A SEARCH FOR MYCOBACTERIAL DNA IN GRANULOMATOUS TISSUES FROM PATIENTS WITH SARCOIDOSIS USING THE POLYMERASE CHAIN-REACTION

We have used the polymerase chain reaction as a tool to detect the presence of mycobacterial DNA from organisms of the Mycobacterium tuberculosis complex and other species of mycobacteria in samples from patients with sarcoidosis. Using systems based on the amplification of a fragment of the gene coding for the 65-kD mycobacterial antigen, which were demonstrated to detect approximately 20 mycobacterial genomes/mu-g total DNA, DNA from M. tuberculosis was reproducibly identified in DNA extracted from granulomatous tissues from two patients with sarcoidosis, but could not be detected in DNA extracted from tissue biopsies (n = 16) or cells recovered by lavage (n = 6) from most sarcoid patients or from control subjects (n = 22). Using a system based on the amplification of a fragment of the IS6110 insertion element, which could reliably detect two genomes of mycobacterial DNA/mu-g total DNA, no additional positive results were observed. in an effort to identity another species of Mycobacterium present in granulomatous tissues from sarcoid patients but not control tissues, a fragment of the 65-kD mycobacterial antigen was amplified and then reamplified using "nested" primers recognizing sequences that are highly conserved among mycobacteria and closely related species, and the amplified DNA products were cloned and sequenced. Amplified DNA was observed in a minority of samples from patients and control subjects (32/84 and 34/77 attempts, respectively, p > 0.2), resulting from amplification of DNA from at least 17 different organisms. Most sequences were identified in both patients and control subjects or only in control subjects; the seven sequences that were identified only in samples from sarcoid patients were identified on only one, or at most, two occasions. We conclude that DNA from M. tuberculosis can be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. if, however, this or another single mycobacterial species plays a pathogenic role in most patients with sarcoidosis, it must be present in established lesions at levels below the threshold of detection reached in these studies (< 15 organisms/10(6) human cells).