{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T13:59:38Z","timestamp":1789999178563,"version":"4.0.1"},"reference-count":51,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP21wm0125004"],"award-info":[{"award-number":["JP21wm0125004"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP23wm0225021"],"award-info":[{"award-number":["JP23wm0225021"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Department of Science and Technology, Ministry of Science and Technology","award":["IF110706"],"award-info":[{"award-number":["IF110706"]}]},{"DOI":"10.13039\/501100001412","name":"Council of Scientific and Industrial Research, India","doi-asserted-by":"publisher","award":["09\/482(0060)\/2014-EMR-I"],"award-info":[{"award-number":["09\/482(0060)\/2014-EMR-I"]}],"id":[{"id":"10.13039\/501100001412","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The massive cholera outbreak in Haiti in 2010 has drawn the attention of both medical professionals and public health officials worldwide. The hypervirulent\n                      <jats:italic toggle=\"yes\">Vibrio cholerae<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">\u2014ctxB<\/jats:italic>\n                      (cholera enterotoxin subunit B),\n                      <jats:italic toggle=\"yes\">tcpA<\/jats:italic>\n                      (toxin-coregulated pilin), and\n                      <jats:italic toggle=\"yes\">rtxA<\/jats:italic>\n                      (repeats-in-toxin)\u2014makes 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\n                      <jats:italic toggle=\"yes\">rtxA<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">tcpA<\/jats:italic>\n                      and combined them with an existing\n                      <jats:italic toggle=\"yes\">ctxB<\/jats:italic>\n                      -specific PCR into a single multiplex PCR assay. We tested this new multiplex PCR on 250 clinical\n                      <jats:italic toggle=\"yes\">V. cholerae<\/jats:italic>\n                      O1 isolates collected from diarrheal patients in Kolkata, India, between 2002 and 2019. Interestingly, we noticed that the first appearance of Haitian\n                      <jats:italic toggle=\"yes\">tcpA, rtxA,<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">ctxB<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">V. cholerae<\/jats:italic>\n                      strains, this multiplex PCR provides a fast, reliable tool for identifying cholera infection and gaining valuable epidemiological insights.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Timely identification of pathogens causing life-threatening diseases is vital in clinical and public health contexts. The emergence and global spread of Haitian\n                      <jats:italic toggle=\"yes\">Vibrio cholerae<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">V. cholerae<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">ctxB<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">tcpA<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">rtxA<\/jats:italic>\n                      , 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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02661-25","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T13:00:19Z","timestamp":1789995619000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A novel SNP-based multiplex PCR profiling divulges contemporary genetic patterns in Haitian variant\n                    <i>Vibrio cholerae<\/i>\n                    O1 clinical isolates"],"prefix":"10.1128","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4612-192X","authenticated-orcid":true,"given":"Prosenjit","family":"Samanta","sequence":"first","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priyanka","family":"Ghosh","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1830-1920","authenticated-orcid":true,"given":"Goutam","family":"Chowdhury","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]},{"name":"Collaborative Research Centre of Okayama University for Infectious Diseases, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sreeja","family":"Shaw","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arindam","family":"Naha","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deboleena","family":"Roy","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayumu","family":"Ohno","sequence":"additional","affiliation":[{"name":"Collaborative Research Centre of Okayama University for Infectious Diseases, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3718-3878","authenticated-orcid":true,"given":"Shin-ichi","family":"Miyoshi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pc6pc55","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University","place":["Okayama, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7999-5036","authenticated-orcid":true,"given":"Thandavarayan","family":"Ramamurthy","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5638-4520","authenticated-orcid":true,"given":"Asish Kumar","family":"Mukhopadhyay","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections","place":["Kolkata, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,21]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.eclinm.2024.103044"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.dcit.2025.100042"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1136\/bmjgh-2024-016168"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrg.2017.105"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aao2136"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1012928"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","unstructured":"Sayeed M Nabil IK Bhattacharjee P Hossain M Akter NJ Akter R Kelley KL Karim M Begum YA Bhuiyan TR Qadri F Khan AI Nelson EJ. 2024. Assembly and performance of a cholera RDT prototype that detects both Vibrio cholerae and associated bacteriophage as a proxy for pathogen detection. Immunology. doi:10.1101\/2024.08.23.609438","DOI":"10.1101\/2024.08.23.609438"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.000783"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid1707.110059"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(22)00330-0"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid2111.150762"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(12)60436-X"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/s0140-6736(03)15328-7"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1080\/29986990.2025.2469919"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature10392"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1207359109"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00387-12"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0112973"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.03042-13"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid1711.110805"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00040-10"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00253-16"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.168.4.1487"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1006\/mpat.1993.1091"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(93)90588-t"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004787"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.272.5270.1910"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-695X.2009.00590.x"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2009.10.003"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.03162-12"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0008128"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00624-12"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.12879"},{"key":"e_1_3_3_35_2","doi-asserted-by":"publisher","DOI":"10.3389\/fpubh.2019.00203"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0002049"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.meegid.2014.03.020"},{"key":"e_1_3_3_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.meegid.2017.06.015"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01227-20"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aad5901"},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10529-005-4688-z"},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijid.2018.06.021"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.4103\/0301-4738.37595"},{"key":"e_1_3_3_44_2","doi-asserted-by":"publisher","DOI":"10.1002\/9780471729259.mc06a05s26"},{"key":"e_1_3_3_45_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.20-1280"},{"key":"e_1_3_3_46_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2016.01.017"},{"key":"e_1_3_3_47_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-102-4_2"},{"key":"e_1_3_3_48_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00831-18"},{"key":"e_1_3_3_49_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.8.1.48"},{"key":"e_1_3_3_50_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0818-3"},{"key":"e_1_3_3_51_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMc2004773"},{"key":"e_1_3_3_52_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00080-20"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.02661-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.02661-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T13:00:20Z","timestamp":1789995620000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.02661-25"}},"subtitle":[],"editor":[{"given":"Kayvan","family":"Zainabad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,21]]},"references-count":51,"alternative-id":["10.1128\/spectrum.02661-25"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.02661-25","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,21]]},"assertion":[{"value":"2025-08-26","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-14","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-21","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e02661-25"}}