Environmental surveillance for typhoidal Salmonellas in household and surface waters in Nepal identifies potential transmission pathways

Introduction Salmonella Typhi and Salmonella Paratyphi, fecal-oral transmitted bacterium have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive for S . Typhi and 70% for S . Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease’s dominant pathway of transmission remains unanswered. Methods We used environmental surveillance to detect S . Typhi and Paratyphi DNA from potential sources of transmission. We collected 1L drinking water samples from a population-based random sample of households between February and October 2019 Between November 2019 and July 2021, we collected monthly 50 mL river water samples from 19 sites along the rivers leading through the Kathmandu and Kavre Districts of Nepal. We processed drinking water samples using a single qpcr and processed river water samples using differential centrifugation and qPCR at time 0 and after 16 hours of culture enrichment. A 3-cycle threshold (Ct) decrease of S . Typhi or S . Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure. Results Among 370 drinking water samples, S . Typhi was detected in 7 samples (1.8%) and S . Paratyphi A was detected in 4 (1.0%) samples. Among 381 river water samples, S . Typhi was detected in 171 (45%) and S . Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive for S . Typhi 12% of the time while samples from locations in and downstream the city had bacterial DNA detected 58% and 67% of the time respectively. Individuals were observed bathing in the rivers, washing clothes, and washing vegetables for sale in Kathmandu markets. Implications These results suggest that drinking water was not the dominant pathway of transmission of S . Typhi and S . Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems srepresent an important source of typhoid exposure in Kathmandu. Author Summary Understanding the dominant route of transmission of a pathogen is important for designing and implementing effective control strategies. Salmonella Typhi and Paratyphi which cause typhoid and paratyphoid fever infect an estimated 10 million people and kill more than 100,000 annually. In Kathmandu prior work suggested that stone spouts where people collected drinking water were contaminated and driving transmission of the diseases. However, many of these spouts no longer function, and people are still getting sick. We tested drinking water from households in this area as well as local river water and found that 13 drinking water samples were positive for S.Typhi and 15 were positive for S. Paratyphi and many river samples tested positive for these bacterium. River water samples were not often positive upstream of Kathmandu city center (12% positive for S.Typhi) but were often positive within the city center (58% positive for S.Typhi) and in rural areas up to 10 km downstream of the city (67% positive for S.Typhi). During sample collection, individuals were observed interacting with rivers by walking in them, washing clothes and washing vegetables for sale in markets. This study shows that drinking water may not be primary driver of typhoid transmission in the Kathmandu valley, but that sewage contaminated river water may be a foci of transmission into the wider population. ### Competing Interest Statement The authors declare they have no actual or potential competing financial interests. IIB consults to BlueDot, a social benefit corporation that tracks emerging infectious diseases, and to the NHL Players? Association. ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Not Applicable The details of the IRB/oversight body that provided approval or exemption for the research described are given below: We recieved IRB approval from the Stanford University IRN, Nepal Health Research Council and Dhulikhel Hospital I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Not Applicable I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Not Applicable I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Not Applicable The protocols used for this publication are available on protocols.io with links in the text. The code used for cleaning data, analysis and visualizations is availible on github and linked in the text and the data used for this publication has been uploaded to dryad . * DNA : Dideoxyribonucleic acid qPCR : quantitative polymerase chain reaction