摘要
目的 调查云南省昭通地区结核分枝杆菌(Mycobacterium tuberculosis,MTB)临床分离株的遗传多样性和耐药分子特征.方法 2022年9月—2023年8月从昆明医科大学附属昭通医院收治的298例涂阳肺结核患者中共采集了MTB 298株,用MeltPro TB测定法通过荧光聚合酶链反应(polymerase chain reaction,PCR)熔解曲线法鉴定所有分离株.用多色熔解曲线分析技术进行间隔区寡核苷酸分型(interval oligonucleotide typing,Spoligotyping).用分枝杆菌散布重复单元(mycobcterial interspersed repetitive units,MIRU)-可变数目串联重复序列分型(variable number of tandem repeat typing,VNTR)(MIRU-VNTR)24位点分型法进行基因分型.使用PCR和靶向测序检测耐药基因突变.结果 北京菌株占所有MTB菌株的69.80％(208/298).其他谱系包括T、LAM、MANU2、CAS、NEW-1和SITVITWEB数据库中未发现的孤儿型,分别占5.70％(17/298)、3.36％(10/298)、0.67％(2/298)、0.34％(1/298)、8.39％(25/298)及11.74％(35/298).北京菌株对左氧氟沙星的耐药率略高于非北京菌株(P=0.042),而北京菌株与非北京菌株2种基因型菌株对异烟肼、利福平、乙胺丁醇、链霉素、吡嗪酰胺、卡那霉素耐药率差异无统计学意义(P>0.05).MIRU-VNTR 24位点分型分析结果显示,33个菌株可分为13株聚类,其聚类率为39.39％.每个等位基因的Hunter-Gaston指数在0～0.814之间,其中18个位点对非北京菌株具有中高鉴别能力.但只有10个位点对北京菌株具有中高鉴别能力.结论 MTB菌株在中国云南省昭通地区表现出较高的遗传多样性,北京菌株是MTB和耐药结核病菌株的优势谱系.
Abstract
Objective To investigate the genetic diversity and drug resistance of clinical strains of Mycobacterium tuberculosis (MTB) in Zhaotong, Yunnan Province. Methods From September 2022 to August 2023, 298 clinical isolates of MTB from smear-positive pulmonary tuberculosis patients in The ZhaoTong Affiliated Hospital of Kunming Medical University were collected. MeltPro TB assay was used to identify all isolates by fluorescence PCR meltdown curve. Spoligotyping analysis was performed using the polychromatic melting curve analysis technique. Genotyping was performed by Mcspoligotping and Mycobacteria interspersing repetitive unit Variable Tandem repeats (MIRU-VNTR) 24-site typing. Drug-resistant gene mutations were detected using PCR and targeted sequencing. Results The Beijing strain accounted for 69.80％ (208/298) of all MTB strains. Other lineages include T, LAM, MANU2, CAS, NEW-1, and orphan types not found in the SITVITWEB database. They were 5.70％ (17/298), 3.36％ (10/298), 0.67％ (2/298), 0.34％ (1/298), 8.39％ (25/298) and 11.74％ (35/298), respectively. The resistance of the Beijing strain to levofloxacin was slightly higher than that of the non-Beijing strains (P=0.042), but there was no significant difference in isoniazid, rifampicin, ethambutol, streptomycin, pyrazinamide, kanamycin and other resistance profiles between the 2 genotypes (P >0.05). The results of MIRU-VNTR 24 locus typing showed that the 33 strains could be grouped into 13 clusters, and the clustering rate was 39.39％. According to the Hunter-Gaston discriminant index, the diversity of each allele varied significantly from 0 to 0.814, with 18 loci having medium-high discriminability against non-Beijing strains. However, only 10 loci had medium-high discriminability against the Beijing strain. Conclusion MTB strains in Zhaotong, Yunnan, China, show a high genetic diversity. The Beijing strain is the dominant lineage of MTB and drug-resistant TB strains.
基金项目
云南省科技厅科技项目(202201AY070001-219)
NETL
NSTL
万方数据