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 and 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, 5, and 3 log10 cfu of seeded ST and SPA in 5, 10, 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.

Evaluation of Sampling and Concentration Methods for Salmonella enterica Serovar Typhi Detection from Wastewater

“…Some samples are filtered through an electropositive ViroCap filter, 0.45-μm membrane filters, or ultrafilters; other methods include centrifugation. 12 , 18 – 22 , 26 , 29 Trap sample methods for Salmonella Typhi have historically used the Moore swab method. 30 , 31 The Moore swab, or Moore cotton tampon, is suspended into the water source for up to 6 days 29 , 32 – 37 to allow for bacteria flowing through water to adsorb on the swab, but not for large debris to get trapped.…”

Evaluation of sampling and concentration methods for Salmonella enterica serovar Typhi detection from wastewater

“…After processing, samples are analyzed using culture-based detection methods and/or quantitative polymerase chain reaction (qPCR). Although culture-based methods have historically been used to isolate Salmonella Typhi, recovery has been inconsistent and culturing is challenging; therefore, qPCR is increasingly used for detection of Salmonella Typhi in wastewater [10,29,38]. Further, when enrichment is not used in sample processing (unless most probable number methodologies are employed), qPCR enables quantification of the organism’s concentration in the original sample and allows for detection of viable but not culturable Salmonella Typhi, whereas culture confirms viability of organism and permits comparative genomic analysis.…”

“…Sample concentration enables a larger volume of wastewater or wastewater-impacted water to be assayed, which is important when low concentrations are anticipated such as is typically the case in environmental samples. Methods for sample concentration also varysome filter samples through an electropositive ViroCap filter, 0.45-μm membrane filters, or ultrafilters; other methods include centrifugation [12,[18][19][20][21][22]26,29]. Trap sample methods for Salmonella Typhi have historically utilized a Moore swab method [30,31].…”

“…Trap sample methods for Salmonella Typhi have historically utilized a Moore swab method [30,31]. The Moore swab, or Moore cotton tampon is suspended into the water source for up to 6 days [29,[32][33][34][35][36][37] to allow for bacteria flowing through water to adsorb on the swab, but not for large debris to get trapped. Swabs are then enriched using different growth/enrichment mediums.…”

Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

“…ipaH-P FAM-AACAGGTCGCTGCATGGCTGGAA-BHQ1 10 −5 ng/µL for genomic DNA templates, 10 −1 CFU/mL for Shigella bacteria culture [stgA-R TATCCGCGCGG TTGTAAAT stgA-P FAM-CCATTACAG CATCTGGCGTAGCGA-BHQ1 0.05-0.005 CFU/mL of wastewater[176] …”