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

medRxiv

Abstract

Abstract 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.

Authors 19

1. Nils Pilotte

   Quinnipiac University

   Affiliation as printed

   Department of Biological Sciences, Quinnipiac University, Hamden, CT, USA

2. Victor Omballa

   University of Washington

   Affiliation as printed

   DeWorm3 Project, Seattle, USA

   Department of Global Health, University of Washington, Seattle, USA

   DeWorm Project, Seattle, USA

3. Leah R. Padgett
   Affiliation as printed

   Quantigen, Fishers, Indiana, USA

4. Malathi Manuel

   Christian Medical College, Vellore

   Affiliation as printed

   The Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore, India

5. Jeanne L. Goodman

   University of Washington

   Affiliation as printed

   DeWorm3 Project, Seattle, USA

   Department of Global Health, University of Washington, Seattle, USA

   DeWorm Project, Seattle, USA

6. D. Timothy J. Littlewood

   Natural History Museum

   Affiliation as printed

   Department of Life Sciences, Natural History Museum, London, UK

7. Zayina Zondervenni Manoharan

   Christian Medical College, Vellore

   Affiliation as printed

   The Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore, India

8. Lisette van Lieshout Aachen Department of Parasitology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

9. Jaco J. Verweij

   Elisabeth-TweeSteden Ziekenhuis

   Affiliation as printed

   Microvida, Laboratory for Medical Microbiology and Immunology, St. Elisabeth Ziekenhuis, Tilburg, The Netherlands

10. Ajith Kumar Muthukumar

    Christian Medical College, Vellore

    Affiliation as printed

    The Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore, India

11. Gretchen Walch
    Affiliation as printed

    Quantigen, Fishers, Indiana, USA

12. Andrew Gonzalez

    Smith College

    Affiliation as printed

    Department of Biological Sciences and Biochemistry, Smith College, Northampton, MA, USA

13. Sean R. Galagan

    University of Washington

    Affiliation as printed

    DeWorm3 Project, Seattle, USA

    Department of Global Health, University of Washington, Seattle, USA

    DeWorm Project, Seattle, USA

14. Sitara Swarna Rao Ajjampur

    Christian Medical College, Vellore

    Affiliation as printed

    The Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore, India

15. Moudachirou Ibikounlé

    Université d'Abomey-Calavi · Institut de Recherche Clinique du Bénin

    Affiliation as printed

    Centre de Recherche pour la lutte contre les Maladies Infectieuses Tropicales (CReMIT/TIDRC), Université d’Abomey-Calavi, Abomey-Calavi, Bénin

    Institut de Recherche Clinique du Bénin, Abomey-Calavi, Benin

    Centre de Recherche pour la lutte contre les Maladies Infectieuses Tropicales (CReMIT/TIDRC), Université d'Abomey-Calavi, Abomey-Calavi, Bénin

16. Steven A. Williams

    Smith College

    Affiliation as printed

    Department of Biological Sciences and Biochemistry, Smith College, Northampton, MA, USA

17. Doug Rains
    Affiliation as printed

    Quantigen, Fishers, Indiana, USA

18. Ushashi Chand Dadwal
    Affiliation as printed

    Quantigen, Fishers, Indiana, USA

19. Judd L. Walson corresponding

    Johns Hopkins University · University of Washington

    Affiliation as printed

    DeWorm3 Project, Seattle, USA

    Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA

    Departments of Global Health, Medicine (Infectious Disease), Pediatrics and Epidemiology, University of Washington, Seattle, USA

    DeWorm Project, Seattle, USA