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Genetic research about Mycobacterium avium complex.

We conducted four genetic studies on the Mycobacterium avium complex (MAC). (1) M. avium genotyping: A total of 70 clinical isolates from patients with pulmonary MAC infections were typed by MATR-VNTR, IS1245-RFLP, and MIRU-VNTR analyses to compare discriminatory powers of these typing methods. To allow a comparison of discriminatory powers, the Hunter-Gaston discriminatory index (HGDI) was calculated, giving a HGDI of 0.960 for IS1245-RFLP, 0.949 for MIRU-VNTR, and 0.990 for MATR-VNTR, demonstrating that MATR-VNTR analysis is the best of the three genotyping methods. (2) Genetic characteristics of M. avium: Japanese clinical isolates of M. avium were subjected to insertion sequence (IS) analyses. First, an analysis of 81 isolates by heat shock protein 65 identified all isolates as belonging to the subspecies of M. avium subsp. hominissuis. Another analysis by IS901 identified about 70% of the isolates as IS901-carriers. IS901 had been thought to be carried by the subspecies that infect birds: M. avium subsp. avium and M. avium subsp. silvaticum. Studies have reported that most human isolates in the U.S. and Europe carry no IS901. The prevalence of IS901-carriers among Japanese clinical isolates of M. avium is thus a significant characteristic. A further analysis of the IS901 showed that compared with M. avium subsp. avium, the clinical isolates shared 60 point mutations of nucleotide sequence. This novel insertion sequence was designated "ISMav6". (3) The CAM-resistance gene in MAC: This study assessed the correlation between CAM-susceptibility and mutation of the gene involved in drug resistance (A DNA sequence analysis identified mutations at positions 2058 and 2059 in domain V of 23S-rRNA). Furthermore, a system was developed to rapidly detect the presence/absence of CAM resistance by ARMS-PCR, a procedure used to detect gene mutations. The utility of this new system was also evaluated. A total of 253 clinical isolates were tested for drug susceptibility, with 227 isolates identified as sensitive and 26 as resistant. Sequence analyses showed that all 28 strains randomly selected for testing from the sensitive strains were wild type, whereas 24 of the 26 resistant strains were mutant type. The rest of the 2 strains were subsequently confirmed to be mutant type after they were isolated from contaminations with sensitive strains. These results showed an association between drug susceptibility and drug-resistant gene mutation. In addition, ARMS-PCR provided a sensitivity of 84.6% (22/26) and a specificity of 100% (28/28) for the detection of gene mutations. The lower sensitive was probably attributable to the fact that every one of the 4 strains was a combination of wild type and mutant type. These results indicated that compared with drug-susceptibility tests, ARMS-PCR provides earlier results on the presence/absence of drug resistance and has the capability of rapid detection even when the specimen contains a mixture of sensitive and resistant strains. (4) Development of a VNTR analysis for M. intracellulare: Bioinformatics analyses were used to develop a VNTR analysis for M. intracellulare and to evaluate the utility of the VNTR analysis. First, the Tandem Repeat Finder (TRF) software was used to conduct a search of TR loci on the genomic data of M. intracellulare ATCC 13950 published in December 2007, resulting in the identification of 16 TR loci, which were used in VNTR analyses of 74 isolates from pulmonary MAC infections. The HGDI was 0.988, suggesting an excellent discriminatory power. Furthermore, a stability evaluation of the VNTR loci was conducted in isolates from patients with long-term bacilli discharge. The VNTR loci were stable without changes for up to 4 years in 14 such patients. These results indicated that this method is useful in M. intracellulare genotyping and in determining whether the cause of recurrence in recurred patients is endogenous from the remnant bacilli or exogenous from another infection of different bacilli, given that the VNTR loci have been confirmed to be stable.