Development of Moore Swab and Ultrafiltration Concentration and Detection Methods for Salmonella Typhi and Salmonella Paratyphi A in Wastewater and Application in Kolkata, India and Dhaka, Bangladesh

Abstract

Enteric fever is a severe systemic infection caused by Salmonella enterica serovar Typhi (ST) and Salmonella enterica serovar Paratyphi A (SPA). Detection of ST and SPA in wastewater can be used as a surveillance strategy to determine burden of infection and identify priority areas for water, sanitation, and hygiene interventions and vaccination campaigns. However, sensitive and specific detection of ST and SPA in environmental samples has been challenging. In this study, we developed and validated two methods for concentrating and detecting ST/SPA from wastewater: the Moore swab trap method for qualitative results, and ultrafiltration (UF) for sensitive quantitative detection, coupled with qPCR. We then applied these methods for ST and SPA wastewater surveillance in Kolkata, India and Dhaka, Bangladesh, two enteric fever endemic areas. The qPCR assays had a limit of detection of 17 equivalent genome copies (EGC) for ST and 25 EGC for SPA with good reproducibility. In seeded trials, the Moore swab method had a limit of detection of approximately 0.05-0.005 cfu/mL for both ST and SPA. In 53 Moore swab samples collected from three Kolkata pumping stations between September 2019 to March 2020, ST was detected in 69.8% and SPA was detected in 20.8%. Analysis of sewage samples seeded with known amount of ST and SPA and concentrated via the UF method, followed by polyethylene glycol precipitation and qPCR detection demonstrated that UF can effectively recover approximately 8 log10 cfu, 5 log10 cfu, and 3 log10 cfu of seeded ST and SPA in 5 L, 10 L, and 20 L of wastewater. Using the UF method in Dhaka, ST was detected in 26.7% (8/30) of 20 L drain samples with a range of 0.11- 2.10 log10 EGC per 100 mL and 100% (4/4) of 20 L canal samples with a range of 1.02 - 2.02 log10 EGC per 100 mL. These results indicate that the Moore swab and UF methods provide sensitive presence/absence and quantitative detection of ST/SPA in wastewater samples, and these two methods can be used jointly or separately for Salmonella Typhi environmental surveillance.

Environmental surveillance for Salmonella Typhi as a tool to estimate the incidence of typhoid fever in low-income populations

“…1 ml of the Ringers lactate solution will then be centrifuged, and the resulting pellet stored at -20°C until DNA extraction. These methods have shown to work adequately for processing Moore swabs and grab samples and for downstream detection of S. Typhi [36]. DNA extraction will be carried out using the QIAamp PowerFecal Pro kit (Qiagen) following the manufacturer's protocol, eluted into 50 µl, and stored at -20°C.…”

“…These methods have shown to work adequately for processing Moore swabs and grab samples and for downstream detection of S . Typhi [36].…”

“…At field ES sites, swabs will be fixed in place using wire or twine tied to a fixed object or stone and suspended in flowing wastewater or sewage for 48-72 hours. Upon retrieval, Moore swabs, including their attachment line, will be placed in containers containing 450 ml of Universal Pre-Enrichment (UPE) broth (BD Difco, Fisher Scientific) [36]. For the grab samples, wastewater/sewage samples will be collected using standard sample collection equipment (e.g.…”