In Bulgaria during 2007–2011, a total of 188 MDR/XDR M. tuberculosis isolates were characterized by drug-susceptibility testing and spoligotyping (31 in 2007, 31 in 2008, 39 in 2009, 47 in 2010, and 40 in 2011) and represent 72% of the 261 MDR/XDR M. tuberculosis isolates identified during that period. The MDR/XDR strains were isolated from sputum of 181 (96.2%) patients, gastric lavage fluid from 3 (1.5%), bronchoalveolar lavage fluid from 2 (1%), pleural fluid from 1 (0.5%), and fistula swab sample from 1 (0.5%). The first MDR/XDR M. tuberculosis strain isolated per patient was analyzed for this study.

Drug susceptibility of these strains was confirmed by the National Reference TB Laboratory, which used liquid culture at concentrations of 0.1 μg/mL for isoniazid, 1 μg/mL for rifampin, 5 μg/mL for ethambutol, 1 μg/mL for streptomycin, 2.5 μg/mL for capreomycin, 1 μg/mL for amikacin, 5 μg/mL for kanamycin, and 2 μg/mL for ofloxacin. Of the MDR/XDR strains, 77 (41%) were resistant to all first-line anti-TB drugs, 51 (27%) were resistant to isoniazid and rifampin; 38 (20%) to isoniazid, rifampin, and streptomycin; and 22 (12%) to isoniazid, rifampin, and ethambutol. Second-line drug-susceptibility testing was performed for 174 (81%) of the MDR strains. Of these, 140 (80%) were sensitive to all second-line anti-TB drugs and 20 (12%) were resistant to ofloxacin. Five percent (n = 9) of XDR strains had combined resistance to ofloxacin, amikacin, kanamycin, and capreomycin.

To detect and genotype the Beijing strains, we used a spoligotyping kit (Isogen Bioscience BV, Maarssen, the Netherlands), and we performed single-nucleotide polymorphism typing by bead-based multiplex ligation-dependent probe amplification (MLPA) (8,9). We also screened for the presence of the Beijing genotype in 117 drug-sensitive strains collected from across the country in 2011, representing a convenience sample of ≈10%. Both methods identified 1 drug-sensitive M. tuberculosis strain of the Beijing genotype (BG_112_11, Table 1). The MLPA test assigned this isolate to the Beijing K1 sublineage (10).

Among the 188 MDR/XDR strains collected during 2007–2011, a total of 6 isolates with the Beijing genotype were identified (prevalence 3.2%, 95% CI 0.7%–5.7%): 2 in 2009 (Beijing SA+/CHIN+/V+), 3 in 2010 (Beijing SA+/CHIN+/V+ and Beijing K1), and 1 in 2011 (Beijing SA+/CHIN+/V+). These 6 drug-resistant strains were isolated from 5 male and 1 female patients. Drug-susceptibility testing and molecular drug-resistance markers confirmed that 1 strain was XDR. A total of 4 strains were Spoligo International Type 1 (SIT1) and 2 strains were SIT265 (Tables 1 and 2). In the neighboring countries of Albania, Greece, Romania, and Turkey, no MDR M. tuberculosis Beijing strains were isolated or prevalence was 1%–3%, mostly of imported origin (11–14). The origins of the MDR/XDR strains and the drug-sensitive strain identified in Bulgaria are described below. Additional methods used and genotyping characteristics found are described in the Technical Appendix.

The first XDR Beijing strain identified (BG_104_10) was from a patient who arrived in Bulgaria from Moldova in 2009 and in whom TB developed a few months later. Before coming to Bulgaria, the patient had had contact with a TB patient in Ukraine. The first M. tuberculosis isolate recovered from this Moldovan patient was confirmed as primary XDR M. tuberculosis. The patient received treatment with second-line TB drugs in 2010, underwent surgery, and in 2011 was considered cured.

The 2 SIT265 Beijing genotype clinical isolates (BG–85-10 and BG–95–10) originated from 2 neighbors. One patient was an alcohol-dependent sailor in whom TB developed after a ship voyage. TB developed in his neighbor (who had diabetes) a few months later. M. tuberculosis transmission most likely occurred between them. The epidemiologic link was supported by identical drug-susceptibility testing results, MLPA, spoligotyping, and variable number–tandem repeat typing profiles with no similarities to other genotypes identified in Bulgaria. Most of the 50 SIT265 Beijing genotype strains, reported in the SITVITWEB database (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE), originated from the United States, Russia, Israel, and Spain (4). Both patients were receiving treatment as of 2012.

The other 3 MDR Beijing isolates had different VNTR patterns but shared the same SIT1 spoligotype and the same MLPA lineage type (SA+/V+/CHIN+) (Table 2). Of these 3 patients, 2 lived in the capital city of Sofia (with strains BG_54_11 and BG_112_11) and the third (with strain BG_07_09) lived in Dobrich, in the northeastern part of the country. The patient was alcoholic, antisocial, and homeless and refused treatment. The MDR TB was diagnosed in 2007, but the strain was lost. In 2009, the patient was hospitalized, and Beijing genotype MDR M. tuberculosis was identified. The patient died that same year.

Another Beijing strain was identified in 2009 from a patient who lived in Sofia (BG_35_09, Tables 1 and 2). The patient’s parents were from Armenia, where the prevalence of the Beijing genotype is no less than 41% (4) and prevalence of MDR TB in 2009 was 22.9% (5). The patient had not been to Armenia, but the possibility of casual contact with migrating Armenians was not excluded. This patient died in 2010.

Among MDR/XDR M. tuberculosis isolates in Bulgaria, prevalence of the Beijing genotype is low. Not all detected cases of TB caused by the Beijing genotype were a result of human migration; MDR/XDR TB transmission within the country was also observed. The M. tuberculosis Beijing genotype strains are considered to be large drivers of international TB transmission and are associated with the emergence and spread of MDR/XDR TB (2). This finding demands organization of wider surveillance in Bulgaria that includes monitoring genotypes of drug-susceptible and drug-resistant M. tuberculosis strains.

Additional methods used and genotyping characteristics found for the identified Mycobacterium tuberculosis Beijing strains isolated in Bulgaria during 2007–2011.