At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia

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1 RESEARCH ARTICLE At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia Igor Mokrousov 1,2, Tatiana Otten 3, Thierry Zozio 2, Eugeni Turkin 4, Vera Nazemtseva 4, Aleksandra Sheremet 3, Boris Vishnevsky 3, Olga Narvskaya 1 & Nalin Rastogi 2 1 Laboratory of Molecular Microbiology, St Petersburg Pasteur Institute, St Petersburg, Russia; 2 Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe; 3 Laboratory of Microbiology of Tuberculosis, The Research Institute of Phthisiopulmonology, St Petersburg, Russia; and 4 Kaliningrad Region Tuberculosis Dispensary, Kaliningrad, Russia Correspondence: Igor Mokrousov, St Petersburg , Russia. Tel.: ; fax: ; imokrousov@mail.ru Received 19 May 2008; revised 2 July 2008; accepted 30 July First published online 17 September DOI: /j X x Editor: Patrick Brennan Keywords Beijing genotype; spoligotyping; MIRU-VNTR; drug resistance; rpob; katg. Abstract The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/ in 2000 to 134/ in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpob and katg. A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region. Introduction Tuberculosis in Russia remains as an important public health issue for the entire country; at the same time, some regions constitute hotspots for tuberculosis epidemics. The Kaliningrad (Königsberg from 1255 to 1946) region is the westernmost part of the Russian Federation and includes and an area on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/ in 2000 to 134/ in This latter figure exceeds both the mean figure for north-west Russia (64/ ) (A. Sheremet, unpublished data) and the country as a whole [106.3/ (Euro TB, 2008)]. The rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients (E. Turkin, unpublished data), a value that is much higher than estimated for the country as a whole [13%; 95% CLs (WHO, 2008)]. Implementation of molecular techniques has considerably benefited the classical epidemiology of bacterial pathogens, including Mycobacterium tuberculosis. In particular, repetitive and insertion sequences have proven useful for studying the epidemiology, evolution and phylogeography of M. tuberculosis, and regularly updated genetic diversity databases are available for this pathogen (El Sahly 2004; Kremer 2004; Mokrousov 2004; Brudey 2006; Weniger 2007). The present study provides a first molecular snapshot of the population diversity of M. tuberculosis in the Kaliningrad region of Russia. We further compare our data with those available for the European part of Russia and for neighbouring countries in the Baltic Sea area.

2 14 I. Mokrousov et al. Materials and methods Study panel The study set included strains randomly selected among those isolated from March to June 2006 in the Regional tuberculosis dispensary in Kaliningrad and sent to the reference laboratory of the St Petersburg Research Institute of Phthisiopulmonology for quality control of the drug susceptibility testing. The 90 M. tuberculosis strains were recovered from 90 adult, epidemiologically unlinked patients with pulmonary tuberculosis who were permanent residents in the Kaliningrad region. Löwenstein Jensen medium was used for cultivation of isolates and susceptibility testing was performed using the absolute concentration method according to the Ministry of Health of the Russian Federation (2003), as recommended by WHO (1998) and as described previously (Viljanen 1998). An isolate was considered to be resistant when bacterial growth occurred at a concentration of 1 mg isoniazid ml 1, 20 mg rifampin ml 1, 5mg streptomycin ml 1 and 2 mg ethambutol ml 1. DNA fingerprinting DNA extraction, spoligotyping and 12-locus mycobacterial interspersed repetitive units (MIRU)-variable-number tandem repeat (VNTR) typing were performed as described previously (van Embden 1993; Kamerbeek 1997; Supply 2001). MIRU-VNTR digital profiles were treated as categorical variables and analysed using PAUP (Swofford, 2002). The Hunter Gaston index (HGI) was used to evaluate discriminatory power of the typing methods and individual diversity of the VNTR loci was calculated as described (Hunter & Gaston, 1988). The individual spoligotyping patterns were entered into an Excel spreadsheet and compared with the international database of the Pasteur Institute of Guadeloupe, which is an updated version of the published SpolDB 4.0 database (Brudey 2006). An identical spoligotype pattern shared by two or more isolates in the database is designated as spoligotype shared type (ST) while a 12-locus MIRU pattern shared by two or more strains is referred to as MIRU international type (MIT). A 2 2 w 2 test was used to detect any significant difference between the two groups. Yates corrected w 2 and P values were calculated with 95% confidence interval using EPICALC 2000 version 1.02 software (Gilman & Myatt, 1998). Resistance mutations detection Mutations in katg315 associated with isoniazid resistance and mutations in the rpob rifampin resistance-determining region (RRDR, codons ) associated with rifampin resistance were analysed using PCR-restriction fragment length polymorphism (RFLP) and modified RIFO assays, respectively, as described previously (Mokrousov 2002, 2006a; Morcillo 2002). To minimize the risk of laboratory cross-contamination during PCR amplification, each procedure (preparation of the PCR mixes, addition of DNA, PCR amplification and electrophoretic fractionation) was conducted in physically separated rooms. Negative controls (water) were included to control for reagent contamination. Results Ninety M. tuberculosis strains from Kaliningrad, Russia, were selected for this study. Seventy-seven of these strains (85.5%) were isolated from newly diagnosed tuberculosis patients and 13 strains were recovered from previously treated tuberculosis patients. Thirteen patients were HIVcoinfected (11 of them were with newly diagnosed tuberculosis) and seven patients were released prison inmates (four of them were with newly diagnosed tuberculosis) (Table 1; supporting Table S1). Genotyping Spoligotyping subdivided all strains into 23 types, including 11 types shared by 2 41 strains and 12 singletons. The largest cluster included 41 strains with a characteristic Beijing genotype profile (the presence of spacers from 35 to 43 only) (van Soolingen 1995; Kremer 2004). Comparison with international spoligotype database SIT- VIT2 allowed us to ascribe a majority of the obtained spoligoprofiles to the shared types already present in the database (Table 2) while three profiles (three strains) did not match any profile recorded in the database and remained orphans. Use of the 12-locus MIRU-VNTR typing achieved a higher differentiation of strains compared with spoligotyping (Table 3). Although a number of clusters identified by the two methods were similar, the total number of types was higher for MIRU typing than for spoligotyping (43 vs. 23). At the same time, the Beijing genotype strains remained closely related and formed a monophyletic group in the MIRU-based tree (cluster I in Fig. 1). Eleven strains of spoligotype ST53 were distributed in very distant branches in the MIRU tree. In one case, an ST53 strain had an MIRU profile identical to one strain with a new profile B, suggesting possible convergent evolution in these loci. Contrary to the ST53 strains that were dispersed across the MIRU-based tree, the ST42 strains constituted a more closely related group (Fig. 1). Interestingly, three strains (6617, 8682 and 2075) had identical MIRU profiles (Fig. 1), and a closer look at their spoligoprofiles revealed their apparent relatedness.

3 Mycobacterium tuberculosis in Kaliningrad, Russia 15 Table 1. Drug susceptibility and prevalence of drug resistance mutations in Mycobacterium tuberculosis strains of Beijing and other genotypes isolated from tuberculosis patients in the Kaliningrad region of Russia All strains, n = 90/new cases, n =77 Beijing genotype, all cases, n = 41/new cases, n =31 Patient status Released prison inmate 7/4 3/2 4/2 HIV coinfection 13/11 6/4 7/7 Strain phenotype Fully susceptible 50/47 8/7 42/40 Rifampin resistant 31/21 29/20 2/1 Isoniazid resistant 37/27 31/22 6/5 Streptomycin resistant 39/30 33/24 6/6 Ethambutol resistant 6/4 6/4 0 MDR 30/21 28/20 2/1 rpob allele 531-TTG 26/18 26/ TGG 1/0 0 1/0 Dwt5 1/0 1/0 0 Wild type 3/3 2/2 1/1 katg 315-ACC 34/25 29/21 5/4 Other genotypes, all cases, n = 49/new cases, n =46 Dwt5 designates an absence of hybridization with wild-type probe #5 in the RIFO assay, i.e. a mutation within rpob codons (Morcillo 2002; Mokrousov 2006a). It may be noted that all three major shared types (ST1, ST53 and ST42) retained their position within three large MIRU superclusters (Fig. 1), although ST1 (Beijing) strains were most closely related while ST53 strains were most divergent. Interestingly, all LAM family strains fall within MIRU supercluster III while all Haarlem family strains were located within MIRU supercluster II (Fig. 1). In summary, the tree shown in Fig. 1 underline the overall concordance of the results obtained by the two genotyping methods. Allelic diversity differed among particular VNTR loci (Table 4). The highest allelic diversity among all strains studied was observed for MIRU26 (0.714), MIRU31 (0.656) and MIRU40 (0.650) and the null allelic diversity was found for the monomorphic loci MIRU24 and MIRU27. However, a closer look at the diversities of the MIRU loci in different phylogenetic lineages revealed that the variation was mainly observed for non-beijing strains. In contrast, the allelic diversity was very low for Beijing genotype strains (Table 4, Fig. 1); only locus MIRU26 was moderately polymorphic: four loci showed low polymorphism and seven loci were monomorphic (Table 4). Strain genotypes vs. patient status The major spoligotype in this study, the Beijing genotype (41/90 strains), was found in 31/77 (40%) strains from newly diagnosed tuberculosis patients and in 10/13 (77%) strains from previously treated tuberculosis patients. Thus, the Beijing genotype was more prevalent in the group of previously treated patients although this difference was only borderline significant [2 2 w 2 = 4.64, OR = 4.95 (95% CI, 1.26; 19.43) P = 0.03]. We additionally compared the HIV status of the tuberculosis patients vs. major genotype groups of the studied M. tuberculosis strains (Table 1). The tuberculosis/hiv coinfection rate did not vary between Beijing- and non-beijinginfected patients [14.6% (6/41) vs. 14.3% (7/49), P = 0.8]. A minor but statistically insignificant difference was observed for a subset of newly diagnosed patients [12.9% (4/31) vs. 15.2% (7/46), P = 0.9). The released prison inmates constituted 7.8% (7/90) of the studied sample (Table 1). No difference in their prevalence between Beijing and non-beijing groups was observed (total sample, P = 0.8; newly diagnosed subset, P = 0.9). Drug resistance analysis We also analysed drug susceptibility profiles and drug resistance mutations in the studied strains. The 40 drugresistant isolates included in the study showed the following resistance profiles: two resistant to streptomycin alone, one resistant to streptomycin and rifampin, one resistant to isoniazid and rifampin, seven resistant to streptomycin and isoniazid, 23 resistant to streptomycin, isoniazid and rifampin, and six resistant to streptomycin, isoniazid, rifampin and ethambutol. Thirty strains (75%) were resistant to both isoniazid and rifampin and thus, were classified as MDR. The drug resistance patterns varied significantly between Beijing and non-beijing strains (Table 1). The Beijing strains were associated with resistance to any particular drug and

4 16 I. Mokrousov et al. Table 2. Geographical distribution of the Mycobacterium tuberculosis spoligotypes identified in this study and in the neighbouring Russian and European regions ST Family European part of the Russian Federation Kaliningrad Tula St Petersburg Karelia Latvia Estonia Poland Germany Finland Sweden 1 Beijing LAM Haarlem T LAM Haarlem Haarlem T T LAM T LAM Haarlem LAM LAM X LAM T Unknown Unknown 5 Orphan A LAM 1 Orphan B Unknown 1 Orphan C Haarlem 1 Total References This study Shemiakin et al. (2002) Narvskaya et al. (2005) Markelov et al. (2007) Tracevska 2003; Krüüner 2001; Sajduda 2004; Niemann unpubl. data; Puustinen 2003; Brudey 2004; is the most recent update of the published database SpolDB4 (Brudey 2006). Beijing genotype constitutes 63% in the Samara region, Russia (Drobniewski 2002), and 44% in Belarus (Vasilenko & Semionov, 2007) of the total Mycobacterium tuberculosis population in these regions. Table 3. Discriminatory power of typing methods in Mycobacterium tuberculosis strains from Kaliningrad, Russia Method, sample No. of types No. of unique isolates No. of clusters Cluster size (range) HGI Spoligotyping All strains Beijing genotype Other genotypes locus MIRU-VNTR typing All strains Beijing genotype Other genotypes multidrug resistance while non-beijing strains were dominated by fully susceptible isolates [2 2 w 2 = 38.6, OR = 50.6 (95% CI, 10.6; 241.0) P =10 6, for association of the Beijing genotype with multidrug resistance]. Analysis of the gene mutations linked to the resistance to the two main antituberculosis drugs, rifampin and isoniazid, revealed that 28 (90.3%) of 31 rifampin-resistant strains had a mutation in the hot-spot region in the rpob gene. A katg 315 AGC 4 ACC mutation was detected in 34 (91.6%) of 37 isoniazid-resistant strains (Table 1, supporting Table S1). No mutations in the targeted loci were detected in drugsusceptible strains.

5 Mycobacterium tuberculosis in Kaliningrad, Russia 17 Fig. 1. The 12-MIRU-VNTR-loci based UPGMA dendrogram of the 90 M. tuberculosis strains from Kaliningrad, Russia, aligned to their spoligotypes, spoligofamilies and spoligotyping profiles. The major spoligotypes are in bold. Discussion This study was undertaken in order to gain first insight into the population structure of M. tuberculosis in the Kaliningrad area of north-western Russia. The population of the area has changed completely since 1946 when the northern part of the former East Prussia was transferred to the USSR as agreed at the Potsdam Conference. The German population

6 18 I. Mokrousov et al. Table 4. Allelic diversity of the 12 MIRU loci in Mycobacterium tuberculosis strains from Kaliningrad, Russia All strains (Beijing genotype strains) VNTR locus No. of alleles No. of repeats, range Allelic diversity MIRU2 2 (1) 2 3 (2) (0) MIRU4 3 (1) 1 3 (2) (0) MIRU10 7 (3) 1 10 (1 4) (0.141) MIRU16 4 (2) 1 4 (2 3) (0.048) MIRU20 2 (1) 1 2 (2) (0) MIRU23 5 (1) 1 6 (5) (0) MIRU24 1 (1) 1 (1) 0 (0) MIRU26 6 (2) 1 7 (5 7) (0.621) MIRU27 1 (1) 3 (3) 0 (0) MIRU31 4 (1) 2 5 (5) (0) MIRU39 2 (2) 2 3 (2 3) (0.048) MIRU40 5 (2) 1 5 (1 3) (0.095) of the province evacuated during World War II and in was subsequently replaced with Slavic migrants from the neighbouring Russian provinces. Virtually closed from foreign contact from 1950 until 1991, Kaliningrad today forms a busy Russian European crossroads, which has had an impact on the epidemiological situation of communicable diseases not only in Russia but also in northern Europe. Mycobacterium tuberculosis population structure in Kaliningrad and regional context Comparison with and the published literature helped to place our data in the global and regional context (Table 2). The M. tuberculosis population of Kaliningrad is dominated by the four large spoligotype families: Beijing (41/90 strains), LAM (16/90 strains), T family (16/90 strains) and Haarlem (7/90 strains). Three major spoligotypes, ST1 (Beijing), ST42 (LAM family) and ST53 (T family), accounted for 57/90 (63%) of the strains in our setting. Their distribution in the M. tuberculosis populations in Kaliningrad, other regions of the European part of Russia and neighbouring countries is plotted in Fig. 2. This clearly shows that the M. tuberculosis population in Kaliningrad shares a joint pool of strains with the European part of Russia and, at the same time, shows some affinity with its northern European neighbours (Table 2, Fig. 2). It appears that two opposing vectors meet in Kaliningrad, thus reflecting an East West interaction. First, the major genotype in our setting, Beijing, is also prevalent in the countries of the former Soviet Union, i.e. both Newly Independent States and the three Baltic countries (Glynn 2002). A visible proportion of the Beijing genotype in Sweden (Table 2) may be explained by immigration from Asia (Brudey 2004) rather than from Russia. Secondly, the other spoligotype (ST53) prevalent in Kaliningrad is more specific for Europe than for Russia (Fig. 2). Finally, ST42 is found virtually in all areas compared here, both former Soviet Union and northern Europe (with the exception of Germany). It is not unlikely that a high prevalence of the Beijing genotype as well as the prevalence of ST42 in Kaliningrad is due to the recent historical and present links with Russia, and, broadly speaking, north-eastern Baltic neighbours (i.e. Lithuania, Latvia and Estonia). By contrast, a noticeable prevalence of ST53 may result from significantly intensified contacts with western neighbours in northern Europe, such as Germany and Poland. Fig. 2. Geographic distribution of the three major spoligotypes identified in M. tuberculosis strains from Kaliningrad. World map ( Courtesy of the University of Texas Libraries, The University of Texas at Austin.

7 Mycobacterium tuberculosis in Kaliningrad, Russia 19 Comparison with global M. tuberculosis database SIT- VIT2 (Institut Pasteur de Guadeloupe) and MIRU database of the Beijing genotype (Mokrousov, 2007, 2008) revealed that the 12-MIRU profile [type MIT17 (SIT- VIT2) or M11 (Mokrousov, 2008)] was found in 27/41 Beijing strains, and profile [type MIT16 () or M2 (Mokrousov, 2008)] in 7/41 Beijing strains in our study. Both types are predominant in different Russian settings although at different ratios. Type M11 (MIT17) is the most globally distributed type within the Beijing genotype, especially in Eurasia, whereas type M2 (MIT16) is more Russia specific (Mokrousov, 2008). For example, in, these profiles and are found in, respectively, 17.7% and 70% of strains from Russia, 39.6% and 11.7% of strains from Europe, and 15.7% and 0.8% strains from Japan. A high prevalence of profile in our study is different from central and north-west Russia and is more similar to the Ural region of Russia (Kovalev 2005) and also to Beijing, China (Jiao 2008). Drobniewski et al. (2005) noted a higher prevalence of profile in prison inmates and of profile in civilians in Samara, central Russia. These profiles differ in only a single locus (MIRU26) and the significance of this finding remains unclear. In our setting, former prison inmates were found in similar proportions in Beijing and non-beijing infected patient subgroups (Table 1). Impact of the Beijing genotype It has been suggested that current transmission of MDRtuberculosis in Russia is considerably influenced by ongoing dissemination of Beijing family strains (Drobniewski 2005; Narvskaya 2005). Contrary to another Russian study that focused on MDR strains, 46% belonged to the LAM family, 42% to the Beijing family and 10% to the Haarlem family (Lipin 2007); however, these authors did not report data on susceptible strains. In our study, a remarkably strong association of MDR as well as of any drug resistance (P =10 6 ) was observed for the Beijing genotype (Table 1). A detailed look at the resistance patterns of non- Beijing strains showed no specific association: six isoniazidresistant and six streptomycin-resistant non-beijing strains belonged to different spoligotypes (supporting Table S1). A comparison of the resistance patterns vs. genotypes in the two groups of previously treated and newly diagnosed patients confirmed findings observed for the entire collection (Table 1). In particular, MDR was identified in 20/31 and 8/10 Beijing genotype strains from newly diagnosed and previously treated patients, respectively. In contrast, 40/46 and 2/3 non-beijing strains from newly diagnosed and previously treated patients, respectively, were fully susceptible. Consequently, the Beijing genotype strains appear to develop drug resistance more readily. Although these strains are not overwhelmingly prevalent in the local population of M. tuberculosis, the current situation of MDR-tuberculosis in the Kaliningrad area in Russia appears to be critically influenced by the high prevalence of the MDR Beijing genotype strains in the local population of M. tuberculosis. Tuberculosis/HIV coinfection is a severe threat compromising tuberculosis control also in the Baltic Sea region (Samarina 2007). The prevalence of HIV infection in the Kaliningrad region is among the highest in Russia and the incidence is almost double the mean figure for the country as a whole (Kuz min 2005; org/pub/2007/05.shtml). We, therefore, further compared the HIV status of tuberculosis patients among major genotype groups of M. tuberculosis strains. However, no statistically significant association with either Beijing or non-beijing strains was found (Table 1). Phenotypic vs. genotypic drug resistance We also looked at the distribution of the rpob and katg mutations in our study and other world regions. The global prevalence of the katg S315T substitution in isoniazidresistant strains highlights the selective advantage conferred by this mutation, which appears to provide the optimal balance between decreased catalase activity and a sufficiently high level of peroxidase activity in KatG. The global prevalence of the katg315 AGC 4 ACC mutation among isoniazid-resistant M. tuberculosis strains is variable but high, for example 47% in Finland (Marttila 2008), 61% in China (Jiao 2007), 71% in Vietnam (Caws 2006), 64% in South Africa (van Rie 2001) and 64% in India (Nusrath Unissa 2008). The frequency of the katg315 mutation in our study (91.9%) was higher than generally worldwide and similar to other and distant Russian regions: St Petersburg, 93.6% (Mokrousov 2002); Siberia, 93 94% (Voronina 2004), central region, Urals and Siberia, 92% (Afanas ev 2007). Accordingly, it appears that katg S315T mutation alone can be used to predict reliably a high proportion of isoniazid-resistant strains in the Kaliningrad region as well as in Russia as a whole. The most frequent rpob mutation was 531TTG, found in 83.9% of the rifampin-resistant strains (Table 1). This result differs from that observed in a study across different Russian regions (Moscow, Urals, Siberia) where this mutation was found in a smaller proportion (64.8%) while other most frequently mutated codons were 526 and 516 (10.3% and 7.7%, respectively) (Afanas ev 2007). By contrast, in the Samara region of Russia, 90% of rifampin-resistant isolates harboured the rpob531 mutation (Nikolayevsky 2004). This variation may be due to a different prevalence of the strain families in the local populations of M. tuberculosis, for example a higher or lower proportion of

8 20 I. Mokrousov et al. the Beijing strains. Our findings corroborate previous observations from Russia (Mokrousov 2003), Kazakhstan (Hillemann 2005) and South Africa (van Rie 2001) that reported an association of the Beijing genotype and the rpob 531TTG allele. However, in other studies, this rpob mutation was reported to be almost equally prevalent in the Beijing vs. non-beijing rifampin-resistant strains from East Asia (Qian 2002; Jou 2005; Mokrousov 2006a) and Latvia (Tracevska 2003), and was even less represented in the Beijing genotype rifampinresistant strains from Korea (Park 2005). The observed variation in the prevalence of the rpob S531L mutation among Beijing strains from different settings may reflect the differential capacity of the different subgroups within the Beijing family to develop drug resistance (Mokrousov 2006b) and, in particular, to acquire certain rpob mutations. Whether a correlation exists between the prevalence of this mutation and/or circulating Beijing subgroups and the specific features of the National tuberculosis control programmes in different countries (e.g. the quality of the drugs used for treatment) remains an open question. In conclusion, the M. tuberculosis population in the Kaliningrad exclave shares a joint pool of strains with the rest of Russia, but at the same time, shares more affinity to the northern European neighbours. The adverse situation as regards MDR-tuberculosis in this Russian region appears to be due to the high prevalence of the MDR Beijing genotype strains in a local M. tuberculosis population. Acknowledgements This study was supported by NATO s Public Diplomacy Division in the framework of the Science for Peace programme (grant SFP ) and by a research fellowship from the European Commission to I.M. (Marie Curie Fellowship contract no. MIF1-CT ). References Afanas ev MV, Ikryannikova LN, Il ina EN et al. 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