A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.Mycobacteria other than Mycobacterium tuberculosis (MOTT) are increasingly recognized as causing human infections (31). Conventional biochemical methods and phenotypic tests for species differentiation are laborious and time-consuming and frequently require specialized testing that is beyond the capacity of clinical laboratories. Genotypic methods for the identification of mycobacteria have been developed in recent years (3,10,23,29). These molecular methods are gaining increasing importance because they yield rapid and, in most cases, unequivocal results.In 1992 Plikaytis et al. (15) developed a method for differentiating among slowly growing Mycobacterium species by PCR and restriction fragment length polymorphism analysis (PRA). A similar approach was used by Telenti et al. (27) for rapid identification of mycobacteria to species level based on evaluation of the gene coding for the 65-kDa heat shock protein (22) by PCR and restriction enzyme analysis. Subsequently, this approach was used for the taxonomic separation of rapidly growing mycobacteria (18, 24), for routine identification of mycobacteria (1,4,7,9,11,12,23,26), and for identifying Mycobacterium leprae (16,25) and Mycobacterium kansasii subspecies (17).In all these studies the algorithm describing the mycobacteria species is based on the use of two restriction enzymes (BstEII and HaeIII) and separation of the restriction fragments on an agarose gel. PRA patterns are then interpreted by converting the running distance in electrophoresis to apparent molecular size (in base pairs). Difficulties in PRA interpretation may stem from similarities in a number of band sizes that are critical for discrimination of species and are not sufficiently resolved by agarose-based gel electrophoresis.In view of the application of PRA-based identification of mycobacteria in our diagnostic laboratory and its application to an increasing number of different species, we conducted the present study in order to propose an algorithm based on 10% polyacrylamide gel electrophoresis (PAGE) of restriction digests to improve the resolu...

A total of 1,830 specimens (75.7% respiratory and 24.3% nonrespiratory) were cultured in parallel with the MB/BacT and BACTEC 460 TB systems and on Lowenstein-Jensen (LJ) medium. Mycobacteria were identified from 173 (6.5%) specimens. The most common species recovered were Mycobacterium tuberculosis complex (65.9%),Mycobacterium avium complex (22.5%), andMycobacterium chelonae (9.2%). The recovery rates by individual systems were 96.5, 99.4, and 95.9% for MB/BacT, BACTEC 460 TB, and LJ medium, respectively, for all mycobacteria; the recovery rates were 99.1, 100, and 98.2%, respectively, for M. tuberculosis complex alone. The difference among the recovery rates for all mycobacteria and those for individual species was not significant. The BACTEC 460 TB system detected M. tuberculosis isolates more rapidly than the MB/BacT system (8 versus 11.8 days for smear-positive specimens [P < 0.01] and 18 versus 21 days for smear-negative specimens [P < 0.05]), whereas the MB/BacT system more rapidly detected the nontuberculous mycobacteria (17.1 versus 12.7 days [P < 0.01]). These results indicate that the nonradiometric MB/BacT system is a rapid, sensitive, and efficient method for the recovery ofM. tuberculosis and nontuberculous mycobacteria from both pulmonary and extrapulmonary clinical specimens.

Mycobacterium genavense is a frequently missed agent of disseminated disease in AIDS patients. The increasing frequency with which such organism is being isolated in Italy suggested a comparison of local survey with data reported in literature. Isolates presumed to belong to the species M. genavense were centralized and identified by means of genomic sequencing and/or HPLC analysis of cell wall mycolic acids; clinical data were obtained from relevant patients' record and collected using a proper questionnaire. In 24 cases in which this organism has been isolated in Italy M. genavense was grown, prevalently from blood, in liquid medium after an average of six weeks of incubation. In overwhelming majority, patients were males, presented other opportunistic diseases and were characterized by very low CD4+ counts (average 23/microl); most frequent symptoms were fever, anemia and weight loss. All but two patients, who died before the mycobacterial infection was diagnosed, were treated with at least three drugs; the mean survival was close to one year. A review of literature reports revealed a wide overlapping of clinical and microbiological features.

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The susceptibility of 115 Mycobacterium tuberculosiscomplex clinical isolates to isoniazid, streptomycin, ethambutol, and rifampin was assessed by the MB/BacT and BACTEC 460TB systems. The correlation between the two tests was 98.3% for isoniazid, 100% for streptomycin and rifampin, and 95.8% for ethambutol. Turnaround times for antimicrobial susceptibility testing ranged from 5 to 11 days (median, 8.5 days) for MB/BacT and from 4 to 8 days (median, 6 days) for BACTEC 460TB.