biotechnology, St. Jude Children’s Research Hospital.

Streptococcus pneumoniae is a leading cause of community acquired pneumonia,bacteremia and meningitis. Nonetheless, scarce information is available in regardsto pneumococcal gene expression during infection. In this study, we examined theexpression of S. pneumoniae genes in vivo using whole genome microarrays avail-able from the Pathogen Functional Genomics Research Center at The Institute forGenomic Research (TIGR). Total RNA was collected from pneumococci isolatedfrom three models of disease including a nasopharyngeal model, blood from septicmice, and cerebrospinal fluid. Pneumococcal RNA was enriched and compared toRNA isolated from in vitro control cultures. Experiments were done in triplicateand accuracy and statistical significance of gene-expression calculated using anANOVA logarithm. Microarray analysis of pneumococcal genes expressed in thesemodels identified global patterns of expression specific to each disease model. Dis-tinct patterns of expression were identified in regards to virulence factors, trans-porters, transcription factors, translation-associated proteins, metabolism, and geneswith unknown function. In regards to the latter, candidate genes with unknown func-tion were assessed by insertion duplication mutagenesis of the genes and challengeof mice with the mutants. We conclude that gene expression in the pneumococcus issite-specific during invasive disease. Moreover, that certain regulons not classicallyassociated with virulence are altered in response to infection. Finally, we concludethat a true understanding of pneumococcal virulence remains distant as we identifyseveral genes with unknown function that contribute substantially to bacterialpathogenesis.


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Oxygen pressure plays a crucial role in membrane composition of Streptococ-cus pneumoniae.

Porat N, Sikron N, Pesakhov S, Dagan R, Kozin-Goldberg I, Cohen Z. Ben-GurionUniv., Beer Sheva, Israel.

Background: Survival of S. pneumoniae in the nasopharynx versus the bloodstreamis associated with different phenotypes. Changes in ambient oxygen concentrationswere shown to affect the regulation of capsular polysaccharide synthesis and H

2O

2

production differently in the 2 phase variants. Peroxides are highly toxic due to theirability to react with membrane lipids.Objective: To show the effect of ambient oxygen pressure and inherent peroxideproduction on membrane composition of S. pneumoniae phase variants.Methods: Using gas chromatography analysis we compared the fatty acid composi-tion generated by S. pneumoniae phase variants under aerobic versus anaerobicconditions, in relation to the amounts of peroxides generated by the organism.Results: High levels of peroxides production by the organism correlated with de-creased proportion of unsaturated fatty acids. The desaturation index (DI - propor-tion of fatty acids, weighted according to their number of double bonds) of a trans-parent, type 2 strain (p210), dropped from 0.44 when grown anaerobically to 0.35under atmospheric pressure, corresponding to a 3-fold increase in H

2O

2 production.

In a p210 SpxB truncated mutant (p878), which produced 100 times less H2O

2, the

DI was extremely high reaching levels of 0.80 and 0.75 at anaerobic and aerobicconditions respectively. In contrast, the DI of a p210 opaque variant under aerobicconditions was low compared to its transparent counterpart, 0.18 versus 0.35 re-spectively, while H

2O

2 production was 40% less. The changes were attributed mainly

to different levels of cis-vaccenic acid (C18:1, w7), indicating de-novo synthesisrather than post-transcriptional modifications.Conclusions: It seems that the opaque phenotype is capable of resisting the delete-rious activity of peroxides and other reactive oxygen species by stringently regulat-ing the biosynthesis of membrane phospholipids, and the activity of membraneanchored enzymes like SpxB, whose activity depends on the membrane phospholi-pid content.

Proteomics of Streptococcus pneumoniae (Pnc) surface proteins display differ-ential expression of proteins in virulent and non virulent strains.

Portnoi M, Ling E, Dagan R, Mizrachi-Nebenzahl Y. Ben Gurion University, BeerSheva, Israel

Aims: The role of surface proteins in Pnc virulence is not clear. We studied patternsof cell wall proteins expression of Pnc strains that induce non-lethal carriage, non-lethal pneumonia and lethal diseases in the mouse model system.Methods: Mouse models of Pnc sepsis, non-lethal pneumonia and carriagewere established. Cell wall proteins were extracted by mutanolysin and sepa-rated by 2-D PAGE from: 1) Highly virulent S. pneumoniae strains, whichinduce lethal disease (serotype 3 strain WU2, serotype 14 [strain14R], andserotype 9V [strain 9VR]). 2) Less virulent strains, which induces non-lethalpneumonia (serotype 14 [stains 14DW].3) Serotype 6B [strain 6BR] which cause asymptomatic nasopharyngeal carriage.Individual protein spots were excised from the gel and sequenced by MALDI-TOF.Results: Three groups of proteins were discovered: 1) proteins, common for allserotypes tested – e.g. glyceraldehydes phosphate dehydrogenase and fructose-biphosphate aldolase, DnaK and GroEL; 2) proteins, expressed only by the highlyvirulent strains – e.g. D-alanine—D-alanine ligase, adenylosuccinate synthetaseand GMP synthase; and 3) proteins, expressed by all disease causing strains – e.g.pyruvate kinase, tyrosyl-tRNA synthetase and glutamyl-tRNA synthase.Conclusions:1) Differences in cell wall proteins expression patterns betweenvirulent and non-virulent strains were discovered. 2) Proteins, specific forhighly virulent strains WU2, 14R and 9VR probably contribute to their in-creased virulence. 3) The role of individual protein in pathogenesis and pro-tection is being studied.


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Invasion of Streptococcus pneumoniae involves Platelet-activating FactorReceptor and �-Arrestin 1

Jana N. Radin12, Carlos J. Orihuela2, Elaine I. Tuomanen2. 1Department of Molecu-lar Sciences, University of Tennessee Health Science Center, Memphis, Tennessee.2Department of Infectious Diseases, St. Jude Children’s Research Hospital, Mem-phis, Tennessee.

Streptococcus pneumoniae is the major cause of community acquired pneumoniaand meningitis. In this study, we examined the eukaryotic factors involved in pneu-mococcal invasion of eukaryotic cells. In vivo experiments with transgenic micelacking platelet-activating factor receptor (PAFr) determined that PAFr is requiredfor bacterial entry into the CSF. Invasion assays with COS cells transfected withdifferent combinations of WT and site-specific mutants of PAFr, and �-arrestin 1further supported a role for PAFr during invasion by showing that PAFr and �-arrestin 1 are simultaneously required for pneumococcal invasion. Confocalmicroscopy of rBCEC6 cells, an endothelial cell line, infected with S. pneumoniaerevealed that PAFr co-localizes with the pneumococci on the surface of the cell.Pre-treatment of cells with chlorpromazine, which prevents formation of vesiclesby inhibiting clathrin assembly, also decreased invasion 3-fold. Western blot analy-sis examining phosphorylation of ERK kinases during invasion showed phosphor-ylation of erk-1 and erk-2. Phosphorylation occurred in presence and absence ofserum. Finally, invasion assays with PD98059, a MAP Kinase inhibitor, also showeda decrease in invasion. We conclude from this study that both the PAFr and �-arrestin 1 are required for pneumococcal invasion of eukaryotic cells and transcytosisof the bacteria from blood to CSF. Additionally, we conclude that pneumococcionce bound to PAFr move into clathrin-coated pits, and endocytosis requires Erksignaling.

An experimental model of pneumococcal meningitis in outbred mice

Ricci S 1, Chiavolini D 1, Tripodi S 2, Parigi R 1, Oggioni MR 1, Blasi E 3, CintorinoM 2, and Pozzi G 1.

Laboratory of Molecular Microbiology and Biotechnology and 2 Institute of Patho-logical Anatomy, University of Siena, Italy, 3 Department of Hygiene, Microbiol-ogy and Biostatistics, University of Modena and Reggio Emilia, Modena, Italy.

Aims: Animal models of pneumococcal meningitis (PM) are useful to study menin-gitis caused by Streptococcus pneumoniae. While the rabbit and the rat have largelybeen used to induce PM, mouse models are more recent. However, available murinemodels of PM are still few and they generally use inbred mice. The purpose of thisstudy was to develop a simple and reproducible method for the induction of PM inoutbred mice.Methods: PM was induced by inoculating bacteria directly into the brain bregma ofmice, and thus this route of infection will be referred to as intracerebral-bregma(i.c.b).Results: The model was tested by inoculating outbred MF1 mice with pneumococciof three different capsular serotypes (types 2, 3 and 4) and with a range of bacterialdoses. Histological analysis clearly showed the establishment of PM closely resem-bling the disease in humans. Survival patterns of animals infected with all threeserotypes were comparable. Following infection with the TIGR4 genome strain,bacterial counts were found to be consistent in brain, spleen and blood of all micetested, and inflammatory cytokines were detected in both serum and brain samplesof infected animals.Conclusions: The proposed method for inducing PM in outbred mice by using thei.c.b. route of infection is easy-to-perform, fast, cost-effective, and reproducibleirrespective of the pneumococcal serotype used. The present model will allow tostudy the pathogenesis of meningitis, the host immune response induced after in-fection, and the efficacy of novel drugs and vaccines. Studies on the role(s) of se-lected pneumococcal virulence factors in PM are in progress.


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S.pneumoniae endocarditis: comparison of pneumococcal adherence proper-ties

Ries J1, Kan B2, Ortqvist Å2, Henriques-Normark B1. Smittskyddsinstitutet1,Karolinska Hospital2, Sweden.

Aims: Eight S.pneumoniae strains found in endocarditis and pericarditis patientswere analysed in regard to their adherence abilities, and compared to eight controlstrains isolated from patients of the age and sex .Methods: Clinical information about the endocarditis patients was examined byÅke Örtqvist and colleagues at the Department of Infectious Diseases, Karolinskahospital. Cell surface hydrophobicity was measured by adherence of bacteria tohexadecane. Bacterial cultures in adherence studies were incubated for 4,8 or 24hours at 37°C (5% CO

2) in 96 well plates coated with collagen I or fibronectin and

the adherence assayed by staining with crystal violet. Adherence to human celllines A549 (lung epithelial), Detroit 562 (nasopharynx epithelial) and PAE (aortaendothelial) was performed in 96 well plates and cfu was determined on blood agarplates. For all adherence assays bacterial cultures were grown in minimal medium.Results: Of the eight isolates from patients with endocarditis or pericarditis fivehad rare serotypes and three patients carried S.pneumoniae serotype 23. In all ad-herence experiments no differences attributed to specific serotypes could be ob-served. In vitro assays of adherence on PVC, polystyrene, fibronectin or collagen Ias well as to human cell lines, showed no differences between clinical isolates caus-ing endocarditis and control strains. While encapsulated strains showed a weak ad-herence, all unencapsulated strains however exhibited a significant adherence in allassays.Conclusions: In all experiments no differences between clinical isolates found inendocarditis and control strains could be found. We would therefore suggest thatpneumococcal strains have no specific adherence capabilities in regard of causingendocarditis. Instead we found a strong correlation between the loss of capsuleexpression and adherence or a possible biofilm formation, as has been reportedbefore. This increased adherence capabilities of non-encapsulated strains could beexplained by higher hydrophobicity or easier accessibility of adherence factors.

Multilocus sequence typing of pneumococci isolated from meningitis in Poland

Sadowy E., Skoczynska A., Hryniewicz W. National Institute of Public Health,Warsaw, Poland.

Aims: The purpose of this study was to characterize the clonal structure of Strepto-coccus pneumoniae population involved in meningitis cases in Poland by multilocussequence typing (MLST).Methods: 156 pneumococcal isolates from the period 1997-2002 were studied us-ing the standard MLST protocol. Allele numbers and sequence types (STs) corre-sponding to particular allelic profiles were assigned using the MLST database(www.mlst.net). The BURST program was applied to estimate the relationshipsamong the isolates.Results: MLST analysis revealed the presence of 89 different allelic profiles amongthe isolates analyzed. Of these, 52 STs represented the STs that previously werereported to the MLST database, 23 allelic profiles consisted of novel combinationsof known alleles and in 14 profiles new allelic variants were observed. BURSTanalysis revealed the presence of 17 clonal groups that contained 91 isolates (58%)and 42 unrelated STs (singletons). While a very good congruence between the STand pneumococcal serotype was observed, clonal groups usually contained isolatesof more than one serotype.Conclusions: Pneumococci involved in meningitis in Poland show a high degree ofvariability and a complex population structure. Further studies are required to es-tablish whether a higher representation of particular clones is caused by their spe-cific virulent properties or high prevalence in the population of S. pneumoniae inPoland in general.


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Clonal properties affect colonization and invasive capacity of Streptococcuspneumoniae

Sandgren A1, Orihuela C2, Albiger B1, Normark, S3, Tuomanen E2, HenriquesNormark B1,3.

Swedish Institute for Infectious Disease Control, Stockholm, Sweden1, St JudeChildren’s Research Hospital, Memphis, USA2, Microbiology and Tumor BiologyCenter, Karolinska Institute, Sweden3.

Aims: To understand the role of clonal properties in the capacity to cause invasivepneumococcal disease using a murine model.Methods: In this study, we compared isolates from patients with invasive diseaseversus isolates from healthy carriers in one geographical area during one specifictime period, by using serotyping, PFGE and MLST. To screen some of these clonesfor virulence and capacity to colonize, we used an intranasal murine model usingthe Xenogen IVIS system.Results: We found a significant difference in the serotype distribution between theinvasive and carriers isolates, with several serotypes uniquely found causing inva-sive disease. We also found certain clones that were only present in invasive diseaseand not among carriers and clones with the opposite properties. The in vivo studiesgave several interesting findings; we have two different 19F clones with one havinghypercolonizing properties and causing bacteremia in 25% of the mice at 48h postchallenge. The other clone was instead cleared from the nose of the mice and thusnot capable of causing bacteremia. Further, we saw that while C57Bl/6 mice aremore susceptible to get pneumonia and sepsis, Balb/c developed more meningitisthan C57Bl/6 (19% of the Balb/c compared to 2% of the C57Bl/6).Conclusions: Clonal properties in addition to the capsular serotype may be an im-portant factor in the ability of pneumococci to cause invasive disease. There aredifferences in capacity to colonize and give bacteremia between clones of the sameserotype. We also found differences in susceptibility to pneumococcal infectionbetween different hosts.

Role of the two component signal transduction system 06 in Streptococcuspneumoniae

SILVA N.A., McCLUSKEY J., TUNNICLIFFE G. & MITCHELL T.J. Division ofInfection and Immunity, Institute of Biomedical and Life Sciences, University ofGlasgow, Glasgow G12 8QQ, Uk.

Aims: The aim of this study is to evaluate the role two component system 06 (TCS06)in the virulence of S. pneumoniae and to identify genes regulated by this system.Methods: We have used virulence assays together with microarray analysis to in-vestigate the importance of TCS06. We constructed rr06 mutation in strain TIGR4by insertion of erythromycin cassette. We compared the growth at different tem-peratures of TIGR4 mutant and the wild type. Murine models were used to studythe effect of mutation in virulence of pneumococcus. Microarray comparison of thetranscriptional profiles of the wild type with the rr06 mutant allowed us to deter-mine the transcriptional changes caused by the mutation of RR06.Results: The in vitro phenotype of TIGR4 wild type and the rr06 mutant are differ-ent. In contrast to the wild-type, the mutant cannot grow at 40ºC and shows a slightdecrease in the growth rate at 37ºC. The mutation decreases the ability of bacteria togrow in the lungs of mice but has no effect on in animal model of bacteraemia.Microarray analysis show differences in expression levels of some genes betweenthe TIGR4 wild type and the mutant, about 1% of the genes are up-regulated and2.5% is down-regulated in the rr06 mutant.Conclusions: TCS06 is important for growth of pneumococcus at high temperatureand also in mouse lung. Microarray analysis identifies candidate genes importantin these situations.


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Dual phases of neuronal apoptosis in pneumococcal meningitis

Smith, S. Hope1, L. Mitchell1, J. Weber2, E. Tuomanen1

1Dept of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Ten-nessee, USA, 2Dept of Neurology, Charite – Universitaetsmedizin Berlin, Berlin,Germany

Recent studies have shown that neuronal damage during pneumococcal meningitisis the result of apoptosis, mainly in the dentate gyrus of the hippocampus. Thecurrent hypothesis is that the host mediates a caspase dependent pathway and thebacteria mediate a caspase independent pathway. The caspase independent path-way is caused by pneumococcal toxins, pneumolysin and H

2O

2 and does not require

the host inflammatory response. We determined the relative time course of the twotypes of apoptosis during meningitis in the mouse. Wild type and caspase 3 knockout mice were challenged with D39 pneumococci and compared for the timing anddegree of hippocampal damage. All infected mice developed meningitis by 15hours after infection based on positive bacterial CSF titers. Infected wild type micebegan to show an increase in apoptotic neurons at 18 hours, and peaked at 21 hours(60±15 vs. 95±7 cells/mm2, p<0.001; t-test). A high level of damage was sustainedthrough 24 hours. The infected caspase 3 knockout mice also showed an increase inapoptotic neurons at 18 hours. However by 24 hours, the number of apoptotic neu-rons declined significantly (84±20.3 vs. 19±9.5 cells/mm2, p<0.001; t-test). His-topathological sections of the hippocampus of the caspase 3 knock out mice at 24hours revealed numerous vacuolated areas suggesting clearance of apoptotic cellswithout continued accumulation of dead cells. In conclusion, our data show thatcaspase 3 is a pivotal caspase involved in neuronal apoptosis during meningitis.Neuronal apoptosis occurs in 2 separate phases. The toxin-mediated, caspase-inde-pendent pathway is implicated in early apoptosis during meningitis and the caspasedependent pathway occurs later in the course of inflammation.

Trafficking of Steptococcus pneumoniae cell wall to the nucleus of endothelialcells

Soulis K, Fillon S, Tuomanen E. Department of Infectious Diseases, St. Jude Chil-dren’s Research Hospital, Memphis, Tennessee 38105, USA.

Streptococcus pneumoniae is the major cause of community-acquired pneumoniaand bacterial meningitis in Europe and US. The pneumococcal cell wall consists ofa multilayered network of peptidoglycan with attached teichoic acid chains. Thesesurface elements have been shown to induce an intense inflammatory response char-acteristic of pneumococcal infection. The phosphorylcholine residue of the teichoicacid binds to the platelet-activating factor receptor (PAFr). The aims of this studywere to determine if pneumococcal cell wall enters the nucleus of eukaryotic cellsand by what mechanism. In vitro experiments with [methyl- 3H choline] labeledpneumococcal cell wall have shown that cell wall enters the nucleus of rat braincapillary endothelial cells (rBCEC

6). Moreover confocal microscopy experiments

using orthogonal projection have revealed that FITC labeled cell wall was locatedin the nucleus of rBCEC

6 cells. Since PAFr and �-Arrestin 1 mediate invasion of

pneumococcus, we tested if cell wall entry utilized the same mechanism. COS-1cells were transfected with different combinations of wild type and non-functionalmutants of PAFr, and �-arrestin 1. Cell wall trafficking was assessed in transfectedcells. Nuclear localization required both PAF receptor and �-Arrestin 1. In conclu-sion, we show that cell wall of Steptococcus pneumoniae enters the nucleus ofeukaryotic cells where it could influence gene expression.


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Cytokine production in human blood mononuclear cells stimulated with Strep-tococcus pneumoniae

Von Ungern-Sternberg M, Miettinen M1, Pitkäranta A2, Julkunen I1, Virolainen A1.National Public Health Institute, Helsinki1, HYKS Helsinki University Hospital2,Finland.

Aims: The purpose of this study was to analyse cytokine production in human pe-ripheral blood mononuclear cells (PBMCs) stimulated with wild type Streptococ-cus pneumoniae in comparison to pneumolysin deficient S .pneumoniae.Methods: Pneumococcal strain L82016 and a pneumolysin deficient mutant of thesame strain (Berry et al., 1996; kindly provided by James Paton, Australia and DavidBriles, USA) were cultured, transferred into liquid medium and grown into loga-rithmic growth phase. Pelleted and washed bacteria were used in 1:1 ratio for invitro stimulation of overnight monolayer cultures of human PBMCs provided byhealthy blood donors. All stimulation experiments were done in duplicates withcells of four different blood donors each time. The supernatants were harvested at 2,4, 6, and 20 hours, and analysed by ELISA for the presence of cytokines TNF-�,IL-1, IL-6 and IL-10.Results: In three independent experiments, pneumolysin deficient strain ofS.pneumoniae induced production of higher levels of TNF-�, IL-6 and IL-10 ascompared to the wild type strain: 27 vs. 11, 35 vs. 11, and 13 vs. 4 ng/ml, respec-tively. In contrast, the production of IL-1 was higher after wild type strain induc-tion: 6 vs. 20 ng/ml.Conclusions: Pneumolysin plays an important role in regulating the production ofinflammatory cytokines in human peripheral blood mononuclear cells stimulatedwith S.pneumoniae. Whether the enhanced production of IL-1 relates to S.pneumoniae-induced apoptosis, remains to be analysed.

CbpA expression and the two component regulatory system RR06 and HK06.

Standish A, Stroeher UH, Paton JC. School of Molecular and Biomedical Science,University of Adelaide, Australia.

Aims: To investigate the effect of deletion mutations in the response regulator RR06and histidine kinase HK06 genes on CbpA expression and virulence of S.pneumoniae.Methods: Levels of the choline binding protein CbpA were investigated usingwestern blotting, whereas the levels of cbpA mRNA were determined by real timeRT-PCR. The relative in vitro adherence of wild type S. pneumoniae D39 and itsisogenic RR06, HK06 and CpbA mutants was examined using A549 (type2pneumocytes) and Detroit 52 (nasopharyngeal) cells. The binding of the responseregulator protein to the promoter of cbpA was studied by gel shift assay using la-belled DNA fragments and cell extracts containing the regulator protein. The globalprotein expression profile was examined using 2-D gel electrophoresis. Protein-protein interactions between RR06 and HK06 were also examined, using an E. coliK-12 LexA-based 2-hybrid system.Results: Deletion of RR06 leads to a reduction in CbpA expression (approx. 3fold), whereas deletion of HK06 leads to an increase in expression (approx. 5 fold).The adherence of the mutants is also significantly reduced compared to the wildtype D39 although not as much as for the cbpA mutant.Conclusions: Expression of CbpA is regulated by the HK06/RR06 two-componentsystem. Other genes regulated by this system are currently being investigated


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Utilization of Putrescine by Streptococcus pneumoniae During Growth in Choline-Limited Medium

Ware D 1, Swaitlo E 1,2. University of Mississippi Medical Center1, Veterans AffairsMedical Center 2, Jackson, Mississippi, USA.

Aims: The purpose of this study was to examine the transcriptional organization of aputative polyamine transporter (Pot) operon in Streptococcus pneumoniae and test forany potential role for utilization of the polyamines putrescine and spermidine during invitro growth of the pnuemococcus in a choline-limited environment.Methods: Using total cellular RNA from pneumococcal strain WU2 grown in syntheticmedia, RT-PCR was performed using primers specific for the 5’ end of the murB geneand the 3’-end of the potD gene. Washed and quantitated stocks of WU2 were grown incompletely defined media (CDM) containing 0.05% putrescine or spermidine in the placeof choline. Gram stains were performed on the polyamine-grown cells to determine cellmorphology. Whole cell lysates of the WU2 grown in the polyamine-containing mediawere blotted onto a nitrocellulose membrane and incubated with primary antibodies againsteither choline or PspA. Pneumococcal cells were grown in CDM containing radiolabledputrescine and fractionated into cell wall components, water-soluble cytoplasmic con-tents, or membranes and insoluble cytoplasmic portions. Each fraction was counted todetermine the location of the radiolabeled polyamine.Results:RT-PCR experiments demonstrated that the four genes encoding the Pot sys-tem are co-transcribed with murB, a gene involved in an intermediary step of peptidog-lycan synthesis. Pneumococci grown in CDM containing putrescine without cholineentered logarithmic phase growth at 36-48 hours, however, culture density at station-ary phase eventually reached that of choline-containing medium. Cells grown in CDM-putrescine formed abnormally elongated chains in which the daughter cells failed toseparate and the choline-binding protein PspA was no longer cell-associated. Experi-ments with CDM containing radiolabled putrescine demonstrated that pneumococciconcentrate this polyamine in the cell walls.Conclusions: A polycistronic mRNA from cells grown in vitro includes four genes thatcomprise the transmembrane Pot complex plus murB, a gene involved in peptidogly-can synthesis. Pneumococci can replicate without choline if putrescine is available,although the lag time until exponential cell growth occurs is extended. Polyaminesmay substitute for aminoalcohols in the cell wall teichoic acids although the exactnature of the association is unknown.

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Mucosal immunity: The first line of defense against pneumococcal infections

*Jonsdottir, I

Landspitali University HospitalDept of ImmunologyHringbraut101 [email protected]

The primordial function of B cells is the defence against encapsulated bacteria

*Carsetti, R

University of Tor Vergata, Research Center OpBGDept. of Publich Health and Cell BiologyVia Montpellier 100133 [email protected]


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Dendritic cell cytokine production is affected by contact with pneumococcalpolysaccharides

Meltzer UA, Goldblatt D. Institute of Child Health, UCL, London, United King-dom

Aims: Dendritic cells (DCs) are critical antigen presentation cells whose function isimpaired in early life. It is in early life too that polysaccharide responses are im-paired resulting in susceptibility to infections with encapsulated bacteria such as S.pneumoniae. We therefore set out to study the interaction between human monocytes-derived DCs and pneumococcal capsular polysaccharides (PPS) in vitro.Methods: Immature DCs were generated from peripheral blood monocytes and in-cubated with FITC-labelled PPS type 9N or 14 for assessment of uptake by FACSand confocal microscopy. They were exposed to PPS type 1, 6B, 9N, 14, 19F or 23Fin the absence or presence of E. coli LPS for assessment of phenotypic DC matura-tion and cytokine production by FACS and ELISA. PPS were obtained from theAmerican Type Culture Collection (Rockville, USA).Results: PPS were taken up by immature DCs and proceeded to HLA-DR+, LAMP-1+ late endosomal compartments. Uptake was reduced in the presence of cytochalasinD and wortmannin, suggesting that both cytoskeletal rearrangements andphosphatidyl inositol 3-kinase activation may be required for internalisation of PPSby DCs. Despite uptake, none of the PPS tested had any effect on DC phenotypeand maturation, as shown by FACS analysis of critical DC surface markers e.g.HLA-DR, CD83 and CD86. Production of IL-12 and IL-10 was not detectable byELISA. However, PPS were capable of modulating the response of the DCs to asecond signal such as LPS. Exposure of DC to PPS in the presence of LPS resultedin an altered cytokine balance with significantly increased IL-10 production andslightly reduced IL-12 production as compared to LPS alone. This effect is not seenusing the control antigen tetanus toxoid.Conclusion: DC-pneumococcal interaction may affect subsequent immune responsesas an altered cytokine balance may have a profound effect on DC-driven T cellpriming. This may partly explain reported negative effects of pneumococcal vacci-nation.

Pulmonary defence against pneumococcus - data from bronchoalveolar lavage (BAL)studies

Gordon S, ea.

See abstracts: IMM-21 (page 188), IMM-22 (page 188) and IMM-23 (page 189)


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Pneumococcal surface proteins PspA & CbpA induce TH1 cytokine responses inadults & children

Thornton R, Pomat W, Langlands J, Richmond P. University of WA School of Paediat-rics & Child Health, Princess Margaret Hospital for Children, Perth, Western Aus-tralia.

Background: Promising pneumococcal protein vaccine candidates which are effica-cious in animal models for the prevention of carriage and invasive disease includepneumococcal surface protein A (PspA) and choline binding protein A (CbpA; PspC).Children are able to mount systemic IgG and mucosal IgA antibody responses to PspA,however little is known about the cellular immune responses to these proteins and theirrole in protection. The purpose of this study was to evaluate cytokine responses ofperipheral blood mononuclear cells (PBMCs) to PspA and CbpA in adults and chil-dren.Methods: Recombinant N-terminal truncated PspA and CbpA proteins expressed inE.coli and purified were used (obtained from J. Paton, Australia). Cryopreserved PBMCswere thawed, resuspended at 0.5 x106 viable cells/ml in AIM-V serum-free mediumwith 2-ME and human AB sera and cultured for 48 hours with medium alone or sup-plemented with optimal concentrations of PspA (5ug/mL), CbpA (2.5ug/mL) or teta-nus toxoid (0.5Lf/mL). After culture, supernatants were collected by centrifugationand stored at -20°C until assayed for IL-10, IL-13 & IFN� via time resolved fluorim-etry.Results: Cytokine responses have been evaluated in 10 children (aged 2-10 years) and10 healthy adults. A strong Th1 response has been noted with all adults &childrenproducing IFN� in response to CbpA and 9 adults & 8 children in response to PspA. Incontrast, only 2 adults & 4 children produced IL-13 in response to CbpA & 1 adult &1 child in response to PspA. Il-10 responses were also seen to both proteins. Cytokineresponses in children to tetanus toxoid were more balanced with ~50% producing IFN& 60% IL-13. Polymyxin did not inhibit cytokine responses suggesting they were notdue to LPS contaminationConclusions: Exposure to pneumococcal carriage appears to prime for vigorous TH1responses in both children and adults to pneumococcal surface proteins, PspA andCbpA.

Polysaccharide and Protein Antibody responses to pneumococcal carriage; alongitudinal study.

Goldblatt D1, Käyhty H2, Hussain M3, Ashton L1, Sundström C2, Melegaro A3, An-drews N3, Pebody R3, George R3, Edmunds J3, Gay N3, Miller E3. 1Institute of ChildHealth, University College London, 2National Institute of Public Health, Helsinki,3Health Protection Agency, London.

Aims: The development and maintenance of immunity to Streptococcus pneumoniae(SPN) in unvaccinated individuals is thought to be due to encounter with the organ-ism or cross reactive antigens. However, few longitudinal studies of carriage andthe consequent immune responses have been conducted using standardised lab tech-niques.Methods: We enrolled 132 families with young children into this study. Nasopha-ryngeal swabs were taken monthly for 10 months, and cultured in a standard fash-ion. The presence of SPN was detected and isolates were serotyped. Venous bloodwas taken from the adults at the beginning (0) and the end (month 10) of the study.Anti-polysaccharide IgG (22F adsorbed) specific for serotypes 1, 4, 5, 6B, 9V, 14,18C, 19F and 23F and antibodies to pneumolysin, Pneumococcal surface protein A(PspA) and Pneumococcal Surface Adhesin A (PsaA) were measured in paired sera.Results: At least one carriage episode was detected over the study period in 84% <5yrs and 42%>5yrs of age. 134 paired sera were available for serological analysis.Serotype specific anti-polysaccharide antibody responses increased significantlyfollowing carriage of serotypes 9V, 14, 18C and 23F by an individual or by a mem-ber of the family. For serotype 14, an antibody titre at the beginning of the study of5�g or above was associated with protection from carriage (p=0.04). Similarly,serotype 6B antibodies protected against the carriage of serotype 6A. There was asmall (17%) but significant increase in antibody titres to PsaA and pneumolysin inresponse to carriage but no change in PspA titres.Conclusion: Adults respond to nasopharyngeal carriage by mounting anti-capsularand weak anti-protein antibody responses. Such encounters are thus likely to main-tain antibody titres. Naturally induced anti-capsular IgG prevent carriage, a mecha-nism that is likely to contribute to their prevention of disease.


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Development of Antibodies Against the Putative Proteinase Maturation Pro-tein A (PpmA) in Relation to Pneumococcal (Pnc) Carriage and Otitis Media

P.V. Adrian1, D. Bogaert1, E. Holmlund2, L. Saarinen2, M. Lahdenkari2, R. de Groot1,H. Käyhty2, and P.W.M. Hermans1

1Erasmus MC-Sophia, Rotterdam, The Netherlands; 2National Public Health Insti-tute, Finland.

Aim. Surface associated Pnc protein PpmA is considered as a protein-based anti-pneumococcal vaccine candidate. The immunogenicity of PpmA during the firsttwo years of life was investigated in a large group of Finnish children who partici-pated in the FinOM Cohort Study (Kilpi ea. PIDJ 2001;20:654-62)Methods. Serum PpmA antibody titers were determined by ELISA in a 50 healthyFinnish children at 6, 12, 18 and 24 months of age, and in 129 healthy adults. ThePnc contacts (carriage and acute otitis media, AOM) of children were determinedduring the follow-up from 2 to 24 mo of age. Sera obtained at 12 months (n=249)and 18 months of age (n=232), were analyzed by PpmA ELISA to investigate theassociation between PpmA antibody titers and risk to develop AOM during thefollowing 6 months.Results. The mean PpmA antibody titers increased with age during the first twoyears of life.The increase was assaocauetd with Pnc contacts. The PpmA antibodytiters in adults were significantly higher than those at 24 months of age. Finally,PpmA antibodies at 18 months of age decreased the risk to develop acute otitismedia, albeit statistically not significantly.Conclusion. The development of PpmA antibodies during the first two years of lifeas well as the association between PpmA antibody titers and decreased risk to de-velop acute otitis media is comparable with recent observations made for naturalantibodies against pneumoccal surface adhesin A, PsaA (Rapola ea. Vaccine;2003;21:3608-13). Whether there is a direct association between the presence ofPpmA (and PsaA) antibodies and protection against acute otitis media remains un-clear.

The role of MyD88 and Toll-Like Receptors in pneumococcal infection

Albiger B1, Sandgren A1, Katsuragi H2, Normark S3 and Henriques Normark B1,3

The Swedish Institute for Infectious Disease Control, Stockholm, Sweden1, NiiponDental University, Niigata, Japan2, Microbiology and Tumorbiology Center, Stock-holm, Sweden3

The Toll-like receptors (TLR) and the adaptor molecule MyD88 have key roles inthe activation of the innate immune system against microbial pathogens.Aims: To understand the role of MyD88 and the TLR in the development of pneu-mococcal invasive disease in vivoMethods: MyD88-deficient (MyD88-/-) and TLR2 (TLR2-/-), TLR4 (TLR4-/-), andTLR6-deficient (TLR6-/-) mice were infected intranasally and intraperitoneally with107 Streptococcus pneumoniae TIGR4.Results: We observed that while infected MyD88-/- mice were more susceptible toinfection than wild-type (wt) mice, TLR2-/-, TLR4-/- and TLR6-/- mice presented thesame survival rate. The bacterial cfu in blood of MyD88-/- mice was significantlyhigher at 48h. There was no TNFa in the sera of the MyD88-/- mice, demonstratingthat higher susceptibility was due to an impaired immune response and an impairedbacterial clearance. No difference in blood cfu or in TNF� level between TLR4-/-

and the wt mice was found, suggesting that TLR4 had no role in the recognition ofpneumococcus. TLR2-/- and TLR6-/- mice had a lower cfu in blood at 24h and 48h,but they presented similar TNF� level as compared to wt mice.Conclusions: Thus while MyD88 has a central role for the control of pneumococcalinvasive infection, TLR4 has no significant role and TLR2 and TLR6 may play amoderate role in vivo. Our studies suggest that other unidentified MyD88-depend-ant pattern recognition receptors are involved in the activation of an innate immuneresponse to pneumococcal invasive disease.


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Host-pathogen interaction during pneumococcal infection in patients withchronic obstructive pulmonary disease

Debby Bogaert1, Paul van der Valk2, Reshmi Ramdin1, Marcel Sluijter1, EvelynMonninkhof2, Ron Hendrix3, Ronald de Groot1, and Peter W.M. Hermans1. 1Depart-ment of Pediatrics, Erasmus MC-Sophia, Rotterdam, The Netherlands. 2Departmentof Pulmonology, Medisch Spectrum Twente, Enschede, The Netherlands. 3RegionalLaboratory of Public Health, Enschede, The Netherlands.

Introduction. Acute exacerbations are a frequent complication of chronic obstruc-tive pulmonary disease (COPD). Recent studies suggest a role for bacteria like Strep-tococcus pneumoniae in the development of acute exacerbations. In this study weinvestigated in COPD patients (i) the epidemiology of pneumococcal colonizationand infection, (ii) the effect of pneumococcal colonization on the development ofexacerbation, and (iii) the immunological response against S. pneumoniae.Methods. We cultured sputa of 269 COPD patients during stable state and exacerbationsof COPD and we characterized the 115 pneumococcal isolates by means of serotyping.Moreover, we studied serum IgG antibodies, avidity and functional antibody titersagainst the seven conjugate-vaccine serotypes in these patients.Results. Colonization with merely pneumococci (monocultures) increased the riskof an exacerbation with a hazard ratio of 2.93 (95% CI 1.41- 6.07). The most preva-lent pneumococcal serotypes found were the serotypes 19F, 3, 14, 9L/N/V, 23A/Band 11. We calculated a theoretical coverage for the 7-, and 11- valent pneumococ-cal vaccine of 60% and 73%, respectively. All patients had detectable IgG levelsagainst the 7 conjugate-vaccine serotypes. These antibody titers were significantlylower than in vaccinated healthy adults. Finally, on average, a 2.5-fold rise in sero-type-specific and functional antibodies was observed during exacerbations with S.pneumoniae positive sputum cultures.Conclusion. Our data indicate that pneumococal colonization in COPD patients isfrequently caused by vaccine serotype strains. Moreover, pneumococcal colonizationis a risk factor for exacerbations of COPD. Finally, our findings demonstrate thatCOPD patients are able to mount a significant immune response to pneumococcalinfection. COPD patients may, therefore, benefit from pneumococcal vaccination.

Multiplex opsonophagocytosis assay (MOPA) is a useful tool for the monitor-ing of the 7-valent pneumococcal conjugate vaccine

D. Bogaert, M. Sluijter, R. de Groot and P.W.M. Hermans. Department of Pediatrics,Erasmus MC-Sophia, Rotterdam, The Netherlands

Introduction. Pneumococcal conjugate vaccination is highly efficacious against in-vasive diseases in young children. Since host protection is mainly mediated by op-sonin-dependent phagocytosis, the in vitro measurement of opsonophagocytic ac-tivity of the anti-capsular antibodies is assumed to be a reliable correlate of protec-tion to monitor vaccine efficacy. Unfortunately, the methods used so far are alltedious to perform and material consuming.Methods. We modified the multi-specificity opsonophagocytosis killing assay(MSOPKA) into a high throughput method, which simultaneously measures theopsonophagocytosis against the seven serotypes covered by the current conjugatevaccine in a single assay.Results. In the so called multiplex opsonophagocytosis assay (MOPA), a mixturecontaining equal numbers of colony forming units (CFUs) of chloramphenicol-resistant serotype 4, spectinomycin-resistant serotype 6B, streptomycin-resistantserotype 9V, erythromycin-resistant serotype 14, rifampicin-resistant serotype 18C,tetracycline-resistant serotype 19F, and trimethoprim-resistant serotype 23F pneu-mococci was used as a target mixture and incubated with serial dilutions of testserum. After opsonophagocytosis by differentiated HL-60 cells in the presence ofcomplement, the samples were spotted onto different blood agar plates containingthe 7 selective antibiotics, respectively. Opsonophagocytosis was calculated as thehighest serum dilution resulting in 90% or more reduction in CFUs. The data ob-tained by this assay correlated well with the data obtained by the MSOPKA.Conclusion. The MOPA simultaneously measures opsonophagocytosis capacity ofserum against the capsular serotypes included in the 7-valent pneumococcal conju-gate vaccine in a high-throughput fashion, requiring low volumes of patient sera.


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Phenotypic and Phagocytic Analyses of HL-60 Cells Utilized in anOpsonophagocytic Killing Assay (OPKA) for Pneumococci.

Burton, R.L.1, and Nahm, M.H.1 University of Alabama at Birmingham, Birming-ham, Alabama, USA1.

Aims: The purpose of this study is to examine the phenotypic and phagocytic changesthat occur during differentiation of HL-60 cells over time. This data may be usefulin optimizing the differentiation protocol.Methods: HL-60 cells (ATCC CRL-1964) were differentiated with 0.8% dimethylformamide (DMF) for a total of 7 days. On each day, the phenotype of the cells wasexamined by FACS analysis using monoclonal antibodies against CD11b, CD16,CD32, CD35, CD64, and CD71. Apoptotic and necrotic cells were enumeratedusing annexin V and propidium iodide, respectively. Also, the differentiated cellswere used as effector cells in a standard OPKA to determine the titer against sero-type 6B. Purified granulocytes were used as controls.Results: CD32 was expressed in 100% of the cells throughout the entire differen-tiation procedure. CD16 and CD64 expression was low prior to differentiation andremained low throughout (<11% and <3%, respectively). Less than 1% of the cellswere apoptotic at all time points.

0 1 2 3 4 5 6 7 Blood GranulocytesCD11b 1 68 94 96 89 87 87 92 100CD35 42 73 98 98 100 99 100 96 99CD71 99 92 68 33 5 6 2 4 2Necrotic 6 7 8 9 17 19 41 53 Not DoneOPKA Titer 170 6800 9800 11600 9500 8700 9700 11600 13700

Conclusions: Cells differentiated for 2-7 days yielded comparable OPKA titers.CD11b expression correlates well with OPKA titer (R2=0.75), and thus may be auseful marker for differentiation.

B-cell receptor-dependent and independent mechanisms regulating serologicalmemory

Capolunghi F.1, Rosado M.M.1, Giorda E.1, Cascioli S.1, Pantosti A.2, Quinti I3 andCarsetti R1. Bambino Gesù Children Hospital1, Istituto Superiore di Sanità2,Università La Sapienza3, Rome, Italy

Aims: The purpose of our study is to dissect the B-cell receptor-dependent and inde-pendent mechanisms regulating the level of protective anti-pneumococcal antibod-ies in the serum.Methods: Serum antibodies constitute the so-called immunological memory includingnatural and specific antibodies. Natural antibodies, mostly of IgM isotype, are presentindependently of intentional immunization, have a broad specificity and have thefunction to limit initial infection. Specific antibodies derive from memory B cells,generated by previous vaccination or infection, have the function to completely clearthe recognised pathogen and to prevent re-infection. It has been recently shown thatserological memory can be maintained by a B-cell receptor-independent aspecificrecall mechanisms: for example, bacterial DNA interacting with TLR9 stimulatesantibody production by memory B cells.We cultured PBL in the presence of different stimuli (intact heat-inactivated pneu-mococci with or without capsule, pneumococcal vaccine polysaccharides and CpG)to evaluate the contribution of B-cell receptor-dependent and independent pathwaysto the maintenance of serological memory and their relative relevance in patientshighly susceptible to pneumococcal infection. We measured the proliferation of dif-ferent B-cell subsets using the CFDA dye and the titer of total and anti-pneumococ-cal Ig.Results: We found that CpG stimulates a strong production of antibodies of differ-ent specificities. Pneumococcus, instead, induces a modest, but specific response.Conclusions: Our experimental approach allows the dissection of B-cell receptor-dependent and independent mechanisms regulating the level of protective anti-pneu-mococcal antibodies in the serum and the identification of the molecular mecha-nisms leading to increased susceptibility to encapsulated bacteria infection.


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Characterization of effector cells for a standardized opsonophagocytic killingassay (OPKA) with a pneumococcal target.

R. A. Fleck1, and M. H. Nahm2. NIBSC, UK1, University of Alabama at Birming-ham, USA2.

Aim: To compare differentiated HL-60 (ATCC CRL-1964) and NB-4 cell lines inan OPKA, and to identify an effector phagocyte cell line for a potential standard-ized OPKA procedure.Methods: Cell lines were differentiated (48-120h) in RPMI-1640, 1mM glutamine,10% FCS, and either 0.8% dimethylformamide (DMF) or 1 x 10-6 M all-trans retinoicacid (ATRA) supplemented with 4 x 10-12 M vitamin D

3 and 30 ng/ml G-CSF. Cells

were examined by flow cytometry, to analyze the progression of differentiation froma pro-myelocytic to a neutrophilic phenotype, and by an OPKA vs. serotype 6Bpneumococci to examine the biological function of the cells.Results: Upon exposure to DMF for 120 h, HL-60 cells increased CD11b and CD16expression (16-fold and 8-fold respectively) and decreased CD71 expression (18-fold). CD32 expression remained high and unchanged during the stimulation. NB-4 cell expression of CD32 was low before the stimulation but increased with theATRA stimulation. NB-4 expression of CD11b, CD16, and CD71 changed as HL-60 did during the ATRA stimulation. For these changes, NB-4 required only 48h ofATRA stimulation.In OPKA, HL-60 cells differentiated with DMF, performed significantly better thanHL-60 cells differentiated with ATRA. Also, NB-4 cells differentiated in DMFwere not as responsive as NB-4 cells differentiated in ATRA. HL-60 cells differen-tiated with DMF for 120 h and NB-4 cells differentiated with ATRA for 48 h gavecomparable OPKA titres.Conclusions: Optimal differentiation was with ATRA for NB-4 and DMF for HL-60. CD11b expression may be useful as a marker of differentiation for both celltypes. Both cell types may be useful as a phagocytic cell line for standardizedOPKA as long as they are differentiated optimally.

Cell line and differentiation, choices in the Pneumococcal OpsonophagocyticKilling Assay (OPKA).

Gillett M.L., Care R, Bygraves J., Fleck R.A., Feavers I.M. NIBSC, EN6 3QG, UK

Aims: To investigate differences between differentiated HL-60 (ATCC CRL-1964),HL-60 (ECACC 98070106) and NB-4 cell lines, used in an OPKA.Methods: Cell lines were differentiated (48-120h) in: RPMI-1640, 1mM glutamine,10% FCS, and either 0.8% dimethylformamide (DMF) or 1 x 10-6 M all-trans retinoicacid (ATRA), supplemented with 4 x 10-12 M vitamin D

3 and 30 ng/ml G-CSF.

Cells were examined by an OPKA using serotype 6B pneumococci.Results: NB-4 and HL-60 cells differentiated with ATRA or DMF produced aconsistent ED50 with varying differentiation time. Differentiation with ATRAproduced similar ED50 values ª1:400. However, the % maximum killing increasedin NB-4 cells after 72 hours and continued to remain 15% higher. Differentiationof HL-60 (ATCC) with DMF resulted in an increased ED50 to 1:600 and the %maximum killing was optimum at 72 hours. Accurate ED50 was difficult to ob-tain in the NB-4 cells differentiated with DMF and was lower than 1:300. Also,the % maximum killing was consistently lower than HL60. NB-4 differs geneti-cally from HL-60. The NB-4 cells have the t(15;17) karyotype resulting in thePML/RARalpha fusion product, whereas the HL-60 does not. HL-60 is also ho-mozygous for the low affinity binding allele R131 of the Fc�IIa receptor whileNB-4 is heterozygous, resulting in increased binding efficiency to IgG2 subclassof antibodies involved in opsonophagocytosis. During this study we discoveredthat an alternate source of HL-60 (ECACC) responded poorly in the OPKA whendifferentiated with both DMF and ATRA.Conclusions: With the careful choice of cell line and differentiation procedure, theOPKA is both reliable and reproducible. However, optimisation of cell differentia-tion is critical in establishing an accurate measure of serum potency. AlthoughED50 estimations remain constant with differentiation time, the maximum numberof bacteria killed varies. This measure of phagocytic potential is important for thegeneration of an accurate ED50.


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Establishing a standardized Opsonophagocytic Killing Assay (OPKA) at NIBSC.

Gillett M.L., Care R., Bygraves J.A., Fleck R.A., Feavers I.M. NIBSC, South Mimms,EN6 3QG, UK

Aims: This project aims to establish a new phagocytic cell line at NIBSC, for use inan OPKA using a standard protocol (Kimet al., 2003). This reference cell line will aid in the international standardization ofthe OPKA.Methods: HL-60 (ATCC CRL-1964), differentiated (120h) with 0.8% DMF in stand-ard culture media (RPMI-1640, 10% FCS, 1% L-Glutamine), was used as the effec-tor cell. Differentiated cells were washed in Hanks Buffer (without and then withCa2+/Mg2+) before resuspension in Opsonisation buffer B (OpB) (1x107cells.ml-1).Serum samples (20�l) were added to a 96-well plate and diluted serially in OpB(10�l). Streptococcus pneumonia (10ml) was added with incubation (RT, 30min),prior to complement (10�l) (Pel-Freez 31038-100) and effector cells (40�l), andfurther incubation (37°C, 60min). Bacterial survival was determined by colony for-mation with automatic enumeration (ProtoCOL, Symbiosis). Alternative sourcesof HL-60 (ECACC 98070106), cell line (NB-4) and differentiation procedure werealso evaluated.Results: The OPKA was easily established at NIBSC, giving robust antibody titres.Key S. pneumoniae serotypes and additional serum sources were successfully evalu-ated. Optimisation of bacterial colony forming units, facilitated the evaluation ofthe ProtoCOL software and development of templates for the enumeration of colo-nies. Optimally differentiated alternative cell lines (determined by CD11b expres-sion), gave comparable antibody titres. However, differences in each cell line’sresponse, when sub-optimally differentiated, indicated that careful control of cellsource, culture and differentiation may be necessary.Conclusion: The results indicate that the OPKA is a robust and reproducible assayand effectively provides antibody titres for serum derived from vaccinees.

Human IgG2 recognizing phosphorylcholine shows cross-reactivity and evi-dence for protection against major bacterial pathogens of the human respira-tory tract.

H.B. Goldenberg, T.L. McCool and J.N. Weiser. Department of Microbiology, Uni-versity of Pennsylvania, Philadelphia, PA

Aims: Phosphorylcholine (ChoP) is an antigenic cell-surface component of a numberof commensal and pathogenic bacteria that reside primarily in the upper airway.Exposure to this common epitope results in high levels of serum antibody to ChoP.Murine anti-ChoP antibody is protective against pneumococcal infections, whilethe effectiveness of human antibosy that recognizes this antigen is unknown. Thecapability of ChoP-specific antibodies found in normal human sera to bind and killHaemophilus influenzae and Streptococcus pneumoniae was determined.Methods: In this study, human ChoP- specific antibody was affinity-purified frompooled serum gamma globulin. The ability of this antibody to kill H. influenzae andS. pneumoniae was assessed with in vitro killing assays. In vivo testing of the pro-tective capabilities of the antibody were performed in a mouse model.Results: This naturally acquired antibody, which was primarily of the IgG2 subtype,recognized ChoP on the lipoteichoic acid of S. pneumoniae and the lipopolysaccha-ride of H. influenzae, two of the leading etiologic agents of infection involving thehuman respiratory tract. Anti-ChoP IgG2 was effective in in vitro killing assaysagainst some clinical isolates of non-typeable H. influenzae and isolates of severalcommon serotypes of S. pneumoniae. Moreover, passively administered humananti-ChoP antibody protected mice against lethal challenge with a transparent iso-late of S. pneumoniae type 6A.Conclusions: The effectiveness of human antibody to this conserved bacterial struc-ture suggests that it could function as a single vaccine antigen targeting multiplehuman pathogens if it can be manipulated to broaden the activity demonstrated inthis study.


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Pulmonary levels of capsule-specific IgG following 7-valent conjugate vaccina-tion are greater in HIV infected than in HIV uninfected adults.

Gordon SB1,2, French N1,2, Kanyanda SE1, Zijlstra EE2, Molyneux ME1,2. Malawi-Liverpool-Wellcome Programme of Clinical Tropical Research, Blantyre1, Univer-sity of Malawi College of Medicine, Blantyre, Malawi2.

Aims: Pneumococcal infections evoke a local humoral immune response in the lungthat is maintained in HIV infected adults. We aimed to compare the immunologicalresponse in serum and lung fluid to injected 7-valent conjugate vaccine (Prevnar®)in both HIV infected and uninfected volunteers.Methods: 42 adult volunteers (20 HIV infected; 22 non-HIV infected - none onantiretroviral therapy) were randomly allocated to receive 7-valent vaccine or pla-cebo. Bronchoscopy to obtain bronchoalveolar lavage (BAL) was carried out on allvolunteers before and 30 days after vaccination. Pneumococcal capsule specificIgG was measured in serum and BAL after 22f adsorption using an ELISA with 23-valent polysaccharide vaccine (Pneumovax®)as the capture antigen.Results: Baseline levels of capsule-specific IgG in BAL were higher in HIV in-fected subjects compared to HIV negative (43 vs 25 ng/ml; p=0.03). There were noincreases in capsule-specific IgG in serum or lung fluid in subjects given placebo.HIV negative subjects showed a non-significant increase in capsule specific IgG inboth serum and BAL following vaccination. HIV infected subjects showed a non-significant increase in serum but a significant increase in BAL capsule-specificIgG following vaccination (mean pre-vaccination vs post vaccination= 43 vs 96 ng/ml; p=0.0001).Conclusions: An anti-pneumococcal capsule-specific pulmonary response to sys-temic 7-valent conjugate vaccination is demonstrable in HIV-infected African adults.Serotype-specific data are required but this preserved pulmonary mucosal responsemay offer a means of optimising vaccination in these subjects.

Alveolar macrophages from Malawian adults with HIV/AIDS show reducedproduction of IL-8 when challenged ex vivo with opsonised Streptococcuspneumoniae.

Gordon SB1,2, Kanyanda SE1, Pridmore AC3, Read RC3, Zijlstra EE2, MolyneuxME1,2. Malawi-Liverpool-Wellcome Programme of Clinical Tropical Research,Blantyre1, University of Malawi College of Medicine, Blantyre, Malawi2, Divisionof Genomic Medicine, University of Sheffield Medical School3.

Aims: HIV infects human alveolar macrophages (AM) and alters their function,often resulting in increased cytokine production. In view of the particular suscepti-bility of HIV infected subjects to pneumococcal infections, however, we tested thehypothesis that AM from HIV infected volunteers would differ in their pro-inflam-matory response to specific challenge ex vivo with opsonised S. pneumoniae fromAM from non-HIV infected control subjects.Methods: 23 adults (12 HIV infected; 11 non-HIV infected - none on antiretroviraltherapy) were studied. Bronchoalveolar lavage (BAL) was obtained from all volun-teers. Alveolar macrophages were challenged ex vivo with opsonised type 1S.pneumoniae. Cytokine responses at 0, 6, 12 and 24 hours (IL-1, IL-6, IL-8) weremeasured by ELISA on supernatant culture media. Cytokine RNA signals are un-der still being analysed using real-time PCR.Results: In response to S.pneumoniae challenge, there was increased IL-1beta, IL-6 and IL-8 production over time in all experiments. As expected, there were highermean IL-1beta and IL-6 levels produced by challenged AM from HIV infected sub-jects compared to non-HIV-infected subjects (p>0.05). In contrast, there was sig-nificantly lower production of IL-8 by AM from HIV infected subjects (area undercurve ranksum p=0.02) compared to non-HIV infected subjects.Conclusions: Reduced IL-8 production by AM from HIV infected subjects in re-sponse to S.pneumoniae may result in poor neutrophil recruitment in these patientsand contribute to the increased susceptibility of these subjects to invasive pneumo-coccal disease. Microarray analysis of these responses is under way.


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HIV viral load, cellular composition and activation markers in bronchoalveolarfluid before and after 7-valent conjugate vaccination in HIV infected Malawianadults.

Gordon SB1,2, French N1,2, D Lindell3, Kanyanda SE1, Zijlstra EE2, Molyneux ME1,2.Malawi-Liverpool-Wellcome Programme of Clinical Tropical Research, Blantyre1,University of Malawi College of Medicine, Blantyre, Malawi2, The University ofMichigan Medical School, Ann Arbor, Michigan, USA3.

Aims: 7-conjugate anti-pneumococcal vaccine produces a specific anti-capsular IgGresponse in the lung fluid of HIV infected adults (ISPPD-4 abstract). We tested thehypotheses that (a) the response might be accompanied by cellular inflammation orincrease in HIV load, and (b) the cellular composition of the alveolar milieu prior tovaccination might predict the level of IgG produced in response to vaccine.Methods: 42 adult volunteers (20 HIV infected; 22 non-HIV infected - none onantiretroviral therapy) were randomly allocated to receive 7-valent vaccine or pla-cebo. Bronchoscopy to obtain bronchoalveolar lavage (BAL) was carried out on allvolunteers before and 30 days after vaccination. A panel of surface markers wasused to describe T cells (CD3, CD4, CD8), B cells (CD19, CD20) and alveolarmacrophages (CD16, CD206). Co-receptors and markers of activation (CD11a,CD11b, CD11c, CD14, CD25, CD40, CD49d, CD54, CD62L, CD69, CD80, CD86,HLA-DR) were measured in these cell populations.Results: HIV infected subjects had a greater percentage of B cells in BAL (0.6% vs0.2% all cells; p=0.003) and a relative CD8 lymphocytosis (65% vs 27% CD8+;p=0.0001) compared to control subjects. There was no evidence of cellular inflam-mation or increased HIV load as a result of 7-valent conjugate vaccination (signrankp=0.89). There were no significant differences in co-receptor expression or mark-ers of activation in T cells, B cells or macrophages between vaccine and placeborecipients in either HIV infected or non-infected groups and flow cytometry param-eters were not predictive of IgG response.Conclusions: These data provide an important benchmark against which any puta-tive inhaled vaccine must be measured given that injected vaccine produces a spe-cific pulmonary IgG response.

Chemokine responses of respiratory epithelial cells to Streptococcus pneumoniaeinfection.

Graham RMA, Paton JC. School of Molecular and Biomedical Science, Universityof Adelaide, South Australia.

Aims: To characterise the chemokine responses of respiratory epithelial cells toStreptococcus pneumoniae infection and to determine the roles that pneumococcalproteins, in particular the surface proteins choline binding protein A (CbpA) andpneumococcal surface protein A (PspA), and the toxin pneumolysin (Ply), play inthis response.Methods: Epithelial cell lines were infected with live WT serotype 2 S. pneumoniaeD39 or isogenic mutants negative for CbpA, PspA, or Ply. Cells were incubatedwith live S. pneumoniae for up to four hours. Alternatively cells were incubated forup to four hours with purified D39 CbpA, PspA or Ply. At 0, 2, and 4 hourssupernatants were collected and assayed for IL-8 by ELISA. Cells were also har-vested and levels of IL-8, MIP-2�, and MIP-2� mRNA were measured using real-time RT-PCR and specific oligonucleotide primers.Results: After incubation with live bacteria, increased levels of IL-8 mRNA andprotein, and MIP-2�, and MIP-2� mRNA were observed in both cell lines. Al-though a moderate increase was seen in the levels of mRNA for these chemokinesafter exposure to WT D39, a significantly greater increase was seen in response toexposure with PspA- and CbpA- mutants.Conclusions: Respiratory epithelial cells upregulate expression of IL-8, MIP-2�and MIP-2� mRNA in response to infection with S. pneumoniae, and the pneumo-coccal proteins PspA and CbpA may have a direct effect on this response.


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Endogenous Interleukin-15 is a crucial factor for the innate immune responseagainst Streptococcus pneumoniae.

Hocke AC1, Lampe M1, Hammerschmidt S2, Witzenrath M1, Schmeck B1, HippenstielS1, Schütte, H1, Suttorp N1 and Rosseau S1. Charité – Faculty of Medicine, HumboldtUniversity and Free University of Berlin, Department of Internal Medicine/InfectiousDiseases1 and Centre of Infection Research, University of Würzburg, Germany2

Introduction: IL-15 is a potent inhibitor of apoptosis, it increases the antimicrobial ca-pacity of phagocytes and it has major impact on NK and NKT cell biology. These fea-tures play a critical role in innate immunity against a variety of microbes, and exogenousIL-15 has been shown to protect mice against septic shock induced by E. coli. The role ofIL-15 in pulmonary host defence and expression of IL-15 in the respiratory tract areunknown. We therefore analysed the expression of IL-15 in the lungs of mice and studiedits regulation after intranasal challenge with S. pneumoniae. Moreover, we focused onthe functional impact of IL-15 on the clinical course of pneumococcal pneumonia.Methods: Mouse model of pneumococcal pneumonia, immunohistochemistry, digitalimage analysis, RT-PCR, western blot, IL-15 neutralization with soluble IL-15 receptoralpha (sIL-15R�).Results: IL-15 was constitutively expressed in mice lungs and immunohistochemistryrevealed expression in bronchial and alveolar epithelial cells, in alveolar macrophages(AM) as well as in vascular and bronchial smooth muscle cells. After intranasal chal-lenge of mice with S. pneumoniae, bronchial and alveolar epithelial cells increased intra-cellular expression of IL-15, and bronchial epithelium as well as AM up-regulated sur-face expression of IL-15. Western blot analysis of lung homogenates confirmed up-regu-lation of IL-15, and RT PCR revealed simultaneous up-regulation of IL-15 mRNA. Neu-tralization of IL-15 bioactivity in vivo dramatically increased mortality of infected mice.Conclusion: IL-15 is constitutively expressed in the lower respiratory tract and lungsof mice. It is expressed intracellularly as well as on the cell surface of epithelial cellsand AM. During pneumococcal pneumonia, expression of IL-15 is markedly up-regu-lated in epithelial cells and it is shuttled to the cell surface of bronchial epithelium andAM. Neutralization of endogenous IL-15 with sIL-15R� revealed a crucial impact ofIL-15 on the innate immune response against S. pneumoniae and the clinical outcomeof pneumococcal pneumonia.Supported by BMBF (CAPNetz to S.R., N.S. and S.Ha. and NBL III to S.Hi.).

Serum antibodies (ab) to the pneumococcal (pnc) surface proteins BVH-3 andBVH-11 in Finnish infants and adults

Holmlund E1, Jaakkola T1, Saarinen L1, Lahdenkari M1, Hamel J2, Brodeur B2, KilpiT1, Käyhty H1. National Public Health Institute, Helsinki, Finland1, Shire Biologics,Québec, Canada2.

Aims: To measure natural ab to the pnc protein vaccine candidates BVH-3 and BVH-11 in infants and adults and to relate the ab concentrations to pnc contacts.Methods: Serum samples taken during the FinOM Cohort Study of 50 healthy in-fants at 6, 12, 18, and 24 mo, 100 healthy adults, 100 children with AOM (acute andconvalescent sera), and appr. 500 infant sera taken at 12 and 18 mo of age wereanalysed for the Ig anti-BVH antibodies by the EIA. Nasopharyngeal carriage (10swabs) and AOM episodes were recorded during the follow-up time from 2 to 24mo of age. The ab concentration at 12 and 18 mo was used in the relative riskcalculation as an explanatory factor for the occurrence of pnc AOM during thefollowing 6 months. The previous pnc contacts were included in multivariate mod-els.Results: The geometric mean concentration (GMC) of infants for anti-BVH-3 and -11 increased from 12 mo of age on (0.5 and 1.1 �g/ml at 12 mo, 1.3 and 2.3 �g/mlat 18 mo and 2.0 and 4.3 �g/ml at 24 mo), but did not reach the adult GMCs (6.9and 17.5 �g/ml) by 24 mo of age. The increase in anti-BVH-3 and -11 was associ-ated with pnc contacts. 4 and 5 out of 28 children with pnc AOM, and 6 and 1 out of72 with non-pnc AOM, had a �2-fold increase in anti-BVH-3 and -11, respectively.At 12 mo, the anti-BVH-3 or -11 concentration did not affect or, at 18 mo slightlyreduced (RR= 0.8 with upper 95% CIs >1) the risk of pnc AOM. Previous pnccarriage was found to reduce and previous pnc AOM to increase the risk for pncAOM in the following 6 mo.Conclusions: Concentrations of anti-BVH-3 and BVH-11 ab increase with age andare strongly associated with pnc exposure. The ab concentration of the children ismarkedly lower than those seen in the adults.


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Effect of absorption with pneumococcal type 22F polysaccharide on maternaland cord blood and infant IgG anti-pneumococcal polysaccharide antibodies.

Inostroza J,1,2 Villanueva S,1 Mason K,3 Leiva L,4 Sorensen R.4 Hospital Regionalde Temuco,1 Universidad de la Frontera, Temuco, Chile,2 School of Public Health,3

School of Medicine, New Orleans, USA4

Aims: To evaluate the effect of absorption with pneumococcal type 22F polysaccha-ride on anti-pneumococcal antibody titers in unimmunized Chilean pregnant womenand on antibodies in their offspring at birth and 3, 6 and 12 months of age.Methods: Sera were obtained from 10 healthy women in the third trimester of preg-nancy and from their offspring cord blood and peripheral blood at 3, 6 and 12 monthsof age. All babies were born at term. IgG antibodies against serotypes 1, 3, 4, 5, 6B,9V, 14, 18, 19F and 23F were measured by a standardized ELISA method. All serawere absorbed with polysaccharide C, and aliquots of each serum were absorbedwith polysaccharide 22F. Antibodies in absorbed and non-absorbed sera were meas-ured simultaneously. Individual results were expressed in �g/ml based on the stand-ard serum pool 89-SF. The geometric means and confidence intervals, the percent-ages of reduction due to absorption and of transplacental transmission were deter-mined according to standard statistical methods and published definitions. Results:Absorption with polysaccharide 22F reduced antibody concentrations in all sam-ples and to all 10 serotypes studied. Reduction was highest in maternal sera and incord blood, but it was also present at 3, 6, and 12 months of age. The percent reduc-tion ranged from 24% for serotype 14 to 56% for serotype 1 in maternal samples.The reduction ranged from 21% for serotype 18C to 52% for serotype 4 in cordblood samples. The percentages of transplacental transmission varied from 27% forserotype 14 to 69% for serotype 3 in absorbed sera pairs. These percentages weresimilar in non-absorbed sera.Conclusions: Absorption with serotype 22F had only a small effect on the percentof transplacental transmission. It had a significant impact, however, on anti-pneu-mococcal antibody concentrations in pregnant women and in cord blood. It alsosignificantly decreased the concentrations of antibodies throughout the first year oflife.

Effect of 1�,25-dihydroxyvitamin D3 on the murine antibody response to pneu-mococcal capsular polysaccharide.

Jeurissen A1, Van Etten E2, Overbergh L2, Mathieu C2, Bossuyt X1. ExperimentalLaboratory Medicine1, Experimental Medicine and Endocrinology2, Catholic Uni-versity Leuven, Leuven, Belgium.

Aims: To study the effect of 1�,25-dihydroxyvitamin D3 (Vit D3) on the murineantibody response to pneumococcal capsular polysaccharides (caps-PS).Methods: Three week old balb/c mice were treated either with Vit D3 or with arachidoil (AO). At the age of 8 weeks, the mice were immunized with caps-PS(Pneumovax®). Fourteen days after immunization, blood was drawn by heart punc-ture and IgM, IgG, and IgG subclasses against the capsular polysaccharide serotype3 were measured using ELISA. Furthermore, some balb/c mice were treated withIL-12 (1 µg/ml on day –1, 0, and 1) and immunized with Pneumovax® on day 0. Tostudy the effect of Vit D3 on the IL-12 production by dendritic cells, murine bonemarrow was cultured in the presence of GM-CSF, IL-4 with or without VitD3. Afterfive days of in vitro culture, MHC II, CD86, CD86, and CD54 were measured usingflow cytometry. Furthermore, IL-12 concentration was measured in the supernatantusing by ELISA.Results: Balb/c mice treated with Vit D3 had a significant decreased IgG2a anti-caps-PS in comparison to the mice treated with AO. Administration of IL-12 to-gether with Pneumovax® to Balb/c mice resulted in a significant increase of IgG2ato caps-PS serotype 3. In vitro culture of bone marrow cells together with Vit D3resulted in a decreased expression of MHC class II, CD86, CD80, and CD54. Fur-thermore, dendritic cells cultured in the presence of Vit D3 had a decreased IL-12secretion.Conclusions: Vit D3 inhibits the IgG2a anti-caps-PS antibody response. This effectis mediated through the inhibitory effect of Vit D3 on the maturation of the den-dritic cells, especially through the suppression of the IL-12 production by dendriticcells.


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CD1 does not play a role in the murine immune response to pneumococcalcapsular polysaccharides.

Jeurissen A1, Wuyts G1, Kasran A2, Boon L3, Ceuppens JL2, Bossuyt X1. Experi-mental Laboratory Medicine1, Experimental Immunology2, Catholic UniversityLeuven, Leuven, Belgium, MacroZyme B.V.3, Amsterdam, The Netherlands.

Aims: To study whether CD1 plays a role in the murine antibody response to pneu-mococcal capsular polysaccharides (caps-PS).Methods: Six to eight week old balb/c mice were treated either with a monoclonalblocking antibody to CD1 (20H2) or with ratIgG and were immunized with caps-PS (Pneumovax®). Fourteen days after immunization, blood was drawn by heartpuncture and anti-caps-PS antibodies were measured using ELISA. Furthermore,CD1 KO mice were immunized with Pneumovax® and after 14 days anti-caps-PSwere measured using ELISA. To study the effect of CD1 on stimulatory effect ofCD4(+) T lymphocytes and the inhibitory effect of CD8(+) T lymphocytes on theanti-caps-PS antibody response, SCID/SCID mice were injected with spleen cellsfrom balb/c mice from which either CD4(+) or CD8(+) T lymphocytes were de-pleted, with or without 20H2 and immunized with Pneumovax¨. Fourteen days afterimmunization, blood was drawn by heart puncture and anti-caps-PS antibodies weremeasured using ELISA.Results: There was no difference in the immune response to caps-PS between micethat were treated with 20H2 or with ratIgG. Furthermore, the anti-caps-PS anti-body response in CD1 KO mice was comparable to that in WT mice. Administra-tion of 20H2 to SCID/SCID mice did not affect the stimulatory effect of CD4(+) Tlymphocytes, or the inhibitory effect of CD8(+) T lymphocytes on the anti-caps-PSantibody response.Conclusions: CD1 does not play a role in the murine immune response to casp-PS.

Effect of IL-12 on the murine antibody response to pneumococcal capsularpolysaccharide is independent of the CD40-CD40L interaction.

Jeurissen A1, Matthys P2, Ceuppens JL3, Bossuyt X1. Experimental LaboratoryMedicine1, Laboratory of Immunobiology (Rega Institute for Medical Research)2,Experimental Immunology3, Catholic University Leuven, Leuven, Belgium.

Aims: To study whether the stimulatory effect of IL-12 on the murine antibodyresponse to pneumococcal capsular polysaccharides (caps-PS) is dependent on theCD40-CD40L interaction.Methods: Six to eight week old balb/c and IFN-� KO mice were treated with IL-12(1 µg/ml on day –1, 0, and 1) and immunized with caps-PS (Pneumovax®) on day0. Fourteen days after immunization, blood was drawn by heart puncture and IgM,IgG, and IgG subclasses against capsular polysaccharide serotype 3 were measuredusing ELISA. The role of the CD40-CD40L interaction was studied by administer-ing a blocking anti-CD40L (MR1) monoclonal antibody.Results: Balb/c mice treated with IL-12 had a significant increased IgG2a anti-caps-PS antibody response in comparison to the control mice, which could not beblocked by the administration of MR1. On the other hand, treatment of IFN-� KOwith IL-12 resulted in a significant increase of IgG1, IgG2b and IgG3 anti-caps-PSantibodies. Again, this increase could not be blocked by MR1 administration.Conclusions: The IL-12 mediated increase of anti-caps-PS IgG2a in balb/c miceand of anti-caps-PS IgG1, IgG2b, and IgG3 in IFN-� KO mice is independent of theCD40-CD40L interaction.


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CD4 T cell involvement in host response to pneumococcal infection

Kadioglu A, Coward W, Andrew PW. Department of Infection, Immunity & Inflamma-tion, University of Leicester, UK.

Aims: We had previously shown that T cells accumulated in peribronchiolar and perivas-cular areas of lungs soon after intranasal infection with Streptococcus pneumoniae. Wenow further investigated the involvement of T cells in pneumococcal disease by usingMHC class-II knockout mice that exhibit a lack of CD4 + T cellsMethods: Streptococcus pneumoniae serotype 2, strain D39 (NCTC 7466) was usedfor intranasal infections. Female MHC class-II knockout (A 0/0) and their isogenicparent strain C57/BL6 mice were infected. All mice were 8-10 weeks old when usedand weighed 30-35 g. These mice did not have detectable levels of anti-type 2 or anti-pneumolysin antibodies. Mice were lightly anaesthetised and fifty microlitres PBScontaining 1 x 106 cfu S. pneumoniae were then administered into the nostrils of mice.At pre-chosen time intervals following infection, groups of mice were deeply anaes-thetised with 5% (v/v) fluothane and blood was collected by cardiac puncture. Subse-quently, the mice were sacrificed by cervical dislocation and the lungs homogenised.Viable counts in homogenates and blood were determined by serial dilution and plat-ing onto blood agar plates.Results: The number of wildtype pneumococci in the lungs of MHC-II ko mice wassignificantly higher than in control mice over the whole 72 hr period post-infection (P<0.01for all time points). Notably, control mice had cleared the infection from their lungs by 48hrs post infection, while MHC-II ko mice still had bacteria in their lungs at 72 hrs. Wild-type pneumococci were detected in the blood of both control and ko mice by 24hrs postinfection, however, ko mice had significantly higher numbers of bacteria over 24-72hrperiod as compared to control mice (P<0.01). Again, control mice had cleared their bacter-aemia by 48 hrs post infection, while MHC-II ko mice still had bacteria in their blood at72 hrs with no indication of declining numbers. By 72 hrs, MHC-II ko mice had becomemoribund and had to be sacrificed. Control mice (C57/BL 6) showed no signs or symp-toms of illness throughout the period of the experiment.Conclusions: We have shown that MHC-II knockout mice, which are effectively CD4T cell negative, are significantly more susceptible to pneumococcal bronchopneumo-nia and septicaemia than their isogenic wildtype parents. These results emphasize theimportant role of the T cell in the host response to pneumococcal infection.

Structure of pneumococcal lipoteichoic acid (LTA) in stimulating Toll-likereceptor 2

Han SH1,2, Kim JH1, Nahm MH1. University of Alabama at Birmingham1, USA,International Vaccine Institute, Korea2.

Aims: To investigate the ability of pneumococcal LTA to stimulate cells via Toll-like receptor (TLR).Methods: Pneumococcal LTA was purified from R36A strain by Bligh-Dyer ex-traction, hydrophobic interaction chromatography, and then ion exchange chroma-tography. LTA with a single acyl chain (LTA-1) was prepared by ion exchange chro-matography at a higher pH. Completely deacylated LTA (LTA-0) was obtained byalkali hydrolysis. Their ability to stimulate TLRs was studied with human periph-eral blood monocytes (PBMCs) and two model cell lines, CHO/CD14/TLR2 andCHO/CD14/TLR4, which express human CD25 proteins in response to TLR2 andTLR4 stimulation, respectively.Results: Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner, but did not stimulate CHO/CD14/TLR4cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureusLTA in stimulating CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcalteichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells evenat high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine,or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However,antibodies against CD14, TLR1, or TLR2 suppressed TNF-� production by PBMCin response to LTA or LTA-1.Conclusions: Pneumococcal LTA stimulates PBMC primarily via TLR2 with thehelp of CD14 and TLR1 and lipid moieties of the LTA are important in TLR2 stimu-lation.


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Development of an elderly mouse model of pneumococcal infection

A.R. Kerr1, P.V. Hermand2 and T.J. Mitchell1. 1Division of Infection & Immunity,IBLS, University of Glasgow, Glasgow, UK and2 GSK Biologicals, Rue de l’Institut,Rixensart Belgium.

Aims: Most cases of pneumococcal infection occur in children under 2yrs or inadults older than 65yrs. The immature status of the immune response in youngchildren is better understood than the immune deficiencies that occur during theaging process. We hope that identification of the differences between aged and youngmice in a murine model of pneumococcal infection will permit the design of moreefficient treatments and vaccines for the aging population.Methods: Various ages of mice were infected intranasally with different serotypesof Streptococcus pneumoniae. Two infectious doses were used. Survival times andbacterial loads were determined. Some animals were passively immunised with anti-capsular antiserum prior to infection.Results: Following intranasal infection, aged mice have significantly shorter sur-vival times and significantly higher bacterial loads within lung airways, lung tissueand the bloodstream. During passive immunisation studies, protection of aged micerequires higher amounts of anti-capsular antiserum than does protection of youngeranimals. This indicates that the immune response of aged mice is impaired in theability to clear pneumococci even when provided with functional antibody.We are currently evaluating the production of inflammatory cytokines and histopa-thology during pneumococcal pneumonia in aged mice as well as comparing theopsonophagocytic activity of cells from young and elderly mice.Conclusions: By using different pneumococcal serotypes, infectious doses and agesof mice we have developed a reproducible model where aged mice are significantlymore susceptible to pneumococcal pneumonia than are young controls. We haveused this model to investigate the differences in the immune response between youngadult and elderly mice during pneumococcal infection.

Phenotypic and Functional Analysis of HL-60 Cells During Induction of Dif-ferentiation for Opsonophagocytic Assay

Kim KH , Seoh JY, College of Medicine, Ewha Woman’s University, Seoul, Korea

Aims: HL-60 can be induced into phagocytes, eliminating the need for human PBLdonors for OPKA. To identify the effect of N-N dimethylformamide(DMF), all-trans retinoic acid(ATRA) and 1�-25-dihydroxycholecalciferol(vitamin D

3) on the

surface marker expression and function to perform opsonophagocytic killing, weinvestigated phenotypic and functional changes as well as apoptosis change duringdifferentiation of HL-60 cells.Methods: After HL-60 cells were induced to differentiate by adding 0.8% DMF, 1�M ATRA or 20�g/mL vitamin D

3, we quantified the expression of immunoglobu-

lin receptors Fc�RI and Fc�RII, LPS receptor CD14, and adhesion molecules CD11c/CD18. We also performed functional studies including respiratory burst andopsonophagocytic assay and apoptotic cell ratio before and 1, 2, 3, 4 and 5 daysafter the stimulation with each inducers.Results: There were no significant differences in Fc RI expression. Fc RII ex-pression increased significantly by all three inducers, which supports that it is themain receptor of immunoglobulins in mature phagocytes. When induced by vita-min D

3, CD14 expression increased significantly. CD11c expression also increased

significantly by all three inducers. DMF stimulated HL-60 cells showed earliestrise of opsonophagocytic titer, however, by day 5 there were no significant differ-ences by the 3 inducers. HL-60 differentiated by ATRA or vitamin D3 showed sig-nificantly higher levels of respiratory burst than DMF. Vitamin D3 protects HL-60cells from apoptosis.Conclusions: The granulocytic differentiation by DMF gave good differentiationwhen compared to ATRA or vitamin D

3 to possess the cell receptors necessary for

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Improved efficiency of pneumococcal opsonophagocytic killing assay bymultiplexing and by colorizing colonies.

Kim KH 1,2 , Yu J 1, Nahm MH 1. University of Alabama at Birmingham, AL, USA1,Ewha Woman’s University, Seoul, Korea2

Aims: In evaluating pneumococcal vaccines, an opsonophagocytic killing assay(OPKA) is useful as a supplement to pneumococcal antibody ELISA. However,evaluations of pneumococcal vaccines require determination of antibody responsesto 7-11 serotypes, and OPKA is tedious to perform and requires more serum thanELISA. Consequently, OPKA is infrequently used in evaluating pneumococcalvaccines. To overcome these limitations, we have developed a simple multiplexed(double-serotype) OPKA.Methods: OPKA was done using antibiotic-resistant pneumococci for nine serotypes.Serotype 6B, 9V, 19A, and 23F strains were made streptomycin-resistant and sero-type 4, 6A, 14, 18C, and 19F strains were made optochin-resistant. The multiplexedOPKA was the same as the single-serotype OPKA except for two changes. First,the target bacteria were a mixture of one streptomycin-resistant strain and oneoptochin-resistant strain. Second, the surviving bacteria of each serotype were enu-merated by plating on Todd-Hewitt agar plates with yeast extract with an agar over-lay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride(TTC). The performance of the multiplexed OPKA was evaluated by analyzing 28serum samples from adults immunized with a 23-valent PS vaccine with the single-serotype and the multiplexed OPKA.Results: The multiplexed OPKA was specific for the desired serotypes. Themultiplexed and conventional OPKAs had comparable assay sensitivity and pro-duced results that were highly correlated (r2 values ranging from 0.92 to 0.98) forall nine serotypes.Conclusions: A simple modification of the conventional OPKA produces amultiplexed assay that greatly reduces effort, reagents, and the necessary amount ofserum.

Sources of variability in results of opsonophagocytic killing assay for antibod-ies to the pneumococcal capsule

Kim KH 1,2 , Nahm MH 1, University of Alabama at Birmingham, AL, USA1, EwhaWoman’s University, Seoul, Korea2

Aims: An opsonophagocytic killing assay (OPKA) measures the protective capacityof antibodies and is useful in evaluating pneumococcal vaccines. With recent im-provements, OPKA has become easier to perform. However, OPKA results are quitevariable and may be more variable than ELISA results. To identify the source(s) ofvariability, we investigated the effect of varying HL-60 cells, bacterial stocks, com-plements or persons on OPKA titers of 4 serum pools.Methods: Four serum pools were made with sera from adults who were immunizedwith one dose of the 23-valent pneumococcal vaccine. OPKA was performed forfour serotypes (serotypes 4, 6B, 9V, and 19F) using a multiplexed OPKA (Kim et al.Clin Diagn Lab Immunol 2003;10:616-21). To determine intra-assay variability,the sera were tested 10 times in one assay run. To determine a short-term inter-assayvariability, sera were tested in 11 independent assays with same lot of reagents. Todetermine the effect of varying assay components, the assay was performed 5 timeswith two different batches of bacterial stock and performed once with three differ-ent batches of complement. To assess inter-personal variability, OPKA was per-formed by three different persons.Results: Coefficient of variation (CV) of OPKA results ranged from 12.4% to 33.9%and this is comparable to the acceptable level of CV of a pneumococcal ELISA,which is 35% or less for at least 85% of the samples (Plikaytis et al. J Clin Microbiol2000;38:2043-50). At this moment, the source of imprecision is mainly “intra as-say”; and reagents or personnel may contribute less for the assay imprecision.Conclusions: Since OPKA CV is independent of reagents lots and operators, theassay may be performed in many laboratories. Thus, standardized OPKA may bepossible.


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Purification of intact and lyso-form of pneumococcal lipoteichoic acid (LTA)with different biological properties

Kim JH1, Han SH1,2, Seo HS1, Sorensen U3 and Nahm MH1. University of Alabamaat Birmingham1, USA, International Vaccine Institute, Korea2, and Department ofMedical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark3.

Aims: To develop a method of purifying pneumococcal LTA with uniform biologi-cal properties.Methods: Pneumococcal LTA from R36A strain was purified by Bligh-Dyer or-ganic solvent extraction, hydrophobic interaction chromatography, and ion exchangechromatography. Biological properties of purified LTA preparations were tested bymeasuring their ability to stimulate human (THP-1) and mouse (RAW 264.7) mac-rophage cell-lines to express TNF-� and IL-1� mRNA using ELISA and RT-PCR,respectively.Results: LTA obtained by ion exchange at pH 9.5 (LTA-9.5) has the mass spectrawith three major peaks of 7248.4, 8547.5, and 9846.2 m/z units, which respectivelycorrespond to LTA with 5, 6, or 7 repeating units and are identical to the resultsobtained with pre-ion-exchange samples. LTA purified by ion exchange at pH 10.5(LTA-10.5) has the mass spectra with three major peaks of 6985.5, 8284.4, and9582.8 m/z units and are found to have lost one acyl chain (i.e., lyso-form). Allbatches of LTA-9.5 efficiently stimulates both human and mouse macrophages.However, LTA-10.5 efficiently stimulates human but not mouse macrophages.Conclusions: A small change in the purification method can yield products withdifferent structure and immune functions. Mouse and human cells can differ intheir responses to LTA.

Early bacterial colonisation, mucosal immune responses and disease suscepti-bility

Kyaw-Myint S M1, Cripps A W 1,2, Kyd J M1, Foxwell A R1, Bowman J 3, Riley T V3,4, Jacoby P 5, Lehmann D. 5 for the Kalgoorlie Otitis Media Research ProjectUniversity of Canberra, ACT1, Griffith University, Gold Coast, Qld2, Division ofMicrobiology & Infectious Diseases, PathCentre, Perth3, Department of Microbiol-ogy, University of Western Australia, Perth4, Telethon Institute for Child HealthResearch, University of Western Australia5, Australia

Aims: This study focuses on the development of salivary antibody responses to 3bacterial pathogens from birth to age two years among children living in an urban/peri-urban setting in an arid part of Australia.Methods: As part of a cohort study investigating causal pathways for otitis media(OM), saliva and nasopharyngeal aspirates/nose swabs were collected at age 1-3weeks and at 6 follow-up visits up to 24 months. We have collated salivary anti-body data from 218 samples from 71 Aboriginal and 481 samples from 144 non-Aboriginal children. Total salivary IgA levels and specific salivary IgA responsesagainst pneumolysin toxoid, whole Streptococcus pneumoniae (Pnc), Haemophilusinfluenzae (Hi) proteins OMP26 and P6, whole Moraxella catarrhalis (Mc) and anM. catarrhalis protein, OMPE, were measured by ELISA.Results: After preliminary analyses of the data the following observations are ap-parent: 1) At ages 3-15 months more Aboriginal children had IgA responses towhole Pnc (~ 70% ) than non-Aboriginal children (~50%); 2) In Aboriginal andnon-Aboriginal children, immune responses to pneumolysin, OMP26 and OMPEwere highest at 1-3 months of age, declining thereafter, while responses to otherantigens did not decline with age; 3) Initial specific antibody responses againstPnc, Hi and Mc appeared to be driven by upper respiratory tract colonisation.It is hypothesised that: 1) Down regulation of the immune response to virulentbacterial antigens occurs following colonisation. 2) This shifts the balance in fa-vour of the bacteria and therefore increases susceptibility to disease. This study isdesigned to test these hypotheses.


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The Immune Response during Pneumococcal Colonization of the Murine Na-sopharynx

T.L. McCool and J.N. Weiser. Department of Microbiology, University of Pennsyl-vania, Philadelphia, PA

Aims: A mouse model of pneumococcal colonization was developed to analyze theimmune mechanisms that lead to clearance of pneumococcal carriage.Methods: C57BL/6 and BALB/c mice were inoculated intranasally with a type 23Fpneumococcal clinical isolate. Colony counts were performed on nasal washes, blood,and lung tissue in order to establish a time course of colonization. Comparison ofthe level of colonization (CFU/ml) with antibody titer and antibody specificities,including pneumococcal surface protein A (PspA), phosphorylcholine, capsularpolysaccharide, and choline binding protein A (CbpA), will be performed at eachtime point.Results: A S. pneumoniae clinical isolate used to experimentally colonize humanssuccessfully colonized mice but did not result in any significant lower respiratoryor invasive disease. Similar to natural clearance observed in humans, C57BL/6 miceclear pneumococci from the nasopharynx within 6 weeks of inoculation. The influ-ence of host genetic background on the ability of pneumococcus to colonize themurine nasopharynx was also studied. The time needed to clear nasal colonizationvaried depending on the mouse strain used. 80% of C57BL/6 mice clear pneumo-coccal nasopharyngeal colonization by 28 days post inoculation, whereas only 40%of BALB/c mice have undetectable numbers of pneumococcus. In addition, thedensity of colonization of C57BL/6 mice is ten-fold less CFU/ml nasal wash thanthe BALB/c mice.Conclusions: A mouse model of experimental pneumococcal colonization was es-tablished that resembles an experimental model of human pneumococcal coloniza-tion. Variation in mouse strain background influences the level of pneumococcalcolonization and the rate at which the pneumococcus is cleared from the murinenasopharynx. Utilization of these mouse models will allow for the identificationand characterization of antibodies that may promote clearance.

Development of Family-specific Antibodies to Pneumococcal Surface ProteinA (PspA) in Relation to Pneumococcal (Pnc) Contacts

Melin M1, Hollingshead S2, Briles D2, Kaijalainen T3, Kilpi T1, Käyhty H1. 1NatlPubl Health Inst, Helsinki, Finland; 2Univ of Alabama, Birmingham, Al; 3Natl PublHealth Inst, Oulu, Finland

Background: PspA is an important virulence factor of S.pneumoniae. PspAs aredivided into two major families, which include variable but cross-reacting proteins.Our previous studies with a truncated family1 PspA antigen suggested that youngchildren develop only low levels of anti-PspA antibodies after Pnc carriage or in-fection. We extended the studies of naturally induced anti-PspA antibodies in chil-dren and in adults by using both family1 and 2 antigens and by family typing thePspAs of the children´s Pnc contacts.Methods: A serum sample from 60 mothers and consecutive sera (2-5; N=142) of47 children under 2 yrs of age with Pnc carriage or acute otitis media (AOM) wereselected from the FinOM Cohort Study. The antibodies to family1 and 2 PspA weremeasured by EIA. Pneumococcal nasopharygeal and middle ear isolates of childrenwere PspA-family typed by whole cell EIA.Results: Children had detectable concentrations of antibodies to PspAs as often asadults (75-83%). The mean antibody concentrations were 5- to 6-fold higher inadults than in children. Antibody concentrations to family1 and family 2 PspA cor-related moderately for adult sera (r=0.55), but poorly for the sera of children (r=0.22).Half of the children had a homologous antibody response to the PspA family theyhad contacted. A simultaneous increase in both anti-family1 and -family2 PspAconcentrations was found in one third of the children, although pneumococci ofonly one family type PspA were isolated.Conclusions: Children can have a relatively family specific anti-PspA response af-ter Pnc contact. The serum anti-PspA antibodies of children are less cross-reactivebetween the families than those of adults. First pneumococcal contacts appear toprime for a higher response after the consecutive contacts. It would be desirable toinclude several types of PspAs in the future pneumococcal vaccine.


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The effect of pneumococcal (Pnc) capsular serotype on the deposition of com-plement protein C3 and on the functional activity of anti-capsular antibodies

Melin M1, Jarva H2, Meri S2, Käyhty H1. 1Natl Publ Health Inst, Helsinki, Finland;2Univ of Helsinki, Helsinki, Finland

Background: The capsule protects pneumococcus from phagocytosis, but anticapsularantibodies in combination with the complement system enhance phagocytosis. In aFinnish vaccine efficacy study Pnc conjugate vaccines protected poorly against acuteotitis media caused by capsule serotype 19F, whereas protection was good againsttype 6B. In the US and South Africa, vaccines protected children equally from inva-sive infections caused by types 6B and 19F. Differences in the capsular structuremay influence the deposition and degradation of C3b on the surface of Pnc, whichplays a role in opsonophagocytosis.Methods: Pneumococcal nasopharyngeal, middle ear and blood isolates (N=18 sofar) of serotypes 6B and 19F were chosen for comparisons. Bacteria were incubatedwith human serum for 1, 3, 10 and 30 minutes and the amount of C3 and iC3bbound on the bacteria was measured by EIA and Western blot. The bacteria wereincubated with factor H and direct binding was measured. The functional activity ofsera of children vaccinated with a Pnc conjugate vaccine against the type 6B and19F strains were determined by opsonophagocytic killing assay (N= 5 sera/strain).Results: Four to 5 times higher anticapsular antibody concentrations were requiredfor 50% opsonophagocytic killing of type 19F strains compared to type 6B. Onaverage, more C3b and iC3b molecules attached to the surfaces of type 6B strainsin comparison to type 19F strains. The binding of factor H was not related to theserotype.Discussion: The binding and degradation of complement protein C3b on pneumo-coccal surface is related to the serotype and may influence the level ofopsonophagocytosis, since the levels of C3b binding are consistent with resultsreceived in the opsonophagocytic assay.

Searching for Antimicrobial peptides against Streptococcus pneumoniae - pos-sible answers for pathology and new strategies against antibiotic resistant bac-teria

Meyer-Hoffert U1,2,3, Harder J2, Schröder JM2, Henriqus Normark B1,3. Swedish In-stitute for Infectious Disease Control, Stockholm, Sweden1; Department of Derma-tology, University Kiel, Germany2, Microbiology and Tumor Biology Centre,Karolinska Institute, Sweden

Aims: The aim of this study is to discover endogenous antimicrobial peptides againstSreptococcus pneumoniae. Antimicrobial peptides might explain control of coloni-zation and exhibit therapeutic potency against antibiotic resistant strains.Methods: We chose two independent strategies to discover antimicrobial peptidesagainst streptococcus. First, we screened natural tissue for fractions with antimicro-bial peptides. After acetic acid extraction, heparin binding and purification by highperformance liquid chromatography (HPLC, Reverse Phase-8 column) we analysedhealthy human skin, a proven source for antimicrobial peptides, for the presence ofantimicrobial fractions against S.pneumoniae. As an alternative approach we specu-lated, whether purified RNase7, a human antimicrobial peptide able to kill variousGram-positive bacteria, exhibits antimicrobial activity against various S.pneumoniaestrains.Results: Two independent fractions from RP-HPLC of healthy skin-derived cati-onic peptides and proteins exhibited antimicrobial activity against S.pneumoniaie(ATCC 33400). Though RNase7 killed different Gram-positive bacteria like Vanco-mycin-resistant Enterococcus faecalis and Staphylococcus aureus it failed to killthe tested S.pneumoniae strains in this study.Conclusions: We conclude that there are antimicrobial peptides against S.pneumoniaepresent in human tissues, which are currently under further characterization. ThoughRNase7 is a potent antimicrobial peptide against Gram-positive bacteriaS.pneumoniae are resistant against RNase7-induced killing. The mechanism of thisresistance has to be explored in further studies.


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New approach to study immunogenecity of pneumococcal proteins

Vainio A1, Fagerlund R1, Melen K1, Lehtinen MJ2, Julkunen I1, Virolainen A1. Na-tional Public Health Institute, Helsinki1, University of Helsinki2, Finland.

Aims: The purpose of this study was to analyse whole genome sequence of Strepto-coccus pneumoniae in order to produce pneumococcal proteins for pathogeneticand immunological studies.Methods: Based on published whole genome of one pneumococcal serotype (Tettelinet al., 2001), 36 genes coding for pneumococcal surface proteins were chosen forhomology search (psi-BLAST) and comparative protein analysis of known con-served domains. Four genes were chosen for amplification by PCR and cloning intoexpression vectors (pCR®II-TOPO®; PproEX™Htb). In vitro translation was doneby TnT® Coupled Reticulote Lysate Systems, and expression by IPTG induction inE.coli BL-21 (DE3). Recombinant S. pneumoniae protein production was con-firmed by SDS-PAGE, and the proteins were purified by preparative SDS-PAGEelution electrophoresis. His-tagged fusion protein was also detected by His-spesificantibody using Western blot. The immunogenecity of produced proteins was testedusing Western blot with human sera from children with or without pneumococcalcarriage or disease.Results: We were able to clone and express several pneumococcal surface proteinsbut only one was produced in E.coli in high enough yields enabling its purification.The DNA sequence of this protein codes for a putative proteinase maturation pro-tein A (PpmA). The denatured protein showed immunogenecity when studied withhuman sera of children with pneumococcal carriage or otitis media by Western blot.Conclusions: Computer assisted analysis of whole genome sequence offers newinsight and possibilities for studying pathogenesis of pneumococcal disease andsearch for new protein vaccine candidates.

Mucosal immunity to pneumococcal capsular polysaccharides in Australianindigenous children

WS Pomat1, Thornton R1, Morris P2, Leach AJ2, Richmond P1. University of West-ern Australia School of Paediatrics & Child Health, Perth1, Ear Health & EducationUnit, Menzies School of Health Research, Darwin2, Australia.

Background: Australian indigenous children in northern Australia have high ratesof chronic persistent otitis media with frequent sequelae. Nasopharyngeal carriagewith otitis media pathogens begins in the first month of life and precedes the devel-opment of otitis media. Little is known about the development of mucosal immu-nity in these children and whether early colonisation leads to impairments in patho-gen specific mucosal immunity and susceptibility to disease.Methods: Infants were recruited in the first 3 months of life into a randomisedtrial of antibiotics for the prevention of otitis media. Children were monitored forotitis media and had nasopharyngeal swabs for bacterial carriage and saliva sam-ples collected at frequent intervals over the first 2 years of life. Salivary IgA re-sponses to pneumococcal capsular polysaccharide serotypes 6B, 14, 19F and 23Fwere evaluated using a standardised ELISA. Laboratory staff remain blinded totreatment groups and outcomes.Results: To date, 175 samples have been analysed from 34 infants for these 4 serotypes.Detectable serotype-specific IgA responses were seen from 3 weeks of age. In thefirst 6 months, 31% (23F), 44% (6B), 45% (14) to 52%(19F) of samples had detect-able IgA. The duration of salivary IgA was generally short lived despite high carriagerates. The proportion of samples with positive IgA from 6 to 24 months of age washigher for 19F (61%) and 23F (37%) but less for serotypes 6B(26%) and 14 (33%).Conclusions: Indigenous infants are able to mount mucosal IgA responses to pneu-mococcal capsular polysaccharides however responses appear short lived. Expo-sure to pneumococcal polysaccharides through carriage in early infancy may im-pair development of mucosal immune memory. Further studies will analyse spe-cific salivary IgA responses to pneumococcal surface proteins and will correlateIgA responses to frequency of carriage, antibiotic use and frequency of otitis mediaand its complication


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Genetic background affects recovery from pneumococcal bronchopneumonia

Preston JA1,2, Beagley KW1,2, Gibson PG2,3 & Hansbro PM1,2. University of Newcas-tle1, Hunter Medical Research Institute2, John Hunter Hospital3, Newcastle, Australia.

Aims: To establish a recovery nonfatal (recovery) model of pneumococcal bron-chopneumonia and to examine the effects of genetic background on recovery.Methods: Several different doses and strains of pneumococci were used to developa nonfatal model. High doses of NC012695 resulted in death of BALB/c mice but1x105 cfu induced mild acute clinical illness from which mice rapidly recovered(within 6h). Symptoms correlated with bacterial colonisation. Pneumococci grewin lung tissue over the first 4h but were completely cleared after 24h. Clearance wasassociated with an influx of inflammatory cells into the lungs, which initially com-prised largely of neutrophils (4-24h) but these were gradually replaced bymacrophages (48h-15d). There was also a gradual lymphocyte influx into the lung,which initially involved mainly CD8 T cells (4-12h), which were later reinforced byCD4 T cells (24h-10d). Inflammatory responses resulted in clearance of the bacte-ria but also lung damage, which was observed by histopathology. This followedimmune cell influx and peaked at 12h but tissues recovered and showed no inflam-mation after 10 days. The pathogenesis of recovery from pneumococcal bronchop-neumonia was then characterised in C57BL/6 mice with a Th1-type genetic back-ground and compared with results from BALB/c mice, which have a Th2-type ge-netic background. Pulmonary and peripheral inflammatory responses (phagocyto-sis & oxidative burst) were also compared.Results: The nonfatal model was used to show that C57BL/6 mice had a reducedimmune cell influx and tissue damage in response to pneumococcal infection com-pared to BALB/c mice. Reduced phagocytosis and oxidative burst was also ob-served in C57BL/6 mice.Conclusions: A nonfatal pneumococcal bronchopneumonia model was developed.C57BL/6 mice had significantly reduced immune and inflammatory responses to lowdoses of pneumococci. This resulted in less inflammatory damage but these responsesare overwhelmed by higher doses [Gingles et al., Infect Immun 2001:69;426-34]. Thusgenetic background affects susceptibility to pneumococcal infection.

THE IMMUNE RESPONSE AGAINST S.pneumoniae: LESSONS FROM PA-TIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY

Quinti I, Rosado MM, Donnanno S, Guazzi V, Soresina AR, Cagliuso M, Plebani A,Aiuti F, Carsetti RDps of Allergy and Clinical Immunology, University of Rome “La Sapienza” andof Peadiatrics, University of Brescia, and Ospedale Pediatrico Bambino Gesù, Rome,Italy

Aim: Recurrent lower respiratory tract infections by encapsulated bacteria lead topermanent organ damage in most CVID patients. Despite the profoundhypogammaglobulinemia, however, some patients do not suffer from bacterialpneumonias. IgM memory B cells and natural antibodies play an important role inthe defence against encapsulated bacteria. We asked the question of whether sus-ceptibility to respiratory infections was correlated to the frequency of IgM memoryB cells and to the level of anti-pneumococcal polysaccharide IgM in CVID pa-tients.Methods: We studied 54 patients: 26 had recurrent bacterial pneumonias and bron-chiectasis (group 1); 22 never had pneumonias and showed no lung lesions (group2); 6 had recurrent pneumonias, but no permanent pulmonary damage (group 3).We analysed B-cell subsets and levels of anti-pneumococcal polysaccharide IgMantibodies and used the Mann-Whitney U-test (p-values) for comparison of results,and the Spearman’s rank correlation coefficient (r-values) to correlate group char-acteristics.Results: CVID patients with and without severe infections differ in the frequency ofIgM memory B cells in the peripheral blood. IgM memory B cells were signifi-cantly reduced only in patients with recurrent pneumonia and their frequency cor-related with the levels of anti-pneumococcal polysaccharide IgM.Conclusions: Our data shows that in CVID patients susceptibility to encapsulatedbacteria infection is associated to a low frequency of IgM memory B cells and to alow level of anti-polysaccharide IgM. Evaluation of these two parameters may helpto plan a more effective therapeutic strategy.


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Pneumococcal carriage in adults induces increases in the functional activity ofantibodies

Sundström C1, Soininen A1, Goldblatt D2, Melegaro A3, Ashton L2, Andrews N3,Pebody R3, George R3, Edmunds J3, Miller L3, Gay N3, Käyhty H1. National Insti-tute of Public Health, Helsinki, Finland1, Institute of Child Health, University Col-lege London2, Health Protection Agency, London3.

Aims: We have determined the opsonophagocytic activity (OPA) of pneumococcalantibodies of adults in relation to contacts with Streptococcus pneumoniae (SPN).Methods: 139 families with young children were enrolled. Nasopharyngeal swabsfor SPN cultures were taken monthly for 10 months. The detected SPN isolateswere serotyped. Venous blood was taken from the parents at the beginning (month1) and in the end (month 10) of the study. We determined the OPA of anti-bodies forSPN of serotypes (ST) 6B, 14, 19F and 23F in selected (N= 81) mo 1 and mo 10sera, which were assigned into groups: 1) carriers of the ST in question (Pnc+ST+),2) carriers of another ST (Pnc+ST-), and 3) no carriage even in the family (Pnc-).Results: Higher OPAs against ST 6B, 19F and 23F were measured in sera of thePnc+ST+ groups than of the Pnc+ST- or Pnc- groups. For ST 14, sera from thePnc+ST- group had higher OPAs than sera from the Pnc+ST+ or Pnc- groups. STspecific OPA titres increased usually during the 1 and 10 month interval, most no-tably for ST 14 in the Pnc+ST+ group. Antibody concentrations (EIA with 22Fneutralization) to ST 6B, 14 and 23F polysaccharides correlated with the OPA ti-tres. Although most of the sera had �1 �g/ml of antibodies to ST 19F, only few hadOPA to type 19F and the correlation between concentration and OPA remainedpoor.Conclusions: In adults, pneumococcal carriage seem to elicit the development offunctional antibodies and thus in part augment in the development of natural immu-nity to SPN. The development of OPA was ST specific with an exception of ST 14;the origin of high ST 14 OPA titres in the Pnc+ST- group needs to be studied fur-ther.

Role of Antibiotics in Release of Pneumolysin and Subsequent InflammatoryReactions.

Thornton J1, Rogers D2, McDaniel L1. University of Mississippi Medical Center,Jackson Mississippi1. University of Tennessee Health Science Center, Memphis,TN2,USA.

Aims: The purpose of this study was to determine host cell response to Streptococ-cus pneumoniae and specifically the virulence protein pneumolysin. Previousmicroarray analysis indicated differential gene regulation in response to pneumolysin.We evaluated the role different classes of antibiotics play in the release ofpneumolysin and the subsequent inflammatory response, as well as the productionof cell adhesion molecules in response to pneumolysin.Methods: Human THP-1 cells were exposed to media alone, Streptococcuspneumoniae D39 (wild type), or an isogenic mutant, PLN, which lacks pneumolysin.During exposure, media contained either no antibiotics or one of the following an-tibiotics at 5 times MIC: penicillin, tetracycline, vancomycin, levoquin. At 3 and 10hrs, cells were pelleted and supernatants were removed and frozen for ELISA analysisto detect IFN-�. THP-1 viability was checked at each time point. Bacterial countswere also taken to assess the number of killed pneumococci which should reflectthe release of intracellular pneumolysin. THP-1 cells were also exposed to purifiedrecombinant pneumolysin and RNA was purified at various time points for realtime PCR analysis using primers specific for cell adhesion molecules.Results: Penicillin and vancomycin led to a greater decrease in colony formingunits of D39 and PLN when compared with tetracycline and levoquin. Exposure toD39 led to higher levels of IFN-� compared with PLN in the presence of penicillin,tetracycline, and vancomycin. Exposure of THP-1 cells to purified pneumolysin ledto an up-regulation of cell adhesion molecules ICAM-1 and MAC-1/LFA-1 (betasubunit).Conclusions: Specific antibiotic treatment of pneumococci results in pneumolysinrelease that can affect the subsequent inflammatory response by triggering releaseof proinflammatory mediators and leading to activation and differentiation of lym-phoid cells.


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Title: Pneumococcal surface protein A and pneumolysin act in synergy to pre-vent complement activation by Streptococcus pneumoniae

Yuste J1, Botto M2, Paton J.C3, Brown J.S1: UCL1 and Imperial College London2,University of Adelaide3.

Aims: Pneumococcal surface protein A (PspA) and pneumolysin (Ply) may affectvirulence by preventing complement activity. We studied the interaction of theseproteins and the complement system using knock out mice with specific deficien-cies in complement components.Methods and Results: We investigated by flow cytometry the C3 deposition on theS. pneumoniae strain D39 and isogenic mutant strains with disruptions in pspA andply. C3 binding to the ply- strain compared to the wild-type strain was increased inserum from wild type (WT) mice but not in C1q-/- serum, indicating that Ply pre-vents classical pathway activity. In contrast, C3 deposition was increased on thepspA- mutant strain in C1q-/- serum suggesting that PspA interferes with the alterna-tive pathway. Loss of pspA and ply was strongly synergistic, with a large increase inC3 deposition on the pspA- / ply- mutant strain in WT serum. We analysed by mixedinfections in WT and complement knock-out mice whether PspA, and Ply preventcomplement activity in vivo. Both pspA- and ply- strains were attenuated in viru-lence compared to the wild-type strain in WT mice (CI of pspA- = 0.025, CI of ply-

= 0.21). In mice deficient of all complement activity (C3-/-), the relative virulence toD39 of both the pspA- and ply- strains improved (CI 0.15 and 0.60 respectively),demonstrating that loss of virulence in these mutant strains is at least partially de-pendent on complement activity. When compared to the ply- mutant the virulence ofthe double pspA- / ply- mutant strain in WT mice was dramatically reduced (CI0.005), but restored in C3-/- mice (CI 1.08), confirming the synergistic protectiveeffect of Ply and PspA against complement activity in vivo.Conclusions: PspA prevents alternative pathway dependent and Ply prevents classi-cal pathway dependent complement activation by S. pneumoniae. Loss of both Plyand PspA has a striking synergistic effect, making S. pneumoniae highly sensitiveto complement activity.

Deficiency of serum amyloid P component results in increased susceptibility toStreptococcus pneumoniae in a mouse model of infection

Yuste J1, Botto M2, Brown J.S1, University College London, England1, ImperialCollege London, England2

Aims: Serum amyloid P component (SAP) is a component of the acute phase reac-tion and may play a role in the immune responses to microbial pathogens. In thiswork we have investigated the role of SAP for innate immunity to S. pneumoniaeusing a genetically engineered mouse strain with SAP deficiency (SAP-/-).Methods and Results: Using a flow cytometry assay of C3 binding to S. pneumoniaewe found that the proportion of S. pneumoniae strain D39 bound with the comple-ment component C3 is reduced in SAP-/- serum (16.5% SD 2.6) compared to serumfrom wild-type mice (38.3% SD 2.2). Furthermore, when assessed by a flowcytometry assay the opsonophagocytosis of S. pneumonaie by a human neutrophilcell line was higher if the bacteria were incubated in wild-type serum compared toSAP-/- serum (71% SD 4.9 vs 12% SD 0.9 at a 1/40 dilution). The functional conse-quences of SAP deficiency were assessed in a mouse sepsis model of S. pneumoniaeinfection. Mice were challenged by intraperitoneal inoculation with D39. Althoughthe mortality of wild-type mice and SAP-/- mice were similar in this infection model,the numbers of bacteria recovered 24 hours after inoculation from both blood andspleen were higher from SAP-/- compared to wild-type mice. To investigate the im-mune response in more detail we analysed the proportion of activated macrophagesand lymphocytes recovered from infected spleens and measured the levels of in-flammatory cytokine in the serum. We observed an increase in activated CD4lymphocytes from SAP-/- spleens and in the levels of the inflammatory cytokinesTNF � and IFN gamma in SAP-/- sera compared with the wild-type, compatiblewith the increased numbers of bacteria recovered from SAP-/- mice.Conclusions: These results suggest that SAP assists innate immunity to S.pneumoniae, possibly mediated by SAP increasing complement binding to the bac-teria increased opsonophagocytosis. In contrast to complement deficiencies down-stream immune responses amongst lymphocytes are not impaired.


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Induction of chemokines and cytokines by pneumococcal proteins in humanfollicular dendritic cells

Zhang Q1, Bernatoniene J1, Mitchell T2 and Finn A1. Department of Clinical SciencesSouth Bristol, University of Bristol, UK1; Division of Infection & Immunity, Instituteof Biomedical & Life Sciences, University of Glasgow, UK2

Follicular dendritic cells (FDCs), reside in the secondary lymphoid follicles, play es-sential roles in the formation of germinal centres (GC) and in presenting intact antigento B cells. One of the principal functions of secondary lymphoid tissue is to bringantigen-presenting cells (APCs) and antigen-specific B and T cells into contact witheach other. FDCs may play a crucial role in this by secreting chemokines and cytokinesin response to microbial stimulation.Aims: To study the production of chemokines and cytokines by human FDC in re-sponse to pneumococcal proteins.Methods: FDC were isolated from human adenoids and purified using anti-humanFDC monoclonal antibody HJ2 and MACS separation. Immunophenotype of FDCswas studied by immunofluorescence staining and microscopy and flow cytometry. FDCswere cultured in RPMI medium with 10% FBS with or without antigen stimulation. Apneumococcal culture supernatant containing secreted pneumococcal proteins (CbpA,PspA, pneumolysin), and recombinant pneumolysin were used as antigen stimulants.A 79 cytokine/chemokine protein array and cytokine ELISAs were used to analysecytokine and chemokine production by FDC after stimulation.Results: The isolated FDCs have characteristics of in vivo FDC, expressing ICAM1(CD54), CD40, VCAM1, CD21 and stain positive for anti-FDC monoclonal anti-bodies CNA42 and HJ2. In an in vitro cell culture model we have shown that this cellline can support the growth and differentiation of adenoidal B cells. The proteinarray showed that stimulated FDCs express a variety of cytokine and chemokinesand that pneumococcal culture supernatant significantly upregulates the productionof ENA-78, GRO, IL-6, IL-8, MCP-1, MIP-1beta, and IP-10. Cytokine and chemokineELISAs confirmed the array results and showed that pneumococcal supernatant in-duces IL-6, IL-8 and MCP-1 production in a dose-dependent manner. Recombinantpneumolysin induced dose-dependent IL-8 production.Conclusion: The induction of chemokines by FDC upon stimulation by antigens de-rived from pneumococcus may be important in recruitment of antigen-specific T andB cells to the GC microenvironment in secondary lymphoid tissue of the humanupper respiratory tract, and thus may play a significant role in antigen-specific im-munity and immune memory.

Regulation of pneumococcal protein -specific antibody responses in adenoidalB cells from children by IFN-� and IL10

Zhang Q1, Bernatoniene J1, Arnaoutakis K1, Paton J2 , Finn A1. Department ofClinical Sciences South Bristol, University of Bristol, UK1, School of Molecularand Biomedical Science, University of Adelaide, Australia2.

Background: Studies in mice have suggested that pneumococcal protein antigensincluding CbpA, PspA, PsaA and pneumolysin may be vaccine candidates againstinvasive disease and/or carriage. We have shown previously that these proteins in-duce antigen-specific antibody responses in adenoidal B cells from children.Aims: To investigate the Th1/Th2 cytokine responses in adenoidal cell cultures afterstimulation with pneumococcal proteins and to determine which cytokines are im-portant in the regulation of antigen-specific antibody production.Methods: Concentrated culture supernatant (CCS) derived from a wild type pneu-mococcus, which contains secreted CbpA, PspA, PsaA and pneumolysin, confirmedby Western blotting, was used as protein antigen stimulant. Adenoidal mononu-clear cells were isolated and cultured in RPMI medium with the CCS. Cell culturesupernatants were collected at day 3 and 8 and tested by immunoassays for Th1/Th2cytokines (day 3) and for pneumococcal protein antigen-specific IgA and IgG anti-bodies (day 9). Cytokine mRNA expression was also assessed using RT-PCR.Results: Pneumococcal CCS stimulation induced significant production of IFN-�and IL-10 in the cell culture which correlated with protein antigen-specific IgGantibody production. There was no significant production of IL-2, IL5, IL12 orTNF� at the protein level, although there was induction of mRNA expression of IL-2 and IL-5. The addition of recombinant IFN-� or IL-10 to the cell culture en-hanced antibody production while neutralising antibodies to IFN-� or IL-10 sig-nificantly reduced antibody production. Recombinant IL-10 also significantly in-hibited IFN-� production by the adenoidal cells.Conclusions: Both IL-10 and IFN-� are important in pneumococcal protein anti-gen-specific antibody production in human adenoidal cell culture. These cytokinesseem to modulate antibody production independently.


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Impact of the 7-valent pneumococcal conjugate vaccine on the clinical courseof otitis media in 3 Australian Aboriginal communities.

Wigger C, MacKenzie G, Tipakalippa P, Latham V, Leach AJ, Morris PS. MenziesSchool of Health Research, Darwin, Australia.

Background: In remote Australian Aboriginal communities, the incidence of inva-sive and respiratory pneumococcal disease (including otitis media) is extremelyhigh. In some communities, 50% of Aboriginal infants have otitis media with effu-sion (OME) by around the age of 30 days and 60% will have experienced a perfo-rated tympanic membrane by the age of 12 months. The 7-valent pneumococcalconjugate vaccine (7vPCV) was introduced into the immunisation schedule for allAboriginal children in June 2001.Aim: The aim of this study is to see if the introduction of 7vPCV was associatedwith a reduction in the prevalence, severity, and duration of OM and pneumococcalcarriage in Australian Aboriginal infants.Methods: A prospective cohort study of OM and pneumococcal carriage was con-ducted in three remote communities from October 2001. All enrolled babies wereseen 2 to 4 weekly from birth to 1 year of age for ear examinations and nasopharyn-geal swabs. Episodes of pneumococcal carriage and all types of OM were docu-mented and changes in carriage serotypes and clinical course of OM described.Results: Carriage of vaccine serotypes comprised around 60% of the carriage typesin infants before the introduction of 7vPCV, compared to 30% in infants in the post-7vPCV group. Carriage of penicillin non-susceptible types has been substantiallyreduced. Overall, pneumococcal carriage has remained high (~80%). Similarly, theoverall rate of OM is unchanged. However, severe infections (AOM/wiP) are onlyaffecting around 30% of children.Conclusions: Pneumococcal carriage and OM remain common in this population.Further studies are needed to understand how the vaccine may affect the severity ofOM, the diversity of pneumococcal strains, and the long-term effect on penicillinnon-susceptible strains.

Dissection of Transition Site-Specific Contributions of Pneumococcal VirulenceFactors to Pathogenesis

Carlos J. Orihuela1, Geli Gao1, Kevin P. Francis2, Jun Yu2, and Elaine I. Tuomanen1.1Department of Infectious Diseases, St. Jude Children’s Research Hospital, Mem-phis, Tennessee, 2Xenogen Corporation, Alameda, California

Streptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media,community-acquired pneumonia, and bacterial meningitis in developed countries.We assessed the ability of mutants deficient in choline binding protein A (CbpA),pneumolysin (Pln), pyruvate oxidase (SpxB), autolysin (LytA), and pneumococcalsurface protein A (PspA) to replicate in the nasopharynx, lungs, and blood of in-fected mice and translocate from one anatomical site to the next. Intranasal, intrat-racheal, and intravenous models of disease were assessed in vivo using D39X, aderivative of D39 transformed with the gram-positive lux transposon cassette Tn4001luxABCDE Kmr. Briefly strains expressing lux are bioluminescent and may be visu-alized in vivo using a CCD camera. Bioluminescent images were supported byquantitation of bacterial titers in the relevant organs. Analysis of the mutants in thethree challenge models determined that CbpA and SpxB were required for pro-longed nasopharyngeal colonization, whereas mutants deficient in CbpA and NanAwere unable to transition from the nasopharynx to the lower respiratory tract. Oncelung infection was established, mutants deficient in Pln, SpxB, and LytA were at-tenuated for bacterial replication in the lungs and translocation into the bloodstream.In the bloodstream Pln and LytA were required for replication of the pneumococcito high titers although CbpA was required for invasion of the CSF. We concludethat transitions between body sites require virulence determinants distinct from or-gan specific replication.


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Rapid Decline of Macrolide Resistance in Streptococcus pneumoniae FollowingIntroduction of the Pneumococcal Conjugate Vaccine: A Population-Based As-sessment

Stephens DS, Zughaier S, Whitney CG, Baughman W, Arnold K, Schuchat A, FarleyMM, and the GA. Emerging Infections Program. Emory University; VA Med. Ctr.;CDC; and the GA. Dept. of Health, Atlanta

Aims and Methods: Using population-based surveillance and molecular typing of iso-lates, macrolide resistant invasive S. pneumoniae disease in metropolitan Atlanta wasassessed before and after the licensure in February 2000 of the 7-valent pneumococcalconjugate vaccine for young children.Results: The overall incidence of invasive pneumococcal disease in Atlanta fell 57%from 30.2/100,000 population (mean annual incidence 1994-1999) to 13.1/100,000 in2002 (p<.001) Declines were most dramatic in children <2 years (82%) (from 278/100,000, 1994-1999, to 50/100,000 in 2002) and 2-4 years (71% decrease), age groupstargeted to receive conjugate vaccine, but significant declines (54%, 25% and 39%,respectively) were also seen in adult populations 20-39, 40-64 and >65 years. Theincidence of macrolide resistant invasive S. pneumoniae disease in Atlanta, after in-creasing steadily from 4.5/100,000 in 1994 to 9.3/100,000 in 1999, declined 69% to(2.9/100,000) in 2002. The decline in incidence of macrolide-resistant invasive S.pneumoniae was led by a rapid decline in incidence of disease caused by mefE (ef-flux)-associated macrolide-resistant serotype 14 isolates. In children <2 years, forexample, the incidence of disease from mefE serotype 14 macrolide resistant isolatesdeclined 96%, from 74.9/100,000 to 2.7/100,000, between 1999 and 2002 (p<.001).Declines in incidence of disease caused by mefE- and erm-mediated macrolide-resist-ant isolates of conjugate vaccine serotypes, 6B, 9V, 19F and 23F, and the vaccine-associated serotype 6A were also observed. Declines, especially serotype 14, weregreatest in African Americans.Conclusions: Disease caused by macrolide-resistant S. pneumoniae, a rapidly increas-ing problem in the 1990s, declined dramatically following the introduction of the pneu-mococcal conjugate vaccine in Atlanta. The decline was a result of the overall de-crease in invasive pneumococcal disease due to macrolide-resistant vaccine or vac-cine-associated serotypes, especially serotype 14. Vaccines can be a powerful strategyfor reducing antibiotic resistance in a community.

Role of Pneumolysin for the development of acute respiratory failure in pneu-mococcal pneumonia

Witzenrath M, Gutbier B, Schmeck B, Hocke AC, Mitchell TJ1, Hippenstiel S,Rosseau S, Suttorp N, Schütte H. Department of Internal Medicine/Infectious Dis-eases, Charité – Faculty of Medicine, Humboldt and Free University Berlin, Ger-many and 1Div. Infection and Immunity, University of Glasgow, UK.

Aims: Acute respiratory failure is an important feature of severe pneumonia. Wecharacterized the role of pneumolysin (PLY), an important virulence factor of Strep-tococcus pneumoniae, for the development of acute lung injury.Methods: Mice lungs were ventilated and ex vivo-perfused with blood-free buffersolution. Recombinant PLY was administered intravascularly or via the airways.PLY-induced changes in pulmonary vascular resistance, endothelial permeabilityand ventilation parameters were monitored. Additionally, immunofluorescence wasperformed on PLY-exposed human umbilical vein endothelial cells (HUVECs).Furthermore, the impact of PLY on electrical cell impedance and hydraulic conduc-tivity of HUVEC monolayers was measured.Results: Intravascular PLY caused a dramatic dose-dependent increase in pulmo-nary vascular resistance and a dose- and time-dependent increase in lung microvas-cular permeability with formation of severe lung edema. PLY administered via theairways dose-dependently increased capillary permeability, but did not affect pul-monary vascular resistance. Permeability of HUVEC monolayers was increased byPLY as determined by changes in electrical cell impedance and hydraulic conduc-tivity. Interestingly, immunofluorescence of PLY-exposed HUVEC monolayersshowed intercellular gap formation and dislocation of tight junction proteins.Conclusions: These results suggest that PLY may play a central role for acute respi-ratory failure in severe pneumococcal pneumonia by causing severe pulmonaryhypertension and impairment of pulmonary microvascular barrier function.


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Pneumococcal vaccination of infants in the developing world: A cost-effective-ness analysis

Sinha A1, 2, Lee G3, Ginsberg G4, Muhib F5, Levine O5, Lieu TA3. Brigham andWomen’s Hospital1; Depts. of Medicine2 and Ambulatory Care and Prevention3,Harvard Medical School and Harvard Pilgrim Health Care, Boston, USA; Dept. ofImmunizations, Vaccines, and Biologicals4, W.H.O., Geneva, CH; Dept. of Interna-tional Health5, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA.

Background: Routine pneumococcal conjugate vaccination in the world’s poorestcountries could significantly reduce child mortality and improve child healthMethods: We constructed a decision analytic model to analyze 4 strategies to pre-vent pneumococcal disease in the 75 poorest countries: (1) No vaccination, (2) naturaluptake of vaccine, (3) accelerated uptake of vaccine, and (4) accelerated with Vac-cine Fund (VF) purchase of vaccine. The model incorporated estimates of childmortality, pneumococcal disease incidence and outcomes, serotype coverage, im-munization rates, and vaccine efficacy from published studies. We extrapolatedmedical and non-medical costs from a recent South African study. Our main out-come measures were deaths prevented, costs, and $US/DALY averted.Results: If no vaccination occurred, the model estimated 1.48 million under-fivedeaths annually in the 75 poorest countries from pneumonia. VF purchase and ac-celerated uptake would prevent 354,665 (24%) of these deaths annually. If vaccinecost $5 per dose, the incremental cost of VF purchase + accelerated uptake wouldbe $US 10.40/ DALY. All other strategies had a less desirable cost-effectivenessratio than Vaccine Fund purchase and accelerated uptake. As vaccine cost increasedto $10 per dose, the cost of an averted DALY increased to $43.60. The model wasalso sensitive to estimates of proportion of pneumonia due to S. pneumoniae, sero-type coverage, and vaccine efficacy.Conclusions: Preliminary analysis suggests that at vaccine costs of $5-10, the pur-chase and accelerated uptake of pneumococcal vaccine for the world’s poorest coun-tries would be an effective and cost-effective intervention.

Immunogenicity and boosting following a reduced number of doses of a Pneu-mococcal Conjugate Vaccine in infants and toddlers.

Goldblatt D1, Ashton L1, Southern J2, Burbidge P1, Burrage M3, Morris R4, BorrowR5, Cartwright K4, Miller E2. 1Institute of Child Health, University College London,Health Protection Agency, 2London, 3Porton Down, 4Gloucerster and 5Manchester.

Aims: The number of doses of pneumococcal conjugate required for optimal pro-tection is not known. We studied the immunogenicity of a 2 versus a 3 dose primaryschedule in infants as well as comparing 1 vs 2 doses in toddlers to assess the utilityof a reduced schedule.Methods: UK infants eligible for routine vaccination at 2,3 and 4 months (m) of agewere recruited and received either 2 (2m & 4m) or 3 doses of a 9 valent pneumococ-cal conjugate vaccine (9VPnC, Wyeth) at the same time as other infant vaccinesfollowed by 9VpnC or a 23 valent polysaccharide vaccine (23PV, Aventis Pasteur)at 12m of age. In a separate study, toddlers (12m) were randomised to receive 1 or2 doses (2m apart) of 9VPnC followed by 23PV at 18m of age.Results: Infants. At 5m of age serotype specific IgG titers were similar irrespectiveof the number of doses received (2 doses n=80, 3 doses n=36). Persistence of titersto 12m of age was also similar, as were responses to a booster. Proportions ofvaccinees achieving titers >0.35�g/ml post primary vaccination did not differ sig-nificantly between the two groups. Toddlers. There was no significant difference inthe primary response to 1 or 2 doses of 9VPnC for 7 of the 9 serotypes. IgG titers toserotypes 6B and 14 were however significantly higher in the 2 vs 1 dose groups.Interestingly, when boosted, responses to 23PV were consistently higher for allserotypes in the recipients of a single dose although this difference was not signifi-cant. Responses to 1 dose in toddlers were consistently higher than those seen ininfants for 7 of the 9 serotypes (excluding 6B and 14).Conclusions: Two doses of 9VpnC are as immunogenic as three doses when admin-istered in the first 4 months of life and prime for memory. Reduced dose schedulesmay be equally protective. Responses to a single dose of 9VpnC in toddlers areconsistently better than infants and this evidence justifies a single dose in catch-upimmunisation programmes for children over 1 year of age.


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Use of a 9-Valent pneumococcal conjugate vaccine (PncCV) to identify therole of bacteria in viral pneumonia.

Madhi SA1, Kuwanda L1, Klugman KP1,2 for The Pneumococcal Vaccine TrialistGroup.1

Respiratory and Meningeal Pathogens Research Unit, South Africa; 2Rollins Schoolof Public Health, Emory University, USA.

Aim: To identify whether PncCV reduces the incidence of severe viral associatedpneumonia.Methods: As part of a phase 3 PncCV efficacy trial, a total of 39 836 children wererandomised to receive either PncCV or placebo at 6, 10 and 14 weeks of age. Thisincluded an estimated 6.02% of children that were HIV infected. Children hospital-ized for a lower respiratory tract infection (LRTI) were investigated for respiratoryviruses using direct viral specific immunofluoresence assays on nasopharyngealaspirates for respiratory syncytial virus (RSV), influenza virus A/B, parainfluenzavirus type 1-3 (PIV1-3) and adenovirus.Results: In the overall ITT analysis, PncCV recipients were less likely to have anepisode of pneumonia due to any of the viruses (reduction: 19%; 95%C.I. 6-30),and specifically those episodes associated with influenza A/B (42% reduction;95%C.I. 14-60) and PIV1-3 (43% reduction; 95%C.I. 11-63). Among HIV uninfectedchildren, there were significant reductions in viral associated pneumonia due toinfluenza virus A/B (40%; 95% C.I.2-63) and PIV1-3 (43%; 95%C.I. 11-63) in theITT analysis; as well as for RSV (31.8%; 95%C.I. 6.4-50.3) in the per protocolanalysis. Although fewer episodes of viral pneumonia occurred among HIV in-fected vaccinees compared to placebo recipients these differences were not signifi-cant.Conclusion: PncCV prevents hospitalization due to RSV, influenza A/B and PIV1-3 associated pneumonia. These data confirm a significant role for bacteria inpneumonias from which only a viral pathogen has been isolated. The vaccine pre-sumably prevents pneumonia by by reducing the incidence of superimposed pneu-mococcal infections amongst children infected by these viruses.


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PSV1-02

Efficacy of the 23-valent polysaccharide vaccine in healthy military recruits

*Russel, K

Naval Health Research Center RespiratoryDisease LaboratoryP.O.Box 85122San Diego, CA 92186-5122UNITED [email protected]

Effect of pneumococcal conjugate vaccine on invasive disease in the U.S.

*Whitney, C

Centers for Disease Control and Prevention1600 Clifton Road NE MS C23Atlanta, GA 30333UNITED [email protected]


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PSV1-04

Follow-up data from the South African pneumococcal conjugate vaccine trial

*Madhi, S

CHB HospitalRespiratory and Meningeal Pathogens Research UnitPO Bertsham, NHLS-room 112013 BertshamSOUTH [email protected]

Efficacy and use of conjugate vaccine in special populations

*Leach, A

Menzies School of Health ResearchP.O.Box 41096Casuarina [email protected]


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PSV2-02

Adding pneumococcal conjugate vaccine to routine infant vaccination in in-dustrialized countries

*Nohynek, H

National Public Health InstituteDepartment of VaccinesMannerheimintie 16600300 [email protected]

Panel discussion: The roles of pneumococcal polysaccharide and conjugatevaccines in developing countries

*Riley, I; Klugman, K

Riley, IThe University of Queensland Medical SchoolTropical Health ProgramHerston Road Qld [email protected]

Klugman, KEmory University1518 Clifton Rd, N.E., Room 764Atlanta, GA 30322UNITED [email protected]

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Similar persisting concentration and functional activity of pneumococcalpolysaccharide antibodies following 1 or 3 doses of 11-valent mixed carrierpneumococcal conjugate vaccine (11-PncTD)

Puumalainen T1, Lucero M2, Jousimies K1, Ugpo J2 , Lindholm N1, Nohynek H1,Käyhty H1 for ARIVAC consortium. 1National Public Health Institute (KTL), Fin-land; 2Research Institute for Tropical Medicine, Philippines

Aims: The11PncTD vaccine (Aventis Pasteur, France) is immunogenic and safe whenadministered simultaneously with EPI (DTwP, OPV, Hib, HBV) vaccines at 6, 10and 14 weeks of age. As an exploratory analysis we determined the concentrationand functional activity of antibodies at 9 months of age in groups who had receivedeither conventional 3-dose schedule (PncTD3) or 1 dose (PncTD1) at 18 weeks ofage.Methods: Healthy infants who visited village health center in Bohol, the Philippinesfor their EPI vaccines were randomised to receive either 3 doses (N=56) or 1 dose(N=54) of aluminium adjuvanted diphtheria toxoid (serotypes: 3, 6B, 14, 18C) ortetanus protein (1, 4, 5, 7F, 9V, 19F, 23F) –conjugated pneumococcal vaccine. Serumsamples collected at 9 months of age were transported frozen to KTL laboratory. IgGantibody concentrations were measured by EIA. The functional activity of antibodieswas measured by flow-cytometric opsonophagocytic assay with HL60 cell line.Results: PncTD3: The geometric mean concentration of antibodies (GMC) for 11serotypes ranged between 0.5-5.0 �g/ml. The percentage of infants with antibodyconcentration � 0.35�g/ml ranged between 43-100% for different serotypes.Opsonophagocytic titer �8 was found in 32%, 45% and 86% of samples for serotypes6B, 19F and 23F. PncTD1: The GMCs ranged between 0.4-5.8 �g/ml. Dependingon serotype 56-100% of infants had antibody concentration � 0.35�g/ml. Theopsonophagocytic titer �8 was found in 40%, 54% and 85% of samples for types6B, 19F and 23F.Conclusions: The persisting concentration and functional activity of antibodies weresimilar in both groups. If proven effective in preventing pneumococcal diseases thefever or alternative doses -schedules of conjugate vaccines could be an affordableoption for countries and families with economic constrains.

Recommendations on Implementation of the Pneumococcal Conjugate Vac-cine (PVC) in the Province of Quebec, Canada

Guay M1,2, De Wals P1,3, Petit G1,4, The Quebec Working Group (QWG) on PCV1,Erickson L4. National Public Health Institute, Quebec1, University of Sherbrooke2,Laval University2, University of Montreal4.

Introduction: The provincial health authorities (PHA) have given the mandate toQWG on PCV to make recommendations on the relevance of implementing PCV inthe routine vaccination program for infants. In Quebec, immunization programs arepublicly funded, and health issues are under provincial jurisdiction.Objective: Describe the recommendation formulation process used.Methods: We used the Erickson and De Wals’s framework for decision making onimmunization programs which implies a systematic approach: review of the illnessand vaccine characteristics, calculation of the clinical and economical burdens ofpneumococcal infections, estimation of the societal costs and cost-effectiveness ofan immunization program using different vaccination schedules, and finally, evalu-ation of the feasibility and acceptability of PCV utilization.Results: For a cohort of children aged 6 months to 9 years and in absence of vacci-nation, 4 deaths, 14 cases of meningitis, 3 830 cases of pneumonia and 57 878 casesof otitis media (OM) would be expected. Vaccination could prevent approximately2 deaths, 9 cases of meningitis, 1 167 cases of pneumonia and 17 795 cases of OM.The societal costs for the different vaccination schedules would vary between $5.2and $15.7 million (CA) while the net societal cost per LYG would vary between$125 000 and $206 000. Vaccination with PCV would be feasible especially if in-cluded to the regular vaccination visits; however, the supplementary injection mayconstitute a barrier to the acceptability of PCV for parents and vaccinators.Conclusion: The QWG recommended in 2003 the implementation of a universaland publicly funded vaccination program targeting all newborns as it would be morecost-effective and easier to implement than targeting older infants. New vaccinesare expensive. The principle that immunization programs should be cost-saving isoutdated, and PHA will have to compare implementation of PCV with other healthinterventions with similar cost-effectiveness ratio values.

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Efficacy of A Pneumococcal Conjugate Vaccine (PCV) against Acute OtitisMedia (AOM) in Children Younger Than 2 Years of Age

Adam D1, Scholz H2, Helmerking M3, Dept. of Antimicrobial Therapy, Dr. v.Haunersches Children’s Hosp., Univ. of Munich, Munich, Germany1, Stra§e 6, Nr.23, 13125 Berlin, Germany2, German Society for Pediatric Infectious Diseases(DGPI) Study Group, DGPI Office, Munich (Strasslach), Germany3,.

Background: Streptococcus pneumoniae is responsible for a wide spectrum of in-fections and a common cause of community-acquired pneumonia and acute otitismedia in children. The pneumococcal conjugate vaccine (Prevenar) has been shownto be effective in reducing the number of episodes of AOM from any cause by 6 to7 %. The German Society of Pediatric Infectious Diseases (DGPI) Study Groupconducted a large-scale study to evaluate the incidence of infections and risk fac-tors in children younger than 2 years of age after immunization with Prevenar.Methods: 4,617 children were randomly assigned, in a 1 : 3 ratio, to receive eitherthe standard immunization alone or in combination with the PCV. Prevenar wasgiven to infants at 2, 3, 4 and 12 to 15 months of age. In this interim analysis theeffectiveness of PCV in reducing episodes of AOM has been evaluated at 12 to 15months of age before additional doses are given.Results: Clinical signs and symptoms of AOM occurred in 492 (10.66 %) of 4,617children. 9.45 % episodes of AOM (331 of 3,502 children) have been documentedin the Prevenar group and 14.44 % (161 of 1,115 children) in the group receivingstandard immunization without PCV. This difference is statistically significant (P <.001).Conclusion: The pneumococcal conjugate vaccine (Prevenar) is safe and effectivein the prevention of acute otitis media.

Immune response to pneumococcal conjugate vaccine in asplenic children andteenagers

Bernatowska E. 1, Käyhty H.2, Mikoluc B. 3, Pietrucha B. 1, Piotrowska-JastrzebskaJ. 3 1The Children’s Memorial Health Institute, Warsaw, Poland, 2National PublicHealth Institute, Helsinki, Finland, 3Medical University in Bialystok, Poland

Aims: Pneumococci are responsible for the most of the invasive infections in asplenicchildren and vaccination with pneumococcal (Pnc) polysaccharide (PPV) and con-jugate vaccine (PCV) has been recommended by ACID. We determined Pnc anti-body concentrations in Polish asplenic children and teenagers before and after vac-cination with a dose of 7-valent PCV.Methods: Twenty asplenic children or teenagers aged 6 months to 19 years wereimmunised with PCV. The cause of asplenia was in 10 cases surgical removal aftertrauma, in the others cystic spleen (4), congenital asplenia (3) and spherocytosis,portal hypertension and tumor. 13 patients had received at least one dose of PPV 1to >10 years before the PCV. One vaccinee had experienced an invasive Pnc dis-ease. The total IgG, IgA and IgM concentrations were at normal level in 19, 18 and17 patients, respectively. The antibody (ab) concentrations against serotypes 4, 6B,9V, 14, 18C, 19F, and 23F in serum samples taken before and 1 to 4 months afterPCV were determined by EIA with a 22F neutralisation step.Results: The previous vaccination with PPV did not have an effect on the pre-PCVantibody concentrations. Geometric mean (GM) pre-PCV concentrations rangedbetween 0.21 (type 6B) to 1.47 (type 19F)�g/ml and 5 (type 19F) to 65 (type 6B)%had <0.35�g/ml. The GM fold increase after PCV ranged from 3 (type 19F) to 11(type 4), and the GM of post-PCV from 1.45 (type 6B) to 14.16 (type 14) �g/ml.Almost all post-PCV concentrations were �0.35 �g/ml. The boy with previousinvasive Pnc disease had a high response to PCV. The cause of asplenia did notaffect on responses to PCV.Conclusion: PCV induces high and most probably protective immune response inasplenic children and teenagers. Those, who have low ab concentrations after PPV,respond satisfactorily to PCV.


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Impact of the Introduction of Pneumococcal Conjugate Vaccine on the Epide-miology of Invasive Disease in Children and Adults within Northern CaliforniaKaiser Permanente (NCKP)

Steven Black, Henry Shinefield, Laura Elvin, Paula Ray, Edwin Lewis, Bruce Fire-man (Kaiser Permanente Vaccine Study Center, Oakland; and Robert Austrian (Uni-versity of PA, Philadelphia).

Objective: Evaluate the impact of routine immunization of children within NCKPwith 7-valent PCV (PNCV7; Prevnar, Wyeth) on the epidemiology of invasivepneumococcal disease in our populationMethod: PNCV7 was licensed and recommended for routine use in children < 2years of age in February 2000. We report on invasive disease in our population ofover three million people through March 2003.Results: Since licensure, 157,471 children have received one or more doses ofPNCV7, but only 24% of those under 2 yo received all four doses due to shortages.The impact of vaccine introduction is shown in the last columns in the followingincidence table (cases/100,000 person-yrs) for vaccine serotype (VST), Non-Vac-cine Serogroup (NVSG). Reporting periods are the 2nd quarter of one year throughthe 1st quarter of the next year regardless of vaccine status. During the same timeperiod, significant reductions in disease incidence for VST were seen in adults 20-40 years of age (58% decrease, p=0.001) and adults over age 60 y.o. (14% decrease,p=0.03) presumably due to a herd effect. In addition, substantial decreases in theproportion of pneumococcal isolates resistant to pen & other abx were seen in thepopulation at large.Conclusion: The PNCV7 vaccine is highly effective in reducing overall diseaseburden in children less than five years of age and there is evidence of herd immu-nity as well as decreases in antibiotic resistant strains causing disease. For invasivedisease, there is no evidence to date of replacement.

2Q96- 2Q97- 2Q98- 2Q99- 2Q00- 2Q01- 2Q02-1Q97 1Q98 1Q99 1Q00 1Q01 1Q02 1Q03

VST < 1yo 51.5 76.3 98.2 69.0 9.4 6.2 0.0 < 5 yo 42.2 46.0 59.6 42.5 17.9 2.8 0.6NVSG < 1 y 9.7 14.7 8.7 9.0 3.1 3.1 6.2 < 5 yo 6.3 6.5 5.1 2.8 2.8 3.9 5.6

Salivary antibody response in children with recurrent acute otitis media immu-nized with pneumococcal conjugate vaccine

D. Bogaert1, R.H. Veenhoven2, R. Ramdin1, I.H.T. Luijendijk1, G.T. Rijkers3, E.A.M.Sanders3, R. de Groot1, and P.W.M. Hermans1. 1Department of Pediatrics, Erasmus MC-Sophia, Rotterdam, 2Department of Pediatrics, Spaarne Hospital Haarlem, 3Depart-ment of Immunology, Wilhelmina Children’s Hospital Utrecht, University MedicalCenter Utrecht, The Netherlands

Introduction: Pneumococcal conjugate vaccination is more than 90% effective againstinvasive pneumococcal diseases. Despite significant systemic IgG responses, the effi-cacy against mucosal diseases such as otitis media caused by conjugate vaccine serotypesis only 57%. This already implies that other mucosal immune responses, possibly IgAantibodies as well as IgG antibodies will be required. In this study, we analyzed themucosal IgA and IgG immune response upon vaccination with the 7-valent pneumo-coccal conjugate vaccine followed by a polysaccharide vaccine booster in childrenwith a history of recurrent acute otitis media.Methods: IgA and IgG antibody concentrations to vaccine serotypes 6B, 14, 18C and19F were measured by enzyme immunoassay in saliva of 38 vaccinated children and45 controls. In the pneumococcal vaccine group, 12 children were sampled prior tovaccination, 12 children 4 weeks after the polysaccharide booster and 15 children 7months after the last vaccination. In the control group 15 children were sampled ateach of these 3 time points.Results: We observed a trend towards increased salivary IgG antibody concentrations14 months after the primary vaccination for the serotypes 14, 18C and 6B in the vacci-nated children but not the control children. As a result of large differences in individualbaseline titers and vaccine responses, this increase in antibodies was only significantfor serotype 14. The IgA antibody titers increased significantly after 8 and after 14months for all serotypes in both pneumococcal vaccine recipients and controls. How-ever, the mean antibody titer increased significantly more in control children com-pared to vaccinated children.Conclusion: Our data suggest that systemic pneumococcal conjugate vaccination re-sults in elevated mucosal IgG antibodies. However, a significant increase in mucosalIgA response is observed in control children compared to vaccinated children. Wehypothesize that this rise in IgA is provoked by ongoing carriage rather then by vacci-nation. We conclude that combined pneumococcal conjugate and polysaccharide vac-cination does not induce a lasting mucosal IgA response.


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Paediatric invasive pneumococcal disease : serotype and sequence type charac-terisation prior to the introduction of pneumococcal conjugate vaccines in theUK

S. C. Clarke 1,2, S.M. McChlery 1 and K. S. Scott 1

1 Scottish Meningococcus and Pneumococcus Reference Laboratory, Glasgow, UK2 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Glas-gow University, UK

Aims: To determine the relationship between pneumococci causing invasive disease inchildren prior to the introduction of pneumococcal pneumococcal conjugate (Pnc)vaccines.Methods: Invasive pneumococci from all children aged less than 5 years were used inthis study. These were characterised by serotyping and multi-locus sequence typing.Sequence analysis was performed using sequence type analysis and recombinationalanalysis software (http://pubmlst.org/software/analysis/start).Results: 92 pneumococcal isolates were used in this study, 84 of which were fromblood and 8 were from CSF. 39 were from children <1 year of age and 53 were fromchildren between 1-4 years of age. There were 25 different serotypes, serotype 14 be-ing the most common accounting for 15 (16.3%) isolates and serotype 18C being thesecond most common accounting for 13 (14.1%) isolates. Overall, Pnc coverage was71%, 73% and 76% for the 7-valent, 9-valent and 11-valent Pnc vaccines respectively.There were 46 different sequence types (ST’s) which grouped into 7 genetic lineagesand 28 singleton types. Serotypes were of limited ST’s except for 8 serotypes whichwere associated with multiple ST’s. In contrast, ST’s were of a single serotype exceptfor ST162 (serotypes 9V and 19F), ST191 (serotypes 7F and 8) and ST 199 (serotypes15 and 19A). Serotypes 7F, 9V and 19F are contained in the licensed 7-valent Pncvaccine whilst serotypes 8, 15 and 19A are not contained in any Pnc vaccine.Conclusions: The 7-valent Pnc vaccine includes the seven most common serotypes inScotland although there is some evidence in the paediatric population that pneumo-cocci can be of different ST’s but the same serotype. This may have implications forthe introduction of Pnc vaccines and enhanced surveillance schemes must be in placebefore and during such vaccine implmentation.

Supression of spontaneous Interferon-gamma (IFN-�) production by periph-eral blood mononuclear cells (PBMC) isolated from 12-month old infants fol-lowing vaccination with a 7-valent pneumococcal-CRM-197 conjugate vaccine.

Clutterbuck, EA.1, Salt, P.1, Marchant, A. 3, Moxon, E.R.1, Beverley, P.2, Pollard, AJ1

1. University of Oxford, Department of Paediatrics. Paediatric Infectious DiseasesLaboratory.2. The Edward Jenner Institute for Vaccine Research, Compton, UK. 3. WeatherallInstitute of Molecular Medicine, Oxford.

Aims: The aim was to quantify the frequency of IFN-� secreting PBMCs followingvaccination of 12-month old infants with a seven-valent pneumococcal-CRM197conjugate vaccine.Methods: Ten 12-month old infants received a single dose of the 7-valent, pneumo-coccal-CRM197-conjugate vaccine. Heparinated blood was obtained pre immuni-sation and 9-11 days later. PBMCs were separated over lymphoprep. PBMCs werecultured for 7 days with tetanus toxoid (TT), diphtheria toxoid (DT), Choline bind-ing protein A (CbpA) and controls. Cells were then harvested and IFN-� secretionwas detected by enzyme linked immunospot assay (elispot). Background IFN-gproduction was subtracted from the antigen specific data. Data were expressed asmean number of IFN� spots per 1x105 PBMCs per well.Results: The number of IFN-� secreting cells detected at day 0 and day 9-11 were asfollows: For medium alone day 0 = 49, day 9-11 = 5 (p=0.004); TT day 0 = 42, day9-11 = 34(p=0.705); DT day 0 = 31, day 9-11 = 26 (p=0.679); CbpA day 0 = 7, day9-11 = 24 (p=0.138) and PHA day 0 = 74, day 9-11 = 132 (p=0.259).Conclusions: There was no significant change in the number of circulating antigen-specific IFN� secreting PBMCs following vaccination in 12-month old infants witha pneumococcal conjugate vaccine. However, there was a highly significant de-crease in the background (medium alone) secretion of IFN� following vaccinationsuggesting that vaccination with the 7-valent vaccine may suppress spontaneousTh1 cytokine production by PBMCs 9-11 days after immunisation.


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Meningitis combination vaccine: preclinical studies

Van Dijken H1, Pillai S2, Van den Dobbelsteen, G.P.J.M.1.1Netherlands Vaccine In-stitute, Bilthoven, The Netherlands,2Wyeth Vaccine Research, Pearl River, USA

Introduction S. pneumoniae (Pn) and N. meningitidis (Men) are both human patho-gens causing severe diseases like meningitis and sepsis in young children. Pneumo-coccal - and Men C conjugate vaccines have been developed and proven to be verysuccessful in protection against invasive disease. A combination vaccine of thesetwo conjugate vaccines has been developed (9vPnC/MnCC) by Wyeth. For Men B,several outer membrane vesicle (OMV) vaccines have been developed and used inclinical studies. HexaMen, a multi-valent PorA OMV vaccine containing six PorAs,is developed at NVI. This vaccine was safe and immunogenic in infants and chil-dren. As there is only room for a limited number of injections in infant vaccinationschedules, the development of combination vaccines should receive strong empha-sis.Aim: To investigate whether the 9vPnC/MnCC vaccine can be combined withHexaMen without immunological interference.Materials en Methods: NIH mice were immunized twice sc. with 1/10 of a humandose on day 0 and 28. The vaccines were combined in one syringe, given separatelyat different injection sites or given individually. Sera were collected on day 42andtested for serotype specific anti-PnPS antibodies by ELISA. The functional activityof the anti-Men B and C antibodies was investigated with a serum bactericidal as-say.Results: Combining 9vPnC/MnCC vaccine with HexaMen has no negative influ-ence on the immune responses against the six PorAs nor on the immunogenicity ofthe Men C vaccine nor on the induced immune response against any pneumococcalserotype. On the contary, in the combination some anti-PnPS titers were enhancedprobably due to the lipopolysaccharides in HexaMen that acts as an adjuvants.Conclusion Preclinical studies in mice indicate that it is possible to combine 9vPnC/MnCC vaccine with HexaMen without causing immunological interference.

Persistence of anti-pneumococcal (Pnc) polysaccharide (PS) antibodies andavidity 3 to 4 years after immunization with pneumococcal conjugate vaccinePncCRM as measured by 22F EIA

Ekström N, Grönholm S, Palmu A, Åhman H, Käyhty H, and The FinOM StudyGroup. National Public Health Institute (KTL), Helsinki, Finland.

Aims: Removal of cross-reactive antibodies by Pnc serotype 22F PS absorptionimproves specificity and correlation between concentration and functionality ofantibodies. The effect of 22F PS absorption on antibody concentration and aviditykinetics was studied until 3 to 4 years after vaccination to relatively frequently car-ried (6B and 19F) and rarely carried (4) serotypes.Methods: Serum anti-Pnc IgG concentration and avidity were determined for 11 to58 children participating in the Finnish Otitis Media Vaccine Trial and receivingPncCRM or hepatitis B (control) vaccine at 2, 4, 6 and 12 months of age. Concen-trations were determined on samples taken at 13 and 24 months and at 4 to 5 yearsof age by EIA with (+22F) and without (-22F) 22F PS absorption and avidity indexwas determined by NaSCN elution EIA for sera with anti-Pnc IgG concentration >0.5 �g/ml after 22F absorption.Results:

Conclusions: The effect of 22F absorption is serotype dependent. Pneumococcalcarriage seems to induce natural boosting of specific antibodies in the vaccinatedchildren. High avidity antibodies persist 3 to 4 years after pneumococcal conjugatevaccination.

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Functional activity of antibodies to pneumococcal serotypes 6A and 19A in theFinnish Otitis Media (FinOM) Vaccine Trial

Ekström N, Haikala R, Lehtonen H, Kilpi T, Åhman H, Käyhty H and The FinOMStudy Group, National Public Health Institute (KTL), Helsinki, Finland.

Aims: Pneumococcal serotypes 6A and 19A are important causes of infections, yetonly 6B and 19F are included in the current pneumococcal conjugate vaccines (PCV).We evaluated whether functional antibodies are produced against serotypes 6A and19A after PCV vaccination in the FinOM Vaccine Trial. Two PCVs tested haddifferent efficacy profiles; PncCRM protected against type 6A acute otitis media(AOM), while PncOMPC did not. Protection against type 19A AOM remained lowfor both PCVs.Methods: 108 infants received 7-valent PCV, either PncCRM or PncOMPC, at 2, 4,6 and 12 months of age. Serum antibody concentrations and opsonophagocytic kill-ing activity (OPA) were determined for serotypes 6B, 6A, 19F and 19A from sam-ples taken at 7, 12, 13 and 24 months of age.Results: The proportion of samples with detectable 6A-OPA was 21% and 4% at 7mo, and 64% and 23% at 13 mo, in the PncCRM and PncOMPC groups, respec-tively. 19A-OPA was found at 7 mo in 0% and 2%, and at 13 mo in 7% and 14% ofthe sera in the PncCRM and PncOMPC groups. The mean anti-6A concentrationand 6A-OPA were lower than for serotype 6B, but correlated with the anti-6B con-centrations. 6A-OPA was found only rarely, if anti-6B concentration was <3 �g/ml.At 13 months the geometric mean of 6A-OPA titer was 39 in the PncCRM, but <8in the PncOMPC group. Anti-19A concentrations and 19A-OPA titers remainedlow in both PCV groups despite relatively high anti-19F concentrations.Conclusions: 6A-OPA was associated with the anti-6B concentrations and was elic-ited more efficiently by PncCRM than PncOMPC. Neither of the PCVs evokedfunctional anti-19A antibodies. Post vaccination mean OPA and mean anti-6A and-19A concentrations were associated with the serotype specific efficacy of the PCVsagainst AOM.

The effect of the 7-valent conjugate pneumococcal vaccine Prevnar on the car-riage and drug resistance of S. pneumoniae in healthy children attending Day-Care Centers (DCCs) in Lisbon: decrease in vaccine serotypes but no changein the frequency of carriage of drug resistant strains

Frazão N1, Simas C1, Nunes S1, Sousa N G1, Mato R1, Saldanha J1, Brito-Avô A1,Santos-Sanches I1, and de Lencastre H1,2. Instituto de Tecnologia Química e Biológica,Portugal1; The Rockefeller University, New York, USA2.

Aims: Prospective study to evaluate the impact of the 7-valent pneumococcal con-jugate vaccine on the composition of the nasopharyngeal flora ofS. pneumoniae (Pn) colonizing healthy children (ages 6m to 6y).Methods: Nasopharyngeal samples were collected between May 2001 and March2003 from 238 children who received Prevnar and a control group of 354 children.Pn carriage, serotypes, drug resistance patterns and genetic background using pulsed-field gel electrophoresis (PFGE) were compared. Antibiotic use and frequency ofrespiratory tract infections (RTI) were evaluated through questionnaires.Results: No significant differences were detected between vaccinees and controlsin the total carriage rate of Pn, in the rate of carriage of penicillin resistant or drugresistant pneumococci (DRPn), and in the frequency of RTI. There was significantreduction in the carriage of vaccine serotypes in DRPn that seemed to have beencompensated by the appearance and increase of DRPn expressing unusual serotypes:6A, 10/10A, 15A and C, 19A, 23A, 33F and non-typable (NT) strains. Most of theDRPn with unusual serotypes also showed novel PFGE patterns. However, one PFGEpattern normally associated with serotype 15A was also detected in a single isolateexpressing serotype 6B and two PFGE patterns mostly present in bacteria of sero-type 6B appeared as well in bacteria with serotype 6A. Antibiotic consumptionremained high in the two groups, which may have been responsible for the undi-minished prevalence of DRPn in spite of the use of Prevnar.Conclusions: Reduction in the carriage of DRPn in colonization may require a com-bination of the conjugate vaccine and a decrease in the antibiotic pressure.


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Safety Of 7-Valent Pneumococcal Conjugate Vaccine In HIV-Infected MalawianAdults Untreated With Antiretroviral Therapy

French N1,2, Gordon SB2, Malamba R2, Kunkeyani M2, Malenje K2, Musaya L2,Molyneux M1,2. 1 Liverpool School of Tropical medicine, UK, Wellcome Trust labo-ratories, Blantyre, Malawi.

Aims: To identify a gross effect of 7-valent conjugate pneumococcal vaccine (CPV)on HIV viral load (VL) and CD4 T-cell count and assess reactoginicity in HIV-infected Malawian adults, prior to undertaking a randomised clinical trial.Methods: After informed consent, 42 volunteers, 22 HIV-uninfected and 20 HIVinfected were randomly assigned to receive two doses of CPV or two doses of pla-cebo (phosphate buffered saline with 1% aluminium phosphate) 4 weeks apart in asingle blind study (patient blind to designation). HIV status was confirmed by test-ing with two rapid HIV tests, viral loads were measured using Roche amplicor¨.Results: Side-effects were confined to self-reported fever which responded to para-cetamol in 3 vaccine recipients (2 HIV-infected) and 2 placebo recipients (1 HIV-infected). Post 2nd vaccine 3 individuals complained of fever (2 HIV-infected 1 HIV-uninfected vaccine recipients). Baseline mean VL in HIV-infected subjects was78,308copies /ml (95% CI 28,342-216,360) with no difference between vaccineand placebo groups. Post-vaccination VL in vaccine recipients was unaltered frombaseline 74,940 (14,874-377,580, n=10, P=0.51). Baseline median CD4 was 223[Range 38-518] and remained unchanged after first (CD4 311 [range 3-1016]) andsecond (CD4 350[range 11-666]) doses of CPV.Conclusion: Although a small study, we are reassured that CPV was well toleratedwith minimal side effects and no gross changes in viral load or CD4 T-cell countwere identified post CPV. A randomised controlled trial of CPV as secondary prophy-laxis in HIV-infected Malawian adults is now underway.

Do different clones of vaccine serotypes differ in their ability to cause acute otitismedia in vaccinated children?

W. P. Hanage1, Arto Palmu2, Jukka Jokinen2, P.Helena Makela2, Terhi Kilpi2 and B.G. Spratt1. Imperial College London1 and National Public Health Institute, Finland2

Aims: To compare breakthrough strains of vaccine type S. pneumoniae causing acuteotitis media (AOM) in children receiving conjugate vaccines with those from con-trols.Methods: The study included isolates of S. pneumoniae obtained by myringotomywith aspiration from middle ear fluid of children suffering from AOM. The childrenin the Finnish Otitis Media Vaccine Trial received one of two 7-valent conjugatevaccines (N=1666), with controls receiving hepatitis B vaccine, (N=831) at 2, 4, 6and 12 months of age. Strains expressing capsular polysaccharides 19F, 23F, 6Band 14 were identified by serotyping and further characterised using multilocussequence typing. The distributions of sequence types (STs) within each of the fourserotypes were compared between breakthrough and control strains using the chi-square test of independence and comparison of Simpson’s index of diversity D.Results: In total, 205 cases of AOM due to the four serotypes occurred in the vac-cine group (85, 79, 22 and 19 strains of the types 19F, 23F, 6B and 14, respectively).For the control group, the number of such AOM-cases was 247 (63 of type 19F, 97of 23F, 57 of 6B, and 20 of 14). The diversity of STs within breakthrough strainswas significantly less than within control strains. For all serotypes, under and over-representations of some STs were observed in breakthrough strains compared tocontrols. Comparison of the distributions of strains in breakthrough cases and con-trols were significantly different for serotype 23F strains (�

(26)2, p<0.025). This

may be due to higher relative frequencies of ST’s 36 and 392 and lower relativefrequencies of ST 507 in cases of AOM due to this serotype in vaccinated children.Conclusions: These data suggest the efficacy of conjugate vaccines against AOMmay vary for the different clones of the vaccine serotypes.

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PSV-24

Post-pneumococcal conjugate vaccine carriage and serotype distribution amongyoung Aboriginal children living in 29 remote communities in the NorthernTerritory of Australia

Hare K, Leach AJ, Beissbarth J, Harrington B, Stubbs E, Mellon G, Wilson C, MorrisPS. Menzies School of Health Research, Darwin, Australia.

Background: The 7-valent pneumococcal conjugate vaccine (7vPCV, Prevenar®) isrecommended at 2, 4 and 6 months of age, followed by the 23-valent polysaccha-ride vaccine (23vPPV, Pneumovax®) at 18 months of age. This schedule is fundedfor all Australian Aboriginal children and a vaccination program including catch-upwas implemented in the Northern Territory (NT) in June 2001.Aims: To describe post-7vPCV pneumococcal carriage and serotype distributionamong Aboriginal children aged 6 to 30 months in 29 remote communities throughoutthe NT.Methods: Pre- and post-7vPCV otitis media surveys were conducted in 2001 and2003. Nasal and ear discharge swabs were also collected during the post-7vPCVsurvey.Results: 645 children in the eligible age range were seen in 2003. All children hadreceived at least one dose of 7vPCV. Nasal swabs from 246 children from 10 com-munities have been cultured; 208 (84.6%) were positive for pneumococcus. Of 227serotypable isolates, 46 (20.3%) were vaccine types (VT), 64 (28.2%) vaccine-re-lated (VRT) and 117 non-vaccine types (nVT). Pneumococci were isolated from14/59 (23.7%) ear discharge swabs from 13/42 (31.0%) children; 3/14 (21.4%)serotypable isolates were VT while none were VRT. 45/227 (19.8%) nasal isolatesand 3/14 ear discharge isolates were 16F.Conclusions: Pneumococcal carriage rates amongst Aboriginal children in the NTremain over 80% post-7vPCV, and over 20% of ear discharge swabs were positivefor pneumococci. The proportion of carriage types that are VT is now less than21%. The proportion of penicillin non-susceptible pneumococci is also likely to below. High carriage rates for serotype 16F, which is absent from both 7vPCV and23vPPV, may be cause for concern.

Critical difference between pneumococcal polysaccharide ELISAs with or with-out 22F inhibition at antibody levels <1µg/mL in pediatric post-immunizationsera

Henckaerts I1, De Grave D1, Ashton L2, Goldblatt D2, Poolman J1. GlaxoSmithKlineBiologicals, Rixensart, Belgium1; Institute of Child Health, London, UK2

Aims: As recommended by WHO, GSK1 has developed a 22F inhibition ELISAaccording to Frasch (Clin.Diagn.Lab Immunol. 8, 266-272, 2001) to measure im-mune responses after pneumococcal conjugate vaccination. A bridging study wasconducted between this 22F inhibition ELISA and the non-22F reference assayemployed at the WHO pneumococcal reference laboratory (ICH2) that is similar tothe assay used to generate data in the three Wyeth’s efficacy trials. The objective ofthe ICH-GSK bridging study was to assess the agreement between the methodsparticularly at lower antibody levels.Methods: The ELISA employed at GSK includes both CPS and 22F PS as inhibi-tors in order to increase the specificity of the assay. Results obtained for a referencepanel were compared to those obtained without 22F adsoption in the WHO refer-ence lab.Results: After conjugate immunization, thirty pediatric post-primary sera with anti-polysaccharide titers ranging from <0.05–15µg/mL were analyzed. Aggregate re-verse cumulative distribution curves, in which antibodies to serotypes 4, 6B, 9V, 14,18C, 19F, and 23F were combined, revealed similar results for both methods atantibody titers >1µg/mL. However, at titers <1µg/mL the distribution curve meas-ured with the 22F inhibition ELISA shifted towards lower titers. This observationwas confirmed by intralaboratory comparisons and indicates that the 22F-basedassay is more specific at low antibody titers. Translation of the critical low titerssuggests that a threshold titer of 0.35µg/mL determined with the WHO-non-22FELISA corresponds to a titer of 0.20 µg/mL with GSK’s 22F inhibition ELISAassay.Conclusions: The 22F inhibition ELISA was more specific at antibody titers <1 µg/mL and should be the established standard pneumococcal assay for pediatric sera.


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PSV-26

Estimation of the Antibody Concentration at Threshold OpsonophagocytosisActivity Using A Sensitive and Robust Opsonophagocytosis Assay

Henckaerts I, Wauters D, Durant N, Poolman J. GlaxoSmithKline Biologicals,Rixensart, Belgium

Aims: Opsonophagocytosis is the primary in vivo mechanism of eliminating invad-ing pneumococci. Hence, an in vitro opsonophagocytosis assay (OPA) is a wellaccepted indicator for the presence of protective antibody levels in a serum sample.The presented work was designed to develop an improved method for OPA whichcan be used in conjunction with the highly specific 22F inhibition ELISA to meas-ure antibody concentrations at threshold opsonic activity.Methods: In order to increase OPA robustness and sensitivity while maintainingspecificity, several modifications of the assay were implemented concerning e.g.the preparation of the bacterial working seeds and the complement concentration inthe assay. GSK strains rather than strains from CDC were used for all serotypesexcept 6B. Antibody concentrations were measured by 22F inhibition ELISA atthreshold opsonic activity in infant sera obtained after conjugate immunization.Results: With the improved assay, opsonophagocytic activity was demonstrated at alower threshold level suggesting agreement of OPA positivity with ELISA levels�0.10 �g/mL for almost all serotypes and �0.2 µg/mL for 19F, which was themost difficult to kill, regardless of target strain. Therefore, a threshold of 0.2 µg/mLby 22F inhibition ELISA can be used for all serotypes although this value seems tobe conservative for most serotypes. It is possible that the lower functional activityfound in vitro for serotype 19F is related to the reduced efficacy observed for thisserotype during clinical conjugate studies.Conclusions: We have developed a new OPA, which allowed identifying criticalassay conditions affecting assay sensitivity and robustness.

Immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PncPS)in Filipino pregnant women and their offspring: Comparison of EIA with andwithout the 22F neutralisation step.

Holmlund E1, Quiambao B2, Grönholm S1, Soriano V2, Nohynek H1, Käyhty H1.National Public Health Institute, Helsinki, Finland1, Research Institute for TropicalMedicine, Manila, The Philippines2

Aims: To measure the immune response to PncPS given during pregnancy and ininfants at 6 or 14 weeks (wk) of age with standard EIA (stEIA) and with a novelEIA including a 22F neutralisation step (22FEIA).Methods: 38 pregnant women were vaccinated with PncPS during the third trimes-ter. Their offspring received PncPS at 6 or 14 wk of age. 20 mothers did not receiveany PncPS during pregnancy; their infants received PncPS at 6 wk. Pre- andpostimmunisation sera of mothers, cord bloods (N=46) and consecutive sera frominfants taken at 6, 10, 14 and 18 wk and 9-10 mo of age were used to determineantibodies to types 1, 5, 6B, 14 and 19F by the stEIA (analyses done in 1995-6) andby the 22FEIA (done in 2003).Results: For the adult preimmunisation sera the GMCs of anti-1, -5, and -6B were 3- 4-fold higher by stEIA compared to 22FEIA, but for anti-14 and -19F equal. Forthe postimmunisation sera the difference was smaller (1.5 - 2.5 -fold) resulting inhigher fold increase (pre- vs. postimmunisation) for types 1, 5, and 6B. Concord-antly, in cord bloods a bigger difference in anti-1, -5, and -6B between immunisedand unimmunised mothers was found by 22FEIA compared to stEIA. For the infantsera the two EIAs gave similar results. Infant responses after PncPS vaccination totypes 1 and 5, but not to the other types, were detected by both EIAs. At 9-10 mo ofage all infants had appr. the same mean concentrations of antibodies regardless ofearlier PncPS vaccinations. The correlation between stEIA and 22FEIA was in gen-eral good, and best for the adult postimmunisation sera (r= 0.75-0.95).Conclusions: 22F EIA reduces the detection of non-specific polyreactive antibod-ies, but there are serotype specific differences in the outcome. The use of 22FEIA isrecommended especially when sera of adults are studied.


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PSV-27

PSV-28

The effectiveness of vaccination in generating serotype-specific antibodiesagainst pneumococcal capsular polysaccharide

Huo Z, Irving D, Harris T*, Sheldon J & Riches P. Division of Biochemistry &Immunology, *Department of General Practice and Primary Care, St. George’sHospital Medical School, London, UK.

Pneumococcal vaccines are intended to produce antibody against pneumococcalcapsule so as to increase host phagocytic capacity against pneumococcus. How-ever, for reasons that are not understood, these vaccines do not protect high riskpatients as effectively as expected on the basis of clinical trials in healthy individu-als. We used an equal-potency ELISA method to measure the IgG antibody againstmulti-serotypes of pneumococcal capsular polysaccharide in order to answer twoquestions: 1) do healthy and high-risk adult groups differ in their serotype-specificresponses to pneumococcus from natural exposure? 2)will the high-risk individu-als acquire the same benefit from pneumococcal vaccines containing multi-serotypesin equal amounts?Results: For all serotypes except S19A no significant differences (T-test) were foundbetween the two groups. In the case of S3, both groups had low antibody levels(<5�g/ml). If a two-fold-increase in antibody level is regarded as a response tovaccination, the % of high-risk individuals making responses varied from 18% to63%, depending on the serotype contained in the vaccine. T test showed vaccinationdid not make any difference in antibody level for S3, 6, 10A and 15B.Conclusion & discussion: the high risk group had a similar profile to the healthyindividuals in antibody response against pneumococcal polysaccharide, which sug-gests factors other than antibody levels could be involved in their susceptibility topneumococcal infection. Individual responses to the same antigen vary, and vaccineswith different structures of serotypes of polysaccharide can also produce differentresponses in the same individual. The current vaccination method must take thesedifferences into account. It cannot be assumed that all patients will make the sameresponse to equal amounts of polysaccharide regardless of its structure, which couldresult in over-evaluation of the serotype-specific immunoprotection from pneumo-coccal vaccination.

Safety of varying dosages of 7-valent pneumococcal conjugate vaccine in adults70-79 years of age

Jackson LA1, Neuzil KM2, Whitney CG3, Shay DK3, Feikin DR3, Baggs J3, CarloneG3, Nahm M4. 1Center for Health Studies and 2VA Puget Sound Medical Center,Seattle, WA. 3CDC, Atlanta, GA. 4Univ of Alabama, Birmingham, AL.

Study design: Sequential dose-escalation study comparing administration of 0.1ml, 0.5 ml, 1.0 ml and 2.0 ml volumes of pneumococcal conjugate vaccine (PCV)(Prevnar®) with administration of 0.5 ml pneumococcal polysaccharide vaccine(PPV) among 220 adults 70-79 years of age previously vaccinated with PPV at age65 years or above and at least 5 years previously. Interim safety analyses were con-ducted following enrolment of the first 20 (of 44) participants in each of the 4 PCVdose phases. These interim results, compared with safety data on the first 16 (of 44)PPV recipients, are presented here.Results: Adverse events reported in the 6 days following vaccination, by study group.

Event type 0.1ml PCV 0.5 ml PCV1.0ml PCV 2.0 ml PCV 0.5 ml PPVn=20 n=20 n=20 n=20 n=16

Any redness atinjection site 15% 10% 15% 25% 38%Any swellingat injection site 5% 10% 5% 25% 38%Arm pain 20% 55% 60% 85% 81%Arm motion limitation 5% 20% 40% 40% 50%Increased armcircumference 5% 35% 25% 25% 44%

Local reactions were generally mild with duration <3 days. There were no reportsof severe arm pain or redness or swelling >15 cm in diameter. No serious adverseevents were reported.Conclusions: Among this study population, increasing dosages of PCV were asso-ciated with a higher frequency of local reactions but the safety profile of the highestdose of PCV was comparable to that of the standard dose of PPV.


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PSV-30

An age-adjusted comparison of the coverage by the 7-valent pneumococcal con-jugate vaccine in carried and invasive isolates from Danish children aged 12 to83 months.

Kaltoft MS1, Madsen J2, Konradsen HB1. WHO Collaborating Centre for Referenceand Research on Pneumococci1 and Department of Biostatistics2, Statens SerumInstitut, Copenhagen, Denmark.

Aims: An age-adjusted comparison of the coverage rate by the 7-valent vaccine(including serotype 6A and 19A) in carried and invasive isolates from Danish chil-dren aged 12 to 83 months of age.Methods: Included were 258 carried isolates collected among 503 children partici-pating in a study in day care centres (DCCs) during the winter of 1999 and 2000,and 683 isolates from episodes of invasive pneumococcal disease (IPD) in children,collected during a 19 years (1981-99) nationwide surveillance study. Specimen col-lection, species identification and serotyping were performed according to stand-ard procedures. Statistical analysis was performed using a multivariable logisticregression model where the GEE method was used for estimation in carriers, as themodel allows for a possible correlation between children attending the same groupwithin DCCs (SAS version 8.2, PROC GENMOD).Results: The coverage by the 7-valent vaccine in carriers and in IPD cases did notvary significantly regarding gender, months of sampling or for carriers size or typeof DCC, but a significantly decrease of coverage by age was observed for both,with and without 6A and 19A included in the analysis. The coverage in carriers fellfrom 77 % in the 12-<24 months age group to 38 % in � 60 months age group. ForIPD cases the figures were 85 % and 44 % respectively. When comparing the twodeclining lines defined by the multivariable logistic regression models, no signifi-cant differences in the parameters of the two lines were observed (p= 0.3662 (theslope) and p=0.2393 (the intercept).Conclusions: The importance of the serotypes included in the 7-valent vaccine bothin IPD cases and among carriers in the youngest childgroups is hereby underlined.

Safety of an 11-valent pneumococcal conjugate vaccine (11PCV) among Fili-pino infants

Lechago M1, Ugpo J1, Ladesma E1, Lacea T1, Puumalainen T3, Heiskanen-KosmaT3, Manson C2, Teuwen D2, Feroldi E2, Nohynek H3, Tallo V1, Lucero M1 for ARIVACconsortium. 1Research Institute for Tropical Medicine, Philippines, 2Aventis Pas-teur, France, 3National Public Health Institute, Finland

Aims: To determine the safety of 11PCV among Filipino infants.Methods: A randomised, double-blind placebo-controlled trial is being conductedon Bohol, Philippines starting from July 2000 to determine the effectiveness of11PCV, containing polysaccharides 1, 4, 5, 7F, 9V, 19F, and 23F (coupled to tetanusprotein) and polysaccharides 3, 6B, 14, and 18C (coupled to diphtheria toxoid)given at 6, 10 and 14 weeks of age in preventing x-ray proven pneumonia. Thevaccine is given with DTwP, OPV, hepatitis B, and Hib vaccines. Serious adverseevents (SAE) are monitored using two complementary passive surveillance sys-tems: immediate (ISAE) and periodic (PSAE). Independent local and internationaldata safety monitoring boards review safety data regularly.Results: Almost 95% of the eligible birth cohort is enrolled. Up to June 2003, 10,662infants received 11PCV or placebo. 2,955 SAE were recorded (280 ISAE and 2,675PSAE). Twelve SAE occurred within 24 hours after administration of the vaccines:febrile convulsion (7), allergic-type reactions (3), abnormal crying (1) and high-grade fever (1). The annual reporting rate of SAE is similar. The 2 most frequentlyreported SAE were acute gastro-intestinal infection (GEA) and pneumonia, with935 and 896 cases, respectively. Fifty deaths were reported, 10 between the 1st and2nd doses, 6 between the 2nd and 3rd doses, and 36 after the 3rd dose. Twenty-sixdeaths were attributed to: GEA (12), sepsis (9) and meningitis (5). No particulartrend was observed. Crude mortality rate during the 3 years of the trial among chil-dren <2 years of age was observed to be much lower when compared to mortalityrates from the period 1996-98 (468 vs 1679/100000).Conclusion: The 11PCV appears to be safe in the light of the present informationcollected.


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PSV-32

Changes in Streptococcus pneumoniae (SP) population structure in a group-randomized clinical trial (GRCT) of the 7-valent conjugate vaccine (PCV)

Lipsitch M 1, Huot H2, O’Brien KL3, Bouchet V2, Santosham M3, Reid R3, PeltonSI2, Goldstein R2 Harvard School of Public Health1, Boston, MA, USA, BostonUniversity Medical Center2, Boston, MA, USA, Johns Hopkins University3, Balti-more, MD, USA

Aims: To characterize the changes in vaccine-serotype (VT) and non-vaccine type(NVT) SP recovered from the nasopharynx in intervention and control communi-ties during a GRCT of PCV. It has been shown that VT were reduced while NVTwere increased in PCV communities; we tested whether these effects were propor-tionate across all VT strains and all NVT strains.Methods: 215 NP isolates from children in the Navajo-Apache GRCT were charac-terized by rRNA flank RFLPs (ribotyped) . Isolates were clustered by neighbor-joining, and “strains” were defined for analysis as clusters with 100%, 93%, 87%,or 78% similarity by Dice coefficients (corresponding to ~0-6 band differences).The hypothesis that PCV selected for a defined subset of VT or a defined subset ofNVT “strains” was tested vs. the null hypothesis that “strain” frequencies wereproportional in both communities.Results: No evidence of preferential outgrowth of particular NVT “strains” wasfound in PCV communities: for all stringencies of clusters defined, the data wereconsistent with the hypothesis that all NVT “strains” were equally increased in thePCV community. Because the PCV is more efficacious against some serotypes thanothers, we expected to find greater reduction in some “strains” than others. Thiswas observed but only at the least stringent (78%) definition of “strain,” and thereonly with p=0.08 (beta-binomial score test).Conclusions: Serotype replacement in NP carriage results from increases in fre-quencies of many unrelated SP “strains,” not from outgrowth of a small number of“strains.” PCV reduces carriage of a broad range of VT “strains”; an expandedstudy is in progress to determine the extent of heterogeneity in this protection.

Radiographic pneumonic consolidation (PC) using standard WHO criteriaamong Filipino infants in a Phase 3 trial of an 11-valent pneumococcal conju-gate vaccine (11PCV)

Lucero M1, DeCampo M1, Lupisan S1, Besa R1, Ocampo A1, Romano R1, Riley I2,Nohynek H3, Simoes E4 for ARIVAC consortium, 1Research Institute for TropicalMedicine, Philippines, 2University of Queensland, Australia, 3National Public HealthInstitute, Finland, 4University of Colorado, USA, 5Imperial College, UK

Aims: To compare 2 methods (standardized criteria set by WHO [RWHO] and theconventional interpretation (RD)) of identifying PC on chest radiographs of Fili-pino children <2 years of age diagnosed with pneumonia in a Phase 3 randomized,double-blind, placebo-controlled trial of 11PCV.Methods: From July 2000 to March 2003, 1997 chest radiographs (AP view) wereobtained from trial children with pneumonia in 4 hospitals in Bohol, Philippines.After extensive training in RWHO, films were read by 2 readers separately andwithout access to clinical history for presence of PC. Films with discordant inter-pretation of PC using RWHO were sent to WHO for arbitration. After 4 months, 1reader read 84% of the same films using more detailed criteria set by trial investiga-tors (RD). In RD, infiltrates were read as alveolar, atelectasis, interstitial, or notpresent.Results: For RWHO, Reader # 1 interpreted 182/1997 as PC, while Reader # 2interpreted 144/1997 (63% agreement, kappa 0.30). With WHO arbitration for dis-cordant PC readings, 176/1997 radiographs (8.8%) were read as having PC. ForRD, 196/1997 (9.8%) were alveolar infiltrates. When RD was compared to RWHO,only 123/176 PC (70%) were read as being alveolar infiltrates.Conclusion: Despite extensive training in RWHO, the interobserver agreement re-mained low. In comparison to RD, RWHO appears more sensitive and at the sametime less specific to the more classical form of radiological pneumonia, i.e. thatindicated by alveolar infiltrates. These methodological limitations need to be keptin mind when using RWHO in interpretation of trial endpoints as well as measuringdisease burden of pneumonia of different etiologies, particularly in poorly resourcedsettings.


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PSV-33

PSV-34

Defining the magnitude of efficacy of a 9-valent pneumococcal conjugate vac-cine (PncCV) in preventing pneumonia among HIV infected and HIV uninfectedchildren.

Madhi SA1, Kuwanda L1, Klugman KP1,2 and The Vaccine Trialist Group.1 Respiratory and Meningeal Pathogens Research Unit, South Africa; 2Emory Uni-versity, USA.

Aim: Define the efficacy of PncCV in preventing clinically and/or radiological con-firmed pneumonia.Methods: 39 836 children, ~6.02% of whom were HIV infected participated in aPncCV efficacy trial. Endpoints were hospitalization with clinical pneumonia (CP)(i.e. radiological alveolar consolidation defined by WHO (WHO-AC) or clinicalcriteria); CP or bronchiolitis= clinically diagnosed lower respiratory tract infec-tions (C-LRTI); bronchiolitis (CP without WHO-AC, but with wheeze);very obvi-ous alveolar consolidation (VOP); ITT efficacy estimates using WHO-AC previ-ously reported by us were (17%, 95%C.I. 4-28) overall, 20% (95%C.I.2-35) amongHIV uninfected and (13%, 95%C.I.-7 –29) among HIV infected children.Results: Based on an ITT analysis, the overall efficacy of the vaccine in preventingCLRTI and CP were 9.3% (95%C.I.2.9-15.3) and 16.2% (95%C.I. 8.8-23.0) re-spectively. Vaccine efficacy against CLRTI and CP were 15.4% (95%C.I.6.0-23.9)and 15.0% (95%C.I. 4.9-24.1) among HIV infected children and 6.6% (95%C.I. -1.4-14.0) and 16.9% (95%C.I. 7.3-25.5) among HIV uninfected children, respec-tively. Reinterpretation of CXRs for VOP yielded unchanged levels of efficacy es-timates compared to WHO-AC estimates among all groups of children. The vaccineattributable reduction (VAR- per 1 000 person years) in episodes of pneumoniavaried depending upon which definition was used. VAR for CLRTI, CP, WHO-ACand VOP were 3.3, 4.0, 1.7 and 0.5 overall; 1.3, 1.9, 1.0 and 0.3 among HIV uninfectedand 26.0, 23.2, 10.7 and 2.4 among HIV infected children respectively.Conclusion: Although the evaluation of efficacy of the PncCV is dependent on theclinical and/or radiological criteria used, PncCV has significant benefits in prevent-ing pneumonia among both HIV infected and HIV uninfected children.

Extended surveillance of the efficacy of a 9-Valent pneumococcal conjugatevaccine (PncCV) in preventing invasive pneumococcal disease (IPD) amongHIV infected and uninfected African children.

Madhi SA1, Kuwanda L1, Klugman KP1,2 and The Pneumococcal Vaccine TrialistGroup. 1 Respiratory and Meningeal Pathogens Research Unit, South Africa; 2RollinsSchool of Public Health, Emory University, USA.

Aim: To define the durability of protection of the PncCV in preventing IPD in theabsence of booster doses of PncCV.Methods: 39 836 children were randomised to receive either PncCV or placebo at 6,10 and 14 weeks of age. This number included an estimated 6.02% of children thatwere HIV infected. Serotypes were considered to be vaccine-type (VT) if includedin the PncCV, vaccine-related (VR) if from the same serogroup of a serotype in-cluded in PncCV and non-vaccine related (NVR) if no such serogroup was includedin PncCV. The mean duration of follow-up for vaccinees and placebo recipients was4.4 years compared to 2.3 years at the time of the previous report.Results: Based on the ITT analysis there was ongoing protection against VT-IPDamong HIV infected (efficacy: 53.6%; 95%C.I.10.8-75.8) as well as HIV uninfectedchildren (88.2%; 95%C.I.49.1-97.3). The reduction in IPD due to ANY serotypewas 51.6% (95%C.I. 25.7-68.5) among HIV infected children and 55.0%(95%C.I.1.2-79.5) among HIV uninfected children. Among HIV infected children,there was a 75% (95%C.I. 39.0-89.7) reduction in VR-IPD, but a non-significantincrease in NVR-IPD (R.R.:1.38; 95%C.I.0.6-3.4). Overall, the efficacy of thevaccine was 66.7% (95%C.I.40.2-81.4) for VT-IPD, 60.0% (95%C.I. 16.8-80.8) forVR-IPD, and 51.2% (95%C.I.28.9-66.6) for IPD due to ANY serotype; whereasthere was a non-signifcant increase in frequency of IPD due to NVR serotypes(33.3%; 95%C.I.-48.4-70.0).Conclusion: The overall potential efficacy of PncCV against IPD among HIVuninfected children is reduced due to trends toward greater number of episodes ofIPD due to non-vaccine serotypes.


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PSV-35

PSV-36

Quantitative antibody responses to a 9-valent pneumococcal conjugate vaccine(PncCV) amongst HIV infected and HIV uninfected children.

Madhi SA1, Kayhty H2, Kuwanda L1, Holm A2, Klugman KP1,3. 1 Respiratory andMeningeal Pathogens Research Unit, South Africa; 2National Public Health Insti-tute, Helsinki, Finland 3 Emory University, USA.

Aim: To compare the antibody responses of HIV infected to that of HIV uninfectedinfants to vaccine serotypes included in a 9 valent PnCV.Methods: 66 HIV infected and 127 HIV–uninfected children were randomized toreceive PncCV or placebo at a mean age (S.D.) of 1.56 ( 0.30)m, 2.63 (0.52)m and3.73 (0.88)m. Serum for antibody concentration measurement were obtained at amean interval of 23.5 (S.D. 5.7) days following the 3rd dose of vaccine. The geo-metric mean antibody concentrations (GMT) were calculated following log-trans-formation of the antibody concentration data.Results: Among vaccinees, HIV infected children had lower GMT responses toserotypes 1 (GMT 3.5; 95%C.I. 2.0-5.9 vs. 7.55; 95%C.I. 6.1-9.4; P=.0001) and18C (GMT: 1.9; 95%C.I.1.0-3.6 vs. 4.6; 95%C.I. 3.7-5.6; P=0.02), whereas therewas no difference in GMT between HIV infected and uninfected children for theremaining 7 serotypes. Among placebo recipients, GMT’s were paradoxically greaterfor serotypes 1, 4, 5, 14, 18C, 19F and 23F among HIV infected compared to HIVuninfected children (P<0.05). The proportion of vaccinated children who had anti-body concentrations of 0.35 ug/ml to indi vidual serotypes varied from 63-93%amongst HIV infected and 79-100% among HIV uninfected children and was onlysignificantly lower among HIV infected children for serotypes 1 (90% vs. 100%,P=0.031) and 18C (83.3% vs. 100%, P=0.003).Conclusion: HIV infected children have similar quantitative antibody responses tomost 9-valent PnCV serotypes compared to HIV uninfected children and have higherbaseline antibody concentrations of unknown functionality.

Multi-valent pneumococcal vaccines do not significantly differ in their cover-age against invasive pneumococcal disease: relationships between serotypes andsequence types

S.M. McChlery1, K. S. Scott1, S. C. Clarke1,2, 1Scottish Meningococcus and Pneu-mococcus Reference Laboratory, Glasgow, UK2 Division of Infection and Immu-nity, Institute of Biomedical and Life Sciences, Glasgow University, UK

Aims: To determine the prevalence of serotypes and sequence types of invasive pneumo-cocci in Scotland prior to the introduction of pneumococcal conjugate vaccines (Pnc).Methods: 577 invasive pneumococci from all age groups were used in this study.These were characterised by serotyping and multi-locus sequence typing. Down-stream sequence analysis was performed using sequence type analysis andrecombinational analysis software (http://pubmlst.org/software/analysis/start/).Results: One hundred and nine different sequence type profiles were identified in thecomplete data set from all agegroups. 74 ST’s were associated with vaccine-relatedserotypes. Sequence types 9 and 124 were most common and are solely associatedwith serotype 14 although they do not belong to the same genetic lineage. BURSTanalysis of the full data set grouped the data into 22 major genetic lineages and 32singleton clones. 61 isolates received were from infants under 5 years of age. The 7most common serotypes, causing 75.4% of IPD in under fives are included in theproposed 7-valent conjugate vaccine. In addition, the 9-valent and the 11-valent vac-cine would cover 77% and 80.3% respectively of IPD cases in this age group, a differ-ence which is not statistically significant (p=0.9231). There was no evidence of sero-type 5 pneumococci (included in 9-valent vaccine) in our data.Conclusions: Administration of a Pnc vaccine would alleviate a large proportion ofdisease. The 7-valent Pnc vaccine includes the 7 most common serotypes in Scot-land; however inclusion of serotype 5 in the 9 and 11-valent vaccines would cur-rently be of limited use as pneumococci of this type have not been identified inScotland in 2003. By mapping the major genetic lineages, closely related non-vac-cine serotypes can be identified which may become more prevalent following Pncimplementation.


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Impact of 7-valent pneumococcal conjugate vaccine on rates of tympanic mem-brane perforation in young Aboriginal children living in remote communitiesthroughout the Northern Territory of Australia: a before and after study.

Mellon G, Wilson C, Hamilton E, Silberberg P, Munkara M, Leach AJ, Morris PS.Menzies School of Health Research, Darwin, Australia.

Background: Rates of tympanic membrane perforation (TMP) among Aboriginalchildren less than 2 years of age in the Northern Territory (NT) were as high as 20-30% in the late 1970s. Despite a decline infant mortality over recent years, therehas been no evidence of improved ear status. The 7-valent pneumococcal conjugatevaccine (7vPCV, Prevenar¨) given at 2, 4, and 6 months plus the 23-valent polysac-charide vaccine (Pneumovax¨) at 18 months, has been funded for all Aboriginalchildren since June 2001.Aims: To describe the prevalence and severity of otitis media, particularly acuteotitis media with perforation (AOM/wiP) and chronic suppurative otitis media(CSOM) in young Aboriginal children in the NT before and after the commence-ment of the 7vPCV vaccination program.Methods: 29 remote communities throughout the NT participated in the study.Aboriginal children aged 6 to 30 months were eligible to be assessed. Each childhad a standardised ear examination including tympanometry, pneumatic otoscopy,and video-otoscopy. They also had their medical records reviewed.Results: 711 of 914 eligible children were examined in the pre-7vPCV survey.Overall, 24% of children had either AOM/wiP or CSOM in their worst ear. Only7% had bilateral normal middle ear status. Post-7vPCV, 646 of 936 eligible Abo-riginal children were seen. Preliminary analyses indicate that overall rates of OMand TMP have not changed substantially.Conclusion: Otitis media remains a major health problem in children living inremote communities in the NT despite the introduction of 7vPCV. Training andsupport for accurate diagnosis and appropriate antibiotic treatment of suppurativemiddle ear infections in this population must continue.

Reactogenicity of Pneumococcal Polysaccharide and Conjugate Vaccines inAdults Aged 55-70 Years

Miernyk K1,2, Bulkow L1, Butler J1, Dentinger C1, Hennessy T1, Hickel J3, KnutsenB3, Parkinson A1, Peters H1,2, Singleton R1,2. AIP/CDC1; ANTHC2; SCF3, Anchor-age, AK.

Background: The value of pneumococcal conjugate vaccines in adults has not beendefined. One proposed use it to prime the immune system with conjugate vaccineand later boost with a dose of polysaccharide vaccine (PPV23). We studied thisstrategy and here we present data on the reactogenicity of using these two vaccinesin combination.Methods: We randomized immunocompetent adults 55-70 yrs who had not previ-ously received any pneumococcal vaccination to receive immunization with either(1) one dose of PPV23, (2) one dose of 7-valent CRM

197 pneumococcal conjugate

vaccine (PCV7) followed two months later by a dose of PPV23, or (3) PCV7 fol-lowed six months later by a dose of PPV23. Local and systemic reactions wererecorded by participants in study diaries during the 4 days after each vaccinationand by study personnel in a phone interview 4-6 days after each dose.Results: Side effects data were obtained from all 144 vaccinations (86 participants)either from a completed diary (83%), a phone interview (88%), or both (72%). Thefrequency of 17 local or systemic reactions was similar for persons receiving one doseof PPV23 and those receiving an initial dose of PCV7 (p>0.1 for each). At 24 hoursafter vaccination with PPV23, persons who had received PCV7 two months earlierwere more likely to report localized swelling (69%) and soreness (56%), than werethose who received either PPV23 six months after PCV7 (35%, p=0.015; 15%, p=0.006,respectively) or those who received PPV23 alone (21%, p<0.001; 13%, p=0.003, re-spectively). The only report of fever �38ºC was by one person after receiving aninitial dose of PCV7. All side effects diminished rapidly over 48-72 hours.Conclusions: PCV7 is well tolerated among adults. To minimize local reactions, itis preferable to give a booster dose of PPV23 at six rather than two months afterpriming with PCV7.


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Immunogenicity Of 7-Valent Protein Conjugate Pneumococcal PolysaccharideVaccine And The Effect Of Past 23-Valent Polysaccharide Vaccine In HIV-In-fected Ugandan Adults.

Miiro G1, Kayhty H2, Watera C1, Tolmie H3, French N3. 1 MRC programme on Aids,Uganda, 2 National Public health Institute, Finland, 3 Liverpool School of TropicalMedicine, UK

Aims: To assess immunogenicity of 7-valent conjugate pneumococcal vaccine (CPV)in HIV-infected antiretroviral treatment naive adults in Uganda and 2. To investi-gate the association of past 23-valent pneumococcal polysaccharide vaccine (PPV)with response.Method: Participants were the surviving members of a randomised controlled trialof PPV in Entebbe cohort study. Following informed consent all participants re-ceived 2 doses of CPV 4, weeks apart. Anti capsular polysaccharide IgG (Anti-PSIgG) was measured to the seven vaccine serotypes using a standard method incor-porating a 22F adsorption step, in pre-vaccine and two post-vaccine serum samples,4 weeks after 1st and 2nd dose respectively.Results: 109 individuals participated (54 past PPV, 55 past Placebo, 84% female).Median CD4 251[range 1-936]. Baseline anti-PS IgG concentration was signifi-cantly directly associated with CD count (p<0.01, ANOVA), and inversely withtime from enrolment in the original study cohort (P�0.02, ANOVA) for all serotypes.There was a significant response to first CPV for all serotypes (P<0.01, paired t-test). There was no difference in response by past PPV exposure (0.14<p<0.97,ANOVA derived). Post-vaccine levels were CD4 associated for all serotypes (p�0.02)except 14 and 18c. Further significant increases in anti-PS were measured for 4(4,6b,19f & 23f) of the 7 serotypes after the 2nd CPV.Conclusion: Past PPV has not been shown to affect response to CPV in this groupof HIV-infected Adults. Response to CPV was significant overall and CD4 T-cellcount dependent. Response to second dose of CPV suggests different dosing sched-ules should be investigated in HIV-infected adults

Conjugate pneumococcal vaccine (PCPV-7) for patients who, post-splenectomy,have invasive pneumococcal disease despite having received 23-valent polysac-charide vaccine (PPV-23)

D. Musher,1,3 H. Ceasar,1,3 E. Kojic,3 B. Musher,3 J. Gathe, Jr.,3 A. White, Jr.2,3 VAMedical Center,1 Ben Taub General Hospital,2 Baylor College Medicine,3 Houston,TX

Background, aims: After splenectomy, patients (pt) have increased risk for over-whelming sepsis and death due to infection with S. pneumoniae (Spn). PPV-23 isstrongly recommended. Some healthy adults who may have genetically mediatedfailure to respond to PPV-23 remain at risk for Spn sepsis. Our aim was to see ifsuch persons develop antibody after receiving PCPV-7 (Prevnar, Wyeth).Methods and Results: We identified 2 post-splenectomy pt who had recurrent Spnsepsis despite �2 doses of PPV-23. Serum in convalescence and after further PPV-23 lacked detectable antibody to Spn capsular polysaccharides (CPS) 4, 6B, 9V, 14,18C, 19F, 23F. After PCPV-7, both pt developed IgG to all CPS contained in thevaccine (see data from representative pt in Table, below). IgG from both subjectsprotected mice against challenge with Spn type 4 (a mouse-virulent strain).Conclusions: There is no clear evidence that adults respond to PCPV-7 with higherlevels of IgG than they do to PPV-23. We have shown that adults who, on a geneticbasis, fail to respond to PPV-23 may develop IgG after receiving PCPV-7, and itseems likely that this is also true of our two pt. Invasive Spn disease after PPV-23 innon-elderly, non-immune-suppressed splenectomized adults might prompt use ofPCPV-7.

Table: Antibody to CPS (ug/ml) in a representative post-splenectomy pt after PPV-23 or PCPV-7

4 6B 9V 14 18C 19F 23F Post-PPV, post-sepsis 0 0 0 0 0 0 0 Post repeat PPV-23 0 0 0 0 0 0 0 Post-PCPV-7 14.3 20.9 4.6 33.6 1.7 2.5 8.6


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Opsonophagocytic activity of antibodies induced by the 11-valent pneumococ-cal conjugate vaccine

Nurkka A1, Poolman J2, Henckaerts I2, Kilpi TM1, Käyhty H1. National Public HealthInstitute, Helsinki1, Glaxo SmithKline, Rixensart, Belgium2.

Aims: The 11-valent pneumococcal conjugate vaccine (11Pn-PD) containing a newcarrier, a recombinant protein D of Haemophilus influenzae, is immunogenic ininfants. We have now extended the studies by determining the opsonophagocyticactivity (OPA) of anti-Pnc PS antibodies induced by the 11Pn-PD.Methods:155 healthy Finnish infants were randomised to receive either 11Pn-PD(N=103) or a hepatitis B vaccine (HBV) (N=52) at 2, 4, and 6 mo of age. At 12-15mo children were boosted either with the 11Pn-PD (all children in the HBV groupand 51 in the 11Pn-PD group) or with 23-valent PS vaccine (N=51 in the 11Pn-PDgroup). OPAs to vaccines serotypes (VST) 6B, 14, 18C, 19F, and 23F and to cross-reactive types, 6A and 19A, were measured by standard killing assay at 7 mo, andbefore and 28 d after the booster. Results: After 3 doses of 11Pn-PD the geometricmean OPA titers to VSTs (the reciprocal of the serum dilution with 50% killingcompared to the controls without serum; detection limit is titer 8) varied between18 (23F) and 209 (6B). Before the booster, over half of the sera were still OPApositive. After the 4th Pnc vaccination almost all sera were OPA positive and after 4doses of 11Pn-PD the mean titers ranged from 42 (23F) to 375 (6B), while in thePnc PS boosted group the mean titers were higher and ranged between 73 (23F) and798 (6B). One dose of 11Pn-PD at the age of 12-15 mo induced low OPA titers; themeans ranged between 4 (23F) and 19 (18C). OPA titers correleted with the IgGanti-PS concentrations (r=0.7-0.9 depending on the serotype and age). 20% hadOPA to the types 6A and 19A at 7 months and 40-60 % after boosting. Conclusions:Antibodies to Pnc VST induced by three or four doses of 11Pn-PD have good OPA.The highest OPA titers were found in the group that received 3 doses of 11Pn-PDand a dose of Pnc PS as a booster. One dose at 12-15 mo induced low OPA re-sponse.

EFFECTIVENESS OF PNEUMOCOCCAL POLYSACCARIDE VACCINE INPATIENTS AGED 65 YEARS OR OLDER.

Ochoa Gondar O; Vila Córcoles A; Llor Vila C; Hospital Guardiola I; Ansa EcheverríaX; and EVAN-65 Study Group.

Catalonian Health Institute-Institut Català de la Salut. Catalonia. Spain.Supported by a grant from the Spanish Government (Fondo de InvestigacionesSanitarias. Ministerio de Sanidad y Consumo. Exp. PI 021117).

Background: Streptococcus pneumoniae causes 20-30% of the community-acquiredpneumonia (CAP). The effectiveness of the pneumococcal vaccination (PPV) withregard to the prevention of pneumococcal infection is currently controversial andappears to vanish among elderly people. However, the indication of vaccination inthis group continues. The aim of this study is to assess the effectiveness of thisvaccine in elderly.Methods: Retrospective study with two cohorts of patients over 65 (301 vaccinatedwith 23-valent pneumococcal polysaccharide vaccine and 301 not vaccinated). Weidentified every CAP succeeded from 1994 to 2000, and compared the incidencerates and severity degree of pneumonia in both groups.Results: The mean incidence rate of pneumonia was 13,29‰ in inhabitants duringthe 6-year period of time (16,6‰ in vaccinated cohort and 9,96‰ in not vaccinatedcohort), with a relative risk for the vaccinated cohort of 1,67 (95%CI: 0,95 – 2,92).No statistical significant differences were found neither in mortality rates (vacci-nated cohort: 13.7% vs. 15.7% in no vaccinated cohort) nor in the severity degreeof pneumonia.The incidence rates were higher when the risk factors for CAP were present (chroniclung disease, smoking habit, diabetes and cardiopathy).Nevertheless, the relative risk was only significant in the smoking habit group (2,88;CI 95%: 1,15-7,01).Conclusions: A protective effect of the pneumococcal vaccine has not been provedin our country, neither to prevent the infection nor to decrease the severity of thepneumococcal pneumonia.


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Immune response to pneumococcal polysaccharide (Pnc PS) vaccine in bonemarrow transplant (BMT) donors and recipients; the effect of 22F neutralisa-tion step in EIA to the results

Parkkali T1, Käyhty H2, Piipponen S2, Holm A2, Lahdenkari M2, Ruutu T1, Ruutu P2.Helsinki University Hospital1 and National Public Health Institute2, Helsinki, Fin-land

Aims: Previous studies using the standard EIA for assessing immune response haveshown that BMT recipients respond poorly to Pnc PS vaccine. The 22F absorptionstep in EIA has been found to increase the specificity of the standard EIA. We havecompared the anti-Pnc PS concentrations measured by the two EIA in sibling BMTrecipients and donors before and after vaccination with Pnc PS vaccine.Methods: 114 patients receiving BMT were included into the study. 54 of the siblingdonors were vaccinated 2 to 12 weeks before BMT with Pnc PS. All patients receivedPnc PS 12 months after the BMT. Serum samples were taken before and 6, 12 and 13months after BMT from recipients and before and after vaccination from donors.Both the standard EIA and 22F EIA were used to measure antibodies to type 6B, 14,19F and 23F PS in sera taken from 83 patients. Opsonophagocytic activity (OPA) of24 sera against type 19F strain was assessed by a standard killing assay.Results: 22F EIA gave lower anti-6B, -19F and 23F, but not -14 concentrations, inboth BMT recipients and donors. The response rate of donors, but not of the recipi-ents, for the the types 6B, 19F and 23F, was higher by 22F EIA than by standardEIA. The concentrations and response rates of the recipients were low by both as-says. The 19F-OPA correlated better with the 22F EIA than with the standard EIA.The difference between the two assays was most notable for sera taken before im-munization.Conclusions: The study confirmed that BMT recipients respond poorly and rarelyto Pnc PS vaccine even when 22F EIA is used for antibody determination. Theresponse rates and fold increases of healthy adults are higher when 22F EIA is used.The future studies among both healthy adults and high risk patient groups shoulduse 22F EIA, because it correlates better with OPA and thus predicts better theclinical protection.

Impact of Booster Dose of PCV on Pneumococcal Colonization, Invasive Dis-ease and Antimicrobial Resistance in Massachusetts.

SI Pelton, CD Marchant, AM Loughlin, KK Hsu, S Karumuri and MassachusettsDPH. Boston University School of Medicine and the Massachusetts Department ofPublic Health, Boston, MA.

Aims: Surveillance of pneumococcal carriage in two communities in MA and ofstatewide invasive disease was initiated following the introduction of PCV in Oct.2000 and 2001, respectively.Results: A decline in nasopharyngeal (NP) carriage of most vaccine serotypes andan increase in non-vaccine serotypes has been observed and continues to evolvemore than 3 years after the introduction of PCV. A greater than 70% reduction ininvasive pneumococcal disease (IPD) has resulted from immunization of childrenwith PCV. Vaccine serogroups 6 and 19 persist in the NP and are now the mostfrequent isolates recovered from vaccine serogroup disease. Twenty-one of 25 casesof vaccine serotype (10 cases) and vaccine-related serotype (15 cases) vaccine fail-ures were caused by serogroup 6 or 19. No substantial changes in the proportion ofdrug-resistant Streptococcus pneumoniae (DRSP) among NP isolates in the com-munity have been observed over the 3-year time period. In contrast to NP isolates,the proportion of DRSP causing IPD has declined over a 2-year time period. Thesedeclines have occurred despite a statewide PCV shortage requiring deferral of thebooster dose in children without specific high-risk medical indications.Conclusions: In May 2003, PCV shortage was resolved and booster dose againrecommended. Ongoing surveillance of both NP carriage in children < than 2 yearsof age and IPD in children < than 18 yrs. will permit evaluation of the impact of thebooster dose on ecology of SP including serotype-specific carriage in children whohave received a primary series as well as those < than 7 months of age, changes inDRSP trends, and serotype-specific incidence and distribution of invasive SP. Thesedata should provide additional understanding of the importance of the booster onpneumococcal disease and carriage in the community.


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Diversity of emerging serotype 16F pneumococci in remote Australian Abo-riginal communities.

Pettit A, Smith-Vaughan H, Hare K, Morris PS, Leach AJ. Menzies School of HealthResearch, Darwin, Australia.

Background: Serotype 16F pneumococci are commonly carried by indigenousAustralian children living in remote communities. Serotype 16F is associated withinvasive pneumococcal disease and otitis media in this population, and has becomethe predominant carriage serotype since infant and catch-up 7vPCV vaccinationprograms commenced in July 2001.Aim: To study antibiotic susceptibility patterns and genetic diversity of the serotype16F pneumococcal populations in 3 remote communities between 1996 and 2003.Methods: Nasopharyngeal carriage isolates were collected from children with oti-tis media. Antibiograms for serotype 16F isolates were determined using disc diffu-sion and E-test. Isolates were typed by BOX-PCR, and multi-locus sequencing typ-ing (MLST) results are pending.Results: Of the 197 isolates tested, all were susceptible to the antibiotics tested,with the exception of resistance to oxacillin which appeared in 1999 and was appar-ent in all 16F isolates by 2003. However, these isolates mantained low minimuminhibitory concentrations for penicillin. Eight different BOX types were found amongthe 26 isolates tested to date. One type predominated throughout the period. Threenew types appeared after the introduction of the 7vPCV vaccination program whichwas accompanied by a fall in vaccine serotype carriage and a rise in prevalence of16F carriage.Conclusions: Serotype 16F pneumococci have become a predominant carriage se-rotype since 7vPCV vaccination commenced in this population. New 16F strainshave appeared and further studies using MLST will reveal the potential associatedmechanisms. Emergence of non-PCV/non-PPV serotypes needs to be monitored,particularly in populations at high risk of invasive pneumococcal disease.

Adult mucosal and systemic immune responses to pneumococcal polysaccha-ride & conjugate vaccines

WS Pomat1, R Thornton1, S Martin1, S Choo2, D Lehmann3, P Richmond1. 1Univer-sity Department of Paediatrics, 2Princess Margaret Hospital, 3Telethon Institute forChild Health, Perth, Western Australia

Background: In order to understand mechanisms of protection against mucosalpneumococcal diseases and optimise use of current vaccines, it is important to beable to accurately measure mucosal immune responses. The aims of this study, wasto evaluate the kinetics of mucosal and systemic antibody responses in healthy adultsto pneumococcal polysaccharide (PPV) and conjugate vaccines (PCV).Methods: 32 healthy adults were randomised to receive either 23-valent PPV(PneumovaxTM) or 7-valent PCV (PrevenarTM) and had blood, saliva and nasal washsamples collected prior to and at 1wk, 2wk, 4wk and 18 months post-vaccination.Serotype specific IgG and IgA were measured using standardised ELISA assays forserotypes 1, 3, 4, 6B, 9V, 14, 18C, 19F and 23F and serotypes 3, 6B, 14, 19F and23F respectively.Results: No serious adverse reactions were reported. Most adults had pre-existingprotective IgG antibody concentrations (� 0.2�g/ml). Both vaccines induced sig-nificant increases in serotype specific serum IgG and IgA antibodies in the respec-tive vaccines. There were no significant differences between vaccines for the PCVserotypes although PCV antibody concentrations peaked at two weeks post vacci-nation whereas antibody responses were maximal four weeks post PPV. SalivaryIgA (sIgA) increased following vaccination although sIgA initially declined fol-lowing PPV at 1 week but not after PCV. Antibody persistence at 18 months wasbetter for IgG compared to IgA with some serotype variation.Conclusions: Both vaccines induce good serum and mucosal antibody concentra-tions, however subtle differences were observed in the kinetics of responses be-tween vaccines. Further studies are ongoing to examine IgG and IgA avidity matu-ration and cellular immune responses to PCV CRM carrier protein and pneumo-coccal surface proteins.


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Multiplexed and Single-Colour Flow Cytometric Opsonophagocytosis Assaysfor Detection of Functional Pneumococcal Antibodies in Infant Sera

Ryynänen M, Jousimies K, Nikkanen N, Käyhty H. and Ekström N. National Pub-lic Health Institute (KTL), Helsinki, Finland.

Aims: Host protection to Streptococcus pneumoniae is mainly mediated by op-sonin dependent phagocytosis and in vitro measured opsonophagocytic activity issuggested to be a reliable correlate of protection. We have compared threeopsonophagocytic assays (OPA); killing assay, single-colour and multiplexed flowcytometric OPA which is capable of simultaneously measuring antibodies to twopneumococcal serotypes.Methods: Panels of 28 or 41 sera from infants immunized with a 7-valent pneu-mococcal conjugate vaccine or a hepatitis B vaccine were used. Theopsonophagocytic activity was evaluated by using 1) standardized killing assay, 2)single-colour flow cytometric assay (SC-OPA), and 3) multiplexed flow cytometricassay (MP-OPA) for which serotype 19F and 23F bacteria were labelled with dif-ferent fluorescent dyes (5,6 carboxy-fluorescein succinimidyl ester and Alexa Fluor“647 succinimidyl ester). Serotypes 4 and 6B were tested by assays 1) and 2), andserotypes 19F and 23F by all three assays.Results: The killing assay and SC-OPA correlated well for all four serotypes (r=0.70-0.88). The correlations between the killing assay and MP-OPA (r=0.64, for sero-type 19F, and r=0.75 for serotype 23F) and between the SC-OPA and the MP-OPA(r= 0.74 for serotype 19F, and r=0.83, for serotype 23F) were good for both serotypestested. All three assays were specific and reproducible.Conclusions: The flow cytometric assays are specific, reproducible and correlatewell with the standardized killing assay. Compared to the killing assay or to theSC-OPA, the MP-OPA is cheaper, increases the overall sample throughput andthus decreases the testing time, is less laborious to perform and requires less sam-ple volume. The multiplexed assay can easily be set up for other serotypes and bedeveloped to measure antibodies to four serotypes simultaneously.

Does nasopharyngeal carriage of S. pneumoniae prime for antibody responsesto pneumococcal polysaccharides during the first year of life?

Salt PM, Oh S, Banner C, Ferry B, Pollard AJ. Oxford Vaccine Group. University ofOxford. England.

Aims: To assess the influence of risk factors for nasopharyngeal carriage of S.pneumoniae during infancy on the immune response to a 7-valent pneumococcalconjugate CRM-197 vaccine.Methods: Forty-five children (age 12-13 months) were immunised with 2 doses ofa 7-valent conjugate pneumococcal vaccine 2 months apart. A blood sample wasobtained before and 9-11 days after the first dose. Presence or absence of risk fac-tors for nasopharyngeal carriage of S pneumoniae were assessed (siblings and day-care attendance), based on data from a longitudinal study of pneumococcal carriagein Oxfordshire infants. Pneumococcal antibody concentration and avidity of anti-body were measured and related to risk factors for nasopharyngeal carriage.Results: Children with no siblings who did not attend day-care (group 1) had simi-lar baseline antibody concentrations to those children who had siblings and alsoattended day-care (group 2) and there was a rise in the geometric mean pneumococ-cal antibody concentration following vaccination in both groups. The post-immuni-sation antibody concentrations against serotype 4 and 23f were significantly higherin sera from children in group 2 than in group 1 (p = 0.0028 and p = 0.0103 respec-tively). Conversely, children with a higher risk for pneumococcal carriage (group 2)did not have higher antibody response to 6b or 19f. No significant difference inavidity of pneumococcal antibody was detected between groups.Conclusion: These data imply that carriage of S. pneumoniae during the first yearof life may prime for B cell responses to serotype 4 and 23f polysaccharides but notclearly for 6b or 19f. The frequently carried serotypes 6b and 19f may be lessimmunogenic, thereby facilitating carriage. Alternatively exposure to these organ-isms may be so frequent in infancy that all children become primed by a year of ageand show evidence of priming regardless of specific risk factors for carriage.Funded by a grant from Wyeth Vaccines.


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Invasive pneumococcal disease (IPD) in Scotland during 2003: genetic rela-tionships between isolates not covered by the 7-valent pneumococcal conjugatevaccine prior to its introduction

SCOTT KJ 1, MCCHLERY SM 1, CLARKE SC 1, 2. Scottish Meningococcus andPneumococcus Reference Laboratory, Glasgow, UK 1 and Division of Infection andImmunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glas-gow, UK 2

Aims: To gain an understanding of the current molecular genetic relationships ex-isting between invasive pneumococci of serotypes not included in the 7-valent pneu-mococcal conjugate vaccine (Pnc) prior to its introduction in Scotland and how itmay affect the pneumococcal population.Methods: Serotyping and multi-locus sequence typing (MLST) was used to charac-terise 255 invasive pneumococci selected by virtue of their serotype not being presentin the licensed 7-valent Pnc vaccine Downstream sequence analysis was performedusing sequence type (ST) analysis and recombinational analysis software availablefrom http://pubmlst.org/software/analysis/start/.Results: Serotyping was able to distinguish 28 different serotypes. MLST catego-rised these isolates into 64 different ST’s, 24 of which were represented by morethen one isolate. BURST analysis grouped the isolates into 13 lineages, with 26singleton sequence types. Comparison of our data with the MLST database revealedthat 12 ST’s included isolates whose serotype was included in the 7-valent vaccine.Two isolates were also found to be of the same lineage, but with a different sero-type, as the PMEN England 14-9 clone which is often associated with erythromycinresistance.Conclusions: Implementation of a Pnc vaccine will likely lead to reduced carriageof included serotypes and also confer herd immunity. Our study provides an initialinsight into the relationships between invasive pneumococci whose serotype is notincluded in the 7-valent Pnc vaccine prior to its introduction in Scotland. Vigilancemust remain over the potential for capsule replacement once Pnc vaccines are im-plemented.

Evaluation Of 9-Valent CRM197

Conjugated Pneumococcal PolysaccharideVaccine In Elderly Adults

Shelly MA1, Schiff G2, Smith V2, O’Brien D1, Nahm M3, Germanson T4, Ewell M4,Treanor JJ1. University of Rochester, Rochester, NY1, Gamble Program for ClinicalStudies, Cincinnati, OH2, and University of Alabama in Birmingham, AL3, EmmesCorp., Potomac, MD4.

Aims: Pneumococcal disease remains a serious problem in the elderly, even with alicensed polysaccharide (PS) vaccine. Protein conjugation improves theimmunogenicity of pneumococcal PS in infants. Therefore, we evaluated theimmunogenicity of protein conjugated PS (CRM-PS) in the elderly and its effect onsubsequent administration of PS vaccine.Methods: 180 healthy adults 65 and older were randomized to receive:

Group Time 0 4 months 8 monthsA CRM-PS Placebo PSB CRM-PS CRM-PS PSC PS Placebo Placebo

Serotype-specific antibody was measured by EIA and opsonization before and 1month after each injection. Results are available for 10 subjects per group; EIAresults will be complete by May 2004.Results: 169 subjects completed the study. Demographics and medical historieswere similar between groups. CRM-PS vaccination was well tolerated and gener-ated no greater local reaction than the PS vaccine. However, PS vaccination 8months after a single CRM-PS vaccination (Group A) resulted in greater frequencyof local reactions than PS vaccine alone (Group C) (induration P=0.005, ery-thema P=0.003). Group A also had higher GMT antibody levels to 8 of 9 serotypes1 month after PS vaccine than seen in Group B or C. The ratio of the differencebetween groups A and C was a median of 1.9 (range, 0.7 to 3.6). Titers of opsoniz-ing antibody rose with either CRM-PS or PS vaccination; differences did not reachstatistical significance in the first 10 subjects.Conclusion: CRM-PS is well tolerated and immunogenic in older adults. Proteinconjugation of pneumococcal PS antigens may prime for a more robust responseto subsequent exposure to PS.


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IgG Subclass Responses to Pneumococcal Polysaccharide (PPS) Booster 6 yearsafter Pneumococcal Conjugate (Pnc) Vaccination in Infancy. Effect of PPSBooster at 13 Months.

Sigurdardottir1 ST, Davidsdottir K2, Jonsdottir I1.1Landspitali University Hospital, Reykjavik, Iceland,2Center for Child Health Services, Reykjavik, Iceland.

Aim: The 8-valent PncT (serotype 3, 4, 6B, 9V, 14, 18C, 19F and 23F, conjugatedwith Tetanus protein) (Aventis Pasteur) was immunogenic when given at 3, 4 and 6month (mo) of age. Booster with 23-valent PPS gave higher serotype specific IgGresponses than PncT at 13 mo1. At 7 years we demonstrated IgG booster responsesto 1/10th dose PPS (2,5 µg/type) for 7 out of 8 vaccine serotypes (VT) not shown incontrol children. No or minimal responses were observed to 3 nonvaccine serotypesNVT2. Now we further analyzed the IgG responses with measurements of IgG1 andIgG2 to 3 VT (6B, 19F and 23F) and 2 NVT (1, 5) in the above group of PncTvaccinated children, boosted with PncT or PPS and unvaccinated controls.Methods: Of 41 child recruited, 32 were primed with PncT, 17 had been boostedwith PncT at 13 months (PncT group), 15 with PPS (PPS group) and 9 had notreceived pneumococcal vaccine. Blood samples were obtained on day 0, 7 and 28and serotype specific IgG, IgG1 and IgG2 responses (ELISA) were evaluated.Results: The IgG responses at 7 years were mostly due to increases in IgG1 al-though only in the PncT group for serotypes 6B and 19F the IgG1 was higher thanIgG2 with IgG1/IgG2 ratio 1.13 and 1.86, respectively on day 28, but 0.86, 0.16,and 0.45 for type 1, 5 and 23F, respectively. In the PPS and control groups the ratiowas 0.58 and 0.28 (serotype 1), 0.19 and 0.08 (serotype 5), 0.84 and 0.15 (serotype6B), 0.77 and 0.51 (serotype 19F) and 0.41 and 0.26 (serotype 23F), respectively.Conclusion: Children immunized with PncT vaccine in infancy still have immuno-logical memory to the vaccine serotypes at 7 years of age. Serotype specific anti-body responses at that age are predominantly IgG1 although IgG2 represents themajority of pneumococcal serotype specific IgG antibodies in all the groups.1Sigurdardottir et al. Pediatr Infect Dis J 2002 2Sigurdardottir et al. [Abstract # G-2049], ICAAC 2003

Nasopharyngeal pneumococcal carriage after combined pneumococcal conjugateand polysaccharide vaccination in children with recurrent AOM.

Veenhoven R1, Bogaert D2, Uiterwaal C3, Rijkers G4, Schilder A4, Hermans P2, SandersE4. 1Spaarne Hospital, Haarlem, 2Sophia Children’s Hospital/Erasmus University Rot-terdam, 3Julius Center for Health Sciences and Primary Care, Utrecht, 4UniversityMedical Center/ Wilhelmina Children’s Hospital, Utrecht, The Netherlands.

Aims. To evaluate the impact of 7-valent pneumococcal conjugate vaccine (PCV7)followed by 23-valent pneumococcal polysaccharide vaccine (PPSV23) on nasopha-ryngeal carriage in children with recurrent AOM.Methods. Prospective, randomised, controlled trial (April 1998 - January 2002). Inclu-sion of 383 children, aged 1-7 years, with � 2 AOM episodes/year. Vaccination schemein pneumococcal vaccine (PV) group: PCV7 [Prevenar®, Wyeth-Lederle] followed 7months later by booster PPSV23 [Pneumune®, Wyeth-Lederle]. Children aged 1-2years received PCV7 twice, older children once. Control vaccine (CV) group: hepatitisvaccines. During follow-up nasopharyngeal swabs were taken at t= 0, 7, 14, 20, and 26months. The nasopharyngeal swabs were cultured by conventional methods and pneu-mococcal serotypes were determined by Quellung reaction. At baseline and one monthafter booster PPSV23, serum IgG to PCV7 serotypes was determined by ELISA in 93children.Results. Prior to vaccination nasopharyngeal carriage of S. pneumoniae was found in49.3% of all children, regardless of age. Of all pneumococcal serotypes, 53% wereincluded in PCV7. In children aged 12-24 months, conjugate vaccine type (CVT) S.pneumoniae decreased significantly after complete pneumococcal vaccination as com-pared to CV group (P<0.001). In older children, carriage of CVT S. pneumoniae didnot differ from controls anymore (P=0.16). After pneumococcal vaccinations high IgGantibody responses were induced against all PCV7 serotypes, except for type 6B.Conclusions: Pneumococcal conjugate vaccinations influence pneumococcal nasopha-ryngeal carriage only in children aged 12-24 months, who received PCV7 twice. Nosignificant influence was observed anymore in older children who received PCV7 onlyonce. Most probably, multiple doses of PCV7 induce more memory B cells as comparedto one dose, resulting in better mucosal immunity. Vaccination with at least twice theconjugate vaccine also after 2 years of age may be mandatory for carriage reduction.


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Immunogenicity of combined pneumococcal conjugate and polysaccharide vacci-nation in children with recurrent AOM.

Veenhoven R1, van Kempen W2, Wiertsema S3, Uiterwaal C4, Schilder A3, Rijkers G3,Dhooge I2, Sanders E3. 1Spaarne Hospital, Haarlem, The Netherlands, 2Ghent Uni-versity Hospital, Belgium, 4Julius Center for Health Sciences and Primary Care,Utrecht, 3University Medical Center/ Wilhelmina Children’s Hospital, Utrecht, TheNetherlands.

Aims: To evaluate the immunogenicity of 7-valent pneumococcal conjugate vaccine(PCV7) followed by 23-valent polysaccharide vaccine (PPSV23) in children with re-current AOM.Methods: Prospective, randomised, controlled trial (April 1998 - January 2002) in457 children in the Netherlands and Belgium. Inclusion of children aged 1-7 years,with �2 AOM episodes/year. Vaccination scheme in pneumococcal vaccine (PV)group: PCV7 [Prevenar®, Wyeth-Lederle] followed 7 months later by PPSV23[Pneumune®, Wyeth-Lederle]. Children aged 1-2 years received PCV7 twice, olderchildren once. Control vaccine (CV) group: hepatitis vaccines. In 122 at randomlyselected children of PV group and 57 children of CV group, pre and post-vaccina-tion serum IgG, IgG1, IgG2 to pneumococcal serotypes included in PCV7 weremeasured by ELISA at baseline and one month after PCV7/control vaccine or boosterPPSV23/control vaccine. Children with IgG anti-pneumococcal antibody titer > 1.0mg/L were considered to be a responder.Results: After pneumococcal vaccinations high IgG antibody responses (>1.0 mg/L)were induced against all PCV7 serotypes, except for type 6B (<1.0 mg/L). Both IgG1and IgG2 antibodies were induced. Children aged 12-24 months responded in > 90%for serotypes 4, 9V, 14, 18C, 19F and 23F. Older children showed same pattern, withexception for serotype 4 (71%).Conclusions: PCV7, followed by booster PPSV23 induced high serum IgG antibodyresponses to all PCV7 pneumococcal serotypes in all children, except for serotype 6B.Not only IgG1, but also IgG2 antibodies were induced. Vaccination with at least PCV7twice, also after 2 years of age, seems to be mandatory for optimal antibody responses.A third conjugate vaccination may be required for improved IgG anti 6B responses.

POLISACCHARYDE PNEUMOCOCCAL VACCINE: CLINIC FACTORSWHICH HAVE INFLUENCE IN THE VACCINATION IN ELDERLY.

Vila Córcoles A; Ochoa Gondar O; Ansa Echeverría X; Llor Vila C; Hospital GuardiolaI; and EVAN Study Group.Catalonian Health Institute-Institut Català de la Salut. Catalonia. Spain.Supported by a grant from the Spanish Government (Fondo de InvestigacionesSanitarias. Ministerio de Sanidad y Consumo. Exp. PI 021117).

Aims: The major aim of this project is to know the level of coverage of the pneumococ-cal vaccine (PPV) among people over 65, and analyse this level of coverage having inmind predictors such as some chronic diseases or risk factors (PRF) to suffer fromcommunity acquired pneumonia (CAP).Methods:design: prospective study, multicentric, observactional and cross-sectional.placed: at Primary Health Care centres.subjects: the sample was composed by peopleover 65 assigned to eight urban Primary Health Care centres (n=11597).intervenctionsand measuraments: with the help of informatic analysis of case-history, vaccinationand risk factors database, an integrated clinic database was created. In the same way,we reviewed in each patient the presence or not of chronic diseases or risk factors(PRF) to suffer from community acquired pneumonia (CAP).statistic analysis: logis-tic regression with the calculation of odds ratio (OR) adjusted for each PRF and estab-lishment of an understanding model with the variables which have influence over thevaccination.Results: The global coverage was 47.1%, without differences between sexe (47.3%males and 46.9% female). The minimum coverage was among people between 65-74(41.3%) and maximum for those between 75-84 (55.5%). This proportion slightly de-creased in people over 85 (50.1%). The coverage of PPV was 56.4% in diabetics,62.2% in lung chronic diseases, and 44.8% in smokers.The age (OR: 1.02; CI 95%: 1.01-1.03), the high blood-pressure (OR:1.92; CI 95%:1.67-2.21), the diabetes (OR: 1.29; CI 95%: 1.03-1.62) and the number of PRF foreach people (OR: 1.46; CI 95%: 1.29-1.65) were factors which have influence in-creasing the probability to be vaccinated. To be smoker was an independent factorwhich decreased significantly the probability of vaccination (OR: 0.57; CI 95%:0.43-0.76).


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Long-term Immune Response to 23-valent Pneumococcal Vaccine in Adultswith Spinal Cord Injury

Ken B. Waites, Kay C. Canupp, Moon H. Nahm, Yu-ying Chen, Michael J. DeVivoUniversity of Alabama at Birmingham, Birmingham, AL USA

Aims: Persons with spinal cord injury (SCI) are predisposed to develop pneumoniabecause of mechanical problems resulting from respiratory paralysis and loss ofneural control mechanisms and pneumonia is the most common cause of death forevery age group and all time periods post-injury. In view of the importance ofStreptococcus pneumoniae as an etiologic agent of community-acquiredpneumonias, we previously demonstrated that persons with SCI who received the23-valent pneumococcal vaccine developed an antibody response to 5 vaccineserotypes that was maintained for up to one year. This study was undertaken toevaluate antibody concentrations for up to 4 years following vaccination in thispopulation.Methods: An enzyme-linked immunosorbent assay was used to measure IgG toselected pneumococcal polysaccharides on sera collected prior to vaccination andyearly for 4 years from 33 adults with SCI.Results: There was a two-fold or greater increase in geometric mean antibody con-centration for serotypes 4, 19F and 23F when measured at 1 year in the study popu-lation. Antibodies against serotypes 3, 4, and 23F declined from their 1 year peakswhen measured after 4 years but were still present in higher concentrations than atbaseline. In contrast, antibody concentrations against serotype 19F were higherwhen measured after 4 years than they were at earlier time points.Conclusions: Antibody response in persons with SCI can persist for at least 4 yearsfollowing vaccination and the magnitude of response and rate of decline vary ac-cording to serotype. Planned studies to continue following these persons to meas-ure antibody concentrations for additional serotypes over longer periods and toevaluate opsonophagocytic killing activity of these sera will provide additional in-formation on the significance of antibody quantitations from a functional stand-point and provide a basis for recommending timing for revaccination if necessary.

Revaccination with the 23-valent Pneumococcal Vaccine in Adults with SpinalCord Injury

Ken B. Waites, Kay C. Canupp, Moon H. Nahm, Yu-ying Chen, Michael J. DeVivoUniversity of Alabama at Birmingham, Birmingham, AL USA

Aims: Persons with spinal cord injury (SCI) are predisposed to develop pneumoniabecause of mechanical problems resulting from respiratory paralysis and loss ofneural control mechanisms and pneumonia is the most common cause of death forevery age group and all time periods post-injury. In view of the importance ofStreptococcus pneumoniae as an etiologic agent of community-acquired pneumonias,we previously demonstrated that persons with SCI who received the 23-valent pneu-mococcal vaccine developed an antibody response to 5 vaccine serotypes that wasmaintained for up to one year. This study was undertaken to determine the effect ofrevaccination on antibody response and the frequency of adverse reactions when asecond vaccination is administered 5 years or more following primary vaccination.Methods: An enzyme-linked immunosorbent assay was used to measure IgG toselected pneumococcal polysaccharides on sera collected just prior to revaccination,1 month and 1 year later in 23 adults with SCI.Results: Preliminary analyses demonstrated a two-fold or greater increase in geo-metric mean antibody concentration for serotypes 4, 19F, and 23F following pri-mary vaccination. After 5 years, antibodies to serotypes 3 and 4 declined to belowpre-vaccination levels. Following revaccination, geometric mean antibody concen-trations to these serotypes rose > two-fold above the second baseline value. Anti-bodies against serotypes 19F and 23F also rose following revaccination but did notachieve a two-fold increment. Three of 23 persons (13%) experienced transient self-limited local swelling and pain at the injection site.Conclusions: Immunity may be maintained for up to 5 years post-vaccination inpersons with SCI. Antibody response and decline over time vary according to sero-type. Revaccination induces a secondary antibody surge of less magnitude than theprimary response. Revaccination appears safe in this population since a minority ofpersons had minor self-limited localized inflammation.


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Efficacy of the pneumococcal 23-valent polysaccharide vaccine in military re-cruits in Russia

Zhogolev S.D.1, Ogarkov P.I.1, Jogolev K.D.2, Lashko K.V.1 Epidemiology Depart-ment1, Immunology Department2, Military Medical Academy, St.Petersburg, Rus-sian Federation.

Aims: To determine the efficacy of the pneumococcal 23-valent polysaccharide vac-cine in military recruits.Methods: In Russia the ‹Pneumo 23› vaccine was first used in servicemen in 1999.By 2001 about 3 thousand of recruits were vaccinated in various regions of thecountry while in November-December of 2002 the number of vaccinated recruitsamounted to over 10 thousand.Results: Preliminary studies allowed us to establish that antigenic composition ofthe vaccine was almost adequate to the stains circulating in military populations,especially those pneumococcal serotypes which cause community-acquired pneu-monia (CAP).The investigation of antigenic activity of the vaccine showed that ithad a high immunogenity. The comparative study demonstrated that CAP incidenceamong the vaccinated persons was 2-3.9 times lower than among unvaccinated sub-jects (efficiency being 62.2-74.2%). Simultaneous use of the influenza virus vac-cine and pneumococcal vaccine appeared to be the most effective (78.5%). Pneu-mococcal vaccine was effective not only in CAP but in acute bronchitis, sinusitis,otitis, which often were pneumococcal etiology. Acute bronchitis among the vacci-nated subjects were observed more seldom than among the unvaccinated persons(2.4-4.8 times, efficiency amounted to 57.5-79.1%) while acute respiratory diseasewas 1.3-2.0 times lower (efficiency – 48.7%). No acute otitis and sinusitis wererecorded or their incidence was 2.5-4.3 times lower compared to the unvaccinatedpersons (efficiency being 59.5-76.7%). In the case of the disease development invaccinated patients the severity of the disease was lower than in unvaccinated per-sons.Conclusions: The pneumococcal 23-valent polysaccharide vaccine is an effectivemeans of preventing pneumococcal infection in military populations.

PSV-58

Pneumococcal conjugate vaccine - herd effect and impact on pneumonia

*Black, S

Kaiser Permanente Vaccine Study Center1 Kaiser PlazaOakland, California 94612UNITED [email protected]


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Efforts for development of pneumococcal protein vaccine

*Hollingshead, S

University Of Alabama at BirminghamBBRB 654/25 Dept. of MicrobiologyBirmingham, AL. 35294UNITED [email protected]

Quadrivalent pneumococcal protein vaccines - clinical and safety investigations

*Maleckar, J

Aventis PasteurDiscovery DriveSwiftwater, PA 18370UNITED [email protected]


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Common Pneumococcal Protein Vaccines: From Discovery to Clinic.

Hamel J, Charland N, Rioux S, Martin D, and Brodeur BR. Vaccine Research Unit,Laval University Medical Center, Sainte-Foy, and Shire Biologics, Laval, Québec,Canada.

The use of the pneumococcal conjugate vaccine has reduced the burden of invasivedisease in young children. Due to the broad diversity of pneumococcal capsularserotypes associated with otitis media in children and pneumonia in adults, as wellas serotype replacement, common protein antigens are promising vaccine alterna-tives. The immunoscreening of a genomic expression library of Streptococcuspneumoniae allowed the identification of protective surface proteins BVH-3 andBVH-11, also known as members of the Pht/Php protein family. Evaluation of mul-tiple pneumococcal strains and isolates demonstrated the molecular and antigenicconservation and ubiquitous expression of these proteins. Comparison of DNA andprotein sequences revealed that BVH-3 and BVH-11 are related, with significanthomologous sequences located at the 5’ and amino termini of the genes and pro-teins, respectively. Immunization via a parenteral route with recombinant BVH-3or BVH-11 proteins elicited protection in mouse models of pneumococcal sepsisand pneumonia. Protection-eliciting epitopes were localized to the C-terminus ofBVH-3 and BVH-11, which corresponds to the protein-specific region. Immuniza-tion with a chimeric protein, comprising the protective region of each protein, im-proved the antibody responses by targeting two surface pneumococcal components.Passive protection studies revealed that antibody alone could prevent experimentaldisease, thus confirming the role of immunoglobulin as a major mechanism of pro-tection. Two phase I clinical trials were recently conducted in humans using eitherBVH-3 or a chimeric recombinant antigen vaccines. Immunization of animals andhumans revealed that BVH-3-based protein vaccines are excellent immunogens.These studies suggest that these particular common protein vaccines could improvedisease prevention by conferring protection against all diseases caused by pneumo-cocci, regardless of serotype.

Killed Noncapsulated Pneumococci (KNP) Given Intranasally (i.n.) with a Non-toxic Adjuvant Confer Multi-serotype Protection of Mice Against PneumococcalColonization and Middle Ear Infection

Malley R1, Morse S1, Leite L2, Areas A2, Ho P2, Kubrusly F2, Almeida I3, Anderson P1.Children’s Hospital, Boston, USA1, Instituto Butantan2 and University of São Paulo3,São Paulo, Brazil.

Aims: KNP given i.n. might induce local and systemic immunity to several surfaceproteins in native configuration and thus be an economical pneumococcal vaccine.We reported that KNP + cholera toxin (CT) protects mice vs nasopharyngeal (NP)colonization (col) by type 6B; but CT alone transiently protects, complicating analy-sis. Here we wished to: a) test additional serotypes, b) examine middle ear (ME)infection, and c) identify adjuvants with lower nonspecific protection and (for fu-ture human use) lower toxicity.Methods: Strain RX1AL- was killed with 70% ethanol to make KNP. C57Bl/6 micewere vaccinated i.n. with saline, adjuvant, or adjuvant + KNP; challenged i.n. with type6B or mixtures of types 14 and 23F; then after 1 week cultured for pneumococci in theNP and ME. Surveyed as adjuvants were: mycoplasma-associated lipoprotein (MALP-2), porcine lung surfactant (PLS) not containing lung surfactant protein A, Bordetellapertussis monophosphoryl lipid A (MPLA), and the binding subunit of CT (CTB).Results: After challenge with type 6B, all saline controls were col in the ME as well asNP, with a polymor- phonuclear response in the ME. KNP plus MALP-2, PLS orMPLA were no more protective than (the marginal effects of) the adjuvants alone. Incontrast, while nonspecific protection by CTB was insignificant, KNP + CTB reducedthe incidence of NP and ME col by 6B (p�0.01 for NP or ME, compared to CTB).Likewise, after challenge with types 23F + 14, all saline controls were col in the NPand ME. Reduction of col by CTB was insignificant, while KNP + CTB protected boththe NP and ME (p=0.036 for both) .Conclusions: The adjuvancy and lack of nonspecific protection by CTB in this modelwill facilitate further studies. CTB is also more likely to be acceptable for humanexperiments. How closely this ME infection of mice models human otitis media re-mains to be investigated; its prevention by KNP commends the approach.


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LACTOCOCCUS LACTIS GHOSTS DISPLAYING MULTIPLE STREPTOCOC-CUS PNEUMONIAE PROTEIN ANTIGENS ELICIT PROTECTIVE IMMU-NITY IN A MURINE PNEUMONIA MODEL

K. Leenhouts1, P.V. Adrian2, S. van Selm2, S. Estevão2, M. van Roosmalen1, J. Neef1, H.Metselaar1, S. Audouy1, E.E.S. Nieuwenhuis2, R. de Groot2, G.T. Robillard1, and P.W.M.Hermans2

1BioMaDe Technology, Nijenborgh 4, 9747 AG Groningen, The Netherlands; 2ErasmusMC-Sophia, Department of Pediatrics, Dr. Molewaterplein 50, 3015 GE Rotterdam,The Netherlands.

Introduction: Streptococcus pneumoniae is the leading etiological agent of severe in-fections such as meningitis, pneumonia and septicemia. The development of an afford-able effective vaccine against invasive pneumococcal disease will have major benefitsin terms of reducing disease burden and health care costs in both developed and devel-oping countries. The current 7-valent capsular carbohydrate conjugate vaccine is veryeffective against invasive pneumococcal disease. However, protection is limited to vac-cine serotypes, and the current unit cost of the vaccine is likely to limit its widespreaduse in developing countries.Methodology: A new approach is the use of protein-based pneumococcal antigens.Lactococcus lactis, a non-pathogenic, non-colonizing food-grade G+-bacterium shar-ing close homology with pathogenic streptococci, is an ideal organism for the produc-tion of pneumococcal vaccine antigens. Of particular importance is the potential toproduce generally affordable mucosal vaccines based on pneumococcal protein anti-gens bound to non-genetically modified non-living lactococci (Ghosts) acting as car-rier and mucosal adjuvant. We have constructed lactococcal Ghost-based multivalentmucosal vaccines containing different combinations of the surface-exposed pneumo-coccal proteins PspA, PsaA, CbpA, PpmA, SlrA and Iga.Results: The vaccines were evaluated for immunogenicity and protective efficacy in anintranasal challenge murine model for pneumococcal pneumonia. The results indicatethat the immune-stimulating potential of the Ghost-based multivalent vaccines is veryhigh, and that mucosal immunization results in an immune-protective response againstinvasive pneumococcal disease.Conclusions: We conclude that the Ghost binding- and display technology has greatpotential to develop a broadly applicable mucosal S. pneumoniae vaccine, which elic-its systemic protection.

Immunization of mice with pneumococcal conjugate vaccine using mutantpneumolysin protein carrier against streptococcus pneumoniae

Chiou C-J, Li Z, Chen M L, Chen M, Chen D-S, Wu M-C. Synergy America, Inc.Rockville MD 20850,USA.

Truncated pneumococcal pneumolysin (PPN) lacking cytolytic activity was expressedin Escherichia coli and purified by DEAE-Sepharose, immunoaffinity and 10 DGgel filtration chromatography. The purified PPN is highly immunogenic in mice.Antibody of mice immunized with either PPN or polysaccharide (PS)-PPN conju-gate reacts positively with wild-type pneumolysin (PL) by enzyme-linkedimmunosorbent assay (ELISA). Moreover, antisera collected from mice immunizedwith 14 (PS)-PPN conjugate not only showed in vitro opsonophagocytic (bacterialkilling) capacity, but also exhibited ability to increase in vivo bacterial clearance inblood of mice after challenge with either homologous serotype 14 or heterologousserotype 7 and 18C pneumococci. These results demonstraed that conjugate usingmutant pneumolysin carrier can provide higher efficacy and broad protection againstpneumococcal infection.


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CTB-PsaA is able to induce anti-PsaA IgG and IgA after intranasal immuni-zation of Balb/C mice

Arêas APM1,2, Oliveira, MLS1, Miyaji EN1, Leite, LCC1,2, Ho PL1,2. InstitutoButantan1, Instituto de Química, Universidade de São Paulo2, Brazil

Aims: The purpose of this study is to evaluate the ability of cholera toxin B subunit(CTB) to improve the immunogenicity of Pneumococcal surface antigen A (PsaA)in a fusion protein.Methods: The psaA gene was amplified by PCR and further, cloned in the 3’ regionof the ctxB gene. The resultant plasmid, pAE-ctxB-psaA, was used to transform E.coli BL21 (SI) competent cells. The pAE is an E. coli expression vector that isbased on T7 promoter. In this system, the protein can be expressed with a 6Xhis tagin the N-terminus, in order to facilitate the purification of the recombinant proteinin a Ni2+ - charged chromatography column. In E. coli BL21 (SI), the T7 RNApolymerase is under the control of the osmotically inducible promoter proU, there-fore the induction was performed by the addition of NaCl. The recombinant proteinwas extracted from the bacterial soluble fraction and purified. The protein was char-acterized by Western Blot and the ability to form pentamers as assessed by 6%SDS-PAGE. Balb/C mice were immunized intranasally with the fusion protein twicea week for three consecutive weeks. After 21 days, serum and saliva were collected,and nasal and bronchial washes were performed. The levels of IgG from the serumand IgA from saliva, and nasal and bronchial washes were analyzed by ELISA.Results: The fusion protein was obtained in a soluble form with a yield of 40 mg ofprotein per liter. It was specifically and strongly recognized by anti-CTB and anti-PsaA polyclonal antibodies. CTB-PsaA was expressed in a functional pentamericform that was able to bind to GM1 receptor. Moreover, it was able to induce highlevels of both IgG and IgA, even better than the positive control, PsaA + CT (chol-era toxin).Conclusions: Since anti-PsaA antibodies can prevent pneumococcal colonization,CTB-PsaA should be evaluated in a colonization model, aiming in the future, to beincluded in a pneumococcal protein vaccine formulation.

Induction of chemokines, known to affect T and B cell function, in human antigenpresenting cells by choline-binding protein A and pneumolysin

Bernatoniene J1, Zhang Q1, Mitchell T2, Finn A1. Department of Clinical Sciences SouthBristol, Institute of Child Health, University of Bristol1, UK; Division of Infection andImmunity, Institute of Biomedical and Life Sciences, University of Glasgow2, UK.

Pneumococcal choline-binding protein A (CbpA) and pneumolysin (Ply) may be goodvaccine candidates against invasive pneumococcal diseases as well as carriage. We havepreviously shown that these proteins induce expression of chemokines by respiratorytract epithelial cell lines and antigen-specific antibody responses in adenoidal B cells. Itis becoming clear that some chemokines may play important regulatory roles in the in-duction of acquired immune responses by modulating dendritic cell (DC) function.Aims: To investigate the effects of CbpA and pneumolysin on chemokine inductionin human monocyte-derived DC (MDDC) in a way which could have important im-plications for their use as vaccines.Methods: Recombinant proteins (CbpA and Ply) and concentrated pneumococcal cul-ture supernatants (PnmCCS) derived from either a wild type 2 pneumococcus (D39),which expresses CbpA and Ply, or from mutant daughter strains lacking CbpA or Plyexpression were used as protein antigen stimulants. Immature DC were generatedfrom human monocytes by negative selection of CD14+ cells using magnetic beadseparation from peripheral blood mononuclear cells (PBMC). DC differentiation wasinduced byadding recombinant human granulocyte-macrophage colony-stimulatingfactor and recombinant human IL-4. mRNA expression and production of chemokinesby iDC were assessed by RT-PCR and ELISA.Results: rCbpA, rPly and PnmCCS significantly up-regulated the mRNA expressionof the CXC and CC chemokines: CXCL10, CCL2, CCL4, CCL5, CCL8 and CCL19,but not CCL3 and CCL22 by immature MDDC when compared with unstimulatedcontrol cells. There was no expression of mRNA of CXCL11 and CXCL12 chemokinesin DC. The up-regulation of mRNA expression was followed by increased secretion ofCXCL8, CCL2 and CCL5 proteins in a dose- and time- dependent manner. PnmCCSderived from Ply and CbpA mutant strains induced significantly less chemkine pro-duction compared to their parent wild type strains.Conclusions: Recombinant and native pneumococcal proteins CbpA, Ply induce sig-nificant expression and production of CXC and CC chemokines by human mono-cyte-derived dendritic cells. This may have important implications for pneumococ-cal protein vaccine research.


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Potential effects of pneumococcal proteins (choline binding protein A andpneumolysin) on human dendritic cell maturation

Bernatoniene J1, Zhang Q1, Mitchell T2, Finn A1. Department of Clinical Sciences SouthBristol, Institute of Child Health, University of Bristol1, UK; Division of Infection andImmunity, Institute of Biomedical and Life Sciences, University of Glasgow2, UK.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role inthe induction and regulation of protective immunity against microorganisms. DC matu-ration is the control point that determines whether an antigen is to become an immuno-gen. It is not known whether pneumococcal protein vaccine candidates choline-bind-ing protein (CbpA) and pneumolysin (Ply) modulate DC maturation.Aims: To determine whether recombinant CbpA and Ply induce DC maturation and thusthe potential efficiency of antigen presentation for protective responses. To assess whetherthese proteins promote dendritic cell permeability and thus necrosis.Methods: Recombinant proteins (rCbpA and rPly) were used as protein antigen stimu-lants. Immature DCs were generated from monocytes purified by negative selection ofCD14+ cells from human peripheral blood mononuclear cells. DC differentiation wasinduced by adding recombinant human granulocyte-macrophage colony-stimulating factorand recombinant human IL-4. As a positive control for DC maturation, DCs were cul-tured with 1 or 0.1 �g/ml bacterial lipopolysaccharide for 2 days. DC differentiation andmaturation were assessed by surface marker immunofluorescent staining and flowcytometry. Effects of rCbpA and rPly on cell viability of DCs at different concentrationswas assessed by staining cells with propidium iodide and FACS analysis.Results: Stimulation with rCbpA and rPly significantly enhanced surface expressionof CD86, CD40 and MHC class II on DCs when compared with unstimulated controls.At higher concentrations, both recombinant proteins can induce cell death (after 2hours). Induction of DC necrosis by rPly and rCbpA was dose dependent. rPly induceda higher percentage of DC death than rCbpA at each concentration.Conclusion: These results suggest that pneumococcal proteins (CbpA and Ply) mayinduce human DC maturation, which may prove important for their immunogenicitywhen used as mucosal or parenteral vaccines in humans.

The use of phage display analysis of Streptococcus pneumoniae (Pnc) cell walllectin (CW-L) proteins for identification of inhibitors of Pnc adhesion to hostmucosa.

Blau K1, Shagan M1, Portnoi M1, Ling E1, Gershoni JM2, Genisova G2, Dagan R1,Mizrachi Nebenzahl Y 1. 1Ben Gurion University of the Negev, Beer Sheva, 2TelAviv University, Israel;

Aims: Pnc adhesion to host mucosa is a prerequisite for carriage and disease devel-opment. Pnc adhesion is probably mediated by CW-L proteins. We used randomphage display peptide libraries to screen Pnc CW-L for identification of bindingpeptide in the attempt to identify their receptors and develop inhibitors of Pnc adhe-sion.Methods: Pnc serotype 3 (WU2) CW-L were purified by fetuin affinity chromatog-raphy, separated by 2D PAGE and sequenced. 20 proteins were detected. Amongthem, fructose-biphosphate aldolase (FBA) and elongation factor G (EFG) pro-teins, previously identified as lectins, were cloned and expressed in E. coli. Anti-FBA and anti-EFG antibodies were produced in mice and rabbits, respectively. Theproteins and the antibodies were used for phage display library screening.Filamentous bacteriophages libraries used in this study are based on the fth1 vectorsystem.Results: 15 EFG and 30 FBA binding peptides were isolated. Five of the phagesexpressing the EFG binding peptides were found to inhibit Pnc adhesion to culturedhuman HaCat epithelial cells. The inhibitory peptides are currently being character-ized.Conclusions: Phage display allows identification of novel peptide inhibitors of Pncadhesion to host mucosa.


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PROT-15

PROT-16

The Interaction of Variants of Pneumococcal Surface Protein C with Factor Hand the Implication For Pneumococcal Survival In Vivo.

Carmicle, S, Dave, S., Quin, L. and McDaniel, L.S., University of Mississippi Medi-cal Center, Jackson, MS, USA

Aims: Pneumococcal surface protein C (PspC), a virulence factor for Streptococcuspneumoniae, interacts with the complement regulator factor H (FH) on the surfaceof pneumococcal cells in vitro. The potential FH-binding site is present in themajority of PspCs, but it is imperfectly conserved. In the current study, we illus-trate the relevance of the interaction between the two proteins in vivo and determinewhether the ability to bind FH is a conserved feature of PspCs in general or charac-teristic of certain PspC group(s).Methods: Pneumococci retrieved from CBA/N mice challenged systemically wereanalysed by flow cytometry for the presence of mouse FH on the surface. Westernblot analysis was used to assay representative strains of pneumococci of the PspCfamily for the ability to bind factor FH in vitro. A blood bactericidal assay was usedto determine the impact of exogenous FH on the survival of pneumococci in wholemouse blood.Results: Under the conditions tested, only cell lysates of PspC group 8 failed tobind biotinylated-human FH. Flow cytometry analysis demonstrated the presenceof mouse FH on the surface of pneumococci indicating that the interaction betweenPspC and FH is relevant in vivo. Furthermore, the addition of exogenous FH topneumococci consistently increases the survival of pneumococci in vitro.Conclusions: The interaction between PspC and FH is important in vivo and mayincrease survival of pneumococci in whole mouse blood. Additionally, the ability tobind FH is largely a characteristic on the PspC family. Examination of the sequenceof the proposed FH binding site revealed that the strains that ineffectively boundFH were among the lowest ranking groups with regard to amino acid sequenceconservation as compared to strain D39.

Immune Response to Immunisation with Two Iron Uptake ABC TransporterProteins, PiaA and PiuA.

Jomaa M1, Brown J.S2 , Yuste J2, Dougan G1. Imperial College London, London1,University College London2, London.

Introduction: PiaA and PiuA are recently identified lipoprotein components of S.pneumoniae iron uptake ABC transporters which are required for full virulence inmouse models of infection. Vaccination of mice with recombinant PiuA and PiaAprotects against invasive S. pneumoniae disease, and these antigens are potentialcandidates for a S. pneumoniae protein vaccine which may overcome the limita-tions of the capsular polysaccharide vaccines. We have investigated the immuno-logical response to these antigens in more detail.Results: We investigated the kinetics of the antibody response to recombinant PiaAand PiuA administered by intraperitoneal injection to BALB/C mice using ELISAsto recombinant protein. The results demonstrated that the antibody titres to bothantigens were increased and prolonged after three vaccinations compared to two orone vaccination. IgG subclass specific ELISAs demonstrated that the antibodyresponses to PiuA and PiaA were predominantly of the IgG1 subset, with only lowlevels of Ig2A, suggesting a Th2 response. PiuA is more immunogenic than PiaA.However, using flow cytometry we have shown that anti-PiaA sera but not anti-PiuA sera improved opsonophagocytosis of FAMSE labelled S. pneumoniae by thehuman neutrophil cell line HL60. In addition mice vaccinated with PiaA when chal-lenged with S. pneumoniae had increased levels of IFN-� compared to the alumvaccinated control group.Conclusions: PiaA elicits protective immunity at least partially by anti-PiaA anti-bodies promoting opsonophagocytosis. The mechanism(s) by which PiuA elicitsimmunity requires further investigation.


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PROT-18

Creation and characterisation of a non-toxic pneumolysin mutant

1L. S. Kirkham, 1A. R. Kerr, 2D.A. Dilts and 1T. J. Mitchell1Division of Infection and Immunity, University of Glasgow, Glasgow, G12 8QQ,Scotland; 2Wyeth Vaccines, Pearl River, NY, 10965, USA

Background: Pneumolysin (PLY) may have an application as a conjugate protein inpneumococcal vaccines, conferring non-serotype specific protection against Strep-tococcus pneumoniae. However, PLY is highly toxic both in vitro and in vivo andalthough the conjugation process should render PLY inactive, a non-toxic form ofPLY would be a more desirable starting point.Aims: To create a non-toxic form of PLY for possible use as a vaccine componentMethods: Site directed mutagenesis was used to create a set of mutations within thePLY gene. Mutants were screened for haemolytic activity using sheep erythro-cytes. Wild type PLY and a non-haemolytic mutant were expressed in Escherichiacoli and purified using hydrophobic interaction chromatography and anion exchangechromatography. Protein purity was visualised by SDS-PAGE and confirmed to bePLY by immunoblotting. Cytotoxicity of the mutant PLY to nucleated cells wasassessed using an L929 murine fibroblast killing assay. Host inflammatory re-sponses to the mutant PLY were analysed in vivo by intranasal administration ofPLY to MF-1 mice. Bronchoalveolar lavage and lung tissue samples were recov-ered and processed and cytokine and total protein levels were analysed.Results: We have created a form of PLY that is non-lytic at concentrations up to 50times that of the native toxin. There was no significant production of IL-6 andTNF-� in bronchoalveolar lavage from mutant PLY treated mice compared to highlevels of these cytokines in wild type treated groups.Conclusion: We have demonstrated that our mutant is non-toxic at high concentra-tions. Use of this mutant in pneumococcal vaccines may add to the efficacy ofcurrent pneumococcal vaccines.

S. pneumoniae (Pnc) glycolytic enzymes are immunogenic in humans and elicitprotective level immune reponses in mice

Ling E1, Daniely D1, Portnoi M1, Feldman G1, Wells J2, Overweg K2, MullhollandF2, Dagan R 1, Mizrachi Nebenzahl Y1; 1Ben Gurion Univ. Beer Sheva, Israel, 2Inst.of Food Res., Norwich, UK

Aims: We have previously shown that there is an age dependent immune response toaround 20 of 150 extracted Pnc cell wall proteins. Vaccination with two of thesepurified antigens, fructose-biphosphate aldolase (FBA) and glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) elicited protective level immune responses in amouse model system whereas immunisation with the less immunogenic Heat shockprotein 70 (DnaK) antigen was not protective (Mizrachi-Nevenzahl et al 43rd ICAAC,2003, G-900). The aim of this study was to evaluate the vaccine potential of anadditional immunogenic protein, 6-phosphogluconate dehydrogenase (6PGD).Methods: BALB/c mice were immunized with 25�g of recombinant Pnc 6PGDwith Alum adjuvant in 50�l PBS or Alum alone as a control and boosted threeweeks later. The ability of 6PGD (n=16) to protect mice from a lethal intranasalchallenge with 108 CFU of Pnc was monitored daily and compared to that of theFBA (n=21), GAPDH (n=23), DnaK (n=20) and control mice (n=20).Results: Immunization of mice with recombinant (r) 6PGD, FBA, GAPDH andHSP70 elicited antibodies reactive to respective native proteins in the Pnc strainsWU2 (serotype 3) and in clinical isolates 14, 9V and 6B. Following challenge,100% of r6PGD and 36% of rFBA or rGAPDH immunized mice survived (p<.05).In contrast all of the control mice or DnaK-immunized mice succumbed to lethalinfection. Cross protection was elicited against the heterologous serotype 9VConclusions: Immunization of mice with r6PGD, rFBA and rGAPDH elicited cross-reactive antibodies in all four Pnc serotypes tested and was protective against lethalchallenge with Pnc via the respiratory route.


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PROT-19

PROT-20

DNA vaccine vector expressing subunit B of Escherichia coli heat-labile toxin(LTB) is a poor adjuvant for DNA vaccination against Streptococcus pneumoniae

Miyaji EN, Ferreira DM, Farias LP, Dias WO and Leite LCC, Instituto Butantan,Brazil

Aims: The aim of this work is to analyze the potential of a DNA vaccine vectorexpressing LTB as adjuvant for DNA vaccines against pneumococcal infectionsbased on PspA (pneumococcal surface protein A).Methods: DNA vaccine vectors expressing either an N-terminal fragment of PspAfrom clade 3 (pSec-PspA3NS) or LTB (pSec-LTB) as secreted proteins were usedfor immunization of BALB/c mice through intradermal inoculation using GeneGunor intradermal needle injection. The induction of anti-PspA antibodies was analyzedthrough ELISA and protection of immunized animals was performed through in-traperitoneal challenge with S. pneumoniae strain St 679/99 (serotype 6B, PspAclade 3).Results: Immunization using GeneGun led to the induction of high anti-PspA anti-body titers. Animals immunized with a mixture of pSec-pspA3NS and pSec-LTBshowed the induction of slightly higher titers, when compared to animals immu-nized with pSec-PspA3NS alone. These higher antibody titers did not correlatewith protection, since only mice immunized with pSec-PspA3NS alone showedsurvival statistically different from animals inoculated with the empty vector. Intra-dermal needle injection of mice with the DNA vaccine vectors led to the inductionof very low antibody titers. In fact, anti-PspA antibodies were only detected insome of the animals immunized with the mixture of pSec-PspA3NS and pSec-LTB,which resulted in slightly higher survival after intraperitoneal challenge.Conclusion: Although we could detect an adjuvant effect of the DNA vaccine vec-tor expressing LTB through the enhancement of the antibody response, this effectwas very poor. Furthermore, this augmentation in the humoral immune responsedid not correlate completely with survival. We will continue our experiments inorder optimize the use of this LTB-expressing vector as adjuvant for DNA vaccines.

Protection Against Pneumococcal Infection by Prime-Boost Immunization withDNA and Protein

Moore, Q , Bosarge, J, McDaniel, L. University of Mississippi Medical Center,Jackson, MS, USA.

Aims: The development of DNA vaccines against extracellular pathogens with apneumococcal immunization / challenge model was evaluated. We focused on thea-helical region of pneumococcal surface protein A (PspA), which has been shownto contain protection-eliciting epitopes. We examined the effect of priming withpspA/EF5668 and boosting with purified recombinant protein. We also tested thehypothesis that the genetic background of the pneumococcus affects the ability ofanti-PspA antibodies to protect in a systemic model.Methods: The DNA fragment encoding the a-helical domain of PspA/EF5668 wascloned into an eukaryotic expression vector designated pJB100EF. PspA-specificantibody was detected in mice immunized with pJB100EF by ELISA and Westernblot.Results: We used a genetically modified variant of WU2 that expresses PspA fromEF5668 as a challenge strain and obtained the following. Mice primed and boostedwith 50�g pspA had serum anti-PspA specific antibodies levels of 5.96�g/ml ±1.45 and had a survival rate of 89%. Mice primed with 50�g pspA and boostedwith 10�g of PspA without adjuvant had serum anti-PspA specific antibodies of1.75�g/ml ± 0.55 and a survival rate of 90%. In mice primed with 10�g of PspAwithout adjuvant and boosted with 50�g pspA, the concentration of anti-PspA spe-cific antibodies was 3.63 �g/ml ± 1.02 and there was a 70% survival rate. Westernblot analysis demonstrates that the immune serum was cross-reactive with PspAfrom several different pneumococcal isolates representing different PspA clades.We also localized the region of cross reactivity to residues 110-288 of PspA/RX1.Conclusions: Survival of immunized mice following pneumococcal challenge dem-onstrated the ability of prime-boost immunizations with the �-helical domain ofPspA to elicit protective immunity.


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PROT-22

DETERMINATION OF PSPA FAMILIES AMONG STREPTOCOCCUSPNEUMONIAE STRAINS FROM INDIA

RekhaPai, Anand Manoharan, Kurien Thomas1, Mary V Jesudason & M.K.LalithaDepartments of Microbiology & Medicine1, Christian Medical College, Vellore

Background: Pneumococcal surface protein A (PspA) is a protection-eliciting sur-face protein found in all pneumococci and is a potential vaccine candidate. PspAproteins are classified into three families that can be further subdivided into sixclades. The commonly circulating PspA families needs to be determined to designthe appropriate vaccine formulationObjective: To determine the PspA families among the invasive and non-invasiveS.pneumoniae strains from this region.Method: PspA PCR typing was undertaken to determine the PspA families amongS.pneumoniae.A total of 310 strains (invasive n=160; non-invasive n=150) ofS.pneumoniae isolated between 1999 & 2001 were studied. PspA PCR was doneusing the following primers: LSM12, SH63, SKH52, SKH41 & SKH42 and theamplified products were detected by gel electrophoresis.Results: The PCR gave clear and discrete bands for both family 1 and family 2, at1000 bp and 1200 bp regions respectively. Three fourths (76.1%) of the study iso-lates belonged to family 1, and 23.2 % to family 2 while none belonged to family 3.A similar distribution was observed among the invasive and non-invasive strainswhen analyzed separately. Only two strains (0.7%), both invasive, were amplifiedby family 1 and family 2 primers. A comparison of the distribution of PspA familieswith serotype distribution showed 93% (28/30) of serotype 1 strains to belong tofamily 1 indicating a significant association (p=0.014).Conclusion: The results show the presence of only two PspA families among strainsin this region supporting the hypothesis that a PspA vaccine covering a few familiescould be effective in this region.

Adherence of Pneumococcal Surface Adhesin A (PsaA)-coated particles to hu-man nasopharyngeal (NP) epithelial cells for the evaluation of anti-PsaA func-tional antibodies

Romero-Steiner S, Caba J, Sampson JS, Hassounah H, Floyd A, Johnson SE, CarloneGM, Ades E. Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

Aims: Development of a specific and reproducible in vitro assay for the measure-ment of functional antibodies to Pneumococcal Surface Adhesin A (PsaA) is neededfor evaluation of future protein vaccine immunogenicity and to be used as a func-tional correlate of protection.Methods: We used carboxylate-modified fluorospheres (Molecular Probes, 1mmin diameter) coated with either recombinant non-lipidated PsaA (rPsaA) or PsaAsynthetic peptides (P1, P2 and P3) for evaluation of the adherence of PsaA to De-troit 562 NP cells. PsaA and peptide beads were compared at a similar inoculum(3100 + 500 beads per well) in adherence assays (measured as mean fluorescentunits (FU) + SE).Results: P1-coated beads showed the highest adherence (52,684 FU + 4,354), fol-lowed by rPsaA-coated beads (44,195 FU + 2,648) and P2-coated beads (17,195FU + 1,044); P3 showed the least adherence with a mean of 9,780 FU (+ 228).Homologous competitions with P1, P2, and P3 peptides resulted in 61%, 43%, and44% reduction in FU, respectively. Heterologous competitions with P1, P2, and P3resulted in inhibition of adherence (88%, 62%, and 75%, respectively) of PsaA–coated beads. Anti-PsaA antibodies in normal adult sera (n = 9) were highly reac-tive with P1 and P2 beads indicating that these two peptides are more immunogenicthan P3. Anti-PsaA Abs capable of inhibiting adherence of PsaA-coated beadshighly correlated (r = 0.75, P < 0.05) with those inhibiting the adherence to P1-coated beads. The mean percent + SD (CV) inhibition of PsaA-bead adherence(n=19 tests) of serum 7074 (1:10 dil.) was 80% + 7.8 (9.8%).Conclusions: These results indicate that PsaA is a Pnc adhesin and that P1 containsa functional epitope for the adherence of PsaA to NP cells. The functional activityof antibodies to a single adhesin or binding epitopes can be evaluated in this in vitromodel of nasopharyngeal adherence.


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PROT-24

Enhancement of pneumococcal CbpA-specific antibody responses by CpG-DNA and EtxB in in vitro culture of adenoidal cells from children

Zhang Q1, Bernatoniene J1, Paton J2, Finn A1. Department of Clinical Sciences SouthBristol, University of Bristol, UK1, School of Molecular and Biomedical Science,University of Adelaide, Australia2.

Pneumococcal choline-binding protein A (CbpA) may be a good protein vaccinecandidate against carriage of pneumococcus. CpG-DNA and E.coli enterotoxinsubunit B (EtxB) enhance mucosal antibody responses in animal studies. It is notknown whether these immunological adjuvants enhance mucosal antibody responsesto pneumococcal antigens in humans.Aims: To study the immune responses to CbpA in adenoidal cells from childrenundergoing adenoidectomy in response to pneumococcal protein stimulation and tosee if CpG-DNA and EtxB can enhance the in-vitro antibody responses to CbpA.Methods: Adenoidal mononuclear cells were isolated using Ficoll gradient centrifu-gation and cultured in RPMI medium with or without CpG-DNA or EtxB followedby the addition of a concentrated culture supernatant (CCS) derived from a wildtype strain of pneumococcus, which has been shown to express CbpA by Westernblotting. A CCS derived from a CbpA-deficient mutant strain was used as a control.Adenoidal cell culture supernatants were collected and tested by immunoassays forCbpA- specific IgA and IgG, total IgA and IgG antibodies, and cells were har-vested for enumeration of antibody secreting cells (ASC) by ELISpot assay.Results: CCS derived from the wild type pneumococcus, but not the CbpA-defi-cient mutant, induced significant CbpA-specific IgG responses in cultured adenoi-dal cells, which was confirmed by increased numbers of IgG ASCs. Both CpG-DNA and EtxB induced a dose-dependent enhancement of CbpA-specific IgG an-tibody responses after stimulation with the pneumococcal CCS. Although total IgGand IgA levels were increased after CpG-DNA stimulation, EtxB did not affect thetotal IgG and IgA concentrations.Conclusions: These results suggest that pneumococcal CbpA may induce mucosalimmune responses in the human upper respiratory tract. Both CpG-DNA and EtxBmay be useful to enhance mucosal responses.

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Author index


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Author Presentation code Page Author Presentation code Page

Aaberge, I S COL-17 107Abboud-Donaldson, R EPI-84 74

EPI-85 75Abrego, P EPI-46 55

EPI-47 56Abucejo-Ladesma, E EPI-11 38

EPI-49 57Adam, D PSV-11 212Adamkiewicz, T V EPI-12 38Adegbola, R DGN-11 84

DGN-21 89EPI-13 39

Ades, E PROT-22 245Adrian, P V GEN-15 152

GEN-16 152IMM-11 183PATH-24 166PROT-05 238

Aebi, C EPI-59 62Aebi, S GEN-14 151

PATH-32 170RES-27 128

Åhman, H PSV-18 215PSV-19 216

Aiuti, F IMM-46 200Albanes, D EPI-36 50Albiger, B IMM-12 183

PATH-41 175Aldana, R EPI-30 47Al-Lahham, A EPI-50 57

EPI-70 67RES-11 120RES-12 120

Alloing, G PATH-24 166Almeida, I PROT-04 237Alonso, P EPI-74 69

EPI-83 74Alonso, P L EPI-76 70Alves, N N RES-36 132Ameyaw, S EPI-13 39Anderson, P PROT-04 237Anderson, R PATH-18 163

PATH-20 164Andes, D R GEN-05 148Andrew, P RES-23 126Andrew, P W IMM-31 193

PATH-14 161PATH-22 165PATH-27 168

Andrews, N IMM-06 182IMM-47 201

Ansa Echeverria, X PSV-54 233Antikainen, J PATH-11 160Aquilar-Ituarte, F EPI-29 47Arcay, J EPI-11 38Ardanuy, C RES-13 121

RES-17 123RES-24 126RES-31 130

Areãs, A PROT-04 237Areãs, A P M PROT-11 240Arguedas, A EPI-67 66Arnaoutakis, K IMM-52 203Arnold, K NEW-03 205Ashton, L IMM-06 182IMM-47 201NEW-06 206PSV-24 218 Audouy, SPROT-05 238Auranen, K COL-20 108EPI-33 49MOD-15 144Azevedo, G M SCOL-14 105 Baggett, HEPI-03 35Baggs, J PSV-28 220Balakrishnan, I PATH-12 160Balraj, V EPI-78 71Balsalobre, L RES-17 123Banner, C PSV-48 230Baqui, A H EPI-50 57Barberino, M G RES-36 132Bar-Ziv, J EPI-21 43

EPI-22 43EPI-23 44

Batt, S L EPI-14 39EPI-15 40RES-19 124

Bättig, P GEN-11 150GEN-14 151

Baughman, W NEW-03 205Beagley, K W IMM-45 200Beall, B EPI-66 65

RES-35 132Begolli, L DGN-14 85Beissbarth, J NEW-01 204

PSV-23 218Benjamin, W H RES-34 131Benjamin, W H Jr PATH-30 169Benson, J DGN-29 93Bentley, S GEN-02 146Bergmann, S PATH-13 161Berisha, L DGN-14 85Bernatoniene, J IMM-51 203

IMM-52 203PROT-12 240PROT-13 241PROT-24 246

Bernatowska, E PSV-12 212Besa, R PSV-32 222Beverley, P PSV-16 214Bingen, E RES-39 134Black, S DGN-29 93

EPI-91 78PSV-13 213PSV-58 235

Blasi, E PATH-38 173Blau, K PROT-14 241Blomberg, C RES-47 138Blue, C E GEN-16 152


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Boëlle, P Y MOD-04 141MOD-14 143

Bogaert, D COL-11 104COL-12 104COL-26 111IMM-11 183IMM-13 184IMM-14 184PSV-14 213PSV-52 232

Boon, L IMM-29 192Boost, M V COL-13 105

RES-14 121RES-15 122RES-16 122

Booy, R EPI-25 45Borrow, R NEW-06 206Bortoni-Rodriques, M E PATH-14 161Bosarge, J PROT-20 244Bossuyt, X IMM-28 191

IMM-29 192IMM-30 192

Botto, M IMM-49 202IMM-50 202

Bouchet, V GEN-17 153PSV-31 222

Bowman, J COL-15 106EPI-18 41IMM-38 196

Braathen, R PATH-17 163Bracht, D PATH-13 161Bramley, J C MOD-13 143Brandão, M A COL-14 105

RES-36 132Brandileone, M C RES-36 132Branny, P RES-23 126Breiman, R EPI-50 57Brett, M EPI-86 75Briles, D EPI-54 59

IMM-40 197Briles, D E PATH-30 169Brilla, E EPI-67 66Brito, D DGN-18 87Brito-Avô, A COL-25 111

PSV-20 216Brodeur, B IMM-26 190Brodeur, B R PROT-03 237Bronsdon, M COL-24 110

EPI-57 61Brooks, W A GAVI-07 97Brown, J S IMM-49 202

IMM-50 202PROT-16 242

Brown, M EPI-86 75Bruden, D EPI-03 35

EPI-38 51EPI-58 61

Brueggemann, A PATH-21 165Brueggemann, A B EPI-06 36

Büchi, W EPI-59 62Bulkow, L EPI-17 41

EPI-37 51PSV-38 225

Burbidge, P NEW-06 206Burrage, M NEW-06 206Burton, R L IMM-15 185Butler, J EPI-02 34

PSV-38 225RES-41 135

Butler, J C EPI-03 35EPI-06 36EPI-17 41EPI-37 51EPI-38 51EPI-58 61

Buzinov, R EPI-82 73Bygraves, J IMM-18 186Bygraves, J A IMM-19 187Caba, J PROT-22 245Cagliuso, M IMM-46 200Caldas, R M RES-36 132Camilli, A PATH-21 165Camilli, R PATH-15 162Canica, M RES-21 125

RES-22 125RES-38 133

Canupp, K C PSV-55 234PSV-56 234

Capolunghi, F IMM-16 185Carapetis, J COL-22 109Cardoso, M R RES-18 123Cardozo, D M COL-14 105Care, R IMM-18 186

IMM-19 187Carlone, G PSV-28 220Carlone, G M COL-03 101

PROT-22 245Carmicle, S PROT-15 242Carneiro, S COL-19 108Carsetti, R IMM-02 180

IMM-16 185IMM-46 200

Cartwright, K EPI-28 46NEW-06 206

Carville, K S COL-15 106EPI-18 41

Castiglia, P EPI-19 42Cauchemez, S MOD-04 141Caugant, D A COL-17 107Ceasar, H PSV-40 226Ceballos, A EPI-81 73Cercenado, E RES-24 126Ceuppens, J L IMM-29 192

IMM-30 192Charalambous, B M COL-05 102

EPI-14 39EPI-15 40PATH-12 160RES-19 124


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Charalambous, S COL-30 113Charland, N PROT-03 237Charles, E R EPI-44 54Cheddie, M EPI-72 68Chen, D-S PROT-06 238Chen, M L PROT-06 238Chen, Y PSV-55 234

PSV-56 234Cheng, A F B RES-28 128Cherian, T DGN-02 80

DGN-29 93EPI-78 71GAVI-09 98

Chiamenti, G P EPI-71 68Chiavolini, D PATH-33 171

PATH-38 173Chiou, C-J PROT-06 238Choo, S PSV-46 229Christie, D EPI-25 45Church, D L COL-21 109Churchyard, G COL-30 113Cil, M Y EPI-70 67

RES-11 120RES-12 120

Cintorino, M PATH-38 173Claes, C MOD-11 142Clarke, S C GEN-18 153

PSV-15 214PSV-36 224PSV-49 231RES-44 136

Claverys, J-P PATH-24 166Clutterbuck, E A PSV-16 214Coates, H PATH-25 167Coen, P G EPI-25 45Cohen, Z PATH-35 172Coovadia, H M COL-23 110

EPI-53 59Corachan, M EPI-76 70Corrah, T DGN-21 89

EPI-13 39Coward, W IMM-31 193Cripps, A W IMM-38 196Crisóstomo, I EPI-73 69

EPI-77 71Crook, D PATH-21 165Crook, D W EPI-06 36Crossette, L EPI-56 60Curfs, J H A J COL-29 113Cutland, C EPI-20 42Cutts, F T DGN-12 84

GAVI-08 97

Dagan, R EPI-21 43EPI-22 43EPI-23 44EPI-67 66PATH-35 172PATH-36 172PROT-14 241PROT-18 243RES-01 116RES-29 129

Daniely, D PROT-18 243Dave, S PROT-15 242Davidsdottir, K PSV-51 232de Gouveia, L DGN-13 85

EPI-04 35EPI-20 42EPI-31 48EPI-32 48EPI-75 70RES-26 127

De Grave, D PSV-24 218de Groot, R IMM-11 183

COL-11 104COL-12 104COL-26 111GEN-15 152GEN-16 152IMM-13 184IMM-14 184PATH-24 166PROT-05 238PSV-14 213

de Jong, A GEN-15 152de la Campa, A G RES-17 123

RES-24 126de Lencastre, H COL-25 111

DGN-18 87EPI-61 63EPI-73 69EPI-77 71PSV-20 216

De Wals, P PSV2-04 211DeCampo, M PSV-32 222Del Grosso, M PATH-15 162

RES-20 124Deloria-Knoll, M GAVI-04 95Dentinger, C PSV-38 225DeVivo, M J PSV-55 234

PSV-56 234Dhooge, I PSV-53 233Di Fabio, J L EPI-24 44

RES-36 132Dias, R RES-21 125

RES-22 125RES-38 133

Dias, W O PROT-19 244Dickens, A L RES-05 118

RES-25 127Dilts, D A PROT-17 243


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Dockrell, D H PATH-28 168Dogan, S PATH-16 162Domínguez, M A RES-13 121

RES-31 130Donnanno, S IMM-46 200Dooley, J S COL-13 105

RES-14 121Dougan, G PROT-16 242Dower, S K PATH-31 170Dowson, C GEN-04 147

GEN-18 153RES-44 136

Dowson, C G RES-04 117du Plessis, M RES-39 134Duarte, J RES-36 132Durant, N PSV-25 219Echenique, J RES-23 126Eckman, J EPI-12 38Edelstein, P EPI-55 60

EPI-56 60Edmunds, J IMM-06 182

IMM-47 201Edmunds, W J MOD-12 142Edwards, G RES-44 136Edwards, K M RES-34 131Eichner, M MOD-15 144Ekdahl, K EPI-39 52Ekström, N PSV-18 215

PSV-19 216PSV-47 230

El Arifeen, S EPI-01 34El Bashir, H EPI-25 45Elm, C PATH-17 163Elsbury, D COL-15 106

EPI-18 41Elvin, L PSV-13 213English, M EPI-27 46

EPI-51 58GAVI-06 96

Enwere, G EPI-26 45Erasmus, M C GEN-16 152Erickson, L PSV2-04 211Erlendsdottir, H EPI-43 54Esparar, G RES-37 133Espinosa de los Monteros, L E COL-18107

EPI-29 47EPI-30 47

Espinoza, A EPI-47 56Espinoza, M EPI-45 55Estevão, S PATH-24 166

PROT-05 238Ewell, M PSV-50 231Facklam, R R EPI-66 65Fagerlund, R IMM-43 199Farias, L P PROT-19 244Farley, J DGN-11 84Farley, M M EPI-12 38

NEW-03 205

Farrington, P MOD-02 140Faustova, M E EPI-64 64Feavers, I IMM-18 186Feavers, I M IMM-19 187Fehnle, K PSV-11 212Feikin, D EPI-27 46

EPI-76 70EPI-83 74

Feikin, D R EPI-35 50PSV-28 220

Feldman, C DGN-17 87PATH-18 163PATH-20 164

Feldman, G PROT-18 243Fenoll, A RES-17 123

RES-31 130Fernebro, J PATH-19 164

RES-42 135Feroldi, E EPI-68 66

PSV-30 221Ferrándiz, M J RES-24 126Ferreira, D M PROT-19 244Ferro, S EPI-84 74

EPI-85 75Ferry, B PSV-48 230Fickl, H PATH-20 164Fillion, P PATH-25 167Fillon, S PATH-44 176Finn, A EPI-28 46

IMM-51 203IMM-52 203PATH-16 162PROT-12 240PROT-13 241PROT-24 246

Finucane, J COL-15 106EPI-18 41

Fireman, B DGN-29 93PSV-13 213

Firth, M EPI-18 41Fishman, N EPI-55 60

EPI-56 60Flannery, B EPI-05 36

EPI-27 46EPI-76 70EPI-83 74

Fleck, R IMM-18 186Fleck, R A IMM-17 186

IMM-19 187Fleites, A RES-24 126Fletcher, L DGN-11 84Fletcher, M EPI-28 46Fletcher, M A EPI-52 58Floyd, A PROT-22 245Foxwell, A R IMM-38 196Francis, K P NEW-02 204Franken, C RES-12 120


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Frazão, N COL-25 111EPI-61 63EPI-73 69EPI-77 71PSV-20 216

Fredlund, H EPI-39 52French, N COL-06 102

IMM-21 188IMM-23 189PSV-21 217PSV-39 226

Fritzell, B EPI-52 58Frøholm, L O COL-17 107Gajee, K COL-23 110

EPI-53 59Galia, E EPI-11 38Gallagher, K EPI-05 36Gallisai, D EPI-19 42Ganesan, A EPI-79 72Gao, G NEW-02 204

PATH-11 160PATH-34 171

García, E GEN-12 150GEN-19 154

Gathe, Jr., J PSV-40 226Gay, N IMM-06 182

IMM-47 201MOD-03 141

Genisova, G PROT-14 241George, R IMM-06 182

IMM-47 201George, R C DGN-24 90Germanson, T PSV-50 231Gershoni, J M PROT-14 241Gibson, P G IMM-45 200Gilbey, A RES-04 117Gilks, C COL-06 102Gillespie, S GEN-04 147Gillespie, S H COL-05 102

EPI-14 39EPI-15 40PATH-12 160RES-05 118RES-19 124RES-25 127

Gillett, M IMM-18 186Gillett, M L IMM-19 187Ginocchio, C C EPI-72 68Ginsberg, G NEW-05 206Giorda, E IMM-16 185Giordano, R C COL-19 108Givon-Lavi, N EPI-21 43

EPI-22 43EPI-23 44

Glasner, J D GEN-05 148Goessens, W H F COL-12 104

COL-26 111

Goldblatt, D COL-23 110DGN-01 80EPI-53 59IMM-04 181IMM-06 182IMM-47 201NEW-06 206PSV-24 218

Goldenberg, H B IMM-20 187Goldstein, R GEN-17 153

PSV-31 222Gómez-Barreto, D COL-18 107

EPI-29 47EPI-30 47

Goncalves, V M COL-19 108Gordienko, T EPI-82 73Gordon, S IMM-03 181

IMM-03 181Gordon, S B IMM-21 188

IMM-22 188IMM-23 189PSV-21 217

Gorgievski-Hrisoho, M EPI-59 62Gove, J EPI-58 61Graham, R M A IMM-24 189Granat, S COL-20 108Grant, A COL-30 113Green, K RES-48 138Greenberg, A J EPI-72 68Greenberg, D EPI-21 43

EPI-22 43EPI-23 44RES-29 129

Grishina, L EPI-82 73Grönholm, S PSV-18 215

PSV-26 219Guay, M PSV2-04 211Guazzi, V IMM-46 200Gudnason, Th EPI-43 54Guerra, M L RES-36 132Guillemot, D MOD-04 141

MOD-14 143Gunnarsdottir, Th EPI-43 54Gutbier, B NEW-04 205Haas, W GEN-13 151

RES-45 137Hadler, J EPI-05 36Haikala, R PSV-19 216Hakenbeck, R RES-02 116Hamel, J IMM-26 190

PROT-03 237Hamilton, E PSV-37 225Hammerschmidt, S IMM-25 190

PATH-13 161PATH-17 163

Hammitt, L L EPI-03 35Han, S H IMM-32 193

IMM-37 196


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Hanage, W P EPI-33 49PSV-22 217

Hansbro, P M IMM-45 200Hansen, J DGN-29 93Harder, J IMM-42 198Hare, K NEW-01 204

PSV-23 218PSV-45 229

Harit, S M EPI-34 49Harker-Jones, M EPI-38 51

EPI-58 61RES-41 135

Harmsen, A PATH-29 169Harrington, B PSV-23 218Harris, T PSV-27 220Harrison, L EPI-05 36Harrison, T G DGN-24 90Hassounah, H PROT-22 245Hathaway, L PATH-32 170Hathaway, L J GEN-11 150

GEN-14 151Hausdorff, W MOD-16 144Hausdorff, W P EPI-35 50Hauser, C RES-27 128Hava, D L PATH-21 165Heath, R PATH-26 167Heffernan, R EPI-05 36Heiskanen-Kosma, T EPI-49 57

EPI-68 66PSV-30 221

Helmerking, M PSV-11 212Hemilä, H EPI-36 50Hemsley, C PATH-21 165Henckaerts, I PSV-24 218

PSV-25 219PSV-41 227

Hendriksen, W T GEN-15 152GEN-16 152

Hendrix, R IMM-13 184Hennessy, T EPI-03 35

EPI-37 51EPI-38 51EPI-58 61PSV-38 225RES-41 135

Hennessy, T W EPI-17 41Henriques Normark, B EPI-39 52

IMM-12 183IMM-42 198PATH-19 164PATH-39 174PATH-41 175RES-42 135RES-47 138

Hermand, P V IMM-33 194Hermans, P PSV-52 232

Hermans, P W M COL-11 104COL-12 104COL-26 111GEN-16 152IMM-11 183IMM-13 184IMM-14 184PATH-24 166PROT-05 238PSV-14 213

Herva, E COL-20 108DGN-22 89EPI-11 38EPI-34 49RES-37 133

Hickel, J PSV-38 225Hill, P EPI-13 39Hippenstiel, S IMM-25 190

NEW-04 205Hirst, R PATH-14 161Hirst, R A PATH-22 165Hjuler, T RES-30 129Ho, P PROT-04 237Ho, P L PROT-11 240Hocke, A C IMM-25 190

NEW-04 205Högberg, L EPI-39 52Høgh Andersen, L RES-30 129Høiby, E A COL-17 107Holden, D H PATH-21 165Hollingshead, S EPI-54 59

IMM-40 197PROT-01 236

Hollingshead, S K PATH-30 169RES-34 131

Holm, A PSV-35 224PSV-43 228

Holmberg, H DGN-28 92Holmlund, E IMM-11 183

IMM-26 190PSV-26 219

Hossain, S EPI-50 57Howgate, J EPI-12 38Hryniewicz, W PATH-40 174Hsu, K K PSV-44 228Huebner, R E RES-32 130Huo, Z PSV-27 220Huot, H GEN-17 153

PSV-31 222Huovinen, M RES-40 134Hurlburt, A RES-41 135Hurlburt, D EPI-03 35

EPI-17 41EPI-37 51EPI-38 51

Hussain, M IMM-06 182IMM-47 20

Hyde, T EPI-58 61


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Iannelli, F PATH-33 171RES-20 124

Ibrahim, Y M PATH-23 166Inostroza, J IMM-27 191Ip, M RES-28 128Irving, D PSV-27 220Ivanova, M V DGN-26 91Jaakkola, T IMM-26 190Jackson, D COL-03 101Jackson, L A PSV-28 220Jacoby, P IMM-38 196Jaka, A DGN-14 85Jakupi, X H DGN-14 85Jalava, J RES-40 134Jarva, H IMM-41 198Jeena, P M COL-23 110

EPI-53 59Jefferies, J RES-44 136Jefferies, J M C GEN-18 153Jesudason, M V PROT-21 245Jetté, L P EPI-40 52Jeurissen, A IMM-28 191

IMM-29 192IMM-30 192

Jimenéz, V EPI-29 47Jogolev, K D COL-31 114

EPI-41 53EPI-64 64PSV-57 235

Johansen, F-E PATH-17 163Johnson, C N RES-34 131Johnson, S E PROT-22 245Jokinen, J EPI-65 65

PSV-22 217Jomaa, M PROT-16 242Jonsdottir, I IMM-01 180

PSV-51 232Jorgensen, J H EPI-66 65Joseph, A EPI-78 71Jousimies, K PSV2-03 211

PSV-47 230Julkunen, I IMM-43 199

PATH-46 177Kadioglu, A IMM-31 193

PATH-14 161PATH-27 168RES-23 126

Kaijalainen, T DGN-16 86DGN-22 89EPI-33 49EPI-34 49EPI-54 59EPI-65 65IMM-40 197

Kaltoft, M S COL-04 101DGN-28 92PSV-29 221

Kamau, T EPI-51 58GAVI-06 96

Kan, B PATH-39 174Kanyanda, S E IMM-21 188

IMM-22 188IMM-23 189

Kaprio, J EPI-36 50Karma, P EPI-65 65Karumuri, S PSV-44 228Kashal, D PATH-34 171Kasran, A IMM-29 192Katsuragi, H IMM-12 183Katz, A RES-29 129Kaushal, D GEN-13 151Käyhty, H EPI-54 59

IMM-06 182IMM-11 183IMM-26 190IMM-40 197IMM-41 198IMM-47 201PSV2-03 211PSV-12 212PSV-18 215PSV-19 216PSV-26 219PSV-35 224PSV-39 226PSV-41 227PSV-43 228PSV-47 230

Keil, T PATH-25 167Kellner, J D COL-21 109Kerr, A R IMM-33 194

PATH-23 166PATH-24 166PROT-17 243

Kerrn, M B DGN-25 91DGN-27 92

Kilpi, T DGN-16 86EPI-54 59EPI-65 65IMM-26 190IMM-40 197PSV-19 216PSV-22 217

Kilpi, T M PSV-41 227Kim, J H IMM-32 193

IMM-37 196Kim, K H IMM-34 194

IMM-35 195IMM-36 195

King, Q PATH-29 169Kirkham, L S PROT-17 243Klemets, P EPI-42 53Klugman, K EPI-20 42

PSV2-02 210RES-35 132


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Klugman, K P COL-30 113DGN-05 82DGN-13 85DGN-17 87EPI-04 35EPI-31 48EPI-32 48EPI-35 50EPI-66 65EPI-75 70NEW-07 207PSV-33 223PSV-34 223PSV-35 224RES-26 127RES-32 130RES-39 134

Knox, K EPI-25 45Knutsen, B PSV-38 225Koch, A RES-30 129Kojic, E PSV-40 226Kõljalg, S RES-46 137Kolkman, M A B GEN-20 154Konradsen, H B COL-04 101

DGN-25 91DGN-28 92PSV-29 221

Korhonen, T K PATH-11 160Kozin-Goldberg, I PATH-35 172Kristinsson, K G EPI-06 36

EPI-43 54Kriwacki, R PATH-26 167Kubrusly, F PROT-04 237Kuipers, O P GEN-15 152Kunkeyani, M PSV-21 217Kurti, A DGN-14 85Kuwanda, L NEW-07 207

PSV-33 223PSV-34 223PSV-35 224

Kvetnaya, A S EPI-34 49Kvist, H GAVI-02 94Kyaw, M H EPI-44 54Kyaw-Myint, S M IMM-38 196Kyd, J M IMM-38 196Lacea, T PSV-30 221Ladesma, E EPI-68 66

PSV-30 221Lagos, R EPI-45 55

EPI-46 55EPI-47 56

Lahdenkari, M IMM-26 190PSV-43 228IMM-11 183

Lainé, C MOD-01 140Lalitha, M K EPI-48 56

EPI-78 71EPI-80 72PROT-21 245

Lampe, M IMM-25 190Langlands, J IMM-05 182

PATH-25 167Laot, T EPI-68 66Lashko, K V PSV-57 235Latham, V NEW-01 204Laxdal, B EPI-43 54Leach, A COL-22 109

PSV1-04 209Leach, A J IMM-44 199

NEW-01 204PSV-23 218PSV-37 225PSV-45 229

Lechago, M PSV-30 221Lee, G GAVI-05 96

NEW-05 206Leenhouts, K PROT-05 238Legood, R EPI-25 45Lehmann, D COL-15 106

EPI-18 41IMM-38 196PSV-46 229

Lehtinen, M J IMM-43 199Lehtonen, H PSV-19 216Leibovitz, E RES-29 129Leinonen, M DGN-06 82

DGN-16 86DGN-22 89EPI-33 49EPI-65 65

Leite, L PROT-04 237Leite, L C C PROT-11 240

PROT-19 244Leiva, L IMM-27 191Lemmens-den Toom, N COL-26 111Leroy, M GEN-17 153Levine, M EPI-74 69

EPI-83 74Levine, M M EPI-45 55

EPI-46 55EPI-47 56

Levine, O EPI-13 39EPI-27 46GAVI-01 94NEW-05 206

Lewis, E PSV-13 213Li, Z PROT-06 238Lieu, T A GAVI-05 96

NEW-05 206Lin, J EPI-89 77Liñares, J RES-13 121

RES-17 123RES-24 126RES-31 130

Lindell, D IMM-23 189Lindgren, M RES-37 133Lindholm, N PSV2-03 211


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Ling, E PATH-36 172PROT-14 241PROT-18 243

Lintu, M EPI-49 57Lipsitch, M COL-01 100

COL-16 106COL-28 112PSV-31 222RES-04 117

Llor Vila, C PSV-42 227PSV-54 233

López, R GEN-12 150GEN-19 154

Loughlin, A M PSV-44 228Low, D E RES-48 138Lozano, J DGN-06 82Lucero, M DGN-19 88

EPI-11 38EPI-49 57EPI-68 66PSV2-03 211PSV-30 221PSV-32 222

Lucero, M G EPI-63 64Luijendijk, A COL-11 104Luijendijk, I H T PSV-14 213Luo, R PATH-26 167Lupisan, S EPI-11 38

EPI-49 57EPI-68 66PSV-32 222RES-37 133

Lutsar, I RES-46 137Lütticken, R EPI-70 67

RES-11 120RES-12 120

Lyon, D J RES-28 128Lyons, M DGN-24 90Lysenko, E PATH-05 158Lyytikäinen, O EPI-42 53MacDonald, J COL-21 109Mackenzie, G COL-22 109

NEW-01 204Madhi, S PSV1-03 209Madhi, S A DGN-05 82

EPI-04 35EPI-20 42EPI-31 48EPI-32 48EPI-75 70NEW-07 207PSV-33 223PSV-34 223PSV-35 224RES-26 127RES-32 130

Madsen, J PSV-29 221Magadla, B COL-30 113Maggi, T PATH-33 171

Mahbubur, R EPI-50 57Maida, A EPI-19 42Maimets, M RES-46 137Mäkelä, P H COL-20 108

DGN-16 86EPI-33 49EPI-65 65PSV-22 217

Malamba, R PSV-21 217Maleckar, J PROT-02 236Malenje, K PSV-21 217Malley, R COL-16 106

COL-28 112PATH-04 157PROT-04 237

Manco, S PATH-27 168Mandomando, I EPI-74 69

EPI-76 70EPI-83 74

Mann, B PATH-11 160Mann, E PATH-26 167Manoharan, M PROT-21 245Manson, C PSV-30 221Maranga, W EPI-51 58

GAVI-06 96Marchant, A PSV-16 214Marchant, C D PSV-44 228Marriott, H M PATH-28 168Martin, D PROT-03 237Martín, R RES-13 121

RES-31 130Martin, S PSV-46 229Martinez, C COL-18 107Martynova, A V RES-33 131Mason, K IMM-27 191Mathieu, C IMM-28 191Mato, R COL-25 111

EPI-61 63EPI-73 69EPI-77 71PSV-20 216

Matthys, P IMM-30 192Mayfield, C A RES-34 131Mbelle, N RES-32 130McChlery, S M PSV-15 214

PSV-36 224PSV-49 231

McCluskey, J PATH-23 166PATH-42 175

McCool, T L IMM-20 187IMM-39 197

McCracken, G H DGN-06 82McDaniel, L IMM-48 201

PROT-20 244McDaniel, L S PROT-15 242McDonagh, M EPI-51 58

GAVI-06 96


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McGee, L EPI-66 65RES-03 117RES-35 132

McGeer, A RES-48 138McHugh, T D EPI-14 39

EPI-15 40McIntosh, E D G EPI-52 58McNally, L M COL-23 110

EPI-53 59McNamee, L PATH-29 169Medley, G MOD-03 141Melbye, M RES-30 129Melegaro, A IMM-06 182

IMM-47 201MOD-03 141MOD-12 142

Melen, K IMM-43 199Melin, M EPI-54 59

IMM-40 197IMM-41 198

Mellon, G PSV-23 218PSV-37 225

Meltzer, U A IMM-04 181Memmi, G PATH-15 162

PATH-33 171Meri, S IMM-41 198Metlay, J EPI-55 60

EPI-56 60Metselaar, H PROT-05 238Meurman, O DGN-20 88Meyer-Hoffert, U IMM-42 198Mhlanga, B EPI-60 62Miayji, E N PROT-19 244Michelow, I C DGN-06 82Miernyk, K PSV-38 225Miettinen, M PATH-46 177Miiro, G PSV-39 226Mikoluc, B PSV-12 212Millar, E EPI-62 63Millar, E V COL-24 110

EPI-57 61Miller, E IMM-06 182

NEW-06 206Miller, L IMM-47 201Mincer, T EPI-55 60Minz, S EPI-78 71Mirza, S PATH-30 169Mitchell, L PATH-43 176Mitchell, T GEN-04 147

IMM-51 203PATH-02 156PATH-16 162PROT-12 240PROT-13 241

Mitchell, T J GEN-16 152GEN-18 153IMM-33 194NEW-04 205PATH-18 163PATH-20 164PATH-23 166PATH-24 166PATH-28 168PATH-42 175RES-44 136

Miyaji, E N PROT-11 240Mizrachi-Nebenzahl, Y PATH-36 172

PROT-14 241PROT-18 243

Moi, K COL-06 102Moloi, V COL-30 113Molyneux, M PSV-21 217Molyneux, M E IMM-21 188

IMM-22 188IMM-23 189

Monck, R COL-15 106EPI-18 41

Monninkhof, E IMM-13 184Montoya, R DGN-15 86Moore, L J PATH-31 170Moore, M EPI-37 51

EPI-58 61Moore, Q PROT-20 244Moreno-Carvalho, O RES-36 132Morfeldt, E RES-42 135

RES-47 138Morris, P COL-22 109

IMM-44 199Morris, P S NEW-01 204

PSV-23 218PSV-37 225PSV-45 229

Morris, R NEW-06 206Morse, S PROT-04 237Moser, S A RES-34 131Moulton, L EPI-62 63Moxon, E R PSV-16 214Muhib, F NEW-05 206Mühlemann, K EPI-59 62

GEN-11 150GEN-14 151PATH-32 170RES-27 128

Mullholland, F PROT-18 243Mulliqi, G J DGN-14 85Mumprecht, V PATH-32 170Munkara, M PSV-37 225Munkundan, S PATH-14 161Muñoz, A EPI-45 55

EPI-46 55EPI-47 56

Murali, N EPI-78 71Murdoch, C PATH-16 162


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Murphy, D COL-15 106Musabeyezu, E DGN-17 87Musaya, L PSV-21 217Musher, B PSV-40 226Musher, D DGN-15 86

PSV-40 226Naaber, P RES-46 137Nahm, M PSV-28 220

PSV-50 231Nahm, M H EPI-89 77

IMM-15 185IMM-17 186IMM-32 193IMM-35 195IMM-36 195IMM-37 196PSV-55 234PSV-56 234

Nanni, A GAVI-03 95Nascimento-Carvalho, C COL-14 105

RES-36 132Neef, J PROT-05 238Nelson, C EPI-60 62Neuberger, N RES-12 120Neuzil, K M PSV-28 220Nieuwenhuis, E E S PROT-05 238Nikkanen, N PSV-47 230Nissinen, A RES-37 133Nohynek, H DGN-19 88

EPI-34 49EPI-49 57EPI-63 64EPI-68 66PSV2-01 210PSV2-03 211PSV-26 219PSV-30 221PSV-32 222

Normark, S IMM-12 183PATH-19 164PATH-41 175RES-42 135

Novakova, L RES-23 126Nunes, S COL-25 111

EPI-61 63EPI-73 69EPI-77 71PSV-20 216

Nuorti, P EPI-42 53Nurkka, A PSV-41 227O’Brien, D PSV-50 231O’Brien, K COL-02 100O’Brien, K L COL-24 110

EPI-17 41EPI-57 61EPI-62 63EPI-84 74EPI-85 75PSV-31 222

O’Callaghan, C PATH-14 161PATH-22 165

Ocampo, A PSV-32 222Ocampo, A F EPI-63 64Ochoa Gondar, O PSV-42 227

PSV-54 233O’Donoghue, M M COL-13 105

RES-14 121RES-15 122RES-16 122

Ogarkov, P I COL-31 114EPI-41 53EPI-64 64PSV-57 235

Ogburn, E GEN-04 147Oggioni, M R PATH-15 162

PATH-33 171PATH-38 173

Olcén, P DGN-28 92Oliveira, M L S PROT-11 240Ollgren, J COL-20 108

DGN-19 88EPI-42 53

Olsen, K DGN-06 82Oluoch, T EPI-51 58

GAVI-06 96Orihuela, C PATH-11 160

PATH-41 175Orihuela, C J NEW-02 204

PATH-34 171PATH-37 173

Oriyo, N COL-05 102Ortqvist, Å PATH-39 174Ossipov, I B RES-29 129Overbergh, L IMM-28 191Overweg, K PROT-18 243Pai, R PROT-21 245Pallarés, R RES-13 121

RES-31 130Palmu, A DGN-16 86

EPI-65 65PSV-18 215PSV-22 217

Pantosti, A IMM-16 185PATH-15 162RES-20 124

Parigi, R PATH-38 173Park, S EPI-37 51

EPI-58 61Parkinson, A EPI-03 35

EPI-37 51EPI-38 51EPI-58 61EPI-62 63PSV-38 225RES-41 135

Parkinson, A J EPI-17 41EPI-57 61

Parkkali, T PSV-43 228


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Parks, D EPI-58 61Parreño, R N EPI-63 64Patel, B PATH-22 165Paton, J IMM-52 203

PROT-24 246Paton, J C IMM-49 202

PATH-27 168PATH-45 177

Pebody, R IMM-06 182IMM-47 201

Pedersen, M K COL-17 107Peled, N RES-29 129Peltola, H DGN-03 81

DGN-19 88Pelton, S GEN-17 153Pelton, S I PSV-31 222

PSV-44 228Perez, E RES-31 130Pérez-Trallero, E RES-17 123

RES-24 126Perovic, O DGN-17 87Pesakhov, S PATH-35 172Peters, H PSV-38 225Petit, G PSV2-04 211Peto, T E A EPI-06 36Pettit, A PSV-45 229Pietrucha, B PSV-12 212Piipponen, S PSV-43 228Piirainen, L COL-20 108Pillai, S PSV-17 215Piotrowska-Jastrzebska, J PSV-12 212Pissarra, C RES-38 133Pitkäranta, A PATH-46 177Platt, A EPI-12 38Plebani, A IMM-46 200Pletz, M W R EPI-66 65Plevneshi, A RES-48 138Poli, A EPI-71 68Pollard, A J PSV-16 214

PSV-48 230Pomat, W IMM-05 182Pomat, W S IMM-44 199

PSV-46 229Pong-Porter, S RES-48 138Poolman, J GEN-03 147

MOD-16 144PSV-24 218PSV-25 219PSV-41 227

Porat, N EPI-23 44EPI-67 66PATH-35 172RES-29 129

Portnoi, M PATH-36 172PROT-14 241PROT-18 243

Powis, J E RES-48 138

Pozzi, G PATH-15 162PATH-33 171PATH-38 173RES-20 124

Preston, J A IMM-45 200Pridmore, A C IMM-22 188

PATH-31 170Puppe, W EPI-87 76Puumalainen, T DGN-19 88

EPI-49 57EPI-68 66PSV2-03 211PSV-30 221

Quan, V EPI-04 35EPI-75 70

Quiambao, B EPI-11 38EPI-68 66PSV-26 219

Quin, L PROT-15 242Quinti, I IMM-16 185

IMM-46 200Radin, J PATH-34 171Radin, J N PATH-37 173Rafundisani, T DGN-17 87

EPI-20 42EPI-31 48EPI-32 48RES-26 127

Rajam, G COL-03 101Raka, L DGN-14 85Ramdin, R IMM-13 184

PSV-14 213Ramirez, M DGN-18 87Ramjee Heera, J DGN-05 82Rantala, M RES-40 134Rashid, H EPI-50 57Rautio, L DGN-19 88Ray, P PSV-13 213Read, R C IMM-22 188

PATH-28 168PATH-31 170

Reasonover, A EPI-38 51EPI-58 61RES-41 135

Reid, R COL-24 110EPI-57 61EPI-62 63EPI-84 74EPI-85 75PSV-31 222

Reinert, R MOD-11 142Reinert, R R EPI-50 57

EPI-70 67EPI-87 76RES-11 120RES-12 120

Reingold, A EPI-05 36


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Ricci, S PATH-38 173Riches, P PSV-27 220Richmond, P IMM-05 182

IMM-44 199PATH-25 167PSV-46 229

Ries, J PATH-39 174Rijkers, G PSV-52 232

PSV-53 233Rijkers, G T COL-12 104

COL-29 113PSV-14 213

Riley, I EPI-11 38EPI-63 64EPI-68 66PSV2-02 210PSV-32 222

Riley, T V COL-15 106EPI-18 41IMM-38 196

Ringberg, H EPI-39 52Ringelstein, A RES-11 120Rioux, S PROT-03 237Robertson, C MOD-13 143Robillard, G T PROT-05 238Roca, A EPI-74 69

EPI-76 70EPI-83 74

Rodríguez, R EPI-30 47Rodríguez, Romeo EPI-29 47Rogers, D IMM-48 201Rohde, M PATH-13 161Romano, G EPI-71 68Romano, R PSV-32 222Romao, S RES-23 126Romero, P GEN-12 150

GEN-19 154Romero-Steiner, S COL-03 101

PROT-22 245Rosado, M M IMM-16 185

IMM-46 200Rosseau, S IMM-25 190

NEW-04 205Rubin, L EPI-72 68Rudolph, K EPI-58 61

RES-41 135Rümke, H C COL-11 104Ruohola, A DGN-20 88Russel, K PSV1-01 208Rüttimann, R EPI-81 73Ruuskanen, O DGN-20 88Ruutu, P EPI-11 38

EPI-42 53PSV-43 228RES-37 133

Ruutu, T PSV-43 228Ryynänen, M PSV-47 230Saaka, M DGN-21 89

Saarinen, L IMM-26 190IMM-11 183

Sadowy, E PATH-40 174Sajith, J EPI-88 76Saldanha, J COL-25 111

PSV-20 216Sá-Leão, R EPI-73 69

EPI-77 71Salmon, J E DGN-24 90Salt, P PSV-16 214Salt, P M PSV-48 230Sam, N E RES-19 124Sampson, J S PROT-22 245San Martín, O EPI-47 56Sanders, E PSV-52 232

PSV-53 233Sanders, E A M PSV-14 213Sanders, L E A M COL-12 104Sandgren, A IMM-12 183

PATH-41 175Santosham, M COL-24 110

EPI-57 61EPI-62 63EPI-84 74EPI-85 75PSV-31 222

Santos-Sanches, I COL-25 111EPI-61 63EPI-73 69EPI-77 71PSV-20 216

Sanz, S EPI-74 69Saukkoriipi, A DGN-06 82

DGN-22 89Scheifele, D COL-21 109Schiff, G PSV-50 231Schilder, A PSV-52 232

PSV-53 233Schilder, A G COL-12 104Schmeck, B IMM-25 190

NEW-04 205Schmitt, H J EPI-87 76Schmitz, F J RES-11 120Scholz, H PSV-11 212Schrag, S RES-35 132Schröder, J M IMM-42 198Schuchat, A EPI-04 35

EPI-05 36EPI-27 46EPI-37 51EPI-58 61EPI-75 70EPI-76 70EPI-83 74NEW-03 205

Schulenberg, J M MOD-11 142Schütte, H NEW-04 205

IMM-25 190


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Scott, A DGN-04 81EPI-27 46EPI-51 58GAVI-06 96

Scott, J A G DGN-23 90Scott, K J PSV-49 231Scott, K S PSV-15 214

PSV-36 224Scotto d’Abusco, A RES-20 124Secka, O DGN-21 89Seo, H S IMM-37 196Seoh, J Y IMM-34 194Shafinoori, S EPI-72 68Shagan, M PROT-14 241Shay, D K PSV-28 220Sheldon, J PSV-27 220Shelly, M A PSV-50 231Sheppard, C L DGN-24 90Shigayeva, A RES-48 138Shinefield, H DGN-29 93

EPI-91 78PSV-13 213

Shoma, S EPI-50 57Siedler, A EPI-70 67Sigauque, B EPI-76 70

EPI-83 74Sigaœque, B EPI-74 69Sigurdardottir, S T PSV-51 232Sikron, N PATH-35 172Silberberg, P PSV-37 225Silk, B EPI-12 38Silva, N A PATH-42 175Silva, N M S COL-14 105Simara, L RES-05 118

RES-25 127Simas, C COL-25 111

EPI-61 63EPI-73 69EPI-77 71PSV-20 216

Simoes, E EPI-11 38EPI-49 57EPI-68 66PSV-32 222

Sinclair, A COL-05 102Singleton, J EPI-44 54Singleton, R EPI-17 41

EPI-37 51PSV-38 225

Sinha, A GAVI-05 96NEW-05 206

Sirkiä, K EPI-34 49Sjöström, K RES-42 135Skoczynska, A PATH-40 174Skovsted, I C DGN-25 91Skripchenko, N V DGN-26 91Slotved, H-C DGN-27 92

Sluijter, M COL-11 104COL-12 104COL-26 111IMM-13 184IMM-14 184

Smith, A GEN-18 153RES-44 136

Smith, A M RES-43 136Smith, S H PATH-43 176Smith-Vaughan, H COL-27 112

PSV-45 229Soininen, A IMM-47 201Sologub, T S EPI-64 64Soma, K EPI-04 35

EPI-75 70Sombrero, L EPI-11 38

EPI-49 57RES-37 133

Sørdal, J O COL-17 107Sorensen, R IMM-27 191Sorensen, U IMM-37 196Soresina, A R IMM-46 200Soriano, V PSV-26 219Soriano-Gabarró, M EPI-76 70

EPI-83 74Sotgiu, G EPI-19 42Soulis, K PATH-44 176Sousa, N G COL-25 111

EPI-61 63EPI-77 71PSV-20 216

Southern, J NEW-06 206Souza, F R COL-14 105Spratt, B G EPI-06 36

EPI-33 49PSV-22 217

Standish, A PATH-45 177Steel, H C PATH-20 164Steinhoff, M EPI-80 72Steinhoff, M C EPI-78 71

EPI-79 72Stenqvist, K EPI-39 52Stephens, D S NEW-03 205Stewart, M G EPI-86 75Stokes, A COL-15 106

EPI-18 41StrŒlin, K DGN-28 92Stroeher, U H PATH-45 177Stubbs, E PSV-23 218Stubbs, L NEW-01 204Sturm, A W COL-23 110

EPI-53 59Stutzmann Meier, P GEN-14 151Sublett, J PATH-34 171

RES-45 137Sundström, C IMM-06 182

IMM-47 201Suosa, N G EPI-73 69


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Suttorp, N IMM-25 190NEW-04 205

Swaitlo, E PATH-47 178Syrjänen, R EPI-33 49Tallo, V PSV-30 221Tamm, E RES-46 137Tanizaki, M M COL-19 108Tapia, R EPI-29 47Tardivo, S EPI-71 68Temime, L MOD-04 141

MOD-14 143Tettelin, H GEN-01 146Teuwen, D PSV-30 221Thomas, K EPI-80 72

PROT-21 245Thompson, C M COL-16 106

COL-28 112RES-04 117

Thornton, J IMM-48 201Thornton, R IMM-05 182

IMM-44 199PATH-25 167PSV-46 229

Timchenko, V N RES-29 129Tipakalippa, P NEW-01 204Tolmie, H PSV-39 226Tomkins, A M COL-23 110

EPI-53 59Tonnaer, E L G M COL-29 113Toschke, M EPI-70 67Treanor, J J PSV-50 231Trefler, R EPI-67 66Tregnaghi, M EPI-81 73Tripodi, S PATH-38 173Trombe, M-C RES-23 126Trzcinski, K COL-16 106

RES-04 117Tsang, L RES-28 128Tubau, F RES-13 121

RES-31 130Tulisov, A EPI-82 73Tunnicliffe, G PATH-42 175Tuomanen, E GEN-13 151

PATH-11 160PATH-26 167PATH-41 175PATH-43 176PATH-44 176RES-45 137

Tuomanen, E I PATH-34 171PATH-37 173

Tvede, M RES-30 129Tyrrell, G COL-21 109Ugpo, J PSV2-03 211

PSV-30 221Uiterwaal, C PSV-52 232

PSV-53 233Ulijasz, A T GEN-05 148

Vainio, A IMM-43 199Valles Casanova, F X EPI-83 74Vallhagen, J RES-42 135

RES-47 138van Belkum, A COL-11 104Van Beneden, C EPI-13 39van den Doppelsteen,G P J M PSV-17 215van der Valk, Paul IMM-13 184van der Zeijst, B A M GEN-20 154van Dijken, H PSV-17 215Van Etten, E IMM-28 191van Kempen, W PSV-53 233van Kuppevelt, T H COL-29 113van Putten, J P M GEN-20 154van Roosmalen, M PROT-05 238van Selm, S GEN-20 154

PROT-05 238Vanderkooi, O G RES-48 138Varon, E MOD-04 141Vasilenko, A J EPI-64 64Veenhoven, R PSV-52 232

PSV-53 233Veenhoven, R H COL-12 104

PSV-14 213Verbrugh, H A COL-11 104Verstraeten, T MOD-16 144Vijayasekaran, S PATH-25 167Vila, C A PSV-54 233Vila Córcoles, A PSV-42 227Villanueva, S IMM-27 191Vilnitz, A A DGN-26 91Viner, R M EPI-25 45Virolainen, A IMM-43 199

PATH-46 177Virtamo, J EPI-36 50Vishnyakova, L A EPI-64 64von Gottberg, A COL-30 113

DGN-13 85DGN-17 87EPI-04 35EPI-20 42EPI-31 48EPI-32 48EPI-75 70RES-26 127

von Kries, R EPI-70 67von Ungern-Sternberg, M PATH-46 177Vuori-Holopainen, E DGN-19 88Waites, K B PSV-55 234

PSV-56 234RES-34 131

Wanahita, A DGN-15 86Wannet, W J W COL-12 104Ware, D PATH-47 178Warren, S EPI-86 75


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Wasas, A COL-30 113DGN-13 85EPI-20 42EPI-31 48EPI-32 48RES-26 127

Watera, C COL-06 102PSV-39 226

Watson, M EPI-86 75Watt, J EPI-13 39

EPI-27 46EPI-62 63

Watt, J P COL-24 110EPI-57 61EPI-84 74EPI-85 75

Wauters, D PSV-25 219Weatherholtz, R EPI-62 63Weber, J PATH-01 156

PATH-43 176Weightman, N C EPI-88 76Weigl, J A I EPI-87 76Weisblum, B GEN-05 148Weiser, J N IMM-20 187

IMM-39 197PATH-05 158

Wells, J PROT-18 243White, Jr., A PSV-40 226Whitney, C PSV1-02 208

RES-35 132Whitney, C G COL-03 101

EPI-05 36EPI-44 54EPI-66 65EPI-84 74EPI-85 75NEW-03 205PSV-28 220

Whitworth, J COL-06 102Whyte, M K B PATH-28 168Wiertsema, S PSV-53 233Wigger, C NEW-01 204Wilson, C PSV-23 218

PSV-37 225Witzenrath, M IMM-25 190

NEW-04 205Wolf-Watz PATH-19 164Wu, M-C PROT-06 238Wuyts, G IMM-29 192Xie, X DGN-11 84Yeoman, E EPI-72 68Yother, J PATH-03 157Yu, J EPI-89 77

IMM-35 195NEW-02 204

Yuste, J IMM-49 202IMM-50 202PROT-16 242

Zack, M EPI-44 54Zagursky, R DGN-11 84Zakaria, M M COL-20 108Zaman, A EPI-90 77Zell, E R EPI-44 54Zhang, Q IMM-51 203

IMM-52 203PATH-16 162PROT-12 240PROT-13 241PROT-24 246

Zhang, Y MOD-15 144Zhao, G M EPI-91 78Zhogolev, S D COL-31 114

EPI-41 53EPI-64 64PSV-57 235

Zijlstra, E E IMM-21 188IMM-22 188IMM-23 189

Zughaier, S NEW-03 205


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