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.

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.

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.

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.

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.

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.

Clinical Profile of SARS-CoV-2 Infection: Mechanisms of the Cellular Immune Response and Immunogenetic Markers in Patients from Brazil.

OBJECTIVES: The aim of this study is to evaluate some mechanisms of the immune response of people infected with SARS-CoV-2 in both acute infection and early and late convalescence phases. METHODS: This is a cohort study of 70 cases of COVID-19, confirmed by RT-PCR, followed up to 60 days. Plasma Samples and clinical data were. Viral load, blood count, indicators inflammation were the parameters evaluated. Cellular immune response was evaluated by flow cytometry and Luminex immunoassays. RESULTS: In the severe group, hypertension was the only reported comorbidity. Non severe patients have activated memory naive CD4+ T cells. Critically ill patients have central memory CD4+ T cell activation. Severe COVID-19 patients have both central memory and activated effector CD8+ T cells. Non-severe COVID-19 cases showed an increase in IL1ß, IL-6, IL-10 and TNF and severely ill patients had higher levels of the cytokines IL-6, IL-10 and CXCL8. CONCLUSIONS: The present work showed that different cellular responses are observed according to the COVID-19 severity in patients from Brazil an epicenter the pandemic in South America. Also, we notice that some cytokines can be used as predictive markers for the disease outcome, possibility implementation of strategies effective by health managers.

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.

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.

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.

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.

Sporotrichosis: A Review of a Neglected Disease in the Last 50 Years in Brazil.

Sporotrichosis is caused by fungi belonging to the genus Sporothrix, which saprophytically are found in plants and organic matter. However, cats are highly susceptible to contamination with fungal spores and, when they become sick, they can transmit it to other animals and to man. The objective of this study is to carry out a systematic review on the emergency, diagnosis, clinical symptoms, therapeutics, and control of zoonotic sporotrichosis. Published data covering the last 50 years using a combination of keywords were selected to answer the question: Why has the zoonotic sporotrichosis been a neglected disease up to now? A total of 135 studies were included in this review. The studies emphasize that in recent decades, Brazil has experienced an unprecedented zoonotic outbreak of sporotrichosis. Advances on the genus Sporothrix allowed one to associate thermotolerance, capacity for melanin synthesis, potential for adhesion to tissue macromolecules, ergosterol peroxide production, and expression of virulence proteins as tools for infection and invasion in S. brasiliensis, the main species involved, although cases with S. schenckii or S. lurei were also reported. Correct diagnosis, early treatment, basic educational measures that emphasize responsible ownership of animals and reproductive control programs for felines can contribute to the control of zoonosis.

Sporothrix brasiliensis as a Causative Agent of Disseminated Sporotrichosis in AIDS.

A 47-year-old woman who lived in the metropolitan area of Rio de Janeiro and had abandoned her Acquired Immune Deficiency Syndrome (AIDS) therapy 3 years prior, was admitted to our hospital due to weight loss, cough, and fever for 2 weeks. The dermatologic examination established normochromic papules with molluscum-like central umbilication on the face (Figure 1) and hands, erythema and infiltration in the oral cavity (Figure 2), desquamative patches on the legs, and ulceration on right calcaneus (Figure 3). Her CD4 (T-cells) count was 47 cells/ mm.3 Direct mycology examination and cultures for several agents were performed. The mycology culture done from her skin and sputum grew Sporothrix 1 week after admission (Figure 4).

Global distribution of animal sporotrichosis: A systematic review of Sporothrix sp. identified using molecular tools.

Sporotrichosis is a subcutaneous mycosis caused by thermodimorphic fungi of the genus Sporothrix. The phenotypic and genotypic differences of the isolates within the genus Sporothrix have been associated with their geographic distribution, virulence capacity, or clinical manifestation of sporotrichosis. Therefore, it is crucial to identify the causative agent of sporotrichosis. However, there are few case reports and studies in animals compared to those in humans, despite the substantial increase in the number of cases of sporotrichosis by zoonotic transmission, especially in endemic areas. Considering the epidemiological importance, taxonomic evolution and worldwide distribution of these fungi in the last decade, there is interest in identifying the species of the genus Sporothrix in different regions of the world. This study aimed to analyze the geographic distribution of animal sporotrichosis in the world, caused by pathogenic species identified by use of molecular tools. This systematic review of articles from 2007 to 2021 analyzed the geographic distribution of species that cause sporotrichosis in cats, dogs and other animals. It demonstrated that the most identified species were S. brasiliensis, isolated from cats in Brazil and S. schenckii isolated from cats in Malaysia. We show the lack of studies in global areas and reinforce the need to use molecular tools to identify and monitor potential pathogens.

Sporothrix brasiliensis and Feline Sporotrichosis in the Metropolitan Region of Rio de Janeiro, Brazil (1998-2018).

Feline sporotrichosis is enzootic in different regions of Brazil, especially in Rio de Janeiro. This study compared the genotype profiles of Sporothrix sp. isolated from cats in Rio de Janeiro between 1998 and 2018 and evaluated their association with clinical and epidemiological characteristics. One hundred nineteen Sporothrix sp. isolates from a cohort of cats with sporotrichosis seen at INI/Fiocruz were included. Clinical and epidemiological data were obtained from the medical records of the animals. T3B PCR fingerprinting was used for molecular identification of the Sporothrix species. All isolates were characterized as Sporothrix brasiliensis, with the observation of low intraspecific variation in 31 isolates (31.3%). The interval between lesion onset and first medical visit at INI/Fiocruz, as well as treatment duration until clinical cure, was longer in cats from the first decade of the epizootic. In addition, the frequency of the variables "good general status" and "presence of lymphadenomegaly" was higher among cats whose strains did not exhibit intraspecific variation. So far, S. brasiliensis has been the only species identified in feline cases of sporotrichosis since the beginning of the epizootic in Rio de Janeiro at INI/Fiocruz.