Diagnosis of smear-negative pulmonary tuberculosis using sequence capture polymerase chain reaction.

Abstract

Techniques based on the polymerase chain reaction (PCR) can be used to rapidly identify DNA from Mycobacterium tuberculosis in clinical samples from patients with tuberculosis, but prior studies evaluating this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis have reported poor sensitivity and/or specificity. We have developed a procedure in which mycobacterial DNA in crude samples is specifically captured prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other inhibitors of the amplification reaction (sequence capture PCR). To evaluate the usefulness of this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis, sequence capture PCR was performed prospectively on samples of bronchoalveolar lavage fluid from consecutive patients suspected of having pulmonary tuberculosis but for whom three consecutive samples of respiratory secretions were smear negative. Of the 27 patients evaluated, active tuberculosis was diagnosed in nine; sequence capture PCR was positive for all of these patients, including the three for whom all specimens submitted for culture were negative. No positive results were obtained for lavage fluid from the 18 patients for whom the diagnosis of active tuberculosis was subsequently excluded or 25 additional patients undergoing bronchoalveolar lavage for evaluation of other pulmonary problems, even though many of these patients had a history of prior tuberculosis or radiographic evidence of prior tuberculous infection. Paucibacillary forms of pulmonary tuberculosis can be rapidly identified with high sensitivity and specificity using sequence capture PCR performed on samples obtained by bronchoalveolar lavage.