incorporation of real time pcr into routine public health ...lucila o. fukasawa instituto adolfo...
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Lucila O. Fukasawa
Instituto Adolfo Lutz
São Paulo - SP
Incorporation of Real Time
PCR into Routine Public
Health Surveillance of
Bacterial Meningitis:
The Brazilian Experience
Confirmed Cases of Meningitis
27,731 cases
11,180 (40.3%)
Bacterial
13,224 (47.7%)
Viral
1,002 (3.6%)
Other meningitis
2,325 (8.4%)
Undetermined
Meningitis
Data: COVER-SINAN/SVS/MS
Brazil - 2006
0
10
20
30
40
50
60
70
80
90
100
2002 2003 2004 2005 2006Year
Undetermined bacteria Other bacteria S.pneumoniae H.influenzae N.meningitidis
%
~50%
Brazil (2002 - 2006)
Data: COVER-SINAN/SVS/MS
Bacterial Meningitis - Etiology
0
10
20
30
40
50
60
70
80
90
100
2002 2003 2004 2005 2006Year
Undetermined Bacteria Other bacteria S.pneumoniae H.influenzae N.meningitidis
%
Data: http://www.cve.saude.sp.gov.br/htm/cve_meni.htlm
~50%
São Paulo State (2002 - 2006)
Bacterial Meningitis - Etiology
Criteria for Confirmation of Cases
Data: COVER-SINAN/SVS/MS
11,180 cases (Brazil) - 2006
45.3%
Cases
confirmed
by
laboratorial
criteria
Culture 28%
6.1% Agglutination Test
1.5% - Counterimmunoelectophoresis
(CIE)
9.7%
Bacterioscopy (Gram Stain)
35.1%
Cytological
Examination of CSF
12.2%
Clinical 7.4%
Unknown
Real Time PCR (RT-PCR)
Triplex
RT-PCR
N. meningitidis
(Men)
ctrA
H. influenzae
(Hi)
bexA
S. pneumoniae
(Spn)
lytA
Carvalho et al, 2007 Corless et al, 2001 Mothershed et al, 2007
Triplex RT-PCR
ABI7500 Slope Efficiency r2
Men
crtA-FAM
-3,517
92,44% 0,996
Spn
lytA-VIC
-3,55 91,29% 0,990
Hi
bexA-NED
-3,599 90,13% 0,997
Recommended Criteria:
Slope (m): -3,6 m -3,1
Efficiency (E): 0,9 E 1,1
r2: 0,990 r2 0,999
Low Limit of Detection (LLD)
Triplex RT-PCR
N. meningitidis (Men)
LLD = 20 fg DNA
S. pneumoniae (Spn)
LLD = 20 fg DNA
Low Limit of Detection (LLD)
Triplex RT-PCR
H. influenzae (Hi)
LLD = 20 fg DNA
Low Limit of Detection (LLD)
Triplex RT-PCR
Sentinel Program
Bacterial Meningitis
9 Sentinel Hospitals – city of São Paulo
3 Sentinel Hospitals – city of Campinas
CSF and/or serum RT-PCR
Clinical and Laboratory Data
Statistical Analysis
Sensitivity
Specificity
Jun/07
Period: Jun/07 – Mar/09
2,403 samples
CSF = 1,194
Sera = 1,209
RT-PCR
Sentinel Program
Bacterial Meningitis
Sacchi CT et al. PLos One, 6(6): e20675, 2011
Triplex RT-PCR
Sensitivity and Specificity
Sacchi CT et al. PLos One, 6(6): e20675, 2011
Positivity
RT-PCR vs. CIE
CIE RT-PCR
Men 136
(18%)
227
(31%)
Spn 0
(0%)
87
(12%)
Positives 136
(18%)
314
(42%)
Negatives 604 426
Total 740 740
Positivity
40%
57%
740 (484 CSF, 256 sera)
Data: Sentinel Program, Instituto Adolfo Lutz, Jun/07-Mar/09
740 (484 CSF, 256 sera)
Data: Sentinel Program, Instituto Adolfo Lutz, Jun/07-Mar/09
Culture RT-PCR
Men 110
(15%)
227
(31%)
Spn 61
(8%)
87
(12%)
Positives 171
(23%)
314
(43%)
Negatives 569 426
Total 740 740
52%
45%
30%
Positivity
RT-PCR vs. Culture
Positivity
RT-PCR in Routine
2011
City of São Paulo
1540
Clinical
Samples
1049 CSF
491 Sera
1214
pacients
Data: Instituto Adolfo Lutz, Jan/11-Dec/11
RT-PCR in Routine
Distribution of etiologic agents identified by RT-
PCR in 1214 cases – city of São Paulo, year 2011
17,5%
7,8%
0,8%
73,9%
Men
Spn
Hi
Neg
Data: Instituto Adolfo Lutz, Jan/11-Dec/11
76,4%
4,7% 0,5% 6,1%
12,3%
B
C
W135
Y
NG
RT-PCR in Routine
Distribution of genogroups of Men (n = 212 cases)
City of São Paulo, year 2011
Data: Instituto Adolfo Lutz, Jan/11-Dec/11
40%
50%
0%10%
a
b
c
d
RT-PCR in Routine
Distribution of genotypes of Hi (n = 10 cases)
City of São Paulo, year 2011
Data: Instituto Adolfo Lutz, Jan/11-Dec/11
Triplex RT-PCR
Introduced in
Routine
Labs that will
be trained SANTO
ANDRÉ
SÃO
PAULO
Triplex RT-PCR
Introduced in
Routine
Received
training
Bacterial Meningitis - Etiology
São Paulo State (2002 -2010)
0
10
20
30
40
50
60
70
80
90
100
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011Year
N. meningitidis H. influenzae S. pneumoniae Other bacteria Undetermined Bacteria
%
~50% 38
%
Sentinel Program
+
RT-PCR Data: www.cve.saude.sp.gov.br/htm/cve_meni.htlm
29
%
23
%
25
% 23
%
Acknowledgments
Universidade de Pittsburgh, PA, EUA
Dr. Lee H. Harrison
Kathleen Shutt
Angela Cruciano
IAL - Centro de Bacteriologia
Dra. Maria Cristina Brandileone
Dra. Maria Cecilia O. Gorla
Dra. Ana Paula S. Lemos
Dra. Samanta C. G. Almeida
Dra. Rosemeire C. Zanella
Dra. Angela P. Brandão
IAL – Lab. Regional de Campinas
Dra. Eneida G. L. Marques
COVISA – Campinas
Dra. Brigina Kemp
CVE – CCD/SES
Dra. Ana Freitas Ribeiro
Dra. Telma Carvalhanas
Dr. Ricardo K. M. Albernaz
CCD/COVISA/SMS/SP
Dra. Raquel M. B. P. Fernandes
Faculdade de Saúde Pública/USP
Dr. Eliseu Waldman
12 Hospitais Sentinelas: I.I. Emílio Ribas, Hosp.
Estadual Grajaú, Hosp. Infantil Menino Jesus,
Hosp. Mun. Tatuapé, Casa Saúde Santa Marcelina,
Complexo Hosp. Mandaqui, Hosp. Regional Sul,
Santa Casa de SP, Hosp. São Paulo, Hosp. Celso
Piero, Hosp. Univers. UNICAMP, Hosp. Mario Gatti
Instituto de Infectologia Emílio Ribas
Dr. Felipe A. S. Gualberto
Dr. Jose Vidal
Dr. Cesar Augusto P. Oliveira
Lucila O. Fukasawa
Instituto Adolfo Lutz
(11) 3068-2899
Thank you !!!
Muchas gracias !!!