Molecular pathologic detection of mycobacteria]

In recent years, significant advances in the diagnosis of mycobacterial infections have been made by the introduction of direct pathogen detection methods. These techniques are usually based on the polymerase chain reaction (PCR) or on a transcription-mediated amplification (TMA) process. The majority of the protocols have been optimized for the detection of mycobacterial nucleic acids in fresh fluid or fresh tissue specimen. Unfortunately pathologists are frequently confronted with the problem that tissues with histologically suspicious lesions have been entirely fixed in formalin. As a result of this routine fixation, DNA and RNA are heavily degraded and the usually high sensitivity of the amplification techniques is greatly impaired. Consequently, only PCR protocols designed for small amplification targets are still suitable for an efficient detection of microbial DNA in formalin-fixed and paraffin-embedded tissues. We therefore adapted PCR assays with amplification products < 200 bp for the detection of M. tuberculosis-complex DNA (targets: IS6110 and 65 kDa-antigen gene) in routine biopsies. Although the sensitivities of the two assays varied significantly with the degree of DNA degradation, we were able to detect M. tuberculosis-complex specific DNA in about 25% of the tissues with a granulomatous inflammation and negative Ziehl-Neelson stain. Recently, we have added a third PCR-assay, which in combination with direct sequencing also allows us to detect DNA from M. leprae and several atypical mycobacteria species. PCR-analysis has significantly improved the diagnosis of mycobacterial infections by supplementing conventional histological examination of formalin-fixed and paraffin-embedded tissues.