Field Evaluation of a Low-cost, Simplified Method to Detect and Characterize Toxigenic V. cholerae O1 from Environmental Water in Lagos, Nigeria
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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.
Author Summary
Standard methods to identify water samples contaminated with V. cholerae serotype O1 are commonly used, but their use for public health practice has been limited due cost, time, and resource requirements. This study describes a convenient, efficient and inexpensive method to detect V. cholerae O1 from environmental water samples and evaluates this method using water samples collected from 17 sites in Lagos, Nigeria during 2025. The method involves filtering one liter of water through a gauze pad, followed by overnight incubation of the gauze in alkaline peptone water (APW) enrichment broth. The APW is then tested for cholera using a rapid diagnostic test (RDT) specific for V. cholerae O1. Among 95 environmental water samples, 54 were positive for V. cholerae O1 using the RDTs. This method provides results within one day at a cost of <$2.50 USD per sample. If there is need for additional studies, RDT positive samples can be sent for culture, and nucleic acid from the APW can be saved by spotting the APW on filter paper. The filter paper can then be stored at room temperature indefinitely. All of the RDT positive samples were confirmed by culture and PCR Among the PCR negative samples, 24 samples were PCR positive for non-O1 V. cholerae, including 11 that were positive for non-toxigenic V. cholerae O139 by qPCR. Whole genome sequencing of the samples found the RDT positive samples to be of the AFR12 lineage, and the DNA contained many genetic markers of antibiotic resistance. Identifying cholera contaminated water quickly provides information to health authorities which can facilitate interventions to reduce transmission and can also provide samples for further epidemiologic and molecular studies.
