Development and validation of a high-throughput qPCR platform for the detection of soil-transmitted helminth infections

Background Historically, soil-transmitted helminth (STH) control and prevention strategies have relied on mass drug administration efforts targeting preschool and school-aged children. While these efforts have succeeded in reducing morbidity associated with STH infection, recent modeling efforts have suggested that expanding intervention to treatment of the entire community could achieve transmission interruption in some settings. Testing the feasibility of such an approach requires large-scale clinical trials, such as the DeWorm3 cluster randomized trial. In addition, accurate interpretation of trial outcomes will require diagnostic platforms capable of accurately determining infection prevalence, particularly as infection intensity is reduced, at large population scale and with significant throughput. Here, we describe the development and validation of a multi-site, high-throughput molecular testing platform. Methodology/Principal Findings Through the development, selection, and validation of appropriate controls, we have successfully created and evaluated the performance of a testing platform capable of the semi-automated, high-throughput detection of four species of STH in human stool samples. Comparison of this platform with singleplex reference assays for the detection of these same pathogens has demonstrated comparable performance metrics across multiple testing locations, with index assay accuracy measuring at or above 99.5% and 98.1% for each target species at the level of the technical replicate and individual extraction respectively. Through the implementation of a rigorous validation program, we have developed a diagnostic platform capable of providing the necessary throughput and performance needed to meet the needs of the DeWorm3 cluster randomized trial and other large-scale operational research efforts for STH. Importance Current models predict that an expansion of intervention efforts to include community-wide treatment strategies could, in certain geographies, result in the elimination of soil-transmitted helminths as a public health problem. Large-scale operational research efforts to reduce the burden of STH such as the DeWorm3 cluster randomized trial have been organized to evaluate these predictions, but such evaluations require a high throughput testing strategy capable of providing sensitive, specific, and accurate results. To meet this need, we have developed a multiplexed diagnostic platform that was rigorously evaluated across multiple testing locations using a novel validation plan with pre-defined performance characteristics. Our successful validation of this platform has provided the community with a tool capable of meeting the diagnostic requirements of large-scale operational research efforts. Furthermore, this strategy provides a blueprint for the development of similar platforms adaptable for use with other neglected tropical disease programs evaluating other intervention strategies. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by a Bill and Melinda Gates Foundation investment to the University of Washington (OPP1129535, PI JLW). A prior award to the Natural History Museum, London, also from the Bill and Melinda Gates Foundation (INV-030049), funded a portion of the work described. There is no involvement of the funders in final decisions regarding the study design and trial procedures or decision to publish the manuscript or in data collection, analysis or publication of study results. The funders reviewed the final decisions regarding study design and trial procedures. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Ethics Committee for Health Research of the Institut de Recherche Clinique au Benin gave ethical approval for this work (002-2017/ CNERS-MS). The College of Medicine Research Ethics Committee at the London School for Hygiene and Tropical Medicine (P.04/17/2161) The Institutional Review Board of the Christian Medical College, Vellore, India gave ethical approval for this work (10392). The Human Subjects Division at the University of Washington gave ethical approval for this work (STUDY00000180). 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Yes All data produced in the present work are contained in the manuscript and supplement.