| Objectives To investigate the distribution of second-line anti-tuberculosis drug resistance phenotypes and gene mutations associated with drug resistance of Mycobacterium tuberculosis in Hebei Province,and to analyze the genotyping characteristics of second-line anti-tuberculosis drug resistant strains.To explore the relationship among phenotypes,drug resistance related gene mutations and genotypes of second line anti-tuberculous drug resistant strains.It provides a scientific basis for understanding the epidemic trend of tuberculosis,formulating prevention and control measures,rapidly diagnosing drug-resistant tuberculosis,and optimizing anti-tuberculosis treatment program.Methods Three hundred and sixteen drug-resistant Mycobacterium tuberculosis isolates(referring to at least resistance to one of first line anti-tuberculous drugs,including isoniazid,rifampicin,ethambutol and streptomycin)and 100 pan-sensitive Mycobacterium tuberculosis isolates were revived.Traditional drug susceptibility test of second-line antuberculosis drugs was performed for all successful resuscitating strains.DNA sequencing technique was used to analyzing mutations in five genes associated with resistance to second-line anti-tuberculosis drugs: gyrA and gyrB for fluoroquinolones;rrs,eis promoter and tlyA for second injectable line drugs.All strains were genotyped by Spoligotyping and MIRU-VNTR.Results 1 A total of 416 Mycobacterium tuberculosis strains were included in this study,of which 80 strains were excluded from the study because of contamination or unsuccessful resuscitation.The remaining 336 Mycobacterium tuberculosis strains were included in this study,including 257 drug-resistant strains and 79 pan-sensitive strains.2 Among 257 drug-resistant strains,51.75% and 1.56% were sensitive and resistant to all five second-line anti-tuberculous drugs,respectively.In addition,the resistance rates of ofloxacin,levofloxacin,kanamycin,amikacin and capreomycin were 32.68%,21.79%,20.23%,7.00% and 6.23%,respectively.3 The resistance rates of fluoroquinolones in newly and re-treated tuberculosis patients from Hebei Province were 22.76% and 45.54%,respectively.4 Of 97 fluoroquinolone-resistant strains,84.54% had gyrA gene mutations and 7.22% had gyrB gene mutations.Besides,double mutations at gyrA and gyrB were detected three isolates.5 Among 63 strains resistant to second-line injectable drugs,the mutation frequencies of rrs,eis promoter and tlyA were 38.10%,7.94% and 4.86%,respectively.6 Compared with drug susceptibility results,the sensitivity and specificity of combining detection of gyrA and gyrB for diagnosis of fluoroquinolones phenotypes was 79.38% and 89.38%,respectively.The sensitivity and specificity of combining detection of rrs,eis promoter region and tlyA for predicting second-line injectable drugs phenotypes was 50.79% and 97.42%,respectively.7 The resistance rates of five second-line antuberculosis drugs between Beijing genotype and non-Beijing genotype had no significance difference on statistical analysis(P>0.05).Furthermore,the mutation frequencies of gene mutation associated with drug resistance to five second-line antuberculosis drugs between Beijing genotype and non-Beijing genotype had no significance difference on statistical analysis(P>0.05).8 Among 124 strains resistant to second-line anti-tuberculous drugs,113 genotypes were found,including 107 unique genotypes and 6 clusters,each cluster contained 2-4 strains.The clustering rate and recent transmission rate were 13.71% and 4.84%,respectively.All the cluster strains were Beijing genotype.Conclusions 1 The drug resistance rate of the five second-line anti-tuberculous drugs was ofloxacin,levofloxacin,kanamycin,amikacin and capreomycin from high to low,in which there was cross resistance between two fluoroquinolones and among three secondline injectable drugs showed cross resistance,but not to the same extent.Therefore,it is necessary to carry out second-line anti-tuberculosis drug susceptibility test for developing a scientific and effective treatment regimen.2 The high drug resistance rate of fluoroquinolones among retreated tuberculosis patients in Hebei Province,indicating that we should strengthen the drug resistance supervision of fluoroquinolone in order to avoid the development of extensively drug-resistant tuberculosis.3 gyrA gene mutation at codons 89-94 was the main mechanism of fluoroquinolone-resistance in our region.Double mutations at gyrA and gyrB can be used as very specific mutations of fluoroquinoloneresistant strains.4 Mutation in rrs A1401 G has a reference value for the rapid diagnosis of second-line injectable drug resistance phenotypes.5 The transmission of second-line antituberculous drug-resistant strains is mainly due to endogenous reburning in Hebei Province,but there is a certain proportion of recent transmission,the recent transmission is mainly on small-scale distribution.Figure 5,Table 14,Reference 125... |
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