Typhinder: Rapid, low-cost colorimetric detection ofSalmonellaTyphi bacteriophages for environmental surveillance
Authors: Da Silva Kesia Esther , Yokoyama Tuya , Naga Shiva Ram , Maharjan Mamata , Pereira dos Santos Paulo César , Fisher Karla , Coulibaly Jean T. , Yang Max Zhang , Nelson Eric Jorge , Charles Richelle C , Shafer Katherine , Igiraneza Brad-Lot , Yusuf Samaila , Mulder Elisabeth , Neuzil Kathleen , Bogoch Isaac I. , Shrestha Rajeev , Tamrakar Dipesh , Andrews Jason R. 
Published: January 2025

AbstractTyphoid 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 programs. We developed a novel colorimetric assay for the detection ofS. Typhi-specific bacteriophages (phages) in environmental water samples, providing an indirect indicator ofS.Typhi contamination and insights into typhoid burden. We collected surface 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 plaque assay. Isolated phages were tested against various bacteria to assess their host range. The colorimetric assay demonstrated high sensitivity (100% concordance with double agar overlay) with a detection limit of 28 plaque-forming units per milliliter (PFU/mL), and results were obtained in 5.5 hours. Phage detection rates were highest in densely populated areas with poor sanitation, particularly in Kathmandu, Nepal (96.6% positivity in river samples) and Abidjan, Côte d’Ivoire (35.1% positivity in drainage samples). The detection ofS. Typhi phages in Côte d’Ivoire is particularly important, as the burden of typhoid in the region was previously undocumented. Phages were not detected in rural and drinking water sources. Host range analyses demonstrated that the isolated phages were specific toS.Typhi, with a small minority of phages (4/30) isolated also capable of infectingS. Paratyphi A. The novel colorimetric assay offers a rapid and sensitive method for detectingS.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.IMPORTANCETyphoid fever, caused bySalmonellaTyphi, remains a significant global health threat, particularly in low-resource settings with inadequate sanitation. Effective control measures, such as vaccines, require precise data on where typhoid is most prevalent, yet current surveillance methods are expensive and limited in scope. This study introduces a rapid, low-cost, and scalable colorimetric assay for detectingS.Typhi bacteriophages (viruses that infect bacteria) in environmental water samples. Unlike traditional methods, this test detects the presence ofS.Typhi indirectly by identifying associated phages, offering a specific and sensitive approach for monitoring typhoid fever circulation. The assay was validated in diverse settings across four countries and demonstrated high accuracy and cost efficiency. By reducing reliance on expensive laboratory equipment and complex procedures, this tool makes typhoid surveillance more accessible, especially in low-income regions, helping prioritize vaccination campaigns and improve public health interventions.