RESUMEN
SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.
Asunto(s)
Leucocitos/inmunología , Esputo/citología , Esputo/inmunología , Tuberculosis Pulmonar/inmunología , Adolescente , Adulto , Brasil , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Masculino , Microscopía , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Adulto JovenRESUMEN
BACKGROUND: Tuberculosis (TB), one of the major airborne infectious bacterial diseases, remains an important health problem worldwide. It is estimated that there are 1700 new cases per year in Santa Catarina State, Brazil. OBJECTIVE: To improve polymerase chain reaction (PCR) sensitivity in detecting Mycobacterium tuberculosis in sputum samples. METHODS: This study proposed the use of glass beads as a modification of the routine protocol for sputum preparation used in the Laboratory of Molecular Biology and Mycobacteria at the Federal University of Santa Catarina, Florianópolis, Brazil. The study comprised 120 sputum samples, 60 of which were treated with the routine protocol, while 60 were treated with the modified protocol using glass beads. RESULTS: Samples treated with the routine protocol had a sensitivity of 56.7% (95%CI 44.1-69.2) in 16S rRNA PCR and 81.7% (95%CI 71.9-91.5) in insertion sequence (IS) 6110 PCR, compared with culture. Samples treated with the modified protocol had a sensitivity of 73.3% (95%CI 62.1-84.5) in 16S rRNA PCR and 100% in IS6110 PCR. CONCLUSION: The modified protocol using glass beads greatly improved mycobacterial detection in sputum samples compared with the routine protocol.