Searching for new antifungals for the treatment of cryptococcosis

ABSTRACT There is a consensus that the antifungal repertoire for the treatment of cryptococcal infections is limited. Standard treatment involves the administration of an antifungal drug derived from natural sources (i.e., amphotericin B) and two other drugs developed synthetically (i.e., flucytosine and fluconazole). Despite treatment, the mortality rates associated with fungal cryptococcosis are high. Amphotericin B and flucytosine are toxic, require intravenous administration, and are usually unavailable in low-income countries because of their high cost. However, fluconazole is cost-effective, widely available, and harmless with regard to its side effects. However, fluconazole is a fungistatic agent that has contributed considerably to the increase in fungal resistance and frequent relapses in patients with cryptococcal meningitis. Therefore, there is an unquestionable need to identify new alternatives or adjuvants to conventional drugs for the treatment of cryptococcosis. A potential antifungal agent should be able to kill cryptococci and "bypass" the virulence mechanism of the yeast. Furthermore, it should have fungicidal action, low toxicity, high selectivity, easily penetrate the central nervous system, and widely available. In this review, we describe cryptococcosis, its conventional therapy, and failures arising from the use of drugs traditionally considered to be the reference standard. Additionally, we present the approaches used for the discovery of new drugs to counteract cryptococcosis, ranging from the conventional screening of natural products to the inclusion of structural modifications to optimize anticryptococcal activity, as well as drug repositioning and combined therapies.

Airborne fungi isolated from different environments of a primary school in the city of Manaus, Amazonas, Brazil / Fungos anemófilos isolados de diferentes ambientes de uma escola primária na cidade de Manaus, Amazonas, Brasil

Introduction: Airborne fungi can cause respiratory diseases, including pulmonary mycoses. The objective of this study was to isolate and identify airborne fungi from external and internal environments at a full-day primary school in Manaus, Brazil, and ascertain the influence of seasonality on the incidence of these microorganisms. Methods: Airborne fungi were collected by exposing Sabouraud agar plates at various external and internal locations in the school. Results: A total of 2,386 fungal colonies were isolated, 1,041 in the rainy season and 1,345 during the dry season. Of these, 1,858 were identified and distributed into 34 genera. The most prevalent were Cladosporium sp. (22.6%); Aspergillus sp. (17.14%); Penicillium sp. (8.55%); Curvularia sp. (6.83%); and Drechslera sp. (5.7%). During the dry season, the most prevalent genre was Aspergillus (19.3%), while in the rainy season, Cladosporium predominated (34.6%). Conclusion: Seasonality influenced fungal incidence, especially of the genus Cladosporium, which increased significantly during the rainy season. Cladosporium can be considered a bioindicator of the rainy season in the Brazilian Amazon.

Introdução: Os fungos presentes no ar, denominados anemófilos, possuem uma ampla diversidade em locais de clima tropical e são causadores de micoses pulmonares e outras doenças do aparelho respiratório. O objetivo do estudo foi isolar e identificar os fungos do ar de uma escola de ensino fundamental de tempo integral, a partir de ambientes externos e internos, e verificar se a sazonalidade influencia a incidência desses microrganismos. Métodos: Para coleta dos fungos do ar, placas de Petri contendo Sabouraud foram expostas nos ambientes externos e internos da escola. Resultados: Foram isoladas 2.386 colônias de fungos, sendo 1.041 na estação chuvosa e 1.345 na estação seca. Foram identificados 1.858 fungos, que puderam ser distribuídos em 34 gêneros. Os gêneros mais frequentes foram Cladosporium sp. (22,6%), Aspergillus sp. (17,14%), Penicillium sp. (8,55%), Curvularia sp. (6,83%) e Drechslera sp. (5,7%). Durante o período seco, o gênero mais frequente foi o Aspergillus (19,21%), e no período chuvoso, o gênero Cladosporium (34,8%). Conclusão: A sazonalidade influenciou principalmente o gênero Cladosporium, que obteve aumento significativo na estação chuvosa, constituindo um biomarcador dessa estação.

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Background The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

Effects of culture filtrates of endophytic fungi obtained from Piper aduncum L. on the growth of mycobacterium tuberculosis

Substances that inhibit the growth of Mycobacterium tuberculosis could potentially be used as antibiotics. These substances could also be added to test culture media to improve the speed of tuberculosis diagnosis. The aim of this work was to investigate the influence of culture filtrates of endophytic fungi isolated from P. aduncum L. on the growth of M. tuberculosis. To achieve this objective, the following methodology was used: a) endophytic fungi were isolated from the leaves and stems of P. aduncum L.; b) the isolated fungi were submitted to submerged bioprocessing; c) culture filtrates from the bioprocess were assayed to evaluate their effect on the growth of M. tuberculosis. We isolated 315 fungal types, which represented 85 morphologies, from different parts of P. aduncum L. The bioassays were performed on 82 culture filtrates and 6 plant extracts and resulted in the detection of 1 culture filtrate that stimulated the growth of M. tuberculosis and 15 that inhibited microbial growth. None of the phytochemical extracts had an effect on the growth of M. tuberculosis. In conclusion, we observed that the endophytic fungi isolated from P. aduncum L. (Piperaceae) produced extracellular metabolites (present in the culture filtrate) that affect the growth of M. tuberculosis. These compounds have the potential to be used as antimicrobials or in the diagnosis of tuberculosis.