{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:02:10Z","timestamp":1791820930276,"version":"4.3.4"},"reference-count":37,"publisher":"Public Library of Science (PLoS)","issue":"3","license":[{"start":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T00:00:00Z","timestamp":1773878400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      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,\n                      <jats:italic>Paracoccidioides<\/jats:italic>\n                      spp., that can be deployed in frontline healthcare facilities.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methodology\/principal findings<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>Paracoccidioides<\/jats:italic>\n                      spp. Primer specificity was evaluated\n                      <jats:italic>in silico<\/jats:italic>\n                      and experimentally against phylogenetically related fungi and clinically relevant pathogens, with no cross-reactivity observed. The assay robustly amplified across multiple\n                      <jats:italic>Paracoccidioides<\/jats:italic>\n                      lineages, and all products were validated by Sanger sequencing. The analytical limit of detection (LoD) was 1 pg of genomic DNA per 8 \u00b5L reaction, demonstrated by UV-based SYBR Green I visualization, agarose gel electrophoresis, and Qubit fluorometric assessment. Key optimizations included reaction miniaturization from 50 \u00b5L to 8 \u00b5L and a simple freeze\u2013boil lysis compatible with crude fungal biomass extracts, avoiding the need for commercial extraction kits, lengthy protocols and expensive equipment.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Conclusions\/significance<\/jats:title>\n                    <jats:p>\n                      This RPA assay offers a rapid, affordable, and operationally simple molecular tool specifically designed for the detection of\n                      <jats:italic>Paracoccidioides<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pntd.0014101","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T17:31:17Z","timestamp":1773941477000},"page":"e0014101","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["A miniaturized, frugal RPA assay for genus-level detection of Paracoccidioides spp. in resource-limited endemic settings"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9092-3879","authenticated-orcid":true,"given":"Melina Noelia","family":"Lorenzini Campos","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ra\u00fal 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