[Mycobacterium shinshuense isolated from cutaneous ulcer lesion of right lower extremity in a 37-year-old woman].

PURPOSE: Second clinical infection case of Mycobacterium shinshuense was presented, we tried the identification of M. shinshuense that is isolated from skin. OBJECT: Mycobacteria species isolated from cutaneous ulcer lesion of right lower extremity in a 37-year-old woman. METHOD: Identification by DNA-DNA Hybridization, 16S rRNA and rpoB method as genomic level and conventional method. RESULT: It did not grow on 1% Ogawa's slant medium at both 37 degrees C and 42 degrees C, but grew at 28 degrees C. It formed yellowish colonies in the dark. It was difficult to distinguish M. shinshuense from M. ulcerans and M. marinum by DNA-DNA hybridization (DDH) and DNA sequencing. To identify that it is M. shinshuense, growth rate, temperature range of mycobacterial growth, light coloration reaction, biochemical and biological tests, and drug susceptibility testing were further explored. Finally it was identified as M. shinshuense based on these CONSIDERATION: For Mycobacteria species which grow 2 weeks after inoculation at 28 degrees C, and which is identified as M. marinum by DDH method, it is necessary to identify with sequence and conventional method.

[Multi-drug resistant lung tuberculosis due to double infection of MDR strain].

The case is 47-year-old, homeless man. He was diagnosed as cavitary, sputum smear positive pan-sensitive lung tuberculosis, and admitted to TB ward of our hospital. At the age of 4-year-old he had tuberculous hilar lymphadenopathy and took medicine. He had no other associated disease, and HIV test was negative. He started standard chemotherapy and 2 months later his sputum culture was converted to negative. His adherence to medicine was thought to be good. But about 2 weeks after the sputum conversion, his sputum culture was re-converted to positive for Mycobacterium tuberculosis. Thereafter, during and after the completion of standard chemotherapy, his sputum culture had been intermittently positive. The drug sensitivity tests of the strain after re-conversion showed multi-drug resistance. RFLP analysis revealed that the strain before conversion was totally different strain from the strain after re-conversion to positive. The case was considered to be caused by the double infection of MDR strain of Mycobacterium tuberculosis during the course of treatment for tuberculosis due to a sensitive strain of Mycobacterium tuberculosis.

A small outbreak of pulmonary tuberculosis in non-close contact patrons of a bar.

Four habitual drinking and smoking patients with pulmonary tuberculosis who were thought to have had no contact with one another were admitted to our hospital. During admission, we found that they were regular visitors of the same bar. To investigate the possibility of outbreak, we analyzed the tuberculosis isolates from them by restriction fragment length polymorphism. Such analysis showed identical chromosomal DNA restriction patterns of these 4 culture isolates. We concluded that these patients were considered to represent a mini-outbreak of pulmonary tuberculosis, although there was little, if any, contact among them while in or out of the bar.

Mutations including IS6110 insertion in the gene encoding the MPB64 protein of Capilia TB-negative Mycobacterium tuberculosis isolates.

A simple immunochromatographic assay, Capilia TB, using anti-MPB64 monoclonal antibodies, is a kit for discriminating between the Mycobacterium tuberculosis complex and mycobacteria other than tubercle bacilli. The sensitivity of the kit was estimated to be 99.2% (381 of 384 samples). The sequencing analysis revealed that all of the Capilia TB-negative isolates had mutations within the mpb64 gene, leading to the production of an incomplete protein as a result of a deletion of the C-terminal region of the protein.

[Molecular epidemiology of Mycobacterium tuberculosis using by RFLP analysis between genomic DNA--its accomplishment and practice].

Recently, epidemiology of M. tuberculosis have been performed by molecular techniques as a probe with the insertion sequence (IS) 6110. In the traditional study of tuberculosis epidemiology, information about social contact of persons and patient's illness history used to be an only relevant basis for elucidating transmission of tuberculosis infection. Therefore, it was very difficult to give a clear conclusion of whether isolates from different patients derived from a common source of infection or not. The subspecies typing of M. tuberculosis strains by IS6110 has become possible, based on the visualization of multiple loci of an insertion sequence (IS6110) that is a relatively stable gene fragment existing in a specific region of the genome. The variability of the number of copies and locations of this IS6110 in a genome is the basis that enables this technique to be used for the above purpose, which is a unique tool applicable to the analysis of M. tuberculosis. Generally, this technique, i.e., restriction fragment length polymorphism (RFLP) analysis, depends on the diversity of pattern of any polymorphic marker found in a genome of a strain. Among various markers so far developed and examined, IS6110 has been proved most appropriate for the purpose of typing strains of M. tuberculosis complex, especially in such circumstances as in Japan where isolated strains' RFLP patterns are similar each with others so that finer subtyping is needed. In this time, I would like to review the following topics based on the world literature of molecular epidemiology and the findings of our own that we have achieved during 1992 through 2001 in our Institute; (1) typing for the tracking of source of infection, (2) diffuse infection, (3) the presence of region-specific influential strains, (4) cross-contamination of strains in the laboratory, (5) the stability of IS6110, (6) phylogeny of tuberculosis (genotypes in Okinawa prefecture), and (7) the distinction between M. tuberculosis and M. bovis BCG, (8) computer-assisted patients management system. We also investigated the mode of transmission and risk factors of tuberculosis, based on the tuberculosis epidemiological data obtained in many parts of the world as well as the findings we gathered in our country from 1992 to 2001.

Heterogeneity of RNA polymerase gene (rpoB) sequences of Mycobacterium gordonae clinical isolates identified with a DNA probe kit and by conventional methods.

In a previous study, we have evaluated genetic identification by using the rpoB gene, which was recently introduced by Kim et al. (J. Clin. Microbiol. 39:2102-2109, 2001; J. Clin. Microbiol. 37:1714-1720, 1999). In this process, we examined the rpoB gene heterogeneity of clinical isolates identified as Mycobacterium gordonae with the conventional biological and biochemical tests and/or a commercially available DNA probe kit. Sequencing of the rpoB gene of 34 clinical isolates revealed that M. gordonae clinical isolates were classified into four major clusters (A, B, C, and D). Interestingly, organisms belonging to cluster D (15 isolates) did not hybridize with M. gordonae ATCC 14470 and specifically possessed urease activity. Therefore, it could be considered to be a novel mycobacterium. The identification of M. gordonae is known to have ambiguous results sometimes. On the other hand, identification of clinical isolates seems to be inconvenient and unsuitable because of a more than 99% 16S rRNA gene similarity value between clusters. These findings suggest that the existence of M. gordonae-like mycobacteria that share similar biochemical and biological characteristics with the 16S rRNA gene of an M. gordonae type strain but less similarity at the genomic DNA level may have complicated the identification of M. gordonae in many laboratories. Furthermore, compared with hsp65 PCR restriction analysis (PRA), rpoB PRA would have the advantage of producing no ambiguous results because of the intracluster homogeneity of the rpoB gene. In this case, rpoB would provide clearer results than hsp65, even if PRA analysis was used. We demonstrated that these M. gordonae-like mycobacteria were easily distinguished by PRA of the rpoB sequence. Additionally, the significance of this M. gordonae-like cluster may help to establish the comparison between the M. gordonae isolates from a clinical specimen and an infectious process in a given patient and to determine the true incidence of infection with this microorganism.

[Molecular epidemiology of Mycobacterium tuberculosis: its accomplishment and future perspective].

In the traditional study of tuberculosis epidemiology, information about social contact of persons and patient's illness history used to be an only relevant basis for elucidating transmission of tuberculosis infection. Therefore, it was very difficult to give a clear conclusion of whether isolates from different patients derived from a common source of infection or not. Recently, the subspecies typing of M. tuberculosis strains has become possible, based on the visualization of multiple loci of an insertion sequence (IS6110) that is a relatively stable gene fragment existing in a specific region of the genome. The variability of the number of copies and locations of this IS6110 in a genome is the basis that enables this technique to be used for the above purpose, which is a unique tool applicable to the analysis of M. tuberculosis. Generally, this technique, i.e., restriction fragment length polymorphism (RFLP) analysis, depends on the diversity of pattern of any polymorphic marker found in a genome of a strain. Among various markers so far developed and examined, IS6110 has been proved most appropriate for the purpose of typing strains of M. tuberculosis complex, especially in such circumstances as in Japan where isolated strains' RFLP patterns are similar each with others so that finer subtyping is needed. In this lecture, I would like to review the following topics based on the world literature of molecular epidemiology and the findings of our own that we have achieved during 1992 through 2001 in our Institute; 1) typing of the isolates for the identification of the infection source, 2) pathogenesis of tuberculosis under low incidence situation, 3) predominance of certain genotypes endemic in an area, 4) cross-contamination of isolates in the laboratory, 5) the stability of IS6110 patterns, 6) phylogeny of M. tuberculosis complex, and 7) differentiation between M. tuberculosis and M. bovis BCG.