PLoS neglected tropical diseases · 2026
A miniaturized, frugal RPA assay for genus-level detection of Paracoccidioides spp. in resource-limited endemic settings.
СтатьяОценочное исследованиеНазвание и аннотация: Перевод в очереди
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Lorenzini Campos Melina Noelia, Acevedo Raúl Maximiliano, Massa Gabriela Alejandra, Valinotto Laura Elena, Corredor Sanguña Luis Hernando, Piz Mario Alberto, Lucero Raúl Horacio, Rojas Florencia, Formichelli Laura Belén, Lösch Liliana Silvina, Mussin Javier Esteban, Giusiano Gustavo
Аннотация
BACKGROUND: 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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
CONCLUSIONS/SIGNIFICANCE: 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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
CONCLUSIONS/SIGNIFICANCE: 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.