Proceedings of the Second International Meeting on Endemic Mycoses of the Americas (IMEMA), and First International Symposium on Implantation Mycoses (ISIM).

The Second International Meeting on Endemic Mycoses of the Americas (IMEMA) and the First International Symposium on Implantation Mycoses (ISIM) took place in Santiago del Estero, Argentina during September 25-27th, 2023. The conference provided a platform for researchers, clinicians, and experts to discuss the latest developments in the field of endemic and implantation mycoses. Topics included epidemiology, diagnostic advances, treatment strategies, and the impact of environmental factors in the spread of these fungal diseases. IMEMA and ISIM contributed to the regional discourse on the mycoses, emphasizing the importance of international cooperation in addressing these public health challenges.

IMEMA/ISIM, held in Santiago del Estero, Argentina, convened experts to discuss endemic and implantation mycoses, covering topics such as epidemiology, diagnostics, treatment, and advocacy. The event highlighted ongoing efforts in combating these diseases.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility.

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Multi-locus sequencing typing reveals geographically related intraspecies variability of Sporothrix brasiliensis.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.

A chromosomal-level reference genome of the widely utilized Coccidioides posadasii laboratory strain "Silveira".

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

New insights into plant natriuretic peptide evolution: From the lysogenic conversion in Xanthomonas to the lateral transfer to the whitefly Bemisia tabaci.

Plant natriuretic peptide-like (PNP) are signaling molecules related to adaptive responses to stress. The Arabidopsis thaliana PNP (AtPNP-A) is capable of modulating catalase 2 (CAT2) and rubisco activase (RCA) activity in some circumstances. Interestingly, many plant-pathogens co-opted PNP-like molecules to their benefit. For instance, the citrus pathogen Xanthomonas citri carries a PNP-like (XacPNP) that can mimic and regulate plant homeostasis, and many phytopathogenic fungi carry effectors (e.g., Ave1 and AvrLm6) that are indeed PNP-like homologs. This work investigates the PNP-like evolution across the tree of life, revealing many parallel gains and duplications in plant and fungi kingdoms. All PNP-like proteins in the final dataset are structurally similar, containing the AtPNP-A active domains modulating CAT2 activity and RCA interaction. Comparative genomics evinced that XacPNP is a lysogenic conversion factor associated with a Myoviridae-like prophage identified in many Xanthomonas species. Surprisingly, a PNP-like homolog was identified in Bemisia tabaci, an important agricultural pest, being to date the second example of lateral gene transfer (LGT) from plant to the whitefly. Moreover, the Bemisia PNP-like homolog can also be considered a potential new effector of this phloem-feeding insect. Noteworthy, the whiteflies infest many plants carrying PNP-like copies and interact with some of their bacterial and fungal pathogens, strongly suggesting complex recipient/donor traits of PNP by LGT and bringing new insights into the evolution of host-pathogen arms race across the tree of life.

Genomic Diversity Analysis Reveals a Strong Population Structure in Histoplasma capsulatum LAmA (Histoplasma suramericanum).

Histoplasmosis is a severe mycotic disease affecting thousands of immunocompetent and immunocompromised individuals with high incidence in Latin America, where the disease agents are Histoplasma capsulatum and Histoplasma suramericanum. In this work, we used whole-genome sequencing to infer the species diversity and the population structure of H. suramericanum in South America. We find evidence for strong population structure and little admixture within the species. Genome-level phylogenetic trees indicate the existence of at least three different discrete populations. We recovered the existence of a previously identified population, LAmB, and confirm that it is highly differentiated along the whole genome. We also find that H. suramericanum is composed of two populations, one in Northern South America, and another in the southern portion of the continent. Moreover, one of the lineages from the southern population is endemic to Rio de Janeiro and there was no association with clinical data and species isolated from patients with histoplasmosis. Our results point out the need to characterize the symptomatology of histoplasmosis caused by different species and lineages of Histoplasma spp.

Mitochondrial genomes of the human pathogens Coccidioides immitis and Coccidioides posadasii.

Fungal mitochondrial genomes encode genes involved in crucial cellular processes, such as oxidative phosphorylation and mitochondrial translation, and the molecule has been used as a molecular marker for population genetics studies. Coccidioides immitis and C. posadasii are endemic fungal pathogens that cause coccidioidomycosis in arid regions across both American continents. To date, approximately 150 Coccidioides isolates have been sequenced to infer patterns of variation in nuclear genomes. However, less attention has been given to the mitochondrial genomes of Coccidioides. In this report, we describe the assembly and annotation of mitochondrial reference genomes for two representative strains of C. posadasii and C. immitis, as well as assess population variation among 77 selected genomes. The sizes of the circular-mapping molecules are 68.2 Kb in C. immitis and 75.1 Kb in C. posadasii. We identify 14 mitochondrial protein-coding genes common to most fungal mitochondria, which are largely syntenic across different populations and species of Coccidioides. Both Coccidioides species are characterized by a large number of group I and II introns, harboring twice the number of elements as compared to closely related Onygenales. The introns contain complete or truncated ORFs with high similarity to homing endonucleases of the LAGLIDADG and GIY-YIG families. Phylogenetic comparisons of mitochondrial and nuclear genomes show extensive phylogenetic discordance suggesting that the evolution of the two types of genetic material is not identical. This work represents the first assessment of mitochondrial genomes among isolates of both species of Coccidioides, and provides a foundation for future functional work.

One Century of Study: What We Learned about Paracoccidioides and How This Pathogen Contributed to Advances in Antifungal Therapy.

Paracoccidioidomycosis (PCM) is a notable fungal infection restricted to Latin America. Since the first description of the disease by Lutz up to the present day, Brazilian researchers have contributed to the understanding of the life cycle of this pathogen and provided the possibility of new targets for antifungal therapy based on the structural and functional genomics of Paracoccidioides. In this context, in silico approaches have selected molecules that act on specific targets, such as the thioredoxin system, with promising antifungal activity against Paracoccidioides. Some of these are already in advanced development stages. In addition, the application of nanostructured systems has addressed issues related to the high toxicity of conventional PCM therapy. Thus, the contribution of molecular biology and biotechnology to the advances achieved is unquestionable. However, it is still necessary to transcend the boundaries of synthetic chemistry, pharmaco-technics, and pharmacodynamics, aiming to turn promising molecules into newly available drugs for the treatment of fungal diseases.

Histoplasmosis Outbreaks in Brazil: Lessons to Learn About Preventing Exposure.

Histoplasmosis is considered the most common invasive opportunistic fungal disease in the Americas, with outbreaks and micro-epidemics reported for over 80 years. In Brazil, this disease has been described since 1946, reaching a remarkable incidence in the population, especially during the HIV-AIDS pandemic. In this study, published and unpublished outbreaks and micro-epidemics of histoplasmosis in Brazil were revisited by accessing different database sources and evaluating epidemiological and clinical features. We have found reports spanning 1946-2017, across 10 Brazilian states and with involvement of 370 humans and 2 dogs, and 13 disseminated cases and 3 deaths were reported. Rio de Janeiro had the largest number of outbreaks (n = 20/40; 50%) reported in this study. The majority of outbreaks and micro-epidemics was reported in caves (n = 21/40; 52.5%), followed by reports in abandoned/deactivated sites (n = 6/40; 15%), mines (n = 5/40; 12.5%), chicken coops (n = 4/40; 10%). Histoplasmosis is a serious health issue in Brazil considering the attractive and growing market of ecotourism throughout more than 7000 caves, and all levels of poultry farming activity are important to raise awareness about how dangerous this neglected disease can be and establish ways to decrease exposure to contaminated environmental sources through adequate preventive measures.

Coccidioidal Meningitis in New York Traced to Texas by Fungal Genomic Analysis.

A child developed hydrocephalus. Sixteen months later, it was discovered to be a complication of coccidioidal meningitis. The infection's source was uncertain until genomic analysis of the fungal isolate identified its origin to be a visit to Beeville, Texas. Improved national reporting of cases of coccidioidomycosis might reduce diagnostic delays.

Mechanisms of copper and zinc homeostasis in pathogenic black fungi.

Black fungi comprise a diverse group of melanized microorganisms, many of which are able to infect humans. One of the recognized diseases that arise with black fungi infection is chromoblastomycosis, a neglected implantation mycosis. Considering their ecology, black fungi may face conditions with distinct metal availability. Zinc and copper are essential transition metals, which become toxic in excess. During the interaction with host, fungi may face either metal deprivation or poisoning. Here we report an in silico analysis of four black fungi genomes concerning zinc and copper homeostasis. Overall, these organisms share apparatus of metal uptake, storage and detoxification with other pathogenic and non-pathogenic fungi. Genes coding plasma membrane and organelle transporters, as well as metal binding proteins were identified. Althought putatives zinc and copper responsive transcription factors have been found in the analyzed genomes, remarkable structural differences were perceived when compared to the already characterized regulators. Black fungi may harbor unique features concerning the regulation of zinc and copper homeostasis, which is probably a result of the niches they can inhabit. The data provided here add knowlegde to a still unexplored aspect of black fungi biology that may be useful in the understanding of their pathogenicity.

Transposable elements and two other molecular markers as typing tools for the genus Paracoccidioides.

Studies comparing Paracoccidioides brasiliensis and Paracoccidioides lutzii have shown that these fungi have significant genomic differences that may have implications in the clinical manifestation, diagnosis, and treatment of paracoccidioidomycosis caused by them. Thus, molecular typing methods are required that can distinguish between various species of Paracoccidioides. The aim of this study was to explore the potential use as molecular markers of the transposable elements Trem A-H recently identified and characterized in the genus Paracoccidioides as a means of differentiating the species. We take advantage of the abundance and distribution of these transposons in the Paracoccidioides genomes to develop a simple and highly reproducible polymerase chain reaction (PCR)-based technique. Furthermore we compare the performance of this test with two other molecular markers already in use to identify these fungi.

Chromosomal polymorphism in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (ß-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.

Phylogenetic analysis reveals a high prevalence of Sporothrix brasiliensis in feline sporotrichosis outbreaks.

Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n =3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.

Pulmonary cavitation and skin lesions mimicking tuberculosis in a HIV negative patient caused by Sporothrix brasiliensis.

A 32-year-old HIV negative male presented with multiple pulmonary cavitation and skin abscesses up to 15 cm in diameter mimicking tuberculosis. Sporothrix brasiliensis was isolated and patient responded well to amphotericin B followed by itraconazole, except the skin lesions that had to be surgical drained to obtain cure.