ISSN 0891�4168, Molecular Genetics, Microbiology and Virology, 2014, Vol. 29, No. 2, pp. 58–62. © Allerton Press, Inc., 2014.Original Russian Text © S.N. Zhdanova, O.B. Ogarkov, A.A. Laz, A.N. Zarbuev, M.V. Badleeva, L.S. Untanova, E.D. Savilov, 2014, published in Molekulyarnaya Genetika,Mikrobiologiya i Virusologiya, 2014, No. 2, pp. 12–16.

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INTRODUCTION

Tuberculosis (TB) has been one of the burningquestions of health care. Detailed identification of TBpathogenic organism strains reveals possibilities fordeeper understanding of fundamental mechanisms ofinfection pathology. In particular, the exposure of his�tory of different pathogen genotypes circulation allowmodeling and predicting further development of epi�demic and infectious TB processes [1]. By itself, itessentially promotes improvement of epidemiologicaland clinical diagnostics of TB and formation of algo�rithms of prognosis disease outcome [2]. The presentresearch is a study of the modern population of myco�bacteria of TB (MTB) circulated among pulmonarytuberculosis sufferers in the Republic of Buryatia. Theaim of the present work is identification of epidemicand precinctive MTB genotypes circulating in the ter�ritory of the Republic of Buryatia.

MATERIALS AND METHODS

The analysis encompasses samples from 283 epi�demically unrelated MTB strains isolated frompatients with different forms of pulmonary tuberculo�sis undergoing medical treatment at the RepublicanClinical TB Dispensary (107 isolates) and in the penalinstitutions of the Republic of Buryatia (176 isolates)in the period from January 2010 to December 2012.

Strains were isolated from the Slavs (183 strains) andBuryats (100 strains). The vast majority of strains—271 specimens—were isolated from men. DNA ofcollected isolates was isolated and genotyped byMIRU�VNTR methods in 12 (283 strains) and 24 loci(31 strains) and by the deletion�analysis method inRD 105 and 207 each, as was mentioned above [3, 4]. Sta�tistical data processing was carried out in MS Excel 7.0and the Statistica package of statistical programs forWindows 6.0. The significance of differences betweenparameters were estimated by the nonparametric cri�terion χ2. The differences were considered statisticalvalues at p < 0.05.

RESULTS AND DISCUSSIONS

Strains of East Asian genetic lines with two delega�tions—RD105 and RD207 [5]—similar to previousstudy [6] were predominant in the studied sample: 188(66.4%) were from 283 isolates, while the remaining95 isolates (33.6%) were related to other families.

All strains were genotyped in 12 loci by theMIRU�VNTR method [4]. Profiles of 183 strains ofEast Asian lines were identified as genotype Beijingin accordance with the international SITVIT data�base (http://www.pasteur�guadeloupe.fr:8081/SITVIT_ONLINE/query). The unique MIRU�VNTRprofiles of five strains of East Asian genetic lines [5]

Identification of Ubiquitous and Endemic Mycobacterium tuberculosis Genotypes in the Republic of Buryatia

S. N. Zhdanovaa, O. B. Ogarkova,b, A. A. Laza, A. N. Zarbuevc, M. V. Badleevad,L. S. Untanovac, and E. D. Savilova,b

aScientific Center for Family Health and Human Reproduction Problems, Siberian Branch, Russian Academy of Medical Sciences, ul. Timiryazeva 16, Irkutsk, 664003 Russia

bIrkutsk State Medical Academy for Continuing Education, microregion Yubileinyi 100, Irkutsk, 664079 RussiacDugarova Republican Clinical TB Dispensary, ul. Klyuchevskaya 40, Ulan�Ude, Buryatia, 670013 Russia

dBuryat State University, ul. Smolina 24a, Ulan�Ude, Buryatia, 670000 Russiae�mail: svetnii@mail.ruReceived June 20, 2013

Abstract—The MIRU�12 and MIRU�24 methods were used for genotyping and estimation of M. tuberculo�sis population structure in Buryatia. It was studied in 283 strains isolated from individuals from the regionalTB dispensary and medical clinics of the penitentiary system of the republic. It was found that the prevalenceof pandemic genotypes Beijing MIT17 and MIT16, as well as subtype Beijing MIT642 (24.5%–46/188), weredominant in the studied area. The isolates of the Beijing MIT642 were common among all groups of patientswith pulmonary tuberculosis with the tendency to formation of MDR and chronic course of the disease.

Keywords: M. tuberculosis, endemic genotype, epidemic genotype, subtype of Beijing genotype, MIT17,MIT642

DOI: 10.3103/S0891416814020086

EXPERIMENTAL WORKS

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IDENTIFICATION OF UBIQUITOUS AND ENDEMIC 59

have no parallel in SITVIT database and MIRU�VNTRplus (http://www.miru.vntrplus.org/MIRU/index.faces). Twenty unclassified MIRU�VNTR pro�files (orphan) were identified, five of which hadhomologs in the SITVIT database. The remainingstrains were identified using the SITVIT database:LAM, 37; Ural, 18; and Haarlem, 10 specimens. Gen�otypes of other families—S�4, T�4, and X�3 strains—were detected in small numbers. In the samplesbrought from the penal institutions, strains of chronicpatients prevailed (68.8%), while strains isolated fromchronic patients in the republican dispensary made up14%. However, reliable genotyping differences of sam�ples were not found.

The frequency of the most widespread Beijing sub�types—MIT17 and MIT1—circulating on the terri�tory of Russia was, for Beijing MIT17, 22.3%(42/188), and for MIT16, 24.0% (45/188), of the totalsample of strains of this genotype. Those two subtypesmade up together 46.3 % (87/188), which was not typ�ical for the structure of the population of MTB of theBeijing genotype circulating on the territory of Russia[4, 7–12].

The pandemic genotypes that are globally wide�spread and exist on the studied territory (epidemic)include Beijing 16 (45 strains), Beijing 17 (42 strains),and LAM (15 strains) (figure). Special attention isdrawn to the studied sample of Beijing 642 (46 strains),which is described as an “endemic” genotype, i.e., isepidemically present on a limited territory—in thiscase, on the territory of Buryatia. The genotype men�tioned was found among isolates from newly diag�nosed patients (7.1%, 20 of the 283 strains of the wholesample), and from patients with chronic TB (9.2%,26 of the 283 strains of the whole sample). At the sametime, the frequency of its detection among patientswith early and chronic TB does not vary significantly(χ2 = 0.621, p = 0.365). The ethnicity of patients withTB caused by the subtype Beijing 642 did not matter(χ2 = 0.864, p = 0.353). An increase of drug resistance

of patients with Beijing 642 was observed in the groupof chronic patients with drug resistance to at least onefirst line drug. The number of isolates from chronicpatients with multiple drug resistance was muchhigher than from newly diagnosed patients (χ2 = 7.6,p < 0.01). This is prominently seen in the case of beinginfected with modern epidemic strains of BeijingMIT17 and MIT16 [13], which suggests that there is asimilarity between the mechanisms of circulation ofBeijing MIT 642, which is endemic to Buryatia. Thetable shows data on the spreading of drug resistanceamong subtypes of Beijing family. Special attention ispaid to the frequent occurrence of multiple drug resis�tance (MDR) in epidemic subtypes of the Beijing fam�ily (MIT 16, 17 and 642) in comparison with minorBeijing subtypes.

The selective developed profile of 31 strains from theabove�mentioned pandemic genotypes of Beijing 16,Beijing 17, and LAM 326 and epidemic Beijing 642 by24 MIRU�VNTR loci did not identify considerabledifferences inside the epidemic clusters (one or tworepetitions in superpolymorphous loci). Isolates ofBeijing 642 differed in number of repetitions in Qub 26,Mtub 04, ETR C, Mtub21, and Qub 4156c loci. Accord�ing to SITVIT database, this genotype was first foundin Japan [14] and has been described among strainsfrom Taiwan [15], with an old circulation of theBeijing genotype being typical for both territories [16].

This testifies to active spreading of the four above�mentioned genotypes in an epidemic fashion in thestudied population. These strains may be epidemio�logically connected with recent contagions from thepopulation infection reservoir despite the absence ofcontact. The antibioticogram results also show resis�tance differences between isolates of MTB insideMIT16 and 17 clusters isolated from chronic patientsand people with newly diagnosed TB. As a sign of asmaller increase of Beijing MIT17 and MIT16 in com�parison with other regions of Russia [4, 7–12], a widerange of Beijing variants was observed. They were

Distribution of drug resistance to main antituberculous medications among identified Beijing subtypes, abs. (%)

Characteristics of sample

Sensitive strains

Monoresistant strains1

Polyresistant (not MDR)

strains2MDR3

χ2, p

General sample (n = 283) 94 (33.2) 24 (8.5) 33 (11.7) 132 (46.6) –

Beijing MIT642 (n = 46) 8 (17.4) 4 (8.7) 7 (15.2) 27 (58.7) χ2 = 9.8;

p < 0.01

BeijingMIT16 (n = 45) 8 (17.8) 4 (8.9) 8 (17.8) 25 (55.6)

Beijing MIT17 (n = 42) 9 (21.4) 5 (11.9) 8 (19.0) 20 (47.6)

Beijing without MIT16, MIT17 and MIT642 (n = 55)

30 (54.5) 6 (10.9) 3 (5.5) 16 (29.1) χ2 = 9.8;

p < 0.011 Monoresistant strains are isolates resistant only to one type of studied antibiotics, 2 polyresistant strains are isolates resistant to two ormore types of antibiotics except for MDR, and 3 MDR indicates strains resistant to main antibiotics (isoniazid and rifampicine). Thisgroup also includes isolates with wide drug resistance, resistant to isoniazid, rifampicine, kanamycin, and fluroquinolone.

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Beijing (1)Beijing 86 (1)Beijing 721 (2)Beijing 858 (1)Beijing 87 (1)Beijing 137 (8)Beijing 82 (2)Beijing 83 (3)Beijing 707 (1)Beijing 16 (45)Beijing 571 (5)Beijing 872 (1)Beijing (3)Beijing 592 (8)Beijing 97 (1)Beijing 17 (42)Beijing 245 (1)Beijing 93 (1)Beijing 135 (2)Beijing (2)Beijing (1)Beijing (1)Beijing (1)Beijing 123 (1)Beijing 121 (1)Beijing (1)Orphan 1561 (1)Beijing (1)Beijing (1)Beijing (2)Beijing 642 (46)Orphan (1)T (1)X 118 (1)S 256 (4)Beijing 203 (1)T 794 (1)X (1)Orphan (1)LAM 1 (16)X 36 (1)Haarlem 741 (1)Haarlem 44 (1)Orphan (1)Haarlem (1)Haarlem 46 (1)Haarlem 247 (1)Haarlem 388 (1)Orphan 148 (1)Orphan (1)Orphan (1)Orphan (1)T 551 (1)Orphan 533 (2)T 125 (2)T 15 (2)Orphan (1)LAM (5)Orphan (1)Orphan (1)Orphan (1)LAM 326 (15)Orphan (1)Orphan (1)Orphan (1)Ural 354 (1)Ural 171 (16)Ural (1)Ural (1)Orphan (1)Orphan (1) 1 2 4 3 1 5 1 5 2 1 2 4

3 2 3 2 2 6 1 6 3 2 2 12 2 7 3 2 5 1 1 3 5 2 22 2 9 2 2 5 1 1 3 5 2 22 2 7 2 2 5 1 1 3 2 2 3

1 2 5 2 2 5 1 5 3 2 2 52 2 6 2 2 5 1 1 3 2 2 3

1 2 4 4 2 5 1 5 3 2 2 31 2 4 3 2 5 1 5 3 2 2 31 2 4 3 2 5 1 5 3 2 2 41 2 4 3 2 5 1 5 3 2 1 41 2 4 3 2 5 1 5 3 2 2 51 2 4 3 2 5 1 3 3 2 2 51 2 3 3 2 5 1 5 3 2 2 51 2 3 3 2 5 1 1 3 2 2 51 2 3 3 2 5 1 5 3 3 2 21 2 3 3 2 5 1 5 3 3 2 41 2 3 3 1 5 1 5 3 3 2 42 2 3 3 1 5 1 4 3 3 2 41 2 6 3 2 5 1 3 3 3 2 31 2 5 3 2 5 1 3 3 3 2 32 2 3 3 2 6 1 5 3 1 2 12 2 4 3 2 3 1 5 3 3 2 22 2 4 3 2 3 1 5 3 3 2 32 2 5 3 2 6 1 5 3 3 2 32 2 5 3 2 5 1 5 3 3 2 42 2 2 3 2 5 1 5 3 3 2 3

0.1

2 2 5 3 2 5 1 5 3 4 2 1

2 2 5 3 2 5 1 5 3 2 2 12 2 5 3 2 5 1 5 3 2 2 3

2 2 4 3 2 5 1 5 3 4 2 32 2 4 3 2 5 1 5 3 2 2 52 2 4 3 2 5 1 5 3 2 2 42 2 3 3 2 5 1 5 3 2 2 52 2 3 3 2 5 1 5 3 2 2 32 2 3 3 2 5 1 5 3 5 2 3

2 2 3 3 2 5 1 4 3 3 2 52 3 3 3 2 5 1 5 3 3 2 5

2 2 3 3 2 5 1 5 3 5 2 52 4 3 3 2 5 1 4 3 3 2 5

2 2 3 3 2 5 1 7 3 4 2 32 2 3 3 2 5 1 7 3 3 2 32 2 5 3 2 5 1 7 3 4 2 32 2 5 3 2 5 1 7 3 3 2 32 2 3 3 2 5 1 4 3 4 2 32 2 3 3 2 5 1 4 3 5 2 32 2 3 3 2 5 1 4 3 3 2 31 2 3 2 2 5 1 5 3 5 3 31 2 3 2 2 5 1 5 3 5 3 11 2 3 2 2 5 1 4 3 5 3 23 2 3 3 2 6 1 4 3 5 3 32 2 2 3 2 5 1 7 3 5 3 4

2 2 3 4 2 5 1 7 3 5 3 32 2 1 3 2 5 1 7 3 5 3 3

2 2 3 3 2 5 1 7 4 5 3 32 2 3 3 2 5 1 7 3 5 3 32 2 3 3 2 5 1 7 3 5 3 22 2 3 3 2 5 1 5 3 4 3 32 2 3 3 2 5 1 5 3 3 3 32 2 4 3 2 5 1 5 3 5 3 32 2 3 3 2 5 1 5 3 5 3 42 2 3 3 2 5 1 5 3 5 3 32 2 3 3 2 5 1 5 3 5 3 12 2 3 3 2 5 1 6 3 5 3 32 2 3 3 2 5 1 2 3 5 3 32 2 3 3 2 5 1 4 3 5 3 32 2 3 3 2 5 1 4 3 4 3 31 2 3 3 1 5 1 7 3 5 3 31 2 3 3 2 5 1 7 3 3 3 3

1 2 3 3 2 5 1 7 3 4 2 31 2 3 3 2 5 1 7 3 4 3 3

UPGMA�Tree, MIRU�VNTR [12]: Categorical

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IDENTIFICATION OF UBIQUITOUS AND ENDEMIC 61

clearly determined by the presence of the followingsubtypes strains in the sample: MIT642 in 24.5%(46/188) of cases, Beijing MIT137 in 4.3% (8/188),and Beijing MIT571 and MIT592 in 2.7% (5/188), aswell as isolates of Beijing MIT82, 83, 86, 87, 93, 97,121, 123, 135, 203, 245, 707, 721, and 1134 at a rate offrom 1 to 3. We discovered unique profiles of MIRU�VNTR of MTB clinical strains, with deletion inRD105/207 being absent in the SITVIT database buthaving been described in the studied territory earlier[4, 13]. In addition to the specimen of the Beijing fam�ily, we discovered isolates belonging to LAM family(MIT1 and MIT326)—11.0% (31/283)—as well asfive LAM�like isolates that were identical in 12 loci.Strains of the Ural family (MIT171) are observed in6.3% of cases (18/283); of the T family (MIT15 andMIT551) and S family (MIT256), in 1.4% each(4/283); and of the X family (MIT36, MIT118,MIT916), in 1.1% each (3/283). Analysis of the distri�bution of the main genotypes of Beijing 16, Beijing 17,LAM 326, and Beijing 642 by ethnicity did not detectreliable differences.

The obtained results testify once again that the epi�demical state of affairs regarding TB infection in eachpart of the studied territory develops as the total sum ofeffects on the sensitive human population of endemicstrains, which as widespread is typical only for the par�ticular territory, and ubiquitous and pandemic MTBstrains that are characterized by a global presence. Itappears that the ability of different MTB genotypes tochange the Th1/Th2 balance (cellular and humoral)of immune response [17] enables a pathogen to persistin a human population at a high epidemic level. Therole of the Ural genotype in TB epidemics on the ter�ritory of the countries of the former Soviet Union isstill obscure. On the one hand, this genotype is wide�spread in Russia and neighboring countries [18]; onthe other hand, strains of this genotype have low viru�lence and are not life�threatening [2]. We suggest thatthe persistence of the Ural genotype in the humanpopulation often proceeds in the form of coinfection,which is not identified by standard bacteriologicalmethods.

ACKNOWLEDGMENTS

This work was conducted with the support of grantof the Russian Foundation for Basic Research no. 12�04�31475 “Research on phylogenetic interrelations ofmain genotypes of Mycobacterium tuberculosis by24 loci of MIRU�VNTR for reconstruction of the his�tory of TB epidemics in the past and present on theterritory of Yakutia, Buryatia, and Irkutsk oblast” andgrant of the Russian Foundation for Basic Research

no. 13�04�00605 “Identification of SNPs specific forlow�virulence genotypes of Mycobacterium tuberculosisby the genome�sequencing method.”

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Translated by N. Beresteska