Preliminary Study On Genotyping Of Clinical Isolates Of Mycobacterium Tuberculosis From Tibet, China

Tuberculosis is one of the infectious diseases which still threats people'shealth heavily. In recent years, because of some causation, such as drugresistance of Mycobacterium tuberculosis (MTB), tuberculosis (TB) controlignored in many countries and areas, and so on, the situation of the contagiousdisease in the world becomes more and more severe. Tuberculosis has been avery important problem of public health in the world now.Epidemiological study has been developed to surveillance the outbreak of infection at the molecular level of pathogen to track the spread of someMycobacterium tuberculosis strains and discover the mechanism of thetransmission of the tuberculosis. Typing identification of the strains from thepatients with tubercolusis plays a key role in tracing the source of infection.Traditional typing methods, including phage-typing, drug sensitivity typing,cell protein electrophoresis, biochemical diversity analysis, multi-loci enzymeelectrophoresis, et al, exist some limitations. With the development ofmolecular biological techniques, the molecular genotyping method hasbecome the major tool in the epidemiological study of tuberculosis.Since 1980, the special identification methods based on sequence for strainshas been gradually built, for example, restriction fragment length polymorphism(RFLP), the analysis of DNA fingerprint pattern, pulsed field of gelelectrophoresis (PFGE), the DNA polymorphism of random-PCR, DNAsequencing and gene chip, et al. IS6110-RFLP is a method based on therestriction fragment length polymorphism of the insertion sequence 6110(IS6110). IS6110 is an insert sequence, only exists in the genome ofMycobacterium tuberculosis complex. The number and distribution of IS6110elements within the genome of M. tuberculosis are different between strains oftuberculosis. Every strain has unique DNA fingerprint pattern, soIS6110-RFLP typing has been recommended the gold standard of DNA typingmethod. Both spacer oligotyping (Spoligotyping), and multiple loci variablenumber tandem repeat analysis (MLVA) built recently are based onpolymerase chain reaction (PCR). These methods are easy and fast.To understand the molecular epidemical character of the genotypedistribution of Mycobacterium tuberculosis collected from Tibet, six areas inTibet were selected to investigate tuberculosis with the unified questionnaire.From Jan. 2005 to Jan. 2006, All suspicious patients infected with TBcoming in the centre for tuberculosis prevention and control were investigated, from which the relation clinical data and the sputum were collected.Mycobacterium tuberculosis was isolated from the sputum samples by meansof the Lowenstein-Jensen culture. Then the bacteria were identified withbiochemical test, The drug susceptibility was tested by absolute concentration.Molecular typing identification of the strains was done by means ofIS6110-RFLP, MLVA, and Spoligotyping.By the biochemical identification, the bacterium of 216 clinical isolates inour study is Mycobacterium tuberculosis, of which 74 strains (34.25%) areabsolutely sensitive to INH, RFP, EMB and SM, the other strains (65.74%) aredrug resistant strains. The drug resistance strains are including 24 strains(11.11%) of single drug resistance, 118 strains (54.63%) more than two drugsresistance or multi-drugs resistance strains; 43 strains (30.28%) of the initialresistance strains and 99 (69.72%)strains the secondary resistance ones iscases.By means of IS6110-RFLP, according to the comparability coefficientamong strains, 216 strains were typed into 14 Gene groups named as Group A,B, C, D, E, F, G, H, I, J, K, L, M and N, displaying 214 genetic types. Therewere 4 strains (1.85%) in Group A, including 4 genotypes; 161 strains (74.07%)in Group C, including 160 genotypes in which one genotype included 2 strains,showing one cluster; 22 strains (10.19%) in Group D, including 22 genotypes;8 strains (3.7%) in Group E, including 8 genotypes; 6 strains (2.78%) inGroup F, including 6 genotypes; 5 strains (2.31%) in Group J, including 5genotypes; 3 strains (1.4%) in Group M, including 2 genotypes in which onegenotype included 2 strains, showing one cluster; 1 strain (0.46%) in Group B,G, H, I, K, L and N, 1 genotype, respectively.According to the results of this study, the number of IS6110 copies inthese strains ranged from 1 to 19 copies. The maximum number of RFLPfingerprinting was 11 copies and 12 copies which were found in 43 strains (19.9%) respectively. The second fingerprinting number was 10 copies and 13copies which were found in 31 strains (14.4%) respectively. The others were 9copies found in 18 strains occupies (8.3%); 14 copies found in 12 strains(5.6%);15 copies found in 11 strains (5.1%);16 copies found in 8 strains(3.7%); 17 copies found in 4 strains (1.9%); 6 copies, 8 copies and 19 copies,found in 3 strains (1.4%) respectively; 5 copies and 2 copies found in 2 strains(0.9%) respectively; 7 copies and 1 copies found in 1 strains (0.5%)respectively. But zero copy was not found.By means of Spoligotyping, 216 strains were typed into 5 Gene groupnamed as GroupⅠ,Ⅱ,Ⅲ,ⅣandⅤ, displaying 24 genotypes. There were 16strains (7.41%) in GroupⅠ, including 9 genotypes in which there were 2, 2, 4,and 3 strains in 4 genotypes, showing 4 clusters respectively; 3 strains (1.38%)in GroupⅡ, including 3 genotypes; 176 strains (81.48%) in GroupⅢ,including 7 genotypes in which there 170 strains in one genotype, showing 1cluster; 19 strains (8.80%) in GroupⅣ, including 4 genotypes in which therewere 17 and 2 strains in 2 genotypes, showing 2 clusters respectively; 2 strains(0.9%) in GroupⅤ, including 1 genotype, showing 1 cluster. The most groupof 5 groups was GroupⅢincluding 176 strains, representing the characteristicof Spoligotyping of which the pattern is the absence spacers of 1 to 34,displaying negative, and that is the characteristics of MTB Beijing family. Thehybridization of 9 spacers from 35 to 43 spacers displays positive responsewhich is the characteristic of Typical Beijing family. Beside, there were 6strains in which the hybridization of 9 spacers from 35 to 43 displays positiveresponse on 8 spacers, while one spacer was negative hybridization,distinguishing from Typical Beijing genotype strains, called Beijing-likegenotype, or Atypical Beijing family.In Beijing family strains, there were 77 strains (43.75%) from the patientswith BCG inoculated, while 99 strains (56.25%) from the patients without BCG inoculated. There was no significant difference in statistics (x~2=1.007, P＞0.05). It was confirmed that there might be no relation betweenBeijin family of Mycobacterium tuberculosis and BCG inoculated. In Beijingfamily strains there were 60 sensitive strains (34.09%), and 116 drugresistance strains (65.91%), there was no significant difference in statistics(x~2=19.209, P＞0.05) between the drug resistant and sensitive, and there wereno significant difference in the distribution of between age groups (x~2=22.296, P＞0.05), sexes (x~2=0.815, P＞0.05), and the areas in Tibet (x~2=0.686,P＞0.05), It was confirmed that That is to say the Beijing family had nocorrelation to the drug resistance, age, sex, and area.By means of MLVA, 216 clinical isolates of mycobacterium tuberculosiswere typed into 19 Gene group named as Group a, b, c, d, e, f, g, h, i, j, k, l, m,n, o, p, q, r and s, displaying 108 genotypes. There were 2 strains (0.9%) inGroup a and Group b, including 2 genotypes respectively; The e group has 4strains (1.8%) in Group e, including 4 genotypes; 2 strains (0.9%) in Group i,including 1 genotype, showing 1 cluster; 187 strains (86.57%) in Group j,including 80 genotypes, in which there were 3, 9, 2, 4, 3, 2, 2, 5, 2, 2, 4, 15, 14,4, 5, 12, 21, 2, 2, 2, 3, 6, 2, 4 and 2 strains in 25 genotypes sharing 132 strains,showing 25 clusters respectively; 2 strains (0.9%) in Group 1, including 2genotypes; 3 strains (1.3%) in Group n, including 3 genotypes; 3 strains (1.3%)in Group o, including 2 genotypes, in which there were 2 strains in 1 genotype,showing 1 cluster; group has only one strain (0.45%) in Group c, d, f, g, h,k, m, p, q, r and s, including 1 genotype respectively.According to the results of this study, in the 20 VNTR loci, Mtub21 andETR-E show high diversity, MIRU26, Mtub30, ETR-A, MIRU10, MIRU23,MIRU16, Mtub02, MIRU39 and MIRU40 show moderate diversity, and theother 7 loci show low diversity. The Mtub02 can identify the Beijing familystrains and non-Beijing family strains, of which the results completely correspond to that by Spoligotyping.The three methods evaluated by HGI: identification on the level of strain,the discriminative power of MLVA is close to that of IS6110-RFLP, the HGIis 97.5%, and 99.99% respectively. While the discriminative power ofSpoligotyping is very low, the Spoligotyping's HGI is 37.63%.The genotype has been studied with 216 clinical isolates ofmycobacterium tuberculosis from Tibet by means of the IS6110-RFLP,Spoligotyping and MLVA respectively. The results showed: by IS6110-RFLP,216 strains could be classified to 214 genotypes, including 212 strains(98.14%) were classified 212 genotypes, 4 strains (1.85%) were classified 2genotypes in which two strains showed one copy of IS6110. It suggests thatIS6110-RFLP is hard to identify strains in which there are few copies on thelevel of strain. By Spoligotyping, 216 strains could be classified to 24genotypes in which there was 1 strain in each type of 16 types. 2 strains inwhich there was one copy of IS6110 were identified into 2 genotypes. Andby MLVA, 216 strains could be classified to 108 genotypes with differentfingerprint pattem of VNTR, showing evident polymorphism of gene, inwhich 80 strains (37.03%) was classified 80 genotypes, 136 strains (62.96%)displayed 28 genotypes. Moreover, 2 strains in which there was one copy ofIS6110 were identified into 2 genotypes. The results of the study showed: Thediscriminative power of Spoligotyping is much lower than that ofIS6110-RFLP and MLVA on the level of strain to identify mycobacteriumtuberculosis. However, if the three methods are combined to use for theidentification of mycobacterium tuberculosis strains, the power is obviouslyhigher than that of them used alone, in 216 strains only 2 strains could not beidentified on the level of strain. In addition, Spoligotyping is good method onidentification of mycobacterium tuberculosis Beijing family.Generally, IS6110-RFLP is the best in the three methods for identification ability on the level of strain, and is recommended as the standard method ofgenotyping technique, but it needs a lot of DNA (about 2μg) and the process iscomplicated, the discriminatory is low when the copy of IS6110 is few in thegenome of a strain. Spoligotyping is the method based on PCR which has themerit of easy and fast, and needing few DNA. It can be used to detect theclinical species directly, and the results are easy to identify. The method hashigh discriminatory ability to detect the strains of few IS6110-RFLP copies.So it is accepted as an valuable method to further detect and identify theMycobacterium tuberculosis, especially to identify the Beijing family strains.The discriminative power of MLVA is close to that of IS6110-RFLP, theadvantaged side of MLVA is that the method based on PCR is easy and fast(It only needs one day to produce the results), needing no special apparatus.Furthermore MLVA can be applied to comparative study between laboratories.By this study, of 216 Mycobacterium tuberculosis clinical isolates Beijingfamily strains occupied 81.48%. It is preliminarily confirmed that Beijingfamily strains is main genotype and main prevalence strain in Tibet. There isno evident relation between Mycobacterium tuberculosis Beijing family andthe vaccination of BCG and drug resistance.