Comparison of two laboratory-developed PCR methods for the diagnosis of pulmonary tuberculosis in Brazilian patients with and without HIV infection.

BACKGROUND: Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of pulmonary tuberculosis (PTB) is cheap and easy to use, but its low sensitivity is a major drawback, particularly in HIV seropositive patients. As such, new tools for laboratory diagnosis are urgently needed to improve the case detection rate, especially in regions with a high prevalence of TB and HIV. OBJECTIVE: To evaluate the performance of two in house PCR (Polymerase Chain Reaction): PCR dot-blot methodology (PCR dot-blot) and PCR agarose gel electrophoresis (PCR-AG) for the diagnosis of Pulmonary Tuberculosis (PTB) in HIV seropositive and HIV seronegative patients. METHODS: A prospective study was conducted (from May 2003 to May 2004) in a TB/HIV reference hospital. Sputum specimens from 277 PTB suspects were tested by Acid Fast Bacilli (AFB) smear, Culture and in house PCR assays (PCR dot-blot and PCR-AG) and their performances evaluated. Positive cultures combined with the definition of clinical pulmonary TB were employed as the gold standard. RESULTS: The overall prevalence of PTB was 46% (128/277); in HIV+, prevalence was 54.0% (40/74). The sensitivity and specificity of PCR dot-blot were 74% (CI 95%; 66.1%-81.2%) and 85% (CI 95%; 78.8%-90.3%); and of PCR-AG were 43% (CI 95%; 34.5%-51.6%) and 76% (CI 95%; 69.2%-82.8%), respectively. For HIV seropositive and HIV seronegative samples, sensitivities of PCR dot-blot (72% vs 75%; p=0.46) and PCR-AG (42% vs 43%; p=0.54) were similar. Among HIV seronegative patients and PTB suspects, ROC analysis presented the following values for the AFB smear (0.837), Culture (0.926), PCR dot-blot (0.801) and PCR-AG (0.599). In HIV seropositive patients, these area values were (0.713), (0.900), (0.789) and (0.595), respectively. CONCLUSION: Results of this study demonstrate that the in house PCR dot blot may be an improvement for ruling out PTB diagnosis in PTB suspects assisted at hospitals with a high prevalence of TB/HIV.

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis.

BACKGROUND: Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M. tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis. FINDINGS: For this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity, positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2% (95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of culture and histopathology of pleural tissue specimens. CONCLUSION: The real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast diagnosis of pleural TB.