Molecular characterization, antifungal susceptibility and virulence of clinical isolates of Cryptococcus spp

A criptococose é uma infecção fúngica causada principalmente pelas leveduras encapsuladas C. neoformans e C. gattii. Entretanto, C. laurentii vem emergindo como agente causador desta infecção que acomente tanto indivíduos imunocomprometidos quanto indivíduos imunocompetentes. Com o aumento de indivíduos portadores do HIV/AIDS, o número de casos de criptococose vem aumentando. Portanto, o isolamento e a identificação correta das espécies são essenciais para o entendimento dos diferentes casos de criptococose. O objetivo do presente estudo foi estudar as características moleculares e fenotípicas dos isolados clínicos de pacientes com criptococose oriundos do HCFMRP-USP. Foram estudados 80 casos de criptococose onde foram identificados 132 isolados clínicos de Cryptococcus spp. os quais representam uma amostragem retrospectiva por conveniência do período 2012 a 2017. Destes isolados clínicos, 113 foram identificados como C. neoformans, sendo a maioria VNI e 6 VNII, 16 C. gattii VGII e 3 C. laurentii. Entre os casos clínicos, 52 são pacientes sorotipo HIV positivo, 23 pacientes sorotipo HIV negativo e 5 pacientes em que o sorotipo HIV não foi determinado. Foram observados 3 pacientes sorotipo HIV negativo com ausência de doença subjacente que justificasse o desenvolvimento da criptococose. Nestes, as espécies isoladas foram C. neoformans VNI e C. gattii VGII. Quanto aos fatores de virulência cápsula polissacarídica e melanina, a porcentagem linear da cápsula polissacarídica dos isolados clínicos C. neoformans e C. gattii foram semelhantes C. laurentii não apresentou cápsula polissacarídica. Entretanto, ao comparar os isolados clínicos considerando o sorotipo HIV do paciente, a porcentagem linear da cápsula polissacarídica dos isolados clínicos de pacientes sorotipo HIV positivo foi maior. Todos os isolados clínicos foram produtores de melanina, porém com diferentes intensidades. A virulência dos isolados clínicos C. neoformans em larvas de G. mellonella foi considerada maior em comparação a C. gattii e C. laurentii. Todos os isolados clínicos foram sensíveis a anfotericina B e ao voriconazol. Para o itraconazol (ITR) foram observados isolados clínicos com Sensibilidade Dose Dependente (SDD), sendo 8 C. neoformans, 7 C. gattii e 1 C. laurentii. Para fluconazol (FLU) também foram observados isolados clínicos com SDD, sendo 1 C. neoformans e 2 C. gattii. Esse perfil foi observado para os tipos moleculares VNI e VGII. Já para 5-flucitosina (5-FC) apenas os isolados clínicos C. laurentii apresentaram resistência. A susceptibilidade entre os isolados clínicos considerando o sorotipo HIV dos pacientes mostrou que ambos sorotipos apresentaram isolados clínicos com SDD para ITR; paciente sorotipo HIV positivo apresentou isolado clínico com SDD para FLU e paciente HIV negativo apresentou 1 isolado clínico com resistência a 5-FC. Portanto, constatamos neste trabalho que não houveram diferenças distintas ao correlacionar os fatores de virulência e a virulência em G. mellonella entre os isolados clínicos estudados. Além disso, a maioria dos isolados clínicos foi sensível aos antifúngicos utilizados na clínicaCryptococcosis is a fungal infection caused by encapsulated yeasts C. neoformans and C. gattii. However, there are other species causing this infection in humans, such as C. laurentii. This infection can affect both immunocompromised individuals and immunocompetent individuals. With the increase in individuals with HIV/AIDS, the number of cases of cryptococcosis has been increasing. In this way, the isolation and correct identification of the species are essential to obtain the different cases of cryptococcosis. The aim of the present study was to study the molecular and phenotypic characteristics of clinical isolates of cryptococcosis patients from HCFMRP-USP. A total of 80 criptococcosis cases were studied in which 132 Cryptococcus sp. strains were isolated. The clinical isolates were a retrospective convenience sampling from the period 2012-2017. Among the clinical isolates, 113 were identified as C. neoformans, being the majority VNI and 6 VNII, 16 C. gattii VGII and 3 C. laurentii. Considering the cryptococcosis cases and HIV serotipe, 52 patients were HIV-positive, 23 HIV-negative and 5 patients with undetermined HIV serotype. Three HIV-negative serotype patients without underlying disease that would justify cryptococcosis were observed. The isolated species were C. neoformans VNI and C. gattii VGII. Regarding the virulence factors, C. neoformans and C. gattii clinical isolates have shown similar linear percentages of the polysaccharide capsule, whereas C. laurentii did not present a polysaccharide capsule. However, when comparing the clinical isolates considering the HIV serotype of the patient, it was observed that the linear percentage of the capsule of the clinical isolates of patients with HIV-positive serotype were higher. All clinical isolates were melanin producers, but with different intensities. The virulence of C. neoformans clinical isolates in G. mellonella larvae was considered higher in comparison to C. gattii and C. laurentii. All clinical isolates were sensitive to amphotericin B and voriconazole. For itraconazole (ITR), clinical isolates with Dependent Dose Sensitivity (DDS) were observed. Among them were 8 C. neoformans, 7 C. gattii and 1 C. laurentii. For fluconazole (FLU), clinical isolates with DDS were also observed (1 C. neoformans and 2 C. gattii). The observed molecular types were VNI and VGII. For flucytosine (5-FC) only the clinical isolates C. laurentii presented resistance. The antifungal susceptibility among the clinical isolates considering the HIV serotype of the patients showed that the ITR presented clinical isolates with DDS for patients with both HIV serologies (positive and negative). FLU presented DDS clinical isolates only form HIV positive serotype patients. 5-FC presented 1 clinical isolate from an HIV negative serotype patient with resistance. Therefore, we verified in this study that there were no differences in the correlation between virulence factors and virulence in G. mellonella among the clinical isolates studied. In addition, the majority of clinical isolates were sensitive to antifungal agents used in the clinical practice for cryptococcosis treatmen

Surveillance of Amphotericin B and Azole Resistance in <i>Aspergillus</i> Isolated from Patients in a Tertiary Teaching Hospital

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives