VPIS Protocol for NIH

AnusakAnusak KerdsinKerdsin

National Institute of HealthNational Institute of Health

DeaprtmentDeaprtment of Medical Sciencesof Medical Sciences

Outline

• Identification / Detection

• Serotyping• Serotyping

• Genotyping

Organism in VPIS

• Corynebacterium diphtheriae

• Streptococcus pneumoniae

• Streptococcus pyogenes and beta-hemolytic

StreptococciStreptococci

• Bordetella pertussis

• Haemophilus influenzae

• Neisseria meningitidis

Laboratory processing in NIH

– Identification by molecular and biochemical methods.

• Multiplex PCR identify of beta-streptococci

• Multiplex PCR for C. diphtheriae and toxin gene

• Real-time PCR directly to nasopharyngeal swab for B.

pertussis.pertussis.

– Serotyping by multiplex PCR for S. pneumoniae,

H. influenzae and N. meningitidis.

– An emm typing for S. pyogenes.

– MLST analysis for C. diphtheriae & H. influenzae.

– PCR confirmation of NTHi

Identification / Detection

- Biochemical test- Biochemical test

- Molecular test

Biochemical testing

• Beta-hemolytic streptococci

– Gram stain, bacitracin, CAMP, SXT

• S. pneumoniae

– Gram stain, optochin, bile solubility

C. diphtheriae• C. diphtheriae

– Gram stain, Elek test

• H. influenzae

– Gram stain, X, V, XV factors

• N. meningitidis

– Gram stain, oxidase

Pertussis Detection

Sample: Nasopharyngeal swab

B. B. B. B. pertussispertussispertussispertussis (81.2(81.2(81.2(81.2----82 82 82 82 ooooCCCC))))

Negative control (74.9 Negative control (74.9 Negative control (74.9 Negative control (74.9 ooooCCCC))))

B. B. B. B. pertussispertussispertussispertussis (81.2(81.2(81.2(81.2----82 82 82 82 ooooCCCC))))

Negative control (74.9 Negative control (74.9 Negative control (74.9 Negative control (74.9 ooooCCCC))))

Thai-NIH Strategy for Pertussis Detection

Clinical specimen (NPS, NPA)

DNA extraction

SYBR Green Real-Time PCR(upstream region of por gene: Bpe)

Multitarget Real-Time PCR (Bpe, Bpa, Bho)

ptxS1 Real-Time PCR (Bpe, Bpa, Bbr)

Interpretation

Thai-NIH Algorithm

por IS481 pIS1001 hIS1001 ptxS1 Interpretation

+ + - - + / - B. pertussis

- + - - + B. pertussis

+ - - - + / - B. pertussis+ - - - + / - B. pertussis

- + - - - Indeterminate

- + - + - B. holmesii

- - - + - B. holmesii

- - + - + B. parapertussis

- - - - + B. bronchiseptica

Multiplex PCR for identification of C. diphtheriae and toxin gene

dtxR

tox : part Btox : part A

Sample: pure culture only

Multiplex PCR identify beta-hemolytic Streptococci

Lane 1 = Streptococcus agalactiae

(Streptococcus group B)

Lane 2 = Streptococcus dysgalactiae

Sample: pure culture only

Lane 3 = Streptococcus pyogenes

Lane 4 = Streptococcus equi subsp.

zooepidemicus

Lane 5 = Streptococcus anginosus group

Lane 6 = Negative control

Molecular Serotyping

- H. influenzae- H. influenzae

- N. meningitis

- S. pneumoniae

Multiplex PCR identify & serotyping of Neisseria meningitidis

Sample: pure culture only

Multiplex PCR identify & serotyping of

Haemophilus influenzae

Sample: pure culture only

Sequential Multiplex PCR Serotyping of S. pneumoniae

Reaction 1 Reaction 2

Sample:

pure

Reaction 3Reaction 4

pure

culture only

PCR identification of S. pneumoniae serotype 6A, 6B, 6C and 6D

Reaction 1 Reaction 2

Sample: pure culture only

Molecular Genotyping

- C. diphtheriae- C. diphtheriae

- S. pyogenes

- H. influenzae

MLST of Diphtheria

An eBURST analysis of C. diphtheriaefrom MLST database

123

243*

258

13

244

245

246254

259

256

246

248

209

247

249

253

255257

260

An emm typing for S. pyogenes

Analysis of nontypable H. influenzae (NTHi)

1. Confirmation of NTHi

- PCR method to determine true NTHi will be done as described by

Binks MJ, et al. (2012).

- The hpd1, hpd3, lgtC, fucK, iga, and ompP2 were used as the

target genes for PCR to confirmation of true NTHi.

Analysis of nontypable H. influenzae (NTHi)

2. MLST analysis: Typable & NT will be analysed.