Wan Kang-lin

National Institute for Communicable Disease Control and Prevention

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Xiuqin Zhao

National Institute for Communicable Disease Control and Prevention

Zhiguang Liu

Northwest A&F University

Haican Liu

National Institute for Communicable Disease Control and Prevention

Xiuqin Zhao

Chinese Academy of Agricultural Sciences

Bing Lü

Affiliated Hospital of Guizhou Medical University

Haiyan Dong

University of Science and Technology of China

Bing Lv

Chinese Center For Disease Control and Prevention

Yi Jiang

Huazhong Agricultural University

Jinghua Liu

Southern Medical University

Machao Li

National Institute for Communicable Disease Control and Prevention

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Capital Medical University

Chinese Academy of Medical Sciences & Peking Union Medical College

National Institute for Communicable Disease Control and Prevention

Fudan University

Sun Yat-sen University

Shanghai Jiao Tong University

Sichuan University

Chinese Academy of Sciences

Chinese Center For Disease Control and Prevention

Center for Disease Control

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Analysis on the 228 Mycobacterium Tuberculosis with MLVA

China Medical Herald (2010)

Wan Kang-lin

Objective:By Multiple locus variable numbers of tandem repeats(MLVA) genotyping,to analyze the genotypes of epidemic Mycobacterium tuberculosis strains in Gansu. Methods:15 standardized MLVA loci were used to type the clinical isolated M. tuberculosis strains of Gansu. Results were analyzed with BioNumerics 4.5. The dendrogram was summarized from the fingerprintings. Results:The result of MLVA showed that 228 strains of M. tuberculosis were classified 4 major genetic groups and 7 main genotypes. The largest genotype included 171(75.0%) strains,the others were 13(5.7%) ,3(1.3%) ,7(3.1%) ,1(0.4%) ,31(13.6%) and 2(0.9%) respectively. Farther particulars,93 strains(40.8%) were belong to unique genotype. The other strains contained 2-10 M. tuberculosis,all 132 geno-clusters. Conclusion:The bacterium strains of M. tuberculosi in Gansu has a rich nucleotide polymorphisms. MLVA is very useful to type the genotype of M. tuberculosi strains. Beijing family genotype strain is the dominant genotype in Gansu.

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Characterization of Mycobacterium tuberculosis isolates from patients in Gansu by multiple locus variable numbers of tandem repeats.

Zhongguo renshougonghuanbing zazhi (2010)

Chongxiang Tong Xiuqin Zhao Jianjun Ma Zhiguang Liu Cao WenJiang

To analyze the genotype of epidemic Mycobacterium tuberculosis strains by multiple-locus variable-number tandem repeat analysis(MLVA)in Gansu from 2004 to 2006,15 standardized VNTR loci were selected to type the clinical isolated M.tuberculosis strains.Results were analyzed by BioNumerics 4.5 and the dendrogram was summarized from finger printings.Result of MLVA showed that the 228 strains of M.tuberculosis were classified into 4 major genetic groups and 7 main genotypes,which including strains of 13(5.7%),3(1.3%),7(3.1%),1(0.4%),171(75%),31(13.6%)and 2(0.9%),respectively.Furtherly,228 strains were divided into 132 geno-clusters,among which there were 93 strains belonged to unique genotype.Results indicate that the bacterial strains of M.tb in Gansu are rich in genetic polymorphism and MLVA is very useful for typing the genotype of M.tuberculosis strains.Beijing family was the dominant genotype in Gansu.

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Establishment and evaluation of a polymerase Chain Reaction technique for detection Mycobacterium tuberculosis in sputum samples from suspect patients with tuberculosis

Zhongguo renshougonghuanbing zazhi (2008)

Wan Kang-lin

To establish the polymerase chain reaction (PCR) for detection of sputum samples of TB, and evaluate its perspective in clinical application. Designed a pair of primers (INS1 INS2) according to the insertion sequence 6110 (IS6110) of Mycobacterium tuberculosis complex (MtbC), and establish PCR reaction condition. The MtbC standard strains, standard Mycobacterium strains and clinical sputum samples of suspected patients with TB were detected by the PCR method. The results were compared to those by smear slide microscopy and culture with chi-square test of statistics analysis. It was found that the minimum detection quantity to M. tuberculosis, M. bovis, BCG was 102,103,103 bacteria/mL respectively, and the MtbC could be detected specifically. Among 574 tested cases, 241 (42%) were positive by PCR. The positive rate of smear slide and culture were 19.69% and 26.31%, respcetively. The positive rate of PCR was much higher than that of bacteriological methods including smear and culture. There was significant difference by means of chi-square statistics analysis (χ2=103.67,P0.01). Using the results of culture as gold standard, the sensitivity(Se)of PCR was 67.53%. It is evident that PCR used for the direction of MtbC appears to be a method for large scale screening and clinical detection of MtbC.

Mycobacterium tuberculosis complex

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Analysis on genotyping of 159 strains of Mycobacterium tuberculosis isolated clinically in some areas of China with MLVA-19-Loci.

Jibing jiance (2009)

Bing Lv Mei Liu Zhaona Li Zhiguang Liu Xiuqin Zhao

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19-VNTR loci used in genotyping Chinese clinical Mycobacterium tuberculosis complex strains and in association with spoligotyping

Journal of Basic Microbiology (2013)

Yi Jiang Haican Liu Huajun Zheng Xiangfeng Dou Biao Tang

Recently, tandem repeat typing has emerged as a rapid and easy method for the molecular epidemiology of the Mycobacterium tuberculosis (M. tuberculosis) complex. In this study, a collection of 19 VNTRs incorporating 15 previously described loci and 4 newly evaluated markers were used to genotype 206 Chinese M. tuberculosis isolates and 9 BCG strains. The discriminatory power was evaluated and compared with that obtained by Spoligotyping. It turned out that 15-locus VNTR could be very useful in M. tuberculosis complex strains genotyping in China. The 4 newly evaluated loci were proved informative and could be useful for future epidemiology studies, especially in Beijing family strains. In addition, a unique pattern of the latter 4 loci were found in Chinese BCG strains.

Molecular Epidemiology

10.1002/jobm.201200230

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Relationship between EMB phenotypic drug resistance and embB mutation

Wan Kang-lin

Objective:To analyze the relationship between genotype resistance and phenotype resistance of Ethambutol(EMB),the correlation between mutation and multi-drug resistant tuberculosis(MDR-TB) and to make use of Met306 mutation to detect MDR-TB.Methods:Totally 301 isolates collected in China were tested by drug sensitive test(DST) and sequencing in the drug resistant determined region(DRDR) of embB,katG and rpoB.Results:The mutation rate between the EMB-resistance(48.5%) and EMB-susceptibility(39.1%) was no significance(P0.05),however it was found statistics difference(P0.05) not only to MDR-TB but also the numbers of drugs.Moreover for MDR-TB,the coincidence rate between Met306 combined rpoB mutation and phenotypic resistance increased from 80% to 93.3%,meanwhile the rate between Met306 combined katG mutation and phenotypic resistance increased from 80% to 86.7%.Met306 combined rpoB and katG could detect 97.8% MDR-TB.Conclusion:Mutations in Met306 could not be the EMB molecular marker.As a new MDR-TB marker,combined rpoB-the acknowledged MDR molecular marker,it would largely improve the checkout rate of MDR.

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Analysis on the characteristics of rpsL gene mutations in Streptomycin resistance isolates of Mycobacterium tuberculosis

Wan Kang-lin

The objective was to understand the characteristics of rpsL gene mutation in Streptomycin resistant clinical isolates of Mycobacterium tuberculosis(M.tuberculosis) in China,and evaluate the application of rapid detection of rpsL gene mutation by PCR.The 302 M.tuberculosis clinical isolates were detected by drug sensitivity test(DST),and the rpsL was detected by PCR-directly sequence(PCR-DS).Of 59 strains were pan-susceptible to 5 drugs including INH,RFP,SM,EMB and PAS,and 61 strains were sensitive to streptomycin(SM) but resistant to the other drugs.The mutation rate of SM susceptible isolates was 5.00%(6/120).In the 182 SM resistant isolates,the rpsL mutation rate was 70.33%,and the mutation rate of codon43 Lys→Arg was 52.20%;there were 4 mutation types in codon 88,and the mutation rate was 20.33%;the mutation rate of codon 86 Arg→Gln was 0.55%.The rpsL gene mutation rate of SM resistant strains was significantly higher than that of SM-sensitive strains χ2=125.04,P0.05.In conclusion,the rpsL mutation in M.tuberculosis is highly associated with Streptomycin resistance,and codon 43 Lys→Arg mutation is the main and important type.Meanwhile,the PCR-DS could be applied to rapid detection of rpsL mutation in clinical M.tuberculosis isolates for identifying SM susceptibility.

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First Insight into the Genotypic Diversity of Clinical Mycobacterium tuberculosis Isolates from Gansu Province, China

PLoS ONE (2014)

Jie Liu Chongxiang Tong Jiao Liu Yuan Jiang Xiuqin Zhao

Background Investigations of Mycobacterium tuberculosis genetic diversity in China have indicated a significant regional distribution. The aim of this study was to characterize the genotypes of clinical M. tuberculosis isolates obtained from Gansu, which has a special geographic location in China. Methodology/Principal Findings A total of 467 clinical M. tuberculosis strains isolated in Gansu Province were genotyped by 15-locus mycobacterial interspersed repetitive units–variable number tandem repeats (MIRU-VNTR) and spoligotyping. The results showed that 445 isolates belonged to six known spoligotype lineages, whereas 22 isolates were unknown. The Beijing genotype was the most prevalent (87.58%, n = 409), while the shared type 1 was the dominant genotype (80.94%, n = 378). The second most common lineage was the T lineage, with 25 isolates (5.35%), followed by the H lineage with 5 isolates (1.07%), the MANU family (0.64%, 3 isolates), the U family (0.43%, 2 isolates) and the CAS lineage with 1 isolate (0.21%). By using the VNTR15China method, we observed 15 groups and 228 genotypes among the 467 isolates. We found no association between the five larger groups (including the Beijing genotype) and sex, age, or treatment status, and there was no noticeable difference in the group analysis in different areas. In the present study, seven of the 15 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. Conclusions/Significance The Beijing genotype is the predominant genotype in Gansu province. We confirm that VNTR15China is suitable for typing Beijing strains in China and that it has a better discriminatory power than spoligotyping. Therefore, the use of both methods is the most suitable for genotyping analysis of M. tuberculosis.

10.1371/journal.pone.0099357

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Genetic diversity of Mycobacterium tuberculosis isolates from Beijing, China assessed by Spoligotyping, LSPs and VNTR profiles

BMC Infectious Diseases (2012)

Bing Lü Ping Zhao Binbin Liu Haiyan Dong Yu Qin

Abstract Background Tuberculosis is one of the most infectious diseases in the world. Molecular typing methods such as spoligotyping, and VNTR (variable number tandem repeats), IS 6110 in the NTF region and LSP (large sequence polymorphisms) analysis are generally useful tools for the resolution of various issues related to the classical epidemiology of Mycobacterium tuberculosis ( M. tuberculosis ). Methods To determine the transmission characteristics of M. tuberculosis strains isolated in Beijing, China, and their genetic relationships, especially those among Beijing family strains, 260 M. tuberculosis strains isolated from patients presenting pulmonary tuberculosis were analyzed by spoligotyping, and by examining 22 VNTR loci and the presence/absence of IS 6110 in the NTF region, RD105 and RD181. Results 81% (211 strains) of the isolates studied were Beijing family strains, 174 (82.5%) of which were identified as modern Beijing strains based on the presence of IS 6110 upstream of the NTF region. RD181 was intact in 9 of the other 37 (17.5%) ancestral Beijing strains. The percentage of Beijing family strains in this study was consistent with previous reports. There are many differences, however, in allele diversity among VNTR loci between reports on strains from different areas. Conclusions The Beijing family is the most prevalent genotype in Beijing city and the predominance of Beijing family strains has not altered in almost twenty years. Differences in the alleles and discrimination ability of VNTR loci between different regions is likely due to population differences in the regions where these M. tuberculosis strains were isolated or to differences in sampling times.

Molecular Epidemiology

10.1186/1471-2334-12-372

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Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China

Journal of Clinical Microbiology (2010)

Haiyan Dong Zhiguang Liu Bing Lv Yuanyuan Zhang Jie Liu

A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.

10.1128/jcm.00549-10

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