Rosely Maria Zancopé-Oliveira

Sporotrichosis is a subacute to chronic mycosis of major public health relevance in endemic regions, affecting both humans and animals, particularly cats. Rapid and accessible diagnostic tools are urgently needed to improve case detection and disease surveillance. Thus, this study aimed to validate a previously described loop-mediated isothermal amplification (LAMP) assay for the diagnosis of sporotrichosis in clinical samples from humans and cats obtained in two highly endemic areas in Brazil. We conducted an observational study including human patients and cats with suspected sporotrichosis. Analytical sensitivity and specificity were initially assessed using reference strains of Sporothrix spp. The diagnostic performance of the LAMP assay was then evaluated in clinical samples at different incubation times and compared with culture results. Sensitivity, specificity, and additional diagnostic parameters were calculated separately for human and cat cases. The LAMP assay was able to detect the three major Sporothrix species associated with sporotrichosis, with higher analytical sensitivity for S. brasiliensis . However, when applied to human clinical samples, the assay showed limited sensitivity and specificity, particularly at earlier incubation times, resulting in poor overall diagnostic performance. In contrast, performance in cat samples was comparatively higher but still affected by false-positive and false-negative results. Despite promising analytical characteristics and previous success in experimental models, this LAMP protocol demonstrated limited diagnostic utility for human and cat sporotrichosis under real-world clinical conditions, highlighting the need for further optimization and rigorous clinical validation before implementation in routine practice.

Sporothrix brasiliensis is the predominant etiological agent of sporotrichosis in Brazil and has been progressively expanding its geographic distribution to other countries in South America. The human patients infected with S . brasiliensis usually respond favorably to antifungal therapy. However, therapeutic failure may occur, and management may be particularly challenging in cases of severe disease. We recently reported that at least seven S. brasiliensis genotypes circulate across South America. However, their biological and clinical relevance of this genetic diversity remains poorly understood. In this study, we investigated whether distinct S . brasiliensis genotypes are associated with reduced susceptibility to the main antifungal agents used in the treatment of human and animal sporotrichosis. A total of 92 clinical, veterinary, and environmental S. brasiliensis strains from Brazil, Paraguay and Argentina, genotyping them using a four-gene MLST scheme. Mating type was assessed as well as antifungal susceptibility according to the M38-A2 CLSI guidelines. Results were interpreted according to the established epidemiological cutoff values for S. brasiliensis . The isolates are clustered into six distinct genotypes, with mating type showing a strong association with phylogenetic clades. Non-wild type strains were identified across four haplotypes (H1, H2, H3 and H4) for all antifungal drugs tested. These non-wild type strains were isolated from six distinct Brazilian states and were not associated to a specific genetic background. Nevertheless, non-wild-type strains were predominantly detected among feline isolates, most of which originated from Minas Gerais and belonged to haplotype H2. However, because only a limited number of H2 isolates were included in this study, additional isolates from this haplotype are need to confirm this association. Together, these findings suggest that decreased antifungal susceptibility in S. brasiliensis is not genotype-restricted but may be influenced by host-related or epidemiological factors, particularly in the context of feline sporotrichosis.

Fungal pneumonias and tuberculosis (TB) remain major causes of mortality among people living with advanced HIV disease (PLWHA). Their overlapping clinical manifestations delay diagnosis in developed and resource-limited settings, with greater impact where access to rapid diagnostic tools is limited. This study evaluated the performance of a multiplex quantitative PCR (qPCR) assay for simultaneous detection of Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jirovecii, and Mycobacterium tuberculosis complex in respiratory samples from PLWHA. A cross-sectional study was conducted at the Instituto Nacional de Infectologia Evandro Chagas (Rio de Janeiro, Brazil) between December 2023 and March 2025. Adults with HIV infection (CD4 < 200 cells/mm³) and clinical or radiological evidence of pulmonary infection were enrolled. Standard diagnostic methods included culture, microscopy, antigen detection (CrAg-LFA, urinary Histoplasma antigen, TB-LAM), GeneXpert® MTB/RIF, and commercial qPCR for P. jirovecii. The multiplex assay targeted the ITS region (H. capsulatum and C. neoformans), mtLSU (P. jirovecii), and IS6110 (M. tuberculosis). Among 198 screened participants, 88 met inclusion criteria (mean CD4 count: 56 cells/µl). Diagnosis included TB (56.8%), pneumocystosis (34.1%), histoplasmosis (10.2%), and cryptococcosis (11.4%); 25% presented coinfections. The multiplex qPCR showed 100% sensitivity for histoplasmosis, 80% for pneumocystosis, and 40% for both cryptococcosis and TB, with specificities ≥ 82% and 100% for TB. Overall accuracy ranged from 66% to 86%. Mortality was 12.5%, highest among cryptococcosis cases (20%). The assay provided accurate detection of H. capsulatum and P. jirovecii, although performance was limited for TB and cryptococcosis, supporting its use as a complementary molecular tool for differential diagnosis in PLWHA.

Cutaneous leishmaniasis (CL) is a vector-borne disease caused by Leishmania spp. Molecular methods such as conventional PCR (cPCR) and real-time PCR (qPCR) show high accuracy but remain restricted to reference centers. Loop-mediated isothermal amplification (LAMP) enables rapid DNA amplification under isothermal conditions. LAMP targeting 18S rDNA using WarmStart^® Colorimetric LAMP 2× Master Mix and Bst 2.0 DNA Polymerase with SYBR^® Green I (BstSg) was evaluated in swab samples (extracted by kit—SW-kit and direct boil—SW-DB) and specimens collected through biopsy from ulcerated lesions of patients attending a reference center in Rio de Janeiro, Brazil. The reference standard was defined as at least one positive parasitological or molecular (cPCR) test plus clinical confirmation. Analytical sensitivity reached 10 fg with WS and 10 pg with BstSg. The diagnostic accuracy of LAMP ranged from 51.8% to 78.6%, with sensitivity from 52.5% to 81% and specificity from 39.6% to 89.4%. LAMP, particularly when applied to swab samples with DNA extracted using the SW-kit protocol and amplified with BstSg, showed promising diagnostic accuracy and operational feasibility as a less invasive approach for CL diagnosis, eliminating the need for skin biopsy and reducing patient discomfort during sample collection.

Background/Objectives: While various clinical manifestations occur in sporotrichosis, cutaneous forms predominate. The recommended sporotrichosis treatment is itraconazole, an antifungal with certain restrictions. In recent years, the observation of reduced treatment effectiveness in some patients has arisen, possibly due to Sporothrix spp. resistance mechanisms. Consequently, there is a growing need for alternative therapeutic approaches. This study investigates the antifungal activity of manuka honey (MH) against pathogenic species of the genus Sporothrix. Methods: In this study, we assessed MH antifungal efficacy across concentrations ranging from 5% to 40% against 26 Sporothrix spp. isolates. In addition, its components were evaluated through chromatography and other in vitro techniques. Results: Minimum inhibitory concentrations of MH were found to be 15–40%, 10–15%, and 5–10% for Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa, respectively. Purified methylglyoxal did not hinder Sporothrix growth. The MH antifungal potential was compromised through treatment with catalase or filtration through a 0.22 µm cellulose membrane. Chromatographic analysis of the volatile organic compounds (VOCs) present in MH identified 40 VOCs, including carbonyl compounds, alcohols, esters, aromatic hydrocarbons, heterocyclic compounds, terpenoids, and carboxylic acids. Additionally, two phenolic compounds were identified as potential markers for the authentication of MH, along with a disaccharide that may contribute to its antifungal activity. Conclusions: MH has demonstrated biological activity against the most significant Sporothrix species with pathogenic impact on humans. This suggests its consideration in future research endeavors focused on novel topical treatments for cutaneous sporotrichosis in both human and animal subjects.

Paracoccidiodomycosis (PCM) is the most important systemic mycosis in Brazil, and is usually associated with rural work. PCM is caused by inhalation of infective propagules of thermodimorphic fungi from the genus Paracoccidioides. In the past, it was believed that Paracoccidioides brasiliensis was the single species responsible for PCM cases. However, recent advances in molecular methods allowed the description of several new species, using phylogenetic concordance as the gold standard. Aside from P. brasiliensis sensu stricto, Paracoccidioides americana is also endemic in Rio de Janeiro state, Brazil. This study aimed to evaluate intraspecific genetic variability of Paracoccidioides isolates from patients diagnosed with PCM at a reference center for endemic mycoses in Rio de Janeiro state, from 2015 to 2021. Among the sixteen retrieved isolates, three (18.75%) were identified as P. americana and thirteen (81.25%) as P. brasiliensis sensu stricto. No intraspecific genetic variation was observed by the M-13 primer in P. americana isolates from this geographic region. However, P. brasiliensis sensu stricto isolates were clustered into two distinct molecular profiles, despite being grouped in a single clade in the phylogenetic tree after partial sequencing of arf and gp43 genes. The results suggest a single P. americana lineage and two P. brasiliensis populations causing PCM in Rio de Janeiro, Brazil.

During the COVID-19 pandemic, fungal infections, especially pulmonary aspergillosis, mucormycosis, and invasive candidiasis, have emerged as a significant health concern. Beyond Candida albicans, the most common cause of invasive candidiasis, other rare ascomycetous yeast species have been described in tertiary care units, potentially posing a broader health threat. We have isolated, from September 2020 to June 2021, nine Diutina catenulata strains from urine samples of six patients. This was intriguing as this fungus had not been previously identified in our institution, nor after June 2021. Therefore, we decided to outline the clinical features of the patients with this rare pathogen, to describe phenotypic characteristics, including antifungal susceptibility profiles, of this yeast species and to identify the genetic makeup through whole-genome sequencing analysis to evaluate if this was a cluster of genetically similar D. catenulata isolates in our institution. The strains were identified through MALDI-TOF MS analyses and Sanger sequencing of two rDNA regions. All patients yielding D. catenulata from urine samples needed ventilator support and used urinary catheters during hospitalization for treatment of COVID-19. None of them had received COVID-19 vaccines. Morphological and biochemical profiles of the nine strains were largely consistent, although fluconazole susceptibility varied, ranging from 4 to 32 μg/mL. Phylogenomic analysis revealed minimal genetic variation among the isolates, with low intrapopulation variation, supported by the identification of only 84 SNPs across all strains. Therefore, we propose that the yeast strains isolated were part of a cluster of D. catenulata funguria in the context of COVID-19.

Over the past two decades, zoonotic sporotrichosis transmitted by naturally infected cats has become hyperendemic in Rio de Janeiro, Brazil. Sporothrix brasiliensis is the main agent involved. However, there are other forms of transmission of sporotrichosis. The aim of this study was to evaluate and associate the epidemiological, clinical and therapeutic data and the susceptibility of Sporothrix spp. to antifungal drugs in 43 non-zoonotic sporotrichosis cases. Forty-three clinical strains of Sporothrix were identified by partial sequencing of the calmodulin gene. An antifungal susceptibility test of amphotericin B, terbinafine, itraconazole, posaconazole and isavuconazole was performed according to the broth microdilution method. Most patients were male (55.8%). Regarding the source of infection, 21 patients (48.8%) reported trauma involving plants and/or contact with soil. Sporothrix brasiliensis was the predominant species (n = 39), followed by S. globosa (n = 3) and S. schenckii (n = 1). Sporothrix brasiliensis was associated with all the sources of infection, reinforcing previous data showing the presence of this species in environmental sources, as well as with all the clinical forms, including severe cases. One clinical strain of Sporothrix brasiliensis was classified as a non-wild-type strain for amphotericin B and another for itraconazole. S. schenckii was classified as non-WT for all the antifungals tested. In this context, it is important to emphasize that non-zoonotic sporotrichosis still occurs in the state of Rio de Janeiro, with S. brasiliensis as the main etiological agent, primarily associated with infections acquired after traumatic inoculation with plants and/or soil contact, followed by S. globosa and S. schenckii. In addition, non-WT strains were found, indicating the need to monitor the antifungal susceptibility profile of these species. It is crucial to investigate other natural sources of S. brasiliensis to better understand this fungal pathogen and its environment and host cycle.