Journal of Infection (2009) 59, 225e231

www.elsevierhealth.com/journals/jinf

REVIEW

Pertussis epidemiology in Argentina: trends over2004e2007

D. Hozbor a,*, F. Mooi b, D. Flores a, G. Weltman c, D. Bottero a, S. Fossati c,C. Lara c, M.E. Gaillard a, L. Pianciola d, E. Zurita a, A. Fioriti a, D. Archuby a,M. Galas c, N. Binsztein c, M. Regueira c, C. Castuma a, M. Fingermann a,A. Graieb a

a Instituto de Biotecnologıa y Biologıa Molecular, Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas,Universidad Nacional de La Plata, CCT La Plata-CONICET, Calles 47 y 115, 1900 La Plata, Argentinab Laboratory for Infectious Diseases and Screening (LIS), Netherlands Centre for Infectious Diseases Control, NationalInstitute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlandsc Servicio de Bacteriologıa Clınica, Departamento de Bacteriologıa, INEI-ANLIS ‘‘Dr. Carlos G. Malbran’’, Av. Velz Sarfield563, CP 1282 AFF, Ciudad Autonoma de Buenos Aires, Argentinad Laboratorio Central, Subsecretaria de Salud de Neuquen, Gregorio Martınez 65, 8300 Neuquen, Argentina

Accepted 17 July 2009Available online 3 August 2009

KEYWORDSPertussis;Epidemiology;South America;Vaccines

* Corresponding author. Tel.: þ54 22E-mail address: hozbor@biol.unlp.

0163-4453/$36 ª 2009 The British Infedoi:10.1016/j.jinf.2009.07.014

Summary Objectives: Pertussis continues causing significant morbidity and mortality world-wide. Although its epidemiology has been studied in many developed countries, the currentpertussis situation in South America is scarcely known. This review summarizes the most impor-tant recent data concerning pertussis in a country of South America, Argentina.Methods: CDC criteria were used for pertussis diagnosis. Proportion of pertussis cases by age,immunization status, and immunization coverage rate evaluated at the Argentinean NationalPertussis Reference Centers was reported. Bordetella pertussis isolates were characterizedand compared with vaccine strains.Results: From 2002 to nowadays, a steady increase of pertussis cases was observed. Most ofthese cases correspond to patients younger than six months old that received less than threedoses of vaccine. However, cases in adolescent and adults have also been detected. For thissituation, which is not peculiar to Argentina, several explanations have been proposed. Amongthem, the inability of current vaccines to induce long-lasting immunity is the most widelyaccepted as a cause of pertussis resurgence. Furthermore, antigenic divergence between localclinical isolates and vaccine strains may have aggravated the effect of waning immunity.

1 425 0497 ext.31; fax: þ54 221 422 6947.edu.ar (D. Hozbor).

ction Society. Published by Elsevier Ltd. All rights reserved.

226 D. Hozbor et al.

Conclusions: Pertussis is an important problem for public health in Argentina. Divergencebetween vaccine strains and local isolates could contribute to the described pertussisepidemiology.ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Pertussis or whooping cough is an acute respiratory tractinfection mainly caused by Bordetella pertussis, a fastidiousGram negative bacterium. This disease, though preventableby vaccination, remains one of the top ten causes of deathby infectious diseases in childhood worldwide. Nowadaysmorbidity of pertussis is significant not only in childrenyounger than one year old but also in adolescents and adul-ts.1e3 Though resurgence or persistence of pertussis hasbeen recognized, the reasons for this phenomenon are stillnot fully elucidated. In some countries (Sweden, Japan) theincrease in pertussis has been recognized as being due totemporary exclusion of pertussis whole-cell immunizationfrom vaccination schedules,4,5 because of an underlyingfear of possible adverse events following immunizationwith such vaccine. Sweden, Japan, the UK and The RussianFederation had active opposition to whole-cell vaccines ethat is, well-organized movements that sought to stop theiruse by means of news stories, television interviews, lec-tures, popular articles, books, and other writings.4,6 Inother countries, changes in surveillance, a decrease in vac-cination coverage, waning of vaccine-induced immunity,and pathogen adaptation are proposed as possible factorsfor the increased incidence of pertussis.7e9 Understandingthe epidemiology of the disease in each country, and com-paring the data obtained from different countries may elu-cidate the possible causes and, what is more important,suggest strategies aimed at improving the control of thedisease. Although many epidemiological studies focusedon pertussis have been published in the United States, Can-ada and Europe, scarce studies are available for SouthAmerica. Here we present data on pertussis epidemiologyin Argentina.

Figure 1 Pertussis incidence per 100,000 habitants registeredin Argentina from 1997 to 2006.

Pertussis trends in Argentina

In Argentina, a country with a 38 million population, thewhole-cell pertussis vaccine was introduced in the 1960sand is still the only kind of pertussis vaccine being usedtoday. Since 1985 the vaccination schedule recommended is2, 4, 6 and 18 months doses with a school entry booster at5e6 years of age. In most parts of the country, the coveragewith three doses during the last years ranged between 90and 95%, although in some jurisdictions it only reached 80%or less.

Routinely in Argentina, clinical cases of pertussis areanalyzed in Public Health Laboratories or in the ArgentineanNational Reference Center. The clinical case definition used isbased on CDC/WHO clinical criteria (www.cdc.gov/ncphi/disss/nndss/casedef/pertussis_current.htm, www.who.int/entity/immunization_monitoring/diseases/pertussis_surveil-lance/en/index.html) that refers to a person with a coughillness lasting at least two weeks with one of the following

symptoms/signs: coughing paroxysms, inspiratory whoop, orpost-tussive vomiting. In patients younger than 6 months ofage, cyanosis and apneas could also be present, and for thisreason these symptoms are also included for pertussis clinicaldiagnosis. A confirmed case of pertussis is defined as:

(1) a person with acute cough illness of any duration, anda positive culture for B. pertussis or

(2) a case that meets the clinical case definition and isconfirmed by PCR, or

(3) a case that meets the clinical case definition and isconfirmed by serology, if the last vaccine dose wasreceived at least four years ago. A significant increaseof overall antibody titer against B. pertussis wholecell (sonicated) in paired sera is used as criteria forserological confirmation.

(4) a case that meets the clinical case definition and isdirectly epidemiologically linked to a laboratoryconfirmed case.

According to the Argentinean National EpidemiologicalSurveillance System (SINAVE), since 1998e2002 a steadydecline in the incidence of pertussis was observed: in-cidence rate per 100,000 inhabitants varied from 2.3 in1998 to 0.7 in 2002 (Fig. 1). However, since 2002, an in-crease in pertussis incidence was observed, which was sus-tained until 2006. Furthermore, in 2005 a pertussisoutbreak in the Argentinean southern province of Neuquenwas registered. The official data for 2007 have not beenpublished yet. Beyond the change observed in 2006, all per-tussis rate records registered during 2003e2006 periodwere higher than those recorded in 2002 (Fig. 1).

This increase was registered by the Public HealthMinistry even before improvements in diagnostic method-ology were introduced (i.e. PCR methodology was incorpo-rated at the end of 2004). The pertussis situation inArgentina will be here discussed in more detail using dataobtained by the Argentinean Pertussis National ReferenceCenter. These data include age, vaccination status andsymptoms of the pertussis confirmed cases obtained during2004e2007. During the mentioned period, 8823 samplesfrom patients with clinical diagnosis of pertussis coming

Pertussis epidemiology 227

from various provinces of our country were analyzed. Themajority of these cases corresponded to the more popu-lated provinces of Argentina such as Buenos Aires, Cordobaand Santa Fe. From these samples, 2102 (23.8%) wereconfirmed as pertussis. In 2005, a pertussis outbreak inNeuquen with 568 confirmed cases was registered. In thispaper, data obtained during this outbreak are analyzedseparately, since they show different epidemiologicalcharacteristics (see below).

The next section will focus on data from Argentinaexcluding the Neuquen outbreak. The annual distributionof confirmed cases showed an increment in the proportionof pertussis cases which was time sustained, at least from2004: 188 in 2004, 206 in 2005, 523 in 2006, and 617 in 2007.Excluding the Neuquen outbreak, 1534 pertussis-suspectedclinical cases were confirmed. Data of patient age wereavailable in 1460 (95.1%) cases. Fig. 2 shows an analysis ofthe distribution of pertussis confirmed cases by age. Clearlymost of the cases were confirmed in patients younger than 6months old (865 of the 1460 cases, 59.2%); the rest was dis-tributed as follows: 16.06% (235) between 6 and 17 monthsof age, 12.8% (188) between 18 months and 6 years old, and11.7% (172 cases) older than 6 years. The large proportion ofcases recorded in patients younger than 6 months old wasnot unexpected, since pertussis is most severe in this agegroup. However, the number of cases registered in otherage groups should not be neglected, especially at the timeof designing a new strategy to improve the control of thedisease. The number of pertussis deaths during the analyzedperiod ranged from 8 to 21 per year (average overall years16), recorded mostly in children less than 1 year old, andregistering the highest value in 2006 with 21 deaths.

Data about patient symptoms and age were available in1130 cases over the 1534 confirmed cases. Table 1 illus-trates the diverse clinical manifestations. As shown in Ta-ble 1, apnea and cyanosis occurred more frequently ininfants aged <6 months, and paroxysms or vomiting aftercough were more common in children older than 6months. As expected, young unvaccinated infants were

Figure 2 Distribution of pertussis confirmed cases by the patient

more likely to have a severe disease, to suffer complica-tions, to require hospitalization, or to die, than older chil-dren and adults.

Another important aspect we have analyzed was thedistribution of pertussis confirmed cases in relation topatient’s age and state of vaccination (Table 2). From thetotal confirmed cases excluding Neuquen outbreak, 64%(984) had complete data about patient’s age and state ofvaccination, and 66% (646) of them were less than 6 monthsold and had received less than 3 vaccine doses. Approxi-mately 23% of pertussis patients had had 3 and 4 vaccinedoses and were between 6 months and less than 6 yearsof age, whereas only 3.5% patients had had at least 5 doses.The same distribution was observed when data were plot-ted by year (not shown). The proportion of cases per yearobserved in vaccinated individuals shows at least in part,an aspect already known: pertussis vaccines are not 100%effective and multiple booster doses are required to im-prove protection against the disease.

The Neuquen pertussis outbreak

During 2005 in the province of Neuquen, which hasapproximately 500,000 inhabitants, a total of 2806 pertus-sis-suspected cases were notified, from which 568 (20.2%)were laboratory confirmed. There were 5 deaths alsoregistered in children under 1 year of age. It is worthmentioning that during this epidemic situation the percent-age of cases per age was different from the previouslyobserved in the rest of the country. Higher rates wereobserved in this outbreak for age groups older than 6months: 132 cases (23%) in patients younger than 6 months,130 (22.8%) between 6 months and 17 months, 143 (25.1%)between 18 months and less than 6 years, and 157 (27.6%)in patients older than 6 years of age. The largest proportionof confirmed cases was observed in the age range from 18months to 6 years. A close analysis of the notified casesshows that a surprisingly large number of symptomaticinfections took place in vaccinated infants (Fig. 3). Out of

’s age. Period from 2004 to 2007 excluding Neuquen outbreak.

Table 1 Pertussis clinical manifestations.

Age ofpatient

Total casesregisteredby age

Paroxysmalcough

Paroxysms andpost-tussivevomiting

Paroxysmal coughand whoop

Cyanosis Apnea Cyanosisand apnea

Cough withcyanosisand apnea

Deaths

0e6 m 816 84(10%) 309(38%) 38(5%) 342(42%) 161(20%) 101(12%) 37(5%) 63a

6e18 m 139 30(22%) 38(27%) 12(9%) 50(36%) 28(20%) 10(7%) 4(3%)18 me6 y 101 31(31%) 42(42%) 21(21%) 32(32%) 9(9%) 5(5%) 3(3%) 16e16 y 50 19(38%) 20(40%) 7(14%) 9(18%) 6(12%) 1(2%)>16 y 24 11(46%) 2(8%) 2(8%) 4(17%) 1(4%) 1 1

Total 1130 175 411 80 437 205 117 44a Corresponds to the total deaths registered in children under 1 year old.

228 D. Hozbor et al.

a total of 568 confirmed cases 352 had data of age and vac-cination status of patients: 101 (28.5%) were less than 6months old, with 0, 1 or 2 doses of vaccine, and 216(61.5%) were between 6 months and less than 6 years ofage with 3 or 4 doses of vaccine (Fig. 3). Clearly, confirmedcases in this outbreak had had more vaccine doses andshifted to older ages compared to the trend in the rest ofthe country (Fig. 3). A similar distribution of cases was pre-viously observed during the 1996e1997 epidemic in theNetherlands.10 Van Boven et al.10 hypothesized that the ob-served age-distribution of pertussis infections indicatedthat the protection time after vaccination decreased forall individuals. This could be explained if the pathogen pop-ulation changed, thus the vaccine used conferred less pro-tection. Given the resemblance of both situations, thisexplanation might also be applied to Argentina. The previ-ous molecular characterization of the Argentinean circulat-ing bacterial population made by our group, seemed toagree with such explanation.11

Genomic analysis of B. pertussis isolatescirculating in Argentina

During the period 1997e2003 the Argentinean NationalReference Center began with the isolation and molecularcharacterization of B. pertussis circulating strains. The firstsigns of divergence in the bacterial population were foundwithin 28 samples studied by fingerprinting using BoxAPCR primers.12 Since 2004, we isolated the causal agentfrom other 145 samples obtained from patients with pertus-sis. Most of these isolates have been genetically character-ized by means of pulse field gel electrophoresis (PFGE) and

Table 2 Distribution of pertussis confirmed cases by age and v

Age of patients Vaccination status

Unvaccinated 1 dose 2

0e2 m 249 (25.31%) 2 (0.21%)2e6 m 137 (13.91%) 208 (21.21%) 56e18 m 6 (0.61%) 7 (0.71%) 318 me6 y 6 (0.61%) 1 (0.11%)6e16 y 1 (0.11%) 1 (0.11%)>16 yNo data available 2 (0.21%)

sequencing the genes for the polymorphic antigens pertus-sis toxin (ptx) and pertactin (prn).13e16 Among B. pertussislocal isolates collected in the periods 1969e1989 and1997e2007, 51 PFGE profiles were identified and classifiedinto six major groups (I, IIIeVII) using UPGMA. The B. per-tussis isolates were grouped in a major group with a mini-mum of 80% overall relatedness among them. The vaccineand the 18323 WHO reference strains were classified ina different PFGE group from all clinical isolates, witha 64% similarity between them.

A large number of clinical isolates from many countrieshave been analyzed for genotyping of pertussis toxin subunit1 (ptxA) and prn and so far, 4 ptxA (ptxA1eptxA4) and 12 prnalleles have been reported. It was observed that the differ-ent alleles in ptxA are due to a single-nucleotide polymor-phism (SNP) resulting in amino acid substitution, and thedifferent alleles in prn are mainly caused by insertion or de-letion of repeating unit GGXXP amino acids.15 From the totalArgentinean clinical isolates analyzed, 91% contained theptxA1 allele, while the remaining strains harboured ptxA2or ptxA3. Two prn alleles were found, prn1 and prn2 with79% of the isolates harboring prn2. A strong correlation be-tween PFGE profile and prn alleles was observed. Since1989 prn2 strains began to prevail reflecting a transitionfrom PFGE IVb and VII to group VI. The predominant allelicvariants that caused Argentinean outbreaks were similar tothe ones previously found in Europe, USA, Canada and Tai-wan. The whole-cell pertussis vaccines used in Argentinasince 1960 were developed with foreign strains which wereisolated in the 1950s. The allelic profiles of these vaccinestrains are ptxA2/prn1 for IM 1416 and ptxA3/prn1 forIM1414. Similar profiles have been found in other B. pertussisstrains used in the production of vaccines.

accination status. Period from 2004 to 2007.

dose 3 dose 4 dose 5 dose

0 (5.08%)6 (3.6%) 120 (12.2%) 1 (0.11%)3 (0.31%) 7 (0.71%) 98 (9.95%) 2 (0.2%)

4 (0.41%) 5 (0.51%) 23 (2.3%)1 (0.11%) 1 (0.11%) 10 (1%)3 (0.31%)

Figure 3 Distribution of pertussis confirmed cases by age in Neuquen outbreak (2005). Vaccination status of patients is indicatedwithin each bar as percentage of the confirmed cases for the respective age group.

Pertussis epidemiology 229

In summary, we observed significant differencesbetween Argentinean B. pertussis clinical isolates andvaccine strains.

Functional implications of the genomicdivergences observed between B. pertussisclinical isolates and vaccine strains

Since in Argentina there are only a few isolates from thepertussis vaccine previous period, it is not possible toelucidate whether the divergence observed between clin-ical isolates and vaccine strains emerged after the in-troduction of vaccination and was the consequence of the

Figure 4 Bacterial lung colonization observed in mice after intramunized with 1/16 of the human dose of a DTwP vaccine of local uslenged either with Tohama strain phase I (panel A) or a local isolatsub-lethal doses (CFU 5� 107). Three independent experiments weshown. Results depicted are mean of four mice per group at each

vaccine selective pressure. However, strains from theprevaccination era have been studied in other countries,and these studies have shown that these divergencesoccurred after the introduction of vaccination, supportingthe hypothesis of the immune selection caused by vacci-nation. The B. pertussis population has been studied ingreat detail in the Netherlands.13,17e20 These studies re-vealed a stepwise difference between vaccine strains andclinical isolates. In the 1960s, strains with vaccine typePtxA were replaced by a novel, non-vaccine type. The pro-cess of antigenic variation continued in the 1980s and1990s, when non-vaccine types of Prn and Fim3 arose.21

An important question to be answered is whether polymor-phism in the B. pertussis population affects the vaccine

nasal challenge. Balb C mice (females 3e4 weeks old) were im-e and ten days after the last immunization the mice were chal-e used as representative of Argentinean collection (panel B) inre performed. Results from one representative experiment aretime.

230 D. Hozbor et al.

efficacy and, as a consequence, contributes to the resur-gence of the disease. This aspect has been studied in mousemodel. Since the intracranial challenge is not the naturalroute of infection, the respiratory models of protectionand challenge have been extensively employed to studypathogenesis and immunity to B. pertussis. This model,by intranasal or aerosol challenge, has proved to be usefulin assessing the efficacy of cellular or acellular vaccines.Both Mills et al.22 and Xing et al.23 demonstrated that theaerosol challenge can be used to distinguish among vac-cines that have shown different efficacies in clinical assays.Moreover, it has been demonstrated that the intranasalchallenge can discriminate between acellular vaccineswith 2 and 3 components with different efficacies.24

Regarding to the scheme of immunization and thechallenge doses, most researchers use 2 immunizationswith a 14-day separation, followed by a challenge 10e14days after the second immunization with 5� 107e1� 108 bacteria.22,24 This scheme of immunization andchallenge allows the correlation of the efficacy of vaccinesin clinical trials with the levels of protection in mice. Vac-cines with 95% efficacy in clinical trials show a completebacterial elimination from mice lungs between 5 and 8days post-challenge.15,22 The 18323 reference strain ob-tained in the USA in 1947, is recommended by the WHOto evaluate the efficiency of vaccines against pertussis.However, 18323 is an atypical B. pertussis strain which isphylogenetically closely related to Bordetella bronchisep-tica and it expresses an unusual type of pertactin (Prn6).For these reasons, since the objective was to evaluatethe role of the polymorphism in this and other antigenswith respect to their protection capacity, we decided touse the Tohama strain (PtxS1B, Prn 1) as a reference in pro-tection assays. This strain was isolated in Japan in 1952,and is used as a source of purified components of acellularvaccines. A group of mice immunized with PBS were used asa negative control. Balb C mice (females 3e4 weeks old)were immunized with 1/16 of the human dose of a localuse DTP vaccine, and ten days after the last immunization,the mice were challenged with a representative local iso-late in sub-lethal doses (CFU 5� 107). Results obtained inthis assay are shown in Fig. 4. Clinical isolate persisted inthe lungs of immunized mice until day 8, while the refer-ence strain could not be recovered from the lungs(p< 0.001) at day 8 after the challenge. These results, inagreement with those published in the Netherlands 15 andPoland,25 indicate that the divergence between the localisolates and the strains used to produce the vaccines, playsa role in protection, at least, under the conditions assayed.Concerning to pertactin, various publications have demon-strated its role in adhesion and in protective immunity.26e28

King et al.15 have analyzed the immunological importanceof pertactin polymorphism, in particular in region 1. Thisis a polymorphic domain at the protein surface close toan RGD motif, involved in the adhesion to host cells. Theseauthors demonstrated that the polymorphic region 1 con-tains the protective epitopes and, as a consequence, var-iations in this domain may affect antibody binding. Otherauthors29,30 have suggested that during the infection andimmunization, antibodies would be generated againsta conformational epitope involving regions 1 and 2 of per-tactin. Even though linear epitopes constituted by the

repetitive sequences of region 1 are capable of inducinga cross reaction, they would still be less effective thanthe conformational antibodies.

Concluding remarks

All the data presented here indicate that pertussis remainsa public health problem in South American countries suchas Argentina, as it does in Europe and North America. In thisregion, the highest incidence rates were also registered inchildren under 6 months of age, unvaccinated or vaccinatedwith insufficient doses to be protected against the in-fection. Increasing numbers of pertussis infections werealso detected in adolescents and adults (187/1460, 12.8%),and this may be due to the fact that vaccines do not inducelong-lasting immunity. The effect of this waning immunitymay be aggravated by the antigenic divergence betweenclinical isolates and pertussis vaccine strains. In relation tothis hypothesis, there is increasing molecular evidence thatB. pertussis circulating strains have changed a number ofvirulence factors, in particular the two protective antigens,pertussis toxin and pertactin. It is proposed that the mis-match observed for these two antigens, between the vac-cine strain and the circulating bacteria, has someadaptative significance and implications on the protectioninduced by the vaccine. Though more data and experimentsare needed to verify this hypothesis, the fact that B. per-tussis may have evolved raises at least, questions about fu-ture vaccines.

Acknowledgements

This work was supported by EU INCO 032296, ANCPyT andCICBA (Argentina) grants to DH and Malbran-UNLP grants toMG and DH. DH is a member of the Scientific Career ofCICBA. DB and EG have fellowships from CONICET. AG hasa fellowship from ANCPyT and CC is supported by an INCO(EU) grant. We thank the healthcare workers involved inpertussis surveillance system.

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