Background: Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. Methodology/Principal findings: This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS. Conclusions/Significance: Development of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers.

The lungs have great importance in patients with paracoccidioidomycosis since they are the portal of entry for the infecting fungi, the site of quiescent foci, and one of the most frequently affected organs. Although they have been the subject of many studies with different approaches, the severity classification of the pulmonary involvement, using imaging procedures, has not been carried out yet. This study aimed to classify the active and the residual pulmonary damage using radiographic and tomographic evaluations, according to the area involved and types of lesions.

Introduction: This study standardized a semi-quantitative dot blotting assay (DB) and a quantitative real-time polymerase chain reaction (qPCR) to detect specific antibodies for Paracoccidioides brasiliensis and its DNA in PCM patients. Methodology: We evaluated 42 confirmed PCM patients upon admission using a serological double agar gel immunodiffusion test (DID), DB, and molecular tests (qPCR in total blood). The control groups included 42 healthy individuals and 37 patients with other infectious diseases. The serological progress during treatment was evaluated in eight patients, and there was a relapse diagnosis in ten patients using the Pb B.339 strain antigen. The cut-off points for the serological tests were determined by a receiver operator characteristic curve. Results: The DB and DID tests showed similar accuracy, but the DB identified lower antibody concentrations. Cross-reactions were absent in the DB assay. In the relapse diagnoses, DB exhibited much higher sensitivity (90%) than DID (30%). Conclusions: A DB assay is easier and faster than a DID test to be performed; DB and DID tests show the same accuracy, while blood qPCR is not recommended in the diagnosis at the time of admission; cross-reactions were not observed with other systemic diseases; DB and DID tests are useful for treatment monitoring PCM patients; and a DB assay is the choice for diagnosing relapse. These findings support the introduction of semi-quantitative DB assays in clinical laboratories.

Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at -80^oC for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at -20^oC for up to six months. Differences of titers higher than one dilution were considered discordant. First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Storage at -80^oC for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at -20^oC.

Background: The increasing incidence of Candida glabrata complex infections in hospitalized patients and their association with high mortality rates prompted the determination of cryptic species among clinical isolates from a tertiary hospital and the evaluation of their susceptibility profiles to commonly prescribed antifungal agents. Methods: This study evaluated 80 C. glabrata isolates obtained from patients admitted to a Brazilian public tertiary hospital. The isolates were recovered from different clinical specimens, predominantly urine and blood, across various medical units. Results: MALDI-TOF MS analysis revealed that all isolates were Candida glabrata sensu stricto. Minimum inhibitory concentrations (MICs), determined via broth microdilution and according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, showed high susceptibility to amphotericin B (AmB), voriconazole (VRC), and echinocandins (ECNs) [caspofungin (CSF), anidulafungin (ANF), and micafungin (MCF)], as well as uniform susceptibility within the "I" category (susceptible, increased exposure) to fluconazole (FLC). Comparisons of resistance profiles revealed higher prevalences of resistance to AmB and VRC than to ECNs, both overall and in urine isolates, with a similar trend observed in blood isolates. Comparative analysis with reference EUCAST C. glabrata data highlighted higher localized MIC values for AmB, ANF, and MCF, lower MICs for FLC, and equivalent distributions for VRC. Conclusion: All recovered isolates were confirmed as Candida glabrata sensu stricto and exhibited different susceptibility profiles from reference EUCAST isolates. These findings establish a strong regional baseline, serving as a useful guide for monitoring therapeutic measures and supporting antimicrobial stewardship.

Background: Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America with marked clinical heterogeneity, suggesting a role for host immunogenetic factors. The NLRP3 inflammasome and IL-1β signaling are important in antifungal immunity, but human genetic evidence remains limited. We evaluated whether single-nucleotide variants (SNVs) in inflammasome-related genes (NLRP1, NLRP3, CARD8, CASP1, and IL1B) are associated with PCM susceptibility, clinical form, and disease severity, including gene–gene interactions. Methods: We conducted a case–control study including 346 individuals (154 people with PCM and 192 gp43-reactive healthy controls). Genetic associations were tested under multiple inheritance models using logistic regression adjusted for age, sex, smoking, and alcoholism. Epistatic interactions were assessed within the inflammasome–IL-1β axis, with Firth penalized logistic regression as a sensitivity analysis. Results: No single-SNV association with PCM susceptibility, clinical form, or severity remained significant after multiple-testing correction. Pairwise epistasis analysis identified an interaction between NLRP3 rs10754558 and IL1B rs1143634 associated with reduced PCM susceptibility (odds ratio [OR] = 0.34; 95% CI, .17–.69; false discovery rate-adjusted P value [pFDR]= 0.015), supported by Firth regression. No significant epistatic interactions were found for clinical form or severity. Conclusions: Genetic interactions within the inflammasome–IL-1β axis may contribute to PCM susceptibility. The interaction between NLRP3 rs10754558 and IL1B rs1143634 warrants validation in larger, independent cohorts.