Five ways to find a parasite: comparison of five molecular assays for the detection of Strongyloides spp.

UNLABELLED: Strongyloidiasis, caused by nematodes of the genus Strongyloides, is a neglected tropical disease with significant public health implications. Accurate diagnostics are essential for disease surveillance and control, yet traditional stool-based and serological methods have limitations in sensitivity and specificity. Molecular assays offer an alternative diagnostic approach. In this study, we compared four probe-based real-time PCR (qPCR) assays and one loop-mediated isothermal amplification (LAMP) assay using human fecal DNA samples from Gabon and Papua New Guinea with both Strongyloides stercoralis and Strongyloides fuelleborni infections. Our results revealed discordance between assays, with no single primer set identifying all positive samples across all assays. One assay was identified as specific for S. stercoralis only, while other assays amplify other Strongyloides spp. This may impact the specificity of molecular diagnostics in epidemiological surveys, particularly with increasing identifications of S. fuelleborni infections in humans from Asia and New Guinea. Of the assays examined, four were probe-based qPCR assays and a LAMP assay. On sequencing positive amplicons, enteric bacterial sequences were identified for two assays. However, subsequent testing and BLAST results identified that including a probe, as the assays were designed, would increase specificity, so that the bacterial sequences would not be amplified. These findings provide a context for the interpretation of results from various Strongyloides molecular assays.

IMPORTANCE: Strongyloidiasis is an underdiagnosed neglected parasitic infection that poses a growing challenge for public health surveillance and control, particularly in regions where multiple Strongyloides species exist and clinical case detection is challenging. Molecular assays are increasingly used as reference diagnostic tools, yet their performance and species specificity have not been systematically evaluated across diverse endemic settings. In this study, we tested the hypothesis that commonly used molecular assays differ in their ability to accurately and consistently detect and discriminate Strongyloides stercoralis and Strongyloides fuelleborni in human fecal samples. By comparing five molecular assays using samples from Gabon and Papua New Guinea, we demonstrate discordance between tests, including species-dependent detection biases. These findings provide evidence that molecular diagnostics may misrepresent species distribution. Our results highlight the necessity for awareness of target species specificity and for regionally validated molecular tools to ensure the reliable interpretation of assay results.