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 high 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.

Associations Between Eight Earth Observation‐Derived Climate Variables and Enteropathogen Infection: An Independent Participant Data Meta‐Analysis of Surveillance Studies With Broad Spectrum Nucleic Acid Diagnostics

“…The etiology of the syndrome encompasses diverse microorganisms, which may be differentially sensitive to environmental conditions, yet clinically undifferentiated, with large reservoirs of asymptomatic transmission. Recent advances in broad spectrum molecular diagnostic technologies that can be deployed on stool samples from population‐based studies (Brown & Cumming, 2019 ; Lappan et al., 2021 ; Liu, Gratz, et al., 2016 ; Liu, Ochieng, et al., 2016 ), as well as climate data products that combine earth and ground‐based observations with model‐based reanalysis offer the potential to address these knowledge gaps (J. M. Colston, Ahmed, Mahopo, et al., 2018 ; J. M. Colston et al., 2019 ; Grace et al., 2015 ; M. C. Levy et al., 2019 ). This analysis is the first to bring together these two sources of data from multiple studies carried out in populations across diverse climate zones, resulting in a database of a scale and scope unprecedented in the study of enteropathogens and findings with implications for potential transmission pathways.…”

Wastewater Surveillance of U.S. Coast Guard Installations and Seagoing Military Vessels to Mitigate the Risk of COVID-19 Outbreaks

“…It is non-invasive and much less resource-intensive relative to mass screening and may be of particular use when monitoring a vaccinated population that is not regularly tested by other means. Our work shows several instances of detection of infections in asymptomatic individuals which resulted in isolation that prevented subsequent spread of the disease, highlighting the value of this important tool in combatting SARS-CoV-2 and other infectious diseases (Lappan et al, 2021; Zahedi, Monis, Deere, & Ryan, 2021).…”

Simultaneous detection of multiple pathogens with the TaqMan Array Card

“…TAC is approximately tenfold less sensitive than equivalent individual qPCR assays [2] , [3] , [4] . However, for applications where there is a need to screen samples for a substantial number of pathogens, this is outweighed by the ability to run 47 custom assays on 8 samples simultaneously.…”

“…The cards are manufactured in the USA. Further details of the custom enteropathogen TAC used in this protocol can be found in Lappan et al (2021) [3] . The primer and probe sequences from each custom assay on this card are reproduced in Supplementary material 1.…”

“…In the context of enteric pathogen detection, multiple viruses, bacteria, protists and helminths contribute to the burden of gastrointestinal disease. Monitoring an array of individual pathogens provides more detailed information on pathogen abundance and transmission than focusing on a few pathogens or relying on faecal indicator organisms to assess environmental contamination [3 , 16] . TAC enables high throughput for both target numbers and sample numbers, is rapid and straightforward to set up, and is cost-effective per pathogen.…”