| Scientific title |
Standardisation of Laboratory Protocol for Detection of Salmonella enterica in Wastewater in Low-Resource Settings |
| Public title |
Lab method for detection of Salmonella in wastewater samples |
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| Background |
Typhoid and paratyphoid fevers, caused by Salmonella enterica serovars Typhi and Paratyphi A, remain major public health concerns in South and Southeast Asia. Traditional clinical surveillance methods—primarily blood and bone marrow cultures—face limitations in sensitivity, accessibility, and cost, especially in low-resource settings. Moreover, asymptomatic carriers can shed the pathogen for extended periods, contributing silently to community transmission. Wastewater environmental surveillance (WES) offers a promising alternative by capturing population-level pathogen shedding, including from asymptomatic individuals.
Despite its potential, WES lacks a standardized laboratory protocol for detecting S. typhi and S. paratyphi A. This research addresses that gap by developing and validating a combined molecular (qPCR/dPCR) and culture-based detection method using spiked wastewater samples. The study emphasizes assay sensitivity, specificity, and feasibility in resource-limited environments. It also explores sampling techniques (grab vs. Moore swab), evaluates primer-probe performance, and determines the limit of detection. The protocol will be tested in collaboration with Gujarat Bio |
| Objectives |
he primary objective of this research is to develop and validate a standardized laboratory protocol for detecting Salmonella enterica serovars Typhi and Paratyphi A in wastewater, with a focus on applicability in low-resource settings. The study aims to establish a proof of concept by integrating both molecular (qPCR/dPCR) and culture-based methods to enhance detection accuracy, sensitivity, and feasibility. A key goal is to optimize primer-probe sets specific to S. typhi and S. paratyphi A, ensuring high specificity and minimal cross-reactivity with non-target organisms.
To achieve this, the protocol will undergo rigorous testing using reference strains, including serial DNA dilutions and spiking experiments in water and wastewater matrices. These experiments will help determine the analytical sensitivity, limit of detection (LOD), and reproducibility of the assays. The study also seeks to compare the performance of qPCR and dPCR platforms, evaluating which offers better detection thresholds and reliability in complex environmental samples.
Another important objective is to validate the protocol under simulated field conditions by spiking wastewater samples collected from urban se |
| Study Methods |
The study follows a structured laboratory-based design to develop and validate a protocol for detecting Salmonella enterica serovars Typhi and Paratyphi A in wastewater. It begins with comparative sample collection using two methods: grab sampling and simulated Moore swab (trap) sampling. Spiked samples are prepared using standard strains to mimic real-world contamination. These samples undergo a concentration step to reduce volume and enrich pathogen content, making them suitable for molecular and culture-based testing.
Testing is conducted in three phases: primer-probe optimization for qPCR/dPCR, validation with clean water spiked with known bacterial concentrations, and validation with actual wastewater samples. The assays are performed in triplicate to assess reproducibility, sensitivity, and specificity. Limit of detection (LOD) is determined through serial dilutions of DNA and bacterial cells. Culture methods are optimized using Salmonella-Shigella agar to confirm organism viability.
Data analysis includes calculating amplification efficiency, Ct values, and reproducibility metrics. Specificity is confirmed by testing against non-target organisms. The study is conducted entirely in a controlled lab setting, with biosafety protocols in place. Findings will inform future field validation and broader implementation in low-resource environments. The study runs from September 2025 to March 2026, with distinct phases for preparation, experimentation, and reporting. |
| Expected outcomes and use of results |
The expected outcome of this study is the development of a validated, standardized laboratory protocol for detecting Salmonella enterica serovars Typhi and Paratyphi A in wastewater, specifically designed for low-resource settings. By integrating molecular (qPCR/dPCR) and culture-based methods, the protocol aims to achieve high sensitivity, specificity, and reproducibility. The study will establish the limit of detection (LOD) for both clean water and complex wastewater matrices, ensuring the method is robust across varied environmental conditions.
Results will include optimized primer-probe sets, validated assay conditions, and performance metrics such as amplification efficiency and intra-assay precision. The protocol will demonstrate reliable detection of target organisms in spiked samples, with minimal cross-reactivity against non-target bacteria. Additionally, the study will confirm the viability of Salmonella through selective culture techniques. These findings will serve as proof of concept, laying the groundwork for future field validation and broader implementation. The research will also produce a scalable budget and operational framework, enabling adaptation in resource-limited environments and expansion to other surveillance sites. Ultimately, the protocol will support early disease detection, inform public health interventions, and enhance environmental surveillance capacity for enteric pathogens. |
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| Keywords |
salmonella, typhoid, wastewater surveillance |
