A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC→ACC (Ser→Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia

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We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.The spread of multidrug-resistant tuberculosis (MDR-TB) has increased worldwide and reached epidemic proportions in many countries. MDR Mycobacterium tuberculosis strains are considered those resistant to at least rifampin (RIF) and isoniazid. RIF is a key component of the World Health Organization DOTS (directly observed therapy, short course) regimen: because RIF monoresistance is extremely rare and development of isoniazid resistance usually precedes that to RIF, resistance to the latter is considered to be the MDR marker (8). It has been shown in many studies that RIF resistance in up to 95 to 98% of resistant strains is caused by mutations in the rpoB gene encoding the RNA polymerase ␤-subunit (20, 21). These mutations are generally described in the short 81-bp region in rpoB (RIF resistance-determining region: codons 507 to 533 [21]). In addition, RIF resistance may be caused by mutations in other parts of rpoB outside the hot spot region, such as codon 176 (146 [21]) in the N-terminal part (9) and codons 541 and 553 (18). Methods used so far to detect rpoB mutations associated with RIF-resistance were direct DNA sequencing, PCR-single-strand conformation polymorphism, heteroduplex mobility, dot spot, RNA-RNA mismatch, and some other assays (reviewed in references 4 and 25). Previously, we have described a multiplex allele-specific PCR (MAS-PCR) assay based on standard PCR and agarose gel electrophoresis to detect mutations in katG315 (15) and embB306 (14) in M. tuberculosis strains. A similar assay to detect rpsL43, rpoB531, and katG315 mutations was very recently published by Victor et al. (24). The MAS-PCR method in our design (14, 15) ...

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Parts of katG and rpoB from 27 RussianMycobacterium tuberculosis isolates were sequenced to detect mutations causing resistance to isoniazid (INH) and rifampin (RMP), respectively. All 24 INH-resistant isolates had a mutatedkatG, and 22 of them (91.7%) carried a mutation coding for a Ser315Thr shift. An rpoB mutation was noted for each of the 21 RMP-resistant isolates, with Ser531Leu being the most prevalent change encoded. Only two isolates had identical IS6110fingerprints.