Background The EDCTP_STOP project is a multicentric clinical trial (ALIVE trial ct.gov: NCT05124691) that aims to interrupt the transmission of soil-transmitted helminths using novel treatment regimens. While cure rate measured by microscopy is the primary efficacy outcome, limitations in sensitivity after successful treatment pose a challenge. Nucleic acid amplification tests are a promising alternative. One objective in the EDCTP_STOP project is to assess real-time polymerase chain reaction (qPCR) as a secondary efficacy outcome, which necessitates implementing an external quality assessment scheme (EQAS).

Methods The Helminth External Molecular Quality Assessment Scheme (HEMQAS), provided by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), was implemented in the study. The sample distribution consists of blinded ethanol-preserved stool samples to assess DNA extraction, and purified DNA samples in stabilizing buffer to assess the amplification technique. Four consortium partners participated in the 2022 assessment. LUMC scored 99% (91/92 targets correctly identified). KEMRI scored 74% (68/92 targets). CISM scored 99% (75/76) and ULE scored 100% (62/62 targets).

Results For stool samples, the outcomes demonstrated that ineffective DNA extraction caused multiple false negative outcomes, particularly for Trichuris trichiura. Pipetting-error during DNA extraction may explain false positive outcomes. For DNA samples, false negative outcomes most likely resulted from handling errors. Systematic errors such as qPCR channels used to detect targets may account for false positive outcomes as spectral overlap in a multiplex qPCR may cause incorrect data interpretation. The use of validated positive control DNA elucidated which primer and probe pairs required optimization.

Conclusion These outcomes facilitated targeted molecular optimization per trial site prior to testing trial samples. Participating in an EQAS facilitates capacity building by identifying training and laboratory validation needs, and ensures reliable reproducible results.