A miniaturized, frugal RPA assay for genus-level detection of Paracoccidioides spp. in resource-limited endemic...

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America, where diagnosis is often delayed due to limited access to rapid, simple confirmatory testing tools in resource-limited settings. This gap creates a critical need for accessible detection methods of its causative agent, Paracoccidioides spp., that can be deployed in frontline healthcare facilities.

We developed a frugal Recombinase Polymerase Amplification (RPA) assay targeting a conserved region of the internal transcribed spacer (ITS) locus for genus-level detection of Paracoccidioides spp. Primer specificity was evaluated in silico and experimentally against phylogenetically related fungi and clinically relevant pathogens, with no cross-reactivity observed. The assay robustly amplified across multiple Paracoccidioides lineages, and all products were validated by Sanger sequencing. The analytical limit of detection (LoD) was 1 pg of genomic DNA per 8 µL reaction, demonstrated by UV-based SYBR Green I visualization, agarose gel electrophoresis, and Qubit fluorometric assessment. Key optimizations included reaction miniaturization from 50 µL to 8 µL and a simple freeze-boil lysis compatible with crude fungal biomass extracts, avoiding the need for commercial extraction kits, lengthy protocols and expensive equipment.

This RPA assay offers a rapid, affordable, and operationally simple molecular tool specifically designed for the detection of Paracoccidioides DNA. Its ability to work with crude lysates and miniaturized reaction volumes supports its implementation in resource-limited endemic areas. Although clinical validation remains necessary, this assay constitutes a practical foundation for expanding molecular diagnostic capacity for PCM in underserved regions. This work demonstrates how frugal methodological strategies can support equitable access to molecular detection tools.