A novel SNP-based multiplex PCR profiling divulges contemporary genetic patterns in Haitian variant Vibrio cholerae O1 clinical isolates

ABSTRACT The massive cholera outbreak in Haiti in 2010 has drawn the attention of both medical professionals and public health officials worldwide. The hypervirulent Vibrio cholerae O1 El Tor strains responsible for this outbreak were not only confined to Haiti but also spread quickly to other cholera-prone regions. Therefore, tracking and understanding these strains in endemic areas have become crucial for timely and effective treatment strategies. The presence of specific genetic mutations in key virulence-related genes —ctxB (cholera enterotoxin subunit B), tcpA (toxin-coregulated pilin), and rtxA (repeats-in-toxin)—makes these Haitian strains distinctive. These genes contain single nucleotide polymorphisms, which act as unique markers for identifying the Haitian variant. However, current detection methods using allele-specific simplex PCRs are time-consuming and can delay critical outbreak responses. To address this, we developed new Haitian allele-specific primers targeting rtxA and tcpA and combined them with an existing ctxB -specific PCR into a single multiplex PCR assay. We tested this new multiplex PCR on 250 clinical V. cholerae O1 isolates collected from diarrheal patients in Kolkata, India, between 2002 and 2019. Interestingly, we noticed that the first appearance of Haitian tcpA, rtxA, and ctxB alleles was in 2003, 2004, and 2006, respectively, years before the Haitian outbreak occurred. Our results matched perfectly with the conventional simplex PCRs and demonstrated 100% sensitivity and specificity. Given the global spread of these Haitian-like V. cholerae strains, this multiplex PCR provides a fast, reliable tool for identifying cholera infection and gaining valuable epidemiological insights. IMPORTANCE Timely identification of pathogens causing life-threatening diseases is vital in clinical and public health contexts. The emergence and global spread of Haitian Vibrio cholerae O1 El Tor strains during cholera outbreaks highlight the urgent need for rapid and reliable diagnostics. To address this, we developed a novel, highly sensitive multiplex PCR assay that delivers results within 2 h from identified isolates.This assay enables efficient detection and differentiation of Haitian V. cholerae strains. Extensive evaluation with a large panel of isolates confirmed its effectiveness in rapid identification and provided valuable insights into the temporal evolution of key virulence-associated genes. Specifically, the assay targets key genetic markers ctxB , tcpA , and rtxA , which are critical for monitoring shifts in virulence and transmission potential. Our findings establish this multiplex PCR as a robust tool for surveillance and diagnosis, especially valuable in outbreak settings where timely intervention is paramount.