Detection and confirmation of toxigenic &lt;i&gt;Vibrio cholerae&lt;/i&gt; O1 in environmental and clinical samples by a direct cell multiplex PCR

Jp Yadava,Ak Goel,M Jain

doi:10.4314/wsa.v39i5.4

Abstract

Epidemic cholera caused by toxigenic Vibrio cholerae O1 is a major health problem in several developing countries. Traditional methods for identifying V. cholerae involve cultural, biochemical and immunological assays which are cumbersome and often take several days to complete. In the present study, a direct cell multiplex PCR was developed targeting the ompW, ctxB and rfbO1 genes for confirmation of V. cholerae , its toxigenicity and serogroup O1, respectively from clinical and environmental samples. The detection sensitivity of the multiplex PCR was 1.9 x 10 3 V. cholerae per PCR reaction. A total of 31 environmental samples and 45 clinical V. cholerae isolates from different outbreaks were examined by the PCR. The assay was simple and specific, as there was no requirement for DNA extraction and no amplification was observed with other homologous bacteria used. The assay can be very useful for rapid surveillance of toxigenic V. cholerae O1 in environmental water samples, as well as for confirmation of clinical isolates.

Highlights

Cholera is an epidemic diarrheal disease that continues to devastate many countries where primary health and sanitation is still a challenge
We have developed a simple, sensitive and specific multiplex PCR that confirms the presence of V. cholerae in environmental and clinical samples, and exhibits its epidemic potential
The reference strain of Vibrio cholerae 20 O1 Ogawa was procured from the National Institute of Cholera and Enteric Disease (NICED), Kolkata, India, and was maintained on Brain Heart Infusion (BHI) agar (Difco, USA) at 22 oC

Summary

Introduction

Cholera is an epidemic diarrheal disease that continues to devastate many countries where primary health and sanitation is still a challenge. Several cholera episodes are reported from developing countries due to contamination of water supplies. In 2010, a total of 48 countries reported 317 534 cases of cholera to the WHO, of which 36% were reported from Africa and 56.6% from the Americas, where a large outbreak was reported in Haiti (WHO, 2011). The true number of cholera cases may be much higher because many cholera cases remain unreported. Cholera is caused by Vibrio cholerae, which is primarily an inhabitant of aquatic environments (Faruque et al, 1998, Islam et al, 1994). Monitoring the presence of V. cholerae in drinking water sources prior to disease outbreak is important to enable effective resource management and public health protection (Choopun et al, 2002)

Methods

Results

Conclusion