DOC

Mycobacterium tuberculosis Beijing Lineage Favors the Spread of

By Troy Nelson,2014-05-30 12:02
10 views 0
Mycobacterium tuberculosis Beijing Lineage Favors the Spread of

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 35443550 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00715-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis Beijing Lineage Favors the Spread of

    Multidrug-Resistant Tuberculosis in the Republic of Georgia (

    1233Stefan Niemann,* Roland Diel, George Khechinashvili, Medea Gegia, 34Nino Mdivani,and Yi-Wei Tang

    1Molecular Mycobacteriology, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany; 2Department of Pneumology, Medical School Hannover, Carl-Neuberg-Str.1, 30625 Hannover, Germany; 3Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia; and Departments of 4Pathology and Medicine, Vanderbilt University Medical Center, Nashville, Tennessee

    Received 7 April 2010/Returned for modication 15 June 2010/Accepted 30 July 2010

    High rates and transmission of multidrug-resistant (MDR) tuberculosis (TB) have been associated with the Mycobacterium tuberculosis complex (MTBC) Beijing lineage, pointing to the importance of pathogen genetic factors for the modulation of infection outcome and epidemiology. We present here an in-depth analysis of the population structure of MTBC strains from the Republic of Georgia, a high-incidence setting at the Black Sea Coast. Phylogenetic lineages were identied based on 24-locus MIRU-VNTR (for mycobacterial interspersed repetitive unit-variable number tandem repeat) and spoligotyping analysis. Clusters of strains with identical genotyping proles were determined as an indicator for the rate of recent transmission. Among the 183 M. tuberculosis isolates investigated, the most prominent lineage found was Beijing (26%), followed by the LAM (18%), Ural (12%), and Haarlem (5%) strains. A closely related previously undened phylogenetic group (62 strains) showed a genotyping pattern similar to laboratory strain H37RV and was denominated as Georgia- H37RV-like. Although isoniazid resistance was found among strains of different lineages, MDR TB was nearly completely restricted to Beijing strains (P < 0.0001). Approximately 50% of the isolates were grouped in clusters, indicating a high rate of recent transmission. Our data indicate that, in addition to the conrmation of the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries, a high-population diversity with strains of the LAM, Ural, Haarlem, and a previously undened lineage repre- sents nearly two-thirds of the strains found in Georgia. Higher rates among previously treated and MDR TB patients point to a higher potential of lineage Beijing to escape therapy and develop MDR TB.

     Drug-resistant Mycobacterium tuberculosis complex (MTBC) rmed the worldwide presence of XDR strains, with rates of strains have emerged worldwide as a serious threat for tuber- up 15% of MDR TB cases (30). culosis (TB) control. Rates of multidrug-resistant (MDR) Considering the difculties and problems associated with the strains (i.e., resistance at least to isoniazid [INH] and rifampin treatment of resistant TB, high levels of MDR and XDR TB [RIF]) have reached levels of up to 14% among patients never have the clear potential to jeopardize TB control on a local or treated and up to 40% among previously treated patients in national level. In addition to various measures for strengthen- several MDR TB hot spots such as such as Karakalpakstan ing TB control such as rapid case detection, proper treatment, (Uzbekistan) and Kazakhstan in Eastern Europe (10, 37). Ev- and rapid detection of drug resistance, the long-term effect of ery year an estimated 489,000 cases of MDR TB arise globally the emergence of drug-resistant strains on the worldwide TB (36). MDR TB is associated with much poorer treatment out- epidemic also depends on the relative tness of the MDR and comes than for drug-susceptible TB, with a much higher risk of XDR strains compared to susceptible strains (3). developing further resistances (2, 7). Prolonged periods of In clinical MTBC strains, drug resistance results from chro- infectivity result in enhanced transmission of drug-resistant mosomal mutations in particular genes that confer resistance, strains, further accelerating the rates of drug resistance (3). which might also have an effect on bacterial tness (5, 38). Even more worrisome is the emergence of a nearly untreatable Although initial experiments indicated a lower tness of e.g., form of TB, namely, extensively drug-resistant TB (XDR TB), INH-resistant strains, recent results conrm that the tness of which is dened as MDR plus additional resistance to any resistant strains depends on the kind of mutations, as well on uoroquinolone and at least one of three injectable drugs (i.e., the strains genetic background (13). Furthermore, the initial amikacin, kanamycin, or capreomycin). A recent survey con- adverse effects on bacterial tness might be reversed by com- pensatory mutations occurring during long-term infection and ongoing transmission. In fact, MDR variants have been de- * Corresponding author. Mailing address: Forschungszentrum Bor- scribed to have even an enhanced tness compared to suscep- stel, National Reference Center for Mycobacteriology, Parkallee 1, tible progenitor strains (13). If these gures are used in models 23845 Borstel, Germany. Phone: 49-4537188762. Fax: 49-4537188311. E-mail: sniemann@fz-borstel.de. for prediction of the MDR TB epidemic, it turns out that even S.N., R.D., and G.K. contributed equally to this study. in the case of a well-functioning TB control system, small ( Supplemental material for this article may be found at http://jcm subpopulations of comparatively t MDR clones might out- .asm.org/. compete susceptible and less-t resistant strains and become Published ahead of print on 11 August 2010.

    3544

     VOL. 48, 2010 M. TUBERCULOSIS BEIJING LINEAGE IN GEORGIA 3545

    dilution of the suspension. All inoculated sets were incubated at 37?C in an the dominant clones in future with dramatic consequences for atmosphere of 5 to 10% COfor 28 days. MDR TB isolates were dened when 2TB treatment and control (5). they were resistant to INH and RIF (14). The most striking association between a mycobacterial ge- DNA techniques. A loopful of colony from the Lowenstein-Jensen medium netic background and drug resistance documented thus far has was suspended in 200 l of RNase-free water and boiled for 10 min as previously been described for strains of the so-called Beijing lineage. described (14). The suspension was centrifuged at 13,000 rpm for 10 min, and the supernatant was stored at 20?C until used. These strains have been found to be involved in outbreaks and All isolates were analyzed by the spoligotyping technique as described previ- the transmission of MDR TB in several areas of the world (15). ously by Kamerbeek et al. (18a). For MIRU-VNTR genotyping, 24 loci were In Eastern Europe, a rising number of studies report a clear amplied by PCR as described previously (31). Briey, analyses were performed association between Beijing genotype infection and drug resis- by using multiplex PCRs, the Rox-labeled MapMarker 1000 size standard (Bio- Ventures, Inc., Murfreesboro, VT), and the ABI 3130 XL sequencer with 16 tance (8, 11, 15, 28). Furthermore, large clusters of dominant capillaries (Applied Biosystems, Foster City, CA). Sizing of the PCR fragments clones have been determined that might indicate the develop- and assignment of the various VNTR alleles were done by using customized ment of highly transmissible MDR Beijing strains circulating GeneScan and Genotyper software packages (Applied Biosystems). in the community (28). Similar observations have been recently The molecular typing data were analyzed with the Bionumerics software (ver- reported from South Africa, where a rapidly spreading highly sion 5.0; Applied Maths, Sint-Martens-Latem, Belgium) as instructed by the manufacturer. Similarities of spoligotyping and MIRU-VNTR patterns were resistant clone represents nearly half of all cases in the George calculated by using the categorical coefcient. A dendrogram was generated by subdistrict (35). using the unweighted pair group method with arithmetic averages (UPGMA). However, the overall picture of the correlation between bac- Minimum spanning tree analysis based on MIRU typing data was done by using terial genotype disease characteristics is incomplete. The ma- the categorical coefcient. The priority rule was to link types rst that had highest number of single-locus variants. Identication of MTBC genotypes was carried jority of studies focused on particular strain types such as the out by using the MIRU-VNTRplus database (1). Beijing genotype only, basically, because they are easy to rec- For the cluster analysis, a cluster was dened as a minimum of two strains ognize by applying genotyping techniques such as IS6110 DNA harboring identical genotype pattern from different patients belonging to the ngerprint and spoligotyping (34). Based on these markers, a study population. variety of strains were not classiable into phylogenetic lin- Statistical analysis. Categorical data were compared by the chi-square test or the Fisher exact test, when expected cell sizes (n) were smaller than 5. The eages or clonal complexes since the genotyping information Wilcoxon rank sum test was performed to determine whether the distribution of was not informative, e.g., due to homoplasy (4, 6). Therefore, age as a continuous variable differed between the two groups. All tests were the presence of particular genotypes might simply be over- performed as two-sided tests. P values below 0.05 were considered statistically looked and, consequently, the association with clinical charac- signicant. Two models were then constructed in a logistic regression analysis with epidemiologically linked membership of a cluster and MDR as the respec- teristics could not be investigated or false associations have tive outcome variables. Odds ratios (OR) and 95% condence intervals (CI) been obtained. Since this question is of scientic and public were calculated by using logistic regression analysis, including demographic (age, health relevance, further studies addressing the population sex, and ethnic group), epidemiologic (previous treatment, history of incarcera- structure of the MTBC applying more appropriate genetic tion in a prison, and internal displacement), and microbiological variables (mul- markers are urgently needed. More recently, a new genotyping tiresistance, infection by a Beijing strain) in order to determine independent risk factors (e.g., adjusted for confounding). Statistical analyses were carried out by techniques based on mycobacterial interspersed repetitive using SPSS software (version 15; SPSS, Inc., Chicago, IL). unit-variable number tandem repeat (MIRU-VNTR) typing was developed that allows the simultaneous high-resolution discrimination of clinical isolates for epidemiological studies RESULTS and a valid phylogenetic strain classication (26, 31). The study sample comprising consecutive MTBC strains iso- In the present study, we used MIRU-VNTR typing and

    spoligotyping to investigate the population structure of strains lated from 196 patients was initially used to evaluate a new obtained from patients living in the Republic of Georgia, rapid test for the detection of drug resistance related muta- where high rates of MDR TB have been recently reported (14, tions (14). We then utilized the available DNA preparations to 20). We specically analyzed the association between Beijing carry out 24-locus MIRU-VNTR and spoligotyping analysis.

    For 13 strains, amplication was not successful, presumably genotype and drug resistance. Furthermore, the data have

    due to degradation of the DNA. For the remaining 183 iso- been used to classify determine the whole variety of strains

    lates, valid genotyping data were obtained and used for further circulating in Georgia and to describe new clonal complexes

    analyses. and/or phylogenetic lineages. Phylogenetic strain classica-

    Among the nal sample of 183 patients, the mean age the tions have been correlated with clinical characteristics.

    standard deviation was 39.1 14.7 (range, 69 years), and most

    patients were male 151/183 (82.5%). Nearly all patients had a MATERIALS AND METHODS

    microscopically conrmed pulmonary TB (175 were sputum Laboratory methods. Consecutive M. tuberculosis isolates recovered from pa- positive, 178 patients had pulmonary TB). Only 8.7% (16/183) tients referred to the National Center of Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia, between January and March 2006 were investi- patients were not of Georgian ethnicity. gated. Collected clinical specimens were processed by standard methods and Overall, 77 patients (42.1%) had a strain showing resistance cultured on Lowenstein-Jensen medium for isolation of mycobacteria. Sample against at least one drug tested. Thirty-nine strains (31.3%) demographic and treatment information was also documented. were resistant to INH, sixteen (8.7%) were resistant to RIF, Susceptibility testing for antimycobacterial drugs was performed by the method of absolute concentration (23). The mycobacterial suspension was de- and thirteen (7.1%) were MDR (Table 1). Patients with MDR termined from the primary culture, and the turbidity was adjusted to 1 McFar- TB did not differ with respect to sex (not signicant), nor with land standard with sterile saline. A series of 10-fold dilutions were prepared, and respect to age (multiresistant [39.9 19.4] versus nonmultire- 0.2 ml was inoculated onto media containing the following rst-line TB drugs: sistant [39.1 14.3; not signicant), nor with respect to the streptomycin (SM; 4 g/ml), RIF (40 g/ml), and ethambutol (EBM; 2 g/ml). proportion individuals with other ethnic groups (12/167 Geor- The INH (0.2 g/ml)-containing media were inoculated with 0.2 ml of a 100-fold

     3546 NIEMANN ET AL. J. CLIN. MICROBIOL.

    TABLE 1. Different categories of drug resistance stratied for MTBC genotypes

    aNo. of strains (%) Strain Total no. S H R E MDR Cluster PT Male 17 (37) 15 (33) 11 (24) 6 (13) 10 (22) 33 (72) 20 (43) 40 (87) 46 Beijing Haarlem 10 3 (30) 2 (20) 0 0 0 2 (20) 1 (10) 6 (60) Georgia-H37Rv-like 62 30 (48) 7 (11) 2 (3) 0 0 26 (42) 15 (24) 47 (76) LAM 34 6 (18) 9 (26) 5 (15) 0 0 0 21 (62) 28 (82) Ural 23 7 (30) 4 (17) 8 (35) 7 (30) 1 (4) 0 1 (4) 22 (96) X-type 1 0 1 0 0 0 0 1 1 M. bovis 1 0 0 0 0 0 0 1 1 No classication 6 1 2 (33) 2 (33) 0 2 (33) 0 2 (33) 6 All 183 64 (35) 39 (21) 16 (9) 6 (3) 13 (7) 86 (47) 57 (31) 151 (82)

    a Abbreviations: H, isoniazid; R, rifampin; E, ethambutol; S, streptomycin; MDR, multidrug resistant; PT, previously treated.

    base. All lineages suspected from dendrogram-based analysis gians versus 1/16 non-Georgians) from non-MDR TB patients.

    were also detected as clonal complexes in the MST (Fig. 2), As expected, previous treatment was signicantly associated

    including the newly described Georgia-H37Rv-like genotype with MDR TB: 9/57 patients with a history of previous treat-

    (Fig. 2). When the genetic diversity among isolates within one ment were MDR, but only 4/126 patients without previous

    clonal complex is considered, it becomes obvious that Beijing treatment (P 0.004 [Fisher exact test]). On the contrary, a

    and LAM strains are more closely related than strains of the prison stay (2/26 versus 11/157; P 0.05) was not associated

    with MDR TB. other genotypes with the lowest intralineage average distances According to the World Health Organization criteria, only (0.10 and 0.13, respectively).

    115 patients (62.8% of all patients) had been treated success- This is also reected by the cluster analysis. Overall, 86 of fully (cured or completed). Although MDR patients had a the 183 strains (47%) shared a genotyping pattern with at least lower treatment success rate (8 of the total of 13 patients were one other isolate and were grouped in 22 clusters (shared neither cured nor was treatment completed), in univariate types) ranging in size from 2 to 19 strains (see Fig. S1 in the analysis this association failed to reach statistical signicance supplemental material). The remaining strains were discrimi- (8/68 without treatment success versus 5/115 with success; P nated into 97 orphan types, all of which were assigned to 0.076 [Fisher exact test]) due to the low total number of MDR MLVA MtbC15-9 types and SpolDB4 nomenclature if possible strains. (see Fig. S1 in the supplemental material). The highest cluster

    rates were observed among Beijing and LAM strains (Table 1). Population structure and cluster analysis. In a rst step,

    Among 23 patients of the 86 patients in clusters (26.7%) 24-locus MIRU-VNTR proles and spoligotyping patterns

    were used to classify the 183 strains from Georgia into main epidemiological links could be veried. Parameters such as

    phylogenetic lineages by using the reference strain collection being an inhabitant of the same region or treated in the same and identication tools available online at www.miru-vntrplus hospital without any additional known personal contact were .org (1). Briey, a stepwise identication procedure was car- not considered to establish an epidemiological link. The largest ried out that based rst on simple match analysis and second cluster (n 19; cluster 19: MLVA MtbC15-9 type 94-32) is on phylogenetic tree identication (Fig. 1). In addition, formed by a Beijing strain, followed by the second largest SpoldB4 shared types were identied (4) and for each 24-locus cluster formed by a strains of the Georgia-H37Rv-like geno- MIRU pattern a unique MLVA 15-9 code was assigned by type (n 9, cluster 5: MLVA MtbC15-9 type 769-15), which using the MIRU-VNTRplus nomenclature. might indicate ongoing transmission of these strains. Overall, Based on these analyses, 46 strains were classied as the M. patients with a Beijing strain were more often cluster members tuberculosis Beijing genotype, 34 were classied as LAM the (33/86 in clusters versus 13/97 not in clusters; P 0.0001), and

    genotype (Latin American Mediterranean), 23 were classied they were often obtained from patients with veried epidemi-

    as the Ural genotype, 10 were classied as the Haarlem geno- ological links within clusters: 15/23 patients with epidemiolog- type, 1 was classied as X-type, and 1 was classied as M. bovis ical link in clusters versus 18/63 patients without epilinks in (Fig. 1, Table 1). Interestingly, 62 strains were found to be clusters (P 0.002). The risk of Beijing TB patients to be in a closely related to laboratory strain H37Rv and appear to form cluster with veried recent transmission within the very short a previously undened branch that was denominated Georgia- study period was nearly 5-fold higher among Beijing patients H37RV-like. Only six strains could not be assigned to a known (OR 4.98; 95% CI 2.4 to 17.1) and 4-fold higher among phylogenetic lineage or the new Georgia-H37Rv-like lineage prison inmates (OR 4.3; 95% CI 1.2 to 27.3), both values (Table 1). being independent predictors for recent transmission. Multi- To conrm this strain classication, we calculated a mini- drug resistance itself was no predictor for clustering (P 0.05).

    mum-spanning tree (MST) based on the 24-locus MIRU- These data focused our interest to a more detailed analysis VNTR data, which uses a maximum-parsimony algorithm to of characteristics of patients with Beijing strain infection. They investigate phylogenetic relationships and to identify clonal did not differ with respect to sex, nor with respect to age 36.7 complexes within a population. The MST (Fig. 2) strikingly 15.4 versus 39.9 14.3, nor with respect to patients with any conrmed the classication according to UPGMA tree-based drug resistance (19/77 [24.7%] with versus 27/106 [25.5%] with- analysis (Fig. 1) and by comparison with the reference data- out any resistance) from patients with non-Beijing strain infec-

     VOL. 48, 2010 M. TUBERCULOSIS BEIJING LINEAGE IN GEORGIA 3547

FIG. 1. Radial UPGMA tree based on the copy numbers of 24 MIRU-VNTR loci (see Materials and Methods). The tree was calculated by

    using the MIRU-VNTRplus server. Abbreviations: LAM, M. tuberculosis Latin American Mediterranean.

    remain independently associated with MDR in multilogistic tion. However, Beijing strains were more often spread in the

    regression procedures. native population (Georgians) than in the few foreign patients

     of other ethnicities (45/167 [27%] natives versus 1/16 [6%]

    non-Georgians); however, this does not reach statistical signif- DISCUSSION icance due to the small numbers (P 0.076). In this study we present the rst in-depth analysis of the Among patients with previous treatment there were more

    patients with Beijing strains (20 Beijing among 57 previously population structure of MTBC strains from a high-incidence treated individuals versus 26 Beijing among 126 not previ- region in the Caucasus that is based on high-resolution 24- ously treated individuals; P 0.037). Interestingly, there locus MIRU-VNTR typing. Our data conrm a highly diverse

    were more Beijing strain infections among patients with a population structure that comprises, in addition to Beijing, prison history than among patients without any history of four main phylogenetic lineages, one of which was not de- incarceration in a prison (15/26 versus 31/157; P 0.0001), scribed before. Although these data indicate that TB epidem- possibly indicating a better transmission of Beijing strains ics in Eastern Europe might be more complex and not only under crowded conditions. This could be conrmed by the driven by strains of the Beijing lineage, we also documented a fact the most patients in the largest cluster (19) have been in clear association between Beijing strain infection and MDR the same prison TB colony. TB. Furthermore, we present evidence of a clear association

    between the Beijing genotype and recent transmission, as well Factors associated with MDR TB. If the distribution of dif-

    ferent categories of drug resistance stratied for strains phy- as a prison stay.

    logenetic lineage is considered (Table 1), it becomes obvious Only few in-depth analyses of the population structure of that although strains of all lineages reach higher levels of INH MTBC strains from high-incidence regions in Eastern Europe or SM resistance (Table 1), the appearance of MDR is nearly are available. Many investigations are limited by sample size, completely restricted to Beijing strains. Accordingly, the MDR available epidemiological information, and/or by the genotyp- risk was 14-fold higher among Beijing patients (OR 14.0, ing technique used. The studies based on IS6110 DNA nger-

    95% CI 3.1 to 63.0). Among several factors analyzed, only printing or spoligotyping described here are hampered by the Beijing genotype infection and a history of previous treatment inherent restrictions of both methods that allow the identi-

     3548 NIEMANN ET AL. J. CLIN. MICROBIOL.

     FIG. 2. Minimum spanning tree based on the diversity of MIRU-VNTR data. The different complexes identied are shaded (maximum neighbor distance: four changes; minimum size: two MIRU-VNTR types) by the set of 24 loci among the 183 MTBC strains analyzed. The size of each circle is proportional to the number of MIRU-VNTR types belonging to a particular complex. LAM, M. tuberculosis Latin American Mediterranean.

    cation of some genotypes but fail to perform a valid phyloge- Using these tools, we were able to classify nearly all strains netic classication of a large proportion of isolates in a certain investigated (97%) to MTBC lineages described before and area due to high rates of homoplasy, especially in the case of one lineage that was discovered in our study. In concordance spoligotyping (6). This has also led to focused analysis of par- with a recently published study from Abkhazia, Beijing strains ticular phylogenetic lineages such as Beijing or Haarlem, since are not as dominant as in other parts of the former Soviet these are easy to identify by spoligotyping. Union such as Uzbekistan, Turkmenistan, or several parts of Here, 24-locus MIRU-VNTR typing offers a clear advantage Russia (9, 12, 22, 27). This might be due to the presence of by providing high-resolution genotyping and valid phylogenetic strains of other genotype families, such as LAM or Ural, that classication, as well as screening for new phylogenetic lineag- were also reported from other Eastern European countries es/clonal complexes (1). Furthermore, it allows for taking ad- (17, 19, 24). In addition, we conrmed the presence of a pre-

    vantage from state-of-the-art analysis tools and the reference viously unknown phylogenetic lineage named Georgia-H37Rv- strain database available online on the MIRU-VNTRplus like that was clearly dened by tree based, as well as by max- server that we have established previously (1). Scientic com- imum-parsimony MST-based analysis. However, comparison munication and tracking of particular strain types is facilitated with other studies is hampered by the fact that they are mainly by the implemented nomenclature server that assigns unique based on IS6110 DNA ngerprint and spoligotyping analysis MLVA 15-9 codes to particular genotyping proles. In addi- hindering a valid analysis of the population structure. Only a tion to tree-based analyses, MIRU-VNTR typing data can also few studies, such as work by Moukrosov et al. (25) or Kovalev be used for generating parsimonious phylogenetic networks, et al. (19) used a combination of MIRU-VNTR and spoligo- e.g., minimum spanning trees (16). typing, although with a reduced set of 12 MIRU-VNTR loci

     VOL. 48, 2010 M. TUBERCULOSIS BEIJING LINEAGE IN GEORGIA 3549

only. As such, the actual picture of MTBC population diversity somehow have evolved unique pathogenic properties favoring

    in Eastern European high-incidence settings is largely incom- efcient transmission.

    The fact that in the present study strains of all major lineages plete and needs a systematic investigation with phylogenetic

    showed higher levels of INH resistance, but only Beijing strains useful genotyping methods. Accordingly, we anticipate a larger

    appear to progress efciently to MDR TB, points to a higher study based on 24-locus MIRU-VNTR typing that shall com-

    capacity of Beijing strains to escape TB treatment in the case prise representative strain sets for several geographical settings

    of underlying monoresistance (or in combination with SM). in Eastern Europe. However, the proof of this hypothesis is difcult since longitu- A rst phylogenetic network of Beijing strains based on 11 dinal population-based studies addressing treatment success MIRU-VNTR loci was recently published by Mokrousov (21). are necessary. In addition to valid documentation of patient When we compare the 11-locus pattern of our the Beijing data, e.g., treatment regimens, it is also mandatory to perform strains of the present study to the reference patterns, it turned molecular typing of follow up culture since exogenous reinfec- out that the main Beijing strains type in Georgia belonged to tion with MDR or even XDR strains is a frequent cause of the M2 clone that is highly prevalent in the Russian popula- treatment failure in high-incidence settings (9, 11). In two tions. Together with LAM, the M2 strains from Georgia recent studies, higher failure rates among patients infected showed the lowest intralineage distances and were rather ho- with Beijing strains were documented in Central Asia (9, 11) mogeneous. This argues for a more recent introduction of both and Indonesia (29). The question of whether Beijing genotype lineages in Georgia (presumably from Russia) that might be infection per se can be seen as an independent risk factor for followed by recent dissemination. treatment failure and might require special treatment proce- Interestingly, Beijing strains were found to be associated dures is challenging and urgently requires further investiga- with recent transmission, especially in a prison setting. The tion. largest cluster formed by a Beijing clone (MtbC15-9 type 94- In conclusion, our study conrms that the Beijing epidemic 32) could be related to an outbreak in a prison colony with, has reached Georgia and is associated with a high rate of however, a spillover in the general population. This nding recent transmission, especially in prison populations, and high illustrates the high risk of accelerated TB transmission in the MDR rates. On the other hand, the highly diverse population prison setting. Similar ndings have been reported previously, structure determined conrms the importance of strains of e.g., from Samara (12) and Kyrgyztan (25), where Beijing other genotypes, such as LAM and Ural, and the presence of strain infection was also found to be strongly associated with a as-yet-undened phylogenetic lineages. Accordingly, the pop- history of imprisonment. Direct comparison of MIRU-VNTR ulation structure of MTBC strains from high-incidence settings proles revealed a striking similarity of Beijing strains (type in Eastern Europe needs a more thorough investigation based M2) spreading in Kyrgyzian and Georgian prison populations, on valid phylogenetic markers. To obtain a more detailed pic- also pointing to the particular propensity of Beijing strains to ture of the landscape of MTBC diversity in Eastern Europe, a spread in a prison environment or in crowded living conditions concerted action of different research groups based on central- in general. Considering prison systems and long-term hospital- ized databases and nomenclature, such as are available on the ization during TB treatment practiced in several Eastern Eu- MIRU-VNTRplus server, is desirable. Based on this, more ropean countries (32), this is likely to provide a selective ad- detailed investigations on genome diversity among strains of vantage that might have contributed to the high rates of Beijing the different lineages and pathobiological consequences can be strains observed throughout former Soviet Union countries. initiated. A further point of consideration is the strong association of REFERENCES Beijing strain infection with a history of previous treatment 1. Allix-Be?guec, C., D. Harmsen, T. Weniger, P. Supply, and S. Niemann. 2008. and MDR TB determined in the present study. This nding is Evaluation and user-strategy of MIRU-VNTRplus, a multifunctional data- in accordance with reports from our group, e.g., from Abkha- base for on-line analysis of genotyping data and phylogenetic identication of Mycobacterium tuberculosis complex isolates. J. Clin. Microbiol. 46:2692 zia, Turkmenistan, or Uzbekistan, or from other investigators, 2699. e.g., from Samara or Archangel Oblast (8, 12, 28, 32, 33). 2. Aziz, M. A., A. Wright, A. Laszlo, A. De Muynck, F. Portaels, A. Van Deun, Taken together, these studies undoubtedly conrm Beijing C. Wells, P. Nunn, L. Blanc, and M. Raviglione. 2006. Epidemiology of antituberculosis drug resistance (the Global Project on Anti-Tuberculosis strains as a major factor driving the MDR TB epidemic in Drug Resistance Surveillance): an updated analysis. Lancet 368:21422154. Eastern Europe. The reasons for this phenomenon, however, 3. Blower, S. M., and T. Chou. 2004. Modeling the emergence of the hot zones: tuberculosis and the amplication dynamics of drug resistance. Nat. are not yet well dened. Considering several reports that dem- Med. 10:11111116. onstrated recent transmission and outbreaks of MDR Beijing 4. Brudey, K., J. R. Driscoll, L. Rigouts, W. M. Prodinger, A. Gori, S. A. Al strains (8, 28), one key point appears to be the capability to Hajoj, C. Allix, L. Aristimuno, J. Arora, V. Baumanis, L. Binder, P. Cafrune, A. Cataldi, S. Cheong, R. Diel, C. Ellermeier, J. T. Evans, M. Fauville- spread after having MDR developed. Hence, tness costs of Dufaux, S. Ferdinand, D. Garcia de Viedma, C. Garzelli, L. Gazzola, H. M. MDR (and XDR) development in Beijing strains might be less Gomes, M. C. Guttierez, P. M. Hawkey, P. D. van Helden, G. V. Kadival, B. N. Kreiswirth, K. Kremer, M. Kubin, S. P. Kulkarni, B. Liens, T. Lille- than in non-Beijing strains and/or compensatory evolution baek, M. L. Ho, C. Martin, C. Martin, I. Mokrousov, O. Narvskaia, Y. F. might be more effective. A remarkable nding is the fact that Ngeow, L. Naumann, S. Niemann, I. Parwati, Z. Rahim, V. Rasolofo-Raza- the strong clonal expansion of some dominant MDR strains, namparany, T. Rasolonavalona, M. L. Rossetti, S. Rusch-Gerdes, A. Sa- jduda, S. Samper, I. G. Shemyakin, U. B. Singh, A. Somoskovi, R. A. Skuce, e.g., documented in Abkhazia (28) or South Africa (18), has D. van Soolingen, E. M. Streicher, P. N. Suffys, E. Tortoli, T. Tracevska, V. the potential to signicantly inuence the MDR epidemic in a Vincent, T. C. Victor, R. M. Warren, S. F. Yap, K. Zaman, F. Portaels, N. Rastogi, and C. Sola. 2006. Mycobacterium tuberculosis complex genetic certain region. These data also indicate that actual conditions diversity: mining the fourth international spoligotyping database (SpolDB4) in high-incidence settings, e.g., treatment policies, favor the for classication, population genetics and epidemiology. BMC. Microbiol. selection of highly transmissible MDR strain variants that 6:23.

3550 NIEMANN ET AL. J. CLIN. MICROBIOL.

5. Cohen, T., and M. Murray. 2004. Modeling epidemics of multidrug-resistant 23. Mokrousov, I., T. Otten, B. Vyshnevskiy, and O. Narvskaya. 2002. Detection Mycobacterium tuberculosis of heterogeneous tness. Nat. Med. 10:1117 of embB306 mutations in ethambutol-susceptible clinical isolates of Myco- 1121. bacterium tuberculosis from Northwestern Russia: implications for genotypic 6. Comas, I., S. Homolka, S. Niemann, and S. Gagneux. 2009. Genotyping of resistance testing. J. Clin. Microbiol. 40:38103813. genetically monomorphic bacteria: DNA sequencing in mycobacterium tu- 24. Mokrousov, I., T. Otten, T. Zozio, E. Turkin, V. Nazemtseva, A. Sheremet, B. berculosis highlights the limitations of current methodologies. PLoS One Vishnevsky, O. Narvskaya, and N. Rastogi. 2009. At Baltic crossroads: a 4:e7815. molecular snapshot of Mycobacterium tuberculosis population diversity in 7. Cox, H. S., S. Kalon, S. Allamuratova, V. Sizaire, Z. N. Tigay, S. Rusch- Kaliningrad, Russia. FEMS Immunol. Med. Microbiol. 55:1322. Gerdes, H. A. Karimovich, Y. Kebede, and C. Mills. 2007. Multidrug-resis- 25. Mokrousov, I., V. Valcheva, N. Sovhozova, A. Aldashev, N. Rastogi, and J. tant tuberculosis treatment outcomes in Karakalpakstan, Uzbekistan: treat- Isakova. 2009. Penitentiary population of Mycobacterium tuberculosis in Kyr- ment complexity and XDR-TB among treatment failures. PLoS One gyzstan: exceptionally high prevalence of the Beijing genotype and its Rus- 2:e1126. sia-specic subtype. Infect. Genet. Evol. 9:14001405. 8. Cox, H. S., T. Kubica, D. Doshetov, Y. Kebede, S. Rusch-Gerdess, and S. 26. Oelemann, M. C., R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann. 2005. The Beijing genotype and drug-resistant tuberculosis in the Niemann, and P. Supply. 2007. Assessment of an optimized mycobacterial Aral Sea region of Central Asia. Respir. Res. 6:134. interspersed repetitive-unit-variable-number tandem-repeat typing system 9. Cox, H. S., S. Niemann, G. Ismailov, D. Doshetov, J. D. Orozco, L. Blok, S. combined with spoligotyping for population-based molecular epidemiology Rusch-Gerdes, and Y. Kebede. 2007. Risk of acquired drug resistance during studies of tuberculosis. J. Clin. Microbiol. 45:691697. short-course directly observed treatment of tuberculosis in an area with high 27. Pardini, M., E. Iona, F. Varaine, H. Karakozian, H. Arzumanian, L. Brunori, levels of drug resistance. Clin. Infect. Dis. 44:14211427. G. Oreci, and L. Fattorini. 2005. Mycobacterium tuberculosis drug resis- 10. Cox, H. S., J. D. Orozco, R. Male, S. Ruesch-Gerdes, D. Falzon, I. Small, D. tance, Abkhazia. Emerg. Infect. Dis. 11:501503. Doshetov, Y. Kebede, and M. Aziz. 2004. Multidrug-resistant tuberculosis in 28. Pardini, M., S. Niemann, F. Varaine, E. Iona, F. Meacci, G. Orru, H. central Asia. Emerg. Infect. Dis. 10:865872. Yesilkaya, T. Jarosz, P. Andrew, M. Barer, F. Checchi, H. Rinder, G. Oreci, 11. Cox, H. S., K. Sibilia, S. Feuerriegel, S. Kalon, J. Polonsky, A. K. Khamraev, S. Rusch-Gerdes, L. Fattorini, M. R. Oggioni, and M. Bonnet. 2009. Char- S. Rusch-Gerdes, C. Mills, and S. Niemann. 2008. Emergence of extensive acteristics of drug-resistant tuberculosis in Abkhazia (Georgia), a high-prev- drug resistance during treatment for multidrug-resistant tuberculosis. alence area in Eastern Europe. Tuberculosis 89:317324. N. Engl. J. Med. 359:23982400. 29. Parwati, I., B. Alisjahbana, L. Apriani, R. D. Soetikno, T. H. Ottenhoff, A. G. 12. Drobniewski, F., Y. Balabanova, V. Nikolayevsky, M. Ruddy, S. Kuznetzov, van der Zanden, J. van der Meer, D. van Soolingen, and R. van Crevel. 2010. S. Zakharova, A. Melentyev, and I. Fedorin. 2005. Drug-resistant tubercu- Mycobacterium tuberculosis Beijing genotype is an independent risk factor for losis, clinical virulence, and the dominance of the Beijing strain family in tuberculosis treatment failure in Indonesia. J. Infect. Dis. 201:553557. Russia. JAMA 293:27262731. 30. Shah, N. S., A. Wright, G. H. Bai, L. Barrera, F. Boulahbal, N. Martin- 13. Gagneux, S., C. D. Long, P. M. Small, T. Van, G. K. Schoolnik, and B. J. Casabona, F. Drobniewski, C. Gilpin, M. Havelkova, R. Lepe, R. Lumb, B. Bohannan. 2006. The competitive cost of antibiotic resistance in Mycobac- Metchock, F. Portaels, M. F. Rodrigues, S. Rusch-Gerdes, A. Van Deun, V. terium tuberculosis. Science 312:19441946. Vincent, K. Laserson, C. Wells, and J. P. Cegielski. 2007. Worldwide emer- 14. Gegia, M., N. Mdivani, R. E. Mendes, H. Li, M. Akhalaia, J. Han, G. gence of extensively drug-resistant tuberculosis. Emerg. Infect. Dis. 13:380 Khechinashvili, and Y. W. Tang. 2008. Prevalence of and molecular basis for 387. tuberculosis drug resistance in the Republic of Georgia: validation of a 31. Supply, P., C. Allix, S. Lesjean, M. Cardoso-Oelemann, S. Rusch-Gerdes, E. QIAplex system for detection of drug resistance-related mutations. Antimi- Willery, E. Savine, P. de Haas, H. van Deutekom, S. Roring, P. Bifani, N. crob. Agents Chemother. 52:725729. Kurepina, B. Kreiswirth, C. Sola, N. Rastogi, V. Vatin, M. C. Gutierrez, M. 15. Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. Fauville, S. Niemann, R. Skuce, K. Kremer, C. Locht, and D. van Soolingen. 2002. Worldwide occurrence of Beijing/W strains of Mycobacterium tubercu- losis: a systematic review. Emerg. Infect. Dis. 8:843849. 2006. Proposal for standardization of optimized mycobacterial interspersed 16. Homolka, S., E. Post, B. Oberhauser, A. G. George, L. Westman, F. Dafae, repetitive unit-variable number tandem repeat typing of Mycobacterium tu- S. Rusch-Gerdes, and S. Niemann. 2008. High genetic diversity among My- berculosis. J. Clin. Microbiol. 44:44984510. cobacterium tuberculosis complex strains from Sierra Leone. BMC Microbiol. 32. Toungoussova, O. S., G. Bjune, and D. A. Caugant. 2006. Epidemic of 8:103. tuberculosis in the former Soviet Union: social and biological reasons. Tu- 17. Ignatova, A., S. Dubiley, V. Stepanshina, and I. Shemyakin. 2006. Predom- berculosis 86:110. inance of multidrug-resistant LAM and Beijing family strains among Myco- 33. Toungoussova, O. S., A. Mariandyshev, G. Bjune, P. Sandven, and D. A. bacterium tuberculosis isolates recovered from prison inmates in Tula Re- Caugant. 2003. Molecular epidemiology and drug resistance of Mycobacte- gion, Russia. J. Med. Microbiol. 55:14131418. rium tuberculosis isolates in the Archangel prison in Russia: predominance of 18. Johnson, R., R. M. Warren, G. D. van der Spuy, N. C. Gey van Pittius, D. the W-Beijing clone family. Clin. Infect. Dis. 37:665672. Theron, E. M. Streicher, M. Bosman, G. J. Coetzee, P. D. van Helden, and 34. van Soolingen, D., K. Kremer, and E. Vynycky. 2003. New perspectives in the T. C. Victor. 2010. Drug-resistant tuberculosis epidemic in the Western Cape molecular epidemiology of tuberculosis, p. 1745. In S. Kaufmann and H. driven by a virulent Beijing genotype strain. Int. J. Tuberc. Lung Dis. 14: Hahn (ed.), Mycobacteria and TB. Karger, Berlin, Germany. 119121. 35. Victor, T. C., E. M. Streicher, C. Kewley, A. M. Jordaan, G. D. van der Spuy, 18a.Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, et M. Bosman, H. Louw, M. Murray, D. Young, P. D. van Helden, and R. M. al. 1997. Simultaneous detection and strain differentiation of Mycobacterium Warren. 2007. Spread of an emerging Mycobacterium tuberculosis drug-re- tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907914. sistant strain in the western Cape of South Africa. Int. J. Tuberc. Lung Dis. 19. Kovalev, S. Y., E. Y. Kamaev, M. A. Kravchenko, N. E. Kurepina, and S. N. 11:195201. Skorniakov. 2005. Genetic analysis of mycobacterium tuberculosis strains 36. World Health Organization. 2008. Anti-tuberculosis drug resistance in the isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping. world. Report 4. World Health Organization, Geneva, Switzerland. Int. J. Tuberc. Lung Dis. 9:746752. 37. Wright, A., M. Zignol, D. A. Van, D. Falzon, S. R. Gerdes, K. Feldman, S. 20. Lomtadze, N., R. Aspindzelashvili, M. Janjgava, V. Mirtskhulava, A. Wright, Hoffner, F. Drobniewski, L. Barrera, S. D. van, F. Boulabhal, C. N. Para- H. M. Blumberg, and A. Salakaia. 2009. Prevalence and risk factors for masivan, K. M. Kam, S. Mitarai, P. Nunn, and M. Raviglione. 2009. Epi- multidrug-resistant tuberculosis in the Republic of Georgia: a population- demiology of antituberculosis drug resistance 200207: an updated analysis based study. Int. J. Tuberc. Lung Dis. 13:6873. of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance. 21. Mokrousov, I. 2008. Genetic geography of Mycobacterium tuberculosis Bei- Lancet 373:18611873. jing genotype: a multifacet mirror of human history? Infect. Genet. Evol. 38. Zhang, Y., and W. R., Jr. Jacobs. 2008. Mechanisms of drug action, drug 8:777785. resistance, and drug tolerance in Mycobacterium tuberculosis: expected phe- 22. Mokrousov, I., O. Narvskaya, A. Vyazovaya, J. Millet, T. Otten, B. Vish- notypes from evolutionary pressure from a highly successful pathogen, p. nevsky, and N. Rastogi. 2008. Mycobacterium tuberculosis Beijing genotype in 323378. In S. H. Kaufmann and E. Rubin (ed.), Handbook of tuberculosis. Russia: in search of informative variable-number tandem-repeat loci. J. Clin. Wiley-VCH Verlag, Weinheim, Germany. Microbiol. 46:35763584.

Report this document

For any questions or suggestions please email
cust-service@docsford.com