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Abstract Background Kazakhstan remains a high-burden TB prevalence country with a concomitent high-burden of multi-drug resistant tuberculosis. For this reason, we performed an in depth genetic diversity and population structure characterization of Mycobacterium tuberculosis complex (MTC) genetic diversity in Kazakhstan with both patient and community benefit. Methods A convenience sample of 700 MTC DNA cultures extracts from 630 tuberculosis patients recruited from 12 out of 14 regions in Kazakhstan, between 2010 and 2015, was independently studied by high-throughput hybridization-based methods, TB-SPRINT (59-Plex, n = 700), TB-SNPID (50-Plex, n = 543). DNA from 391 clinical isolates was successfully typed by two methods. To resolve the population structure of drug-resistant clades in more detail two complementary assays were run on the L2 isolates: an IS6110-NTF insertion site typing assay and a SigE SNP polymorphism assay. Results Strains belonged to L2/Beijing and L4/Euro-American sublineages; L2/Beijing prevalence totaled almost 80%. 50% of all samples were resistant to RIF and to INH., Subtyping showed that: (1) all L2/Beijing were “modern” Beijing and (2) most of these belonged to the previously described 94–32 sublineage (Central Asian/Russian), (3) at least two populations of the Central Asian/Russian sublineages are circulating in Kazakhstan, with different evolutionary dynamics. Conclusions For the first time, the global genetic diversity and population structure of M. tuberculosis genotypes circulating in Kazakhstan was obtained and compared to previous local studies. Results suggest a region-specific spread of a very limited number of L2/Beijing clonal complexes in Kazakhstan many strongly associated with an MDR phenotype.

The aim of this study is to develop an innovative and alternative nucleic acid-based method for multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) diagnostics, such as the Cepheid GeneXPert or the Hain–Line Probe Assay methods, to simultaneously identify MDR-TB (patient benefit) and to provide clues as to MDR-TB transmission (community benefit) at a reasonable cost to the national public health programs. The DPO (Dual-Priming Oligonucleotide) principle was used to initially develop a 4-Plex multiplexed polymerase chain reaction (PCR) that simultaneously amplifies: (1) the CRISPR; (2) the rpoB hotspot, the katG and the inhA genes. The current maximal version of the assay allows the characterization of up to 68 markers, 59 of which are used routinely in one step (for the Luminex 200) or two-step methods (for the MagPix). Spoligotyping requires 43 markers, and rpoB516, 526, 531, katG315, inhA-8, -15, gyrA94, rrs1401, 1402, 1484 requires 25 markers. For each primer couple, one is biotinylated. Hybridization is performed after a PCR reaction on a microbead-based suspension array device with individually detectable oligonucleotide-coupled beads (Luminex 200 or MagPix systems) and detection proceeds through Streptavidin–Phycoerythrin labelling of the biotinylated-hybridized PCR products. This study shows that this technique provides 100% specificity and sensitivity for rpoB and 100% specificity and 90% sensitivity for isoniazid resistance on DNA extracted from cultures, compared with phenotypic DST. The method was fully validated against sequencing. The method is currently in the experimental validation phase on DNA extracted from biological material and preliminary results will be shown. This technique was recently upgraded to detect fluoroquinolone and aminoglycoside resistance by including 2 new PCR primer couples and 9 more probes on the rrs and gyrA genes. It is suggested that population studies using this laboratory method could provide a simpler and cheaper way to perform national TB-resistance surveys and would also allow to delineate more precisely, without further enquiries, MDR-TB transmission risks. In conclusion, these methods appear to be both economically and technically very innovative. The extension from a Research Use Only to a CE-marked In-Vitro Diagnostics assay is in progress.