Juan Guillermo McEwen

The genomics of human pathogenic thermally dimorphic fungi has advanced significantly thanks to international initiatives such as the 1000 Fungal Genomes Project, which has reported a wide range of complete genomes in public repositories. This has driven the genomic characterization of genera such as Histoplasma, Paracoccidioides, and Sporothrix, including clinically relevant Colombian isolates. Genomic studies have revealed key aspects of their diversity, evolution, and host adaptation, while enabling comparative analyses based on orthologous genes that highlight both conservation and divergence among lineages. Beyond their description, the availability of complete genomes has had direct applications in diagnostics through systematic comparison algorithms that identify unique genomic regions serving as highly specific molecular targets. This has facilitated the design and validation of innovative PCR assays for the above-mentioned genera, overcoming the limitations of traditional candidate-gene approaches. In Colombia, significant challenges remain, including the limited availability of clinical isolates, biosafety requirements, and difficulties in timely diagnosis. In this context, we aimed to analyze the current state of assembled genomes of these dimorphic fungi available in public databases for Colombia and their use in the development of highly specific molecular diagnostic tools.

Paracoccidioides is a genus of dimorphic fungal pathogens endemic to Latin America. We generated long-read de novo assemblies for 11 isolates representing four species of the brasiliensis complex ( P. brasiliensis, P. americana, P. restrepiensis, P. venezuelensis ) and P. lutzii . These include the first complete telomere-to-telomere assemblies for P. brasiliensis (Pb18) and P. americana (Pb03), each with five chromosomes. Comparative analyses revealed chromosomal fusion and fission events distinguishing P. brasiliensis and P. americana , and a 90 kb tandem duplication in P. americana containing siderophore biosynthesis genes ( sid1, sid3, sid4 ), a cluster of putative virulence factors. Mitochondrial genomes showed conserved gene order but a phylogenetic topology inconsistent with the nuclear tree, suggesting mitochondrial introgression between P. lutzii and P. venezuelensis . RNA transposable elements were enriched near telomeres, correlated with genome size, and most abundant in P. lutzii . These assemblies provide key resources for understanding genome evolution and introgression in Paracoccidioides .

The genus Aspergillus comprises a diverse group of fungi that can cause a range of health issues, including systemic infections and allergic reactions. In this regard, A. fumigatus has been recognized as the most prevalent allergen-producing species. This genus taxonomic classification has been subject to frequent updates, which has generated considerable difficulties for its classification when traditional identification methodologies are employed. To demonstrate the feasibility of this approach, we sequenced the whole genomes of 81 Aspergillus isolates and evaluated a WGS-based pipeline for precise species identification. This pipeline employed two methodologies: (i) BLASTn web using four barcode genes and (ii) species tree inference by OrthoFinder. Furthermore, we conducted a prediction of allergenic capacity based on a homology analysis across all the isolated species and confirmed by RT-qPCR the expression of three orthologous allergens (Asp f 1, Asp f 3 and Asp f 22) in fifteen different Aspergillus species. The species-level identification rate with the barcoding and the species tree were calculated at 64.2% and 100%, respectively. The results demonstrated that A. fumigatus, A. flavus and A. niger were the most prevalent species. The species A. hortae, A. uvarum, A. spinulosporus, A. sydowii, A. westerdijkiae, A. amoenus and A. rhizopodus identified in this study represent the inaugural report of their presence in our region. The results of the homology analysis indicated the presence of orthologous allergens in a wide range of non-fumigatus species. This study presents a novel approach based on WGS that enables the classification of new species within the genus Aspergillus and reports the genomic sequences of a great diversity of species isolated in our geographic area that had never been reported before. Additionally, this approach enables the prediction of allergens in species other than A. fumigatus and demonstrates their genetic expression, thereby contributing to the understanding of the allergenic potential of different species within this fungal genus.

The Aspergillus genus comprises fungi that are widely distributed in nature. The Terrei section is particularly important due to its functions in recycling organic matter. Aspergillus hortae is a species from this section that has been isolated from clinical samples, but its role as a pathogenic agent is unclear. In this study, three clinical A. hortae isolates from Colombia, initially identified as A. terreus, were characterised by micro and macro-morphological analysis at 26°C and 37°C. A bioinformatic pipeline was used for molecular typing, which included analysis of whole genome sequences for accurate species identification. Susceptibility assays were also conducted using the microdilution broth method (EUCAST). It was confirmed that this species is intrinsically resistant to Amphotericin B with a minimum inhibitory concentration (MIC) of 2-4 mg/L, and is also thermotolerant. We obtained the in vitro susceptibility profiles for three azole drugs (MIC, 0.25-1 mg/L). The three isolates showed phylogenetic divergence from the reference genome strain. A mutation (M769K) in the MelA ortholog of a hypopigmented isolate, which had the lowest MIC value for AmB (2 mg/L), was identified. This study presents the morphological characterization, molecular typing through whole-genome analysis and identification of susceptibility profiles to azoles and amphotericin B of three clinical isolates of A. hortae from Colombia.

La paracoccidioidomicosis es una micosis sistémica endémica en Latinoamérica. La presentación más frecuente compromete crónicamente los pulmones, la piel y las mucosas. Al inicio, este paciente presentó, por varios años, una lesión única en la mucosa oral que, en ausencia de otros síntomas, se relacionó con una neoplasia maligna, específicamente con un carcinoma escamocelular.La diferenciación entre los dos diagnósticos se hace mediante un examen directo, un estudio histopatológico y cultivos iniciales y subsecuentes. Sin embargo, tales estudios no fueron concluyentes. Después de varias consultas y pruebas, con los resultados del examen directo, la inmunodifusión y la PCR en tiempo real se confirmó el diagnóstico de paracoccidioidomicosis crónica multifocal.Este caso alerta sobre la ausencia de sospecha clínica de micosis endémicas, dada la presencia de lesiones mucocutáneas que pueden ser producidas por hongos como Paracoccidioides spp, y la importancia de considerarlas entre los diagnósticos diferenciales.

Introduction. For over a century, Sporothrix schenckii was considered the sole species responsible for sporotrichosis. In 2007, scientific community confirmed the disease could be caused by various Sporothrix species. These species differed in their virulence factors and their antifungal sensitivity.Objective. This study aims to characterize 42 Colombian clinical isolates of Sporothrix spp. phenotypically and genotypically.Material and methods. Forty-two clinical isolates were characterized using phenotypic methods. It involved various culture media to determine their growth range at different temperatures and to assess the type and distribution of pigment and colony texture. Microscopic morphology was evaluated through microcultures, as well as the conidia diameter, type of sporulation, and morphology. Additionally, the assimilation of carbohydrates was selected as a physiological trait for species identification. Genotyping of 40 isolates was performed through partial amplification of the calmodulin gene, followed by sequence analysis.Results. Molecular studies enabled the identification of 32 isolates of S. schenckii and 8 isolates of S. globosa. The combination of phenotypic and genotypic methods eased these species characterizations and the recognition keys development based on parameters such as growth diameter at 25 and 30 ºC, colony texture (membranous or velvety) on potato dextrose agar, and microscopic morphology with predominance of pigmented triangular, elongated oval globose, or subglobose conidia.Conclusions. Confirmation of the phenotypic characteristics and molecular analysis is crucial for identifying Sporothrix species and determining adequate treatment. This study represents the first phenotypical and genotypical characterization of clinical isolates of Sporothrix spp. reported in Colombia.

Genus Paracoccidioides now encompasses five species, P. lutzii, P. brasiliensis sensu stricto, P. americana, P. venezuelensis, and P. restrepiensis. All the paracoccidioidomycosis (PCM) cases reported in Colombia have been classified as P. restrepiensis, which was named in honor of Angela Restrepo, a leading researcher in the field of the biology of the fungus. Previous assessments of genetic diversity have suggested that Paracoccidioides species have differences in their level of polymorphism. To infer changes in effective population size, we used the Pairwise sequentially Markovian coalescent (PSMC) by generating two pseudodiploids for P. restrepiensis and one for P. brasiliensis sensu stricto. We found that P. restrepiensis diverged from its sister species recently, the divergence time from P. venezuelensis being 125,000 (± 42,000) years. The analyses using PSMC show a systematic reduction in the effective population size of P. restrepiensis with a rapid decrease in genetic variability compared to P. brasiliensis sensu stricto, which indicates that P. restrepiensis has undergone a systematic population bottleneck. None of the other two species show the dramatic effective population size reduction observed in P. restrepiensis. These comparisons suggest that the trajectory of P. restrepiensis is somehow different from the other Paracoccidioides species and poses the question regarding the biogeographic events that have led to such a dramatic population pattern.

: Histoplasmosis, one of the most frequent endemic mycoses in the Americas, is caused by the inhalation of airborne conidia of Histoplasma capsulatum. Better understanding of the distribution of this fungus in the environment is important for the development of appropriate public health measures to prevent human infections. Previously, we used Hc100 nested polymerase chain reaction (PCR) to identify H. capsulatum DNA in 10% of environmental samples in Colombia. Here, we validate a 100-kDa real-time PCR assay for the detection of this fungus in the environment. Using this method, we identified H. capsulatum DNA in 80% of samples of raw organic materials, such as chicken manure, soil from caves, and bird and bat guano, as well as in 62% of samples of organic fertilizer that underwent the composting process. We demonstrated that 100-KDa real-time PCR is a useful tool for environmental surveillance that can be used to identify the potential reservoirs of H. capsulatum and to prevent outbreaks, especially in people with the higher risk of exposure, such as spelunkers, farmers, poultry manure collectors, and anyone who handle organic fertilizers or bat and bird excreta.