可变数目串联重复序列和全基因组测序在一起学校结核传播中的应用

Application of VNTR and WGS in tracing the transmission of Mycobacterium tuberculosis in a tuberculosis outbreak in a school setting

目的利用可变数目串联重复序列(variable number tandem repeats,VNTR)和全基因组测序(whole genome sequencing,WGS)分析一起学校结核病疫情的传播情况,探讨2种基因分型方法在处理学校结核传播中的作用。方法对2019年北京市首起学校耐多药肺结核聚集性疫情中获得的6株菌株分别进行菌种鉴定、间隔区寡核苷酸分型(Spoligotyping)、VNTR以及WGS分析,分析菌株的分型特征,确定传播链。结果6株阳性培养菌株共涉及2个年级4个班的6名学生,其中1人为首发病例,3人是首发病例的密切接触者,另外2人为一般接触者。经菌种鉴定6株菌株均为结核分枝杆菌。Spoligotyping分型结果显示5株菌株为北京基因型,另1株无结果。VNTR基因分型将6株菌株分成3个簇,菌株成簇率为66.7%,最大的一簇包含4株菌株,基因型相同,表明存在一定程度的近期传播。其余2株为单一菌株,分别在1~2个位点上与其他4株菌相差1.0~1.6个拷贝。WGS结果显示6株菌株间基因组单核苷酸多态性(single nucleotide polymorphisms,SNP)差异均>12个SNP,按照本研究分子生物学鉴定标准,6株菌株间异质性较大,不存在同源性。结论WGS较VNTR基因分型方法在判断结核病近期传播方面有更高的精度和优势。在学校发生结核病疫情,尤其是有耐药结核病例时,WGS能更加精准的鉴定结核病近期传播情况,应作为传统流行病学的补充。

Objective To evaluate the application of variable number tandem repeats(VNTR)and whole genome sequencing(WGS)in tracing the transmission of Mycobacterium tuberculosis in a tuberculosis outbreak in school,and explore the roles of these genetic methodologies in addressing tuberculosis transmission in school environments.Methods Identification of mycobacterial strains,spacer oligonucleotide typing(Spoligotyping),VNTR,and WGS were performed on six Mycobacterium tuberculosis strains obtained from TB cases in the first multidrug-resistant tuberculosis outbreak happened in a school in Beijing.Genotyping characteristics were analyzed to identify the transmission chain.Results The 6 culture-positive strains involved 6 students from 4 classes across 2 grades.One of them was the first case,three were close contacts of the first case,and the other two were general contacts.All 6 strains were identified as Mycobacterium tuberculosis.Spoligotyping results indicated that 5 of the strains were of the Beijing genotype;the other one had no result.VNTR genotyping divided the 6 strains into 3 clusters with a clustering rate of 66.7%.The largest one of the 3 clusters contained 4 strains with the same genotype,indicating a significant level of recent transmission.The remaining two strains,differing by 1.0-1.6 copies at 1-2 loci from the other four strains,were identified as individual strains.The results of WGS showed that the genomic SNP differences among the 6 strains were greater than 12 SNPs.According to the molecular biology identification criteria of this study,the strains exhibited significant heterogeneity with no homology.Conclusions WGS offers higher accuracy and advantages over VNTR genotyping in evaluating the recent transmission of tuberculosis.WGS can more accurately characterize recent TB transmission in the case of TB outbreaks in schools,especially when there are drug-resistant TB cases,and should be used as a supplement to traditional epidemiology.