we analyzed culture - positive cases of mdr tb reported to the california tb registry from january 1 , 2004 , through december 31 , 2007 . california state law ( health and safety code title 17  2505 ) requires reporting of all verified cases of tb , submission of all m. tuberculosis isolates to local public health laboratories , and submission of all mdr m. tuberculosis isolates to the california department of public health microbial diseases laboratory . testing for first- and second - line drug susceptibilities was performed at local laboratories or at the microbial diseases laboratory by using bactec 460 ( becton dickinson diagnostic instruments , sparks , md , usa ) , mgit 960 ( becton dickinson ) , or the agar proportion method . some isolates were forwarded to the centers for disease control and prevention ( cdc ; atlanta , ga , usa ) for additional second - line drug testing or for confirmation of drug resistance . patients were included in the study if mdr m. tuberculosis was identified on > 1 isolate . demographic and clinical information for all patients with mdr tb was abstracted from state tb surveillance forms ( report of a verified case of tuberculosis ) , which have high validity ( 18 ) . all protocols were approved by the california committee for the protection of human subjects and university of california , san francisco , committee for the protection of human subjects . for each isolate , genomic dna was extracted from solid media ( lowenstein - jensen slants , middlebrook 7h10 or 7h11 agar ) , liquid media ( bactec 12 b or mgit [ becton dickinson ] ) , or smear - positive sputum sediments . a real - time pcr assay with 6 molecular beacon probes was performed by using an iq5 icycler instrument ( bio - rad , hercules , ca , usa ) to screen for mutations associated with isoniazid and rifampin resistance ( 19 ) . two molecular beacons that targeted katg ( codon 311317 ) and the inha promoter were used to detect isoniazid resistance  conferring mutations , and 4 molecular beacons that targeted the core region of rpob were used to detect rifampin resistance  conferring mutations . isolates with mutations in katg detected by the wild - type probe were further tested with another molecular beacon that specifically targeted katg s315 t ( agc  acc ) . when molecular beacon analysis did not show katg s315 t or -c15 t inha promoter mutations , the entire fura - katg locus ( h37rv : 21536262156657 , 3,031 bp ) was sequenced as described ( 6 ) . sequence data were generated by using abi bigdye v3.1 dye terminator sequencing chemistry and the abi prism 3730xl capillary dna analyzer ( applied biosystems , foster city , ca , usa ) at the genomic core facility , university of california , san francisco ( www.genomics.ucsf.edu/sequencing/index.aspx ) , and were analyzed with clustalw ( www.ebi.ac.uk/tools/clustalw/index.html ) . spacer oligonucleotide typing ( spoligotyping ) and mycobacterial interspersed repetitive unit ( miru ) typing were performed in accordance with the centers for disease control and prevention universal genotyping program procedures ( 20 ) . spoligotyping was performed by using luminex - based methods to detect 43 known spacer sequences in the direct repeat locus ( 21 ) . a capillary sequencer , ceq 8000 ( beckman , fullerton , ca , usa ) , was used to analyze the number of repeated sequences at each of the 12 loci . is6110-based restriction fragment length polymorphism ( rflp ) genotyping was performed following standardized methods ( 23 ) . rflp patterns were compared by using bioimage whole band analyzer software version 4.2.1 ( bioimage corp . , ann arbor , mi , usa ) ( 24 ) . rflp patterns with < 20 identical bands , or > 20 bands and differing by no more than a single band , were considered matched . is6110 rflp band assignment was edited by 2 independent readers , and the cluster assignment was confirmed visually spoligotype families h , lam , and t , x , s were considered to be of euro - american lineage ; beijing of east - asian lineage ; eai of indo - oceanic lineage ; and cas of east african - indian lineage ( 5 ) . cases were defined as clustered if > 2 isolates from cases reported during the study period shared the same miru and spoligotype , matched is6110 rflp , and had specific drug resistance  conferring mutations for isoniazid and rifampin . clustering was assumed to represent both transmission of m. tuberculosis and progression to active disease , leading to secondary case generation within the period of the study . cases not in a cluster were considered to be the result of reactivation of latent infection . patients with the earliest case report date within a cluster were regarded as index cases . the proportion of clustered mdr tb cases was analyzed as the number of clustered cases divided by the total number of culture - positive cases that occurred during the study period . because of limited sample size , the independent effects of katg s315 t and phylogeographic lineage on clustering were estimated by using exact logistic regression methods . refugee status and refugee resettlement during the study period could bias our results in that overrepresentation of ethnic groups or geographic locations with a high prevalence of particular strain - specific factors ( phylogeographic lineage or drug - resistance mutations ) could confound the association under study . to examine this influence and potential clustering of tb cases within households or communities related to refugee resettlement , sensitivity analysis was conducted by reestimation of study results after excluding 1 ) patients immigrating from refugee settings within the past 3 years and 2 ) the single largest patient cluster , which accounted for 40% of all clustered cases . in a separate analysis , standard logistic regression was used to estimate the effect of katg s315 t and phylogeographic lineage on sputum smear positivity , again controlling for refugee status . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated to measure associations of interest . categorical data ( e.g. , sex , foreign vs. us birth , homelessness ) were compared by using fisher exact tests . the wilcoxon rank - sum test was used to determine differences in the distribution of continuous variables ( e.g. , age , time from entry into the united states to tb diagnosis ) . interaction between the independent variables was assessed separately for each factor by stratification and statistical testing ( breslow - day with the tarone correction   and zelen test ) , with p<0.2 assumed to indicate the presence of interaction . analyses were performed by using stata 10 ( statacorp . , college station , tx , usa ) and statexact 8 ( cytel software corp . , cambridge , ma , usa ) . for each isolate , genomic dna was extracted from solid media ( lowenstein - jensen slants , middlebrook 7h10 or 7h11 agar ) , liquid media ( bactec 12 b or mgit [ becton dickinson ] ) , or smear - positive sputum sediments . a real - time pcr assay with 6 molecular beacon probes was performed by using an iq5 icycler instrument ( bio - rad , hercules , ca , usa ) to screen for mutations associated with isoniazid and rifampin resistance ( 19 ) . two molecular beacons that targeted katg ( codon 311317 ) and the inha promoter were used to detect isoniazid resistance  conferring mutations , and 4 molecular beacons that targeted the core region of rpob were used to detect rifampin resistance  conferring mutations . isolates with mutations in katg detected by the wild - type probe were further tested with another molecular beacon that specifically targeted katg s315 t ( agc  acc ) . when molecular beacon analysis did not show katg s315 t or -c15 t inha promoter mutations , the entire fura - katg locus ( h37rv : 21536262156657 , 3,031 bp ) was sequenced as described ( 6 ) . sequence data were generated by using abi bigdye v3.1 dye terminator sequencing chemistry and the abi prism 3730xl capillary dna analyzer ( applied biosystems , foster city , ca , usa ) at the genomic core facility , university of california , san francisco ( www.genomics.ucsf.edu/sequencing/index.aspx ) , and were analyzed with clustalw ( www.ebi.ac.uk/tools/clustalw/index.html ) . spacer oligonucleotide typing ( spoligotyping ) and mycobacterial interspersed repetitive unit ( miru ) typing were performed in accordance with the centers for disease control and prevention universal genotyping program procedures ( 20 ) . spoligotyping was performed by using luminex - based methods to detect 43 known spacer sequences in the direct repeat locus ( 21 ) . a capillary sequencer , ceq 8000 ( beckman , fullerton , ca , usa ) , was used to analyze the number of repeated sequences at each of the 12 loci . is6110-based restriction fragment length polymorphism ( rflp ) genotyping was performed following standardized methods ( 23 ) . rflp patterns were compared by using bioimage whole band analyzer software version 4.2.1 ( bioimage corp . , ann arbor , mi , usa ) ( 24 ) . rflp patterns with < 20 identical bands , or > 20 bands and differing by no more than a single band , were considered matched . is6110 rflp band assignment was edited by 2 independent readers , and the cluster assignment was confirmed visually . the phylogeographic lineage of strains was determined from spoligotyping results . spoligotype families h , lam , and t , x , s were considered to be of euro - american lineage ; beijing of east - asian lineage ; eai of indo - oceanic lineage ; and cas of east african - indian lineage ( 5 ) . cases were defined as clustered if > 2 isolates from cases reported during the study period shared the same miru and spoligotype , matched is6110 rflp , and had specific drug resistance  conferring mutations for isoniazid and rifampin . clustering was assumed to represent both transmission of m. tuberculosis and progression to active disease , leading to secondary case generation within the period of the study . cases not in a cluster were considered to be the result of reactivation of latent infection . patients with the earliest case report date within a cluster were regarded as index cases . the proportion of clustered mdr tb cases was analyzed as the number of clustered cases divided by the total number of culture - positive cases that occurred during the study period . because of limited sample size , the independent effects of katg s315 t and phylogeographic lineage on clustering were estimated by using exact logistic regression methods . refugee status and refugee resettlement during the study period could bias our results in that overrepresentation of ethnic groups or geographic locations with a high prevalence of particular strain - specific factors ( phylogeographic lineage or drug - resistance mutations ) could confound the association under study . to examine this influence and potential clustering of tb cases within households or communities related to refugee resettlement , sensitivity analysis was conducted by reestimation of study results after excluding 1 ) patients immigrating from refugee settings within the past 3 years and 2 ) the single largest patient cluster , which accounted for 40% of all clustered cases . in a separate analysis , standard logistic regression was used to estimate the effect of katg s315 t and phylogeographic lineage on sputum smear positivity , again controlling for refugee status . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated to measure associations of interest . categorical data ( e.g. , sex , foreign vs. us birth , homelessness ) were compared by using fisher exact tests . the wilcoxon rank - sum test was used to determine differences in the distribution of continuous variables ( e.g. , age , time from entry into the united states to tb diagnosis ) . interaction between the independent variables was assessed separately for each factor by stratification and statistical testing ( breslow - day with the tarone correction   and zelen test ) , with p<0.2 assumed to indicate the presence of interaction . analyses were performed by using stata 10 ( statacorp . , college station , tx , usa ) and statexact 8 ( cytel software corp . , cambridge , ma , usa ) . during the study period , 11,395 cases of tb were reported in california , of which 9,037 ( 79% ) had positive cultures . of these , of the 141 ( 2% ) incident mdr tb cases , 123 ( 87% ) had isolates available for miru , spoligotyping , and is6110 rflp analysis . isolates unavailable for genotyping ( n = 18 ) were more often from los angeles county ; other demographic and clinical characteristics were similar to those of analyzed cases . twenty - five mdr m. tuberculosis isolates were aggregated in 8 clusters ( 1 cluster of 10 cases , 1 cluster of 3 cases , and 6 clusters of 2 cases ) for an overall cluster proportion of 20% ( 25/123 ) . excluding the 8 index cases , 14% ( 17/123 ) of all cases were considered to have resulted from recent transmission and rapid progression to disease ( secondary cases ) . within clusters , a median of 3 months elapsed between successive secondary cases ( range 020 months ) . of the 123 total cases , 113 ( 92% ) occurred among foreign - born patients , with more than half ( 56% ) occurring among immigrants from mexico , philippines , the people s republic of china , or vietnam ( table 1 ) . seven of 8 index cases occurred either in mexican immigrants ( 3/8 ) or recent refugees from thailand , lao people s democratic republic , or india ( 4/8 ) . median time from patient arrival in the united states to tb diagnosis was approximately twice as long for clustered as for nonclustered cases ( 4.3 years vs. 2.4 years ) and 3 times as long for cases in mexican - born persons as for cases in persons from other countries ( 7.3 years vs. 2.3 years ) . * mdr tb , multidrug - resistant tuberculosis ; or , odds ratios ; ci , confidence interval ; iqr , interquartile range ; afb , acid - fast bacilli . ors describe the association between the characteristic of interest and mdr tb case - clustered status . afghanistan ( 1 ) , burma ( 1 ) , cambodia ( 5 ) , ethiopia ( 1 ) , indonesia ( 1 ) , mongolia ( 1 ) , nepal ( 1 ) , peru ( 2 ) , south korea ( 5 ) , and ukraine ( 1 ) . # nonclustered cases : cervical lymph node ( 5 ) , bone ( 1 ) , other ( 1 ) ; clustered cases : pleural ( 1 ) .  * * culture positive after > 8 months of treatment ; limited to pulmonary tb patients who were alive at diagnosis and had an initial positive sputum culture . younger persons were more likely than older persons to harbor strains involved in mdr tb clusters ( table 1 ) . hiv infection was unusual in this patient population ; only 3 ( 4% ) of 75 patients with known hiv status were hiv - infected . twenty - eight percent ( 35/123 ) of patients reported a history of active tb ; this proportion did not vary between clustered and nonclustered cases ( p = 0.75 ) . time to culture negativity ( 2.4 vs. 2.8 months , p = 0.95 ) , treatment failure , or death did not differ between clustered and nonclustered cases ( p = 0.57 ) among 105 ( 85% ) of 123 cases for which data were available . seventy - five percent of mdr m. tuberculosis strains harbored the isoniazid resistance  conferring mutation katg s315 t , including all ( 100% ) clustered strains ( table 2 ) . when we controlled for strain lineage and refugee status , katg s315 t was inversely associated ( or 0.28 , 95% ci 0.090.89 , p = 0.03 ) with sputum smear positivity ( table 3 ) . most rifampin resistance  conferring mutations were found between positions 529 and 534 of the rpob gene , likely indicative of the serine to leucine substitution at position 531 ( s531l ) of the rpob gene ( 27 ) . the association between this mutation and clustering did not reach statistical significance ( or 2.2 , 95% ci 0.87.4 ; p = 0.16 ) . * mdr tb , multidrug - resistant tuberculosis ; s315 t , serine - to - threonine substitution at position 315 . novel mutations detected : y413stop , t314 t ( silent ) , w161 g , d61e ( fur a ) , r145p , p325l , and v633f . inha promoter mutation was concomitant with 4/91 ( 4% ) isolates harboring the katg s315 t and 2/8 ( 25% ) isolates with katg mutations other than s315 t . no mutations detected by molecular beacons ; sequencing was not possible for these isolates because of degraded dna . rifampin resistance  conferring mutations were not detected by the molecular beacon assay for 4 isolates . * mdr tb , multidrug - resistant tuberculosis ; s315 t , serine - to - threonine substitution at position 315 ; or , odds ratio ; ci , confidence interval . mdr m. tuberculosis isolates were distributed among east - asian ( 47% ) , euro - american ( 30% ) , indo - oceanic ( 20% ) , and east african  indian ( 3% ) phylogeographic lineages . lineage could not be established for 11 cases . on univariate analysis , east - asian strain lineage was associated with clustering ( table 4 ) , but not with adverse outcome ( death or treatment failure ) . mdr , multidrug - resistant tuberculosis ; or , odds ratio ; ci , confidence interval . clustering was independently associated with katg s315 t ( or 11.2 , 95% ci 2.2- ; p = 0.004 ) and refugee status ( or 6.0 , 95% ci 1.236.2 ; p = 0.03 ) in exact multivariate analyses in which strain lineage and sputum smear positivity were controlled for ( table 5 ) . the estimated association between katg s315 t and case clustering did not appreciably change in sensitivity analyses that excluded either all recently arrived refugees or the single largest patient cluster . * mdr tb , multidrug - resistant tuberculosis ; or , odds ratio ; ci , confidence interval . in this 4-year population - based molecular epidemiologic study , transmission followed by secondary case generation contributed to 14% , or 1 of every 7 , mdr tb cases in california . clustered cases occurred more often among younger persons and persons who had emigrated from mexico and refugee settings in southeast asia . in addition , pathogen - specific factors were associated with clustering of mdr tb cases , independent of traditional clinical and demographic risk factors . the proportion of mdr tb cases attributed to transmission in california was higher than that reported by other investigators in most low incidence settings ( 2832 ) . the largest clusters in our study resulted from mdr tb outbreaks in california after resettlement of hmong refugees in 20052006 ( 33 ) and resettlement of tibetan refugees in 20012006 . the associations between pathogen - specific factors and case clustering could be due to regional differences in strain prevalence and preferential migration of persons with specific strains to california . we attempted to control for these factors by using highly stringent criteria to define clustered cases and by adjusting for refugee status in our multivariate model . however , without detailed contact information and contact tracing , we can not be certain of the extent to which transmission or progression to active disease occurred within or outside california . given that most clustered cases occurred among persons residing in the united states for > 3 years and that us - born persons were involved in 3 of 8 clusters , at least some proportion of mdr tb transmission is likely to have occurred in california . this observation suggests that although most mdr tb cases in the united states are related to the migration of persons already infected with drug - resistant m. tuberculosis , a small but notable proportion may be due to ongoing transmission . heterogeneity in the reproductive success of drug - resistant m. tuberculosis is now well established ( 34,35 ) . in this study , the katg s315 t mutation was the only isoniazid resistance  conferring mutation found among clustered mdr tb cases . the high prevalence of katg s315 t among mdr strains ( 12,14,3638 ) and the association of this mutation with increased secondary case generation among isoniazid - monoresistant strains ( 6,7 ) have been documented . we report that the katg s315 t isoniazid resistance  conferring mutation retains an independent effect on clustering of mdr tb cases despite the presence of mutations that confer resistance to additional drugs . in particular , secondary case generation did not significantly vary according to site of rpob mutations that confer rifampin resistance , which supports the hypothesis that these are no - cost mutations or that compensatory mutations commonly exist ( 39 ) . the katg s315 t mutation is thought to preserve fitness through the relative preservation of catalase - peroxidase production ( 9 ) , although whether this mutation is associated with different clinical phenotypes is unknown . in this study , we noted an inverse association between presence of the katg s315 t mutation and sputum smear positivity . in addition , the katg s315 t mutation was associated with case clustering , independent of sputum smear status . these findings suggest that the katg s315 t mutation may preserve the ability of m. tuberculosis to transmit and cause secondary cases through mechanisms unrelated to conventional indices of disease severity , such as the presence of abundant acid - fast bacilli in sputum . first , mdr tb in california is occurring predominantly among patients who were not born in the united states , with some cases from recent transmission and rapid progression to disease . our study suggests that in california , younger persons and persons who have emigrated from mexico and from refugee settings may be at higher risk for transmitting mdr m. tuberculosis . likewise , our findings reinforce the need for giving priority to screening and prevention activities in immigrant communities and us investment in international tb control ( 40 ) . second , if our results are verified in other settings , tb - control programs should consider pathogen - specific factors such as isoniazid resistance  conferring mutations when planning the intensity of contact investigation and secondary case - finding activities . first , our estimates of case clustering are imprecise because of the limited number of mdr tb cases observed during the study period . however , these estimates are the best currently available , given that the data make up the largest population - based tb registry in the united states . second , although our definition of genotypic clustering was highly rigorous , the lack of detailed epidemiologic information precluded confirmation of transmission within california . third , because our study did not include pan - susceptible or isoniazid - monoresistant strains , we can not comment directly on mdr m. tuberculosis pathogenicity relative to these groups . lastly , our analyses implicitly assume independence of outcome events , and household or community - level factors potentially associated with clustering were not available . future studies should be designed so that statistical methods can be used that are able to accommodate the possible effects of within - household clustering . we found a substantial proportion of mdr tb cases and case clustering in california among non  us - born persons , and the katg s315 t mutation was independently associated with clustering . validation of these findings in larger cohorts and in different population settings may have crucial public health consequences .
toc summary : type of isoniazid resistance  conferring mutation may be a determinant of genotypic clustering .