insights into the origin, emergence, and current spread of ... · cient application of modern...

Insights into the Origin Emergence and Current Spread of aSuccessful Russian Clone of Mycobacterium tuberculosisIgor Mokrousov

Laboratory of Molecular Microbiology St Petersburg Pasteur Institute St Petersburg Russia

SUMMARY 342INTRODUCTION 342

Genome Markers of M tuberculosis 343Population Structure and Clinical Relevance 343Hypothesis and Questions 345

SAMPLING AND METHODS 345Selection of Studies 345Methods of Analysis 345

Genotyping 345Statistical and phylogenetic analysis 345Comparison with human history 346

GENETIC FEATURES OF BEIJING B0W148 346PATHOBIOLOGY OF BEIJING B0W148 347

Virulence and Transmissibility 347Pathological features 347Animal and macrophage-based studies 347Clustering 348Bacterial population growth 348Circulation in special settings 349

Drug Resistance 351HUMAN POPULATION CONTEXT 352

Geographic Diversity and Place of Origin of B0W148 352Timing of Origin and Primary Dispersal of B0W148 354Driving Forces behind Emergence and Spread of B0W148 355

CONCLUSIONS AND PERSPECTIVES 355ACKNOWLEDGMENTS 356REFERENCES 356AUTHOR BIO 360

SUMMARY

Mycobacterium tuberculosis variant Beijing B0W148 is regardedas a successful clone of M tuberculosis that is widespread in theformer Soviet Union and respective immigrant communitiesUnderstanding the pathobiology and phylogeography of thisnotorious strain may help to clarify its origin and evolutionaryhistory and the driving forces behind its emergence and currentdissemination I present the first review and analysis of allavailable data on the subject In spite of the common percep-tion of the omnipresence of B0W148 across post-Soviet coun-tries its geographic distribution shows a peculiar clinal gradi-ent Its frequency peaks in Siberian Russia and to a lesserextent in the European part of the former Soviet Union Incontrast the frequency of B0W148 is sharply decreased in theAsian part of the former Soviet Union and it is absent in au-tochthonous populations elsewhere in the world Placing themolecular clinical and epidemiological features in a broadhistorical demographic and ecological context I put forwardtwo interdependent hypotheses First B0W148 likely origi-nated in Siberia and its primary dispersal was driven by a mas-sive population outflow from Siberia to European Russia in the1960s to 1980s Second a historically recent phylogeneticallydemonstrated successful dissemination of the Beijing B0W148strain was triggered by the advent and wide use of modern

antituberculosis (anti-TB) drugs and was due to the remark-able capacity of this strain to acquire drug resistance In con-trast there is some indication but not yet systematic proof ofan enhanced virulence of this strain

I am I and my circumstanceJ Ortega y Gasset Meditations on Quixote

INTRODUCTION

Paradoxically and contrary to its etymology a ldquocircumstancerdquolies within no less than beyond and the last word in the above

famous maxim may well be replaced with a much more earthlyone microbes During the long course of its evolution the speciesHomo sapiens has acquired a ldquosecond genomerdquo ie the humanmicrobiome defined as the sum of all our resident microbeswhich coevolved with their hosts From the phylogeographic

Address correspondence to igormokrousovyahoocom

Supplemental material for this article may be found at httpdxdoiorg101128CMR00087-12

Copyright copy 2013 American Society for Microbiology All Rights Reserved

doi101128CMR00087-12

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point of view the human microbiome may be regarded as analternative source of data for deciphering human migrations andorigins Reciprocally knowledge about human migration and de-mographics may explain under certain limitations the origin anddispersal of human pathogens

Evolutionary histories of H sapiens and human pathogens areat least partly comirrored and coshaped However horizontallyepidemically transmitted pathogens (eg influenza virus andHIV) are less likely to mirror the population structure of the hu-man host instead their population structures reflect a rapid out-burst spreading of particular genotypes Accordingly microbe-aided tracing of the events in human history should rely onnonepidemically or mainly latently manifested microorganismswith a familyhousehold mode of transmission and sufficientprevalence in a population Regarding human tuberculosis (TB)historically short devastating periods of horizontal epidemicspread have been followed by longer time spans of relativelypeaceful coexistence of the surviving human populations and thefamilyhousehold-transmitted pathogen Therefore Mycobacte-rium tuberculosis is a suitable candidate for tracing human migra-tions since remarkably useful features of M tuberculosis include(i) a high prevalence (30) of latent tuberculosis in humansand hence a mainly familyhousehold mode of transmission dur-ing most of human history and (ii) an extreme rarity of lateralgene transfer and hence less risk of diffusing the phylogeographicsignal

Genome Markers of M tuberculosis

The M tuberculosis genome contains various insertion sequences(IS6110 is the most widely used) and repeat loci that have beenused widely to study the genetic diversity of the species The mostfrequently used of the latter include one CRISPR (historicallynamed a direct repeat [DR] [1]) locus and multiple variable-num-ber tandem-repeat (VNTR) loci (Fig 1)

The CRISPR locus in M tuberculosis as in other bacteria con-sists of minisatellite alternating exact direct repeats and variablespacers (Fig 1A) Its evolution in M tuberculosis currently occursvia neutral consecutive deletions of either single units or contigu-ous blocks (2) A method to study variation in the CRISPR locus inM tuberculosis is named spoligotyping which in its classical andusually macroarray-based format detects the presence or absenceof 43 particular spacers (Fig 1A) A more comprehensive analysisof the CRISPR locus relies on 68 different spacers which givessome added discriminatory value (2) As a whole the method issimple and portable but lacks sufficient discriminatory power Forthis concern more discriminatory methods of M tuberculosisstrain typing deal with more polymorphic IS6110 and VNTR loci

IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing is based on analysis of the insertion sequenceIS6110 which is found in most isolates of the M tuberculosis com-plex (Fig 1D) Until relatively recently this was the most widelyapplied and standardized molecular typing method for M tuber-culosis complex isolates The method was considered to be themost discriminatory and was thus the most widely used method inepidemiological studies (3) Unfortunately it is cumbersome andtime-consuming and requires large quantities of DNA and inter-laboratory comparison of results is difficult

Mycobacterial interspersed repetitive unit-VNTR (MIRU-VNTR) loci are scattered throughout the genome and isolates canbe typed based on the number of copies of repeated units (4)

(Fig 1B) Implementation of the large number of carefully evalu-ated loci has been expected to achieve a high level of discrimina-tion Since 1998 the VNTR typing of M tuberculosis has under-gone a remarkable development Whereas the first schemecomprised only 6 exact tandem-repeat loci (5) a more recentlyimplemented but already classical MIRU set involved 12 loci (4)and finally the new format for MIRU typing includes 24 loci (6)The necessity of further adjusting this scheme to particular set-tings by adding so-called hypervariable loci was recognized mostrecently (7ndash9) This method was shown to possess a higher dis-criminatory power than spoligotyping and depending on thenumber of VNTR loci tested a power roughly similar to that ofIS6110-RFLP typing (6 7) The other apparent advantage of theVNTR approach (compared to IS6110 typing) is its portable for-mat and hence its easy international exchange and use in databasedevelopment

A number of other molecular markersmethods such as single-nucleotide polymorphisms (SNPs) large deletions (regions of dif-ference [RD]) and whole-genome new-generation sequencingshould be mentioned They vary in discriminatory power al-though they demonstrate a clear potential to reconstruct reliablephylogenies and may aid in high-resolution epidemiological in-vestigations (10) However they are not relevant to the topic ofthis review and thus are not discussed further here

Population Structure and Clinical Relevance

It is very common yet true saying that M tuberculosis is a majorhuman killer However a closer look at the population structureof the pathogen reveals that some of its lineages show a greatercapacity to spread and cause active disease Consequently effi-cient application of modern antituberculosis treatment regimensis hampered by dissemination of such M tuberculosis strains Mtuberculosis has a clonal population structure and several geneticfamilies have been identified within this species eg East-Afri-can-Indian CASDelhi Beijing Haarlem Manu and LAM Ini-tially endemic within specific geographical areas some of thesefamilies have become ubiquitous An example of such an omni-present lineage is the Beijing familygenotype It was identified forthe first time in the Beijing area of China (11) but strains of thislineage are now found on all continents although at differentrates and thus may be defined as endemic epidemic or sporadic(12 13)

In addition to the lineagefamily-associated strain properties ithas been recognized that some of the clonal clusters (sublineagesand subgenotypes) within the same family may be more transmis-sible than others In particular while the Beijing genotype as awhole is frequently (but not always) marked by drug resistanceand hypervirulence (reviewed in reference 14) some of its variantsdemonstrate even more remarkable pathogenic properties espe-cially in areas with a high burden of drug-resistant TB (15) Thereare several classifications of Beijing genotype strains in particularby subdividing them into ancient or ancestral and modern groups(16ndash18) which are suggested to differ in mechanisms of adapta-tion to drug-induced selective pressure (19) In China the mod-ern Beijing strains are more likely than the ancient sublineages tobe clustered (20) and an increasing circulation of the modernBeijing sublineage was recently demonstrated in Japan (21)

While discrimination into modern and ancient Beijing strainsprovided a rough phylogenetically meaningful subdivision high-resolution typing permitted the definition of more homogeneous

Successful Russian Clone of M tuberculosis

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FIG 1 Positions of particular genomic loci on a circular map of M tuberculosis H37Rv along with their structure and analysis (A) DRCRISPR locus and43-spacer-based spoligotyping (B) Multiple VNTR loci including 24 MIRU-VNTR (in bold) and 3 hypervariable loci and their analysis using agarose gelelectrophoresis M 100-bp ladder (C) Rv2664-Rv2665 region in H37Rv and W-148 strains (D) IS6110-RFLP typing with hybridization of PvuII-digested DNAsof M tuberculosis strains with an IS6110-derived probe Beijing genotype strains are shown by asterisks The characteristic double band in the profile of strainB0W148 is shown by short arrows M strain Mt14323 used as a molecular size marker

Mokrousov

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clonal clusters or strains of clinical and epidemiological interestNotorious examples include strain W which caused the multi-drug-resistant TB (MDR-TB) epidemic in New York City in theearly 1990s (22) the highly transmissible Beijing strain in GranCanaria Island whose circulation is still ongoing (23 24) and theRussian strain Beijing B0W148 (25ndash28) the subject of this re-view

Hypothesis and Questions

A clonal group named B0 or W148 (27 28) has been reported forabout one-fourth of the Beijing genotype isolates in different partsof Russia and the former Soviet Union and in Russian immigrantsin the United States and Europe (25ndash31) It has been hypothesizedthat the Beijing B0W148 clone spread historically recentlythroughout the former Soviet Union due to its special pathogenicproperties and thus may represent a successful clone of M tuber-culosis (8) However an integral picture of the time and location oforigin evolutionary history and biological properties of the Bei-jing B0W148 strain remains obscure Did it originate within theborders of Russia or the former Soviet Union or was it importedfrom abroad for example from East Asia where the Beijing ge-notype is a prevalent lineage or from other parts of Asia Whatwas the driving force of its dissemination more specifically whathuman movement served unwittingly as a vehicle What patho-biological properties underlie its spread I tried to answer thesequestions by searching the published literature for the presenceand properties of Beijing B0W148 strains in local populations ofM tuberculosis A systematic and critical review reanalysis andnew analyses of these data are presented below

SAMPLING AND METHODS

Selection of Studies

The M tuberculosis Beijing family isolates were defined based onspoligotyping isolates showing hybridization to at least three ofspacers 35 to 43 and an absence of hybridization to spacers 1 to 34were defined as having the Beijing genotype (32) (Fig 1A)

M tuberculosis Beijing B0W148 isolates were defined based onvisual similarity of their IS6110-RFLP profiles to the prototypeprofile (8 27 28) (Fig 1D) One should note that no freely acces-sible database of IS6110-RFLP profiles exists and that the searchfor profiles was done directly in the original articles More detailson genetic features of B0W148 are provided below

For literature search the Google Google Scholar Scopus andMedline search engines and databases were used The primarykeywords used were ldquotuberculosisrdquo ldquoBeijing genotyperdquo ldquoB0rdquoldquoW148rdquo ldquoIS6110rdquo ldquofingerprintingrdquo ldquospoligotypingrdquo ldquotypingrdquoand country names in English and Russian

The primary definition of the W148 clone was based on theIS6110-RFLP profile in this sense the inclusion of data on IS6110-RFLP profiles was the key feature of the article to be considered forfurther analysis In fact very few studies mention this clone in theabstract and searches of raw data and other analyses were per-formed directly on the information found in or extracted from thefull-text articles A per-country search was based on the generalperception of the geographic specificity of B0W148 to the formerSoviet Union ie the search of IS6110-RFLP profiles in areasbeyond Eurasia was representative but not exhaustive The B0W148 isolates recovered from immigrants from the former SovietUnion in Europe and the Americas make a special case and were

studied separately these studies were not included in geographicalmapping (Fig 2) but information on patientsrsquo country of originwas used in some kinds of analysis (see Table S1 in the supplemen-tal material) Data on the Beijing genotype as a whole are shownonly for Eurasia where B0W148 is circulating and only for loca-tions with available data on IS6110-RFLP profiles

Thus in total 63 studies were retained based on visual inspec-tion of the content and were used for the different aspects of anal-ysis presented in this review (15 25ndash27 29ndash31 33ndash88) (see TableS1 in the supplemental material)

The limitations of this review should be acknowledged as it wasbased on published studies it depended on their own limitationsand potential biases First not all studies used all molecular meth-odsmarkers necessary for identification of the B0W148 strainand its further subtyping Second only a few studies made a spe-cific focus of the phenotypic features of the B0W148 strain andmost studies did not show data on B0W148 alone but as part ofthe Beijing family Third not all studies provided data on individ-ual isolatespatients so it was not possible to extract the precisestrainpatient-specific details

All available publications dealing with genotyping data andpublished since the early 1990s were (re)analyzed in order to es-tablish the rate of Beijing B0W148 in local populations of Mtuberculosis (see Table S1 in the supplemental material) The ob-tained information was further interpreted in light of the mean-ingful clinical epidemiological and phenotypic strainpatientproperties The summarized data are shown in Table S1 and in Fig2 In spite of a bias of some studies certain special settings such asprisons offered additional interesting clues to a better under-standing of the epidemiology of the Beijing B0W148 variant

Methods of Analysis

Genotyping Typing data obtained by classical and more recentmethods ie IS6110-RFLP typing spoligotyping and MIRU-VNTR typing performed according to international protocols (689 90) were considered in the analysis

The international databases MIRU-VNTRplus (wwwmiru-vntrplusorg) (91) and SITVIT Web (httpwwwpasteur-guadeloupefr8081SITVIT_ONLINE) (92) were consulted forattributing international codes to the MIRU-VNTR profiles

Statistical and phylogenetic analysis The Hunter-Gaston in-dex (HGI) was used as an estimate of the diversitydiscriminatorypower of a molecular markermethod The HGI was calculatedusing the following formula

HGI 1 1

NN 1j1

s

njnj 1

where N is the total number of strains in the typing scheme s is thetotal number of distinct patterns discriminated by the typingmethod and nj is the number of strains belonging to the jth pat-tern (93)

The minimum spanning tree (MST) and neighbor-joining treebased on VNTR types were built using the PAUP package (94)and the PARS routine of the PHYLIP 36 package (95) respec-tively VNTR alleles were treated as categorical variables

Meta-analysis was carried out using the Review-Manager 43(Oxford United Kingdom) and STATA version 10 (Stata Corp)programs The inconsistency index (I2) was calculated and a 2-based Q-statistic test was performed to evaluate between-studyheterogeneity (96 97) I2 values of 50 and P values of 01



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indicated significant heterogeneity A Z test was used to test thesignificance of the odds ratio (OR) (P values of 005 were con-sidered significant) Beggrsquos rank correlation method was used tostatistically assess publication bias (no bias was present if the Zvalue was 196 and the P value was 005) (98)

Comparison with human history Searches for major flows ofhuman migration and historical links between the areas targetedin this work were done by searching Google Entrez Pubmed (httpwwwncbinlmnihgovsitesentrez) and Russian Demoscope(httpdemoscoperuweekly) search resources The primary key-words used were ldquohuman migrationrdquo ldquotuberculosisrdquo ldquohistoryrdquoldquophylogeographyrdquo ldquocoevolutionrdquo and relevant geographic namesused alone and in combination in English and Russian This pro-cedure was followed by further sorting and mining of the largebody of retrieved information and if necessary additional search-ing using more specific keywords covering bilateral relations be-tween particular regions and countries Although this method isneither exhaustive nor quite systematic a quantitative approachto comprehensively study large events in human history does notyet exist to the best of my knowledge

GENETIC FEATURES OF BEIJING B0W148

The Beijing B0W148 variant was initially defined using IS6110-RFLP typing (25ndash28) and is marked by a characteristic doubleband (71 and 92 kb) in the upper part of the profile (Fig 1D)Strains with the Beijing B0W148 sensu stricto profile and similarprofiles sharing the same characteristic double band were more

recently defined as the B0W148 cluster (8) Thus IS6110-RFLPtyping is by definition the ldquogold standardrdquo method for definingB0W148 cluster isolates

B0W148 isolates have a nine-signal 43-spacer spoligotypingprofile that is characteristic of the whole Beijing family (16 27 4853) (Fig 1) More comprehensively based on 68-spacer spoligo-typing the CRISPR locus in B0W148 isolates consists of 15 unitsalthough this was shown only for isolates from northwestern Rus-sia (16) and is a feature common to other Beijing genotype vari-ants A discussion about ldquopseudo-Beijingrdquo strains (99) is beyondthe scope of this article

The evolution of the VNTR loci of the Beijing B0W148 isolatesin Russia is discussed in more detail below In St Petersburg innorthwestern Russia most of these isolates had the 24-MIRU pro-file 244233352644425173353723 (8) the loci are listed accordingto their clockwise order in the genome (Fig 1 see Table S2 in thesupplemental material) In the international MIRU-VNTRplusdatabase this 24-locus profile is designated 100-32 Interestinglythis profile also falls within the 12-MIRU type M11 (digital profile223325173533 [17 33]) also named MIT17 (SITVIT Web data-base) which is the largest type in East Asia and the second (but notfirst) largest in Russia (13 100) Three hypervariable lociQUB3232 VNTR3820 and VNTR4120 were characterized by 1414 and 10 copies respectively in most of the B0W148 clusterisolates in the St Petersburg area of Russia (see Table S2) Thepresence of seven copies each of MIRU26 and QUB26 was initiallysuggested to be specific for B0W148 in northwestern Russia (8)

FIG 2 Global distribution of M tuberculosis Beijing genotype and Beijing B0W148 strains in autochthonous populations in Eurasia Data are not shown for (i)prison settings and (ii) immigrant communities in Europe Data on the Beijing family as a whole are shown only if the frequency of the B0W148 strain could beverified by an IS6110-RFLP or multiplex PCR method (33) For Azerbaijan and Kyrgyzstan no information on the Beijing family rate is shown and estimatedrates of B0W148 prevalence are shown (see Table S1 in the supplemental material for details) Numbers inat circles show percentages of the Beijing family andB0W148 strain in the M tuberculosis population in a given area circle sizes are proportional to these percentages

Mokrousov


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However in Novosibirsk Siberia the B0W148 isolates hadmainly 8 as well as 6 and 7 copies of QUB26 (44) and the use ofthis two-locus signature to identify the B0W148 cluster isolates isapparently limited to the St Petersburg area of Russia In sum-mary although the aforementioned profile 100-32 was prevalentamong these strains in St Petersburg no single consensus 24-MIRU profile exists for B0W148

A complete genome sequence of the first M tuberculosis strainof the B0W148 cluster was released in 2011 (strain W-148GenBank accession no ACSX000000001) This was an extremelyhelpful achievement toward a more in-depth study of the strainsof this cluster and a better understanding of their pathogenomicsIn particular this opened new perspectives for the search of newmolecular targets for rapid diagnostics for this variant IS6110-RFLP typing is the gold standard method for detecting B0W148cluster isolates however it is admittedly cumbersome and time-consuming In silico analysis of the complete genome sequence ofstrain W-148 helped to identify positions and surrounding se-quences of IS6110 insertions (33) The reversed IS6110 in contig139 (positions 19418 to 20772) was found located within the9162-bp PvuII digestion fragment which corresponded to thelargest band of the B0W148 IS6110-RFLP profile (Fig 1C versusD) This insertion corresponds to positions 2982598 and -9 in thecomplete genome of strain H37Rv (GenBank accession noNC_0009622) in a short intergenic region between the Rv2664and Rv2665 genes This intergenic region is also both intact andidentical in all other published complete genomes of M tubercu-losis complex strains of different genotypes available in GenBankThe only exceptions are (i) M bovis and M bovis BCG strains withan A iexcl G substitution 179 nucleotides upstream of this IS6110insertion in strain W-148 (position 2935950 in the complete ge-nome sequence of M bovis BCG strain Pasteur 1173P2 [GenBankaccession no NC_0087691]) and (ii) M canetti in whose genomeno similarity was found Both the Rv2664 and Rv2665 genes en-code hypothetical proteins of unknown function (ldquohypotheticalproteinrdquo and ldquohypothetical arginine-rich proteinrdquo respectively)and are located within the phiRv2 prophage Their functions re-main unknown even after a recent reanalysis and reannotation offunctions for the hypothetical open reading frames (ORFeome) ofthe M tuberculosis genome (101) Therefore whether this partic-ular IS6110 insertion has a biological meaning andor conferssome hypothetical advantage is unclear

Whatever its function this Rv2664-Rv2665IS6110 inser-tion was used as a target for a multiplex PCR assay to detect theB0W148 strain (33) This method was proven to be sensitiveand specific and was recommended for simple and reliable de-tection and surveillance of B0W148 isolates in areas of epi-demic circulation such as Russia and other countries of theformer Soviet Union and in areas receiving immigrants fromthese countries It will also be interesting to apply it to retro-spective strain or DNA collections and archival samples fromdifferent locations To date only one study has used this novelmethod to assess the prevalence of B0W148 strains in retro-spective strain collections representing Russia Belarus ChinaVietnam and Brazil (33)

PATHOBIOLOGY OF BEIJING B0W148

The pathobiology of microorganisms encompasses those biolog-ical properties that may cause under certain hostpathogenenvi-ronment-related conditions pathological changes in the host

organism andor host population An understanding of pathobi-ology is crucial for elucidating the driving forces underlying thetempo and mode of dissemination of a particular strain or clone

Virulence and Transmissibility

Pathological features To date a sufficient number of studies fo-cusing on the Beijing family have analyzed patient-related infor-mation and pathological features such as the course and particu-larities of the disease progression (duration and severity of diseasefebrile response and disease site eg pulmonary versus extrapul-monary TB) treatment outcomes (such as failure or relapse) pa-tient age (younger versus older) BCG vaccination status etc Un-fortunately this kind of clinically meaningful data is extremelyscarce for the Beijing B0W148 strain

Contrasting pulmonary versus spinal TB one very recent studyin St Petersburg analyzed the genetic diversity of M tuberculosisstrains from adult TB spondylitis patients coming from differentRussian regions (35) In particular a 748 rate of prevalence ofthe Beijing family in the studied population was found (comparedto an average 50 rate among pulmonary TB patients across Rus-sia [16 27 36ndash48]) At the same time the rate of B0W148 in theBeijing population was 30 (35) Accordingly when recalculatedthe rate of B0W148 prevalence was 224 of all M tuberculosisisolates from TB spondylitis patients which is double the 128average rate of B0W148 prevalence in St Petersburg (Fig 2 seealso Table S1 in the supplemental material]) BCG vaccinationwhich is recognized to best protect against disseminated TB (al-though indeed in children [14 102 103]) is obligatory in RussiaIn my opinion this finding suggested an association of B0W148and spinal TB and thus can be interpreted as a propensity of thisstrain to cause extrapulmonary TB disease in the BCG-protectedpopulation ie as an indication of increased virulence

Animal and macrophage-based studies Understanding thetransmissibility of a strain in particular that of an M tuberculosisstrain may perhaps be achieved through gaining knowledge aboutits virulence manifested through host-pathogen interactions atthe individual and population levels In this regard studies usinganimal models and macrophage-based models may provide a rel-atively direct proof of virulencetransmissibility properties

Use of animal models of TB has previously highlighted inter-strain variations and although these findings cannot be extrapo-lated directly to human infection they have aided in the study ofthe pathogenesis and manifestation of the disease Animal modelstudies have shown that highly prevalent and clustered strains aremore virulent than unclustered sporadic isolates and that highlyvirulent strains can induce nonprotective immune responses withsome correlation with clinical and epidemiological characteristics(104) Macrophages are a primary target of M tuberculosis andthe ability of the organism to multiply rapidly within these cells isimportant for pathogen survival A number of properties havebeen linked with highly virulent mycobacteria such as the capac-ity to kill infected macrophages by cell necrosis favoring bacterialsurvival and liberation (105) and the induction of a low level ofproinflammatory tumor necrosis factor alpha (TNF-) and a highlevel of inhibitory inerleukin-10 (IL-10) production by infectedmacrophages (106)

It should be noted that differently designed macrophage-basedstudies produced concordant results and demonstrated an in-creased virulence of the B0W148 variant compared to other Bei-jing variants (15 107 108) A comparison of Russian strains of



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different genetic families demonstrated that Beijing genotype iso-lates were more viable than LAM and Ural strains in a murinemacrophage model irrespective of the infecting dose Interest-ingly B0W148 isolates showed even higher viability in murinemacrophages than that of other Beijing strains (108 109)

A comparative study of MDR Beijing genotype strains fromRussia and Brazil found that Russian B0W148 isolates demon-strated an increased fitness and growth in THP-1 macrophage-likecells (15) The Beijing strains from Russia (including B0W148strains) were significantly more cytotoxic than other Beijingstrains and they induced a distinct pattern of cytokine expressionfavoring immunosuppression a reduction over time of TNF-production and a high level of IL-10 secretion In contrast MDRBeijing isolates from Brazil showed reduced fitness and a lack ofelevated IL-10 production

Unlike macrophage-based studies studies using animal mod-els generated contrasting results by two Russian groups (109ndash111) Vishnevskiı et al (110) demonstrated an increased virulenceof the B0W148 isolates in noninbred albino mice Their M tu-berculosis isolates did not show a genotype versus virulenceviabil-ity correlation but there was a lower rate of decreased virulence inmultidrug-resistant isolates of the Beijing B0W148 genotype thanin the isolates of other genotypes (110) A limitation of the studywas a control group that included non-Beijing genotypes but notother Beijing family strains In contrast a more recent Russianstudy revealed a heterogeneity of the Beijing genotype isolates interms of inbred C57BL6 mouse survival and a lack of specialvirulence properties of the B0W148 isolates compared to otherBeijing isolates and isolates of other genotypes (109 111)

Indirect indications of the increased transmissibility of a strainmay include (with certain important limitations) a high level ofclustering as proof of ongoing active transmission increased cir-culation in prison versus community settings an increasing prev-alence in the total population in the survey area and involvementin an outbreak This kind of information may be gained throughepidemiological studies Below I review and discuss the availableevidence related to these issues

Clustering A clustering rate (when calculated based on high-resolution typing data) may serve to estimate transmissibility es-pecially in the case of clustered MDR isolates from newly diag-nosed patients However it is difficult to apply this directly toB0W148 versus other Beijing variants based on IS6110-RFLP typ-ing data because the B0W148 cluster is by definition a cluster ofstrains with similar profiles dominated by the B0W148 sensustricto profile In the Archangel civilian study in northern Russiathe B0 cluster included two IS6110-RFLP profiles that differed in asingle band and were shared by 5 and 15 isolates (38) Likewise theArchangel prison study showed only 3 B0 cluster profiles sharedby 19 4 and 1 isolate (39) In the Abkhazia study 23 B0W148isolates included 14 B0W148 sensu stricto isolates 2 small clus-ters of 2 isolates each and 5 singletons all of which were closelyrelated (51)

Meta-analysis is known to increase the effective sample size bypooling of data from individual studies thereby enhancing thestatistical power to assess an association A meta-estimation of theabove clustering data (as a measure of recent transmission) wasperformed through comparison of the rates of IS6110-RFLP-clus-tered isolates among all Beijing B0W148 cluster isolates (cases)versus other Beijing genotype strains (controls) (Fig 3) A funnelplot was applied for comparison of cases versus controls in an OR

analysis Beggrsquos test provided no evidence of funnel-plot asymme-try (Z 013 P 09) (Fig 3B) The studies were not heteroge-neous but differed in study design so both fixed- and random-effect models were tested As a result the overall effect was foundto be significant for both models (Fig 3A) which confirms anincreased clustering of the B0W148 cluster isolates thus suggest-ing their enhanced capacity to be transmitted While caution isrequired in interpretation of this result due to the possibility offalse clustering by IS6110-RFLP typing such an error would prob-ably equally influence both groups (B0W148 and other Beijingstrains) and hence would not probably produce a significant effecton the outcome

VNTR typing data (ie VNTR-based discrimination withinthe B0W148 cluster) might be helpful to more adequately assessthe true level of clustering although this kind of information islimited with regard to B0W148 cluster isolates A study in StPetersburg demonstrated that the B0W148 cluster is a truemonophyletic cluster by both IS6110-RFLP typing and high-res-olution 27-locus VNTR typing that included 24 MIRU and 3 hy-pervariable loci (8) This was not the case for the other majorRussian IS6110-RFLP variant of the Beijing genotype named A0which was distributed in different and distant branches of thesame VNTR dendrogram (8) A lower level of VNTR diversity forB0W148 than for A0 cluster strains should also be noted (HGI 054 versus 087) Furthermore a star-like VNTR-based network(Fig 4) was suggested to mirror a recentoutburst disseminationof these strains in the survey area ie northwestern Russia (8)Indeed a large core type is linked to almost all other small types bya single locus variation remarkably involving different loci Con-sequently from the phylogenetic point of view the B0W148 clus-ter may indeed represent a successful clone of M tuberculosis

Bacterial population growth An increasing frequency of a ge-netic variant in the total local population over time may mirror itsincreased transmissibility (if confounding factors such as humanmigration are ruled out) For example Wirth et al (112) appliedBayesian statistics to the 24-locus VNTR diversity data in order toestimate the change of effective population size of the differentlineages within M tuberculosis As a result they demonstrated thata human population explosion in the last 200 years was the pri-mary reason for growth of all lineages but in the case of the Beijinggenotype the estimated growth rate was higher than the averagefor other lineages this finding was interpreted as proof of theenhanced transmissibility of the Beijing genotype (112)

Unfortunately this kind of data is relatively abundant for theBeijing genotype as a whole but rather limited for the B0W148variant For example the proportion of Beijing genotype strainsamong MDR strains from Germany has changed markedly from192 in 1995 to 583 in 2001 however a clear role of immigra-tion from the countries of the former Soviet Union has been sug-gested (30) and thus this population growth of the Beijing geno-type in Germany was due to an influx of the human populationnot to increased transmission of this strain In Novosibirsk (Sibe-ria Russia) the data on samples from 2000 and 2009 did not showa difference in the prevalence rates of the Beijing genotype in thelocal population with values of 515 and 47 respectively (4547) However information on the Beijing B0W148 variant in theabove studies was available only for a single time point and it wasnot possible to trace its change over time

The data on St Petersburg since the early 1990s show no visibleincrease in the rate of B0W148 in the Beijing genotype popula-

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tion with rates of 267 in 1993 to 1995 243 in 1997 to 2002and 288 in 2008 to 2010 (27 33 34) or in the total M tubercu-losis population these studies were carried out in the same setting(Hospital of the Research Institute of Phthisiopulmonology) andthus are comparable In contrast the rate of B0W148 increasedover time from 2003 to 2005 in Abkhazia (51) As no significantchange in population size and structure was reported for Abkhaziafor the years 2003 to 2005 (113 114) the data of Pardini et al (51)may be considered proof of increasing circulation of the B0W148MDR strain due to its increased transmissibility On the otherhand demographic data on St Petersburg (1996 to 2010) show nosignificant change in population size (115) thus confirming a lack ofpopulation growth of the B0W148 strain over a long survey periodand hence a lack of an enhanced capacity to be transmitted

Circulation in special settings Other indirect evidence oftransmissibility may rely on the involvement of a strain in noso-comial or community outbreaks or an enhanced circulation of a

strain in prison versus civilian settings An argument that a strain mayhappen to be in the right place and right time and thus just coinci-dentally cause an outbreak is reasonable but not exactly correct underparticular conditions of the local population structure of the patho-gen The Beijing genotype comprises up to 90 of isolates in the cityof Beijing and surrounding provinces in North China (11 19) and itwould hardly be justified to investigate if a Beijing strain is moreresponsible than other strains for outbreaks including nosocomialoutbreaks in this area In contrast the Beijing genotype is found at a40 to 60 frequency across Russian regions which is far from theoverwhelming dominance Furthermore other genetic families of Mtuberculosis in Russia such as the LAM family were suggested to belinked to MDR (41) An increased circulation of Beijing strains (com-pared to strains of other M tuberculosis genotypes) in prison environ-ments has been demonstrated in most but not all settings (see refer-ence 36 and references therein)

Thus the Russian M tuberculosis population is heterogeneous

FIG 3 Meta-analysis of the association between M tuberculosis Beijing B0W148 and clustering measured as the rate of IS6110-RFLP-clustered isolates amongall isolates in a given group Error bars indicate 95 confidence intervals (95 CI) Solid squares represent each study in the meta-analysis The solid blackdiamond represents the pooled OR the gray diamond shows the fixed-effect model pooled OR (in a test for overall effect Z 403 [P 00001]) Both civilianand prison samples were considered for the study of Dubiley et al (41) and drug-resistant strains were considered for the study of Kruumluumlner et al (29) (B) Beggrsquosfunnel plot with pseudo-95 confidence limits The references in the figure correspond to references 31 41 27 51 52 38 39 and 29



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without an overwhelming predominance of a single M tuberculo-sis strain in any setting Nonetheless it was the B0W148 strainthat caused a nosocomial outbreak in the above-mentioned TBhospital in St Petersburg (116) this outbreak concerned oneindex case (TB patient) and four contacts (three TB patientsand a nurse) At the same time to the best of my knowledge noother studies supported by classical epidemiological investiga-tions on outbreaks or chains of transmission caused by B0W148 or other strains in Russian settings have been publishedTherefore it is hardly possible to make a statistically robustconclusion about increased transmissibility of the B0W148strain (compared to other Beijing family strains) based on thiskind of data only

On the other hand transmission under overcrowded andstress-causing prison conditions makes a special case of continu-ous outbreak The prisons in the countries of the former SovietUnion in the 1990s were marked by overcrowding thus facilitat-ing transmission of airborne pathogens A comparison of prisonversus civilian settings may thus help to elucidate the issue oftransmissibility of a strain

It is noteworthy that one of the first papers that drew attentionto the M tuberculosis B0W148 variant published in 1999 con-cerned prison settings in Siberia the B0W148 strain was found in5 patients coming from 5 different prisons in the Kemerovo oblast(117) A very high notification rate of TB due to a single clonenamely B0W148 was reported for a West Siberian prison as thisstrain was recovered from 190 prisoners who were newly diag-nosed TB patients in 1998 to 2000 (28) The spread of B0W148 inthe Siberian prison was suggested to be due partly to an ineffectivepublic health infrastructure the lack of a system for drug suscep-tibility testing and inadequate treatment of drug-resistant TB(118) At the same time nothing was reported about the rate of

B0W148 in the corresponding local civilian population and it isnot possible to adequately compare the two settings

On the other hand B0W148 isolates were found at similarrates in M tuberculosis populations in prison and civilian settingsin Archangel North Russia (38 39) with rates of 211 and168 (P 05) respectively This lack of a significant differenceis especially apparent in light of the significantly higher rate of theBeijing family as a whole in the local prison versus civilian popu-lations with rates of 763 versus 445 (P 0000002) Likewisein the central Russian region of Tula (41) B0W148 constitutedsimilar proportions of the local Beijing genotype populations inthe civilian and prison settings namely 237 and 163 respec-tively ie the rate was still somewhat higher in prison but stillnonsignificantly (P 04) Interestingly in the earlier Azerbaijanprison study (52) B0W148 was found at a similar (compared tothat for Tula Russia) rate (217) among Beijing isolates On theother hand looking at the rate of B0W148 prevalence in the totallocal populations it was significantly more prevalent in the Tulaprison setting (OR [95 confidence interval] 299 [124 717])When meta-estimation of the B0W148 rate in M tuberculosispopulations in prison versus civilian settings was performed onthe two available locations Tula and Archangel the pooled OR(95 confidence interval) under a random-effect model was 188(085 417) and the overall effect was nonsignificant (P 012)Thus no marked difference in transmission of B0W148 versusother Beijing strains was observed in Russian settings although aslightly higher rate of B0W148 in prison than in civilian settingsshould be noted and further accumulation of this kind of datamay eventually clarify this issue

To sum up this section there is an indication but not yet asystematic proof of the significantly enhanced virulence andincreased transmission capacity of the Beijing B0W148 strain

FIG 4 Minimum spanning tree of the VNTR types of B0W148 cluster isolates from St Petersburg and other locations in northwestern Russia based on thecombined use of 24 MIRU-VNTR and 3 hypervariable VNTR loci Circle size is roughly proportional to the number of strains VNTR digital profiles are shownin Table S2 in the supplemental material The 24-MIRU component of the central type V1 corresponds to profile 100-32 in the MIRU-VNTRplus database(Adapted from reference 8)

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Drug Resistance

The association with drug resistance appears to be the most avail-able and reliable proxy to assess the extent of danger posed by thecirculation of the B0W148 strain since the published data arerelatively abundant The first article on Russian strains isolated inthe mid-1990s already showed MDR (defined as simultaneousresistance to rifampin and isoniazid two key anti-TB drugs) to behighly prevalent among B0W148 isolates (34) B0W148 isolatesconstituted 372 of drug-resistant Beijing strains in Estoniawhile no susceptible B0W148 isolates were found in that studywhich enrolled strains isolated in 1994 (29) More recently B0W148 isolates were found to be associated with multidrug resis-tance in an epidemiologically meaningful subsample of newly di-agnosed patients in St Petersburg in northwestern Russia (P 00001) (26) and the Tula oblast in central Russia (P 001) (41)In Abkhazia 22 of 23 B0W148 isolates were MDR while otherBeijing genotype isolates included a notable number of pansus-ceptible isolates (10 of 55 isolates) (51) In prison settings this

association was even more remarkable all 11 B0W148 isolates inTula and 8 of 10 B0W148 isolates in Azerbaijan were MDR incontrast to the more diverse samples of other Beijing genotypeisolates which also included susceptible and monoresistant iso-lates (42 52)

A meta-analysis was performed to systematically summarize theavailable studies and to provide a meta-estimate of the associationbetween the Beijing B0W148 variant and multidrug resistance(Fig 5) A control group included other Beijing family strainssince multiple previous studies demonstrated that Beijing familystrains in the countries of the former Soviet Union includingRussia are associated with drug resistance (29 36 43) Beggrsquos testprovided no evidence of funnel-plot asymmetry (Z 136 P 017) (Fig 5B) ie there was a lack of publication bias The eightstudies were not significantly heterogeneous but differed in studydesign which is why both random- and fixed-effect models weretested Both showed similar outcomes and the overall effect ie astronger association of the B0W148 strain and multidrug resis-

FIG 5 (A) Meta-analysis of the association between M tuberculosis Beijing B0W148 variant and multidrug resistance Error bars indicate 95 confidenceintervals (95 CI) Solid squares represent each study in the meta-analysis The solid black diamond represents the pooled OR the gray diamond shows thefixed-effect model pooled OR (in a test for overall effect Z 564 [P 00001]) (B) Beggrsquos funnel plot with pseudo-95 confidence limits The references inthis figure correspond to references 41 42 29 36 51 34 52 and 27



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tance was significant in both models (Fig 5A) Meta-analysis wasadditionally done for case-control comparisons in which B0W148 (case group) was compared to either all other M tubercu-losis strains or non-Beijing genotypes Under the random-effectmodel the overall effect ie increased odds of being MDR forB0W148 was found to be significant for both kinds of compari-sons (P 00005) and the pooled ORs (95 confidence intervals)were 658 (256 1692) and 997 (276 3605) respectively

HUMAN POPULATION CONTEXT

Geographic Diversity and Place of Origin of B0W148

A precise determination of the place of origin of the B0W148strain remains elusive The location with the highest prevalencerate and highest level of diversity may provide a helpful indication

The geographical distribution of the Beijing genotype and Bei-jing B0W148 strains in different populations is shown in Fig 2The prevalence of the Beijing B0W148 variant varies across Rus-sian regions but in most cases it is above 10 being in particular16 in northern European Russia (Archangel) and 19 to 22 inSiberia Slight decreases are observed in the westward peripheralregions of European Russia (79 in Pskov) in Russiarsquos neighbors(75 in Estonia to the northwest and 7 in AbkhaziaGeorgiato the southwest) and in Moscow Furthermore the rate of B0W148 prevalence sharply decreases in former Soviet Central Asiabeing only 36 in Kazakhstan and 24 in the more distantcountries Uzbekistan and Turkmenistan

As described in Sampling and Methods the B0W148 clusterprofiles were searched in publications on other areas where theBeijing genotype is endemic such as East Asia and South Africa aswell as in Russianex-Soviet neighbors in Eastern Europe andCentral Asia The B0W148 variant was not found in East Asiancountries marked by endemicity and a high prevalence of the Bei-jing genotype or in a country with its own particular subset ofBeijing strains (South Africa) (see Table S1 in the supplementalmaterial) No or rare isolates of the Beijing family were recoveredfrom permanent residents of Eastern European countries such asPoland Bulgaria and Romania (75 76 119 120) and it is reason-able to assume a negligible if any presence of the Beijing B0W148 variant in this part of Europe None of the above countriesreceived a significant influx of immigrants from the former SovietUnion and this situation is not unexpected The Russian (ex-Soviet) emigration has been directed toward the developed worldin regions of western Europe and North America (121) Indeedthe B0W148 strain has been isolated from Russian immigrants inthe United States (28) and while reports from Sweden and Ger-many described Beijing genotype strains recovered from immi-grants of different origins the B0W148 strain was isolated onlyfrom TB patients coming from the former Soviet Union (30 80)In Germany the B0W148 cluster included mainly patients fromcountries of the former Soviet Union first of all Russia In con-trast other Beijing genotype isolates represented more countriesof origin without a bias toward a single country (30) (see TableS1) In total the B0W148 strain was isolated more frequentlyfrom Russian immigrants than from other immigrants thoughnot significantly apparently due to the small sample size (P 009) In further contrasting immigrants coming from Russia andthe European part of the former Soviet Union with those fromAsian countries of the former Soviet Union B0W148 was preva-lent among the former (529 versus 167 P 02) Likewise a

Swedish study of drug-resistant TB (80) described Beijing geno-type strains from immigrants of different origins but the BeijingB0W148 strain was isolated only from immigrants from the for-mer Soviet Union especially from those coming from Russia andthe European part of the former Soviet Union (see Table S1)

In summary B0W148 is specifically prevalent in the Europeanpart of the former Soviet Union and at the same time it is found athigh rates in Russian Siberian settings (Novosibirsk and Tuva)making up 19 to 22 of local M tuberculosis populations and 33to 40 of the Beijing subpopulations This highlights an intrigu-ing gradient of prevalence of the Beijing B0W148 variant withthe highest rate in the Siberian part of Russia and somewhat lowerrates in the European part of the former Soviet Union from Esto-nia in the north to Azerbaijan in the south and including Euro-pean Russia Significantly or nearly significantly higher rates ofB0W148 in total populations in comparably designed studies inSiberian versus European Russian settings should be noted (seeTable S1 in the supplemental material) ie for Tuva versus Pskov(218 versus 78 P 006) and Novosibirsk versus Pskov(194 versus 78 P 005) At the same time B0W148 wasalso more prevalent in Archangel in northern European Russiathan in Pskov with rates of 168 versus 78 a nearly significantdifference (P 008)

Furthermore to estimate the diversity in B0W148 strainsacross Russia I used the published VNTR data on St Petersburgin northwestern European Russia (8) and Novosibirsk West Si-beria (44 45) to build phylogenies of the Beijing genotype and theBeijing B0W148 variant These two settings were selected basedon the available information on high-resolution VNTR loci typedfor these strains No other studies from other locations that metthese criteria were available As a whole both trees show a cleardivergence of the isolates from these distant Russian locations(Fig 6) but the nuances in topology are distinctive and intriguingIn the Beijing dendrogram the subsample from Novosibirskmakes a compact subtree compared to the more diverse samplefrom St Petersburg (Fig 6A) the simplest explanation is thelarger sample size of the latter collection However and in contrastto the all-Beijing tree (Fig 6A) the Beijing B0W148 minimumspanning tree shows not only a divergence of the two settings butalso strikingly different topologies of their subtrees (Fig 6B) Oneshould note that evolution by loss rather than gain of repeats hasbeen suggested as a major mode of evolution of the VNTR loci inM tuberculosis based on mathematical modeling (122) In thissense the MST shown in Fig 6B appears to corroborate this hy-pothesis and is therefore valid note the allelic changes on theedges of the tree which in most cases are reductions of the numberof repeats By VNTR typing 45 B0W148 isolates from St Peters-burg were subdivided into 9 types with a single predominant type(star-like phylogeny) thus suggesting a recentoutburst dissemi-nation In contrast 14 B0W148 isolates from Novosibirsk weresubdivided into 6 types without a single predominant profile andpresenting a more chain-like phylogeny thus suggesting a longerperiod of evolution (Fig 6B) The Hunter-Gaston indexes (esti-mations of the VNTR-based diversity) of the St Petersburg andNovosibirsk B0W148 samples were 043 and 079 respectivelyTaken together these findings ie (i) the highest rates of B0W148 strain prevalence in Novosibirsk and Tuva (ii) the highestlevel of diversity of B0W148 in Novosibirsk and (iii) the longerevolution of B0W148 in Novosibirsk suggest the (West) Siberian

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FIG 6 Phylogenetic analysis of 10-VNTR-locus profiles of Beijing strains from Novosibirsk West Siberia and St Petersburg northwestern European Russia (A)Neighbor-joining tree for Beijing genotype strains (B) Minimum spanning tree for Beijing B0W148 strains Type numbers are for convenience only Digital VNTRprofiles show copy numbers for the following loci ETR-A ETR-C MIRU31 MIRU23 QUB26 QUB11b Mtub29 Mtub39 MIRU40 and Mtub21 Each arm depictsone locus change


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area of Russia to be a hypothetical area of origin of the B0W148variant

Timing of Origin and Primary Dispersal of B0W148

Clearly B0W148 did not originate in the two most recent de-cades in such a case it would not be found in Tuva which wasmarked with not an influx but a sharp outflow of ethnic Russiansexactly since the 1990s (123) Furthermore in the earlymid-1990s B0W148 isolates had already been described from acrossthe former Soviet Union in locations as distant as northwesternRussia Estonia and Azerbaijan Sinkov et al (124 125) recentlyproposed an intriguing hypothesis about Gulag-driven importa-tion of the Beijing genotype into and its primary dispersal acrossthe former Soviet Union beginning in the 1930s Although its firstpenetration into Russia is open to question (see the work of Mok-rousov et al [17] for an earlier timing of the first penetration of theBeijing genotype into northern Eurasia) the Beijing genotype wasindeed most likely disseminated throughout the country due to amassive population mixing in the Soviet time of industrializationurbanization and concentration camps of the Gulag penitentiarysystem However could this also be the case for the B0W148variant The Archangel area in North Russia and the studied Si-berian settings (Novosibirsk Tuva and the Mariinsk prison in theKemerovo oblast [see above]) represent the past (Gulag) and cur-rent penitentiary systems seemingly reemphasizing an acceler-ated transmission of the Beijing B0W148 strain under prisonconcentration camp conditions However the Gulag systemcovered almost all of Russia not only the northern and Siberianareas but also Kazakhstan and central Asia (126) The Gulag sys-tem began shrinking in 1954 and was closed in 1960 In this viewthe Gulag system could not be the source of dissemination of theB0W148 strain throughout the country Furthermore the B0W148 variant could not have started its emergence before 1960 Insuch a case it would have spread across all of the former SovietUnion to both its European and Asian parts similar to the spreadof the Beijing genotype as a whole A speculative explanation isthat B0W148 originated in Siberia before 1960 but not inside theGulag system and remained confined to its unknown nichehostpopulation until some social event triggered its emanation beyondSiberia

A closer look at the data on Soviet demographics reveals twolandmark events that involved mass human movement in the for-mer Soviet Union in the 1950s to 1980s (i) a virgin land campaign(tselina) from 1955 to 1960 and (ii) population outflow from Si-beria especially West Siberia in the 1960s to 1980s (127ndash129)(Fig 5)

A mass migration from European Russia (but not Siberia) toKazakhstan from 1955 to 1960 within the frame of the virgin landcampaign dramatically decreased the proportion of Kazakhswithin the population from 57 in 1926 to 30 in 1959 Further-more after the end of the campaign 6 million ethnic Russians andUkrainians stayed in Kazakhstan (130 httpenwikipediaorgwikiVirgin_Lands_Campaign) and hence their interaction withthe local population continued in the coming decades If B0W148had been circulating in European RussiaUkraine before 1960 itwould have been brought to Kazakhstan in 1955 to 1960 andwould have spread there Apparently this was not the casesince its rate in Kazakhstan is only 36 It should be kept inmind that the mass virgin land campaign of 1954 to 1960 tar-geted not only Kazakhstan but also less inhabited and less de-

veloped areas in Ural and West Siberia (131) Consequently asit was not brought to Kazakhstan from European Russia duringthe virgin land campaign B0W148 could not have beenbrought to Siberia from European Russia either To sum upB0W148 isolates did not circulate across European Russia be-fore 1960

A remarkable population outflow from Siberia and the Far Eastto the central and northwestern regions of Russia was another andopposite (with respect to the virgin land campaign) process thatstarted in the 1960s Eight hundred thousand persons migratedfrom West Siberia to European Russia from 1961 to 1970 (127ndash129 132) I speculate that this may have been the first dispersal ofthe B0W148 variant beyond its hypothetical area of origin WestSiberia (Fig 7)

The pair of St Petersburg and Novosibirsk represents both geo-graphically and phylogenetically (in terms of B0W148 frequencyand diversity) distant regions Both are also areas with high levelsof population density and urban development In contrast themuch less populated and geographically marginal sites of Archan-gel North Russia and Tuva West Siberia make another and pe-culiar pair that deserves no less attention since it permits a view ofthe manifestation of the Beijing B0W148 strain in another socio-ecological context

The Beijing B0W148 cluster is found at a high rate in TuvaHistorically Tuva (formerly known as Tannu Uriankhai) was in-fluenced by Mongolians and to some extent the Chinese but inboth cases this interaction was limited to political relations withlittle human exchange and hence little probability of pathogenexchange In contrast Russian colonists settled there only begin-ning at the end of the 19th century and furthermore settled in thesparsely populated northern and central parts hence interpopu-lation transmission of infectious agents was less probable at leastin the first decades (133) Accordingly B0W148 even if it existedat that time would not have been spread by Russians in Tuva

FIG 7 Direction and timing of major flows of human migration associatedwith origin and dispersal of the Beijing B0W148 strain The thickness of ar-rows roughly reflects the volume of migration Red arrows indicate the hy-pothesized dissemination of the B0W148 strain

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Tannu Uriankhai was definitely included in the Soviet Union in1944 The Russian population there increased from 20 in 1918to 1940 to 36 to 40 in 1960 to 1980 (123 133) However thepopulation of ethnic Russians in Tuva has been reduced sharplyto 16 in the last 2 decades hence the B0W148 strain shouldhave penetrated to Tuva prior to the 1990s Indeed the Russianpopulation increased in Tuva in the 1960s to 1980s up to 40and this may have correlated with the first spread of the B0W148strain in this region

Two thousand miles away from Tuva the Archangel province islocated in the Russian North a specific area of the White Sea andBarents Sea region that is still in the limits of European Russia butmarked by a severe climate and a low population density By allmeans Archangel cannot be placed within Siberia But unlike thecase in other parts of European Russia (and the former SovietUnion) the rate of the B0W148 strain is high in the civilian pop-ulation of Archangel and nearly significantly higher than inPskov the westernmost region of mainland (European) Russialocated at the Russian-Latvian border (Fig 2) To explain thissituation the following sometimes opposing and complex factsmay be considered First a moderate but permanent populationoutflow from North Russia has taken place since 1990 (134)hence B0W148 must have been brought to this region before1990 Second the population in Archangel almost doubled over aspan of 30 years from 256000 in 1959 to 415000 in 1989 (httpfrwikipediaorgwikiArkhangelsk) which could have been dueat least partly to incoming migration from other Russian regions(129) Third a rotation work system has been practiced in NorthRussia and temporary workers could in principle come fromeither Siberia or European Russia Migration from Siberia wouldseem to be a less significant factor as people in this area tend tochangesoften their climate conditions rather than move to a sim-ilar hard climate Hence rotation migrants more likely moved toArchangel from European Russia and furthermore the newcom-ers in the harsh climate and hard living conditions of the Northmay have experienced stress leading to increased morbidity (135)Fourth the low genetic diversity of the B0W148 cluster in Arch-angel (38) reflecting its recent evolution in situ thus readily cor-roborates the hypothesis about a mainly secondary Europeansource of these strains in the Russian North To summarize theB0W148 strain was likely brought to Archangel not from its pri-mary focus Siberia but rather from European Russia followingthe secondary wave of its dispersal across the country (Fig 7)Reactivated through climate conditions the B0W148 clone hasultimately reached a high rate there higher than in its possiblesource area central Russia

To sum up based on the historical demographic and ecologi-cal knowledge and molecular data on the diversity and frequencyof the Beijing B0W148 strain across the former Soviet Unionmost parsimoniously this M tuberculosis variant likely originatedin Siberia and before 1960 Primary dispersal of B0W148 outsideSiberia started after 1960 driven by the population outflow fromSiberia to European Russiaformer Soviet Union but not to for-mer Soviet Central Asia This hypothetical scenario is summarizedin Fig 7

Driving Forces behind Emergence and Spread of B0W148

The above reasoning links particular human migration flows tothe dissemination of B0W148 in Russia since the 1960s The nextquestion is as follows why has its spread become so dramatically

successful (as exemplified by the star-like phylogeny in Fig 4)What event along with human migration triggered the emer-gence in and wide propagation of this strain from Siberia acrossthe country and within new areas in European Russia I hypoth-esize that the advent of modern drugs for anti-TB treatment wassuch a catalyzing circumstance As a brief reminder the relativelyrecent milestones within the course of the history of anti-TB che-motherapy are in particular the introduction of ethambutol inthe 1960s and the recognition of the crucial role of isoniazid andrifampin in the 1970s and of pyrazinamide in the 1980s (136) Allof these events correspond to the above-hypothesized timeline ofthe emergence and dispersal of B0W148 beyond Siberia Whileproof of the enhanced virulence of the B0W148 strain is unsys-tematic and controversial it is remarkable that the above meta-analysis demonstrated a strong association of B0W148 with mul-tidrug resistance In this sense the primary spread of the B0W148strain outside Siberia since the 1960s was not just coincidence butmost likely was catalyzed and fueled by a finalization of the mod-ern anti-TB regimen and wide use of the key first-line anti-TBdrugs

In contrast a link between spread of the B0W148 strain and itscapacity to evade the protective effect of BCG vaccination is muchless apparent although not completely impossible Regarding theBeijing family as a whole it has previously been hypothesized thatthese strains have acquired mechanisms to circumvent BCG-in-duced immunity nonetheless the association of the emergence ofthe Beijing genotype and BCG vaccination remains inconclusive(14) It should be noted that BCG vaccination was widely imple-mented in the Soviet Union in both rural and urban areas begin-ning in the 1930s On the other hand BCG vaccination of new-borns became mandatory in 1952 (137) thus coinciding with thehypothesized emergence of the B0W148 variant in Siberia and itsprimary dispersal to European Russia since the 1960s

CONCLUSIONS AND PERSPECTIVES

To sum up in this review placing clinical epidemiological andmolecular data in a broad historical demographic and ecologicalcontext I put forward two interdependent hypotheses First theBeijing B0W148 strain likely originated in Siberia and its pri-mary dispersal was driven by a massive population outflow fromSiberia to European Russia in the 1960s to 1980s Second a his-torically recent emergence and dissemination of B0W148 weretriggered by the advent and wide use of modern anti-TB drugs andwere due to the remarkable capacity of this variant to acquire drugresistance In contrast there is some indication but not yet a sub-stantial and systematic proof of enhanced virulence of this strainNonetheless the star-like topology of the high-resolution VNTRnetwork does suggest a historically recent andor outburst dissem-ination of this clonal cluster in its new secondary focus north-western Russia thus supporting the designation of B0W148 as asuccessful clone Furthermore the historical spread of the BeijingB0W148 variant across Russia in the last 50 years is currentlyevolving toward a global propagation of this strain by Russianemigrants and its circulation in respective immigrant communi-ties in Western Europe and the United States This situationshould be taken into consideration by national TB control pro-grams especially in view of the remarkable propensity of B0W148 to develop multidrug resistance In this regard a recentlydescribed simple PCR-based method to rapidly detect this strain



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(33) may become a useful complement to the currently used mo-lecular methods of M tuberculosis diagnostics and typing

As demonstrated in this review in spite of the common per-ception about the omnipresence of B0W148 across all post-So-viet space a closer look at its geographic distribution reveals apeculiar clinal gradient Its rate peaks in Siberian Russia and to alesser extent in the European part of the former Soviet Union Incontrast the rate of B0W148 is sharply decreased in the Asianpart of the former Soviet Union One explanation is that an ordi-nary human exchange per se is not enough to introduce a newtuberculosis strain into an indigenous population even in thepresent time of urbanization and overcrowding There may be akind of human resistance developed in the local populationthrough its coexistence with historically established clones coun-teracting an invasion of new ldquoalienrdquo strains For example theBeijing family as a whole is present at high rates in Latvia andEstonia most likely as a result of the large influx (demic diffusion)of Russian migrants from 1944 to 1991 In contrast the Beijinggenotype is not found in autochthonous populations in EasternEurope while the Eastern European countries were part of theWarsaw Treaty they did remain independent also keeping a sin-gularity of their microbial pools Similar to the RussianEuropeanhistory of the Beijing family dissemination of the Beijing B0W148 strain apparently required a massive human movementsuch as the population outflow from Siberia to the European partof the former Soviet Union In the absence of such a significanthuman flow from Siberia to former Soviet Central Asia the B0W148 strain is still found at negligible rates in Kazakhstan Kyr-gyzstan Uzbekistan and Turkmenistan In this regard a study ofhuman candidate genes with relationships to host susceptibility totuberculosis caused by Beijing B0W148 compared to other Mtuberculosis strains in different ethnic groups is warranted A num-ber of previous studies demonstrated a coadaptive interaction ofcertain human alleles and endemic M tuberculosis genotypesmanifested as different clinical outcomes in different hosts in-fected with the same strain (138ndash142) For example a lethal effectof the Beijing genotype that is exerted more frequently on CD209336G male carriers in Slavic and East Asian populations hasbeen hypothesized (142)

The especially high rate of multidrug resistance among activelycirculating Beijing B0W148 isolates suggests that certain com-pensatory genome mutations may have been retained throughsecond-order selection mechanisms that permit a rapid acquisi-tion of drug resistance without reducing the pathogenic capacityof a strain To study the biological fitness of the B0W148 strain afar-from-exhaustive list of target genes includes 3R genes (recom-bination replication and repair genes) (143) secondary mem-bers of the drug resistance-associated groups of genes (such asrpoA and rpoC [144] and the embCAB operon) and the virulence-related eis mce and esx gene families Other studies of relativelysimple design may be envisaged to elucidate the fitness-relatedfeatures of the B0W148 strain eg studies of the biological costof drug resistance mutations through evaluations of the growthrate and mixed-infection model studies to see if this strain canoutcompete other strains

Last but not least efforts to fight TB also include the develop-ment of new vaccines and novel anti-TB drugs Both should un-doubtedly show their efficacy against the predominant strains oftubercle bacilli in order to be adopted for therapeutic use in thenext decade In this sense Beijing B0W148 is an important can-

didate for inclusion in the panel of successful M tuberculosisstrains that may be ideal candidates for future studies on vaccinedevelopment and for assessing the therapeutic efficacy of newdrugs

Future studies on more isolates from new locations and mostimportantly new time points (ie focused on archival specimens)will increase the density of data and the resolution of analysis testthe proposed hypothesis about the place and time of origin of theM tuberculosis Beijing B0W148 strain and shed more light on thepathogenomics-related features lying behind its emergence andspread in the human population

ACKNOWLEDGMENTS

I am grateful to Francisco Ayala for a critical reading of an early version ofthe manuscript and to the seven anonymous reviewers for valuable com-ments

REFERENCES1 Hermans PW van Soolingen D Bik EM de Haas PE Dale JW van

Embden JD 1991 Insertion element IS987 from Mycobacterium bovisBCG is located in a hot-spot integration region for insertion elements inMycobacterium tuberculosis complex strains Infect Immun 592695ndash2705

2 van Embden JD van Gorkom T Kremer K Jansen R van Der ZeijstBA Schouls LM 2000 Genetic variation and evolutionary origin of thedirect repeat locus of Mycobacterium tuberculosis complex bacteria JBacteriol 1822393ndash2401

3 van Soolingen D 2001 Molecular epidemiology of tuberculosis andother mycobacterial infections main methodologies and achievementsJ Intern Med 2491ndash26

4 Supply P Lesjean S Savine E Kremer K van Soolingen D Locht C2001 Automated high-throughput genotyping for study of global epide-miology of Mycobacterium tuberculosis based on mycobacterial inter-spersed repetitive units J Clin Microbiol 393563ndash3571

5 Frothingham R Meeker-OrsquoConnell WA 1998 Genetic diversity in theMycobacterium tuberculosis complex based on variable numbers of tan-dem DNA repeats Microbiology 1441189 ndash1196

6 Supply P Allix C Lesjean S Cardoso-Oelemann M Rusch-Gerdes SWillery E Savine E de Haas P van Deutekom H Roring S Bifani PKurepina N Kreiswirth B Sola C Rastogi N Vatin V Gutierrez MCFauville M Niemann S Skuce R Kremer K Locht C van SoolingenD 2006 Proposal for standardization of optimized mycobacterial inter-spersed repetitive unitndashvariable-number tandem repeat typing of Myco-bacterium tuberculosis J Clin Microbiol 444498 ndash 4510

7 Iwamoto T Yoshida S Suzuki K Tomita M Fujiyama R Tanaka NKawakami Y Ito M 2007 Hypervariable loci that enhance the discrim-inatory ability of newly proposed 15-loci and 24-loci variable-numbertandem repeat typing method on Mycobacterium tuberculosis strains pre-dominated by the Beijing family FEMS Microbiol Lett 272282ndash283

8 Mokrousov I Narvskaya O Vyazovaya A Millet J Otten T Vish-nevsky B Rastogi N 2008 Mycobacterium tuberculosis Beijing genotypein Russia in search of informative VNTR loci J Clin Microbiol 463576 ndash3584

9 Allix-Beacuteguec C Wahl C Hanekom M Maeda S Martin C MokrousovI Niemann S Nikolayevskyy V Rastogi N Samper S Sng L-H WangS-X Warren R Supply P 2011 Typing Mycobacterium tuberculosisBeijing isolates with hypervariable loci a multicentric consensus studyabstr OP58 Abstr 32nd ESM Congr Luumlbeck Germany 26 to 29 June2011

10 Roetzer A Diel R Kohl TA Ruumlckert C Nuumlbel U Blom J Wirth TJaenicke S Schuback S Ruumlsch-Gerdes S Supply P Kalinowski JNiemann S Whole genome sequencing versus traditional genotypingfor investigation of a Mycobacterium tuberculosis outbreak a longitudinalmolecular epidemiological study PLoS Med 10e1001387 doi101371journalpmed1001387

11 van Soolingen D Qian L de Haas PEW Douglas JT Traore HPortaels F Quing Z Enkhasaikan D Nymadawa P van Embden JDA1995 Predominance of a single genotype of Mycobacterium tuberculosisin countries of East Asia J Clin Microbiol 333234 ndash3238

Mokrousov


on Septem


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12 European Concerted Action on New Generation Genetic Markers andTechniques for the Epidemiology and Control of Tuberculosis 2006BeijingW genotype Mycobacterium tuberculosis and drug resistanceEmerg Infect Dis 12736 ndash743

13 Mokrousov I 2012 Human migratory history through the looking-glass of genetic geography of Mycobacterium tuberculosis p 317ndash341 InCrawford MH Campbell B (ed) Causes and consequences of humanmigration Cambridge University Press Cambridge United Kingdom

14 Hanekom M Gey van Pittius NC McEvoy C Victor TC Van HeldenPD Warren RM 2011 Mycobacterium tuberculosis Beijing genotype atemplate for success Tuberculosis (Edinb) 91510 ndash523

15 Lasunskaia E Ribeiro SC Manicheva O Gomes LL Suffys PN Mok-rousov I Ferrazoli L Andrade MR Kritski A Otten T Kipnis TL daSilva WD Vishnevsky B Oliveira MM Gomes HM Baptista IFNarvskaya O 2010 Emerging multi-drug resistant Mycobacterium tu-berculosis strains of the Beijing genotype circulating in Russia express apattern of biological properties associated with enhanced virulence Mi-crobes Infect 12467ndash 475

16 Mokrousov I Narvskaya O Otten T Vyazovaya A Limeschenko ESteklova L Vyshnevskyi B 2002 Phylogenetic reconstruction withinMycobacterium tuberculosis Beijing genotype in northwestern RussiaRes Microbiol 153629 ndash 637

17 Mokrousov I Ly HM Otten T Lan NN Vyshnevskyi B Hoffner SNarvskaya O 2005 Origin and primary dispersal of the Mycobacteriumtuberculosis Beijing genotype clues from human phylogeography Ge-nome Res 151357ndash1364

18 Ebrahimi-Rad M Bifani P Martin C Kremer K Samper S Rauzier JKreiswirth B Blazquez J Jouan M van Soolingen D Gicquel B 2003Mutations in putative mutator genes of Mycobacterium tuberculosisstrains of the W-Beijing family Emerg Infect Dis 9838 ndash 845

19 Mokrousov I Jiao WW Sun GZ Liu JW Valcheva V Li M NarvskayaO Shen AD 2006 Evolution of drug resistance in different sub-lineageswithin Mycobacterium tuberculosis Beijing genotype Antimicrob AgentsChemother 502820 ndash2823

20 Yang C Luo T Sun G Qiao K Sun G DeRiemer K Mei J Gao Q2012 Mycobacterium tuberculosis Beijing strains favor transmission butnot drug resistance in China Clin Infect Dis 551179 ndash1187

21 Iwamoto T Fujiyama R Yoshida S Wada T Shirai C Kawakami Y2009 Population structure dynamics of Mycobacterium tuberculosis Bei-jing strains during past decades in Japan J Clin Microbiol 473340 ndash3343

22 Bifani J Mathema B Kurepina NE Kreiswirth BN 2002 Globaldissemination of the Mycobacterium tuberculosis W-Beijing familystrains Trends Microbiol 1045ndash52

23 Caminero JA Pena MJ Campos-Herrero MI Rodriguez JC Garcia ICabrera P Lafoz C Samper S Takiff H Afonso O Pavon JM TorresMJ van Soolingen D Enarson DA Martin C 2001 Epidemiologicalevidence of the spread of a Mycobacterium tuberculosis strain of the Bei-jing genotype on Gran Canaria Island Am J Respir Crit Care Med1641165ndash1170

24 Millaacuten-Lou MI Alonso H Gaviacuten P Hernaacutendez-Febles M Campos-Herrero MI Copado R Cantildeas F Kremer K Caminero JA Martiacuten CSamper S 2012 Rapid test for identification of a highly transmissibleMycobacterium tuberculosis Beijing strain of sub-Saharan origin J ClinMicrobiol 50516 ndash518

25 Narvskaya O Mokrousov I Otten TF Vyshnevskiy BI 1999 Geneticmarking of polyresistant Mycobacterium tuberculosis strains isolated inthe North-West of Russia Probl Tuberk 1999(3)39 ndash 41 (In Russian)

26 Narvskaya O 2003 Genome polymorphism of Mycobacterium tubercu-losis and its role in epidemic process DSc dissertation Institute of Ex-perimental Medicine St Petersburg Russia (In Russian)

27 Narvskaya O Mokrousov I Otten T Vishnevsky B 2005 Molecularmarkers application for studies of Mycobacterium tuberculosis popula-tion in Russia p 111ndash125 In Read MM (ed) Trends in DNA fingerprint-ing research Nova Science Publishers New York NY


29 Kruumluumlner A Hoffner SE Sillastu H Danilovits M Levina K SvensonSB Ghebremichael S Koivula T Kaumlllenius G 2001 Spread of drug-resistant pulmonary tuberculosis in Estonia J Clin Microbiol 393339 ndash3345

30 Kubica T Ruumlsch-Gerdes S Niemann S 2004 The Beijing genotype is

emerging among multidrug-resistant Mycobacterium tuberculosis strainsfrom Germany Int J Tuberc Lung Dis 81107ndash1113

31 Cox HS Kubica T Doshetov D Kebede Y Ruumlsch-Gerdess S NiemannS 2005 The Beijing genotype and drug resistant tuberculosis in the AralSea region of Central Asia Respir Res 6134

32 Kremer K Glynn JR Lillebaek T Niemann S Kurepina NE Kre-iswirth BN Bifani PJ van Soolingen D 2004 Definition of the Bei-jingW lineage of Mycobacterium tuberculosis on the basis of geneticmarkers J Clin Microbiol 424040 ndash 4049

33 Mokrousov I Narvskaya O Vyazovaya A Otten T Jiao WW GomesLL Suffys PN Shen AD Vishnevsky B 2012 Russian ldquosuccessfulrdquoclone B0W148 of Mycobacterium tuberculosis Beijing genotype multi-plex PCR assay for rapid detection and global screening J Clin Micro-biol 503757ndash3759

34 Marttila HJ Soini H Eerola E Vyshnevskaya E Vyshnevskiy BIOtten TF Vasilyef AV Viljanen MK 1998 A Ser315Thr substitution inKatG is predominant in genetically heterogeneous multidrug-resistantMycobacterium tuberculosis isolates originating from the St Petersburgarea in Russia Antimicrob Agents Chemother 422443ndash2445

35 Vyazovaya A Solovieva N Mokrousov I Starkova D Otten T Vish-nevsky BV Narvskaya O 2012 Molecular characterisation of Mycobac-terium tuberculosis strains from patients with tuberculous spondylitisInt J Tuberc Lung Dis 16S75

36 Mokrousov I Vyazovaya A Otten T Zhuravlev V Pavlova E Tarash-kevich L Krishevich V Vishnevsky B Narvskaya O 2012 Mycobac-terium tuberculosis population in northwestern Russia an update fromRussian-EULatvian border region PLoS One 7e41318 doi101371journalpone0041318

37 Markelov Y Narvskaya O 2010 Circulation of multidrug-resistanttuberculosis pathogen strains in the Republic of Karelia Probl TuberkBolezn Legk 2010(2)54 ndash56 (In Russian)

38 Toungoussova OS Sandven P Mariandyshev AO Nizovtseva NIBjune G Caugant DA 2002 Spread of drug-resistant Mycobacteriumtuberculosis strains of the Beijing genotype in the Archangel oblast Rus-sia J Clin Microbiol 401930 ndash1937

39 Toungoussova OS Mariandyshev A Bjune G Sandven P CaugantDA 2003 Molecular epidemiology and drug resistance of Mycobacte-rium tuberculosis isolates in the Archangel prison in Russia predomi-nance of the W-Beijing clone family Clin Infect Dis 37665ndash 672

40 Maumlkinen J Marjamaumlki M Haanperauml-Heikkinen M Marttila H En-dourova LB Presnova SE Mathys V Bifani P Ruohonen R ViljanenMK Soini H 2011 Extremely high prevalence of multidrug resistanttuberculosis in Murmansk Russia a population-based study Eur JClin Microbiol Infect Dis 301119 ndash1126

41 Dubiley S Ignatova A Mukhina T Nizova A Blagodatskikh S Ste-panshina V Shemyakin I 2010 Molecular epidemiology of tuberculo-sis in Tula area central Russia before DOTS Clin Microbiol Infect161421ndash1426

42 Ignatova A Dubiley S Stepanshina V Shemyakin I 2006 Predomi-nance of multi-drug-resistant LAM and Beijing family strains amongMycobacterium tuberculosis isolates recovered from prison inmates inTula region Russia J Med Microbiol 551413ndash1418

43 Drobniewski F Balabanova Y Ruddy M Weldon L Jeltkova KBrown T Malomanova N Elizarova E Melentyey A Mutovkin EZhakharova S Fedorin I 2002 Rifampin- and multidrug-resistant tu-berculosis in Russian civilians and prison inmates dominance of theBeijing strain family Emerg Infect Dis 81320 ndash1326

44 Surikova OV Voitech DS Kuzmicheva G Tatkov SI Mokrousov IVNarvskaya OV Rot MA van Soolingen D Filipenko ML 2005 Effi-cient differentiation of Mycobacterium tuberculosis strains of the W-Beijing family from Russia using highly polymorphic VNTR loci Eur JEpidemiol 20963ndash974

45 Surikova OV Voıtykh DV Kuzrsquomicheva GA Tatrsquokov SI MokrousovIV Narvskaia OV Filippenko ML 2005 Differentiation of the Myco-bacterium tuberculosis W-Beijing family strains from the Russian Feder-ation by the VNTR-typing Mol Gen Mikrobiol Virusol 2005(3)22ndash29 (In Russian)

46 Norkina OV Kinsht VN Mokrousov IV Kurunov Iu N Krasnov VAFilipenko ML 2003 The genetic diversity of Mycobacterium tuberculosisand an assessment of risk factors of tuberculosis spread in Russiarsquos Sibe-rian region by molecular epidemiological methods Mol Gen Mikro-biol Virusol 2003(3)9 ndash18 (In Russian)

47 Dymova MA Kinsht VN Cherednichenko AG Khrapov EA Svistel-



on Septem


rasmorg

Dow

nloaded from

nik AV Filipenko ML 2011 Highest prevalence of the Mycobacteriumtuberculosis Beijing genotype isolates in patients newly diagnosed withtuberculosis in the Novosibirsk oblast Russian Federation J Med Mi-crobiol 601003ndash1009

48 Matrakshin AG Mesrsquoko EM Beliakova NK Andreevskaia SN Smir-nova TG Larionova EE Kuzrsquomin AV Mokrousov IV Pospelov LEChernousova LN 2004 Genotypic characteristics of Mycobacterium tu-berculosis strains from the Republic of Tyva Probl Tuberk Bolezn Legk2004(3)37ndash 40 (In Russian)

49 Vasilenko N Vyazovaya A Mokrousov I Limeschenko E Semenov VNarvskaya O 2006 Spacer oligonucleotide typing of drug-resistant My-cobacterium tuberculosis circulating on the territory of Belarus Immu-nopat Allergol Infektol 2006(4)70 ndash74 (In Russian)

50 Dymova MA Liashenko OO Poteiko PI Krutko VS Khrapov EAFilipenko ML 2011 Genetic variation of Mycobacterium tuberculosiscirculating in Kharkiv oblast Ukraine BMC Infect Dis 1177 doi1011861471-2334-11-77

51 Pardini M Niemann S Varaine F Iona E Meacci F Orrugrave G YesilkayaH Jarosz T Andrew P Barer M Checchi F Rinder H Orefici GRuumlsch-Gerdes S Fattorini L Oggioni MR Bonnet M 2009 Charac-teristics of drug-resistant tuberculosis in Abkhazia (Georgia) a high-prevalence area in Eastern Europe Tuberculosis (Edinb) 89317ndash324

52 Pfyffer GE Straumlssle A van Gorkum T Portaels F Rigouts L Mathieu CMirzoyev F Traore H van Embden JD 2001 Multidrug-resistant tuber-culosis in prison inmates Azerbaijan Emerg Infect Dis 7855ndash861

53 Kubica T Agzamova R Wright A Aziz MA Rakishev G Bismilda VRichter E Ruumlsch-Gerdes S Niemann S 2005 The Beijing genotype is amajor cause of drug-resistant tuberculosis in Kazakhstan Int J TubercLung Dis 9646 ndash 653

54 Mokrousov I Valcheva V Sovhozova N Aldashev A Rastogi NIsakova J 2009 Penitentiary population of Mycobacterium tuberculosisin Kyrgyzstan exceptionally high prevalence of the Beijing genotype andits Russia-specific subtype Infect Genet Evol 91400 ndash1405

55 Jiao WW Mokrousov I Sun GZ Guo YJ Vyazovaya A Narvskaya OShen AD 2008 Evaluation of new variable-number tandem-repeat sys-tems for typing Mycobacterium tuberculosis with Beijing genotype iso-lates from Beijing China J Clin Microbiol 461045ndash1049

56 Kam KM Yip CW Tse LW Wong KL Lam TK Kremer K Au BKvan Soolingen D 2005 Utility of mycobacterial interspersed repetitiveunit typing for differentiating multidrug-resistant Mycobacterium tu-berculosis isolates of the Beijing family J Clin Microbiol 43306 ndash313

57 Dou HY Tseng FC Lin CW Chang JR Sun JR Tsai WS Lee SY SuIJ Lu JJ 2008 Molecular epidemiology and evolutionary genetics ofMycobacterium tuberculosis in Taipei BMC Infect Dis 8170 doi1011861471-2334-8-170

58 Jou R Chen HY Chiang CY Yu MC Su IJ 2005 Genetic diversity ofmultidrug-resistant Mycobacterium tuberculosis isolates and identifica-tion of 11 novel rpoB alleles in Taiwan J Clin Microbiol 431390 ndash1394

59 Choi GE Jang MH Song EJ Jeong SH Kim JS Lee WG Uh Y RohKH Lee HS Shin JH Ryoo NH Kim YR Jeong J Kim JH Lee SMYi J Hwang SH Kim HH Lee EY Chang CL Kim MB Kim YD 2010IS6110-restriction fragment length polymorphism and spoligotypinganalysis of Mycobacterium tuberculosis clinical isolates for investigatingepidemiologic distribution in Korea J Korean Med Sci 251716 ndash1721

60 van Crevel R Nelwan RH de Lenne W Veeraragu Y van der ZandenAG Amin Z van der Meer JW van Soolingen D 2001 Mycobacteriumtuberculosis Beijing genotype strains associated with febrile response totreatment Emerg Infect Dis 7880 ndash 883

61 Mathuria JP Sharma P Prakash P Samaria JK Katoch VM Anu-purba S 2008 Role of spoligotyping and IS6110-RFLP in assessing ge-netic diversity of Mycobacterium tuberculosis in India Infect Genet Evol8346 ndash351

62 Singh UB Suresh N Bhanu NV Arora J Pant H Sinha S AggarwalRC Singh S Pande JN Sola C Rastogi N Seth P 2004 Predominanttuberculosis spoligotypes Delhi India Emerg Infect Dis 101138 ndash1142

63 Varma-Basil M Kumar S Arora J Angrup A Zozio T Banavaliker JNSingh UB Rastogi N Bose M 2011 Comparison of spoligotypingmycobacterial interspersed repetitive units typing and IS6110-RFLP in astudy of genotypic diversity of Mycobacterium tuberculosis in DelhiNorth India Mem Inst Oswaldo Cruz 106524 ndash535

64 Mistry NF Iyer AM Drsquosouza DT Taylor GM Young DB Antia NH2002 Spoligotyping of Mycobacterium tuberculosis isolates from multi-

ple-drug-resistant tuberculosis patients from Bombay India J Clin Mi-crobiol 402677ndash2680

65 Banu S Gordon SV Palmer S Islam MR Ahmed S Alam KM ColeST Brosch R 2004 Genotypic analysis of Mycobacterium tuberculosis inBangladesh and prevalence of the Beijing strain J Clin Microbiol 42674 ndash 682

66 Storla DG Rahim Z Islam MA Plettner S Begum V Mannsaaker TMyrvang B Bjune G Dahle UR 2006 Heterogeneity of Mycobacteriumtuberculosis isolates in Sunamganj District Bangladesh Scand J InfectDis 38593ndash596

67 Rahim Z Zaman K van der Zanden AG Moumlllers MJ van SoolingenD Raqib R Zaman K Begum V Rigouts L Portaels F Rastogi N SolaC 2007 Assessment of population structure and major circulating phy-logeographical clades of Mycobacterium tuberculosis complex in Bangla-desh suggests a high prevalence of a specific subclade of ancient Myco-bacterium tuberculosis genotypes J Clin Microbiol 453791ndash3794

68 Doroudchi M Kremer K Basiri EA Kadivar MR Van Soolingen DGhaderi AA 2000 IS6110-RFLP and spoligotyping of Mycobacteriumtuberculosis isolates in Iran Scand J Infect Dis 32663ndash 668

69 Farnia P Masjedi M Mirsaeidi M Mohammadi F Jallaledin-GhanaviV Bahadori M Velayati AA 2006 Prevalence of Haarlem I and Beijingtypes of Mycobacterium tuberculosis strains in Iranian and AfghanMDR-TB patients J Infect 53331ndash336

70 Bicmen C Esen N Graviss EA Williams-Bouyer N Ramaswamy SVYulug N 2007 Molecular characterization of Mycobacterium tuberculo-sis isolates from Izmir Turkey New Microbiol 30229 ndash240

71 Otlu B Durmaz R Gunal S Sola C Zozio T Rastogi N 2009BeijingW and major spoligotype families of Mycobacterium tuberculosisstrains isolated from tuberculosis patients in Eastern Turkey New Mi-crobiol 32255ndash263

72 Prodinger WM Bunyaratvej P Prachaktam R Pavlic M 2001 Myco-bacterium tuberculosis isolates of Beijing genotype in Thailand EmergInfect Dis 7483ndash 484

73 Rienthong D Ajawatanawong P Rienthong S Smithtikarn SAkarasewi P Chaiprasert A Palittapongarnpim P 2005 Restrictionfragment length polymorphism study of nationwide samples of Myco-bacterium tuberculosis in Thailand 1997ndash1998 Int J Tuberc Lung Dis59576 ndash581

74 Phyu S Stavrum R Lwin T Svendsen Oslash Ti ST Grewal HM 2009Predominance of Mycobacterium tuberculosis EAI and Beijing lineages inYangon Myanmar J Clin Microbiol 47335ndash344

75 Valcheva V Mokrousov I Narvskaya O Rastogi N Markova N 2008Molecular snapshot of drug-resistant and drug-susceptible Mycobacte-rium tuberculosis strains circulating in Bulgaria Infect Genet Evol8657ndash 663

76 Panaiotov S Zozio T Evans JT Brankova N Karcheva A LevterovaV Kantardjiev T Hawkey P Rastogi N Sola C 2005 Biodiversity ofMycobacterium tuberculosis in Bulgaria assessed by spoligo- and MIRU-typing p 37 Abstr 26th Annu Congr Eur Soc Mycobacteriol Istan-bul Turkey

77 Kubin M Mezenskyacute L Prikazskyacute V Kremer K van Soolingen D 2001Family outbreaks of tuberculosis in South Moravia a low incidence areaof the Czech Republic Int J Tuberc Lung Dis 5426 ndash 431

78 Kubiacuten M Havelkovaacute M Hyncicovaacute I Svecovaacute Z Kaustovaacute J KremerK van Soolingen D 1999 Multidrug-resistant tuberculosis microepi-demic caused by genetically closely related Mycobacterium tuberculosisstrains J Clin Microbiol 372715ndash2716

79 Marttila HJ Maumlkinen J Marjamaumlki M Ruutu P Soini H 2008Molecular genetics of drug-resistant Mycobacterium tuberculosis isolatesin Finland 1995ndash2004 Int J Tuberc Lung Dis 12338 ndash343

80 Ghebremichael S Groenheit R Pennhag A Koivula T Andersson EBruchfeld J Hoffner S Romanus V Kaumlllenius G 2010 Drug resistantMycobacterium tuberculosis of the Beijing genotype does not spread inSweden PLoS One 5e10893 doi101371journalpone0010893

81 Agonafir M Lemma E Wolde-Meskel D Goshu S Santhanam AGirmachew F Demissie D Getahun M Gebeyehu M van SoolingenD 2010 Phenotypic and genotypic analysis of multidrug-resistant tu-berculosis in Ethiopia Int J Tuberc Lung Dis 141259 ndash1265

82 Cooksey RC Abbadi SH Woodley CL Sikes D Wasfy M CrawfordJT Mahoney F 2002 Characterization of Mycobacterium tuberculosiscomplex isolates from the cerebrospinal fluid of meningitis patients at sixfever hospitals in Egypt J Clin Microbiol 401651ndash1655

83 Abbadi S El Hadidy G Gomaa N Cooksey R 2009 Strain differen-

Mokrousov


on Septem


rasmorg

Dow

nloaded from

tiation of Mycobacterium tuberculosis complex isolated from sputum ofpulmonary tuberculosis patients Int J Infect Dis 13236 ndash242

84 Warren RM van der Spuy GD Richardson M Beyers N Booysen CBehr MA van Helden PD 2002 Evolution of the IS6110-based restric-tion fragment length polymorphism pattern during the transmission ofMycobacterium tuberculosis J Clin Microbiol 401277ndash1282

85 Hanekom M van der Spuy GD Gey van Pittius NC McEvoy CRNdabambi SL Victor TC Hoal EG van Helden PD Warren RM2007 Evidence that the spread of Mycobacterium tuberculosis strains withthe Beijing genotype is human population dependent J Clin Microbiol452263ndash2266

86 Vinhas SA Palaci M Marques HS Lobo de Aguiar PP Ribeiro FKPeres RL Dietze R Gomes HM Suffys PN Golub JE Riley LW NoiaMaciel EL 2012 Mycobacterium tuberculosis DNA fingerprint clustersand its relationship with RD(Rio) genotype in Brazil Tuberculosis (Ed-inb) [Epub ahead of print] doi101016jtube201209001

87 Gilpin C Fyfe J 2004 MIRU the national tuberculosis genotypingstrategy in Australia Microbiol Australia 2534 ndash35

88 Yuen LK Leslie D Coloe PJ 1999 Bacteriological and molecularanalysis of rifampin-resistant Mycobacterium tuberculosis strains iso-lated in Australia J Clin Microbiol 373844 ndash3850

89 van Embden JDA Cave MD Crawford JT Dale JW Eisenach KDGicquel B Hermans P Martin C McAdam R Shinnik TM Small P1993 Strain identification of Mycobacterium tuberculosis by DNA finger-printing recommendations for a standardized methodology J Clin Mi-crobiol 31406 ndash 409

90 Kamerbeek J Schouls L Kolk A van Agterveld M van Soolingen DKuijper S Bunschoten A Molhuizen H Shaw R Goyal M vanEmbden J 1997 Rapid detection and simultaneous strain differentia-tion of Mycobacterium tuberculosis for diagnosis and tuberculosis con-trol J Clin Microbiol 35907ndash914

91 Weniger T Krawczyk J Supply P Niemann S Harmsen D 2010MIRU-VNTRplus a Web tool for polyphasic genotyping of Mycobacte-rium tuberculosis complex bacteria Nucleic Acids Res 38W326 ndashW331

92 Demay C Liens B Burguiegravere T Hill V Couvin D Millet J Mok-rousov I Sola C Zozio T Rastogi N 2012 SITVIT WEBmdasha publiclyavailable international multimarker database for studying Mycobacte-rium tuberculosis genetic diversity and molecular epidemiology InfectGenet Evol 12755ndash766

93 Hunter P Gaston M 1988 Numerical index of the discriminatoryability of typing systems an application of Simpsonrsquos index of diversityJ Clin Microbiol 262465ndash2466

94 Swofford DL 2002 PAUP phylogenetic analysis using parsimony (andother methods) Sinauer Associates Sunderland MA

95 Felsenstein J 2004 PHYLIP (phylogeny inference package) version36b Department of Genome Sciences University of Washington Seat-tle WA

96 Lau J Ioannidis JP Schmid CH 1997 Quantitative synthesis in sys-tematic reviews Ann Intern Med 127820 ndash 826

97 Higgins JP Thompson SG Deeks JJ Altman DG 2003 Measuringinconsistency in meta-analyses BMJ 327557ndash560

98 Begg CB Mazumdar M 1994 Operating characteristics of a rank cor-relation test for publication bias Biometrics 501088 ndash1101

99 Fenner L Malla B Ninet B Dubuis O Stucki D Borrell S Huna TBodmer T Egger M Gagneux S 2011 ldquoPseudo-Beijingrdquo evidence forconvergent evolution in the direct repeat region of Mycobacterium tuber-culosis PLoS One 6e24737 doi101371journalpone0024737

100 Mokrousov I 2008 Genetic geography of Mycobacterium tuberculosisBeijing genotype a multifacet mirror of human history Infect GenetEvol 8777ndash785

101 Doerks T van Noort V Minguez P Bork P 2012 Annotation of the Mtuberculosis hypothetical orfeome adding functional information tomore than half of the uncharacterized proteins PLoS One 7e34302 doi101371journalpone0034302

102 Garciacutea-Rodriacuteguez JF Aacutelvarez-Diacuteaz H Lorenzo-Garciacutea MV Marintildeo-Callejo A Fernaacutendez-Rial acuteA Sesma-Saacutenchez P 2011 Extrapulmonarytuberculosis epidemiology and risk factors Enferm Infecc MicrobiolClin 29502ndash509

103 Lalvani A Sridhar S 2010 BCG vaccination 90 years on and still somuch to learn Thorax 651036 ndash1038

104 Hernaacutendez-Pando R Marquina-Castillo B Barrios-Payaacuten J Mata-Espinosa D 2012 Use of mouse models to study the variability in viru-

lence associated with specific genotypic lineages of Mycobacterium tuber-culosis Infect Genet Evol 12725ndash731

105 Park JS Tamayo MH Gonzalez-Juarrero M Orme IM Ordway DJ2006 Virulent clinical isolates of Mycobacterium tuberculosis grow rap-idly and induce cellular necrosis but minimal apoptosis in murine mac-rophages J Leukoc Biol 7980 ndash 86

106 Theus SA Cave MD Eisenach KD 2005 Intracellular macrophagegrowth rates and cytokine profiles of Mycobacterium tuberculosis strainswith different transmission dynamics J Infect Dis 191453ndash 460

107 Andreevskaia SN Chernousova LN Smirnova TG Larionova EEKuzrsquomin AV 2007 Impact of Mycobacterium tuberculosis genotype onsurvival in mice with experimental tuberculosis Probl Tuberk BoleznLegk 2007(7)45ndash50 (In Russian)

108 Andreevskaia SN Chernousova LN Smirnova TG Larionova EEKuzrsquomin AV 2006 Examining the ex vivo growth in the macrophages ofMycobacterium tuberculosis of various genotypic clusters Probl TuberkBolezn Legk 2006(12)43ndash 48 (In Russian)

109 Chernousova LN Andreevskaia SN Smirnova TG Zemskova ZSLarionova EE 2008 Biological properties of Mycobacterium tuberculosisW cluster strains Probl Tuberk Bolezn Legk 2008(10)45ndash50 (In Rus-sian)

110 Vishnevskiı BI Narvskaia OV Vasilrsquoeva SN Sapozhnikova NV Muk-rousov IV Otten TF 2002 Virulence of Mycobacterium tuberculosisProbl Tuberk 2002(10)33ndash36 (In Russian)

111 Zemskova ZS Andreyevskaya SN Smirnova TG Larionova YYChernousova LN 2010 Experimental tuberculosis caused by Mycobac-terium tuberculosis strains of genotypical W AI and HD clusters ProblTuberk Bolezn Legk 2010(3)41ndash 46 (In Russian)

112 Wirth T Hildebrand F Allix-Beacuteguec C Woumllbeling F Kubica TKremer K van Soolingen D Ruumlsch-Gerdes S Locht C Brisse S MeyerA Supply P Niemann S 2008 Origin spread and demography of theMycobacterium tuberculosis complex PLoS Pathog 4e1000160 doi101371journalppat1000160

113 Tskitishvili I 2012 On the demographic situation in Abkhazia Demo-scope Wkly 2012517ndash518 httpdemoscoperuweekly20120517gazeta029php (In Russian)

114 Georgia Department for Statistics 2005 Statistical yearbook of Georgia2005 population p 33 Department for Statistics Tbilisi Georgia

115 Chistyakova N 2004 Population of Northern capital Demoscope Wkly2004163ndash164 httpdemoscoperuweekly20040163tema01php (InRussian)

116 Narvskaya O Otten T Limeschenko E Sapozhnikova N Graschen-kova O Steklova L Nikonova A Filipenko ML Mokrousov I Vysh-nevskiy B 2002 Nosocomial outbreak of multidrug-resistant tubercu-losis caused by a strain of Mycobacterium tuberculosis W-Beijing family inSt Petersburg Russia Eur J Clin Microbiol Infect Dis 21596 ndash 602

117 Portaels F Rigouts L Bastian I 1999 Addressing multidrug-resistanttuberculosis in penitentiary hospitals and in the general population ofthe former Soviet Union Int J Tuberc Lung Dis 3582ndash588

118 Kimerling ME 2000 The Russian equation an evolving paradigm intuberculosis control Int J Tuberc Lung Dis 4S160 ndashS167

119 Dela A Sajduda A Pawlowska I Dziadek J 2006 Molecular charac-terization of Mycobacterium tuberculosis isolates from Lodz Polandanalysis by IS6110 restriction fragment length polymorphism and dou-ble-repetitive-element PCR J Infect 52346 ndash353

120 Muntean IS Adriana D Homorodean D Hoffner S 2012 Spoligo-typing of M tuberculosis strains from MDR-TB patients from pneu-mophthisiology hospital Brasov isolated between 2006 ndash2011 p 95 Ab-str 33rd Annu Congr Eur Soc Mycobacteriol Brasov Romania

121 Ministry of Foreign Affairs of Russian Federation 2003 Russian lan-guage in the world Ministry of Foreign Affairs of Russian FederationMoscow Russia httpwwwlnmidruBrp_4nsfarhB6BE784B3E2ABD1343256DF8003AC21COpenDocument (In Russian)

122 Grant A Arnold C Thorne N Gharbia S Underwood A 2008Mathematical modelling of Mycobacterium tuberculosis VNTR loci esti-mates a very slow mutation rate for the repeats J Mol Evol 66565ndash574

123 McMullen R 1993 Tuva Russiarsquos Tibet or the next Lithuania EurSecur 2451ndash 460

124 Sinkov VV Savilov ED Ogarkov OB 2011 Reconstruction of theepidemic history of the Beijing genotype of Mycobacterium tuberculosis inRussia and former Soviet countries using spoligotyping Mol Gen Mik-robiol Virusol 2011(3)25ndash29 (In Russian)

125 Sinkov VV Savilov ED Ogarkov OB 2012 Epidemiology of tubercu-



on Septem


rasmorg

Dow

nloaded from

losis in Russia molecular and historical evidence in support of dissemi-nation of the M tuberculosis Beijing genotype in the 20th century ProblTuberk Bolez Legk 2012(3)57ndash 62 (In Russian)

126 Solzhenitsyn AI 1974 The Gulag archipelago Harper amp Row NewYork NY

127 Perevedentsev VI 1965 Modern migration of population of West Sibe-ria West-Siberian Book Publishing Novosibirsk Russia (In Russian)

128 Perevedentsev VI 1989 Who are we How many are we Myslrsquo Mos-cow Russia (In Russian)

129 Zayonchkovskaya ZA 1991 Demographic situation and populationmovement p 72ndash73 Nauka Moscow Russia (In Russian)

130 Zimovina EP 2003 Dynamics and composition of the population ofKazakhstan in the 2nd half of the 20th century Demoscope Wkly 2003103ndash104 httpdemoscoperuweekly20030103analit03php (In Rus-sian)

131 Ilichev EA 1978 Tselina p 1408 ndash1411 In Bolshaia Sovetskaia entsiklo-pediaya vol 46 Sovetskaya Entsiklopediya Moscow Russia

132 Horie N 2003 Forced migration in West Siberia Far East Stud 269ndash87133 Vainshtein SI Mannai-Ool MK (ed) 2001 History of Tuva Nauka

Novosibirsk Russia (In Russian)134 Trunova N 2007 North cities of Russia Ross Ekspert Obozrenie

2007(4 ndash5)34 ndash36 httpprotownruinformationarticles3352html135 Rodionovskii YV 2002 Features of the population formation in Euro-

pean North example of the Nenets autonomous district PhD thesisInstitute of Social and Political Research Moscow Russia (In Russian)

136 Iseman MD 2002 Tuberculosis therapy past present and future EurRespir J 3687sndash94s

137 Lapina AI 1970 The development of tuberculosis control in the USSRBull Int Union Tuberc 43188 ndash192

138 Caws M Thwaites G Dunstan S Hawn TR Lan NT Thuong NTStepniewska K Huyen MN Bang ND Loc TH Gagneux S vanSoolingen D Kremer K van der Sande M Small P Anh PT ChinhNT Quy HT Duyen NT Tho DQ Hieu NT Torok E Hien TT Dung

NH Nhu NT Duy PM van Vinh Chau N Farrar J 2008 Theinfluence of host and bacterial genotype on the development of dissem-inated disease with Mycobacterium tuberculosis PLoS Pathog4e1000034 doi101371journalppat1000034

139 Herb F Thye T Niemann S Browne EN Chinbuah MA Gyapong JOsei I Owusu-Dabo E Werz O Ruumlsch-Gerdes S Horstmann RDMeyer CG 2008 ALOX5 variants associated with susceptibility to hu-man pulmonary tuberculosis Hum Mol Genet 171052ndash1060

140 Intemann CD Thye T Niemann S Browne EN Amanua ChinbuahM Enimil A Gyapong J Osei I Owusu-Dabo E Helm S Ruumlsch-Gerdes S Horstmann RD Meyer CG 2009 Autophagy gene variantIRGM-261T contributes to protection from tuberculosis caused by My-cobacterium tuberculosis but not by M africanum strains PLoS Pathog5e1000577 doi101371journalppat1000577

141 Thye T Niemann S Walter K Homolka S Intemann CD ChinbuahMA Enimil A Gyapong J Osei I Owusu-Dabo E Rusch-Gerdes SHorstmann RD Ehlers S Meyer CG 2011 Variant G57E of mannosebinding lectin associated with protection against tuberculosis caused byMycobacterium africanum but not by M tuberculosis PLoS One6e20908 doi101371journalpone0020908

142 Ogarkov O Mokrousov I Sinkov V Zhdanova S Antipina S SavilovE 2012 lsquoLethalrsquo combination of Mycobacterium tuberculosis Beijing ge-notype and human CD209-336G allele in Russian male population In-fect Genet Evol 12732ndash736

143 Dos Vultos T Mestre O Rauzier J Golec M Rastogi N Rasolofo VTonjum T Sola C Matic I Gicquel B 2008 Evolution and diversity ofclonal bacteria the paradigm of Mycobacterium tuberculosis PLoS One3e1538 doi101371journalpone0001538

144 de Vos M Muumlller B Borrell S Black P van Helden P Warren RGagneux S Victor T 2013 Putative compensatory mutations in therpoC gene of rifampin-resistant Mycobacterium tuberculosis are associ-ated with ongoing transmission Antimicrob Agents Chemother 57827ndash 832

Igor Mokrousov is Leading Researcher in theLaboratory of Molecular Microbiology at the StPetersburg Pasteur Institute St PetersburgRussia He is an editor for Infection Geneticsand Evolution PLoS One and BMC Microbiol-ogy In the last 10 years Dr Mokrousov haspublished 79 peer-reviewed papers (59 in Eng-lish) and 9 book chapters (in particular forbooks published by Cambridge University Pressand SpringerHumana Press) and he holds 4patents Dr Mokrousov received a ScientificPrize from the International Union Against Tuberculosis and Lung Diseasein 2004 and Honor Awards from the Russian Ministry of Health in 2005 and2009 His major achievements in tuberculosis research include the globalphylogeography of the Mycobacterium tuberculosis Beijing and Ural familiesin light of human migration the concept of ancient and modern sublineageswithin the Beijing genotype the development of a new measure of geneticdistance between bacterial populations VNTR database development for theBeijing genotype reevaluation of embB306 mutations as markers of etham-butol resistance in M tuberculosis and development of multiplex allele-specific PCR (MAS-PCR) assays to detect resistance to the main antituber-culosis drugs

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Insights into the Origin Emergence and Current Spread of a Successful Russian Clone of Mycobacterium tuberculosis

point of view the human microbiome may be regarded as analternative source of data for deciphering human migrations andorigins Reciprocally knowledge about human migration and de-mographics may explain under certain limitations the origin anddispersal of human pathogens

Evolutionary histories of H sapiens and human pathogens areat least partly comirrored and coshaped However horizontallyepidemically transmitted pathogens (eg influenza virus andHIV) are less likely to mirror the population structure of the hu-man host instead their population structures reflect a rapid out-burst spreading of particular genotypes Accordingly microbe-aided tracing of the events in human history should rely onnonepidemically or mainly latently manifested microorganismswith a familyhousehold mode of transmission and sufficientprevalence in a population Regarding human tuberculosis (TB)historically short devastating periods of horizontal epidemicspread have been followed by longer time spans of relativelypeaceful coexistence of the surviving human populations and thefamilyhousehold-transmitted pathogen Therefore Mycobacte-rium tuberculosis is a suitable candidate for tracing human migra-tions since remarkably useful features of M tuberculosis include(i) a high prevalence (30) of latent tuberculosis in humansand hence a mainly familyhousehold mode of transmission dur-ing most of human history and (ii) an extreme rarity of lateralgene transfer and hence less risk of diffusing the phylogeographicsignal

Genome Markers of M tuberculosis

The M tuberculosis genome contains various insertion sequences(IS6110 is the most widely used) and repeat loci that have beenused widely to study the genetic diversity of the species The mostfrequently used of the latter include one CRISPR (historicallynamed a direct repeat [DR] [1]) locus and multiple variable-num-ber tandem-repeat (VNTR) loci (Fig 1)

The CRISPR locus in M tuberculosis as in other bacteria con-sists of minisatellite alternating exact direct repeats and variablespacers (Fig 1A) Its evolution in M tuberculosis currently occursvia neutral consecutive deletions of either single units or contigu-ous blocks (2) A method to study variation in the CRISPR locus inM tuberculosis is named spoligotyping which in its classical andusually macroarray-based format detects the presence or absenceof 43 particular spacers (Fig 1A) A more comprehensive analysisof the CRISPR locus relies on 68 different spacers which givessome added discriminatory value (2) As a whole the method issimple and portable but lacks sufficient discriminatory power Forthis concern more discriminatory methods of M tuberculosisstrain typing deal with more polymorphic IS6110 and VNTR loci

IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing is based on analysis of the insertion sequenceIS6110 which is found in most isolates of the M tuberculosis com-plex (Fig 1D) Until relatively recently this was the most widelyapplied and standardized molecular typing method for M tuber-culosis complex isolates The method was considered to be themost discriminatory and was thus the most widely used method inepidemiological studies (3) Unfortunately it is cumbersome andtime-consuming and requires large quantities of DNA and inter-laboratory comparison of results is difficult

Mycobacterial interspersed repetitive unit-VNTR (MIRU-VNTR) loci are scattered throughout the genome and isolates canbe typed based on the number of copies of repeated units (4)

(Fig 1B) Implementation of the large number of carefully evalu-ated loci has been expected to achieve a high level of discrimina-tion Since 1998 the VNTR typing of M tuberculosis has under-gone a remarkable development Whereas the first schemecomprised only 6 exact tandem-repeat loci (5) a more recentlyimplemented but already classical MIRU set involved 12 loci (4)and finally the new format for MIRU typing includes 24 loci (6)The necessity of further adjusting this scheme to particular set-tings by adding so-called hypervariable loci was recognized mostrecently (7ndash9) This method was shown to possess a higher dis-criminatory power than spoligotyping and depending on thenumber of VNTR loci tested a power roughly similar to that ofIS6110-RFLP typing (6 7) The other apparent advantage of theVNTR approach (compared to IS6110 typing) is its portable for-mat and hence its easy international exchange and use in databasedevelopment

A number of other molecular markersmethods such as single-nucleotide polymorphisms (SNPs) large deletions (regions of dif-ference [RD]) and whole-genome new-generation sequencingshould be mentioned They vary in discriminatory power al-though they demonstrate a clear potential to reconstruct reliablephylogenies and may aid in high-resolution epidemiological in-vestigations (10) However they are not relevant to the topic ofthis review and thus are not discussed further here

Population Structure and Clinical Relevance

It is very common yet true saying that M tuberculosis is a majorhuman killer However a closer look at the population structureof the pathogen reveals that some of its lineages show a greatercapacity to spread and cause active disease Consequently effi-cient application of modern antituberculosis treatment regimensis hampered by dissemination of such M tuberculosis strains Mtuberculosis has a clonal population structure and several geneticfamilies have been identified within this species eg East-Afri-can-Indian CASDelhi Beijing Haarlem Manu and LAM Ini-tially endemic within specific geographical areas some of thesefamilies have become ubiquitous An example of such an omni-present lineage is the Beijing familygenotype It was identified forthe first time in the Beijing area of China (11) but strains of thislineage are now found on all continents although at differentrates and thus may be defined as endemic epidemic or sporadic(12 13)

In addition to the lineagefamily-associated strain properties ithas been recognized that some of the clonal clusters (sublineagesand subgenotypes) within the same family may be more transmis-sible than others In particular while the Beijing genotype as awhole is frequently (but not always) marked by drug resistanceand hypervirulence (reviewed in reference 14) some of its variantsdemonstrate even more remarkable pathogenic properties espe-cially in areas with a high burden of drug-resistant TB (15) Thereare several classifications of Beijing genotype strains in particularby subdividing them into ancient or ancestral and modern groups(16ndash18) which are suggested to differ in mechanisms of adapta-tion to drug-induced selective pressure (19) In China the mod-ern Beijing strains are more likely than the ancient sublineages tobe clustered (20) and an increasing circulation of the modernBeijing sublineage was recently demonstrated in Japan (21)

While discrimination into modern and ancient Beijing strainsprovided a rough phylogenetically meaningful subdivision high-resolution typing permitted the definition of more homogeneous



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insights into the origin, emergence, and current spread of ... · cient application of modern...

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