Prevalence of pulmonary mycoses in smear-negative patients with suspected tuberculosis in the Brazilian Amazon.

BACKGROUND: Pulmonary mycoses resemble clinically and radiologically chronic pulmonary tuberculosis. Studies describing the prevalence, etiology and clinical features of pulmonary mycosis are of crucial importance in the Brazilian Amazon. AIMS: To estimate the frequency of pulmonary mycoses in smear-negative tuberculosis patients; to describe their demographic, epidemiological, and clinical characteristics; and to evaluate diagnostic methods. METHODS: A cross-sectional study was conducted at two tuberculosis reference institutions in Amazonas, Brazil. We included 213 patients and collected clinical data, blood and induced sputum to perform serological, direct microscopy, microbiologic culture and PCR-based assays to identify infections caused by Aspergillus fumigatus, Paracoccidioides brasiliensis, Histoplasma capsulatum, Cryptococcus, and HIV. Chest computed tomography was also performed. RESULTS: Pulmonary mycoses were diagnosed in 7% (15/213) of the cases, comprising ten aspergillosis cases, three cases of paracoccidioidomycosis and one case each of histoplasmosis and cryptococcosis. Among the patients with pulmonary mycoses, 86.7% were former tuberculosis patients. The most significant clinical characteristics associated with pulmonary mycoses were cavity-shaped lung injuries, prolonged chronic cough and hemoptysis. CONCLUSIONS: Our study confirmed the high prevalence of pulmonary mycoses in smear-negative tuberculosis patients in the Brazilian Amazon.

MLST reveals a clonal population structure for Cryptococcus neoformans molecular type VNI isolates from clinical sources in Amazonas, Northern-Brazil.

Cryptococcosis is considered endemic in Amazonas state, occurring more frequently in individuals with AIDS, who are predominantly infected by Cryptococcus neoformans molecular type VNI. Infections by Cryptococcus gattii VGII predominate in immunocompetent hosts from the American continent and are associated with outbreaks in North America, particularly the subtypes VGIIa and VGIIb, which are also present in the Brazilian Amazon region. Despite few environmental studies, several aspects of the molecular epidemiology of this disease in Amazonas remain unclear, including the limited use of multilocus sequence typing (MLST) to evaluate the genetic population structure of clinical isolates, mainly C. neoformans. Therefore, we used MLST to identify the sequence types of 38 clinical isolates of C. neoformans VNI and C. gattii VGII and used phylogenetic analysis to evaluate their genetic relationship to global isolates. Records of 30 patients were analyzed to describe the current scenario of cryptococcosis in the region and their associations with the different subtypes. Broth microdilution was also performed to determine the susceptibility profile to the antifungals amphotericin B, fluconazole and itraconazole. MLST identified that patients with HIV (n = 26) were exclusively affected by VNI strains with ST93, and among the VGII strains (n = 4), three STs (ST5, ST172 and the new ST445) were identified. An in-hospital lethality of 54% was observed in the HIV group, and there were no significant differences in the clinical aspects of the disease between the HIV and non-HIV groups of patients. In addition, all isolates were susceptible to the antifungals tested. Therefore, in Amazonas state, VNI isolates are a genetically monotypic group, with ST93 being highly important in HIV individuals.

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.