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A polymerase chain reaction (PCR) method, which amplifies a fragment of the 16S ribosomal RNA (rRNA) gene present in all mycobacterial species, was developed and tested on 84 formalin-fixed paraffin-embedded tissue specimens from 51 patients with human immunodeficiency (HIV) infection. The PCR products were characterized either by sequencing or by hybridization with nonradioactive oligonucleotide probes specific for Mycobacterium tuberculosis complex, M. avium, or M. genavense. Sequencing was successful for 26 samples compared with the 45 samples for probe hybridization. The sensitivity of DNA amplification compared with microscopic examination was 79.5%. A mixed infection was detected with M. genavense for only one patient who was infected with M. tuberculosis complex. In the group of 22 control patients, where no diagnosis of mycobacterial infection was made during life and no acid-fast bacteria were seen during the autopsy, four samples of one patient were positive by hybridization with the M. tuberculosis probe. This patient had a clinical history compatible with tuberculosis. This PCR method may be a powerful tool for the precise diagnosis of mycobacterial infections from histopathologic material, provided that several sections from the same specimen block are tested.