Detection of soil-transmitted helminths and Schistosoma spp. by nucleic acid amplification test: Results of the first 5 years of the only international external quality assessment scheme

Abstract

Background: Infections with soil-transmitted helminths (STH) and schistosomiasis (SCH) result in a significant global health burden, particularly in rural communities in low and middle-income countries. While microscopy remains the primary diagnostic method for STH and SCH in resource-limited settings, nucleic acid amplification tests (NAATs) are gaining prominence as tools for evaluation of public health control programs in endemic countries, and individual diagnosis in high-income countries. Despite the high sensitivity and specificity of NAATs, previous research has highlighted inter-laboratory variations, both in technical and clinical performance, justifying the need for continuous proficiency testing.

Methodology: Results from 5 rounds over a 5-year period of the so far only longitudinal international Helminth External Molecular Quality Assessment Scheme (HEMQAS), coordinated by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), were examined in order to (i) assess the diagnostic proficiency of laboratories in detecting helminths in stool and (ii) identify potential factors contributing to variations in performance.

Outcome and conclusions: Thirty-six laboratories, from 18 countries and 5 continents, participated in HEMQAS. The overall diagnostic performances were satisfying, with remarkably low numbers (<2%) of false-positive results. False-negative results were more often reported for stool (15%) than for DNA (5%) samples. False-negative results varied largely between targets (the highest number (29%) for Trichuris trichiura). Twenty-five laboratories provided a sufficient number of results for a robust comparison between participating laboratories, which confirmed substantial inter-laboratory variability in quantitative NAAT results (Cq-values). This variability likely arises from differences in pre-treatment, DNA isolation and DNA-target amplification procedures. This study emphasizes the complexity of molecular diagnosis for STH and SCH, highlighting the critical role of proper stool preparation and DNA isolation methods. The results underscore the necessity for laboratory professionals and public health decision-makers to recognize these complexities and continuously undertake external quality assessment schemes to ensure accurate and reliable performance in molecular diagnosis.

Fig 1. Difference in reported Cq-values for stool and DNA samples positive for Ascaris spp.

Each point represents the reported Cq-value of the individual participant subtracted by the median of reported Cq-value of all participants and each horizontal line represents the median difference in reported Cq-value. Points and lines were only shown if the participants reported four or more Cq-values for either stool or DNA (educationals excluded). The table below represents the number of false negatives, false positives and false negative educationals (*) are displayed. Some numbers were placed in italics, this means the participant included ’positive’ results without reporting a Cq-value. Each letter on the the X-axis represents a different participant, these participants are similarly ordered throughout Fig 1–5. For the target Trichuris (Fig 5), most samples were assigned as educationals.

Fig 2. Difference in reported Cq-values for stool and DNA samples positive for Schistosoma spp.

For detailed explanation of the figure, see legend of Fig 1.

Fig 3. Difference in reported Cq-values for stool and DNA samples positive for Strongyloides stercoralis.

For detailed explanation of the figure, see legend of Fig 1.

Fig 4. Difference in reported Cq-values for stool and DNA samples positive for Necator americanus.

For detailed explanation of the figure, see legend of Fig 1.

Fig 5. Difference in reported Cq-values for stool and DNA samples positive for Trichuris trichiura.

For detailed explanation of the figure, see legend of Fig 1.