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Journal of Clinical Microbiology, August 2008, p. 2766-2773, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00424-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluation and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping Vibrio parahaemolyticus: an International Multicenter Collaborative Study{triangledown}

Kai Man Kam,2* Cindy K. Y. Luey,2 Michele B. Parsons,6 Kara L. F. Cooper,6 G. B. Nair,1 M. Alam,1 M. Atiqul Islam,1 Danny T. L. Cheung,2 Y. W. Chu,2 T. Ramamurthy,3 G. P. Pazhani,3 S. K. Bhattacharya,3 H. Watanabe,4 J. Terajima,4 E. Arakawa,4 O.-A. Ratchtrachenchai,5 S. Huttayananont,5 Efrain M. Ribot,6 Peter Gerner-Smidt,6 Bala Swaminathan,6 for the Vibrio parahaemolyticus PulseNet PFGE Protocol Working Group

International Centre for Diarrhoeal Diseases Research, Dhaka, Bangladesh,1 Public Health Laboratory Centre, Kowloon, Hong Kong,2 National Institute of Cholera and Enteric Diseases, Kolkata, India,3 National Institute of Infectious Diseases, Tokyo, Japan,4 National Institute of Health, Bangkok, Thailand,5 PulseNet Methods Development and Validation Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia6

Received 3 March 2008/ Returned for modification 7 May 2008/ Accepted 3 June 2008

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.

* Corresponding author. Mailing address: Public Health Laboratory, 7/F, Public Health Laboratory Centre, Rm. 731, 382 Nam Cheong Street, Shek Kip Mei, Kowloon, Hong Kong. Phone: 852 23198302. Fax: 852 27761446. E-mail: kmkam{at}dh.gov.hk

{triangledown} Published ahead of print on 25 June 2008.

Journal of Clinical Microbiology, August 2008, p. 2766-2773, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00424-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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