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

## TL;DR

A low-cost, simple molecular test for detecting Paracoccidioides, the fungus causing a neglected disease in Latin America, was developed for use in resource-limited areas.

## Contribution

A frugal RPA assay for genus-level detection of Paracoccidioides spp. with no cross-reactivity and minimal equipment requirements.

## Key findings

- The RPA assay detected Paracoccidioides DNA with high specificity and no cross-reactivity with related pathogens.
- The assay achieved a detection limit of 1 pg of genomic DNA in 8 µL using simple visualization methods.
- Miniaturization and freeze–boil lysis reduced costs and eliminated the need for commercial extraction kits.

## Abstract

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.

Paracoccidioidomycosis (PCM) primarily affects rural populations in Latin America. Rapid, accessible tools for detecting its causative agent, Paracoccidioides spp., are urgently needed in endemic regions. We developed a simple and low-cost molecular test based on Recombinase Polymerase Amplification (RPA) that detects Paracoccidioides DNA. Validated with DNA from fungal cultures after a freeze-boil cycle, this approach overcomes the need for commercial extraction kits, lengthy protocols, or specialized equipment. The assay operates at a constant low temperature, yields results within minutes, and requires only a basic UV lamp for visualization, making it suitable for small frontline healthcare facilities with limited budgets. It demonstrates high specificity, no cross-reactivity with related pathogens and reliable performance using miniaturized reaction volumes that significantly reduce per-test costs. Although evaluation with clinical samples remains a future step, this frugal and equipment-minimal assay represents a practical advance toward equitable molecular detection of this pathogen in resource-limited settings. We believe this work could meaningfully contribute to improving the diagnosis of this long-neglected disease.