from november 2008 through may 2010 , m. tuberculosis isolates were cultured during routine care of adults > 18 years of age with primary tb and no history of treatment . the patients were from 2 regional referral centers , the irkutsk regional tb - prevention dispensary and the research practice center for phthisiatry ( yakutia ) ; the study was approved by the institutional review boards at the university of virginia and irkutsk state medical university . initial pretreatment isolates were grown on lowenstein - jensen agar slants and identified to species in accordance with world health organization recommendations . drug susceptibility was tested by absolute concentration method on agar slants ; drugs tested were rifampin ( critical concentration 40 g / ml ) , isoniazid ( 1 g / ml and 10 g / ml ) , ethambutol ( 2 g / ml ) , streptomycin ( 10 g / ml ) , ethionamide ( 30 g / ml ) , and kanamycin ( 30 g / ml ) . dna extraction was performed on all isolates , followed by 12-loci mycobacterial interspersed repetitive unit variable number tandem repeat ( miru - vntr ) analysis ( 7 ) and further lineage definition by region of difference deletions , or for ural strains as described ( 5 ) . phylogenetic tree construction was based on the miruvntrplus database ( 8) , and vntr international type numbers were confirmed on the sitvit database ( 9 ) . dna from mdr isolates was amplified and sequenced for the known drug - resistance determining regions katg , inha , rpob , embb , gyra , rrs , and eis by using methods described by the centers for disease control and prevention ( 10 ) . sequences were compared with published sequences for m. tuberculosis h37rv by using genedoc version 2.7.0 . among 235 patients with primary tb ( 130 from yakutia , 105 from irkutsk ) , isoniazid monoresistance was found in isolates from 16 ( 12% ) from yakutia and 19 ( 18% ) from irkutsk ( p = 0.27 ) . multidrug resistance was found for 61 patients ( 36 [ 28% ] from yakutia and 25 [ 24% ] from irkutsk ) ( p = 0.55 ) . mean age (  sd ) for these 61 patients was 33 (  12 ) years , 40 ( 66% ) were male , and these characteristics did not differ significantly between patients from irkutsk and from yakutia . however , no hiv - infected patients were identified from yakutia compared with 11 ( 44% with mdr tb ) from irkutsk ( p<0.001 ) . twelve mdr tb patients from irktutsk died ( outcome unknown for the other 13 patients ) , including all with hiv , compared with 4 ( 11% ) from yakutia who died ( p = 0.002 ) . follow - up varied and was limited mostly to inpatients . among all 235 patients with primary tb , strains of the beijing family were significantly more common among those from irkutsk ( 70 [ 67% ] ) than from yakutia ( 40 [ 31% ] ) ( p<0.001 ) . however , strains found in yakutia ( s 256 [ 11% ] , t 8 [ 7% ] , and ural 171 [ 5% ] ) were not found in irkutsk ( table 1 ) . the cluster of s 256 ( miru profile 233325153325 ) was the most common among primary mdr tb isolates from yakutia and was fully 86% mdr ( table 1 ; technical appendix ) . * miru - vntr ,  mycobacterial interspersed repetitive unit  variable number tandem repeat ( original 12-loci profile ) . included genotypes found in > 5 isolates only ; mit ,  miru  vntr international type ; mdr ,  multidrug - resistant tuberculosis ( conventional resistance to isoniazid and rifampin ) ; na , not applicable . significance determined by  analysis with yates correction or fisher exact test when appropriate . among isolates from patients with primary mdr tb , 51 ( 84% ) were available for dna sequencing : 27 from yakutia and 24 from irkutsk ( table 2 ) . among isoniazid - resistant isolates , the mutation in codon 315 of katg was present in 91% . among rifampin - resistant isolates , mutations in the resistance - determining region of rpob ( codons 511533 ) were present in only 79% . the pnca mutation was common across genotypes from both sites , occurring in 62% of isolates amplified . notably , both isolates with mutation in eis from yakutia occurred in mdr strains with the s 256 genotype and without rrs mutation . * all inha mutations were associated with a ser315thr mutation in katg except for 1 isolate in which katg did not amplify . excluding 25 silent pnca mutations ( ser32ser most common , n = 14 ) . for all 3 mutations of eis associated with kanamycin resistance , rrs was wild type . in eastern siberia , > 25% of primary tb was mdr , equivalent to the highest proportion reported from the russian federation ( 2 ) . however , regionally specific genotypic patterns and resistance mutations were identified . as expected , in irkutsk primary mdr tb was driven by strains of beijing lineage ( 5,6 ) . yet in the more geographically isolated population of yakutia , a strain previously unidentified in the russian federation , s 256 , had a miru profile recently found among canadian aboriginal populations ( 11 ) . in yakutia , s 256 was highly drug resistant and was the most common genotype among patients with primary mdr tb . although rpob mutations were found in only 79% of rifampin - resistant isolates , these findings are consistent with those in a recent report from novosibirsk oblast , which similarly included non - beijing and s - family strains and found a sensitivity of only 63% for the rpob mutation ( 12 ) . lack of phenotypic correlation can result from alternate mechanisms of resistance or imperfect conventional susceptibilities in lowenstein - jensen medium or from use of old drug stock . such discrepancy necessitates urgent clarification because substitution of conventional susceptibility testing with molecular probe  based methods such as genexpert mtb / rif ( cepheid , ca , usa ) has been strongly advocated but would lead to dramatically different results and treatment regimens ( 13 ) . of note , isolates of the s 256 strain accounted for a proportion of the cases in which mutation in the promoter region of eis was associated with kanamycin resistance , but rrs was wild type . commercial assays have focused on the rrs locus , which has greater sensitivity for amikacin , as the sole target for the class of injectable agents ( 14 ) , yet in eastern siberia , the injectable agent available is kanamycin . furthermore , we found a range of reported and unreported mutations across the entire pnca gene ; most were point mutations resulting in amino acid substitution , but some strains had mutations that resulted in deletion or frameshift . phenotypic methods and assays of functional pyrazinamidase activity should be performed in this region because results might have major implications for novel mdr tb drugs that work best with pyrazinamide ( 15 ) . we were unable to obtain detailed clinical information about all patients with primary tb , thus preventing adequate comparison of nongenotypic risk factors for mdr tb or establishment of definitive epidemiologic links among clustered isolates . furthermore , lack of conventional fluoroquinolone or pyrazinamide susceptibility testing limited comparison with gyra and pnca mutations , respectively . despite these limitations , this work characterizes severe isoniazid monoresistant and mdr tb in eastern siberia among patients with no history of tb treatment . the regionally distinct phylogenetic patterns and certain drug - resistance mutations necessitate careful application of novel diagnostics and empiric therapeutic strategies . phylogenetic trees of mycobacterium tuberculosis from patients with primary tuberculosis , yakutia and irkutsk , russian federation .
of 235 mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the irkutsk oblast and the sakha republic ( yakutia ) , eastern siberia , 61 ( 26% ) were multidrug resistant . 
 a novel strain , s 256 , clustered among these isolates and carried eis - related kanamycin resistance , indicating a need for locally informed diagnosis and treatment strategies .