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

## TL;DR

This study evaluates a portable PCR system for diagnosing intestinal parasitic worms in Mozambique, showing it performs as well as traditional PCR methods.

## Contribution

The study introduces a cartridge-based real-time PCR system (diaxxoPCR) suitable for low-resource settings to detect three soil-transmitted helminths.

## Key findings

- The diaxxoPCR system showed high sensitivity and specificity (above 97% and 94%) for detecting Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis.
- Quantitative results from the diaxxoPCR system correlated strongly with those from qPCR, indicating reliable performance.
- The system correctly detected all three targets in a multiplex design, demonstrating its potential for simultaneous testing.

## Abstract

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.

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).

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.

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.

Intestinal parasitic worms remain an important cause of disease in low- and middle-income countries, particularly in tropical regions with poor access to adequate water, sanitation, and hygiene. Microscopy of stools is the cornerstone to assess the necessity and the success of control programs but is limited in use mainly due to the need for expert microscopists and its shortcomings in diagnostic accuracy. Molecular diagnostics, including real-time PCR (qPCR), offer a highly sensitive and specific alternative, but accessibility in poor-resource settings remains a challenge. In this study we evaluated a portable and affordable cartridge-based real-time PCR system (diaxxoPCR) for the diagnosis of 3 intestinal parasitic worms and compared its performance to a well-established qPCR protocol. Overall, the diaxxoPCR system produced qualitative and quantitative results that were analogous to those obtained with qPCR, which shows that it has the potential to improve the accessibility of molecular diagnostic procedures in poor-resource settings, where these parasites are a public health problem. The diaxxoPCR system can be adapted for the purpose of STH species-specific population-based surveys or the management of individual patients.