incorporation of real time pcr into routine public health ...lucila o. fukasawa instituto adolfo...

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

lucilaof@gmail.com

(11) 3068-2899

Thank you !!!

Muchas gracias !!!