The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It Rifampin is an important component of effective multidrug therapies for tuberculosis and leprosy; however, widespread use has led to the emergence of rifampin-resistant (Rif) strains, threatening its usefulness in treating mycobacterial diseases (4-6, 8, 26, 27). Rapid information about drug susceptibility patterns is critical to the treatment of individuals with mycobacterial disease for which rifampin is indicated. Since conventional drug susceptibility testing can require 2 to 4 weeks after growth detection (Mycobacterium tuberculosis) or up to a year (Mycobacterium leprae) in mouse footpads, improvements are needed to yield accurate analysis in a shorter time. DNA diagnostic assays have the potential to provide rapid analysis of rifampin resistance in mycobacteria because of their high degree of sensitivity and specificity and the fact that they do not rely on in vitro growth for results. Shortening the time between diagnosis and the onset of effective therapy should improve patients' survival (tuberculosis) or decrease physical deformities and ocular manifestations resulting in disabilities and blindness (leprosy).Developing such assays requires knowledge of the molecular basis of Rif' in pathogenic mycobacteria. Mutations resulting in the Rif' phenotype in prokaryotes have been mapped to the gene encoding the 1-subunit of the DNA-dependent RNA polymerase (rpoB gene) (10, 11). Recently, the entire rpoB genes of M. leprae (7) resistance have been identified in both species (8,12,28,29). To further characterize mutations associated with the Rif phenotype in M. tuberculosis, M. leprae, and other pathogenic mycobacteria, we developed a rapid PCR-based, DNA sequencing protocol targeted to a 305-bp region of rpoB. By direct DNA sequencing of PCR products, the nucleic acid sequence within this region was determined in 4 rifampinsusceptible (Rifs) and 4 Rif' strains of M. leprae and in 12 Rif' and 110 Rif' strains of M. tuberculosis. In addition, mutations were identified in this region of Rif' strains of Mycobacterium africanum and Mycobacterium avium, the latter causing frequent opportunistic infections in immunocompromised hosts. On the basis of these results we have established conditions for a PCR-heteroduplex formation assay (PCR-HDF) for the rapid detection of the Rif' phenotype in pathogenic mycobacteria. MATERUILS AND METHODSMycobacterial strains. Rifampin-susceptible and -resistant strains of M. leprae were isolated initially from homogenates of skin biopsy samples from lepromatous leprosy patients not responding to antileprosy therapy, which included rifampin, and were subsequently defined as resistant to rifampin by the standard mouse footpad drug susceptibility assay (23). These strains were amplifie...

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The 81-bp region of the rpoB gene in 66 Rif r Mycobacterium 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.

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.To eradicate tuberculosis, it is important to improve diagnostic techniques so that active tuberculosis can be treated at an early stage before tuberculous bacilli can be detected in the sputa. The tuberculin skin test is not useful in subjects with a previous history of active tuberculosis or Mycobacterium bovis BCG vaccination. Gene technology utilizing nucleic acid amplification has been successfully introduced for rapid diagnosis of pulmonary tuberculosis. Clinically, however, its usefulness is reduced in patients without sputum expectoration. In fact, in previous papers, including a report of the American Thoracic Society (ATS) Workshop in 1997, the sensitivity of gene diagnosis was reported to be about 50% in patients with acid-fast bacillus smear-negative pulmonary tuberculosis (4, 6, 8) and was particularly low (5 to 20%) in smear and culture-negative patients with active pulmonary tuberculosis (5, 12). A further drawback is that this test is expensive. Therefore, there has been strong demand for the development of rapid, reliable, and less costly diagnostic methods for the detection of pulmonary tuberculosis.We previously developed an enzyme immunoassay in which the glycolipid antigen trehalose 6,6Ј-dimycolate (TDM) purified from Mycobacterium tuberculosis H37Rv was used as an antigen for detecting antituberculosis immunoglobulins and showed that a glycolipid was an effective antigen for serodiagnosis (13, 16). Subsequently, by mixing TDM with more hydrophilic glycolipids, we constructed a new tuberculous glycolipid (TBGL) antigen and successfully established a sensitive serodiagnostic kit for tuberculosis using this ant...

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.Mycobacterium tuberculosis has been known to secrete more than 33 different proteins (3). One of the predominant proteins is MPB64, a 24-kDa protein initially isolated from culture filtrates of Mycobacterium bovis BCG Tokyo (5,11,20). The MPB64 antigen was found in the culture fluid of only strains of the M. tuberculosis complex and some substrains of M. bovis BCG (1, 5, 10, 11) and has shown potential for diagnostic use [1, 12, 13; T. Tomiyama, K. Matsuo, and C. Abe, abstract, Int. J. Tuberc. Lung Dis. 1(Suppl. 1): S59, 1997]. Recently, a simple immunochromatographic assay, Capilia TB (TAUNS, Numazu, Japan), using anti-MPB64 monoclonal antibodies for rapid discrimination between the M. tuberculosis complex and mycobacteria other than tubercle bacilli (MOTT), was developed [1; Tomiyama et al., Int. J. Tuberc. Lung Dis. 1(Suppl. 1): S59, 1997]. The test strip consists of a sample pad, a reagent pad, a nitrocellulose membrane, and an absorbent pad. Because the test sample applied in the sample well flows laterally through the membrane, the antibody-colloidal gold conjugate binds to the MPB64 antigen in the sample. The complex then flows further and binds to the monoclonal antibodies on the solid phase in the test zone, producing a red-purple band within 15 min. Some evaluation studies have demonstrated that the Capilia TB is very useful for rapid confirmation of the M. tuberculosis complex in liquid cultures without any troublesome sample preparation in a laboratory [1, 2, 6, 8; Tomiyama et al., Int. J. Tuberc. Lung Dis. 1(Suppl. 1):S59, 1997]. The kit is commercially available in Japan (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) and is routinely used in most clinical laboratories in hospitals or commercial laboratory centers.More recently, Capilia TB-negative M. tuberculosis strains have been isolated in some districts of Japan; therefore, the sensitivity of the Capilia TB test was reexamined by using a large number of clinical specimens in this study. A total of 13,942 clinical specimens, mostly sputum, were used for the study. Patients were admitted to Fukujuji Hospital (Japan Anti-Tuberculosis Association, Kiyose-shi, Tokyo) with symptoms of pulmonary tuberculosis from September 2001 to October 2002. Of these, 784 (5.6%) were culture positive for Mycobacteria. All mycobacterium-positive cultures were differentiated and identified by the Capilia TB, and the results were compared with those of the Accuprobe-M. tuberculosis complex culture confirmation test (Gen-Probe, San Diego, Calif.) and the DN...

We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.

In Mycobacterium smegmatis and a limited number ofMycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, ≧200 μg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying therrs gene and the intervening sequence between therrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI andTsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI orDdeI.