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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A large diphtheria epidemic in the 1990s in Russia and neighboring countries underlined the importance of permanent surveillance of the circulating and emerging clones of Corynebacterium diphtheriae, and hence there is a need for highly discriminatory, simple and portable typing methods. In the complete genome sequence of C. diphtheriae strain NCTC13129, we previously identified in silico two clustered, regularly interspaced short palindromic repeat (CRISPR) loci, and developed a macroarray-based method to study polymorphism in the larger DRB locus. We named this method spoligotyping (spacer oligonucleotide typing), analogously to a similar method of Mycobacterium tuberculosis genotyping. Here, we included in the analysis novel spacers of the other CRISPR locus in C. diphtheriae (DRA); both loci were simultaneously co-amplified and co-hybridized against the membrane with 27 different immobilized spacer-probes. The use of additional DRA spacers improved strain differentiation and discriminated within large DRB clusters. The 156 Russian strains of the epidemic clone were subdivided into 45 combined spoligotypes compared to 35 DRB-spoligotypes and only two ribotypes ('Sankt-Peterburg' and 'Rossija'). The spoligotyping method allows digital presentation of profiles and therefore it is perfectly suitable for interlaboratory comparison and database management; it may become a powerful tool for epidemiological monitoring and phylogenetic analysis of C. diphtheriae.