A Novel Molecular Assay for Point-of-Care Diagnosis of Paracoccidioidomycosis from Clinical Samples Using Recombinase Polymerase Amplification Coupled with a Lateral Flow Assay

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Its diagnosis is often delayed due to the limited availability of accessible and rapid molecular tools in endemic settings. In this study, we developed and conducted a preliminary evaluation of a recombinase polymerase amplification assay coupled with a lateral flow readout (RPA-LFA) for genus-level detection of Paracoccidioides spp. using reference strains and DNA extracted from clinical specimens. The assay targets the ITS1–5.8S–ITS2 ribosomal region using dual-labeled primers, which enable visual detection on LFA strips. Analytical specificity, analytical sensitivity, and clinical concordance were evaluated using reference strains, non-target microorganisms, and an exploratory set of clinical samples. No cross-reactivity was observed with Coccidioides posadasii, Emmonsia crescens, Histoplasma capsulatum, or Leishmania braziliensis. The preliminary analytical limit of detection (LOD) was 100 copies/µL. Visual results were obtained within 35 min, including 20 min of amplification at 39 °C and 15 min for LFA readout. In the clinical sample set analyzed, the assay showed complete concordance with direct microscopy. The RPA-LFA approach addresses several operational limitations of conventional molecular methods by combining isothermal amplification, a short turnaround time, visual interpretation, and low equipment requirements. This work provides a proof of concept for a point-of-care-oriented molecular approach for Paracoccidioides detection in clinical specimens. Further validation in larger and more diverse clinical cohorts is required to establish its diagnostic performance and potential implementation in endemic settings.