Field Evaluation of a Low-cost, Simplified Method to Detect and Characterize Toxigenic V. cholerae O1 from Environmental Water in Lagos, Nigeria

Cholera is caused by toxigenic Vibrio cholerae serogroups O1 or O139 and is transmitted through contaminated water or food. Conventional detection methods for V. cholerae in environmental water samples, including membrane filtration and culture, typically require about three days, specialized equipment, and technical expertise. A simple and rapid method for detecting the V. cholerae O1 in water could support outbreak prevention, environmental surveillance, and studies of persistence and transmission. Here, we describe a low-cost, streamlined method for detecting toxigenic V. cholerae O1 in environmental water which can detect V. cholerae O1 within 24 hours. We filter one liter of the water through gauze, incubate the gauze in alkaline peptone water (APW) overnight at 37?C, and test the APW sample with a serogroup O1-specific rapid diagnostic test (RDT). To preserve nucleic acid material from APW broth, we place two drops on each circle of a filter paper (Whatman 903), dry it, and later analyze the DNA using PCR and whole genome sequencing. We evaluated this method using 95 environmental samples collected from 17 sites in Lagos, Nigeria. 54 samples were RDT positive and each of these samples were confirmed by culture and by PCR targeting ompW and ctxA as toxigenic V. cholerae O1. Whole-genome sequencing identified these isolates as belonging to the AFR12 lineage. Among the RDT-negative samples, PCR detected 24 samples positive for ompW but negative for ctxA, consistent with non-toxigenic non-O1 V. cholerae, and 11 of these were further identified as non-toxigenic serogroup O139 by qPCR using a commercial assay (Bioperfectus). The cost for the RDT test was <$2.50 per water sample. This low-cost APW enrichment and RDT-based approach enables identification of environmental water contaminated with toxigenic V. cholerae O1 within 24 hours and enables the collection of samples useful for further bacterial molecular characterization.