2020年凉山州彝族地区结核分枝杆菌全基因组序列情况分析

目的 分析凉山州彝族地区获得的30株结核分枝杆菌全基因组序列情况，为凉山州结核病防控工作开展提供科学参考。方法 将凉山州彝族地区昭觉县、越西县、美姑县3县2020年的30株结核分枝杆菌进行全基因组测序，使用结核分枝杆菌全基因组序列分析平台分析其菌型、耐药情况、分型特征等，并使用MEGAX完成菌株的进化树构建。结果30株结核分枝杆菌，18株属于Lineage2（东亚系），12株属于Lineage4（欧美系）；25株未检测到已知耐药突变，为敏感菌株，5株检测到已知耐药突变，主要为利福平耐药。30菌株的进化树形成2个大簇，分属于L2和L4,L2谱系中有3株菌株全基因组序列具有一致性，L4谱系菌株中有2株菌株的全基因组序列具有一致性，此5例菌株都来自于学生人群。结论 该地区流行的结核分枝杆菌为L2和L4谱系菌株，以L2为主，L4为辅，且近期存在学校结核病聚集性事件，该地应加强学校结核病防控工作，控制结核病近期传播。     

上海市静安区2010-2015年肺结核传播状况及影响因素分析

目的 了解上海市静安区肺结核传播状况及其影响因素。方法 收集2010-2015年上海市静安区结核病定点医院诊治的肺结核患者中分离到的结核分枝杆菌进行药物敏感性试验,采用12个位点（QUB11b、QUB18、Mtub21、Miru26、QUB26、Mtub04、Miru31、Miru40、VNTR2372、VNTR3820、3232、4120）结核分枝杆菌可变数目串联重复序列（MIRU-VNTR）多态性分析,结合现场流行病学调查,分析结核分枝杆菌的成簇特征及其影响因素。结果 80株结核分枝杆菌菌株总耐药率为28.75%（23/80）,总耐多药率为16.25%（13/80）。MIRU-VNTR基因型分型结果显示,58例患者的分离株为单一基因型（72.50%,58/80）,22例为成簇菌株,成簇比为27.50%（22/80）,共分7个簇,每簇含2~10例菌株。结核分枝杆菌近期传播影响多因素分析结果显示,耐多药（OR=35.799,95% CI：4.239~302.346）和合并症（OR=7.695,95% CI：1.421~41.658）与近期传播有关。成簇病例的现场流行病学调查发现,1个簇患者含有10例耐多药结核病患者（MDR-TB）,9例患者相互间认识,有确定的联系,1例患者有可能的联系。结论 2010-2015年上海市静安区肺结核患者中存在一定比例的近期传播,耐多药和合并症是结核病近期传播的重要危险因素。     

利用全基因组测序解析耐多药结核病传播路径

目的应用全基因组测序技术解析耐多药结核分枝杆菌的传播路径。方法对2009—2012年上海市一起经VNTR分型鉴定的耐多药结核病传播案例中8例簇病例进行全基因组测序,描述耐多药结核分枝杆菌的传播路径。结果该起传播案例中源病例是1例复发耐多药结核病患者,在3年内传播耐药菌株导致另外至少7例患者发病。结论全基因组测序技术可以弥补传统流行病学调查与基因分型技术的不足,准确地描述结核病传播的方向与细节,鉴定传染源与缺失病例,有助于准确揭示耐多药结核病的传播路径。     

2020年南充市一中学结核病疫情临床分离菌株情况分析

目的 通过对一中学校结核病疫情中得到的临床分离菌株进行全基因组测序和分析，确定其耐药、分型特征及传播特点，探讨全基因组测序技术在学校结核病疫情处置中的应用价值。方法 对2020年南充市一中学结核病疫情中得到的17株菌株进行全基因组测序，并使用结核分枝杆菌全基因组数据库分析平台SAM-TB对其菌型、耐药、分型特征进行分析，并结合患者信息分析其传播特点。结果 该学校结核病疫情得到的17株临床菌株，13株为结核分枝杆菌（Mycobacterium Tuberculosis,MTB）,2株为脓肿分枝杆菌（Mycobacteroides abscessus）,2株为支气管戈登氏菌（Gordonia bronchialis）,13株MTB中12株属于L2.2.1,1株属于L4.5。L2.2.1谱系的12株菌株中10株为同一株菌，且来自于发生聚集性疫情的班级，由此确定该次疫情由此株菌传播引起。结论 该次学校结核病疫情是由谱系为L2.2.1的结核分枝杆菌引起的聚集性传播事件，全基因组测序技术在学校结核病疫情处置中具有确定传播链的重要作用。     

北京市昌平地区2011-2015年结核分枝杆菌的流行特征

目的 了解昌平地区结核分枝杆菌不同基因型流行特征,以及不同基因型和耐药性的关系,探讨基因分型用于评价本地区结核病控制中的应用价值。方法 选取北京市昌平区2011-2015年所有培养阳性结核分枝杆菌1 099株,采用Spoligotyping和Gao等推荐的12位点VNTR进行基因分型,同时检测上述菌株对利福平、异烟肼、乙胺丁醇、链霉素、阿米卡星和氧氟沙星的耐药性。结果 2011-2015年,昌平地区结核对氧氟沙星耐药率提高（2.9%比8.9%,P=0.01）。在所有菌株中,北京基因型菌株总计976株,占88.8%,非北京基因型菌株总计123株,占11.2%,其中北京基因型菌株中还包括189株（17.2%）古代北京基因型和787株（71.6%）现代北京基因型。北京基因型所占比例在5年期间无显著性变化,从2011年的81.1%到2015年的82.0%。采用VNTR基因分型后,仅有2株菌成簇,成簇率为0.1%。北京基因型对阿米卡星的耐药率（1.7%）低于非北京基因型（4.9%,P=0.02）;而古代北京基因型对链霉素耐药率（28.0%）高于现代北京基因型（15.7%,P=0.01）。结论 近5年来,昌平地区结核对氧氟沙星耐药率提高。北京基因型所占比例在5年期间无明显变化,且菌株成簇率较低,提示近期传播率低。     

福建省结核分枝杆菌多位点可变数目串联重复序列基因分型研究

目的 了解福建省结核分枝杆菌的多位点可变数目串联重复序列基因分型(MLVA)的特征.方法 选择15个可变数目串联重复位点(VNTR),检测福建省30个耐药监测点临床分离的结核菌株,结果使用BioNumerics (Version 4.5)软件进行聚类分析.结果 313株结核菌被分为9个基因群(Ⅰ～Ⅸ),分别包含220、9、48、2、1、3、10、10、10株菌,以Ⅰ群为主(70.3％,220/313);Ⅰ群菌株异烟肼、链霉素、乙胺丁醇和耐多药的耐药率与其他基因群的差异无统计学意义(P＞0.05),但利福平(RFP)耐药率为33.2％(73/220),明显高于其他群菌株RFP的耐药率20.4％(19/93),差异有统计学意义(P＜0.05).结论 福建省结核分枝杆菌菌株存在明显的基因多态性,以Ⅰ群菌株为主,并与RFP耐药性具有相关性,应加强此类菌株流行的监测.     

青海省结核分枝杆菌临床分离株可变数目串联重复序列基因多态性研究

目的 初步了解青海省结核分枝杆菌临床分离株基因多态性和基因分型特征。方法 2009-2012年收集青海省疾病预防控制中心分离的结核分枝杆菌临床分离株,提取DNA,对15个可变数目串联重复序列(VNTR)位点进行PCR扩增和产物电泳分析,使用BioNumerics软件对菌株进行聚类分析。结果 共检测251株结核分枝杆菌临床分离株的15个VNTR位点,显示这些菌株有明显的基因多态性,15个VNTR位点中Hunter-Gaston指数>0.6的VNTR位点有6个,位点分辨能力最高的是MIRU26,经聚类分析,可分为4个基因群,238个基因型。4个基因群分别占4.9%、91.9%、1.6%和1.6%。结论 青海省流行的结核分枝杆菌菌株存在明显的VNTR基因多态性。     

基于全基因组测序的我国耐多药结核分枝杆菌耐药突变特征分析

目的 利用全基因组测序数据分析中国耐多药结核分枝杆菌的耐药相关基因突变谱及主要突变类型与菌株基因型的相关性。方法 查询并下载NCBI数据库中截至2019年8月公开发表的中国结核分枝杆菌全基因组测序数据,利用全基因组数据预测菌株分子药敏结果,统计不同药物耐药相关基因的突变类型,并分析耐药突变类型与菌株基因型的相关性。结果 根据分子药敏结果从2 019株菌株中鉴定出1 024株耐多药菌株,对常用抗结核药物的耐药相关基因主要突变类型分别为katG S315T（73.2%,异烟肼）、rpoB S450L（63.1%,利福平）、rpsL K43R（70.0%,链霉素）、embB M306V（37.4%,乙胺丁醇）、pncA启动子区T（-11）C（7.9%,吡嗪酰胺）、gyrA A90V（32.3%,氟喹诺酮类）、rrs A1401G（67.7%,二线注射类）、fabG1启动子区C（-15）T（87.0%,乙硫异烟胺）、folC I43T（30.4%,对氨基水杨酸）。其中,katG S315T、rpsL K43R、embB M306V、gyrA D94G在L2系菌株中的频率显著高于L4系菌株,folC I43T仅在L2系菌株中发现;katG S315T在古典北京型菌株中比例较高,而rpsL K43R在现代北京基因型菌株中的比例更高,其差异均有统计学意义（均P<0.05）。结论 本研究提供了基于全基因组测序的我国耐多药结核分枝杆菌对多种常用抗结核药的耐药相关基因主要突变类型,为研发敏感、特异的快速分子检测方法提供了依据;同时也发现多种耐药相关基因主要突变类型与菌株基因型有关。     

宜昌地区结核分枝杆菌北京基因型菌株的鉴定及VNTR分型

 目的 研究宜昌地区结核分枝杆菌北京基因型菌株的多态性和流行特征,揭示该地区结核病的分子流行病学特征,为结核病防治提供科学依据。方法 选择2018年1月-2019年12月具有完整病例信息的结核分枝杆菌298株,进行人型结核分枝杆菌的鉴定,用扩增RD105缺失片段的多重PCR (DTM-PCR)鉴定北京基因型菌株;应用优化的9位点数目可变串联重复序列技术(VNTR)分析北京基因型菌株的多态性。结果 298株结核分枝杆菌中,260株为人型结核分枝杆菌,DTM-PCR鉴定发现北京基因型菌株140株(53.85%),非北京基因型菌株120株(46.15%)。北京基因型菌株耐药率(32.14%)高于非北京基因型菌株耐药率(10.83%),差异有统计学意义(P<0.05)。VNTR-9位点对北京基因型菌株的分辨率为0.998 5,成簇率为12.15%。结论 北京基因型菌株在宜昌地区呈较高流行趋势,且北京基因型菌株耐药率更高。VNTR-9位点基因分型方法用于该地区结核分枝杆菌北京基因型菌株的鉴别,能准确反映宜昌地区结核分枝杆菌的分子流行病学特征。     

结核分枝杆菌传播的分子流行病学特征

目的探讨结核分枝杆菌传播的基因型特征及其对于耐药结核菌近期传播的影响。方法选取13个社区作为监测点,应用分枝杆菌散在重复单位(MIRU)技术分析结核分枝杆菌DNA多态性。结果 558株结核分枝杆菌的12个MIRU位点检测产生143个基因型,其中成簇基因型66个,成簇率86.2%;74.6%的菌株属于北京家族,177个菌株(31.7%)属于山东基因型;耐多药菌株的近期感染估计值(46.7%)明显低于全敏感菌株(72.7%)。结论山东基因型菌株具有较强的在人群中传播的能力、较高的耐药性以及对抗督导化疗的能力;利用基因分型技术开展结核病监测可以有效促进结核病控制工作。     

基于全基因组测序估算结核分枝杆菌分子钟

目的 基于全基因组测序数据,通过内源性激活期间菌株基因组突变分析,开展结核分枝杆菌（MTB）分子钟研究。方法 筛选MTB内源性激活全基因组研究文献,下载全基因组测序数据,提取初治-复发配对样本单核苷酸多态性（SNP）差异和菌株分离时间,运用Poisson回归模型拟合初治-复发时间间隔与差异SNP的关系,计算MTB分子钟,估算突变率。结果 若MTB传代时间为18 h,0~2年短期内源性激活突变率为6.47×10^-10（95%CI：5.59×10^-10~7.44×10^-10）,明显高于2~14年长期内源性激活突变率（3.27×10^-10,95%CI：2.88×10^-10~3.69×10^-10）。0~、1~、2~、3~、5~、7~14年突变率分别为7.10×10^-10、6.06×10^-10、4.24×10^-10、5.34×10^-10、2.59×10^-10、1.26×10^-10。结论 内源性激活复发期间,MTB突变率随初治-复发时间间隔增加而下降,本研究从微观层面验证了临床实践观察到的初治结核病2年内易复发现象。     

Distinct clinical and epidemiological features of tuberculosis in New York City caused by the RDRio Mycobacterium tuberculosis sublineage

BackgroundGenetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RD^Rio M. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001–2005) to: (1) affirm the position of RD^Rio strains within the M. tuberculosis phylogenetic structure, (2) determine its prevalence, and (3) define transmission, demographic, and clinical characteristics associated with RD^Rio TB.MethodsIsolates classified as RD^Rio or non-RD^Rio M. tuberculosis by multiplex PCR were further classified as clustered (?2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RD^Rio was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics.ResultsRD^Rio strains were found to: (1) be restricted to the Latin American–Mediterranean family, (2) cause approximately 8% of TB cases in NYC, and (3) be associated with heightened transmission as shown by: (i) a higher cluster proportion compared to other prevalent lineages, (ii) a higher secondary case rate, and (iii) cases in children. Furthermore, RD^Rio strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance.ConclusionsThe RD^Rio genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RD^Rio case and exposure identification could be of benefit due to its association with heightened transmission.     

Insight into the drug resistance whole genome of Mycobacterium tuberculosis isolates from Khyber Pakhtunkhwa,Pakistan

Whole genome sequencing (WGS) is one of the most reliable methods for detection of drug resistance, genetic diversity in other virulence factor and also evolutionary dynamics of Mycobacterium tuberculosis complex (MTBC). First-line anti-tuberculosis drugs are the major weapons against Mycobacterium tuberculosis (MTB). However, the emergence of drug resistance remained a major obstacle towards global tuberculosis (TB) control program 2030, especially in high burden countries including Pakistan. To overcome the resistance and design potent drugs, genomic variations in drugs targets as well as in the virulence and evolutionary factors might be useful for better understanding and designing potential inhibitors. Here we aimed to find genomic variations in the first-line drugs targets, along with other virulence and evolutionary factors among the circulating isolates in Khyber Pakhtunkhwa, Pakistan. Samples were collected and drug susceptibility testing (DST) was performed as per WHO standard. The resistance samples were subjected to WGS. Among the five whole genome sequences, three samples (NCBI BioProject Accession: PRJNA629298, PRJNA629388) harbored 1997, 1162, and 2053 mutations. Some novel mutations have been detected in drugs targets. Similarly, numerous novel variants have also been detected in virulency and evolutionary factors, PE, PPE, and secretory system of MTB isolates. Exploring the genomic variations among the circulating isolates in geographical specific locations might be useful for future drug designing. To the best of our knowledge, this is the first study that provides useful data regarding the insight genomic variations in virulency, evolutionary factors including ESX and PE/PPE as well as drug targets, for better understanding and management of TB in a WHO declared high burden country.     

结核分枝杆菌毒素-抗毒素伴侣系统基因多态性初步研究

目的 研究结核分枝杆菌毒素-抗毒素伴侣(TAC)系统中higA、higB和Rv1957在结核分枝杆菌及其不同亚型菌株中的基因多态性,并探讨其生理意义。方法 选取183株结核分枝杆菌临床菌株,经间隔区寡核苷酸方法进行基因分型,同时分析TAC系统基因higA、higB和Rv1957的PCR扩增及序列,利用I-Mutant 2.0软件预测非同义突变对蛋白结构和功能的影响。结果 183株中138株(75.41%)属北京家族,45株(24.59%)属非北京家族。共149株菌(81.42%)的TAC系统发生突变,包括2种同义突变和6种非同义突变:同义突变发生于higA基因,仅见于北京家族菌株;3个基因均可见非同义突变,其中2种非同义突变仅见于非北京家族菌株,其余4种突变仅见于北京家族菌株。6种非同义突变中有4种突变可能影响蛋白的功能。位于higA基因的CAC121CAT突变位点在单耐链霉素和单耐利福平菌株中的突变频率高于敏感株,且差异有统计学意义(P<0.05)。结论 结核分枝杆菌中的TAC系统具有一定的基因多态性,其中北京家族呈现更高的多态性水平,可能更有利于适应不同的宿主环境。     

The tempo and mode of molecular evolution of Mycobacterium tuberculosis at patient-to-patient scale

A total of six polymorphisms were identified by comparing the genomes of the first and the last isolate of a well-characterized transmission chain of Mycobacterium tuberculosis involving five patients over a 12 and a half year period. The six polymorphisms consisted of four single nucleotide changes (SNPs), a tandem repeat polymorphism (TRP) and a previously identified IS6110 transposition event. These polymorphic sites were surveyed in each of the isolates from the five patients in the transmission chain. Surprisingly, five of the six polymorphisms accumulated in a single patient in the transmission chain; this patient had been non-compliant to tuberculosis treatment.This first insight into the tempo and mode of molecular evolution in M. tuberculosis at the patient-to-patient level suggests that the molecular evolution of the pathogen in vivo is characterized by periods of relative genomic stability followed by bursts of mutation. Whatever the mechanism for the accumulation of mutations, this observation may have profound consequences for the application of vaccines and therapeutic drugs, the management and treatment of disease outbreaks of M. tuberculosis, the most important bacterial pathogen of humans.     

DNA fingerprinting of Mycobacterium tuberculosis: from phage typing to whole-genome sequencing

Current typing methods for Mycobacterium tuberculosis complex evolved from simple phenotypic approaches like phage typing and drug susceptibility profiling to DNA-based strain typing methods, such as IS6110-restriction fragment length polymorphisms (RFLP) and variable number of tandem repeats (VNTR) typing. Examples of the usefulness of molecular typing are source case finding and epidemiological linkage of tuberculosis (TB) cases, international transmission of MDR/XDR-TB, the discrimination between endogenous reactivation and exogenous re-infection as a cause of relapses after curative treatment of tuberculosis, the evidence of multiple M. tuberculosis infections, and the disclosure of laboratory cross-contaminations. Simultaneously, phylogenetic analyses were developed based on single nucleotide polymorphisms (SNPs), genomic deletions usually referred to as regions of difference (RDs) and spoligotyping which served both strain typing and phylogenetic analysis. National and international initiatives that rely on the application of these typing methods have brought significant insight into the molecular epidemiology of tuberculosis. However, current DNA fingerprinting methods have important limitations. They can often not distinguish between genetically closely related strains and the turn-over of these markers is variable. Moreover, the suitability of most DNA typing methods for phylogenetic reconstruction is limited as they show a high propensity of convergent evolution or misinfer genetic distances. In order to fully explore the possibilities of genotyping in the molecular epidemiology of tuberculosis and to study the phylogeny of the causative bacteria reliably, the application of whole-genome sequencing (WGS) analysis for all M. tuberculosis isolates is the optimal, although currently still a costly solution. In the last years WGS for typing of pathogens has been explored and yielded important additional information on strain diversity in comparison to the classical DNA typing methods. With the ongoing cost reduction of DNA sequencing it is possible that WGS will become the sole diagnostic tool in the secondary laboratory diagnosis of tuberculosis for identification, drug susceptibility testing and genetic characterization.     

Added epidemiologic value to tuberculosis prevention and control of the investigation of clustered genotypes of Mycobacterium tuberculosis isolates

The Centers for Disease Control and Prevention established the US National Tuberculosis Genotyping and Surveillance Network to study the utility of genotyping Mycobacterium tuberculosis isolates for prevention and control. From 1998 to 2000, four sites performed conventional contact investigations and epidemiologic investigations of cases with genotypically matched M. tuberculosis isolates, called cluster investigations. The authors compared cluster pairs (two cases with M. tuberculosis isolates having identical genotypes) whose epidemiologic linkages were discovered only during cluster investigation with those whose epidemiologic linkages were discovered during conventional contact investigation. Among the 2,141 reported culture-positive tuberculosis cases, 2,055 (96%) M. tuberculosis isolates were genotyped. By itself and at a minimum, cluster investigation added 43 (38%) of the 113 total epidemiologic linkages discovered. Of the epidemiologic linkages discovered during conventional contact investigation, 29% of tuberculosis case pairs were not supported by genotyping data. The linkages discovered only during cluster investigation were more likely discovered in nontraditional settings and relationships and among larger clusters (cluster size of >5: adjusted odds ratio = 57.6, 95% confidence interval: 31.8, 104.6). Information gained from genotyping M. tuberculosis isolates should initiate cluster investigations of tuberculosis cases not previously discovered as being epidemiologically linked during conventional contact investigation. Cluster investigations will play a crucial role in predicting recent tuberculosis transmission more accurately, as we move toward tuberculosis elimination in the United States.     

Transmission of tuberculosis in Gironde: epidemiologic investigation by genomic analysis of Mycobacterium tuberculosis

BACKGROUND: Over the past few years, epidemiologic surveys of tuberculosis have been strengthened by new biologic technology, in particularly using RFLP (Restriction Fragment Length Polymorphism). This technique, which identifies Mycobacterium tuberculosis patterns, has allowed to study thoroughly tuberculosis bacilli transmission and pathogenesis. First applied on tuberculosis epidemics in at risk groups, RFLP has now an interest in the epidemiologic molecular survey of urbans populations. The aim of this study is to identify, in a French department, the proportion of clustering cases of tuberculosis, suspected of recent contamination. METHODS: An active surveillance of tuberculosis allows to record systematically the cases of tuberculosis-disease in Gironde. All M. tuberculosis isolates from the patients reported in this surveillance system were processed through IS6110 based RFLP analysis. Patients were interviewed face to face before this analysis, using a standardised data collection instrument. RESULTS: 102 patients were included in 1997; the RFLP analysis of all available strains identifies a high degree of polymorphism with 71 unique patterns; twelve groups with clustering patterns were found, grouping two (nine clusters), three (two clusters) and seven patients (one cluster) each. Those cases suspected of recent transmission were younger (age<60 years) and lived in poorer conditions. Epidemiologic links were confirmed in only 35% of the 31 patients clustered. CONCLUSION: This community survey analysis has allowed to identify at risk groups for tuberculosis transmission and to strengthen tuberculosis control in Gironde.