Monitoring of diverse enteric pathogens across environmental and host reservoirs with TaqMan array cards and standard qPCR: a methodological comparison study

Rachael Lappan,Rebekah Henry,Steven L Chown,Stephen P Luby,Ellen E Higginson,Lamiya Bata,Thanavit Jirapanjawat,Christelle Schang,John J Openshaw,Joanne O'Toole,Audrie Lin,Autiko Tela,Amelia Turagabeci,Tony H F Wong,Matthew A French,Rebekah R Brown,Karin Leder,Chris Greening,David Mccarthy

doi:10.1016/s2542-5196(21)00051-6

Abstract

SummaryBackgroundMultiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmited via several pathways through human, animal, and environmental reservoirs. Individual qPCR assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan array card (TAC), which allows simultaneous detection of multiple enteropathogens, has only previously been validated in human clinical samples.MethodsIn this methodological comparison study, we compared the performance of a custom 48-singleplex TAC relative to standard qPCR. We established the sensitivity and specificity of each method for the detection of eight enteric targets, by using spiked samples with varying levels of PCR inhibition. We then tested the prevalence and abundance of pathogens in wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji using both TAC and qPCR.FindingsBoth methods exhibited similarly h specificity (TAC 100%, qPCR 94%), sensitivity (TAC 92%, qPCR 100%), and quantitation accuracy (TAC 91%, qPCR 99%) in non-inhibited sample matrices with spiked gene fragments. PCR inhibitors substantially affected detection via TAC, though this issue was alleviated by ten-fold sample dilution. Among samples from informal settlements, the two techniques performed similarly for detection (89% agreement) and quantitation (R2 0·82) for the eight enteropathogen targets. The TAC additionally included 38 other enteric targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR.InterpretationThe two techniques produced similar results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel enabling a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs could allow better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load.FundingWellcome Trust Our Planet, Our Health programme.

Highlights

Diarrhoeal disease due to inadequate sanitation and poor water quality is a major public health issue and a target of one of the UN Sustainable Development Goals (SDG 6)
We show that TaqMan array card (TAC) is similar to standard qPCR for the monitoring of enteropathogens across sample types from multiple reservoirs
Specificity was very high for both assays, with no false positives detected via TAC (100%, 16/16) and one false positive detected by qPCR (94%, 15/16)

Summary

Introduction

Diarrhoeal disease due to inadequate sanitation and poor water quality is a major public health issue and a target of one of the UN Sustainable Development Goals (SDG 6). This problem disproportionately affects lower-income and middle-income countries, especially people living in urban informal settlements.[1,2] Approximately 500 000 children under the age of 5 years die from diarrhoeal disease each year,[3,4,5] despite the potential to prevent an estimated 360 000 child deaths annually by improvements to water, sanitation, and hygiene (WASH).[6] Various non-diarrhoeal pathogens, most notably helminths, contribute to enteric disease burden and malnutrition.[7] asymptomatic or subclinical carriage of various enteropathogens impacts child growth.[8] Recent evidence has suggested that traditional household-level WASH interventions such as pit latrines, handwashing with soap, and chlorination of water deliver suboptimal reductions in enteric disease in environments that are densely populated,[9] highly contaminated,[10] or have a high prevalence of diarrhoea.[11] This finding is probably due to the inability of these interventions to address the many pathways that connect environmental enteropathogens to community residents Humans, animals, and their surrounding environments can serve as extensively interconnected reservoirs for enteropathogens. Unified one health and planetary health approaches are needed to identify pathogen exposure pathways and Lancet Planet Health 2021; 5: e297–308

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