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

Abstract

Background: Multiple 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.

Methods: In 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.

Findings: Both 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.

Interpretation: The 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.

Funding: Wellcome Trust Our Planet, Our Health programme.

Figure 1

Quantitation of spiked genetic material in nuclease-free water by TAC and standard qPCR Ten different combinations of spiked material were tested in a randomised double-blinded manner. Figure shows samples spiked randomly in different combinations (samples 1, 3, 4, 6, 9, 10); those spiked at consistent concentrations of 10 copies per μL (sample 7), 100 copies per μL (sample 2), or 1000 copies per μL (sample 8); or not spiked at all (sample 5; a blank control). For each target, the quantity of material spiked (white circle), the copies detected by standard qPCR (blue circle), and the copies detected by TAC (yellow circle) are shown. TAC=TaqMan array card. EPEC=enteropathogenic Escherichia coli. STEC=Shiga toxin-producing Escherichia coli.

Figure 2

Concordance between standard qPCR and TAC in detecting pathogens in animal scats, child stool, soil, and water collected from informal settlements of Suva, Fiji Agreement between the methods with respect to the number of positive detections of targets (A) and the measured target quantity in log₁₀ gene copies per μL of extracted DNA (with a pseudocount of 1 added before log₁₀ transformation; (B). The regression lines with associated 95% CIs are shown for the subset of data where a target was quantified by both methods (blue, R² 0·815). Across all datapoints, R² 0·668 (grey). TAC=TaqMan array card. EPEC=enteropathogenic Escherichia coli. STEC=Shiga toxin-producing Escherichia coli.

Figure 3

Pathogen and indicator targets detected via TAC in Melbourne wastewater samples and animal scats, child stool, soil, and water collected from informal settlements of Suva, Fiji Heatmaps represent the prevalence (percentage of positive samples [A]) and abundance (mean value of log₁₀ gene copies per ng of DNA across positive samples [B]) of each target by sample type. White represents a zero value, and 18S rRNA quantitation was unavailable. The number of pathogens or indicators detected per sample is represented by histograms (C), also by sample type. This excludes the 16S rRNA and 18S rRNA targets and counts pathogens with multiple gene targets (ie, Campylobacter spp, Shigella spp, EAEC, ETEC, STEC, EPEC and Entamoeba spp) only once. TAC=TaqMan array card. EAEC=enteroaggregative Escherichia coli. ETEC=enterotoxigenic Escherichia coli. STEC=Shiga toxin-producing Escherichia coli. EPEC=enteropathogenic Escherichia coli.