Reconstruction of the Epidemic History of the Beijing Genotype of Mycobacterium tuberculosis in Russia and Former Soviet Countries Using Spoligotyping

Transcription

1 ISSN , Molecular Genetics, Microbiology and Virology, 011, Vol. 6, No. 3, pp Allerton Press, Inc., 011. Original Russian Text V.V. Sinkov, E.D. Savilov, O.B. Ogarkov, 011, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 011, No. 3, pp EXPERIMENTAL WORKS Reconstruction of the Epidemic History of the Beijing Genotype of Mycobacterium tuberculosis in Russia and Former Soviet Countries Using Spoligotyping V. V. Sinkov a, E. D. Savilov b, c, and O. B. Ogarkov b, d a Irkutsk Regional Diagnostic Center, Irkutsk, Russia b Institute of Epidemiology and Microbiology, Siberian Branch, Russian Academy of Medical Sciences, Irkutsk, Russia c Irkutsk Institute for Medical Advanced Studies, Irkutsk, Russia d Irkutsk TB-prevention Dispensary, Irkutsk, Russia Received January 14, 011 All known genotypes (395 spoligoprofiles of M. tuberculosis) were selected using the SITVIT database in nine countries, including countries of the former Soviet Union: Russia, Latvia, Estonia, Poland, Finland, Italy, Portugal, Japan, and Vietnam. The programs SpolTools and DESTUS were used to construct consensus networks to identify the epidemic genotypes of M. tuberculosis in the studied countries. In Russia, Latvia, and Estonia, the Beijing strain was determined as the main epidemic genotype. In Finland, Poland, Portugal, and Italy, all epidemic genotypes belonged to other families. The hypothesis on the explosive nature of the spreading of the Beijing genotype of M. tuberculosis was put forward for Russia and other former Soviet countries in the 0th century. The basic idea of the hypothesis was that the pattern of dissemination of the Beijing genotype occurred at the CER (Chinese Eastern Railway), in the Gulag, and in the civilian society of Soviet Union. The Beijing genotype of M. tuberculosis affected Russian builders of the CER during the end the 19th and early 0th centuries. With the repression of CER builders in the Soviet Union, the Beijing genotype was spread among Gulag prisoners; after 1953, it had spread throughout the civilian society of the entire country. The distribution of epidemic genotypes of M. tuberculosis in the studied countries was interpreted as evidence of the suggested hypothesis. Keywords: spoligotyping, M. tuberculosis DOI: /S INTRODUCTION Today, tuberculosis remains one of the most challenging problems of practical healthcare worldwide. 1 According to WHO data, more than two billion people, i.e., one-third of the Earth s population, are infected with Mycobacterium tuberculosis, and one in ten infected people experience the tuberculosis disease during their lifetime [9]. In 009, 9.4 million cases of tuberculosis were registered in the world [9], with an average frequency of 139 disease cases per people [9, 9]. In 008, 1.8 million people died of tuberculosis, including HIV-infected ones, which is an average of 4500 deaths per day [9]. Moreover, the incidence rate has been shown to be increasing not only in developing countries, but also in Western Europe and United States. Russia, where a marked rise in all epidemiological markers of tuberculosis has been observed in recent decades, is not an exclusion [1]. Indeed, in 007, the incidence rate of tuberculosis among the adult population of Russia was 8.6 per people, with a lethality rate of 18.1 per people, which accounts for 85% of lethal cases of all infectious and parasitic diseases [7]. In comparison with 1990s (the beginning of the increase in the incidence rate), the lethality of tuberculosis has increased by.6 times [7]. Tuberculosis is characterized by an irregular geographical distribution. Minimal incidence rates have been observed in North and South America (3%) and Europe (5%), while maximal incidence rates have been observed in southeast Asia (55%) and Africa (31%) [5]. Presently, the distribution of tuberculosis is characterized as pandemic [14], the most important aetiological agent of which is genetically a similar group of strains called the Beijing genotype [8]. It should be noted that in Russia this subfamily prevails over all registered genotypes [, 3, 8, 11, 19, 6], making Russia considerably different from other European states [17]. However, it is important to note that newly formed European states founded on the basis of former republics of the USSR (Latvia, Lithuania, and Estonia) demonstrate a situation similar to that in Russia [16, 0, 7]. The genetic diversity of M. tuberculosis has been formed as a result of population migrations. Evidently, the Beijing genotype of M. tuberculosis occurred in northern China more than 000 years ago and pene- 10

2 RECONSTRUCTION OF THE EPIDEMIC HISTORY OF THE BEIJING GENOTYPE 11 trated to other regions via population migrations from southeast Asia [6]. I. Mokrousov et al. suggest that the predominance of the Beijing genotype in Russia originates from the medieval epoch and is connected with the Mongol Tatar invasion []. This was the time when contact between Slavs and Europeans with the Beijing genotype would have first taken place. However, this hypothesis fails to explain the phenomenon of the high similarity between M. tuberculosis genotypes in Russia and the Baltic states, which are former Soviet Union republics [16, 0, 7]. It is also impossible to explain the differences in this genotype distribution in the former Soviet states in comparison with other European states, including former Soviet states, within the framework of the medieval model [6, 8, 16, 19, 0, 6, 7]. Therefore, it seems that the scenario of the Beijing genotype s distribution must be different. It may be hypothesized that the distribution of the Beijing genotype of M. tuberculosis reached its modern level in Russia and the Baltic states (Latvia, Lithuania and Estonia) in a relatively short period of the USSR, whereas in neighboring states the distribution of the Beijing genotype remained the same, possibly at medieval levels. In order to verify this hypothesis, a model of evolutionary relationships among the strains of M. tuberculosis in the studied states was composed, and epidemiologically important genotypes were identified. Use of this model allowed us to obtain statistically significant data about the differences between the most important genotypes of tuberculosis etiological factors circulating in Russia, the Baltic states, and other European states. The present study was aimed at the formulation of the Beijing genotype s distribution scenario in the USSR region in the 0th century on the basis of molecular modeling results for the processes of occurrence of epidemiologically important M. tuberculosis genotypes in the studied states. MATERIALS AND METHODS Known genotypes of M. tuberculosis obtained by spoligotyping were used for the study. Spolygotyping profiles were obtained from the online database SITVIT [1]. In total, 395 genotypes (906 unique variants), which were isolated from the patients from Russia, Latvia, Estonia, Poland, Finland, Italy, Portugal, Japan, and Vietnam, were studied. The information about each spoligotype was presented as a numerical sequence of 43 numerals, where a deletion was marked as 0 and the absence of deletion was marked as 1. All spoligotypes were saved as.rsf files (rich spoligotype format) for further analysis with the Spol- Tool program [5]. A consensus spoligoforest network was composed on the basis of SpolTool spoligotypes [4]. The size of each cluster in the consensus network corresponded to the number of isolates of identical spoligotype. Signs between the clusters of the pictures show evolutionary interconnections between spoligo- States, number of strains and spoligotypes used in the study State Number of strains Number of spoligotypes Russia Finland Poland Latvia Estonia Portugal Italy Japan Vietnam Tota types and the calculated direction of mutation event ancestors to descendants. To ensure the correctness of the choice of an epidemiologically important genotype, data verification algorithms aimed at controling for the absence of false-positive interconnections with multiple data comparison were used (Bonferroni rule) with the DESTUS (Detecting Emerging Strains of Tuberculosis Using Spoligotypes) program [5]. The stability of spoligotrees was assessed using the Benjamini Hochberg, Storey, and Dunn Sidak algorithms, which calculate false discovery rate (FDR). Genotypes which demonstrated values of p < 0.01 in the sequential analysis with all three algorithms were considered epidemiologically significant. The relationships of epidemiologically significant genotypes to the known M. tuberculosis subfamilies were estimated using the spoligotype database SpolDB4 in accordance with their ST numbers [15]. RESULTS AND DISCUSSION In order to perform a comparative assessment, countries which were both historically and geographically related to Russia (Finland, Poland, Latvia, and Estonia) and were well-represented in the SpolDB4 database [15] were chosen. The fact that Lithuania was omitted is due to its poor representation in the SpolDB4 database. The other group included contrast countries which were poorly connected with Russia both historically and geographically. European states (Portugal and Spain) where the Beijing genotype is insignificantly distributed, as well as Asian states (Japan and Vietnam) where it dominates, were also chosen [13, 18]. The list of countries studied, number of M. tuberculosis strains, and the number of spoligotypes used in the study are shown in the table. States where a single genotype obviously dominates were not chosen for the study. The main parameters for a spoligotree analysis were cluster sizes and the number of ancestors [3]. For example, large clusters with numerous descendants were thought to evidence the ancientness of a genotype. Such a spoligotype could MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 6 No

3 1 SINKOV et al. Rank Spoligotype Subfamily p log(p) Spoligoforest Russia 1 Beijing 9.913e ST53 T1.7714e ST5 LAM e ST4 LAM ST54 T5_RUS e H e ST6 H e ST35 H4 1.13e Estonia 1 Beijing e (467) ST8 (8) ST3134 (18) (50) ST54 (50) ST53 (67) ST35 (18) ST4 (5) ST5 (5) Latvia 1 Beijing.1067e (75) Japan 1 Beijing.308e (169) Vietnam 1 39 EA14_VN M 1.688Be Beijing (181) (387) Note: Here and in Fig., the genotype rank shows the calculated epidemic significance of a genotype in comparison with that of other genotypes that circulate in a country; number in the SpolDB4 database [15]; subfamily name [15]; p value calculated by DESTUS program [5]; logarithms calculated with the same program, which provide the basis for assessment of epidemic significance (rank) of a genotype; spoligoforest of epidemiologically significant genotypes for each state. Fig. 1 States where the Beijing genotype () was shown to be epidemiologically significant. have been distributed in a population for a long period and should have had sufficient time to accumulate a good deal of mutations. However, from an epidemiological point of view, genotypes which can be distributed in a population faster than the natural process of mutation accumulation are of much more interest. These genotypes are characterized by larger cluster sizes and a minimal number of descendants and must be considered epidemiologically significant [3]. As a result of modeling, a consensus spoligoforest network which, after performing the Bonferroni test, allowed statistically significant isolation of the epidemic genotype [5] was obtained for each country [4]. Data on the full structure of the spoligotype consensus network for each country are not shown in the paper due to their bulkiness. The described modeling revealed epidemiologically significant genotypes with the confidence level p < The revealed genotypes are shown in Figs. 1 and. Connections between the clusters in Fig. 1 and show the presence of a final mutation step number between epidemic genotypes, which allows the Spol- MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 6 No

4 RECONSTRUCTION OF THE EPIDEMIC HISTORY OF THE BEIJING GENOTYPE 13 Rank Spoligotype Subfamily p log(p) Spoligoforest Finland 1 ST49 H e ST47 H e ST53 T e ST50 H e ST6 H4 1.94e LAM9 4.95e (181) (387) Poland H1.638e ST891 LAM e (16) ST891 (1) Portugal 1 ST0 LAM1.88e ST4 LAM e ST0 (49) ST4 (44) Italy 1 ST33 LAM3.3044e ST53 T1.4319e CAS1-DELHI e ST50 H e ST6 H e ST73 T-T e ST53 (54) 346 (14) ST33 (16) ST50 (30) ST73 (11) ST6 (14) Fig.. States from the comparison group, where the Beijing genotype was not shown to be epidemiologically significant. Tool program to calculate the virtual ancestor of epidemic genotypes; in contrast, the absence of these connections indicates significant genetic differences between these genotypes. Figure 1 shows countries (except Vietnam) where the Beijing genotype () is epidemiologically significant; i.e., it demonstrates the first rank of epidemic significance. Figure shows the countries of the comparison group. The Beijing genotype was shown to be the only epidemiologically significant genotype for the Baltic States (Estonia had 119 strains and Latvia had 140 strains) (Fig. 1). At the same time, the neighboring countries of Finland and Poland demonstrated a totally different spectrum of epidemic genotypes, in which strains of LAM, T, and Haarlem prevailed (Fig. ). The Russian epidemic genotype profile (Fig. 1) demonstrates both European (LAM and T) and Asian (Beijing) traits. The Haarlem 4 subfamily, which is more correctly called Ural, was also found among Russian epidemic genotypes [8, 19]. Apparently, this group of closely related genotypes [8] (Fig. 1 shows them all connected with each other) has different origins than other epidemic genotypes shown in Figs. 1 and. Therefore, the results of modeling of the formation processes of genotypes of M. tuberculosis strains from three states of the former USSR (Russia, Estonia, and Latvia), two neighboring states (Finland and Poland), and two remote European states (Portugal and Italy) may suggest that the Beijing genotypes identified in Russia, Estonia, and Latvia originate from a single source. Moreover, the absence of other epidemic genotypes in the studied Baltic states allows us to hypothesize relatively recent epidemics induced by the spoligotype. It goes without saying that it is not possi- MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 6 No

5 14 SINKOV et al. ble to exclude the probability that the Beijing genotype was presented earlier in the Russian Empire, as for example in the comparison states [16, 17, 0, 7]. However, the Beijing genotype () distribution pattern in Russia and the Baltic states suggests a single moment and an explosive distribution of this subfamily s strains. The scenario for such an epidemiological process could be as follows: the Beijing genotype is endemic to China and neighboring countries [14], and its history dates back several centuries [6]. However, contacts between the Russian Empire and China were very limited during this period, and only in the 0th century did a small Russian-speaking society form in Chinese territory due to the building of the China Eastern Railway and the development of neighboring lands [1]. Later, in the period of cooling of relations between the USSR and China, workers of the China Eastern Railway were forced to return to the USSR, which coincided with the period of mass repressions [1]. Tens of thousands of repatriates were jailed in correctional labor colonies, which led to the wide distribution of Beijing subfamily tuberculosis strains first among Gulag prisoners, and later, after the amnesty in 1953, in the civilian society of the Soviet Union. Within the framework of the unified state (USSR) and, more importantly, the unified penitentiary system, the soligotype was distributed throughout the entire territory of the Soviet Union, including the Baltic states. At the same time, in the countries of the Eastern Bloc, the profile of epidemic strains of tuberculosis did not change significantly or changed in accordance with the principles observed in other European states in the 0th century. These observations were even more surprising in light of the fact that both Finland and Poland were either totally or partially integrated into the Russian Empire: Finland from 1809 to 1917 [10] and the Polish Kingdom (with a population of.7 million) from 1815 to 1915 [4]. Similarly, both Baltic states studied were integrated into the Russian Empire: Estonia from 171 to 1918 and Latvia from 1796 to 190 as a part of the Province of Courland [4]. Therefore, the middle of the 0th century is considered to be the most likely date for the massive distribution of the spoligotype in the Baltic states. Evi- dently, the possibility of distribution of tuberculosis strains across the borders of states under the regular conditions of civilian society is much smaller than is traditionally supposed. It is suggested that even the relatively short historical period of the unified penitentiary system drastically affected the process of distribution of tuberculosis strains of the Beijing subfamily. It should be noted as a conclusion that the methods of molecular epidemiology are doubtlessly important for tuberculosis studies. The identification of epidemiologically important genotypes and the analysis of their structure in different countries and continents allow us to retrace the historical paths of epidemic distribution and identify the epicenters of highly active genotypes. This, in turn, provides the basis for antiepidemic and therapeutic approaches aimed against tuberculosis infection. REFERENCES 1. Ablazhei, N.N., Emigration from Russia (SSSR) to China and Reimmigration in the First Half of the XX Century, Extended Abstract of Doctoral (Histor.) Dissertation, Moscow, Balabanova, Ya.M., Nikolaevskii, V.V., and Raddi, M., Probl. Tub., 006, no., pp Baranov, A.A., Mar yandyshev, A.O., Markelov, Yu.M., et al., Ekol. Chel., 007, vol. 7, pp Bokhanov, A. and Gorinov, M., Istoriya Rossii s nachala XVIII do kontsa XIX veka (History of Russia from the Beginning of XVIII to the End of XIX Century), Sakharov, A.N., Ed., Moscow, Global naya bor ba s tuberkulezom (doklad) (Global Tuberculosis Control (Report)), WHO, Mokrousov, I.V., Genetic Diversity and Evolution of Mycobacterium tuberculosis, Extended Abstract of Doctoral (Biol.) Dissertation, St. Petersburg, On the Number of Tuberculosis Cases. Letter no. 01/ dated Federal Service on Surveillance of Consumer s Right Protection and Human Wellbeing, 008. URL: httr:// dosuments/letters/74/. 8. Ogarkov, O.B., Medvedeva, T.V., and Zozio, G., Mol. Med., 007, no., pp Onishchenko, G., Vestn. Ross. Akad. Med. Nauk, 008, no. 3, pp Osmo, Yu., Velikoe knyazhestvo Finlyandskoe (The Grand Duchy of Finland), Rumyantsev, A., Ed., Helsinki, Savilov, E.D., Sin kov, V.V., and Ogarkov, O.B., Epid. Infekts. Bol., 010, no. 4, pp Ftiziatriya. Natsional noe rukovodstvo (Phthisiology: National Guidance), Perel man, M.I., Ed., Moscow, Anh, D.D., Borgdor, M.W., Van, I.N., et al., Emerg. Infect. D, 000, vol. 6, no. 3, pp Bifani, P.J., Mathema, В., Kurepina, N.E., and Kreiswirth, B.N., Trends Microbiol., 00, vol. 10, no. 1, pp Brudey, K., Driscoll, J.R., Rigouts, L., et al., BMC Microbiol., 006, vol. 6, p Devaux, I., Kremer, K., Heersma, H., and Soolingen, D.V., Emerg. Infect. Dis., 009, vol. 15, no. 7, pp Harvard Medical School O. S. I. The Global Impact of Drug-Resistant Tuberculosis, Heymann, D.L., Ed., Boston, Iwamoto, T., Kekkaku, 009, vol. 84, no. 1, pp Kovalev, S.Y., Kamaev, E.Y., Kravchenko, M.A., et al., Int. J. Tuberc. Lung D, 005, vol. 9, no. 7, pp MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 6 No

6 RECONSTRUCTION OF THE EPIDEMIC HISTORY OF THE BEIJING GENOTYPE Kröner, A., Hoffner, S.E., Sillastu, H., et al., J. Clin. Microbiol., 001, vol. 39, no. 9, pp Liens, В., Sola, C., Brudey, K., and Rastogi, N., 6th Annu. Congr. Eur. Soc. Mycobacteria, Turkey, Istambul, June 6 9, Mokrousov, I., Ly, H.M., Often, T., et al., Genome Res., 005, vol. 15, pp Reyes, J.F., Francis, A.R., and Tanaka, M.M., BMC Bioinformatics, 008, vol. 9, p Tanaka, M.M. and Francis, A.R., Proc. Natl. Acad. Sci. USA, 006, no. 41, pp Tang, C., Reyes, J.F., Luciani, F., et al., Bioinformatics, 008, vol. 4, no. 0, pp Toungoussova, O.S., Sandven, P., Mariyandyshev, A.O., et al., J. Clin. Microbiol., 00, vol. 40, no. 6, pp Tracevska, Т., Jansone, I., Baumanis, V., et al., Int. J. Tuberc. Lung Dis., 003, vol. 7, no. 11, pp Van Soolingen, D., Qian, L., de Haas, P.E., et al., J. Clin. Microbiol., 1995, vol. 33, pp WHO Stop ТВ Partnership Update Tuberculosis Facts, 009. URL: tbfactsheet_009update_one_page.pdf SPELL: 1. healthcare,. spoligotype, 3. antiepidemic MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 6 No