Spoligotype-Based Comparative Population Structure Analysis of Multidrug-Resistant and Isoniazid-Monoresistant Mycobacterium tuberculosis Complex Clinical Isolates in Poland

The spoligotyping-based population structure of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated in Poland (n = 46), representing all culture-positive MDR tuberculosis (MDR-TB) cases, was compared to that of isoniazid (INH)-monoresistant strains (n = 71) isolated in 2004. The latter data set from a previous study (E. Augustynowicz-Kopeć, T. Jagielski, and Z. Zwolska, J. Clin. Microbiol. 2008, 46:4041-4044) represented 87% of all INH-monoresistant strains. The clustering rates and genotypic-diversity indexes for the 2 subpopulations were not significantly different (P = 0.05). The results were entered in the SITVIT2 database to assign specific shared type designations, corresponding genotypic lineages, and geographical distributions and compared to available data from neighboring countries (Germany, n = 704; Czech Republic, n = 530; Sweden, n = 379; Kaliningrad, Russia, n = 90) and strains from previous studies in Poland (n = 317). MDR strains resulted in 27 patterns (20 unique strains within the study and 7 clusters containing 2 to 6 isolates per cluster with a clustering rate of 56.5%) and belonged to the following genotypic lineages: ill-defined T family (28.3%), Haarlem (17.4%), Latin American and Mediterranean (LAM) (13%), Beijing (8.7%), S family (4.35%), and the X clade (2.17%). Comparison of the genetic structure of the MDR strains with that of INH-monoresistant strains showed that a total of 9 patterns were shared by both groups; these represented 1/3 of the MDR strains and 2/3 of the INH-monoresistant strains. Interestingly, 76.1% of the MDR isolates and 71.8% of the INH-resistant isolates yielded spoligotypes that were previously reported from Poland. The observation that nearly half of the spoligotypes identified among both MDR (48.1%) and INH-monoresistant (43.3%) M. tuberculosis isolates were present in Poland''s neighboring countries suggested that a significant proportion of MDR and INH-resistant TB cases in Poland were caused by strains actively circulating in Poland or its neighbors. Our results corroborate the leading role of the T and Haarlem genotypes in the epidemiology of drug-resistant TB in Poland. Nevertheless, the LAM and Beijing family strains that infected, correspondingly, 13% and 9% of patients with MDR-TB were absent among the strains from patients with INH-monoresistant TB, suggesting that a proportion of MDR-TB cases in Poland are due to ongoing transmission of MDR clones exhibiting specific genotypes. Study of the population genetic relationships between MDR and INH-monoresistant strains by drawing minimum spanning trees showed that ill-defined T1 sublineage strains (1/3 of all INH-monoresistant strains), represented by its prototype, SIT53, constituted the central node of the tree, followed by strains belonging to the well-defined H3, H1, and S subgroups. However, the MDR group, in addition, contained LAM (n = 6) and Beijing (n = 4) lineage isolates. With the exception of the 4 Beijing lineage strains in the latter group and a single orphan isolate in the INH-monoresistant group, none of the remaining 112/117 isolates belonged to principal genetic group 1 (PGG1) in our study. Given the high rate of clustering and the near absence of immigrants in the study, the persistence of MDR-TB in Poland seems to result from active transmission of MDR strains within the autochthonous population, the bulk of it caused by evolutionarily recent tubercle bacilli.Tuberculosis (TB) remains a substantial global health problem. With more than 9 million new cases and almost 2 million deaths every year (50), TB holds one of the leading positions among infectious causes of morbidity and mortality worldwide. One of the reasons for the continuing TB prevalence is the emergence and spread of drug-resistant (DR) and, especially, multidrug-resistant (MDR) (i.e., resistant to at least isoniazid [INH] and rifampin [RMP]) Mycobacterium tuberculosis strains. According to a recent World Health Organization (WHO) report, the global population weighted percentages for any resistance and MDR among all TB cases are 20% and 5.3%, respectively (49). Poland, the largest and most populous country in Central Europe (38,530,000 inhabitants in 2002), has witnessed a dramatic fall in the incidence of TB over the last few decades. While in 1957 the notification rate for all clinical forms was 290.4 per 100,000 population, it had dropped almost 10-fold by the end of 1990s (43). However, the TB notification rate in Poland is still relatively high, nearly twice the mean European Union (EU) rate (24.6/100,000 versus 12.8/100,000 in 2004) (14).Since the early years of chemotherapy for TB, the prevalence of primary drug-resistant TB in Poland has been closely monitored. In 1994, Poland joined the WHO/International Union against Tuberculosis and Lung Disease (IUATLD) global project on anti-TB drug resistance surveillance, and during 1996 and 1997, the first national survey on primary and acquired drug resistance was performed. The subsequent study, completed in 2001, revealed a 2-fold increase in the primary resistance rate in comparison with that in 1997, i.e., 6.1% versus 3.6%, respectively. Likewise, the prevalence of primary multidrug resistance doubled, from 0.6% in 1997 to 1.2% in 2000 (3). In 2004, there was a slight decrease to 5.6% in the percentage of TB cases with primary drug resistance and quite a significant fall to 0.3% in the share of multidrug-resistant tuberculosis (MDR-TB) cases compared to 2000 (49). These figures place Poland among the countries with low to moderate DR-TB rates in the world. However, inadequacies in the registration of TB patients may contribute to an underestimation of drug-resistant cases. The most detrimental impact on the epidemiology of TB in Poland might be the spread of DR-TB from outside the country. This is because Poland shares borders with countries of the former Soviet Union, such as Lithuania, Ukraine, and Russia, where the incidence of DR-TB, including MDR-TB, is exceptionally high (49). Given the geographical epidemiological context, the surveillance capacity for TB in Poland has to be particularly well established.Studying genetic relationships among M. tuberculosis strains has provided important insights into the spread and transmission of TB (8, 15, 26, 30, 31, 34). Spoligotyping targeting the polymorphism of the chromosomal direct-repeat locus is one of the most extensively used techniques for genotypic differentiation between M. tuberculosis strains (27). Primarily used for first-line screening of outbreaks (22, 40), spoligotyping is ideally followed by more discriminatory second-line IS6110 restriction fragment length polymorphism (RFLP) or mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (9, 24). It has also proven useful for tracking laboratory cross-contaminations (36) and describing the global spread of TB (9, 17). Major advantages of the spoligotyping technique are its simplicity, rapidity, reproducibility, and portability, due to the digital nature of the generated results. Of particular importance is the existence of the international spoligotype database, which provides useful information on the epidemiology and phylogeny of the circulating genotypes of tubercle bacilli worldwide (9).This study aimed to investigate the spoligotyping-based population structure of MDR M. tuberculosis clinical isolates isolated in Poland in 2004 compared to that of INH-monoresistant strains isolated in the same year. Indeed, as reviewed by Dye and Espinal (12), a high proportion of TB patients carrying INH-monoresistant strains acquire RMP resistance after treatment failure, suggesting that the development of INH resistance usually precedes that to RMP.