Screening of Candida spp. in wastewater in Brazil during COVID-19 pandemic: workflow for monitoring fungal pathogens.

Fungal diseases are often linked to poverty, which is associated with poor hygiene and sanitation conditions that have been severely worsened by the COVID-19 pandemic. Moreover, COVID-19 patients are treated with Dexamethasone, a corticosteroid that promotes an immunosuppressive profile, making patients more susceptible to opportunistic fungal infections, such as those caused by Candida species. In this study, we analyzed the prevalence of Candida yeasts in wastewater samples collected to track viral genetic material during the COVID-19 pandemic and identified the yeasts using polyphasic taxonomy. Furthermore, we investigated the production of biofilm and hydrolytic enzymes, which are known virulence factors. Our findings revealed that all Candida species could form biofilms and exhibited moderate hydrolytic enzyme activity. We also proposed a workflow for monitoring wastewater using Colony PCR instead of conventional PCR, as this technique is fast, cost-effective, and reliable. This approach enhances the accurate taxonomic identification of yeasts in environmental samples, contributing to environmental monitoring as part of the One Health approach, which preconizes the monitoring of possible emergent pathogenic microorganisms, including fungi.

A novel report on the emerging and zoonotic neurotropic fungus Trichosporon japonicum in the brain tissue of the endangered Brazilian guitarfish (Pseudobatos horkelii) off the southeastern coast of Brazil.

Yeast infections have gained significant attention in the field of marine biology in recent years. Among the broad diversity of marine organisms affected by these infections, elasmobranchs (sharks and rays) have emerged as highly susceptible, due to climate change effects, such as increasing water temperatures and pollution, which can alter the composition and abundance of fungal communities. Additionally, injuries, or compromised immune systems resulting from pollution or disease may increase the likelihood of fungal infections in elasmobranchs. Studies are, however, still lacking for this taxonomic group. In this context, this study aimed to screen yeast species in cell cultures obtained from the brain of artisanally captured Pseudobatos horkelii, a cartilaginous fish that, although endangered, is highly captured and consumed worldwide. Fungi were isolated during an attempt to establish primary cultures of elasmobranch neural cells. Culture flasks were swabbed and investigated using morphological, phenotypic, and molecular techniques. Two isolates of the emerging opportunistic pathogen Trichosporon japonicum were identified, with high scores (1.80 and 1.85, respectively) by the MALDI-ToF technique. This is the first report of the basidiomycetous yeast T. japonicum in Pseudobatos horkelii in Brazil. This finding highlights the need for further research to determine the potential impact on elasmobranch health, ecology, as well as on commercial fisheries.

Cerebrospinal fluid: a target of some fungi and an overview.

Meningitis is a potentially life-threatening infection characterised by the inflammation of the leptomeningeal membranes. The estimated annual prevalence of 8.7 million cases globally and the disease is caused by many different viral, bacterial, and fungal pathogens. Although several genera of fungi are capable of causing infections in the central nervous system (CNS), the most significant number of registered cases have, as causal agents, yeasts of the genus Cryptococcus. The relevance of cryptococcal meningitis has changed in the last decades, mainly due to the increase in the number of people living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and medications that impair the immune responses. In this context, coronavirus disease 19 (COVID-19) has also emerged as a risk factor for invasive fungal infections (IFI), including fungal meningitis (FM), due to severe COVID-19 disease is associated with increased pro-inflammatory cytokines, interleukin (IL)-1, IL-6, and tumour necrosis factor-alpha, reduced CD4-interferon-gamma expression, CD4 and CD8 T cells. The gold standard technique for fungal identification is isolating fungi in the culture of the biological material, including cerebrospinal fluid (CSF). However, this methodology has as its main disadvantage the slow or null growth of some fungal species in culture, which makes it difficult to finalise the diagnosis. In conclusions, this article, in the first place, point that it is necessary to accurately identify the etiological agent in order to assist in the choice of the therapeutic regimen for the patients, including the implementation of actions that promote the reduction of the incidence, lethality, and fungal morbidity, which includes what is healthy in the CNS.

A comprehensive assessment of leachate contamination at a non-operational open dumpsite: mycoflora screening, metal soil pollution indices, and ecotoxicological risks.

The final disposal of municipal solid waste (MSW) in dumpsites is still a reality worldwide, especially in low- and middle-income countries, leading to leachate-contaminated zones. Therefore, the aim of this study was to carry out soil and leachate physicochemical, microbiological, and toxicological characterizations from a non-operational dumpsite. The L-01 pond samples presented the highest physicochemical parameters, especially chloride (Cl; 4101 ± 44.8 mg L-1), electrical conductivity (EC; 10,452 ± 0.1 mS cm-1), and chemical oxygen demand (COD; 760 ± 6.6 mg L-1) indicating the presence of leachate, explained by its close proximity to the landfill cell. Pond L-03 presented higher parameters compared to pond L-02, except for N-ammoniacal and phosphorus levels, explained by the local geological configuration, configured as a slope from the landfill cell towards L-03. Seven filamentous and/or yeast fungi genera were identified, including the opportunistic pathogenic fungi Candida krusei (4 CFU) in an outcrop sample. Regarding soil samples, Br, Se, and I were present at high concentrations leading to high soil contamination (CF ≤ 6). Pond L-02 presented the highest CF for Br (18.14 ± 18.41 mg kg-1) and I (10.63 ± 3.66 mg kg-1), while pond L-03 presented the highest CF for Se (7.60 ± 1.33 mg kg-1). The most severe lethal effect for Artemia salina was observed for L-03 samples (LC50: 79.91%), while only samples from L-01 were toxic to Danio rerio (LC50: 32.99%). The highest lethality for Eisenia andrei was observed for L-02 samples (LC50: 50.30%). The applied risk characterization indicates high risk of all proposed scenarios for both aquatic (RQ 375-909) and terrestrial environments (RQ > 1.4 × 105). These findings indicate that the investigated dumpsite is contaminated by both leachate and metals, high risks to living organisms and adjacent water resources, also potentially affecting human health.

Geographic Expansion of Sporotrichosis, Brazil.

Brazil has experienced geographic expansion of zoonotic sporotrichosis. Social problems in the country contribute substantially to the expansion. A comprehensive sporotrichosis control program is beyond the sphere of public health. A One Health approach is needed to control the disease in animals and humans.

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii.

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 °C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.

Feline sporotrichosis: associations between clinical-epidemiological profiles and phenotypic-genotypic characteristics of the etiological agents in the Rio de Janeiro epizootic area.

BACKGROUND: Sporotrichosis is caused by species of the genus Sporothrix. From 1998 to 2015, 4,703 cats were diagnosed at the Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil. Even after the description of the Sporothrix species, the characterisation of feline isolates is not performed routinely. OBJECTIVES: To characterise the clinical isolates from cats at the species level and correlate them with the clinical and epidemiological characteristics of the cats. METHODS: Forty seven Sporothrix spp. isolates from cats assisted at Fiocruz from 2010 to 2011 were included. Medical records were consulted to obtain the clinical and epidemiological data. The isolates were identified through their morphological and physiological characteristics. T3B polymerase chain reaction (PCR) fingerprinting was used for molecular identification of the species. FINDINGS: In phenotypic tests, 34 isolates were characterised as S. brasiliensis, one as S. schenckii and 12 as Sporothrix spp. PCR identified all isolates as S. brasiliensis. MAIN CONCLUSIONS: S. brasiliensis is the only etiological agent of feline sporotrichosis in Rio de Janeiro to date. None association was found between the isolates and the clinical and epidemiological data. In addition, we strongly recommend the use of molecular techniques for the identification of isolates of Sporothrix spp.

Successful Treatment of Canine Sporotrichosis with Terbinafine: Case Reports and Literature Review.

Sporotrichosis occurs worldwide, and the metropolitan region of Rio de Janeiro, Brazil, is a main endemic area, with a large number of human and animal cases in the last 19 years. This mycosis is more frequently described in cats rather than in dogs. There are a limited number of oral antifungal agents for the treatment of sporotrichosis in animals. In this context, the effectiveness of terbinafine in the treatment of sporotrichosis in humans, as well as the promising results of in vitro susceptibility tests, inspired us to use this drug in the therapy of this mycosis in dogs. We reported for the first time the use of terbinafine in the treatment of two dogs with sporotrichosis caused by Sporothrix brasiliensis. Moreover, we provided an overview of therapeutic features of canine sporotrichosis cases reported since the 1960s. One of the dogs presented the fixed cutaneous form of the disease, while the other patient presented hyperemia of the nasal mucosa and respiratory signs only. Terbinafine showed high antifungal activity in vitro against the canine Sporothrix isolates. The dogs were successfully treated with terbinafine, with remission of all clinical signs initially presented. The current reports indicate that this drug can emerge as a therapeutic option for canine sporotrichosis.

Refractory sporotrichosis due to Sporothrix brasiliensis in humans appears to be unrelated to in vivo resistance.

Sporotrichosis is a subacute to chronic infection caused by members of the Sporothrix schenckii complex. Itraconazole is the first choice antifungal drug for treating this infection, with terbinafine and potassium iodide as alternatives and amphotericin B used in cases of severe infections. Correlation of antifungal susceptibility data with the clinical outcome of the patients is scarce. The aim of this study was to correlate clinical and mycological data in patients with refractory sporotrichosis. In this work, antifungal susceptibilities, determined according to the reference M38-A2 CLSI protocol, of 25 Sporothrix strains, isolated from seven human cases of sporotrichosis with adversities in the treatment, are presented. Tested drugs included itraconazole, ketoconazole, posaconazole, voriconazole, terbinafine, and amphotericin B. Fungi were identified using the T3B PCR fingerprinting. This method identified all strains as Sporothrix brasiliensis and also demonstrated a high degree of similarity between the strains. In general, voriconazole was ineffective against all strains, and elevated minimal inhibitory concentrations (MICs) were observed for amphotericin B. High itraconazole and terbinafine MICs were not observed in S. brasiliensis isolates from patients of this study. Moreover, a significant increase in itraconazole and terbinafine MIC values from strains isolated from the same patient in different periods was not observed. The results suggest that the antifungal susceptibility to terbinafine and itraconazole determined by the reference method does not play an important role in therapeutic failure of sporotrichosis and that acquisition of resistance during prolonged antifungal treatment is not likely to occur in S. brasiliensis.

Minimal inhibitory concentration distributions and epidemiological cutoff values of five antifungal agents against Sporothrix brasiliensis.

BACKGROUND: Sporothrix brasiliensis is the most virulent sporotrichosis agent. This species usually responds to antifungal drugs, but therapeutic failure can occur in some patients. Antifungal susceptibility tests have been performed on this species, but no clinical breakpoints (CBPs) are available. In this situation, minimal inhibitory concentration (MIC) distributions and epidemiological cutoff values (ECVs) support the detection of identification of resistant strains. OBJECTIVES: To study the MIC distributions of five antifungal drugs against S. brasiliensis and to propose tentative ECVs. METHODS: MICs of amphotericin B (AMB), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), and terbinafine (TRB) against 335 S. brasiliensis strains were determined by the Clinical and Laboratory Standards Institute broth microdilution method. FINDINGS: The proposed ECV, in µg/mL, for AMB, ITR, KET, POS, and TRB were 4.0, 2.0, 1.0, 2.0, and 0.25, respectively. Percentages of wild-type strains in our population for the above antifungal drugs were 98.48, 95.22, 95.33, 100, and 97.67%, respectively. MAIN CONCLUSIONS: These ECVs will be useful to detect strains with resistance, to define CBPs, and to elaborate specific therapeutic guidelines for S. brasiliensis. Rational use of antifungals is strongly recommended to avoid the emergence of resistant strains and ensure the therapeutic effectiveness of sporotrichosis.

Paracoccidioides brasiliensis PS2: First Autochthonous Paracoccidioidomycosis Case Report in Rio de Janeiro, Brazil, and Literature Review.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by pathogenic dimorphic fungi of the Paracoccidioides brasiliensis complex. It is the most important systemic mycosis in Latin America, mainly in Brazil. Despite its severity and high mortality rates, it is considered a neglected disease. Species within the genus Paracoccidioides present genetics and morphological variations with probable clinical, diagnostic and therapeutic consequences. In fact, there are a very small number of detailed case reports with molecular identification of these fungal agents. Here, it is reported a case of PCM due to Paracoccidioides brasiliensis PS2. Molecular identification of the isolate was performed by amplification and sequencing of the arf and gp43 genes. Clinical cases and strain reports with molecular identification in the literature are also reviewed. The case herein presented is the first autochthonous report of PCM due to Paracoccidioides brasiliensis PS2 species in the state of Rio de Janeiro, Brazil, an important endemic area. The patient presented a chronic pulmonary form of PCM and had a satisfactory response to sulfamethoxazole/trimethoprim although sequelae such as adrenal insufficiency and dysphonia were observed. This study may contribute to improve the knowledge about this severe disease, its causative cryptic species and their consequences to patients.

Systematic review of literature to evaluate global distribution of species of the Sporothrix genus stored in culture collections.

Introduction: Sporotrichosis is a subcutaneous mycosis caused by fungi of the genus Sporothrix sp. Phenotypic and genotypic differences have been associated with their geographic distribution, virulence, or clinical manifestation of sporotrichosis. In the past decade, the interest in identifying species of the Sporothrix sp. has been increasing, due to its epidemiological importance and, in consequence, is important to know how to preserve them for future studies, in culture collection. Aims: The purposes of this study were to analyze the global distribution of environmental isolates and/or causal agents of sporotrichosis identified by polyphasic taxonomy, with mandatory use of molecular identification, and to evaluate the percentages and distribution of isolates stored in culture collections. Methods: A systematic review of articles on animal and human sporotrichosis and/or environmental isolation of the fungus, from 2007 to 2023, was done. Results: Our results demonstrated that, S. globosa, S. schenckii, and S. brasiliensis were the most identified species. With respect to the deposit and maintenance of species, we observed that only 17% of the strains of Sporothrix sp. isolated in the world are preserved in a culture collection. Conclusions: This systematic review confirmed a difficulty in obtaining the frequency of Sporothrix species stored in culture collection and insufficient data on the molecular identification mainly of animal sporotrichosis and isolation of Sporothrix sp. in environmental samples.

Rapid Molecular Diagnosis of Sporotrichosis Directly from Biological Samples from a Reference Center in Brazil.

The gold standard diagnosis of sporotrichosis is the isolation of Sporothrix sp. in culture media, but this is a time-consuming test that is susceptible to contamination and can be affected by the fungal load. Molecular methods such as nested PCR are gaining more ground in the management of several infections as they are tools for the rapid and accurate identification of microorganisms from pure cultures or directly from biological samples. This study aimed to apply a nested PCR molecular protocol for the rapid detection of Sporothrix spp. directly from clinical samples. Thirteen samples-six from skin biopsies, five from skin exudates, and two from conjunctival secretions-were obtained from patients diagnosed with sporotrichosis due to S. brasiliensis. Calmodulin gene sequencing identified all the isolates as S. brasiliensis. Nested PCR was able to detect all the Sporothrix sensu lato directly from clinical samples as well as the CBS 120339 reference strain. The nested PCR protocol stands out as a diagnostic alternative, as it allows the identification of Sporothrix spp. directly from clinical samples without the need for fungal isolation.

Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil.

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

Colonized patients by Candida auris: Third and largest outbreak in Brazil and impact of biofilm formation.

Objective: To describe the clinical-epidemiological features of patients colonized by Candida auris in the largest outbreak in Brazil and to show the biofilm formation capacity of yeast strains. Methods: Clinical yeasts suspected of C. auris isolated from urine and surveillance samples were seeded on chromogenic media at 30°C and Sabouraud agar at 42°C. matrix-assisted laser desorption/ionization-time of flight mass spectometry was used for reliable identification. After proteomic confirmation, the genomic approach and culture on Chromagar Candida Plus media were carried out. Biofilm formation was investigated based on metabolic activity, and the clinical-epidemiological profile of patients was described. Results: A total of 11 C. auris clinical yeasts from nine patients were identified between the end of December 2021 and March 2022. Two clinical yeasts were isolates from urine and nine clinical yeasts were isolates from axillary and inguinal surveillance swabs. No case is related to previous Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, all the yeasts showed a high ability of biofilm formation. Conclusion: C. auris requires great vigilance as its high capacity to colonize and form biofilms contributes to its dissemination. The rapid and precise identification of this species is essential for the management, control, and prevention of infections.

A Novel One Health Approach concerning Yeast Present in the Oral Microbiome of the Endangered Rio Skate (Rioraja agassizii) from Southeastern Brazil.

The current climate change scenario caused by anthropogenic activities has resulted in novel environmental pressures, increasing the occurrence and severity of fungal infections in the marine environment. Research on fungi in several taxonomic groups is widespread although not the case for elasmobranchs (sharks and rays). In this context, the aim of the present study was to screen the oral fungal microbiota present in artisanally captured Rioraja agassizii, a batoid that, although endangered, is highly fished and consumed worldwide. Oropharyngeal samples were obtained by swabbing and the samples were investigated using morphological and phenotypic methods by streaking on Sabouraud Dextrose Agar (SDA) and subculturing onto CHROMagar Candida (BD Difco) and CHROMagar Candida Plus (CHROMagarTM), as well as molecular techniques by amplification of the ITS1-5.8S-ITS2 ribosomal DNA region and a MALDI-TOF MS assessment. The findings indicated the presence of Candida parapsilosis (seven isolates), Candida duobushaemulonii (one isolate) and Rhodotorula mucilaginosa (three isolates), several of these reported for the first time in Rioraja agassizii. In addition, a 100% agreement between the MALDI-TOF results and partial ITS region sequencing was noted, demonstrating that the MALDI-TOF MS is a rapid and effective alternative for yeast identification in Rioraja agassizii isolates and potentially in other elasmobranch species. These findings highlight the need for further research to determine the potential impact on elasmobranch health, ecology, and commercial fisheries. Furthermore, this research is paramount in a One Health framework and may be employed to predict elasmobranch responses to an evolving ocean, keep healthy populations in check, monitor species, and assess the public health consequences of consuming these species.

The Mating Type (MAT) and Virulence of Sporothrix schenckii sensu stricto Isolates Maintained in Culture Collection.

In an attempt to determine the mating type of different Sporothrix schenckii sensu stricto isolates that remained viable after a long period of preservation in a culture collection and to correlate them with the degree of virulence/pathogenicity, a PCR technique using primers designed for the sequences of MAT1-1-1 and MAT1-2-1 genes and a murine experimental model were used. The results showed that there was no correlation between the mating type and virulence among the isolates. Furthermore, different degrees of virulence/pathogenicity, ranging from high to low, were found among them based on different virulence parameters. It was assumed that the long period of preservation favored the changes, yielding the isolation of variants. Thus, we believe that new technologies for studies on factors can improve our knowledge of the pathogenesis of sporotrichosis.

Candida palmioleophila: A New Emerging Threat in Brazil?

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal diseases. In addition to climate alterations, the onset of the COVID-19 pandemic has also been associated with the emergence of fungal pathogens. Fungi showed that an inability to grow at high temperatures limits the capacity of fungi to infect mammals. However, fungi can develop thermotolerance, gradually adapting to rising temperatures due to climate change, and generating a greater number of disease-causing organisms. In the present study, we reported the detection and identification of Candida palmioleophila isolates recovered from raw sewage samples in Niteroi city, Rio de Janeiro State, Brazil, during a monitoring program for measuring SARS-CoV-2 presence and concentration. Using polyphasic taxonomy to identify the species and evaluating some virulence aspects of this species, such as biofilm formation and extracellular enzyme production, our data highlight this species as a possible emerging pathogen in Brazil, especially in the pandemic context.

First report of fungal meningoencephalitis by Penicillium chrysogenum in Brazil.

INTRODUCTION: Fungal infections of the central nervous system present a variety of clinical syndromes, such as meningitis, encephalitis, raised intracranial pressure with a nonspecific presentation, and, in the last two decades, have increased the incidence of these fungal infections. Fungal meningoencephalitis is frequently associated with Cryptococcus, but this report stands out for presenting one species of Penicillium genus. OBJECTIVES: Here, we present the first case of meningoencephalitis associated with brain injury caused by Penicillium chrysogenum, in a patient who is immunocompetent and was admitted to Hospital Naval Marcílio Dias, Rio de Janeiro, Brazil. METHODS: To identify the fungal species, we performed phenotypic and genotypic methodologies, from the culture to the sequencing of internal transcribed spacer region, and ß-tubulin gene, a rare fungus in cerebrospinal fluid cultures, belonging to the genus Penicillium, was identified. CONCLUSION: We highlight the importance of the first report of meningoencephalitis caused by P. chrysogenum in a patient who is immunocompetent, registered in Brazil. We also emphasize the need for further studies to determine an effective treatment with the least possible side effects for patients infected by fungi that are rarely related to the most severe forms of invasive infections.