Validation of the performance of a point of care molecular test for leprosy: from a simplified DNA extraction protocol to a portable qPCR

Abstract

The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the16S rRNAand RLEP targets ofM. lepraewere established using synthetic DNA, purified DNA fromM. leprae, and pre-characterized clinical samples. In the simplified extraction step, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by depositing the lysed material on FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD95%for the optimized reactions was 113.31 genome-equivalents/μL for16S rRNAand 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the applicability of the portable platform in the detection ofM.leprae in low infrastructure settings.