Fluorescence in situ hybridization assay using peptide nucleic acid probesfor differentiation between tuberculous and nontuberculous mycobacterium species in smears of Mycobacterium cultures

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of t he Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The tes t. is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examinati on. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Den mark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic spec ificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic spec ificity of 100% in both studies. The low sensitivity of the NTM-specific PN A probe in the Thai study was due to a relatively high prevalence of Mycoba cterium fortuitum, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (n = 46) or NTM (n = 17), whereas the remaining 9 mere negative with both probes and thus the result s were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.