Análise de restrição enzimática do gene hsp65 de isolados clínicos de pacientes com suspeita de tuberculose pulmonar em Teresina, Piauí / Restriction enzyme analysis of the hsp65 gene in clinical isolates from patients suspected of having pulmonary tuberculosis in Teresina, Brazil

OBJETIVO: Identificar as espécies de micobactérias encontradas no escarro de pacientes com suspeita de tuberculose pulmonar e analisar o impacto dessas identificações na abordagem terapêutica. MÉTODOS: Foram avaliados 106 pacientes com suspeita de tuberculose pulmonar encaminhados para o serviço de pneumologia de um hospital público em Teresina, Piauí. Espécimes de escarro matinal foram avaliados quanto à presença de micobactérias por baciloscopia e cultura. Foram utilizadas PCR e análise de restrição enzimática do gene hsp65 (PRA-hsp65) para a identificação das cepas de micobactérias isoladas em cultura. RESULTADOS: Foram analisadas 206 amostras de escarro. A idade dos pacientes variou de 15 a 87 anos, sendo 67 por cento do gênero masculino. Tosse ocorreu em 100 por cento dos casos. O padrão radiográfico predominante foi de lesão moderada, observada em 70 por cento. A positividade no esfregaço foi de 76 por cento, e isolamento em cultura ocorreu em 91 por cento das culturas executadas. Testes tradicionais identificaram micobactérias não tuberculosas (MNT) em 9 por cento dos isolados. O método PRA-hsp65 confirmou esses dados, mostrando sete padrões de bandas capazes de identificar as espécies de MNT isoladas: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5 e M. gordonae 7. Todos os pacientes com MNT tinham mais de 60 anos, e observaram-se bronquiectasias em 88 por cento das radiografias. Houve dois casos de reinfecção, identificados inicialmente como infecção por M. abscessus e M. kansasii. CONCLUSÕES: As MNT causam infecção pulmonar em pacientes imunocompetentes, e a identificação das MNT é importante para estabelecer o diagnóstico correto e a decisão terapêutica mais adequada. O método PRA-hsp65 é útil para identificar espécies de MNT e pode ser implantado em laboratórios de biologia molecular não especializados em micobactérias.

OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67 percent were male. There was cough in 100 percent of the cases. The predominant radiographic pattern was moderate disease, observed in 70 percent. Smear positivity was 76 percent, and isolation in culture occurred in 91 percent of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9 percent of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88 percent of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7 percent) were human immunodeficiency virus (HIV)-positive and 26 (41.3 percent) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7 percent) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5 percent), and 24/63 (38.1 percent) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8 percent). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6 percent) of the HIV-positive subjects and in 13/26 (50 percent) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Coccidioidomycosis: an unusual cause of acute respiratory distress syndrome

A male farmer, 20 years old, from the countryside of the State of Piauí, developed acute respiratory infection. Despite adequate antimicrobial therapy, his conditions worsened, requiring mechanical ventilation. His X-rays showed diffuse pulmonary infiltrates. His PaO2/FiO2 ratio was 58. Direct microscopy and culture of tracheal aspirates showed the presence of Coccidioides immitis. Autochthonous cases of coccidioidomycosis have only recently been described in Brazil, most of them from the State of Piauí. C. immitis has been isolated from humans, dogs and armadillos (Dasypus novemcinctus), and also from soil samples of armadillo's burrows. Failure to respond to antimicrobial therapy and a patient's origin from recognized endemic areas should alert to the possibility of acute pulmonary coccidioidomycosis.