VPIS Protocol for NIH Anusak Anusak Kerdsin Kerdsin National Institute of Health National Institute of Health Deaprtment Deaprtment of Medical Sciences of Medical Sciences
VPIS Protocol for NIH
AnusakAnusak KerdsinKerdsin
National Institute of HealthNational Institute of Health
DeaprtmentDeaprtment of Medical Sciencesof Medical Sciences
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
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
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
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
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.