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Background: We evaluated the results of examining a single faecal sample for gastrointestinal parasites (GIP) using a combination of traditional methods with multiplex qPCR for helminths and protozoa, compared to a reference standard of examining three faecal samples from each person using traditional diagnostic methods alone., Methods: Three faecal samples were collected at weekly intervals from 596 healthy Nepalese men. Each sample underwent formalin-ethyl acetate (FEA) concentration and light microscopy, and charcoal culture. The combined results of these investigations for all three stool samples were designated the reference standard. The first sample was also analysed using a multiplex TaqMan™ qPCR assay, screening for five helminths and three protozoa. We compared sensitivity and specificity of analysing the first faecal sample with qPCR alone, or a hybrid approach combining qPCR with traditional methods, to the reference standard. Additionally, a serum sample was taken from each participant for Strongyloides stercoralis IgG ELISA., Results: The reference standard identified 139 GIP infections in 133 (22.3%) participants. Use of qPCR alone in one stool identified 176 infections in 147 (24.8%) participants, rising to 187 infections in 156 (26.3%) when combined with FEA microscopy and charcoal culture. The sensitivity of this latter hybrid approach was 100% for Strongyloides spp., 90.9% for Trichuris trichiura, 86.8% for hookworm species and 75% for Giardia duodenalis compared to the reference standard. The hybrid approach increased the detected prevalence of G. duodenalis by 4.5% (27 cases) overall, T. trichiura by 2.9% (17 cases), Strongyloides spp. by 1% (6 cases), and hookworm by 0.5% (3 cases), compared to the reference standard., Conclusion: Examination of a single faecal sample using qPCR alone showed superior or equivalent sensitivity to traditional methods for most GIP infections when both were compared to the reference standard. Combining molecular and traditional methods to analyse a single stool improved the detection rate for most studied parasites. This approach has value in settings where repeated sampling and/or faecal culture for helminths is impractical, but molecular diagnostics are available., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)