Evaluation of nested polymerase chain reaction for detecting mycobacterial DNA in pleural fluid

Abstract

A protocol based on the polymerase chain reaction (PCR) is the most sensitive method for detecting mycobacteria in clinical samples. However, few studies have assessed the usefulness of this method in the diagnosis of tuberculous effusion. We developed a highly sensitive and specific nested PCR method, that amplifies the bovine tuberculous MPB70 gene and the mycobacterial 16S rRNA gene for use in detecting Mycobacterium tuberculosis (M. tuberculosis) and mycobacteria, respectively, in clinical samples. We determined the sensitivity of this method for detecting mycobacteria in samples containing known amounts of mycobacterial DNA and in DNA extracted from pleural effusions obtained from 10 patients with pulmonary tuberculosis in whom standard microbiological techniques had detected mycobacteria in sputum but not in pleural effusion. The nested PCR method for the bovine tuberculous MPB70 gene and the mycobacterial 16S RNA gene was able to detect M. tuberculosis and mycobacterial genomes only if there were at least 2 copies per sample. Positive results for M. tuberculosis and the mycobacterial genomes were obtained by nested PCR in 2 of 10 and in 3 of 10 samples of pleural fluid, respectively but no mycobacteria were detected in malignant effusions obtained from 9 patients with lung cancer. The nested PCR method represents a rapid means for detecting mycobacteria in some pleural effusions previously found to be negative by culture. We speculate that the reaction of the host against mycobacteria is more important than the mycobacteria themselves in the pathogenesis of pleural effusion in which mycobacteria are not detected.