Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water

Development of Environmental Surveillance Methods for the Detection of Salmonella enterica serovar Typhi in Wastewater

Thesis (Master's)--University of Washington, 2020Typhoid fever continues to be a leading cause of global morbidity and mortality, especially in developing countries with limited access to safe drinking water and adequate sanitation. Environmental surveillance – the process of examining samples contaminated with fecal matter for the presence of pathogens - is a useful tool for monitoring the circulation of Typhoid fever in populations. Many common methods for the detection of Salmonella enterica serovar Typhi (S. Typhi) in environmental samples benefit from an enrichment step. Selenite-based media - the enrichment media commonly recommended for S. Typhi - poses occupational and environmental health concerns. This study proposes an alternative enrichment media that does not contain hazardous material. Additionally, this new enrichment media outperforms Selenite F media for the enrichment of both pure cultures of S. Typhi and wastewater cultures seeded with S. Typhi. This study also reports the estimated limit of detection for the combination of a common wastewater sampling method – membrane filtration – with an enrichment step utilizing this new media. Finally, this study presents a dynamic computational model, designed to predict the concentration of S. Typhi bacteria and the probability of detection of S. Typhi at various sampling locations in a wastewater system. This model is utilized in conjunction with a quantitative microbial risk model to estimate the occupational risk of contracting Typhoid fever for environmental samplers handling contaminated wastewater in the field