title: Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.
abstract: The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.
authors.affiliation: Public Health Laboratory, 7/F, Public Health Laboratory Centre, Rm. 731, 382 Nam Cheong Street, Shek Kip Mei, Kowloon, Hong Kong. kmkam@dh.gov.hk
authors.forename: Kai Man
authors.initials: KM
authors.lastname: Kam
authors.forename: Cindy K Y
authors.initials: CK
authors.lastname: Luey
authors.forename: Michele B
authors.initials: MB
authors.lastname: Parsons
authors.forename: Kara L F
authors.initials: KL
authors.lastname: Cooper
authors.forename: G B
authors.initials: GB
authors.lastname: Nair
authors.forename: M
authors.initials: M
authors.lastname: Alam
authors.forename: M Atiqul
authors.initials: MA
authors.lastname: Islam
authors.forename: Danny T L
authors.initials: DT
authors.lastname: Cheung
authors.forename: Y W
authors.initials: YW
authors.lastname: Chu
authors.forename: T
authors.initials: T
authors.lastname: Ramamurthy
authors.forename: G P
authors.initials: GP
authors.lastname: Pazhani
authors.forename: S K
authors.initials: SK
authors.lastname: Bhattacharya
authors.forename: H
authors.initials: H
authors.lastname: Watanabe
authors.forename: J
authors.initials: J
authors.lastname: Terajima
authors.forename: E
authors.initials: E
authors.lastname: Arakawa
authors.forename: O-A
authors.initials: OA
authors.lastname: Ratchtrachenchai
authors.forename: S
authors.initials: S
authors.lastname: Huttayananont
authors.forename: Efrain M
authors.initials: EM
authors.lastname: Ribot
authors.forename: Peter
authors.initials: P
authors.lastname: Gerner-Smidt
authors.forename: Bala
authors.initials: B
authors.lastname: Swaminathan
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods / Q000592:standards*; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field / Q000592:standards*; D005838:Genotype; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods / Q000592:standards*; D014736:Vibrio parahaemolyticus / Q000145:classification* / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases