First Insight into the Molecular Epidemiology of...

Research ArticleFirst Insight into the Molecular Epidemiology ofMycobacterium tuberculosis Isolates from the MinorityEnclaves of Southwestern China

Lianyong Chen12 Yu Pang3 Li Ma12 Huijuan Yang12 Haohao Ru12 Xing Yang12

Shuangqun Yan12 Mao Jia12 and Lin Xu12

1Yunnan Provincial Center for Tuberculosis Research Yunnan Provincial Center for Disease Control and PreventionKunming China2Yunnan Provincial Dispensary for Tuberculosis Control and Prevention Yunnan Provincial Center for DiseaseControl and Prevention Kunming China3National Clinical Laboratory on Tuberculosis Beijing Key Laboratory on Drug-Resistant Tuberculosis ResearchBeijing Chest Hospital Beijing Tuberculosis and Thoracic Tumor Institute Capital Medical University Beijing China

Correspondence should be addressed to Lin Xu 123316859qqcom

Received 13 December 2016 Accepted 9 March 2017 Published 17 May 2017

Academic Editor Isabel Portugal

Copyright copy 2017 Lianyong Chen et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Yunnan is a province located in southwestern ChinaThe aim of this study was to investigate the genetic diversity ofMycobacteriumtuberculosis (MTB) strains circulating in Yunnan Province We used spoligotyping and a 12-locus high-resolution VNTR set toidentify a total of 271 MTB isolates collected from six prefecturesrsquo Yunnan All the 271 patients were classified as 11 different ethnicgroups including 133 Han patients (491) and 138 minority patients (509) Spoligotyping analyses revealed that the largest twospoligotypes were SIT1 (Beijing family 119899 = 136) and SIT53 (T family 119899 = 35) Statistical analysis indicated that the proportion ofBeijing genotype in Qujing was significantly higher than that in the ethnic enclaves (119875 le 001) Compared with the percentage ofBeijing-family isolates from patients under 25 years of age (727) those from patients aged 45sim64 years (446 119875 lt 001) andolder than 64 years (481) (119875 = 004) were significantly lower Beijing genotype strains (238 36151) showed higher clusteringrate than non-Beijing genotype strains (16120 133 119875 = 0029) In conclusion our data demonstrated that the Beijing genotypewas the predominant genotype in Yunnan Province The distribution of Beijing genotype strains showed geographic diversity Inaddition Beijing genotype was associated with recent transmission rather than drug resistance

1 Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosiscomplex (MTBC) remains amajor global health concern andranks as the second leading cause of death from infectiousdiseases [1] The World Health Organization (WHO) esti-mates that TB caused 96 million new cases and 15 milliondeaths in 2014 [2] Only behind India and Indonesia Chinahas the worldrsquos third-largest amount of TB patients with anapproximately 10 million new cases reported annually [2]According to the most recent national TB epidemiologicalsurvey the prevalence of pulmonary TB was 442 cases per100000 population in China [3] Despite the decreased trendin the incidence and mortality rate from 1990 to 2010 TB is

still one of the most important issues threatening the publichealth and social development in China [3 4]

Molecular epidemiological investigation has been provedto be a useful tool for TB control and surveillance whichallows us to better understand the dynamic of diseasetransmission and distinguish between relapse and infection[5] In addition there is increasing evidence that certaingenotypes of M tuberculosis are associated with drug resis-tance virulence and disease progression [6] Hence theknowledge regarding strain diversity within the patients hasspecial contemplation during TB epidemics or suspectedoutbreaks thereby providing new insights for establishingstrategic measures for TB control and prevention [7 8]

HindawiBioMed Research InternationalVolume 2017 Article ID 2505172 9 pageshttpsdoiorg10115520172505172

2 BioMed Research International

Yunnan is located in southwestern China with an area of394100 square kilometers and a population of 469 millionpeople in 2013 Out of all inhabitants there are remarkable140 million inhabitants belonging to 55 different ethnicminority groups accounting for one-third of the wholeinhabitants In addition most of these minority inhabitantslive along the border with Myanmar Laos and VietnamAccording to the data from provincial surveillance systemfor TB the overall incidence of pulmonary TB was 56 casesper 100000 population in Yunnan [9] The diversity of TBburden was also observed across the different regions andthe TB incidences in several regions of ethnic enclaves werethree times higher than the average incidence [9] Thereforeit is meaningful to investigate the patientrsquos structure and drugsusceptibility ofMTB isolates circulating in Yunnan Provinceto highlight the geographic diversity of TB in ethnic enclavesUnfortunately limited data have been reported on this issueIn this study our aim was to characterize the genotypes ofMTB strains collected in Yunnan and to investigate the trans-mission profile of the predominant TB genotypes in this area

2 Materials and Methods

21 Ethics Statement This study was approved by the EthicsCommittee of the Yunnan Provincial Center for DiseaseControl and Prevention Patients were enrolled in the studyafter agreeing to and signing an Informed Consent form

22 Bacterial Isolates All smear-positive pulmonary TBpatients seeking health care at six prefectural TB dispensariesin 2014 were continuously enrolled in this study includingQujing Lijiang Dehong Lincang Puer and XishuangbannaDemographic information was collected from InformedConsent forms Two sputum samples from each patient wereused for mycobacteria culturing with Lowenstein-Jensen(L-J) medium One strain from each patient was collected forfurther molecular epidemiological study

23 DNA Extraction The crude genomic DNA of MTBisolates were extracted as previously reported [5] Brieflya loopful of colonies from L-J slants were transferred toa microcentrifuge tube containing 500 120583L Tris-EDTA (TE)buffer Followed by heating at 95∘C water bath for 30minthe suspension was centrifuged at 12000 rpm for 10min TheDNA in the supernatant was pipetted into a microcentrifugetube for further PCR amplification

24 Drug Susceptibility Testing (DST) The proportionalmethod was performed to determine the in vitro drugsusceptibility of MTB isolates against isoniazid (INH) andrifampicin (RFP) according to the recommendations bythe World Health Organization [4 9] The concentrationsof INH and RFP in the L-J medium were 02 120583gmL and40 120583gmL respectively The resistance of a strain against adrug was declared when the growth rate was higher than1 compared with the control The Yunnan Provincial TBReference Laboratory has passed the DST proficiency tests ofthe National TB Reference Laboratory since 2007 Multiple

drug resistant tuberculosis (MDR-TB) isolates were definedas strains being resistant to both INH and RFP

25 Genotyping Spoligotyping was performed using com-mercial kit manufactured by Isogen Bioscience BV (Maar-ssen Netherlands) [4]The genomicDNAwas amplified withprimers DRa (51015840-CCGAGAGGGGACGGAAAC-31015840) andDRb (51015840-GGTTTTGGGTCTGACGAC-31015840) The ampliconswere hybridized with a membrane and the final image wasdetected with a chemiluminescence system (AmershamBuckinghamshire United Kingdom) The original binarydata were entered in the SITVITWEB database to obtainthe Spoligotyping International Type (SIT) patterns and thesublineages of MTB isolates [10] In addition all the isolateswere genotyped with the 12-locus VNTR set with highdiscriminatory power described by Luo et al [11] The PCRproducts were analyzed using 15 agarose electrophoresis at5 Vcm for 1 hour The 100 bp DNA ladder and amplicons ofH37Rv were loaded per 7 lanes as a size marker to calculatethe repeat number of each locus The Hunter-GastonDiscriminatory Index (HGDI) was used to analyze thediscriminatory power of each VNTR locus [12] In additionthe genotyping patterns were clustered with BioNumericssoftware version 50 (Applied Maths Sint-Martens-LatemBelgium)

26 Data Analysis A chi-square test was performed to detectany significant difference between the two groups and thestatistical results were expressed as odds ratios (ORs) with95 confidence intervals (CIs) Two-sided 119875 value of lt005was declared as statistically significant All calculations wereanalyzed in SPSS 140 (SPSS Inc USA)

3 Results

31 Drug Susceptibility Profiles and Demographic Character-istics of Han and Minority Patients A total of 271 MTBisolates represented patients from different districts acrossYunnan Province (Figure 1) Out of these patients 196(723) were males and 75 were females The mean age ofthe patients was 498 plusmn 121 years (range 16ndash76 years) Inaddition all the 271 patients were classified as 11 differentethnic groups including 133 Han patients (491) and 138minority patients (509) Of these minority groups HaniYi and Dai were the most frequently observed accountingfor 111 (30271) 103 (28271) and 96 (26271) of all thepatients respectively We further analyzed the distribution ofdemographic and drug susceptibility characteristics betweenHan and minority patients As shown in Table 1 statisticalanalysis indicated that the proportion of minority patientsin Qujing (20 149) were significantly lower than that inPuer (713 6794) Xishuangbanna (700 2840) Lijiang(636 1422) Dehong (628 235) and Lincang (194631) respectively (119875 lt 005) Compared with Han patientsthe minority patients had higher proportion of farmers(884 versus 782 119875 = 002) In addition we foundthat the Han patients were significantly associated with RIFresistance (119875 = 003) while the rate of INH resistance showed

BioMed Research International 3

Dehong (35)

Lijiang (22)

Lincang (31)

Puer (94)

Qujing (49)

Xishuangbanna (40)

Beijingnon-Beijing

Figure 1 Distribution of Beijing genotype MTB isolates enrolled inthis study The number presents the absolute number of isolates inthis region

no statistically significant difference Although all the MDR-TB patients belonged to Han patients the difference in theprevalence of MDR-TB between Han and minority patientswas not significant (Table 3) which was more likely to beattributed to small sample size Demographic characteristicsincluding gender age and treatment history showed nostatistical difference between Han and minority patients

32 Spoligotyping The 271 MTB isolates were analyzed byspoligotyping in this study The 271 genotyped strains weredivided into 16 types shared by 2 to 136 isolates and 24orphans (genotypes found only once in study) Out of thesespoligotypes SIT1 was the largest lineage (502 136271)which belonged to the classical Beijing genotype In additionthe second largest type was SIT53 from T1 family thatincluded 35 isolates (129 Table 2) Eleven types containing21 isolates were identified for the first time

Based on spoligotyping 151 (557) MTB strains belon-ged to the Beijing genotype while the other 120 (443) werefrom non-Beijing families indicating that Beijing genotypewas the predominant sublineage in Yunnan Strains assignedto non-Beijing family included 45 from T1 family (166)34 from T3 family (125) 6 from LAM9 family (22) 5from the T2 family (18) 4 fromT2-T3 family (15) 2 fromMANU2 family (07) 1 from T5 family (04) 1 from H3family (04) 1 from X2 family (04) and 21 of undefinedgenotypes (77 Table 2)

33 Drug Susceptibility Profiles and Demographic Charac-teristics of Beijing and Non-Beijing MTB Isolates We com-pared the proportion of drug susceptibility profiles anddemographic characteristics of patients identified as infectionwith Beijing and non-Beijing genotypes Statistical analysisindicated that the distribution of Beijing genotype showedgeographic diversity and the proportion of Beijing genotype

in Qujing was significantly higher than that in the ethnicenclaves (119875 le 001) Compared with the percentage ofBeijing-family isolates from patients under age of 25 yearsthose from patients aged 45sim64 years [OR (95 CI) 030(014sim066) 119875 lt 001] and older than 64 years [OR (95CI) 035 (013sim095) 119875 = 004] were significantly lowerWith regard to the gender distribution of Beijing genotypestrains the percentage of female patients infectedwithBeijinggenotype strains was significantly higher than that of malepatients indicating that women might be at high risk forthe infection of Beijing genotype strains [OR (95 CI) 204(117sim357) 119875 = 001] Our data revealed that occupationtreatment history and drug resistance showed no statisticallysignificant association with the Beijing genotype (Figure 1and Table 3)

34 VNTR Using the 12-locus high-resolution VNTRmeth-od the 271 isolates were differentiated into 30 clusters (2 to 17isolates per cluster) and 177 unique genotypes showing aclustering rate of 236 (Figure 2 and Table S1 in Supple-mentary Material available online at httpsdoiorg10115520172505172)We also analyzed the clustering rate accordingto different patients Our data revealed that the clusteringrate of Han patients (263 35133) was significantly higherthan that of minority patients (152 21138 119875 = 0024)In addition Beijing genotype strains (238 36151) showedhigher clustering rate than non-Beijing genotype strains(16120 133 119875 = 0029) Interestingly the largest clusterwith 6 3 8 2 4 8 5 3 3 8 4 2 VNTR profile contained 17strains which were all from Han patients in Qujing districtAs shown in Table 4 eight out of the 12 VNTR loci includingQub11b Qub18 Qub26 MIRU26 Mtub21 Mtub04 ETR-F and MIRU31 were highly discriminating loci the allelicdiversity for which was higher than 06 [13] All the other4 loci (MIRU10 Qub4156 MIRU40 and Qub1895) showedmoderate discrimination (03 le HGDI le 06) In additionwe found that the loci exhibited different discriminating abil-ity to distinguish Beijing and non-Beijing genotype strainsQub26 Mtub04 andMIRU40 showed higher discriminatingpower for non-Beijing genotype strains while Mtub21 andQub4156 were more effective in identifying Beijing genotypestrains (Table 4)

4 Discussion

This study provides the first insight into the molecularepidemiological characteristics of MTB strains circulatingin Yunnan Province China Our data revealed that Beijinggenotype is still the predominant genotype throughout Yun-nan accounting for 557 of the MTB strains in the presentstudy The prevalence of Beijing genotype is similar to that inFujian (573) [14] and that in Guangxi (619) [15] althoughit is lower than most regions in China ranging from 767to 919 [16ndash19] All these findings were consistent with arecent study by Pang et al (2012) [4] which revealed that thedistribution of Beijing differed from one geographic region toanother in China and the Beijing genotype constitutes 765of the MTB strains currently epidemic in north China withonly 532of those in southChinaAlthough the exact reason


Table 1 Differences of characteristics between Han and minority population

Characteristics Total number of isolatesNumber () of isolates

OR 95 CI 119875 valueHan Minority(119899 = 133) (119899 = 138)

Resistance toINH 14 (52) 9 (68) 5 (36) 052 017sim159 024RIF 5 (18) 5 (38) 0 (00) 096 093sim099 003MDR 4 (15) 4 (30) 0 (00) 097 094sim100 006

GenderMen 196 (723) 94 (707) 102 (739) 10 (Ref) mdash mdashWomen 75 (277) 39 (293) 36 (261) 118 069sim200 055

Age group (years)lt25 44 (162) 21 (158) 23 (167) 10 (Ref) mdash mdash25ndash44 108 (399) 54 (406) 54 (391) 110 054sim221 08045ndash64 92 (339) 45 (338) 47 (341) 105 051sim215 090gt64 27 (100) 13 (98) 14 (101) 102 039sim265 097

OccupationFarmer 226 (834) 104 (782) 122 (884) 047 024sim091 002Others 45 (166) 29 (218) 16 (116) 10 (Ref) mdash mdash

RegionQujing 49 (181) 48 (361) 1 (07) 10 (Ref) mdash mdashDehong 35 (129) 13 (98) 22 (159) 001 000sim010 lt001Lijiang 22 (81) 8 (60) 14 (101) 001 000sim010 lt001Lincang 31 (114) 25 (188) 6 (43) 009 010sim076 001Puer 94 (347) 27 (203) 67 (486) 001 000sim007 lt001Xishuangbanna 40 (148) 12 (90) 28 (203) 001 000sim007 lt001

Treatment HistoryNew case 246 (908) 120 (902) 126 (913) 10 (Ref) mdash mdashRetreated 25 (92) 13 (98) 12 (87) 114 050sim259 076

for the relative low prevalence of Beijing genotypes in southChina remains elusive it is believed to be associated with thenatural environment living habit and the evolution historyof Beijing genotype [4 20]

In addition we also found that the distribution of Beijinggenotype showed remarkable geographic diversity inYunnanwhich may be attributed to the different composition ofminority patients among these regions The prevalence ofBeijing genotype in the Han enclaves was significantly higherthan that in the minority enclaves which was consistentwith the observation from Luo and colleagues [20] InYunnan as opposed to Han patients the minority patientsgenerally live in themountainous areas located in the nationalboundary lines rather than the plain areas Hence the lowerpatient densities ofmountainous areasmay play an importantrole in decreasing the transmission of the epidemic Beijinglineage In addition due to living in the regions borderingVietnam and Myanmar the minority patients prefer tomove across national boundary lines for commercial tradeConsidering the frequent patient communication betweenminority patients and neighboring foreigner the relatively

low prevalence of Beijing genotype in Vietnam (35) [21]and Myanmar (32) [22] may be another potential reasonresponsible for our findings

Several previous studies demonstrated that the Beijinggenotype is associated with young patients (aged less than25 years) [21 23] while researchers from other countriesobserved no association between the infection with theBeijing genotype and a patient age [24] In line with theformer results we also found that young age was a riskfactor for the infection with Beijing genotype strain On onehand compared with the older patients children and youngadults undergo higher level of social activities [25] In viewof the increased transmissibility of Beijing genotype [26]the more frequent social interactions may cause the recenttransmission of Beijing strains in these patients On the otherhand BCG vaccination was included in the National Immu-nization Program of China since 1980s [27] Previous studieshave hypothesized that Beijing strains could escape frominsufficient protection induced by BCG [28 29] which maybe responsible for their emergence in the BCG-vaccinatedyoung patients In addition to age we observed that infection


Table 2 Spoligotypes of the 271M tuberculosis isolates enrolled in this study

SITa Lineageb Number () Spoligotype description binary1 Beijing 136 (502)190 Beijing 6 (22)269 Beijing 3 (11)1674 Beijing 2 (07)632 Beijing 1 (04)265 Beijing 1 (04)743 Beijing 2 (07)53 T1 35 (129)7 T1 2 (07)86 T1 1 (04)120 T1 1 (04)154 T1 1 (04)205 T1 1 (04)373 T1 1 (04)1105 T1 1 (04)1166 T1 1 (04)2170 T1 1 (04)52 T2 2 (07)848 T2 1 (04)1797 T2 1 (04)1890 T2 1 (04)73 T2-T3 4 (15)37 T3 34 (125)44 T5 1 (04)183 H3 1 (04)42 LAM9 1 (04)249 LAM9 5 (18)583 MANU2 2 (07)137 X2 1 (04)Orphan NA 5 (18)Orphan NA 4 (15)Orphan NA 3 (11)Orphan NA 2 (07)Orphan NA 1 (04)Orphan NA 1 (04)Orphan NA 1 (04)Orphan NA 1 (04)Orphan NA 1 (04)Orphan NA 1 (04)Orphan NA 1 (04)aSIT from SITVITWEB databasebRepresenting spoligotype lineages annotated in SITVITWEB databaseNumber of strains with the same SITOrphan represents the spoligotyping type which is not found in SITVITWEB database

with Beijing genotype was associated with gender while theexact reason for this difference still remains unknown

A recent report from Yang and colleagues (2015) hasdemonstrated that Beijing genotype strains favor transmis-sion in China [26] which is consistent with our obser-vation that the rate of Beijing genotype was significantly

higher than that of non-Beijing genotype Considering thelower prevalence of Beijing genotype strains in the minoritypatients it also serves as the dominant contributor to thelower clustering rate of TB strains in these patients Inter-estingly we also identified a suspected outbreak in Qujing ofYunnan Province which included 17 Beijing genotype strains


Table 3 Differences of characteristics between Beijing and non-Beijing genotype


OR 95 CI 119875 valueBeijing Non-Beijing(119899 = 151) (119899 = 120)



Age group (years)lt25 44 (162) 32 (212) 12 (100) 100 (Ref) mdash mdash25ndash44 108 (399) 65 (430) 43 (358) 057 026sim122 01445ndash64 92 (339) 41 (272) 51 (425) 030 014sim066 lt001gt64 27 (100) 13 (86) 14 (117) 035 013sim095 004


EthnicityHan 133 (491) 76 (503) 57 (475) 100 (Ref) mdash mdashHani 30 (111) 11 (73) 19 (158) 043 019sim098 004Yi 28 (103) 10 (66) 18 (150) 042 018sim097 004Dai 26 (96) 13 (86) 13 (108) 075 032sim174 050Others 54 (199) 41 (272) 13 (108) 237 116sim482 002

RegionQujing 49 (181) 41 (272) 8 (67) 100 (Ref) mdash mdashDehong 35 (129) 28 (185) 7 (58) 078 025sim240 067Lijiang 22 (81) 12 (79) 10 (83) 023 008sim072 001Lincang 31 (114) 10 (66) 21 (175) 009 003sim027 lt001Puer 94 (347) 46 (305) 48 (400) 019 008sim044 lt001Xishuangbanna 40 (148) 14 (93) 26 (217) 011 004sim028 lt001


sharing the same VNTR pattern Further epidemiologicalinvestigation will help us to identify the ldquosuperspreaderrdquo ofthis outbreak Recently numerous studies have declared thatVNTR can not capture the full level of genetic diversitywithina single MTB genotype while Whole-Genome Sequencing(WGS) serves as a useful tool to identify MTB isolates associ-atedwith tuberculosis outbreak due to its inherent advantagesexceeding conventional molecular epidemiologic techniques[30 31] Nevertheless our genotyping data using VNTRreflect the potential ongoing community transmission inYunnan which highlights the urgent need for early diagnosisof the infectious TB cases to avoid a large TB outbreak Inrecent years more than 70 of TB laboratories in Yunnan

have been equipped with the molecular diagnosis toolsincluding GeneXpert and LAMP and further the applicationofmolecular techniqueswith high sensitivity for TBdiagnosiswill increase access to testing and decrease diagnostic delaysin a resource-limited county thereby decreasing the trans-mission of TB in the community

In conclusion our data demonstrated that the Beijinggenotype was the predominant MTB genotype in YunnanProvince The prevalence of Beijing genotype strains inthe Han enclaves was significantly higher than that in theminority enclaves indicating that distribution of TB strainshowed geographic diversity In addition we observed thatBeijing genotype was more likely to contribute to recent


848

111111111111111111111111

190190190190190190

111111111111111111111111111111111111111111111

265111111111

16741674

1111111111111111

632111mdash1mdash111111111111111111

26911

269111111111111111111

2691166

37373737373737373737373737mdash53535353

Orphan373737373737

1545353373773737337mdashmdash373753

Orphan53373737mdash375337

5832055353535353

5833737537

5337

Orphan1890

4452mdashmdash7mdashmdash5353

1203753mdash535353mdashmdashmdash538653

1835353425353

Orphan375373

2495353

249249249249

44444

179752

13753

11052170

535353

373

SIT Strain numberSpoligotype

Y121Y118Y133Y113Y73

Y116Y258Y135Y105

Y3Y218Y256Y54Y5Y4

Y19Y18Y17Y16

Y120Y114Y255Y96

Y115Y156Y151Y225Y216Y272Y274Y130Y87

Y124Y141Y254Y241Y235Y127Y95Y88Y94Y2

Y268Y49Y24

Y164Y140Y183Y179Y173Y163Y158Y155Y153Y149Y148Y146Y145Y144Y143Y142Y139Y138Y137Y178Y159Y154Y168Y150Y110Y111Y152Y104Y131Y129Y81

Y112Y62

Y205Y64Y48Y43

Y248Y220Y259Y174Y169Y197Y103Y134Y45

Y177Y182Y165Y172Y161Y212Y195Y193Y192Y242Y223Y83

Y227Y69Y66

Y157Y38

Y171Y12Y52

Y184Y222Y136Y123Y228Y125Y265

Y9Y8Y7Y6

Y261Y20Y15Y10

Y263Y249Y126Y47Y35

Y199Y210Y201Y200Y232Y26Y28Y27Y33Y77Y74Y25

Y217Y119Y132Y109Y170Y167Y39

Y102Y122Y58Y34

Y219Y215Y203Y204Y191Y189Y188Y186Y185Y214Y211

Y1Y252Y251Y250Y238Y90Y79Y72Y42Y23Y37Y36

Y253Y107Y234Y176Y162Y181Y175Y166Y84

Y262Y224Y226Y147Y266Y70

Y237Y13

Y240Y239Y86

Y245Y91Y78Y76Y82Y14Y32Y97Y31

Y246Y40

Y271Y269Y80

Y243Y89

Y273Y63

Y257Y98Y41Y30Y29

Y244Y22Y21Y61Y57

Y233Y260Y55

Y270Y56

Y198Y128Y202Y213Y208Y247Y59

Y264Y75Y85

Y108Y106Y11

Y101Y117Y71

Y236Y67

Y180Y93Y92

Y160Y53Y51Y50

Y209Y190Y207Y206Y187Y46

Y194Y196Y229Y267Y221Y68Y60Y44Y99

Y100

Figure 2 Dendrogram of 271 MTB isolates from Yunnan Province The phylogenetic tree was generated with the 12-VNTR loci set


Table 4 Allelic diversity of 12 different VNTR loci amongMycobac-terium tuberculosis strains (119899 = 271)

Locus HGDIAll strains Beijing Non-Beijing

Qub11b 0830 0731 0714Qub18 0854 0813 0672Qub26 0800 0632 0907MIRU26 0799 0655 0832Mtub21 0790 0705 0469Mtub04 0647 0397 0604ETR-F 0633 0451 0507MIRU31 0630 0277 0334MIRU10 0570 0162 0274Qub4156 0533 0625 0228MIRU40 0476 0243 0639Qub1895 0363 0368 0356

transmission rather than drug resistance The suspected TBoutbreak identified by VNTR highlights the urgent need forearly diagnosis of the infectious TB cases to avoid a large TBoutbreak

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

LianyongChen andYuPang contributed equally to this paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81560544) The authors are grateful toall staff from six prefectural TBdispensaries for their excellentwork in sputum collection They also would like to thank themembers from National Tuberculosis Reference Laboratoryfor their technical assistance

References

[1] V Lisdawati N Puspandari L Rif rsquoati et al ldquoMolecular epi-demiology study ofMycobacterium tuberculosis and its suscep-tibility to anti-tuberculosis drugs in Indonesiardquo BMC InfectiousDiseases vol 15 no 1 article 366 2015

[2] World Health Organization Global Tuberculosis Report 2015World Health Organization Geneva Switzerland 2015

[3] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[4] Y Pang Y Zhou B Zhao et al ldquoSpoligotyping and drugresistance analysis of Mycobacterium tuberculosis strains fromnational survey in Chinardquo PLoS ONE vol 7 no 3 Article IDe32976 2012

[5] D Zhang J An J Wang et al ldquoMolecular typing and drugsusceptibility of Mycobacterium tuberculosis isolates fromChongqing Municipality Chinardquo Infection Genetics and Evolu-tion vol 13 no 1 pp 310ndash316 2013

[6] Z Zhang J Lu M Liu et al ldquoGenotyping and molecular char-acteristics of multidrug-resistant Mycobacterium tuberculosisisolates from Chinardquo Journal of Infection vol 70 no 4 pp 335ndash345 2015

[7] J Singh M M Sankar P Kumar et al ldquoGenetic diversityand drug susceptibility profile of Mycobacterium tuberculosisisolated fromdifferent regions of Indiardquo Journal of Infection vol71 no 2 pp 207ndash219 2015

[8] C L Nogueira R I Prim S G Senna et al ldquoFirst insight intothe molecular epidemiology of Mycobacterium tuberculosis inSanta Catarina southern Brazilrdquo Tuberculosis vol 97 pp 57ndash64 2016

[9] World Health Organization Anti-Tuberculosis Drug Resistancein theWorld Fourth Global Report World Health OrganizationGeneva Switzerland 2008

[10] C Demay B Liens T Burguiere et al ldquoSITVITWEBmdasha pub-licly available international multimarker database for studyingMycobacterium tuberculosis genetic diversity and molecularepidemiologyrdquo Infection Genetics and Evolution vol 12 no 4pp 755ndash766 2012

[11] T Luo C Yang Y Pang Y Zhao J Mei and Q GaoldquoDevelopment of a hierarchical variable-number tandem repeattyping scheme for Mycobacterium tuberculosis in chinardquo PLoSONE vol 9 no 2 Article ID e89726 2014

[12] P R Hunter and M A Gaston ldquoNumerical index of thediscriminatory ability of typing systems an application ofSimpsonrsquos index of diversityrdquo Journal of Clinical Microbiologyvol 26 no 11 pp 2465ndash2466 1988

[13] I Mokrousov O Narvskaya E Limeschenko A VyazovayaT Otten and B Vyshnevskiy ldquoAnalysis of the allelic diversityof the mycobacterial interspersed repetitive units in Mycobac-terium tuberculosis strains of theBeijing family practical impli-cations and evolutionary considerationsrdquo Journal of ClinicalMicrobiology vol 42 no 6 pp 2438ndash2444 2004

[14] Q-F Liang Y Pang Q-Y Chen et al ldquoGenetic profile oftuberculosis among the migrant population in Fujian ProvinceChinardquo International Journal of Tuberculosis and Lung Diseasevol 17 no 5 pp 655ndash661 2013

[15] R Lan C Yang L Lan et al ldquoMycobacterium tuberculosisand non-tuberculous mycobacteria isolates from HIV-infectedpatients in Guangxi Chinardquo International Journal of Tuberculo-sis and Lung Disease vol 15 no 12 pp 1669ndash1674 2011

[16] Q Liu D Yang W Xu et al ldquoMolecular typing of mycobac-terium tuberculosis isolates circulating in Jiangsu ProvinceChinardquo BMC Infectious Diseases vol 11 article 288 2011

[17] J Wang Y Liu C-L Zhang et al ldquoGenotypes and character-istics of clustering and drug susceptibility of Mycobacteriumtuberculosis isolates collected in Heilongjiang Province ChinardquoJournal of Clinical Microbiology vol 49 no 4 pp 1354ndash13622011

[18] Y Liu M Tian X Wang et al ldquoGenotypic diversity analysisof mycobacterium tuberculosis strains collected from beijing in2009 using spoligotyping and VNTR typingrdquo PLoS ONE vol 9no 9 Article ID e106787 2014

[19] H Dong L Shi X Zhao et al ldquoGenetic diversity of Mycobac-terium tuberculosis isolates fromTibetans in Tibet ChinardquoPLoSONE vol 7 no 3 Article ID e33904 2012


[20] T Luo I Comas D Luo et al ldquoSouthern East Asian originand coexpansion of Mycobacterium tuberculosis Beijing familywith Han Chineserdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 112 no 26 pp 8136ndash8141 2015

[21] T N Buu M N Huyen N T N Lan et al ldquoThe Beijinggenotype is associated with young age and multidrug-resistanttuberculosis in rural Vietnamrdquo International Journal of Tuber-culosis and Lung Disease vol 13 no 7 pp 900ndash906 2009

[22] S Phyu R Stavrum T Lwin Oslash S Svendsen T Ti and HM Grewal ldquoPredominance ofMycobacterium tuberculosis EAIand Beijing lineages in Yangon Myanmarrdquo Journal of ClinicalMicrobiology vol 47 no 2 pp 335ndash344 2009

[23] Y Pang Y Song H Xia Y Zhou B Zhao and Y Zhao ldquoRiskfactors and clinical phenotypes of Beijing genotype strains intuberculosis patients in Chinardquo BMC Infectious Diseases vol 12article 354 2012

[24] D D Anh M W Borgdorff L N Van et al ldquoMycobacteriumtuberculosis Beijing genotype emerging in vietnamrdquo EmergingInfectious Diseases vol 6 no 3 pp 302ndash305 2000

[25] J Wallinga P Teunis and M Kretzschmar ldquoUsing data onsocial contacts to estimate age-specific transmission parametersfor respiratory-spread infectious agentsrdquo American Journal ofEpidemiology vol 164 no 10 pp 936ndash944 2006

[26] C Yang X Shen Y Peng et al ldquoTransmission of Mycobac-terium tuberculosis in China a population-based molecularepidemiologic studyrdquo Clinical Infectious Diseases vol 61 no 2pp 219ndash227 2015

[27] Y PangW Kang A Zhao et al ldquoThe effect of bacille Calmette-Guerin vaccination at birth on immune response in ChinardquoVaccine vol 33 no 1 pp 209ndash213 2015

[28] F Abebe and G Bjune ldquoThe emergence of Beijing family geno-types of Mycobacterium tuberculosis and low-level protectionby bacille Calmette-Guerin (BCG) vaccines is there a linkrdquoClinical and Experimental Immunology vol 145 no 3 pp 389ndash397 2006

[29] A G Tsolaki S Gagneux A S Pym et al ldquoGenomic deletionsclassify the BeijingW strains as a distinct genetic lineage ofMycobacterium tuberculosisrdquo Journal of Clinical Microbiologyvol 43 no 7 pp 3185ndash3191 2005

[30] P W Smit T Vasankari H Aaltonen et al ldquoEnhanced tuber-culosis outbreak investigation using whole genome sequencingand IGRArdquo European Respiratory Journal vol 45 no 1 pp 276ndash279 2015

[31] J L Gardy J C Johnston S J Ho Sui et al ldquoWhole-genomesequencing and social-network analysis of a tuberculosis out-breakrdquo New England Journal of Medicine vol 364 no 8 pp730ndash739 2011

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Yunnan is located in southwestern China with an area of394100 square kilometers and a population of 469 millionpeople in 2013 Out of all inhabitants there are remarkable140 million inhabitants belonging to 55 different ethnicminority groups accounting for one-third of the wholeinhabitants In addition most of these minority inhabitantslive along the border with Myanmar Laos and VietnamAccording to the data from provincial surveillance systemfor TB the overall incidence of pulmonary TB was 56 casesper 100000 population in Yunnan [9] The diversity of TBburden was also observed across the different regions andthe TB incidences in several regions of ethnic enclaves werethree times higher than the average incidence [9] Thereforeit is meaningful to investigate the patientrsquos structure and drugsusceptibility ofMTB isolates circulating in Yunnan Provinceto highlight the geographic diversity of TB in ethnic enclavesUnfortunately limited data have been reported on this issueIn this study our aim was to characterize the genotypes ofMTB strains collected in Yunnan and to investigate the trans-mission profile of the predominant TB genotypes in this area

2 Materials and Methods

21 Ethics Statement This study was approved by the EthicsCommittee of the Yunnan Provincial Center for DiseaseControl and Prevention Patients were enrolled in the studyafter agreeing to and signing an Informed Consent form

22 Bacterial Isolates All smear-positive pulmonary TBpatients seeking health care at six prefectural TB dispensariesin 2014 were continuously enrolled in this study includingQujing Lijiang Dehong Lincang Puer and XishuangbannaDemographic information was collected from InformedConsent forms Two sputum samples from each patient wereused for mycobacteria culturing with Lowenstein-Jensen(L-J) medium One strain from each patient was collected forfurther molecular epidemiological study

23 DNA Extraction The crude genomic DNA of MTBisolates were extracted as previously reported [5] Brieflya loopful of colonies from L-J slants were transferred toa microcentrifuge tube containing 500 120583L Tris-EDTA (TE)buffer Followed by heating at 95∘C water bath for 30minthe suspension was centrifuged at 12000 rpm for 10min TheDNA in the supernatant was pipetted into a microcentrifugetube for further PCR amplification

24 Drug Susceptibility Testing (DST) The proportionalmethod was performed to determine the in vitro drugsusceptibility of MTB isolates against isoniazid (INH) andrifampicin (RFP) according to the recommendations bythe World Health Organization [4 9] The concentrationsof INH and RFP in the L-J medium were 02 120583gmL and40 120583gmL respectively The resistance of a strain against adrug was declared when the growth rate was higher than1 compared with the control The Yunnan Provincial TBReference Laboratory has passed the DST proficiency tests ofthe National TB Reference Laboratory since 2007 Multiple

drug resistant tuberculosis (MDR-TB) isolates were definedas strains being resistant to both INH and RFP

25 Genotyping Spoligotyping was performed using com-mercial kit manufactured by Isogen Bioscience BV (Maar-ssen Netherlands) [4]The genomicDNAwas amplified withprimers DRa (51015840-CCGAGAGGGGACGGAAAC-31015840) andDRb (51015840-GGTTTTGGGTCTGACGAC-31015840) The ampliconswere hybridized with a membrane and the final image wasdetected with a chemiluminescence system (AmershamBuckinghamshire United Kingdom) The original binarydata were entered in the SITVITWEB database to obtainthe Spoligotyping International Type (SIT) patterns and thesublineages of MTB isolates [10] In addition all the isolateswere genotyped with the 12-locus VNTR set with highdiscriminatory power described by Luo et al [11] The PCRproducts were analyzed using 15 agarose electrophoresis at5 Vcm for 1 hour The 100 bp DNA ladder and amplicons ofH37Rv were loaded per 7 lanes as a size marker to calculatethe repeat number of each locus The Hunter-GastonDiscriminatory Index (HGDI) was used to analyze thediscriminatory power of each VNTR locus [12] In additionthe genotyping patterns were clustered with BioNumericssoftware version 50 (Applied Maths Sint-Martens-LatemBelgium)

26 Data Analysis A chi-square test was performed to detectany significant difference between the two groups and thestatistical results were expressed as odds ratios (ORs) with95 confidence intervals (CIs) Two-sided 119875 value of lt005was declared as statistically significant All calculations wereanalyzed in SPSS 140 (SPSS Inc USA)

3 Results

31 Drug Susceptibility Profiles and Demographic Character-istics of Han and Minority Patients A total of 271 MTBisolates represented patients from different districts acrossYunnan Province (Figure 1) Out of these patients 196(723) were males and 75 were females The mean age ofthe patients was 498 plusmn 121 years (range 16ndash76 years) Inaddition all the 271 patients were classified as 11 differentethnic groups including 133 Han patients (491) and 138minority patients (509) Of these minority groups HaniYi and Dai were the most frequently observed accountingfor 111 (30271) 103 (28271) and 96 (26271) of all thepatients respectively We further analyzed the distribution ofdemographic and drug susceptibility characteristics betweenHan and minority patients As shown in Table 1 statisticalanalysis indicated that the proportion of minority patientsin Qujing (20 149) were significantly lower than that inPuer (713 6794) Xishuangbanna (700 2840) Lijiang(636 1422) Dehong (628 235) and Lincang (194631) respectively (119875 lt 005) Compared with Han patientsthe minority patients had higher proportion of farmers(884 versus 782 119875 = 002) In addition we foundthat the Han patients were significantly associated with RIFresistance (119875 = 003) while the rate of INH resistance showed


Dehong (35)

Lijiang (22)

Lincang (31)

Puer (94)

Qujing (49)

Xishuangbanna (40)

Beijingnon-Beijing








4 Discussion





































848

111111111111111111111111

190190190190190190

111111111111111111111111111111111111111111111

265111111111

16741674

1111111111111111

632111mdash1mdash111111111111111111

26911

269111111111111111111

2691166

37373737373737373737373737mdash53535353

Orphan373737373737

1545353373773737337mdashmdash373753


5832055353535353

5833737537

5337

Orphan1890



1835353425353

Orphan375373

2495353

249249249249

44444

179752

13753

11052170

535353

373


Y121Y118Y133Y113Y73

Y116Y258Y135Y105

Y3Y218Y256Y54Y5Y4

Y19Y18Y17Y16

Y120Y114Y255Y96

Y115Y156Y151Y225Y216Y272Y274Y130Y87

Y124Y141Y254Y241Y235Y127Y95Y88Y94Y2

Y268Y49Y24


Y112Y62

Y205Y64Y48Y43

Y248Y220Y259Y174Y169Y197Y103Y134Y45

Y177Y182Y165Y172Y161Y212Y195Y193Y192Y242Y223Y83

Y227Y69Y66

Y157Y38

Y171Y12Y52

Y184Y222Y136Y123Y228Y125Y265

Y9Y8Y7Y6

Y261Y20Y15Y10

Y263Y249Y126Y47Y35

Y199Y210Y201Y200Y232Y26Y28Y27Y33Y77Y74Y25

Y217Y119Y132Y109Y170Y167Y39

Y102Y122Y58Y34

Y219Y215Y203Y204Y191Y189Y188Y186Y185Y214Y211

Y1Y252Y251Y250Y238Y90Y79Y72Y42Y23Y37Y36

Y253Y107Y234Y176Y162Y181Y175Y166Y84

Y262Y224Y226Y147Y266Y70

Y237Y13

Y240Y239Y86

Y245Y91Y78Y76Y82Y14Y32Y97Y31

Y246Y40

Y271Y269Y80

Y243Y89

Y273Y63

Y257Y98Y41Y30Y29

Y244Y22Y21Y61Y57

Y233Y260Y55

Y270Y56

Y198Y128Y202Y213Y208Y247Y59

Y264Y75Y85

Y108Y106Y11

Y101Y117Y71

Y236Y67

Y180Y93Y92

Y160Y53Y51Y50

Y209Y190Y207Y206Y187Y46

Y194Y196Y229Y267Y221Y68Y60Y44Y99

Y100











Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Dehong (35)

Lijiang (22)

Lincang (31)

Puer (94)

Qujing (49)

Xishuangbanna (40)

Beijingnon-Beijing








4 Discussion





































848

111111111111111111111111

190190190190190190

111111111111111111111111111111111111111111111

265111111111

16741674

1111111111111111

632111mdash1mdash111111111111111111

26911

269111111111111111111

2691166

37373737373737373737373737mdash53535353

Orphan373737373737

1545353373773737337mdashmdash373753


5832055353535353

5833737537

5337

Orphan1890



1835353425353

Orphan375373

2495353

249249249249

44444

179752

13753

11052170

535353

373


Y121Y118Y133Y113Y73

Y116Y258Y135Y105

Y3Y218Y256Y54Y5Y4

Y19Y18Y17Y16

Y120Y114Y255Y96

Y115Y156Y151Y225Y216Y272Y274Y130Y87

Y124Y141Y254Y241Y235Y127Y95Y88Y94Y2

Y268Y49Y24


Y112Y62

Y205Y64Y48Y43

Y248Y220Y259Y174Y169Y197Y103Y134Y45

Y177Y182Y165Y172Y161Y212Y195Y193Y192Y242Y223Y83

Y227Y69Y66

Y157Y38

Y171Y12Y52

Y184Y222Y136Y123Y228Y125Y265

Y9Y8Y7Y6

Y261Y20Y15Y10

Y263Y249Y126Y47Y35

Y199Y210Y201Y200Y232Y26Y28Y27Y33Y77Y74Y25

Y217Y119Y132Y109Y170Y167Y39

Y102Y122Y58Y34

Y219Y215Y203Y204Y191Y189Y188Y186Y185Y214Y211

Y1Y252Y251Y250Y238Y90Y79Y72Y42Y23Y37Y36

Y253Y107Y234Y176Y162Y181Y175Y166Y84

Y262Y224Y226Y147Y266Y70

Y237Y13

Y240Y239Y86

Y245Y91Y78Y76Y82Y14Y32Y97Y31

Y246Y40

Y271Y269Y80

Y243Y89

Y273Y63

Y257Y98Y41Y30Y29

Y244Y22Y21Y61Y57

Y233Y260Y55

Y270Y56

Y198Y128Y202Y213Y208Y247Y59

Y264Y75Y85

Y108Y106Y11

Y101Y117Y71

Y236Y67

Y180Y93Y92

Y160Y53Y51Y50

Y209Y190Y207Y206Y187Y46

Y194Y196Y229Y267Y221Y68Y60Y44Y99

Y100











Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































848

111111111111111111111111

190190190190190190

111111111111111111111111111111111111111111111

265111111111

16741674

1111111111111111

632111mdash1mdash111111111111111111

26911

269111111111111111111

2691166

37373737373737373737373737mdash53535353

Orphan373737373737

1545353373773737337mdashmdash373753


5832055353535353

5833737537

5337

Orphan1890



1835353425353

Orphan375373

2495353

249249249249

44444

179752

13753

11052170

535353

373


Y121Y118Y133Y113Y73

Y116Y258Y135Y105

Y3Y218Y256Y54Y5Y4

Y19Y18Y17Y16

Y120Y114Y255Y96

Y115Y156Y151Y225Y216Y272Y274Y130Y87

Y124Y141Y254Y241Y235Y127Y95Y88Y94Y2

Y268Y49Y24


Y112Y62

Y205Y64Y48Y43

Y248Y220Y259Y174Y169Y197Y103Y134Y45

Y177Y182Y165Y172Y161Y212Y195Y193Y192Y242Y223Y83

Y227Y69Y66

Y157Y38

Y171Y12Y52

Y184Y222Y136Y123Y228Y125Y265

Y9Y8Y7Y6

Y261Y20Y15Y10

Y263Y249Y126Y47Y35

Y199Y210Y201Y200Y232Y26Y28Y27Y33Y77Y74Y25

Y217Y119Y132Y109Y170Y167Y39

Y102Y122Y58Y34

Y219Y215Y203Y204Y191Y189Y188Y186Y185Y214Y211

Y1Y252Y251Y250Y238Y90Y79Y72Y42Y23Y37Y36

Y253Y107Y234Y176Y162Y181Y175Y166Y84

Y262Y224Y226Y147Y266Y70

Y237Y13

Y240Y239Y86

Y245Y91Y78Y76Y82Y14Y32Y97Y31

Y246Y40

Y271Y269Y80

Y243Y89

Y273Y63

Y257Y98Y41Y30Y29

Y244Y22Y21Y61Y57

Y233Y260Y55

Y270Y56

Y198Y128Y202Y213Y208Y247Y59

Y264Y75Y85

Y108Y106Y11

Y101Y117Y71

Y236Y67

Y180Y93Y92

Y160Y53Y51Y50

Y209Y190Y207Y206Y187Y46

Y194Y196Y229Y267Y221Y68Y60Y44Y99

Y100











Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















First Insight into the Molecular Epidemiology of...

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