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.

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.

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.

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.

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.

Soil samples from sporotrichosis transmission belt area: Searching for fungal species and their antagonistic activity against Sporothrix brasiliensis.

Since 1998, the state of Rio de Janeiro, Brazil, has become a public health problem regarding sporotrichosis, a disease caused by Sporothrix spp. involving contact with infected cats. Efforts to isolate these species from environmental sources are not always successful. In our study, soil from residences situated in cities of Rio de Janeiro where cats with sporotrichosis live was collected and cultured an attempt to isolate Sporothrix spp. but it was not successful. However, other saprophytic fungal species were isolated from soil and identified and among them Purpureocillium lilacinum was the most frequent. From there, we decided to study the in vitro interaction of this species with S. brasiliensis, the principal agent that causes sporotrichosis in this state. The results showed that ten isolates of P. lilacinum inhibited the radial mycelial growth of S. brasiliensis with different percentage of inhibition. The interaction between them revealed the pattern described as overgrowth by antagonist. In conclusion, our data suggest that fungal species with very fast growth and capable of producing metabolites could hinder the growth of Sporothrix spp., it also opens the way for the identification of secondary metabolites with biological activity that could be tested against pathogenic fungi.

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.

Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil.

Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial ß-tubulin gene sequencing and phylogenetic analysis.

Preservation Methods in Isolates of Sporothrix Characterized by Polyphasic Approach.

Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by eight pathogenic species of the Sporothrix genus. Different ex situ preservation methods are used around the world to maintain the survival, morphophysiological and genetic traits of fungal strains isolated from patients with sporotrichosis for long terms. The main aim of the present study was to evaluate the survival, phenotypic and genotypic stability of Sporothrix strains after preservation on PDA slant stored at 4 °C, sterile water and cryopreservation at -80 °C, for a period of time of 6, 12, 18 and 24 months of storage. Eight clinical Sporothrix isolates were identified based on a polyphasic approach consisting of classical macro- and micro-morphological traits, biochemical assays, proteomic profiles by MALDI-TOF MS and molecular biology. According to the final identification, one strain was identified as S. schenckii (CMRVS 40428) and seven strains were re-identified as S. brasiliensis (CMRVS 40421, CMRVS 40423, CMRVS 40424, CMRVS 40425, CMRVS 40426, CMRVS 40427 and CMRVS 40433). In addition, it was observed that the isolates survived after the different time points of storage in distilled water, PDA slant and cryopreservation at -80 °C. For fungi preserved in water, low polymorphisms were detected by the partial sequencing of ß-tubulin. Cryopreservation at -80 °C induced morphological changes in one single isolate. The proteomic profiles obtained by MALDI-TOF MS after preservation showed differences among the methods. In conclusion, preservation on agar slant stored at 4 °C was the most effective method to preserve the eight clinical Sporothrix strains. This method produced less change in the phenotypic traits and kept the genetic integrity of all strains. Agar slant stored at 4 °C is a simple and inexpensive method and can be especially used in culture collections of limited funding and resources.

Clinical and Anatomopathological Evaluation of BALB/c Murine Models Infected with Isolates of Seven Pathogenic Sporothrix Species.

BACKGROUND: Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by seven pathogenic species of Sporothrix genus: S. schenckii sensu stricto, S. brasiliensis, S. globosa and S. luriei (clinical clade), and the species S. mexicana, S. pallida and S. chilensis (environmental clade). Isolates of the same species of Sporothrix may have different pathogenicities; however, few isolates of this fungus have been studied. Thus, the aim of this work was to analyze the clinical and anatomopathological changes in immunocompetent and immunosuppressed BALB/c mice infected with clinical and environmental isolates of seven different species of Sporothrix, from both clades. One human clinical isolate of S. schenckii sensu stricto, S. brasiliensis, S. globosa, S. luriei, S. mexicana and S. chilensis species and one environmental isolate of S. pallida were inoculated subcutaneously in immunocompetent mice and the same isolates of S. brasiliensis and S.schenckii sensu stricto were inoculated in immunossupressed mice. Clinical manifestations as external lesions, apathy, and alopecia were observed. At 21, 35, and 49 days after fungal inoculation, four mice from each group were weighed, euthanized and necropsied for evaluation of splenic index, recovery of fungal cells, macroscopic and histopathological analysis of livers, lungs, kidneys, and hearts. The survival assessment was observed for 50 days following inoculation. Our results demonstrated that, clinical S. schenckii isolate, followed by clinical S. mexicana, and environmental S. pallida isolates, the last two, species grouped in the environmental clade, were capable of inducing greater anatomopathological changes in mice, which was reflected in the severity of the clinical signs of these animals. Thus, we reinforce the hypothesis that the pathogenicity of Sporothrix is not only related to the species of this fungus, but also shows variation between different isolates of the same species.

Skin Immune Response of Immunocompetent and Immunosuppressed C57BL/6 Mice After Experimental Subcutaneous Infection Caused by Purpureocillium lilacinum.

Hyalohyphomycosis is a fungal infection characterized by the presence of a hyaline mycelium in the host. It is caused by several agents, such as Purpureocillium lilacinum. Our study aimed to evaluate some cell subsets and inflammatory markers involved in the in situ immune response to subcutaneous hyalohyphomycosis by P. lilacinum in C57BL/6 murine models. The fungal isolate was inoculated in mice randomly distributed in immunocompetent/infected (CI) and immunosuppressed/infected (SI) groups. Mice were evaluated on days 1, 3, 5, and 7 after inoculation. Histopathological studies showed several lesions in the site of infection as well as the formation of multifocal and mixed inflammatory infiltrates, which differed between the CI and SI groups. This analysis also revealed conidia and hypha-like structures in subcutaneous tissues of mice of both groups. The immunohistochemical analysis showed lower percentages of macrophages and neutrophils in the SI group compared to those in the CI group. Moreover, the intensity of interleukin (IL)-1ß and nitric oxide synthase 2 production by cells of immunosuppressed mice was discreet, compared to immunocompetent mice that ranged from moderate to intense over time. The quantitative interference of dexamethasone in the response to the fungus was also demonstrated. We concluded that our results can be useful not only to broaden the knowledge on P. lilacinum but also, based on this host-parasite relationship, to contribute to the understanding of the mechanisms of infection.

Tregs in the immune response of BALB/c mice experimentally infected with species of the Sporothrix genus.

Background: Sporotrichosis occurs through contact with contaminated soil and plant. However, the incidence of sporotrichosis as a zoonotic epidemic has increased, particularly in Rio de Janeiro. Aim: In this work, we decided to evaluate some T-cell phenotypes involved in the immune response. Materials & methods: We used flow cytometry to quantify TCD4+ and TCD8+ and Treg from immunocompetent and immunosuppressed mice infected with Sporothrix species with different levels of virulence and pathogenicity. Results: It was demonstrated the predominance of TCD4+ over the TCD8+ cells in both groups, inoculated with all the species, and percentages of Treg observed in infected immunocompetent mice. Conclusion: This regulatory phenotype can be associated with a protective immunity in the initial periods of infection.

A case of sporotrichosis caused by different Sporothrix brasiliensis strains: mycological, molecular, and virulence analyses.

BACKGROUND: Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES: The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS: Genotypic analyses by partial sequencing of the calmodulin, ß-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS: These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.