Performance evaluation of the diaxxoPCR system for rapid and user-friendly stool-based diagnosis of trichuriasis, ascariasis and strongyloidiasis in Mozambique

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BACKGROUND: Nucleic acid amplification tests have shown promising results for the diagnosis of soil-transmitted helminths (STH). The implementation of real-time PCR (qPCR) in low-resource settings is, however, still hampered by multiple logistical challenges. In this study, we assessed the diagnostic performance of a cartridge-based real-time PCR system (diaxxoPCR) for the detection of DNA of Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis in clinical samples, using qPCR as the reference test. METHODOLOGY: Initially, a technical validation of the diaxxoPCR system in a singleplex pod (cartridge) design was performed using 37 predefined DNA samples (Study A), followed by a diagnostic comparison between the diaxxoPCR system (singleplex) and qPCR on DNA samples extracted from 325 stools collected in a clinical trial in a rural area of Mozambique (Study B). Finally, one negative and one positive DNA sample were used to demonstrate the technical performance of a multiplex pod design as a potential use-case for the diaxxoPCR system (Study C). PRINCIPAL FINDINGS: Study A demonstrated that the diaxxoPCR system performed reliably for each of the three STH targets, with minimal intra- and inter-assay variation and sufficient output reproducibility. Study B, performed in Mozambique, showed a positive qPCR result in 57.5% (187), 15.4% (50), and 0.3% (1) of the 325 DNA trial samples for T. trichiura, A. lumbricoides, and S. stercoralis, respectively. The diaxxoPCR system demonstrated sensitivities and specificities above 97% and 94% for each target, resulting in nearly perfect to perfect qualitative agreements with the reference test. Quantitatively, significant and positive associations were seen between the Ct-values (qPCR) and Cq-values (diaxxoPCR). In Study C, the diaxxoPCR system correctly detected all 3 targets in the multiplex pod design. CONCLUSION: With refinements regarding faecal DNA extraction procedures, the diaxxoPCR system has potential to provide accurate and easy-to-use real-time molecular diagnostics of STH in low resource laboratories.