| Objective: To identify the genetic polymorphism and molecular epidemiological characteristics of the Mycobacterium tuberculosis clinical isolates in Shijiazhuang. To understand the current situation and genotype features of first-line anti-TB drug resistance of Mycobacterium tuberculosis clinical isolates in Shijiazhuang. This study provides basic science evidence to strengthen the prevention and control of tuberculosis in Shijiazhuang.Methods: We collected the Mycobacterium tuberculosis strains isolated from TB patients in the Fifth Hospital of Shijiazhuang, including the background information of patients, during January 2014 to October 2014. Then the strains were identified by differential PNB/TCH medium and multi-locus PCR method. Drug susceptibility testing of first-line anti-TB drug(streptomycin, isoniazid, rifampin, ethambutol) was performed using the L-J proportion method. Spacer oligonucleotide typing(Spoligotyping) and 15-loci VNTR(variable number tandem repeat) analysis were applied to genotype the strains. Cluster analysis was done by Bio Numerics software5.0 and SPSS software16.0 was used to conduct Hunter-Gaston distinguish Index(HGDI) and process the Chi-square test and t test.Results:1 Three hundred and seventy-three Mycobacterium isolates were finally collected, of which 367 strains were identified as Mycobacterium tuberculosis(MTB) and 6 strains were Non-tuberculous mycobacteria(NTM) by the differential PNB/TCH medium and multi locus PCR method.2 SpoligotypingThree hundred and sixty-seven MTB strains presented obvious genetic polymorphism. According to Spol DB4.0 database, 367 strains were divided into 2 genitic groups, Beijing family and non-Beijing family, with the proportion of 91.3%(335/367) and 5.7%(21/367), respectively. However, 11(3.0%) strains couldn’t be identified by Spol DB4.0 database, which might be new genotypes in Shijiazhuang. Among the Beijing family strains, 99.4%(333/335) was typical Beijing family, consisting of the absence of the first 34 spacer oligonucleotides and the presence of spacers 35 to 43. And the other strains were atypical Beijing family or Beijing-like family with 1 to 4 absence of spacers 35 to 43. Non-Beijing family showed high level of genetic polymorphism, including T, MANU, LAM and H family for 66.7%, 14.3%, 14.3% and 4.7% each. All the TB strains were classified into 29 genotypes, including 10 clusters and 147 unique patterns. The largest cluster was Beijing family genotype containing 331(90.2%) strains.3 VNTRA total of 194 different VNTR types were identified among the 367 strains. Two hundred twenty-one(60.22%) strains were grouped into 48 clusters and 146(39.78%) strains were unique patterns. Beijing family strains were grouped into 161 genotypes, of which 219(65.37%) strains were classified into 45 clusters and 116(34.63%) were unique patterns. The HGDI value of 15-loci VNTR for all stains was 0.9838, and it reached 0.9853 when combined with Spoligotyping.4 The total rate of TB drug resistance in Shijiazhuang was 31.61%(116/367). The initial and the acquired drug resistance rate were 24.47%(69/282) and 55.29%(47/85), respectively. The acquired drug resistance rate was statistically significantly higher than the initial drug resistance rate(χ2=28.71, P<0.05). Among the drug resistant strains, 36 strains(9.80%) were resistant to just one drug and 80 strains(21.81%) were resistant to at least 2 drug.5 According to the results of cluster analysis, 367 strains were divided into cluster type and individual type. By the drug sucpetibility result, all strains were grouped into sensitive group and resistant group. The result showed that the distribution of resistant group strains in cluster type and individual type were statistically significantly different(χ2=0.084, P=0.773). There was no correlation between cluster type and individual type in gender, age and treatment history, respectively(P>0.05).Conclusion:1 The Mycobacterial tuberculosis strains in Shijiazhuang exhibit high genetic polymorphism and the Beijing family is the dominant epidemic genotype. We discovered 11 new Spoligotyping genotypes, which could enrich the information of Spol DB4.0 database.2 15-loci VNTR genotyping has higher discriminatory power than Spoligotyping. The combination of 15-loci VNTR and Spoligotyping possesses the highest discriminatory power and can comprehensively analyze genetic polymorphism of Mycobacterium tuberculosis. The 15 VNTR locus we applied are suitable for genotyping analysis of TB strains in Shijiazhuang.3 The total drug resistance rate and initial drug resistance rate of Shijiazhuang are flat with the national average level, but the acquired drug resistance rate is at a high level. The drug resistant situation of TB strains in Shijiazhuang remains serious and we should strengthen the momitoring and management of drug-resistant TB population and improve patient treatment compliance. At the same time, standardized clinical use of antibiotics should be emphasized to reduce the incidence of acquired drug resistance of TB patients.4 For all strains, there is no correlation between the cluster type and the individual type in gender, age and treatment history of patients. The result prompts that there may be some other factors, which has not been found at present, led to the widespread of the TB strains and further research is needed in the future. |
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