Evaluation of a rapid dipstick test for identifying cholera cases during the outbreak.

Background & objectives: Intermittent cholera outbreaks are major problem in many of the states of India. It is essential to identify cholera at the earliest for timely mobilization of public health responses and to abort the outbreaks. The present study was a part of a diarrhoeal outbreak investigation in Secunderabad, India, during May 2009 where the usefulness of Crystal VC rapid dipstick kit was assessed for detecting the aetiologic agent of the outbreak. Methods: Stool specimens were collected from 15 hospitalized patients with acute watery diarrhoea and analyzed for detection of cholera vibrios using Crystal VC rapid dipstick kit and the usefulness of the kit was determined by comparative analysis of the same set of specimens using both microbiological and real-time PCR (RT-PCR) based assays. Results: Detection of Vibrio cholerae O1 from 10 of 15 specimens was recorded using dipstick assay. Microbiological methods detected V. cholerae O1 positivity among 11 specimens. However, RT-PCR based assay showed all 15 specimens positive for the presence of V. cholerae O1. In addition, the same assay showed that the pathogen load in the dipstick as well as RT-PCR positive specimens ranged from 106 colony forming units (cfu)/ml or more. Interpretation & conclusions: Crystal VC kit had the potential to identify cholera cases in 10 min in field conditions without having good laboratory support. Therefore, dipstick kit may be considered as cholera detecting tool in diarrhoeal outbreak investigations. Specimens from clinically typical cholera cases, if negative by dipstick, should be reanalyzed by culture based methods.

Biotyping of Vibrio cholerae O1: time to redefine the scheme.

Considering the recent emergence of "hybrid biotype" and "El Tor variant", we propose to redefine the biotyping scheme for Vibrio cholerae O1 serogroup. The existing biotyping scheme has limitations and causes confusion as many of the hybrid biotype and El Tor variant strains have phenotypic and genetic changes. A revised biotyping scheme will play a significant role to understand the ecology, epidemiology and nature of infection of V. cholerae O1 strains in future.

Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.

Cluster-analysis & patterns of dissemination of multidrug resistance among clinical strains of Vibrio cholerae in Calcutta, India.

BACKGROUND & OBJECTIVES: Antimicrobial resistance among Vibrio cholerae has been monitored for several years in Calcutta. To investigate the changing trends in multidrug resistance (MDR) among different serogroups of V. cholerae and to perform software assisted cluster analysis the current study was undertaken. METHODS: Strains isolated from patients with cholera and "cholera-like" diarrhoea admitted in the Infectious Diseases Hospital, Calcutta were analysed. Eight hundred and forty V. cholerae strains isolated from 1992 through 1997 were tested for susceptibility to 11 antibiotics. Cluster analysis was done using SPSS software. RESULTS: Most of the strains exhibited MDR with fluctuating trends as the resistance profile diverged each year. A total of 119 different resistance profiles exhibited by V. cholerae O1, O139 and non-O1, non-O139 serogroups were analysed by cluster combination method. During 1993 and 1994, 53 per cent of V. cholerae O139 and 82 per cent of V. cholerae O1 serogroups, respectively, exhibited maximal number of new resistance patterns. The frequency of new resistance patterns among V. cholerae non-O1, non-O139 was constantly high (33-47%) during 1995 to 1997. INTERPRETATION & CONCLUSIONS: With a few exceptions, preponderance of the resistance profiles was generally not confined to any serogroup. The cluster analysis depicted dissemination of some of the resistance patterns commonly found among V. cholerae non-O1, non-O139 belonging to different serogroups to the O139 serogroup in the succeeding years. In this study we have shown that the V. cholerae strains are resistant to several antibiotics with constant change in the MDR profiles. It is imperative to define the susceptibility pattern of the strains to determine the effective drug of choice for the treatment of cholera.