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.

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.

Figures

RPA assay optimization and specificity evaluation using DNA from cultures.(A) Specificity testing in the 50 µL reaction format. Lane 1: non-template control (NTC); lane 2: 1 ng of Paracoccidioides brasiliensis S1 (IMR-M-Pb 369, Argentina); lane 3: 0.1 ng of P. brasiliensis S1 (IMR-M-Pb 369); lane 4: Leishmania sp.; lane 5: Coccidioides sp.; lane 6: Emmonsia sp.; lane 7: Histoplasma sp.; lane 8: Fusarium sp.; lane 9: 100 bp DNA ladder (Inbio Highway, Argentina). Background from viscous RPA mixture is visible in lane 1. (B) Assay performance of the miniaturized 8 µL reactions with approximately 100ng of DNA input. Lane 1: NTC; lane 2: P. lutzii (Pb01, Brazil); lanes 3-4: P. brasiliensis clinical isolates (IMR-Pb-473 and IMR-Pb-611, Northern Argentina); lane 5: P. brasiliensis PS3 (Pb339, Brazil); lane 6: P. brasiliensis PS3 (Pblish, Colombia); lane 7: P. brasiliensis PS3 (ATCC 60855, Colombia); lane 8: P. brasiliensis IMR-Pb-243 (Peru); lane 9: 100 bp DNA ladder (Inbio Highway, Argentina). This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Analytical performance of the endpoint miniaturized RPA assay for Paracoccidioides spp.(A) Direct fluorescence detection under UV light after SYBR Green I addition into the 8 µL reaction tubes. Tubes contain: 1, non-template control (NTC); 2, negative control (1 ng of Leishmania sp. DNA); 3 to 10, ten-fold decreasing series of Paracoccidioides genomic DNA inputs: 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg per reaction, respectively. Fluorescence was clearly distinguishable down to 1 pg. (B) Agarose gel electrophoresis (2%) of amplification products from reactions with decreasing DNA inputs. Lanes 1 and 12: 50 bp DNA ladder (Inbio Highway, Argentina); Lane 2: NTC; Lane 3: negative control (1 ng of Leishmania sp. DNA); Lanes 4 to 11: 10 ng to 1 × 10 ⁻ ⁶ ng (1 fg) of Paracoccidioides DNA. The 242 bp target band is visible from lane 4 (10 ng) down to lane 8 (1 pg). (C) Fluorometric assessment (Qubit) of technical triplicates; RFU values decreased progressively with lower DNA inputs, confirming successful amplification across the series. The dashed horizontal line indicates the limit of blank (LoB = 20,148 RFU), calculated as mean(NTC) + 3 × SD(NTC). (D) Fluorometric signal trend across DNA inputs. The observed decrease in RFU values with lower DNA input, corroborated by a strong positive correlation (r = 0.96; p = 0.0077), demonstrates the assay’s consistent performance across the tested range. The empirical LoD (3/3 positives) corresponds to 1 pg per 8 µL RPA reaction. Negative controls remained below the LoB. This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

RPA assay directly from culture extracts.(A) SYBR Green I fluorescence under UV light observed directly in reaction tubes. Tube: 1, non-template control (NTC); tubes 2-3: Paracoccidioides brasiliensis S1 (1 and 3 freeze-boil cycles); tubes 4-5: P. brasiliensis PS3 (1 and 3 cycles); tubes 6-7: P. lutzii (3 and 1 cycles). (B) Agarose gel electrophoresis of corresponding amplification products. Lane 1: 100 bp DNA ladder (Inbio Highway, Argentina); lane 2: NTC; lanes 3-4: P. brasiliensis S1 (1 and 3 cycles); lanes 5-6: P. brasiliensis PS3 (1 and 3 cycles); lanes 7-8: P. lutzii (3 and 1 cycles). The expected 242 bp amplicon is indicated. Electrophoresis conditions: 2% agarose gel stained with GelRed, 100 V for 55 min. This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Full text

1. Introduction

Paracoccidioides, a thermodimorphic fungus comprising the P. brasiliensis complex (S1, PS2, PS3, PS4) and P. lutzii, is the causative agent of paracoccidioidomycosis (PCM), considered a neglected systemic mycosis endemic to Latin America [1,2]. In 2022, the World Health Organization (WHO) included Paracoccidioides genus in its priority pathogens list [3]. In Argentina, the S1 lineage is prevalent and a PS3 case was detected [4]. PCM imposes a substantial clinical burden, primarily affecting rural populations with limited access to specialized healthcare. Infection typically begins with the inhalation of fungal propagules, leading to pulmonary conversion to the yeast phase and potential dissemination. Diagnostic delays, often due to limited access to health services or diagnostic tools in some regions, but also to low clinical suspicion given the nonspecific clinical manifestations, become especially critical in acute/subacute presentations, which progress rapidly in children and young adults [5–8]. Biological factors further compound these challenges, including the fungus’s robust cell wall [9,10], low fungal burden in some clinical specimens, and the slow, technically demanding growth of cultures [11].

Current PCM diagnosis relies on a combination of conventional methods, each of which has significant limitations. Direct microscopic examinations are quick and inexpensive but require an experienced operator. The sensitivity of this method depends on the type of clinical sample, sometimes the fungal load is low and obtaining such a sample is not always feasible. Fungal recovery in culture is also a reference for definitive diagnosis. However, this process can take several weeks, has low sensitivity, and is often unavailable in low-complexity laboratories. Histopathology provides valuable information on tissue involvement but requires invasive sampling and specialized expertise. Likewise, turnaround times are generally not short. There are no commercial tests for antibody detection. In house serological assays are used in most referral centers in endemic areas and are also useful for monitoring. However, cross-reactivity, variable sensitivity among clinical forms, and reduced accuracy in immunocompromised patients can affect their diagnostic performance. Furthermore, standardized antigens are lacking, and sensitivity can vary when using antigens from different species or non-autochthonous strains [5,6].

Taken together, these limitations underscore the need for rapid, specific, and less infrastructure- and personnel-dependent complementary diagnostic approaches. Molecular methods offer the potential to overcome several of these limitations by enabling the direct, objective detection of fungal DNA, thus supporting timely detection.

In this context, isothermal molecular amplification methods such as recombinase polymerase amplification (RPA) presents a promising molecular approach for such settings. This isothermal technique operates at a constant low temperature (typically 37–42°C) and delivers results in 20–30 minutes, requiring minimal equipment [12]. Its compatibility with direct visual detection and successful application across diverse pathogens make it particularly suitable for resource-limited contexts [13–20].

While loop-mediated amplification (LAMP) has been explored for Paracoccidioides [21,22] as an isothermal alternative, RPA-based detection has not been reported for this fungus. Here, we present the development and optimization of a frugal RPA assay for the detection of Paracoccidioides spp. DNA, targeting a 242 bp fragment of the ITS region. This integrates a simplified sample preparation method using crude fungal biomass from routine cultures, a miniaturized reaction volume that reduces reagent consumption, and SYBR Green I for direct UV-based visualization from reaction tubes.

2. Materials and methods

2.1. Fungal isolates and DNA extraction

Four clinical isolates of Paracoccidioides spp., two from acute/subacute forms (IMR-M-Pb 369 and IMR-M-Pb 473) and two from chronic forms (IMR-M-Pb 243 and IMR-M-Pb 611) were obtained from the culture collection of the Mycology Department at the Instituto de Medicina Regional, Universidad Nacional del Nordeste, Argentina. The strain IMR-M-Pb 369, sequenced and classified as S1 (GenBank accession number: JBICBQ000000000), served as positive control. Pb01 (P. lutzii), Pb339 (P. brasiliensis PS3), PbCNH (P. brasiliensis PS3), ATCC 60855 (P. brasiliensis PS3) were also included. For isolate metadata, see S1 Table.

Genomic DNA was extracted using our previously established protocol [11]. To minimize overall assay time and reagent use, a simplified extraction protocol for direct amplification from fungal biomass was adapted from Daddy Gaoh et al. [23] and Yang et al. [17]. Briefly, 0.3 g of yeast-phase biomass was resuspended in 500 µL of nuclease-free water in a 2 mL screw-cap tube and subjected to one freeze-boil cycle (20 min at -20 °C, then 10 min at 100 °C). After cooling and clarification, 1 µL of supernatant was used directly as RPA template. A detailed protocol is provided in S1 File.

2.2. Primer design and specificity evaluation

Following the TwistAmp Basic Kit (TwistDx, UK) guidelines, a pair of oligonucleotides for RPA was designed using NCBI Primer-BLAST [24], targeting a 242 bp genus-specific internal transcribed spacer (ITS) fragment from Paracoccidioides. Primer specificity was evaluated in silico against the NCBI nucleotide (nt) database, using the BLASTn tool [25]. Particular attention was paid to potential matches with Homo sapiens and relevant microorganisms, including Leishmania sp., Coccidioides sp., Histoplasma sp., Aspergillus sp., Sporothrix sp., Mycobacterium tuberculosis, Emmonsia sp., and Fusarium sp. Amplification was empirically tested using genomic DNA from different Paracoccidioides species. To assess potential cross-reactivity, DNA from phylogenetically related fungi (Emmonsia sp. and Fusarium sp.), as well as from common pathogens causing similar clinical presentations in PCM-endemic regions (Leishmania sp., Coccidioides sp., and Histoplasma sp.), was included.

2.3. Primer validation and RPA assay miniaturization

Primers were initially tested by conventional PCR using the T-Plus Free DNA Polymerase 500U Kit (Inbio Highway, Argentina). Reactions contained: 5 µL of TAS buffer (10X), 1 µL of MgCl₂ (25 mM), 1 µL of Taq DNA polymerase (5000 U/ml), 1 µL of dNTP mix (10 mM), 0.5 µL of each primer (10 µM), DNA template (approximately 100 ng), and water to a final volume of 25 µL. Thermocycling conditions were: 95 °C for 2 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; final extension at 72 °C for 5 min. After PCR validation, primers were tested in RPA reactions using the TwistAmp Basic Kit (TwistDx, UK), following the manufacturer’s protocol: 29.5 µL of rehydration buffer, 2.4 µL of primers F and R (10 µM), DNA template and water to 13.2 µL, and 2.5 µL of MgOAc (280 mM). Reactions were incubated for 20 min at 37°C [15]. Subsequently, reaction volumes were scaled down from 50 µL to 8 µL to enhance cost-effectiveness. Amplification products were analyzed by 2% agarose gel electrophoresis and confirmed by Sanger sequencing (Macrogen, Korea). Base-calling reliability was assessed by the Phred quality score (Q) using Ridom TraceEdit [26], and alignments were performed with BioEdit [27] and Aliview v1.28 [28]. Genus-level identity was evaluated by BLAST analysis against the NCBI database.

2.4. Robustness assessment

RPA assay robustness was evaluated by testing performance under two conditions: direct amplification from crude fungal extract (Section 3.1) and volume miniaturization (Section 3.3). Additionally, to assess whether increased cell wall disruption improved DNA release and assay performance, the standard one freeze-boil cycle was compared against three consecutive freeze-boil cycles. For this comparison, 1 µL of lysate was added to 7 µL of RPA master mix, resulting in a final reaction volume of 8 µL.

2.5. Direct SYBR Green I-based visual detection and limit of detection (LoD) assessment

To further reduce consumables and equipment requirements, endpoint detection was performed directly from RPA reaction tubes. For this, SYBR Green I (Invitrogen, USA) was used at a final concentration of 50X [18], with primers adjusted to 0.12 μM each, following SYBR Green I-optimized RPA work by Zheng et al. [12]. To determine the LoD, 10-fold serial dilutions of Paracoccidioides genomic DNA (10–1 × 10^−6^ ng/µL), non-template controls (NTC), and a heterologous negative control containing Leishmania DNA (1 ng/µL) were prepared. From each, 1 µL was used as template in the final 8 µL RPA reaction. All reactions were performed in triplicate, following the empirical approach described by Jarvi et al. [29]. Fluorescence was inspected visually under natural and UV illumination [18] and assessed as Relative Fluorescence Units (RFU) using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to Oshiki et al. [30]. The limit of blank (LoB) was calculated from NTC raw RFU values as: LoB = mean(NTC) + 3 × SD(NTC), following Jarvi et al. [29]. Correlation analysis was performed on RFU versus log₁₀-transformed total DNA input per reaction, across a five-point range (10–1 × 10^−3^ ng). Analyses were completed in Python 3.11 [31] and cross-checked in InfoStat v2020 [32]. Graphical designs were adapted from Daddy Gaoh et al. [23] and Oshiki et al. [30]. The empirical LoD was defined as the lowest total DNA input per reaction resulting in detectable amplification in all technical replicates (3/3), in accordance with Jarvi et al. [29]. Presence of the 242 bp amplicon was confirmed on 2% agarose gel electrophoresis stained with GelRed (Genbiotech, Argentina).

3. Results

3.1. Primer design and in silico specificity

Genus-specific primers were successfully designed to target the ITS region of Paracoccidioides. In silico analysis confirmed their ability to generate a 242 bp amplicon specific to Paracoccidioides entries in NCBI databases. Although minor homology was observed with Emmonsia and Fusarium species, discriminatory sequence positions were selected during primer design to ensure high specificity for the target genus. Nonetheless, these heterologous fungi were included as critical negative controls to experimentally validate assay specificity. The primer sequences are as follows:

Forward: 5′-CGTGCCCGCCGGGGACACCGTTGAACTTCTGGTTC-3′ andReverse: 5′-CGCTTGAGGGTTGAAATGACGCTCGGACAGGCATG-3′

3.2. RPA assay specificity and optimization

Our RPA assay demonstrated high specificity when tested against a panel of phylogenetically related and clinically relevant pathogens, with no cross-reactivity detected against DNA from Leishmania sp., Coccidioides sp., Histoplasma sp., Fusarium sp., or Emmonsia sp. (Fig 1A). Successful amplification was achieved for all tested Paracoccidioides spp. strains, including P. brasiliensis S1 (IMR-M-Pb 369), P. brasiliensis PS3, and P. lutzii (Fig 1B). Furthermore, scaling down the reaction volume from 50 µL to 8 µL reduced reagent costs per sample while eliminating non-specific background observed in larger volumes (Fig 1A, 1B).

RPA assay optimization and specificity evaluation using DNA from cultures.(A) Specificity testing in the 50 µL reaction format. Lane 1: non-template control (NTC); lane 2: 1 ng of Paracoccidioides brasiliensis S1 (IMR-M-Pb 369, Argentina); lane 3: 0.1 ng of P. brasiliensis S1 (IMR-M-Pb 369); lane 4: Leishmania sp.; lane 5: Coccidioides sp.; lane 6: Emmonsia sp.; lane 7: Histoplasma sp.; lane 8: Fusarium sp.; lane 9: 100 bp DNA ladder (Inbio Highway, Argentina). Background from viscous RPA mixture is visible in lane 1. (B) Assay performance of the miniaturized 8 µL reactions with approximately 100ng of DNA input. Lane 1: NTC; lane 2: P. lutzii (Pb01, Brazil); lanes 3-4: P. brasiliensis clinical isolates (IMR-Pb-473 and IMR-Pb-611, Northern Argentina); lane 5: P. brasiliensis PS3 (Pb339, Brazil); lane 6: P. brasiliensis PS3 (Pblish, Colombia); lane 7: P. brasiliensis PS3 (ATCC 60855, Colombia); lane 8: P. brasiliensis IMR-Pb-243 (Peru); lane 9: 100 bp DNA ladder (Inbio Highway, Argentina). This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

3.3. Limit of detection (LoD)

Endpoint visualization by direct addition of SYBR Green I to reaction tubes enabled clear detection of amplification under UV illumination down to 1 pg of DNA per 8 µL reaction (Fig 2A). Agarose gel electrophoresis reproducibly showed the expected 242 bp amplicon down to 10 pg per reaction, with light bands observable at 1 pg (Fig 2B). Qubit fluorometric assessment confirmed successful amplification, with RFU values progressively decreasing with lower DNA input (Fig 2C). This trend was corroborated by a strong positive correlation (r = 0.96; p = 0.0077) between RFU values and the logarithm of the DNA input (Fig 2D), which demonstrates the assay’s consistent performance across the tested range. The limit of blank (LoB) was calculated as 20,148 RFU. The empirical LoD was established at 1 pg per reaction, the lowest DNA input yielding positive results in all replicates. Heterologous negative controls (Leishmania sp.) remained below the LoB, confirming specificity. Importantly, due to its consistent fluorescence and superior sensitivity, the UV-based detection method using SYBR Green I at a 50X concentration was selected for all subsequent experiments. In contrast, ambient-light visualization of results was inconsistent and insufficient for reliable endpoint determination, as it required significantly higher concentrations of amplicon and dye, thereby increasing assay cost without guaranteeing a clear result.

Analytical performance of the endpoint miniaturized RPA assay for Paracoccidioides spp.(A) Direct fluorescence detection under UV light after SYBR Green I addition into the 8 µL reaction tubes. Tubes contain: 1, non-template control (NTC); 2, negative control (1 ng of Leishmania sp. DNA); 3 to 10, ten-fold decreasing series of Paracoccidioides genomic DNA inputs: 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg per reaction, respectively. Fluorescence was clearly distinguishable down to 1 pg. (B) Agarose gel electrophoresis (2%) of amplification products from reactions with decreasing DNA inputs. Lanes 1 and 12: 50 bp DNA ladder (Inbio Highway, Argentina); Lane 2: NTC; Lane 3: negative control (1 ng of Leishmania sp. DNA); Lanes 4 to 11: 10 ng to 1 × 10 ⁻ ⁶ ng (1 fg) of Paracoccidioides DNA. The 242 bp target band is visible from lane 4 (10 ng) down to lane 8 (1 pg). (C) Fluorometric assessment (Qubit) of technical triplicates; RFU values decreased progressively with lower DNA inputs, confirming successful amplification across the series. The dashed horizontal line indicates the limit of blank (LoB = 20,148 RFU), calculated as mean(NTC) + 3 × SD(NTC). (D) Fluorometric signal trend across DNA inputs. The observed decrease in RFU values with lower DNA input, corroborated by a strong positive correlation (r = 0.96; p = 0.0077), demonstrates the assay’s consistent performance across the tested range. The empirical LoD (3/3 positives) corresponds to 1 pg per 8 µL RPA reaction. Negative controls remained below the LoB. This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

3.4. Assay robustness: performance with crude extracts in miniaturized reactions

The RPA assay coupled with the rapid extraction protocol successfully detected the genus Paracoccidioides across cultures from different lineages, including P. lutzii, P. brasiliensis PS3, and the P. brasiliensis S1 positive control IMR-M-Pb 369 (Fig 3A), demonstrating broad applicability to routine isolates. Moreover, this approach reduced processing time compared with the conventional DNA extraction method and maintained robust performance in a miniaturized 8 µL reaction format without non-specific background. Notably, occasional increased signal intensity was observed following three consecutive freeze-boil cycles compared to a single cycle, suggesting that additional cell-wall disruption can moderately improve DNA release in certain cases (Fig 3B).

RPA assay directly from culture extracts.(A) SYBR Green I fluorescence under UV light observed directly in reaction tubes. Tube: 1, non-template control (NTC); tubes 2-3: Paracoccidioides brasiliensis S1 (1 and 3 freeze-boil cycles); tubes 4-5: P. brasiliensis PS3 (1 and 3 cycles); tubes 6-7: P. lutzii (3 and 1 cycles). (B) Agarose gel electrophoresis of corresponding amplification products. Lane 1: 100 bp DNA ladder (Inbio Highway, Argentina); lane 2: NTC; lanes 3-4: P. brasiliensis S1 (1 and 3 cycles); lanes 5-6: P. brasiliensis PS3 (1 and 3 cycles); lanes 7-8: P. lutzii (3 and 1 cycles). The expected 242 bp amplicon is indicated. Electrophoresis conditions: 2% agarose gel stained with GelRed, 100 V for 55 min. This figure was created specifically for this study by the corresponding author and is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

3.5. Sequence identity confirmation of RPA amplicons

The identity of all RPA amplicons was confirmed by Sanger sequencing, including those from both genomic DNA extracted using our prior protocol, and from crude fungal lysates processed with the freeze-boil method. Base quality assessed by Ridom TraceEdit was consistently high, with the majority of bases achieving quality scores above Q30. The few bases with quality lower than Q30 were manually checked and resolved by electropherogram inspection. All sequences were confirmed as belonging to the Paracoccidioides genus by subsequent BLASTn analysis and were deposited in GenBank (accession numbers: PX684487 - PX684497). Fungal isolate metadata are provided in S1 Table. Multiple sequence alignment of the ITS fragments confirmed a high degree of sequence conservation across the phylogenetic lineages tested, as demonstrated in S2 File.

4. Discussion

The diagnosis of PCM remains critically hindered by reliance on conventional methods with well-documented limitations [5]. In this context, molecular methods have increasingly been proposed as valuable complementary tools to classical mycological and serological approaches. Studies have demonstrated that PCR- and RT-PCR–based assays can improve diagnostic sensitivity and support case confirmation, particularly in complex clinical scenarios and in non-endemic areas where clinical suspicion is low [33–35]. However, despite their analytical performance, most reported molecular assays for PCM rely on thermocycler-dependent platforms, quantitative instrumentation, or multi-step workflows, limiting their routine implementation in low-complexity laboratories in endemic regions. This gap underscores the need for alternative molecular approaches that combine specificity with operational simplicity and affordability. With this objective, we have developed an RPA assay that prioritizes minimal equipment usage, cost-effectiveness, and operational simplicity, while maintaining robust analytical performance.

RPA has been successfully used to detect multiple infectious agents, including bacterial and parasitic pathogens, in both clinical and field settings [13–20]. In these contexts, RPA-based assays have been employed as rapid confirmatory tools or screening methods, particularly when conventional molecular platforms are not readily available. These prior applications establish RPA as a mature, and adaptable molecular amplification strategy, providing a relevant framework for its extension to neglected systemic mycoses such as PCM.

Isothermal amplification methods represent a practical alternative to conventional PCR, as they eliminate the need for a thermocycler, reducing initial costs and enabling portable use. LAMP has been successfully explored for the detection of Paracoccidioides brasiliensis [21,22], and offers advantages such as a rapid turnaround time, simple equipment requirements and user-friendly readouts. However, RPA provides a distinct and complementary operational profile. LAMP requires more primers, operates at higher temperature (63 °C), and involves longer overall assay time. Our RPA-based approach achieves robust genus-specific amplification at a lower, constant temperature (approximately 37 °C) and delivers amplification in shorter timeframes, simplifying reagent complexity and implementation while reducing equipment needs and consumable expenses. This is particularly advantageous in resource-constrained endemic settings where precise temperature control and thorough sample preparation can be challenging. Furthermore, RPA requires only two primers, which simplifies assay design and optimization compared to LAMP, which typically relies on four to six primers and more complex interactions. The choice between LAMP and RPA will depend on local infrastructure, operational constraints, and economics.

The cornerstone of our assay’s reliability lies in its primer design, which is particularly critical for the Paracoccidioides genus given the significant genomic heterogeneity among its species [1]. This complexity is further underscored by recent genomic analysis [36], which supports the highly dynamic nature of the nuclear genome, shaped by both large-scale chromosomal rearrangements, such as fusions and fissions, and the differential activity of transposable elements. By targeting conserved regions within the ITS locus, we achieved comprehensive detection across major phylogenetic lineages while maintaining high specificity against common cross-reactive pathogens. This strategic target selection addresses a notable limitation in the molecular identification of Paracoccidioides, where conventional targets like gp43 exhibit excessive variability that can compromise reliable genus-level detection [4]. In this context, the consistent amplification across multiple species and geographic origins validates our bioinformatic design and supports the assay’s applicability throughout endemic regions. Critically, Sanger sequencing confirmed the identity of all RPA amplicons as belonging to the Paracoccidioides genus and the conserved nature of the targeted region, providing essential verification. This is crucial given RPA’s tolerance to primer-template mismatches, which can promote non-specific amplification [37]. The high-quality chromatograms (Q30+) confirm that our optimized conditions mitigated these risks across amplification from both purified DNA and crude lysates. Multiple-sequence alignment further confirmed the expected conservation of the ITS region. However, validation gaps remain: the absence of PS2 and PS4 species in our sample set may introduce uncertainty regarding universal detection. Given the high conservation of the targeted ITS region, the detection of these lineages is expected, but clinical work should prioritize the evaluation of these lineages, present in other Latin American regions but not yet reported in Argentina [1].

Furthermore, we believe our methodological optimizations exemplify a frugal science approach, strategically balancing cost and performance to develop a practical tool for low-resource endemic settings. The substantial reduction to 8 µL reaction volumes provided dual benefits: minimizing non-specific amplification artifacts while drastically cutting per-test costs. This is in accordance with Lai & Lau [20], who demonstrated that volumes as low as 6.25 µL maintain amplification efficiency. The combination of miniaturization and UV-based SYBR Green I detection establishes an economically sustainable system. The one-time investment in a simple UV lamp provides greater cost-effectiveness compared to recurring expenses associated with specialized single-use detection formats. Notably, Jiang et al. [18] report that very low SYBR Green I concentrations, such as 0.16X, can be used for UV detection, which would further reduce costs per reaction. However, due to resource constraints that limited our capacity for extensive optimization, we prioritized the 50X concentration identified by these authors as the most robust condition. This finding nevertheless underscores the potential for even greater cost reduction, making this approach cost-effective compared to other molecular detection alternatives.

A practical advantage for resource-limited settings is the assay’s compatibility with simplified sample processing. We demonstrated that reliable detection is achievable directly from crude freeze-boil lysates, bypassing costly extraction kits and reducing processing time associated with conventional or commercial DNA extraction methods. Improvements in signal intensity observed after additional freeze-boil cycles suggest an accessible optimization strategy for difficult-to-lyse isolates. This approach leverages the routine fungal cultures already established in endemic areas, creating a powerful molecular confirmation step without requiring substantial new infrastructure.

With a limit of detection of 1 pg of DNA per 8 µL reaction, the assay is less sensitive than ITS1-targeted qPCR [33]. Nevertheless, it remains a practical alternative as it enables rapid and robust amplification from minimal crude culture extracts. This aspect is particularly relevant given the slow growth of Paracoccidioides cultures [5]. By prioritizing workflow simplicity and cost reduction, the design is well-suited for frontline use. The strong correlation between template input and fluorescence signal (r = 0.96, p = 0.0077) confirms the assay’s reliable performance across the tested range.

As with all nucleic acid amplification techniques, the proposed RPA-based assay has technical and operational limitations that must be considered. One potential cause of false-positive results is carryover contamination, especially in workflows involving the handling of open tubes. However, the short assay time of this technique and the endpoint visualization directly in closed reaction tubes, reduce post-amplification handling and can mitigate this risk compared to conventional PCR. Conversely, false-negative results could occur when using clinical samples with a very low fungal load or inefficient DNA release, or when amplification inhibitors are present in complex biological matrices.

Other limitations include potential variability related to reagent stability, batch-to-batch consistency, and workflow standardization. These limitations can affect reproducibility when the assay is implemented in different laboratories or field settings. It is important to note that, since this study focuses on analytical development and specificity using culture-derived DNA, diagnostic sensitivity, specificity, and reproducibility in clinical samples have not yet been formally established. These parameters will require systematic evaluation during subsequent clinical validation with well-characterized patient samples.

This assay was primarily designed for future use in low-complexity laboratories where access to molecular diagnostics is limited. In this context, the assay was conceived as an analytical development and feasibility assessment, aimed at establishing technical robustness, specificity, and operational simplicity. While clinical metadata of the isolates is provided, this study does not evaluate diagnostic sensitivity across clinical forms or tissue types, because amplification was performed on culture-derived DNA. Therefore, the next essential phase of this research is clinical validation using samples from patients with acute and chronic PCM. This validation should include a comparison with established diagnostic methods and an estimation of diagnostic performance parameters, such as sensitivity and specificity. The future integration of a frugal DNA extraction strategy for clinical samples with our RPA assay would further enable a fully accessible and affordable diagnostic pipeline for PCM in resource-limited endemic settings.

Finally, we believe that this work goes beyond technical optimization and serves as a case study in developing context-appropriate methods. By systematically addressing the economic, infrastructural, and operational constraints of PCM-endemic regions, we developed a practical tool tailored to the environments where it is needed most. We hope that our frugal RPA assay represents a significant step toward equitable molecular diagnostics for this neglected disease, demonstrating that strategic, context-aware innovation can help bridge the gap between novel techniques and the underserved populations who bear the greatest burden.

5. Conclusion

This study presents an RPA assay specifically developed for the genus-level detection of Paracoccidioides spp., tested across major phylogenetic lineages, with no cross-reactivity to related pathogens. By targeting a conserved ITS region, the assay achieves a sensitive, specific, and robust workflow suitable for laboratories in resource-limited endemic settings.

Through a frugal and context-appropriate scientific approach, we designed a cost-effective solution while preserving analytical performance. This was achieved by combining reaction miniaturization to 8 µL with UV-based endpoint detection and a simple freeze-boil lysis strategy that substantially reduces per-test costs. A LoD of 1 pg of total DNA input per 8 µL reaction was consistently demonstrated through three independent readouts: direct tube visualization, gel electrophoresis, and fluorometry.

While validated on culture-derived material, the assay’s core function, the specific detection of Paracoccidioides DNA, positions it as a versatile tool ready for integration with future frugal DNA extraction methods from clinical samples. Overall, this assay represents a meaningful step toward expanding molecular diagnostic capacity for PCM in underserved regions.

Supporting information

S1 FileRapid DNA extraction from Paracoccidioides spp. yeast cultures using a freeze–boil method.Detailed protocol describing a simplified thermal shock–based DNA extraction procedure used prior to RPA assays.(DOCX)

S2 FileMultiple sequence alignment of the RPA-amplified ITS target region from Paracoccidioides isolates after trimming primer sequences.The conserved region and single nucleotide polymorphisms (SNPs) are shown.(DOCX)

S1 TableMetadata of Paracoccidioides spp. isolates used in this study and clinical characteristics of the corresponding patients.The table includes isolate origin, species or lineage assignment, DNA extraction method, GenBank accession numbers, and relevant clinical information.(DOCX)