Identificação molecular de espécies de micobactérias por PCR-RFLP hsp65 e implicações clínicas do diagnóstico convencional

Abstract

Introduction: The establishment of the therapeutic regimen for mycobacteriosis depends on the accurate identification of the species of mycobacteria and the misdiagnosis can result in inappropriate treatment and increased mortality rate of patients. Differentiation between mycobacterial species has been made by conventional tests that analyze phenotypic and biochemical characteristics after a long culture period. The molecular diagnosis of mycobacteria allows fast detection and identification of the species, although it is not available in public health programs. Objective: To standardize the use of molecular technique PCR-RFLPhs^65 in species identification of mycobacteria, test method in culture samples from patients diagnosed with mycobacteriosis compared to the conventional techniques and to determine the clinical implications of erroneous diagnosis based on symptoms, culture and bacilloscopy. Methodology: The PCR-RFLP hsp65 technique was standardized in samples already identified in reference centers and subsequently tested in 55 culture samples from patients with mycobacterial infections occurred in 2013 and 2014, comparing the results with the clinical diagnosis. Results: All patients were clinically diagnosed with tuberculosis, but only 63.7% (35/55) of culture samples were confirmed as M. tuberculosis by PCR-RFLP and the rest, 36.3% (20/55) of the patients were diagnosed by molecular method as atypical mycobacteria The molecular method identified 18 positive samples (32.7%) for nontuberculous mycobacteria (M avium type1, M. avium type 2, M. kansasii type1, M. intracellulare type 1, M. mucogenicum, M. chelonae, M. terrae type 3 and one NTM without pattern known in literature) and 3.7% (2 samples) were negative. Concerning to the treatment only 11% (6/55) had substitute treatment in the case of failure of the standard treatment and suspected or confirmed infection with atypical mycobacteria. Conclusion: PCR-RFLPhs^65 differentiated complex tuberculosis of non-tuberculous mycobacteria classifying them at the species level, showed be discriminatory, fast and economical. Species identification in mycobacterial infections is essential to choose the correct therapeutic regimen and the use of this technique for diagnosis can assist public health programs increasing accuracy in diagnostic and reducing inappropriate treatment.