Typhoid fever remains a global public health challenge, especially in low- and middle-income countries where poor sanitation and limited access to clean water facilitate transmission. The lack of data on disease burden poses a significant barrier to adopting effective interventions such as vaccination. We developed a novel colorimetric assay for the detection of S. Typhi-specific bacteriophages in environmental water samples, providing an indirect indicator of S. Typhi contamination and insights into typhoid burden. We collected water samples from Brazil, Côte d'Ivoire, Nepal, and Niger, covering urban, peri-urban, and rural areas. We evaluated the colorimetric assay efficiency against agar overlay assay. Isolated phages were tested against various bacteria to assess host range. The colorimetric assay demonstrated high sensitivity (100% concordance with double agar overlay) with a detection limit of 28 PFU/mL, and results were obtained in 5.5 hours. Phage detection rates were highest in densely populated areas with poor sanitation, particularly in Nepal (96.6%) and Côte d’Ivoire (35.1%). The detection of S. Typhi phages in Côte d’Ivoire is particularly important, as the burden of typhoid in the region was previously undocumented. Host range analyses demonstrated that the isolated phages were specific to S. Typhi, with a small minority of phages isolated also capable of infecting S. Paratyphi A. The novel colorimetric assay offers a rapid and sensitive method for detecting S. Typhi bacteriophages in environmental water. The scalability, low cost (~$2.40 USD per sample), and minimal equipment requirements, suggest that this could be effective tool for typhoid surveillance in resource-limited settings.

IIB consults to the Weapons Threat Reduction Program at Global Affairs Canada. The other authors have no potential conflicts of interest.

This work was supported by the Bill and Melinda Gates Foundation (OPP 1217093) and the Stanford Center for Innovation in Global Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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