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Mycobacterium tuberculosis resistance to rifampin results from nucleotide changes in the gene encoding the beta-subunit of the RNA polymerase (rpoB). We developed a reverse hybridization-based line probe assay (LiPA; the INNO-LiPA Rif. TB) carrying one oligonucleotide probe for the detection of M. tuberculosis complex strains and nine probes designed to detect nucleotide changes in the relevant part of rpoB. This assay was evaluated with 107 M. tuberculosis isolates with known rpoB sequences, 52 non-M. tuberculosis complex strains, and 61 and 203 clinical isolates found to be sensitive and resistant, respectively, by in vitro testing. The results indicated that (i) the M. tuberculosis complex probe was 100% specific, (ii) when compared to the results of nucleotide sequencing, no discrepancies with the results of INNO-LiPA Rif. TB were observed, (iii) all strains sensitive by in vitro susceptibility testing were correctly identified, and (iv) among the strains resistant by in vitro susceptibility testing, only 4 (2%) yielded conflicting results. The INNO-LiPA Rif. TB is therefore a reliable and widely applicable assay and a valuable tool for routine diagnostic use, given its simplicity and rapid performance.

In an attempt to clarify the taxonomy of the Mycobacterium avium complex, the relationship between IS1245 RFLP, growth temperature, 16S rDNA signature sequences and the 16S-23S rDNA internally transcribed spacer (ITS) of 160 M. avium-complex isolates from different sources was investigated. All 70 isolates identified as M. avium by INNO-LiPA MYCOBACTERIA (Innogenetics, Belgium), a DNA probe test that targets the ITS, and by 16S rDNA analysis carried multiple copies of IS1245. Three isolates with multiple copies of IS1245 were identified by 16S rDNA analysis as Mycobacterium intracellulare and by LiPA as M. intracellulare (n = 1) and M. avium-intracellulare complex (n = 2). A dichotomy among the M. avium isolates was found on the basis of a C and a G signature nucleotide at position 228 of the 16S-23S rDNA spacer sequence, and this grouping was largely confirmed on the basis of similarities in IS1245 RFLPs. Strains with the characteristic three-band IS1245 'bird-type', as well as M. avium subsp. silvaticum or 'wood-pigeon' strains, invariably contained the C signature. A third characteristic that separated the M. avium bird-type isolates from M. avium isolates from humans and other mammals was growth-temperature tolerance: in contrast to bird isolates, human/porcine isolates grew at 24 and 45 degrees C. Based on differences in IS1245 RFLP, 16S-23S rDNA ITS and growth temperature, M. avium isolates originating from birds should be considered as a separate, evolutionarily conserved taxon. Because all M. avium isolates from birds are invariably of this type, the designation M. avium subsp. avium should be reserved for these bird-type strains. For clarity in the epidemiology of M. avium-related disease, isolates from humans and pigs with multibanded IS1245 RFLPs merit a separate designation. The designation 'M. avium subsp. hominissuis' is suggested for this group of bacteria.

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species-or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n ‫؍‬ 18) or M. intracellulare (n ‫؍‬ 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n ‫؍‬ 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.To date, more than 70 mycobacterial species have been identified, and occasionally, isolates with unknown characteristics, mostly from immunodeficient patients, are being described. Differentiation of pathogenic and nonpathogenic mycobacteria other than Mycobacterium tuberculosis (MOTT) from members of the M. tuberculosis complex (MTC) is needed for patient management, considering that many MOTT are resistant to the antibiotics used for treatment of tuberculosis (27).Identification of mycobacterial isolates to the species level is performed by analysis of phenotypic and biochemical characteristics of the organisms after culture in solid media, which is a time-consuming process, or by high-pressure liquid chromatography analysis, which requires expensive equipment. Development of molecular tests has speeded up diagnosis, but most methods suffer from specific drawbacks. The AccuProbe system (Gen-Probe) differentiates only the MTC, M. avium, M. intracellulare, M. gordonae, and M. kansasii. In-house PCRbased identification systems have been developed but either identify a limited number of mycobacterial species or are difficult to use on a routine basis (4, 11). Restriction enzyme analysis of PCR products of specific genes is still widely used for identification of mycobacteria to the species level (7,19,23) and on clinical isolates (...