Of the validated species in the genus Mycobacterium, M. tuberculosis is the most common and important pathogen and causes 2 million deaths and over 8 million cases of tuberculosis annually worldwide (2-4, 7). In addition to the multidrug resistant strains of M. tuberculosis, NTM (non-tuberculosis mycobacteria) infection can cause other serious clinical problems. Moreover, because the drugs of choice for the treatment of NTM infection are heavily dependent on taxonomic statuses (15,19,20), it is of importance that we are able to differentiate these at the species level during early diagnostic procedures.The classical identification of mycobacteria based on cultural and biochemical tests may take several weeks, and sometimes, even then, may fail to provide a precise identification. To resolve such disadvantages of conventional methods, various PCR-mediated methods have been applied for the rapid detection and identification of mycobacteria species. Among these, PCRrestriction analysis (PRA) is preferred, since it offers an easy, rapid, and inexpensive means of identifying mycobacteria species. Thus, a number of PRA methods targeting several chronometers, such as, 16S rDNA (6, 8), hsp65 (5, 17), dnaJ (16), and rpoB (11), have been developed. However, although these PRA techniques have been successfully applied to mycobacteria identification at the culture level, problems are encountered when they are applied directly to clinical specimens, especially sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, because these bacteria produce complex PRA patterns that are difficult to interpret.Previously, we reported that a novel PRA method that targets 644 bp hsp65 DNA is useful for the differentiation of mycobacterial species at the culture level (12). In particular, by only the use of a single restriction enzyme, AvaII, it can differentiate the three most clinically important mycobacteria species, i.e., M. tuberculosis and two MACs, M. avium and M. intracellulare. Moreover, the simplicity of this PRA method might enable the direct detection of pathogenic mycobacteria from clinical specimens such as sputum.In the present study, to evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 sequence Abstract: To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS6110 PCR. Although this method showed a sensitivity slightly lower than IS6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.
