GoKawiilEthics statement
Study procedures for the Bangladesh 2014–2018 surveillance study and 2% icddr,b Dhaka Hospital Surveillance study were approved by the Research Review Committee and Ethical Review Committee of icddr,b. Participants provided informed written consent for data and sample collection. The study conducted in North India was approved by the Institute Ethics Committee and the Institute Collaborative Committee of PGIMER, Chandigarh. Written informed consent was obtained from all participants before data and sample collection.
Sample collection and sequencing in Bangladesh
Study procedures for the 2014–2018 surveillance study were outlined in detail in ref. 42 and approved by the Research Review Committee and Ethical Review Committee of icddr,b. Sentinel surveillance was carried out at ten sites from 2014 to 2016, interrupted from January to May 2016 because of a gap in funding and subsequently expanded to 22 sites in 21 districts from May 2016 onwards. Informed written consent was obtained from all adult participants or from legal guardians for children younger than 18 years old. A stool sample was collected for detection of V. cholerae O1/O139. At the ten sites where surveillance was established in 2014, samples were also tested for enterotoxigenic Escherichia coli, Salmonella and Shigella species. Among 26,221 patients with acute watery diarrhoea, 6.2% (n = 1,604) were confirmed as V. cholerae O1 cases, of which 1,526 underwent whole-genome sequencing (Supplementary Fig. 7). Similarly, from the 2% icddr,b Dhaka Hospital Surveillance, in which every 50th patient visiting the icddr,b Hospital in Dhaka was enrolled and tested for enteric pathogens, 63 V. cholerae samples collected from 2022 to 2023 were also sequenced.
From the sentinel sites, stool samples were transported in Cary–Blair medium to icddr,b within 15 days of collection42. Samples were streaked directly onto taurocholate–tellurite gelatin agar and enriched in alkaline peptone water (pH 8.6) for 18 h before plating on taurocholate–tellurite gelatin agar. These were incubated overnight at 37 °C. Suspected V. cholerae colonies were serotyped using O1 Ogawa-specific, O1 Inaba-specific and O139-specific antibodies. A subsample (every fifth V. cholerae-positive culture) underwent antibiotic susceptibility testing (doxycycline, n = 311; tetracycline, n = 195; erythromycin, n = 366; azithromycin, n = 350; ciprofloxacin, n = 350; trimethoprim/sulfamethoxazole; n = 195; nalidixic acid, n = 140) using commercially available antibiotic discs (Oxoid) following the guidelines of the Clinical and Laboratory Standards Institute43. In 2017, testing for ampicillin (n = 187), ceftriaxone (n = 171) and cefixime (n = 171) was introduced. E. coli American Type Culture Collection 25922 susceptible to all antimicrobials was used as a control strain.
Genomic DNA was extracted from 5-ml cultures of V. cholerae incubated overnight at 37 °C in Luria–Bertani medium using the Wizard Genomic DNA Kit (Promega). DNA integrity was confirmed by agarose gel electrophoresis, and purity was evaluated with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). For the 2014–2018 surveillance study, 150-bp paired-end sequencing was carried out at the Wellcome Sanger Institute using the HiSeq 2500 platform (Illumina). Read data have been deposited in the European Nucleotide Archive (ENA) database under study accession ERP112767. For the 2% icddr,b Dhaka Hospital Surveillance study, paired-end sequencing was carried out using NextSeq 2000. Read data have been deposited in the ENA database under study accession ERP167534. FASTQ files were trimmed using fastp v.0.23.4, moving a sliding window from the 5′ and 3′ ends of the reads and trimming bases with a mean quality below 20. Read quality was verified using FastQC v.0.11.8 and MultiQC v.1.8.
Sample collection and sequencing in North India
This study was approved by the Institute Ethics Committee of PGIMER, Chandigarh. Stool and water samples were collected during an outbreak investigation in affected areas of Chandigarh and neighbouring states in North India. Additionally, water samples were obtained from freshwater sites, including rivers and ponds. The stool and water samples were transported in Cary–Blair medium and under a cold chain, respectively, to PGIMER, Chandigarh, for processing.
Water samples were filtered using 0.22-μm nitrocellulose acetate filters, followed by vortexing in phosphate-buffered saline to release adherent cells. Both filtered water samples (100-μl aliquots) and stool samples were enriched in alkaline peptone water at 37 °C for 6–8 h. This was followed by subculturing the enriched samples onto blood agar and thiosulfate–citrate–bile salts–sucrose agar for further incubation at 37 °C for 18–24 h. The collected samples were also tested for other enteric pathogens, such as Shigella and Salmonella. Colonies resembling V. cholerae or other enteric pathogens were identified using matrix-assisted laser desorption/ionization–time of flight. Genomic DNA was extracted as described above. High-throughput genome sequencing was carried out on the Illumina platform to generate 150-bp paired-end reads, and quality control of the sequencing data was performed as described above. Read data have been deposited in the ENA database under study accessions ERP188886 and ERP188887.
Phylogenetic analysis
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