MIRU-VNTR typing of drug-resistant tuberculosis isolates in Greece

Therapeutic Advances in Respiratory Disease Original Research MIRU-VNTR typing of drug-resistant tuberculosis isolates in Greece Nikoletta Rovina, Simona Karabela, Pantelis Constantoulakis, Vassiliki Michou, Konstantinos Konstantinou, Vassileios Sgountzos, Charis Roussos and Nikolaos Poulakis Abstract: The increasing immigration rate in Greece from countries with a high prevalence of Mycobacterium tuberculosis (MTB) and multidrug-resistant tuberculosis (MDR-TB) may have an impact on the number of MDR-TB cases in Greece. The aim of this study was to genotypically characterize the MTB isolates from patients with pulmonary drug-resistant tuberculosis (DR-TB) in Greece, and to determine whether there is any association between the prevalent genotypes and drug resistance. Fifty-three drug-resistant MTB strains isolated from culture specimens of clinical material from native Greeks and immigrant patients with pulmonary tuberculosis were genotyped using the mycobacterial interspersed repetitive units variable number of tandem repeats (MIRU-VNTR) method. The phylogenetically distinct groups of isolates identified were: the Beijing (34%), the LAM (11%), the Haarlem (24.5%), the Uganda I (9.4%), the Ural (3.8%), the Delhi/CAS (9.4%) and the Cameroon (3.8%) families. Greek patients were more likely to have monoresistant and polyresistant TB with the most prevalent isolates belonging to the Haarlem family. Among foreign-born patients with MDR-TB, the most prevalent genotypes belonged to the Beijing family. MIRU-VNTR rapidly obtained clinically useful genotyping data, by characterizing clonal MTB heterogeneity in the isolated strains. Our results underline the need for more effective antituberculosis control programs in order to control the expansion of DR-TB in Greece. Keywords: Mycobacterium tuberculosis, multidrug-resistant tuberculosis, MIRU-VNTR Introduction Tuberculosis (TB) remains a major threat to human health, especially in developing countries. In recent years, Greece has become the destination or crossroads of a number of immigrants (who are transported legally or smuggled into the country illegally) pursuing a better standard of living in this country or in the rest of Europe (by passing through Greece). Most of them come from countries with high rates of TB and multidrug-resistant TB (MDR-TB), such as Pakistan, Bangladesh, India, Russia, sub-saharan countries and the countries of Eastern Europe. This tendency has led to an increase in TB cases in Greece and, most importantly, of MDR-TB [World Health Organization, 2010]. Under these circumstances, molecular epidemiological studies have been proved as useful tools in tracing the transmission patterns of Mycobacterium tuberculosis (MTB), enabling the community to take effective measures for the containment of the disease. Genotyping is an accurate method for the diagnosis of MTB infection from nontuberculous mycobacterial disease. It can differentiate whether second episodes of tuberculosis are due to relapse or reinfection by another tuberculous strain and allow for the evaluation of isolates with different patterns of drug susceptibility. Genotyping can also be used to evaluate an outbreak, delineate the extent and guide public health measures to limit disease transmission [Barnes and Cave, 2003]. The mycobacterial interspersed repetitive units variable number of tandem repeats (MIRU-VNTR) method is based on detection of independent minisatellite-like loci scattered throughout the MTB genome and has been shown to be a reliable and reproducible typing method with high discriminatory power [Sola et al. 2003; Cowan et al. 2002; Supply et al. 2001; Dale et al. 1999] for studying the MTB population structure in different countries [Sola et al. 2003; Supply et al. 2003, 2001]. Ther Adv Respir Dis (2011) 5(4) 229 236 DOI: 10.1177/ 1753465811402120! The Author(s), 2011. Reprints and permissions: http://www.sagepub.co.uk/ journalspermissions.nav Correspondence to: Nikolaos Poulakis, MD, FCCP 1st Pulmonary Department, SOTIRIA District Chest Diseases Hospital, 152 Mesogion Street, 11527 Athens, Greece mpoulaki@otenet.gr Nikoletta Rovina, MD, PhD 1st University Pulmonary Department, Medical School, National and Kapodistrian University of Athens SOTIRIA District Chest Diseases Hospital, 152 Mesogion Street, 11527 Athens, Greece Simona Karabela, MD National Center of Tuberculosis, Microbiology Department, SOTIRIA District Chest Diseases Hospital, 152 Mesogion Street, 11527, Athens, Greece Pantelis Constantoulakis, MSc, PhD Vassiliki Michou, MSc Locus Medicus Research Center, Athens, Greece KonstantinosKonstantinou, MD Vassileios Sgountzos, MD Clinic of Multi-drug Resistant Tuberculosis, SOTIRIA District Chest Diseases Hospital, 152 Mesogion Street, 11527 Athens, Greece Charis Roussos, MD, MSc, PhD, MRS, FRCP National and Kapodistrian University of Athens, Pulmonary Department, SOTIRIA District Chest Diseases Hospital, 152 Mesogion Street, 11527 Athens, Greece and National and Kapodistrian University of Athens, Department of Critical Care and Pulmonary Services, General Hospital Evangelismos, 3 Ploutarhou Street, 10675 Athens, Greece http://tar.sagepub.com 229

Therapeutic Advances in Respiratory Disease 5 (4) The typed strains are expressed by a 12-digit numerical code, corresponding to the number of repeats at each locus [Mazars et al. 2001; Supply et al. 2000]. This numerical code is easy to compare and exchange at interlaboratory and intralaboratory levels. The discriminatory power of MIRU genotyping is almost as great as that of IS6110-based genotyping [Mazars et al. 2001; Supply et al. 2001, 2000]. More recently a set of 24 MIRU-VNTR loci was reported to have greater discriminatory power than the original 12 loci system and may exceed that of restriction fragment length polymorphism (RFLP) when combined with spoligotyping [Christianson et al. 2010; Maes et al. 2008]. Unlike IS6110- based genotyping, MIRU analysis can be automated and can thus be used to evaluate large numbers of strains, yielding intrinsically digital results that can be easily catalogued on a computer data base. MIRU genotyping is technically simpler than IS6110-based genotyping and can be applied directly to MTB cultures without DNA purification. It may replace IS6110-based genotyping in the future, particularly if the evaluation of additional loci increases the discriminatory power. The discriminatory power of MIRU-VNTR analysis is proportional to the number of loci evaluated. Twelve-loci-based MIRU-VNTR analysis has been used in a number of molecular epidemiologic studies and to elucidate the phylogenetic relationship of clinical isolates [Kremer et al. 2005; Sun et al. 2004; Warren et al. 2004; Sola et al. 2003; Supply et al. 2003]. Since Greece has recently been the hub for a high number of economic immigrants transferring from countries with a high prevalence of DR-TB, and especially MDR-TB (from the former Soviet Union, Asia and Africa), the need to genetically track the MTB strains in order to evaluate the import of new drug-resistant strains in the country is mandatory. So far, very little is known regarding the genotypic prevalence of DR-TB and MDR-TB in Greece, and this is the first record of this prevalence using molecular techniques. The aim of this study was to genotypically characterize the MTB isolates from patients with pulmonary DR-TB in Greece, and to determine whether there is any association between the prevalent genotypes and drug resistance. Material and methods Patients Molecular analysis was conducted on the MTB isolates of 53 patients (33 males, 15 females and five of unreported sex) who were treated for DR-TB in SOTIRIA Hospital, in Athens Greece, from January 2007 to December 2008. Fifty-one patients were adults (range of age, 18 69 years), and two were children 2 and 5 years old, respectively. Since this was a retrospective analysis of frozen culture specimens from smears positive to MTB sputa, subjects were not asked to give their informed consent for participating in the study; however, the Hospital ethics committee approved the study. Bacterial strains Fifty-three patients were found to be drug resistant among the newly diagnosed TB patients living in Greece for at least 6 months. MTB isolates were tested for susceptibilities to isoniazid, rifampin, ethambutol and streptomycin using the proportion method [Caws et al. 2006]. If the number of colonies growing on drug-containing medium (isoniazid, 0.2 mg/l; rifampin, 40.0 mg/l; ethambutol, 2.0 mg/l; and streptomycin, 4.0 mg/l) was 1% of that growing on drug-free medium, the isolate was considered to be resistant. Isolates resistant to one or more first-line drugs (i.e. isoniazid, rifampicin, ethambutol and streptomycin) were included in this study. These isolates were further tested for drug resistance to the following anti-tb agents: ofloxacin, amikacin, kanamycin, capreomycin and rifabutin. The isolates were characterized as monoresistant (resistant to one anti-tb drug), polyresistant (resistant to more than one anti-tb drugs except to the combination of rifampicin and isoniazid), MDR-TB (resistance to a combination of rifampicin and isoniazid) and extensively drug-resistant strains (XDR-TB; resistance to a combination of rifampicin and isoniazid plus a quinolone and one injectable agent), according to their resistance pattern. 230 http://tar.sagepub.com

N Rovina, S Karabela et al. Molecular typing analysis Fifty-three stored MTB isolates were selected for MIRU-VNTR genotyping. DNA was extracted from mycobacterial cultures cultivated on Löwenstein Jensen medium, as follows: a loop full of bacterial colonies was suspended into 200 ml mm Tris-HCl, 1 mm EDTA (ph 7.0), and incubated at 95 C for 45 min, the suspension was then centrifuged for 1 min at 15,000 g, the supernatant containing the DNA was collected and stored at 20 C until further use. MIRU-VNTR genotyping was performed by amplifying the 24 MIRU-VNTR loci as described previously in a technical guide [Supply, 2005] with the following modification: 5% DMSO in addition to Q solution was used in all Loci. The sequencing was performed in ABIPRISM 3100 genetic analyser (Applied Biosystems), for each MIRU depending on their base pair product, a locus convention allele was assigned according to the technical guide [Supply, 2005]. To confirm our results, the samples were also sent for amplification and sequencing at Genoscreen (Lille, France, http:// www.genoscreen.com). The genotype of MTB was then determined by applying the 24 MIRU-VNTRs of each sample, in the MIRU- VNTR plus database (http://miru-vntrplus.org). Results MIRU-VNTR typing of resistant MTB clinical isolates Thirty out of 53 patients were Greek, 21 were foreigners (four Pakistanis, four Albanians, three Russians, two Bulgarians, two Georgians and one each came from Kazakhstan, Korea, Poland, Romania, Iran and Moldavia) and two were of unknown nationality. The foreigners had been living and working in Greece for at least 6 months. All 53 stored MTB isolates that were selected for MIRU-VNTR genotyping were confirmed as belonging to the MTB complex. The 24-loci MIRU-VNTR analysis detected a total of 53 MIRU patterns out of 63 strains (Figure 1). Eighteen patients (34%) were infected by the Beijing strain, 13 (24%) by the Haarlem strain, six (11%) by the LAM strain, five (9%) by the Delhi/CAS strain, five (9%) by the T2-Uganda I strain, two (5%) by the Ural strain, and one patient was infected by two different strains concomitantly (a LAM10-Cameroon and a Haarlem strain). In two patients the isolates were unclassified. The most prevalent genotype among patients born in Greece (30/53, 57%) was Haarlem, followed by Beijing, T2-Uganda, LAM and Delhi/ CAS, while two of the isolates remained unclassified. The most prevalent isolates among foreign-born patients belonged to the Beijing family, followed by Delhi/CAS, LAM, Ural, and Haarlem families. Table 1 shows the genotyping patterns of drug-resistant strains (monoresistant, polyresistant and MDR) isolated in patients born in Greece and in foreigners. Drug resistance of MTB isolates Determination of drug susceptibility showed that 33 out of 53 strains (62%) were phenotypically monoresistant or polyresistant to anti-tb drugs, while 18 out of 53 (34%) were MDR strains, including three XDR strains (6%). Table 2 shows the distribution of MIRU genotypes of clinical MTB according to their drug resistance profiles. The specific genotype, the drug resistance patterns and the nationality of patients with monoresistance or polyresistance to the first-line MIRU-VNTR convention alias number of repeats 580 2996 802 960 1644 3192 424 577 2165 2401 Lineage (n=53) Beijing (18) 2 5 3 3 3 5 4 4 3 4 3 2 6 5 8 2 5 3 2 1 3 3 2 3 Haarlem (13) 2 5 3 5 3 3 2 3 3 4 3 3 7 3 4 2 5 2 2 1 3 2 2 3 LAM (6) 2 5 4 4 3 2 3 2 2 1 2 2 2 3 6 1 5 2 2 1 3 4 2 3 Delhi/CAS (5) 2 4 3 4 4 5 3 2 4 2 3 4 2 4 7 2 5 3 2 3 4 2 3 T2-Uganda (5) 2 4 2 3 4 3 2 3 2 2 3 2 5 2 5 2 5 2 2 1 3 4 2 3 Ural (2) 2 1 3 7 2 2 3 5 4 4 3 3 2 3 6 2 5 2 2 1 3 4 2 3 Cameroon (2) 2 5 2 3 1 3 2 4 3 2 3 2 4 2 5 2 5 2 2 1 3 4 2 3 Unclassified 1 3 4 5 3 3 4 3 4 4 2 3 2 2 1 3 2 5 2 2 1 3 4 2 3 Unclassified 2 3 3 5 3 3 3 3 4 3 2 3 2 1 7 2 5 2 2 1 3 4 2 3 Figure 1. The 24 MIRU-VNTR repeats of each Lineage isolated from multidrug-resistant tuberculosis patients. 3690 4156 2163 1955 4052 154 2531 4348 2059 2687 3007 2347 2461 3171 http://tar.sagepub.com 231

Therapeutic Advances in Respiratory Disease 5 (4) Table 1. Genotyping patterns of drug resistant strains (monoresistant, polyresistant and multidrug resistant) isolated in patients born in Greece and in foreign-born patients. MTB strain n (%) Born in Greece n (%) Foreign born n (%) Beijing 18 (34) 8 (44.4) 10 (55.5) Haarlem 13 (24.5) 12 (92.3) 1 (7.7) LAM 6 (11.3) 3 (50) 3 (50) Delhi/CAS 5 (9.4) 1 (20) 4 (80) S2-Uganda 5 (9.4) 4 (80) 1 (20) Ural 2 (3.8) 0 2 (100) Cameroon 2 (3.8) 2 (100) 0 Unclassified 2 (3.8) 2 (100) 0 MTB, Mycobacterium tuberculosis. Table 2. Drug susceptibility of Mycobacterium tuberculosis isolates. MIRU genotype Monoresistant isolates n (%) Polyresistant isolates n (%) MDR isolates n (%) XDR isolates n (%) Beijing (n ¼ 18) 0 8 (44.4) 9 (50) 1 (5.6) Haarlem (n ¼ 13) 5 (38.5) 7 (53.8) 1 (7.7) 0 LAM (n ¼ 6) 0 0 4 (66.6) 2 (33.3) Delhi/CAS (n ¼ 5) 4 (80) 1 (20) 0 0 S2-Uganda (n ¼ 5) 3 (60) 0 2 (40) 0 Cameroon (n ¼ 2) 1 (50) 1 (50) 0 0 Ural (n ¼ 2) 0 0 2 (100) 0 Unclassified (n ¼ 2) 0 2 (100) 0 0 Total (n ¼ 53) 13 (24.5) 19 (35.8) 18 (34) 3 (5.7) MIRU, mycobacterial interspersed repetitive unit; MDR, multidrug resistant; XDR, extensively drug resistant. Table 3. Genotyping and drug monoresistance patterns to first-line antituberculous drugs. Drug to which the strain was monoresistant MTB lineage Sex Nationality Isoniazid (n ¼ 6) Uganda I F Greek Delhi/CAS M Pakistani Delhi/CAS M Pakistani Delhi/CAS M Pakistani Haarlem M Greek Haarlem M Greek Streptomycin (n ¼ 5) Haarlem F Greek Haarlem M Greek Delhi/CAS F Greek Uganda I F Greek LAM F Greek Ethambutol (n ¼ 1) Cameroon, Haarlem M Greek MTB, Mycobacterium tuberculosis; M, male; F, female. anti-tb drugs are shown in Tables 3 and 4, respectively. It should be noted that the majority of polyresistant strains belonged to the Beijing and Haarlem families (44.4% and 53.8%, respectively). The majority of the Haarlem strains (12 out of 13 isolates or 92.3%) were isolated from native Greeks. Genotyping and drug sensitivity patterns of MDR and XDR groups are shown in Table 5. 232 http://tar.sagepub.com

N Rovina, S Karabela et al. Table 4. Patterns of drug resistance and genotyping in polyresistant tuberculosis patients (n ¼ 20). MTB lineage Sex Nationality Drug resistance Haarlem F Greek Isoniazid, streptomycin Haarlem M Greek Isoniazid, streptomycin Haarlem M Romanian Isoniazid, streptomycin Haarlem M Greek Isoniazid, amikacin Haarlem F Greek Isoniazid, ethambutol Haarlem M Greek Rifampicin, streptomycin, rifambutin, capreomycin Haarlem M Greek Rifampicin, streptomycin, capreomycin Beijing M Unknown origin Isoniazid, streptomycin Beijing M Albanian Isoniazid, streptomycin Beijing M Albanian Isoniazid, streptomycin Beijing M Greek Isoniazid, ethambutol, streptomycin, amikacin Beijing M Russian Isoniazid, ethambutol, streptomycin, amikacin, capreomycin Beijing M Greek Isoniazid, ethambutol Beijing M Greek Isoniazid, streptomycin, amikacin Beijing M Russian Streptomycin, amikacin, capreomycin Delhi/CAS M Pakistani Isoniazid Delhi/CAS M Pakistani Isoniazid, streptomycin Cameroon F Greek Isoniazid, streptomycin, amikacin Unclassified M Greek Streptomycin, amikacin, capreomycin Unclassified M Greek Isoniazid, streptomycin amikacin MTB, Mycobacterium tuberculosis; M, male; F, female. Table 5. Genotyping and drug sensitivity patterns in the MDR/XDR-TB groups. MTB strain Sex Nationality Drug sensitivity Haarlem M Unknown OF, RIB AMIK Haarlem M Greek LAM M Greek EM, OF, AMIK, CAP LAM F Polish OF, RIB LAM F Bulgarian OF, RIB LAM F Bulgarian OF, RIB, AMIK Uganda I M Greek SM, EM, OF, RIB, AMIK,CAP Uganda I M Iranian OF Beijing M Greek SM, EM,OF,CAP Beijing M Albanian OF, RIB, AMIK Beijing M Kazakhstan EM, OF Beijing F Greek OF, CAP Beijing F Russian OF, RIB Beijing M Georgian OF Beijing M Georgian OF Beijing F Greek OF Ural M Albanian OF XDR-TB Beijing M Greek CAP LAM M Moldavian EM LAM M Greek MTB, Mycobacterium tuberculosis; XDR-TB, extensively drug-resistant tuberculosis; M, male; F, female; OF, ofloxacin; RIB, rifambutin; AMIK, amikacin; EM, ethambutol; CAP, capreomycin; SM, streptomycin. The genotypes of Beijing, LAM and Ural families were associated with MDR-TB. Discussion In our study, 53 drug-resistant MTB samples isolated from patients with pulmonary TB were analyzed using MIRU-VNTR fingerprinting. To the best of the authors knowledge, this is the first study regarding the molecular analysis of drug-resistant MTB in patients with pulmonary TB in Greece. The main findings of this study are: (1) the high variety of genotype patterns of http://tar.sagepub.com 233

Therapeutic Advances in Respiratory Disease 5 (4) MDR-TB cases in Greece, with the major phylogenetically distinct groups of strains belonging to the Beijing and Haarlem families; and (2) the differences in genotype patterns and drug resistance between patients born in Greece and immigrants. The predominant genotype in Greeks was that of Haarlem lineage. The Haarlem type is found in about 25% of the Europe isolates and it is one of the three major families (Haarlem, LAM and T) that are the most frequent in Europe, Africa and South America [Brudey et al. 2006]. Most of the Greek patients had disease with monoresistant and polyresistant strains. It is noteworthy that only one patient with Haarlem genotype strain had MDR-TB. This is important information concerning the epidemiology of the area, since in a recent report from Tunisia the epidemic potential of this prevalent genotype is highlighted, as this represents 90% of all MDR-TB cases of the northern part of this country for the period 2001 2003 [Mardassi et al. 2005]. In the foreign-born patients of our study, the Beijing strain was the most prevalent genotype and was exclusively found in the polyresistant TB and MDR-TB. Furthermore, three of the MDR-TB Beijing strains were sensitive only to ofloxacin (i.e. closer to the XDR strains) indicating the proclivity of the strains of this family for drug resistance. Beijing strains are known to have been disseminated globally in recent years, and have been associated with drug resistance [Brudey et al. 2006]. The spread of Beijing strains in Greece might pose a potential major health problem by the expansion of polyresistant TB and MDR-TB cases if proper action is not taken, something that has already been seen in other parts of the world [Phyu et al. 2009]. Beijing strains are predominant in other regions of the world such as Far East Asia, but also in the Middle East, Central Asia and Oceania (45.9%, 16.5% and 17.2%, respectively). It has been endemic in China, and for a long time has been emerging in some other parts of the world, especially in countries of the former Soviet Union, and to a lesser extent in the Western world. In our study the origin of almost all of the Beijing isolates were from countries of the former Soviet Union. The prevalence of the Beijing isolates in Russia, both in prisons and communities, and the high rate of MDR-TB associated with this specific genotype, is well recognized [Ignatova et al. 2006; Glynn et al. 2002]. This may reflect the importation and dissemination of new MTB strains in Greece during the huge migration of people from countries of the former Soviet Union, as well as from the neighbouring Balkan countries. The CAS1-Delhi family is essentially localized in the Middle East and Central Asia, more specifically in South Asia, and preferentially in India as well as in other countries of this region such as Iran and Pakistan [Sola et al. 2001]. In our study, most of the Delhi/CAS isolates were monoresistant to isoniazid or to streptomycin and none were MDR-TB. These strains were mainly found in Pakistani patients who live and work in Greece; however, isolates of this strain were also found in Greek Caucasian patients, a finding that indicates the spreading of Delhi/CAS family into the Greek population. A surprising finding was that two patients with isolates belonging to the Cameroon family and four out of five patients with isolates belonging to Uganda I* family were Greek Caucasians who had never been to Africa. The Cameroon subfamily is predominant in Cameroon, other Western African countries (Benin, Senegal and the Ivory Coast) and in the Caribbean area [Niobe-Eyangoh et al. 2003]. The Uganda I* subfamily [Niemann et al. 2002] is endemic in Kampala, Uganda and represents 70% of all isolates of MTB from this area. Although limited data is available regarding the epidemiology and resistance patterns of these African origin strains, it is worth mentioning that in our study two out of five Uganda I family isolates were MDR. Furthermore, two patients in our study were infected by Ural strains: an Albanian with MDR-TB sensitive only to ofloxacine and a Moldavian with XDR-TB sensitive only to ethambutol. This indicates the high risk of expansion of new, difficult to treat tuberculosis cases in the Greek community. The Ural genotype represents 15.2% of the total isolates and 13.9% of the drug-resistant strains of the Ural region in Russia [Niobe-Eyangoh et al. 2003]. Isolates of this subfamily have also been found in Kazakhstan, Georgia and Saudi Arabia. This finding indicates that new MDR-TB strains have already entered into Greek communities and this is an alarming warning for the expansion of MDR-TB in Greece. 234 http://tar.sagepub.com

N Rovina, S Karabela et al. The LAM family isolates in our study, originating from Germany and former Soviet Union, accounted for the 25% of MDR-TB and were equally distributed between Greeks and foreignborn patients. It is worth mentioning that all LAM isolates except one were MDR, a finding that denotes the emergence of drug-resistant strains of this family in the Greek community. The presence of the LAM family is highest in Venezuela, in the Mediterranean basin and in the Caribbean region [Brudey et al. 2006]. Although, limited data are available on epidemiological and molecular characteristics of the LAM family, recent reports point out that MDR isolates of the LAM family are widely spread in Central Russia and represent the second predominant genotype of MDR-TB in prisons of this area [Ignatova et al. 2006; Kovalev et al. 2005; Shemyakin et al. 2004]. This finding is suggestive of a possible spread of this MDR genotype in Greece with the migratory transfer of many people of Greek descent from this Russian area to Greece. The major limitation of this study is the modest sample size analyzed and the lack of information concerning all isolates of MTB during the study period (sensitive and drug-resistant strains). This fact precludes the possibility of analyzing the specific association between the prevalent genotypes and drug resistance in Greece. However, SOTIRIA Hospital is a reference hospital for tuberculosis in Athens, a city of 4.5 million people (half of the population of the whole country) and the descriptive data presented here reflect at large the drug-resistance patterns of MTB in the country at this time period. Conclusion In conclusion, we have found that drug-resistant TB in Greece is due to strains of great genotypic diversity, reflecting the recently growing migratory transfer of people from countries with a high burden of TB. As drug-resistant TB is an emerging global problem, Greece does not represent an exception and needs to take action in order to control the expansion of drug-resistant MTB strains. The use of MIRU-VNTR typing is a powerful tool for molecular epidemiological studies of MTB. It allows the establishment of national databases which give the picture of the transmission pathways of MTB strains throughout the countries, reveal regional patterns of causative agents and the sources of infection, and facilitates the implementation of a global action for a more cooperative and effective anti- TB control program. Acknowledgements The authors wish to thank the chest physicians Dr A. Papavassiliou and Dr V. Tamvakis of the Clinic of Multi-drug Resistant Tuberculosis, SOTIRIA District Chest Diseases Hospital, for helping with the evaluation of patients. Funding This work was funded by a grant from the Hellenic Thoracic Society. Conflict of interest statement There are no conflicts of interest for the authors to declare. Authors contributions NR contributed to the design of the study and the interpretation of the results and has been involved in drafting and revising the manuscript. SK had a significant contribution in the acquisition and microbiological analysis of patient data. PC and VM performed the molecular analysis of the samples, and have been involved in the statistical analysis, drafting and revising of the manuscript. KK and VS provided material for MIRU analysis and feedback in manuscript preparation. CH supervised the work and manuscript preparation. NP conceived of the idea and designed the study, had the main contribution in the interpretation of the results, and drafted and revised the version of the manuscript to be published. References Barnes, P.F. and Cave, M.D. (2003) Molecular epidemiology of tuberculosis. N Engl J Med 349: 1149 1156. Brudey, K., Driscoll, J.R., Rigouts, L., Prodinger, W.M., Gori, A., Al-Hajoj, S.A. et al. (2006) Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6: 23. Caws, M., Thwaites, G., Stepniewska, K., Nguyen, T.N., Nguyen, T.H., Nguyen, T.P. et al. (2006) Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis. J Clin Microbiol 44: 3934 3939. Christianson, S., Wolfe, J., Orr, P., Karlowsky, J., Levett, P.N., Horsman, G.B. et al. (2010) Evaluation of 24 locus MIRU-VNTR genotyping of http://tar.sagepub.com 235

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