Chiyoji Abe

rpoB Genotypes of Mycobacterium tuberculosis Beijing Family Isolates from East Asian Countries

ABSTRACT The 81-bp region of the rpoB gene in 66 RifrMycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.

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

ABSTRACT 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.

Clinical Evaluation of Anti-Tuberculous Glycolipid Immunoglobulin G Antibody Assay for Rapid Serodiagnosis of Pulmonary Tuberculosis

ABSTRACT Previously we reported the development of a highly sensitive enzyme-linked immunosorbent assay specific for anti-tuberculous glycolipid (anti-TBGL) for the rapid serodiagnosis of tuberculosis. In this study, the usefulness of an anti-TBGL antibody assay kit for rapid serodiagnosis was evaluated in a controlled multicenter study. Antibody titers in sera from 318 patients with active pulmonary tuberculosis (216 positive for Mycobacterium tuberculosis in smear and/or culture tests and 102 smear and culture negative and clinically diagnosed), 58 patients with old tuberculosis, 177 patients with other respiratory diseases, 156 patients with nonrespiratory diseases, and 454 healthy subjects were examined. Sera from 256 younger healthy subjects from among the 454 healthy subjects were examined as a control. When the cutoff point of anti-TBGL antibody titer was determined as 2.0 U/ml, the sensitivity for active tuberculosis patients was 81.1% and the specificity was 95.7%. Sensitivity in patients with smear-negative and culture-negative active pulmonary tuberculosis was 73.5%. Even in patients with noncavitary minimally advanced lesions, the positivity rate (60.0%) and the antibody titer (4.6 ± 9.4 U/ml) were significantly higher than those in the healthy group. These results indicate that this assay using anti-TBGL antibody is useful for the rapid serodiagnosis of active pulmonary tuberculosis.

Biological and Molecular Characteristics of Mycobacterium tuberculosis Clinical Isolates with Low-Level Resistance to Isoniazid in Japan

ABSTRACT We reevaluated the BACTEC MGIT 960 antimicrobial susceptibility testing system (MGIT 960 AST) by using 1,112 isolates of Mycobacterium tuberculosis. When the results of MGIT 960 AST were compared with that of the proportion method using Ogawa medium (Ogawa PM), discrepant results were obtained for 30 strains with isoniazid, all resistant by MGIT 960 AST but susceptible by Ogawa PM. For 93% of the strains that produced discrepant results, the MIC was 0.4 or 0.8 μg/ml, showing resistance by the proportion method using Middlebrook agar plates. Furthermore, it was also established by analyses of the katG and inhA genes that strains resistant only by MGIT 960 AST have a low level of isoniazid (INH) resistance, indicating that MGIT 960 AST is a reliable method. Ninety-six strains were resistant to 0.1 μg/ml INH by MGIT 960 AST. When they were divided into three groups, Low-S (susceptible at 0.2 μg/ml), Low-R (resistant at 0.2 μg/ml), and High-R (resistant at 1.0 μg/ml), by Ogawa PM, 43.3% of the Low-S strains had mutations in the promoter region of inhA and no mutations were detected in katG codon 315, while 61.7% of the High-R strains had katG codon 315 mutations or a gross deletion of katG. These results suggest that mutations in inhA are associated with low-level resistance to INH and katG codon 315 mutations are associated with high-level resistance to INH. In addition, the analyses demonstrated some relationship of mutations in the inhA gene with ethionamide resistance for the Low-S strains, but not for the High-R strains.

Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan

Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of MboII restriction digestion. Thirty‐three out of 54 (61·1%) streptomycin‐highly resistant isolates (MIC  >  200 μg ml−1) were not digested by MboII. By contrast, the remaining 21 of 54 (38·9%) streptomycin‐highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 μg ml−1  <  MIC  ≤  200 μg ml−1), and all of 77 streptomycin‐sensitive isolates, were restricted. Thus, all isolates resistant for MboII digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed. Eighteen out of 19 (94·7%) streptomycin‐highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene  ; 10 out of 17 (58·8%) streptomycin‐resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene. In the total 36 streptomycin‐resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin‐resistant isolates, corresponding to 77·8%, whereas none of the streptomycin‐sensitive isolates had mutations in either the rpsL or rrs gene.

Heterogeneity of RNA Polymerase Gene (rpoB) Sequences of Mycobacterium gordonae Clinical Isolates Identified with a DNA Probe Kit and by Conventional Methods

ABSTRACT 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.

Evaluation of BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA): compared with the results of pyrazinamidase assay and Kyokuto PZA test

The fully automated BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA) was evaluated using 101 Mycobacterium tuberculosis clinical isolates. The results obtained with the system were compared with those of the pyrazinamidase (PZase) assay and the Kyokuto PZA test based on a broth culture, which is commercially available in Japan. The overall concordance rate was 90.1% (91/101) among the three methods in the initial test. The concordance rates between the BACTEC MGIT 960 PZA medium vs the PZase assay, the BACTEC MGIT 960 PZA medium vs the Kyokuto PZA test, and the PZase assay vs the Kyokuto PZA test were 93.1, 91.1, and 96.0%, respectively. On the repeat test of the 10 strains with discrepant results among the three methods, the concordance rates reached over 97% between each of the two systems. The results of the repeat test were confirmed by MIC testing and sequencing analysis of the pncA gene encoding PZase of M. tuberculosis. The mean turnaround times from incubation for PZA susceptibility testing were almost similar for the two methods based on liquid media, the BACTEC MGIT 960 PZA medium and the Kyokuto PZA test (7.7 and 7.4 days, respectively). These results indicate that both methods based on liquid media, the fully automated BACTEC MGIT 960 PZA medium and the Kyokuto PZA test for susceptibility testing to PZA, are useful for rapid diagnosis of PZA resistant tuberculosis.