Background: Various primers are being tested for the detection of Mycobacterium tuberculosis DNA. The accuracy of the polymerase chain reaction (PCR) depends on the target sequence used and whether the test will be performed in culture or in clinical specimens. Objectives: To identify DNA sequences, specifically those commonly reported as targets for diagnosis of tuberculosis (TB), in clinical samples of M. tuberculosis strains. Method: Eighty-one clinical samples from suspected TB patients were initially processed and submitted to bacilloscopy (smear) and culture, and PCR was performed with specific primers for the following targets: IS 6110, 65 kDa, 38 kDa and MPB64. Results: Smear and culture results were negative in 24 samples, as was the PCR. The 19 samples testing smear positive, as well as the isolated strains, were 100% positive on PCR, with the exception of the 89.4% result from PCR with MPB64 primers. In the 38 smear negative and culture positive samples, PCR results were inconsistent. The primers specific for amplifying the 123 bp IS 6110 fragment gave the highest positivity (92.1%), diagnostic agreement (0.9143), co-positivity (94.7%) and co-negativity (100%). Conclusion: The IS 6110, 38 kDa, MPB64 and 65 kDa sequences were found in the genome of all M. tuberculosis strains isolated in patients from the state of Amazonas. The protocol for processing the clinical samples prior to PCR analysis and the specific primers used to amplify the 123bp IS 6110 fragment showed a greater efficiency in diagnosing pulmonary (paucibacillary) tuberculosis in comparison to published data.

Keywords: Primers/PCR. Diagnosis/Tuberculosis. Mycobacterium tuberculosis.

Objective: To evaluate the accuracy of bacteriological methods and of polymerase chain reaction (with primers specific for IS6110 of the Mycobacterium tuberculosis complex) in testing sputum samples from indigenous (Amerindian) and non-indigenous patients. Methods: A total of 214 sputum samples (154 from indigenous patients and 60 from non-indigenous patients) were analyzed in order to determine the accuracy of smear microscopy (direct and concentrated versions) for acid-fast bacilli, culture, and polymerase chain reaction. Results: Both microscopy methods presented low sensitivity in comparison with culture and polymerase chain reaction. Specificity ranged from 91% to 100%, the concentrated acid-fast smear technique being the least specific. Nontuberculous mycobacteria were isolated three times more frequently in samples from indigenous patients than in those from non-indigenous patients. False-positive and false-negative polymerase chain reaction results were more common in the indigenous population. Conclusion: Positivity and isolation of nontuberculous mycobacteria in the acid-fast smear in conjunction with polymerase chain reaction positivity raise the following hypotheses: nontuberculous mycobacteria species with DNA regions homologous to, or even still possessing, the M. tuberculosis IS6110 exist in the Amazon; colonization of the oropharynx or of a tuberculous lesion accelerates the growth of the nontuberculous mycobacteria present in the sputum samples, making it impossible to isolate M. tuberculosis; A history of tuberculosis results in positivity for M. tuberculosis DNA. The absence of bacteriological positivity in the presence of polymerase chain reaction positivity raises questions regarding the inherent technical characteristics of the bacteriological methods or regarding patient history of tuberculosis.

Keywords: Tuberculosis, pulmonary/diagnosis; Mycobacterium tuberculosis, Polymerase chain reaction/methods; Indians, south american

Objective: To propose a methodology for characterizing proficiency in sputum smear microscopy for acid-fast bacilli (AFB) in the diagnosis of tuberculosis and to determine the number of microscopies necessary to establish this proficiency, as well as the quality of the transcription of results, the causes of the discrepancies in the readings (rater or microscope used), and the criterion for classification of microscopy results that poses the most difficulty in characterizing proficiency. Methods: Four hundred sputum smear microscopies for the diagnosis of tuberculosis were analyzed through double-blind readings by six professionals who usually read/supervise microscopies performed in public health care facilities. The sample was stratified to obtain, at least, a reliability of 90% in the double-blind readings, an α error of 5%, and a precision of 3%. The results were analyzed using observed reliability and the kappa index. Results: Thirteen errors (0.27%) were found in the transcription of results. Reliability increased when the three distinct categories of positive results (AFB+, AFB++, and AFB+++) were grouped or when inconclusive results were excluded from the analysis. The quantification of the bacterial load was the classification criterion that posed the most difficulty in establishing proficiency. Using higher quality microscopes increased reliability. Reliability values stabilized only from the reading of 75 microscopies onward. Conclusions: Double-blind sputum smear microscopy readings using a panel containing 75 slides (36 negative, 4 inconclusive, and 35 positive) proved to be appropriate for characterizing proficiency in sputum smear microscopy for the diagnosis of tuberculosis when such proficiency is intended to reproduce laboratory routine.

Keywords: Tuberculosis; Microscopy; Reproducibility of results.