Rearing and Maintenance of Galleria mellonella and Its Application to Study Fungal Virulence

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided

Atividades diárias e sociais influenciam a qualidade de vida de pacientes renais crônicos em hemodiálise

Objective: This study aimed to evaluate the physical and social factors that influence the quality of life of patients with chronic kidney disease (CKD) on hemodialysis (HD). Study Design: Descriptive and cross-sectional. Methods: There were 149 patients with CKD routinely submitted to HD that answered a questionnaire adapted (Medical Results Survey; SF-36) about the presence of symptoms during and after the HD sessions, main diseases concomitant, and the presence of factors that influence the quality of life. Results: According to the multivariate analysis, the presence of symptoms after the HD contributes to a decrease in the quality of life (p=0.04). However, emotional well-being was positively associated with social activities (p=0.01). The daily activities and the absence of concomitant diseases are associated with good health and, therefore, a better quality of life for patients with CKD (p<0.05). Patients with chronic diseases such as hypertension and diabetes should be better observed, since such illnesses are always associated with renal dysfunction. Conclusion: The results of the present study demonstrate that the chronic renal patients should be inserted into social environments and should perform daily activities despite the limitations of the treatment since an active lifestyle contributes positively to their well-being and quality of life.Objetivo: O objetivo deste estudo foi avaliar os fatores físicos e sociais que influenciam a qualidade de vida de pacientes com doença renal crônica (DRC) em hemodiálise (HD). Modelo de estudo: Descritivo e transversal. Métodos: Foram incluídos 149 pacientes com DRC rotineiramente submetidos a HD que responderam ao questionário adaptado (Medical Results Survey; SF-36) sobre a presença de sintomas durante e após as sessões de HD, principais doenças concomitantes e presença de fatores que influenciam a qualidade de vida. Resultados: De acordo com a análise multivariada, a presença de sintomas após a HD contribui para uma diminuição da qualidade de vida (p=0,04). Entretanto, o bem-estar emocional foi positivamente relacionado com as atividades sociais (p=0,01). As atividades diárias e a ausência de doenças concomitantes estão associadas com uma boa saúde e assim, a uma melhor qualidade de vida dos pacientes com DRC (p<0,05). Pacientes com doenças crônicas como hipertensão e diabetes devem ser melhor acompanhados, uma vez que, estas doenças estão constantemente associadas a disfunção renal. Conclusão: Os resultados do presente estudo demonstram que os pacientes crônicos renais devem ser inseridos no ambiente social e devem realizar atividades cotidianas, apesar das limitações do tratamento, uma vez que um estilo de vida ativo contribui positivamente para a melhora da qualidade de vida

Case Report: Disseminated Sporothrix brasiliensis Infection with Endocardial and Ocular Involvement in an HIV-Infected Patient

Disseminated sporotrichosis occurs in individuals with impaired cellular immunity, such as in cases of neoplasia, transplantation, diabetes, and especially, acquired immunodeficiency syndrome. This report presents a 32-year-old Brazilian human immunodeficiency virus (HIV)-infected patient who developed a protracted condition of disseminated sporotrichosis with endocarditis, bilateral endophthalmitis, and lymphatic involvement. He needed cardiac surgery to replace the mitral valve. Sporothrix brasiliensis isolates were recovered from cultures of subcutaneous nodules and mitral valve fragments. Species identification was based on classical and molecular methods. the patient received amphotericin B for 52 days and subsequently, oral itraconazole. He remains asymptomatic, and he is on maintenance therapy with itraconazole. Despite his positive clinical outcome, he developed bilateral blindness. To our knowledge, this case is the first report of endocarditis and endophthalmitis caused by S. brasiliensis.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Fed Univ Tricingulo Mineiro, Hosp Clin, Infect & Parasit Dis Unit, Uberaba, MG, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Unit, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Unit, São Paulo, BrazilFAPEMIG: PQ1813/09Web of Scienc

Genotyping Analysis of <i>Cryptococcus deuterogattii</i> and Correlation with Virulence Factors and Antifungal Susceptibility by the Clinical and Laboratory Standards Institute and the European Committee on Antifungal Susceptibility Testing Methods

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world

Fatal Case of Fungemia by Wickerhamomyces anomalus in a Pediatric Patient Diagnosed in a Teaching Hospital from Brazil

In recent decades, emerging fungal infections have changed the clinical mycology scenario as a consequence of the advances in medical diagnostics and therapeutic procedures, long hospitalization times, and the growing number of individuals with debilitating chronic diseases and impaired immune systems. This report presents a 19 months old Brazilian female patient who developed a severe fungal sepsis by an uncommon yeast. She was admitted at the intensive care unit with severe pneumonia, bronchopulmonary dysplasia, and weight-for-age z score of less than &minus;2. She remained more than 30 days in the intensive care unit where she had a femoral venous catheter placement, enteral nutrition, broad-spectrum antibiotic therapy, and prophylaxis with fluconazole. Moreover, pericardiocentesis was performed due to cardiac tamponade. She had a previous history of prematurity, cardiac surgery due to patent ductus arteriosus, and a long period of hospital stay. Despite the antifungal prophylaxis, two yeast isolates were recovered from blood and then identified by classical mycological methods and internal transcribed spacer (ITS) sequencing as Wickerhamomyces anomalus. Both isolates exhibited susceptibility to amphotericin B, ketoconazole, itraconazole, voriconazole, and fluconazole. Her clinical state worsened, presenting anasarca, epistaxis, and hemorrhagic suffusions in the mouth, sclera, oliguria, and bradycardia. Two days after the first positive culture, she presented a gradual reduction of the white blood cells count, with severe leukopenia and neutropenia. She died five days after

Comparative Genomics of Histoplasma capsulatum and Prediction of New Vaccines and Drug Targets

Histoplasma capsulatum is a thermodymorphic fungus that causes histoplasmosis, a systemic mycosis that presents different clinical manifestations, ranging from self-limiting to acute lung infection, chronic lung infection and disseminated infection. Usually, it affects severely immunocompromised patients although immunocompetent patients can also be infected. Currently, there are no vaccines to prevent histoplasmosis and the available antifungal treatment presents moderate to high toxicity. Additionally, there are few options of antifungal drugs. Thus, the aim of this study was to predict possible protein targets for the construction of potential vaccine candidates and predict potential drug targets against H. capsulatum. Whole genome sequences from four previously published H. capsulatum strains were analyzed and submitted to different bioinformatic approaches such as reverse vaccinology and subtractive genomics. A total of four proteins were characterized as good protein candidates (vaccine antigens) for vaccine development, three of which are membrane-bound and one is secreted. In addition, it was possible to predict four cytoplasmic proteins which were classified as good protein candidates and, through molecular docking performed for each identified target, we found four natural compounds that showed favorable interactions with our target proteins. Our study can help in the development of potential vaccines and new drugs that can change the current scenario of the treatment and prevention of histoplasmosis

Cytokine profiles at admission can be related to outcome in AIDS patients with cryptococcal meningitis.

Cryptococcal meningitis (CM) remains as common life-threatening AIDS-defining illness mainly in resource-limited settings. Previous reports suggested that baseline cytokine profiles can be associated to fungal burden and clinical outcome. This study aimed to evaluate the baseline cytokine profiles in AIDS patients with CM and its relation with the outcome at weeks 2 and 10. Thirty AIDS patients with CM diagnosed by cerebrospinal fluid (CSF) Cryptococcus neoformans positive culture, India ink stain and cryptococcal antigen test were prospectively evaluated. As controls, 56 HIV-infected patients without CM and 48 non-HIV individuals were included. Baseline CSF and sera levels of IL-2, IL-4, IL-8, IL-10, IL-12p40, IL-17A, INF-γ and TNF-α were measured by ELISA. Of 30 CM patients, 24 (80%) were male, median age of 38.1. The baseline CSF high fungal burden and positive blood culture were associated with a positive CSF culture at week 2 (p = 0.043 and 0.029). Most CSF and sera cytokines presented higher levels in CM patients than control subjects (p < 0.05). CSF levels of IL-8, IL-12p40, IL-17A, TNF-α, INF-γ and sera TNF-α were significantly higher among survivors at weeks 2 and 10 (p < 0.05). Patients with increased intracranial pression exhibited CSF IL-10 high levels and poor outcome at week 10 (p = 0.032). Otherwise, baseline CSF log10 IFN-γ and IL-17A were negatively correlated with fungal burden (r = -0.47 and -0.50; p = 0.0175 and 0.0094, respectively). The mortality rate was 33% (10/30) at week 2 and 57% (17/30) at week 10. The severity of CM and the advanced immunodeficiency at admission were related to a poor outcome in these patients. Otherwise, the predominant Th1 cytokines profile among survivors confirms its pivotal role to infection control and would be a prognostic marker in cryptococcal meningitis