Investigation of fluconazole heteroresistance in clinical and environmental isolates of Cryptococcus neoformans complex and Cryptococcus gattii complex in the state of Amazonas, Brazil.

Heteroresistance, defined as the occurrence of apparently homogeneous subpopulations of microbial cells showing different levels of antimicrobial susceptibility is a problem that has been associated with therapeutical failure in cryptococcosis. The purpose of the study was an investigation on the level of heteroresistance to fluconazole (LHF) as observed in clinical and environmental C. neoformans/C. gattii complex species isolates from Amazonas State (AM), Brazil. A total of 45 isolates and 9 type strains were analyzed. The assessments comprised testing for minimal inhibitory concentrations (MICs), for LHFs, for the strains' capacity of adaptation to high fluconazole (FLC) concentrations above the LHF, and for the stability of the heteroresistance phenomenon. The mean MICs for clinical isolates of C. gattii (6.4 µg/ml) were higher than those observed for environmental C. gattii strains (1.7 µg/ml) and clinical (3.7 µg/ml) as well as environmental (1.5 µg/ml) C. neoformans isolates. The phenomenon of heteroresistance to FLC was recorded for all isolates. On average, the LHF (8-256 µg/ml) of the isolates was 16 times higher than the FLC MICs (0.5-16 µg/ml) and a proportion of 85% isolates showed LHFs ≥ 16 µg/ml, 40% even ≥ 32 µg/ml. According to the adaptation assay, a considerable number of isolates (58%) showed the capacity of adaptation to MICs even higher than the initially recorded LHF. After the adaptation experiment, the adaptative-LHF values (8-512 µg/ml) were about 60 times higher than the original MIC values. After nine subsequent passages in drug-free broth, the isolates had their adaptative-LHF reduced. However, the LHF did not revert to the initially measured level. Our findings challenge the clinical interpretation of the antifungal MIC testing and motivate future studies correlating the levels of heteroresistance and parameters like LHF and adaptative-LHF with cryptococcosis-associated morbidity and mortality. LAY SUMMARY: Cryptococcosis affects many people and is caused by fungi of the Cryptococcus neoformans/Cryptococcus gattii complexes. These agents appear to become more resistant to antifungals when exposed to increasing concentrations of antifungals due to a phenomenon called heteroresistance.

Antimicrobial Potential of Metabolites in Fungal Strains Isolated from a Polluted Stream: Annulohypoxylon stygium WL1B5 Produces Metabolites against Extended-Spectrum Beta-Lactamase-Positive Escherichia coli.

The emergence of multidrug resistance in bacterial pathogens is a growing public health concern requiring solutions including the discovery of new antimicrobial drugs. Fungi have been used for decades as a source of antimicrobials. Ongoing screenings for newly characterized fungal strains producing antimicrobials include environments that are difficult to access like the deep sea, glaciers, wastewaters and environments polluted due to human activity. In the present study, fungal microorganisms were isolated from water samples taken from a polluted stream in the city of Manaus, AM, Brazil, and screened for antimicrobial effects against Escherichia coli. Using extracts from five isolates (Annulohypoxylon stygium WL1B5, Colletotrichum fructicola WL3B9, Clonostachys rosea WL5B18, Clonostachys rosea WL8B28 and Trichoderma harzianum WL9B49), antimicrobial activity against the reference strains Escherichia coli ATCC 25922 as well as E. coli NCTC 13353, an extended-spectrum beta-lactamase-positive strain, was observed. Inhibition zones ranged from 1 to 35.9 mm and a minimum inhibitory concentration of 400 µg/mL could be demonstrated. Assessments of the metabolites of Annulohypoxylon stygium WL1B5 allowed us to identify nodulisporone and daidzein, which have already been associated with antimicrobial activity. The findings confirm the feasibility of isolating fungal strains from polluted sites producing metabolites that can serve as potential future alternatives for the treatment of multidrug-resistant bacteria.

Modifications of antifungal sensibility testing as suggested by CLSI document M27-A4: proposal for using different culture medium and buffer.

A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.

Cryptococcal meningitis in non-HIV patients in the State of Amazonas, Northern Brazil.

Cryptococcosis is a life-threatening fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii species complex. Most cases are recorded in patients suffering from HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome). However, this infection also occurs in non-HIV patients with a proportion of 10-30% of all cases. The study aimed at the clinical and molecular characterization of non-HIV patients diagnosed with cryptococcosis at the Tropical Medicine Foundation (FMT-HVD) from July 2016 to June 2019. Medical records of respective patients were analyzed to describe the course of cryptococcosis in non-HIV patients. In addition, multi-locus sequence typing (MLST) was applied to identify the sequence types of the isolated Cryptococcus strains, to perform phylogenetic analysis, and to evaluate the isolates' genetic relationship to global reference strains. Antifungal susceptibility profiles to amphotericin B, fluconazole, and itraconazole were assessed by broth microdilution. From a total of 7 patients, 4 were female, the age range varied between 10 and 53 years (median of 36.3 years). Cryptococcal meningitis was the common clinical manifestation (100%). The period between onset of symptoms and confirmed diagnosis ranged from 15 to 730 days (mean value of 172.9 days), and the observed mortality was 57.1%. Of note, comorbidities of the assessed cryptococcosis patients comprised hypertension, diabetes mellitus, and intestinal tuberculosis. Genotyping applying PCR-RFLP of the URA5 gene identified all clinical isolates as C. gattii genotype VGII. Using MLST, it was possible to discriminate the sequence types ST20 (n = 4), ST5 (n = 3), and the newly identified sequence type ST560 (n = 1). The antifungals amphotericin B, fluconazole, and itraconazole showed satisfactory inhibitory activity (microdilution test) against all C. gattii VGII strains.

Factors influencing susceptibility testing of antifungal drugs: a critical review of document M27-A4 from the Clinical and Laboratory Standards Institute (CLSI).

Due to the increasing numbers of fungal infections and the emergence of drug-resistant fungi, optimization and standardization of diagnostic methods for the measurement of antifungal susceptibility are ongoing. The M27-A4 document by the US Clinical and Laboratory Standards Institute (CLSI) is presently used for the interpretation of minimum inhibitory concentrations of major opportunistic yeast species as measured by broth microdilution testing in many countries. Although microdilution is considered a benchmark for reproducible and accurate results, increased testing capacity, and limited human bias, the method is often inaccessible to routine clinical laboratories and researchers, especially in low-income countries. Furthermore, several studies suggest that there are still a considerable number of factors that make the estimation of in vitro activity of antifungal agents challenging. This review article summarizes the limitations of the M27-A4 standard which, despite the advances and improvements obtained by the standardization of antimicrobial resistance testing methods by CLSI, still persist.

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.

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.

Micoses superficiais na cidade de Manaus, AM, entre março e novembro/2003 / Superficial mycoses in the City of Manaus/AM between March and November/2003

FUNDAMENTOS: Micoses superficiais estritas são infecções fúngicas que se localizam nas camadas superficiais da pele e seus anexos. As micoses superficiais cutâneas representadas pelas dermatofitoses e candidíases podem ultrapassar a camada córnea da pele. Na região amazônica possuem incidência elevada. OBJETIVOS: Estudar as micoses superficiais, estritas e cutâneas, diagnosticadas sob o ponto de vista epidemiológico e micológico. PACIENTES E MÉTODOS: Pacientes com suspeita clínica de micoses superficiais submetidos a exame micológico no período de março a novembro de 2003 no Laboratório de Micologia Médica/CPCS/INPA. RESULTADOS: Foram realizados 394 exames, tendo 256 apresentado diagnóstico positivo. As micoses mais incidentes foram onicomicoses (135) e pitiríase versicolor (98). Malassezia spp. (77) e Candida spp. (72) foram os agentes fúngicos mais isolados. Tinea capitis apresentou maior ocorrência nos pré-escolares (3), e onicomicoses em adultos (94). O sexo feminino foi o mais acometido (91). Todas as classes sociais foram infectadas, com predominância da C (37). CONCLUSÃO: Onicomicoses e pitiríase versicolor acometeram sobretudo adultos. A Tinea capitis ocorre principalmente, em crianças. As micoses superficiais apresentaram mais incidentes nas mulheres. Malassezia spp. e Candida spp. foram os agentes mais isolados.