Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from high prevalence tuberculosis states in Mexico

Mexico is one of the most important contributors of multidrug resistance tuberculosis (MDR-TB) in Latin-America, however little is known about the molecular characteristics of these strains. For this reason, the objective of this work was to determine the genotype and characterize polymorphisms in genes associated with resistance to rifampicin, isoniazid, and second-line drugs in isolates from two regions of Mexico with high prevalence of drug resistant tuberculosis.Clinical isolates from individuals with confirmed MDR-TB were genotyped using MIRU-VNTR 12 loci. To characterize the polymorphisms in genes associated with resistance to rifampicin, isoniazid and second-line drugs; rpoB, katG, inhA, rrs, eis, gyrA, gyrB and tlyA were sequenced.22 (41%) of the 54 MDR-TB isolates recovered were from the state of Baja California, while 32 (59%) were from Veracruz. The results show the katGS315T mutation was observed in 20% (11/54) of the isolates, while rpoBS315L was present in 33% (18/54). rrs had three polymorphisms (T1239C, ntA1401C and ntA1401G), gyrB presented no modifications, whereas gyrA showed five (S95T, F60Y, A90V, S91P and P124A), eis two (G-10A and A431G) and tlyA one (insertion at codon 67). Only 20% (11/54) of isolates were confirmed as MDR-TB by sequencing, and no mutations at any of the genes sequenced were observed in 43% (23/54) of the strains. Two isolates were recognized with the proper set of mutations like pre-XDR and one was XDR-TB. Eighteen isolates were classified as orphans and the remaining thirty-six were distributed in fourteen lineages, the most frequent were S (11%), Haarlem (9%), Ghana (9%) and LAM (7%). Out of the fourteen clusters identified, seven included unknown genotypes and nine had lineages.This is one of the most detailed analyses of genotypic characteristics and mutations associated with drug resistance to first and second-line drugs in MDR-TB isolates from Mexico. An important genetic variability and significant discrepancy between phenotypic tests and polymorphisms was observed. Our results set the need to screen additional loci as well as implement a molecular epidemiological surveillance system of MDR-TB in the country.     

Molecular characterization of pncA gene mutations in Mycobacterium tuberculosis clinical isolates from China

A sample of 35 pyrazinamide (PZA)-resistant and 30 PZA-susceptible clinical isolates recovered from Beijing and Taiyuan City, China were characterized by SSCP and sequence analysis for mutations in the pncA gene that encodes the Mycobacterium tuberculosis PZase. The purpose of this study was to understand the molecular basis and the characteristics of pncA gene mutations and its relation to PZA resistance in M. tuberculosis strains from China. Several mutations with base changes leading to amino acid substitutions were found in the PZA-resistant isolates. No mutations were seen in the 243 PZA-susceptible isolates. Among the 35 PZA-resistant isolates, 32 isolates (91.4%) had nucleotide substitutions, insertions and deletions that resulted in amino-acid substitution; or frameshifts in some strains. Other previously uncharacterized mutations were found as follows: Asn118->Thr, CG insertion at position 501; CC insertion at nucleotide position 403; a 8 base-pair deletion at start codon; Pro54->Thr; AG insertion at 368; Tyr41->His, Ser88->stop, and A insertion at nucleotide position 301. IS6110 subtyping revealed that each strain was unique; indicative of the epidemiologic independence of the isolates.     

重庆市结核患者耐药结核杆菌embB306分子突变特征研究

目的 了解重庆结核杆菌的embB306突变特征,评价其作为诊断乙胺丁醇(ethambutol,EMB)耐药结核杆菌分子标记物的临床应用价值.方法 采用DNA测序方法分析重庆市结核患者痰中分离的51株EMB耐药结核杆菌和50株EMB敏感株的embB基因突变特征,与传统药敏实验进行诊断学试验评价embB306突变作为EMB耐药株分子标记物的临床应用价值.结果 embB306突变率在51株EMB耐药和50株敏感结核杆菌中分别为75.5%、6%;其中来自复治病例的EMB耐药菌株的embB306突变率是87.5%,高于来自初治病例的48.1%;embB306突变率随菌株耐药数量增加而升高,embB306在EMB耐药的耐多药菌株(Multidrug-Resistant Tuberculosis,MDR-TB)中的突变率高于EMB耐药的非MDR-TB和EMB敏感的MDR-TB的突变率;以embB306突变诊断EMB耐药菌株的诊断学试验评价主要指标为:灵敏度为66.7%;特异度为94.0%;准确度为80.2%;Youden指数为60.7%.结论embB306突变是重庆地区结核杆菌产生EMB耐药性的主要分子机制,且与耐药数量和治疗时间有关,其作为EMB耐药菌株诊断的分子标记物具有一定临床应用价值.     

深圳市结核分枝杆菌异烟肼耐药流行株的基因分析

异烟肼(INH)和利福平(RFP)是抗结核药中的一线药物,近年来出现了大量耐药结核分枝杆菌,在中国INH-耐药率高达17.6%[1].INH耐药相关的最主要基因为katG,其次为inhA、ahpC和inhA94.为了解深圳地区INH耐药流行株的主要基因突变类型,本研究对深圳地区303株结核分枝杆菌的katG、inbA、oxyR-ahpC基因进行序列分析.     

Molecular characterization of Mycobacterium bovis isolates from patients with tuberculosis in Baja California,Mexico

The incidence of tuberculosis (TB) from Mycobacterium bovis in humans is likely to be underestimated and in some cases even ignored in most developing countries. This may be due to the difficulty of differentiating TB caused by either Mycobacterium tuberculosis or M. bovis.Our objectives were to determine the prevalence of M. bovis human disease among the patients referred for study to the Tuberculosis Laboratory of the Tijuana General Hospital in Baja California, Mexico and to characterize molecularly the clinical isolates using 8 loci of MIRU-VNTR.A cross-sectional analysis of all culture-proven cases of tuberculosis was conducted during the period from January 1, 2011 through June 30, 2013. Clinical isolates that exhibited resistance to pyrazinamide (Z) were submitted for molecular analysis.A total of 2699 clinical samples were cultured during the study period and 600 (22%) that tested positive were processed for drug susceptibility for first line drugs. Sixty-four (10.7%) of the tested isolates tested were resistant to Z, and 27 (4.5%) of those were subsequently identified molecularly as M. bovis. Three of the M. bovis isolates were polyresistant to Z, isoniazid (H), ethambutol (E) and rifampicin (R) (Z + H + E, Z + E and Z + R); the rest were only resistant only to Z. VNTR typing, based on the 8 VNTR loci commonly tested for M. bovis, detected 12 allelic profiles (genotypes).The real burden of M. bovis cases among the total reported human tuberculosis cases can only be known from especially designed studies in which, during a specific period, all specimens submitted to tuberculosis diagnosis in one or more laboratories are cultured on the appropriate media and the isolated mycobacteria are analyzed to differentiate M. bovis from M. tuberculosis and other Mycobacterium species.     

Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland

Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (‘U’ clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI > 0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.     

Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India.

Spoligotyping was performed on 540 Mycobacterium tuberculosis isolates in order to evaluate the genetic biodiversity of tubercle bacilli in India. One hundred and forty seven patterns were unique and 393 were grouped in 48 clusters. Comparison with an international spoligotype database showed that the most predominant clades among tuberculosis (TB) isolates were Central Asian (CAS) and East-African Indian (EAI) with shared-types (ST) ST26 and ST11 alone being responsible for 34% of all TB cases. Twenty one (3.8%) isolates belonged to the Beijing genotype. Marked variations were observed among circulating strains, STs belonging to CAS family predominated in the North, whereas the EAI family was more common in the Southern India. TB in India is predominantly caused by strains belonging to the principal genetic group 1 (PGG1), suggesting that most of the TB burden in India may be traced to ancestral clones of the tubercle bacilli. This study gives an insight into the global M. tuberculosis genetic biodiversity in India, the predominant spoligotypes and their impact on disease transmission.     

Molecular epidemiology of Mycobacterium tuberculosis isolates from Kerala,India using IS6110-RFLP,spoligotyping and MIRU-VNTRs

Tuberculosis (TB) continues to be a major health problem in India, and there is very little information about the prevalent genotypes of tubercle bacilli that cause TB in India, especially in Kerala. Our aim was to study the different circulating strains of Mycobacterium tuberculosis (MTB) that are prevalent in Kerala, India. We analyzed 168 MTB isolates from as many pulmonary TB patients using IS6110-RFLP, spoligotyping and MIRU-VNTRs. The results of IS6110-RFLP revealed that majority of isolates had null copy (10.89%) or single copy (44.87%) of IS6110 insertion. Low copy (<6) isolates accounted for 71.5% in the isolates studied. Genotypic clade designations were done by comparing with the SITVIT2 database which showed 68 patterns; of which 51 corresponded to different shared types whereas 17 patterns were orphans. Among the 51 SITs recorded, 42 SITs matched a preexisting SIT in the SITVIT2 database, whereas 9 SITs were newly-created. Majority of the isolates (64.28%) belonged to the ancestral East-African Indian (EAI) lineage. MIRU-40 and 31 (HGDI = 0.6555 and 0.6524) showed highest discrimination, while MIRU-2 and 20 (HGDI = 0.0354 and 0.0696) had the least discriminatory power. ETR-A and B (HGDI 0.7382 and 0.6743) discriminated better as compared to other MIRU loci. The overall HGDI for MIRU-VNTRs at 0.9735 (calculated for 166 isolates) showed a better discriminatory power than spoligotyping used alone. This study of MTB genotypic diversity was useful by providing a first snapshot of circulating MTB genotypic clones in Kerala.     

Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Sri Lanka

The strain diversity of 100 Mycobacterium tuberculosis isolates collected over a period of 18 months from tuberculosis (TB) cases in Sri Lanka was studied by spoligotyping. When compared to the international spoligotyping database, 43 spoligotype patterns were identified, of which 20 were previously described. The majority of isolates (72.45%) were clustered into major genetic group 1, and the most common spoligotype pattern belonged to the Beijing (ST1) strain family. All the Beijing strain isolates belonged to more recently evolved sublineages of M. tuberculosis. The characterization of Sri Lankan M. tuberculosis isolates by spoligotyping shows a heterogeneous pattern. The physical separation from the main Indian peninsula may be responsible for the different patterns observed between the two countries. An in-depth field study is needed to understand the spread and the true epidemiology of this infection.     

重庆市结核病人耐药结核杆菌katG315分子突变特征研究

目的 了解重庆结核分支杆菌的katG315突变特征,评价其作为诊断异烟肼(Isoniazide,INH)耐药结核杆菌分子标记物的临床应用价值.方法 采用DNA测序方法 .分析重庆市结核病人痰中分离的97株INH耐药、敏感结核杆菌菌株间的katG基因突变特征,与传统药敏实验进行诊断学试验评价katG315突变作为INH耐药株分子标记物的临床应用价值.结果 49株INH耐药结核杆菌中katG315突变率为75.5%,而48株INH敏感结核杆菌中未见katG315突变;INH高度耐药菌株中katG315的突变率为88.5%,显著高于INH低度耐药株的60.9%(P＜0.01);耐多药结核杆菌中katG315突变率为89.7%,显著高于单耐INH的结核杆菌的55.0%(P＜0.05),耐2种药物及以上药物的结核杆菌中的katG315突变率均显著高于单耐INH菌株(P＜0.05);来自复治病例的耐INH菌株中的katG315突变率为95%,显著高于来自初治病例的INH耐药菌株的62.1%(P＜0.05);katG315突变诊断INH耐药菌株的诊断学试验评价主要指标为:灵敏度=75.5%;特异度=100%;准确度=87.6%;Youde指数=75.5%.结论 katG315突变是重庆地区结核杆菌产生INH耐药性的主要分子机制,且与耐药程度和耐药数量呈显著有关,其作为INH耐药菌株诊断的分子标记物具有良好的临床应用价值.     

Molecular typing and drug susceptibility of Mycobacterium tuberculosis isolates from Chongqing Municipality,China

China’s tuberculosis (TB) burden is second only to that of India worldwide. In Chongqing, the largest municipality in southwestern China, although the prevalence of both TB and drug-resistant TB is higher than in other municipalities, the molecular characteristics and drug susceptibility phenotypes are poorly known. In this study, 297 Mycobacterium tuberculosis isolates from Chongqing were genotyped with spacer oligonucleotide typing (spoligotyping) and 28-locus MIRU-VNTR (24-locus MIRU-VNTR scheme and 4 other loci). Spoligotyping results were compared with drug-resistant profiles. Patients who showed clustering by both spoligotyping and 28-locus MIRU-VNTR were interviewed to investigate their detailed contact history. Our data demonstrated that the Beijing genotype was the most prevalent genotype, and ST1 was the most predominant lineage in Chongqing. The Beijing genotype was significantly associated with ethambutol resistance and multidrug-resistant phenotypes. A combination of the 10 most polymorphic loci permitted to achieve higher discriminatory power than 24-VNTR. In addition, a presumed transmission pathway was observed in a cluster of patients with the same MIRU-VNTR profile. The 10-VNTR locus set is suitable for genotyping of Mycobacterium tuberculosis in Chongqing.     

三种不同的DNA分型技术在158株结核杆菌研究中的应用

目的评价IS6110限制性片段长度多态性(RFLP)、间隔区寡核苷酸分型(Spoligotyping)及分枝杆菌散在重复单位(MIRU)三种分型方法在结核病流行病学研究中的应用。方法对158株结核分枝杆菌临床分离株应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行鉴定。结果应用三种分型方法产生的类型数分别为118、20和105个。IS6110RFLP的分辨率大于Spoligotyping,MIRU的分辨能力与IS6110RFLP接近。在MIRU的12个区中,重复区4、10、26、40具有较高的多态性。广东地区与其他地区成簇率和北京基因型所占比例差异有统计学意义(P<0.05),广东地区成簇率和北京基因型所占比例均显著低于其他地区。结论应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行结核病流行病学研究具有重要意义且非常有效,可以发现中国不同地区菌株的不同特点。     

结核分枝杆菌间隔区寡核苷酸分型法分析

目的 应用间隔区寡核苷酸分型(spoligotyping)方法分析甘肃省结核分枝杆菌临床分离株的分子型别特征。方法 用spoligotyping基因分型方法,对甘肃省兰州市肺科医院住院或门诊确诊患者分离的结核分枝杆菌进行分型,采用BioNumerics 4.5软件进行聚类(cluster)分析,对聚类结果与国际间隔寡核苷酸分型数据库(SpolDB4)对比。结果 215株临床分离株被分成3个基因群22种基因型,其中成簇菌株形成11个基因型共204株,独特基因型菌株11株;北京家庭基因型结核分枝杆菌占86.51%(186/215),T4占4.19%(9/215)、H1占1.86%(4/215)、MANU占1.40%(3/215)等基因型;Logistic多元回归分析结果表明,北京家庭基因型与患者性别、年龄、职业、抗结核治疗史、耐药、发病情况和菌种耐药性、来源地等均无关联。结论 甘肃省结核分枝杆菌临床分离株基因具有多态性,北京家族基因型结核分枝杆菌为该地区主要流行株,其他罕见的基因型结核分枝杆菌也值得重视。     

合肥地区异烟肼耐药结核分枝杆菌KatG基因突变的分子特征

目的 了解合肥地区结核分枝杆菌异烟肼(Isoniazid,INH)耐药株katG基因突变特点.方法 应用PCR-直接测序法对合肥地区肺结核病人痰液中分离的86株结核分枝杆菌的katG基因731bp序列进行测定分析.结果 86株结核分枝杆菌中67株对INH耐药,19株对INH敏感;67株耐INH临床分离株中PCR成功扩增61株,其中56株发生katG基因突变,突变率为91.8％ (56/61);katG基因发生联合突变的有30株,突变率为49.2％ (30/61),26株发生katG基因单位点突变,突变率为42.6％ (26/61).突变位点主要集中在463位精氨酸和315位丝氨酸,突变率分别为93.4％(57/61)和42.6％ (26/61).而在19株INH敏感株中发现有78.9％ (15/19)株存在Arg4631eu突变,突变形式与INH耐药株相同.结论 在合肥地区,KatG基因315位密码子突变是菌株INH表型耐药的重要分子基础,以Ser315Thr错义突变为主.KatG基因463位密码子突变与INH耐药表型没有相关性,不是结核杆菌INH耐药的分子标志.该位点突变可能是基因多态性,与结核菌治疗带来的选择性压力无关.     

Molecular epidemiology of tuberculosis in Prague and South Moravia, Czech Republic: genetic analysis of Mycobacterium tuberculosis isolates by IS6110-RFLP fingerprinting and spoligotyping

OBJECTIVES: To genetically characterize and compare Mycobacterium tuberculosis isolates among culture-confirmed TB cases in two regions in the Czech Republic in 1998. METHODS: Consecutive M. tuberculosis isolates from 111 TB patients in Prague and 120 patients in the South Moravia region were genotyped using the standardized IS6110 Southern blot hybridization method and by spoligotyping. RESULTS: Eighty of the Prague patients (72.1%) had isolates with unique RFLP patterns, while 31 (27.9%) had isolates which belonged to 10 clusters. Seventy-eight (64.7%) of the South Moravia strains displayed unique RFLP pattern and 42 (35.3%) were assigned into 15 clusters. The spoligotype profiles previously identified in the U.S. were found in 69 (33%) samples and newly identified Czech spoligotypes in 24 (11.4%) of the total number of examined strains. CONCLUSIONS: The present population-based molecular epidemiological study performed in two regions of the Czech Republic in 1998 demonstrated the distribution of individual genotypes as well as clustered strains of M. tuberculosis isolated from TB patients, and confirmed the similarity between the Czech strain collection and the European Community TB Database, that includes countries with low TB rate. The sporadic import of TB cases from foreign countries and recent transmission events probably do not play significant roles in the epidemiological situation in the Czech Republic.     

Predominance of ancestral lineages of Mycobacterium tuberculosis in India

Although India has the highest prevalence of tuberculosis (TB) worldwide, the genetic diversity of Mycobacterium tuberculosis in India is largely unknown. A collection of 91 isolates originating from 12 different regions spread across the country were analyzed by genotyping using 21 loci with variable-number tandem repeats (VNTRs), by spoligotyping, by principal genetic grouping (PGG), and by deletion analysis of M. tuberculosis-specific deletion region 1. The isolates showed highly diverse VNTR genotypes. Nevertheless, highly congruent groupings identified by using the 4 independent sets of markers permitted a clear definition of 3 prevalent PGG1 lineages, which corresponded to the "ancestral" East African-Indian, the Delhi, and the Beijing/W genogroups. A few isolates from PGG2 lineages and a single representative of the presumably most recent PGG3 were identified. These observations suggest a predominance of ancestral M. tuberculosis genotypes in the Indian subcontinent, which supports the hypothesis that India is an ancient endemic focus of TB.     

用变性高压液相色谱检测结核分枝杆菌耐药株

目的利用变性高压液相色谱(DHPLC)技术快速检测结核分枝杆菌gyrA基因的突变,对结核分枝杆菌是否耐受氟喹诺酮类药物做出初步判断。方法提取试验菌株基因组DNA,扩增gyrA基因的氟喹诺酮药物耐受决定区(QRDR)核酸片段;分别与H37Rv标准株(或者含有单纯Ser284突变的临床分离株作为标准)的相应PCR产物混合、杂交后,用WAVE核苷酸片段分析系统对各杂交产物进行DHPLC检测;序列测定结果与相应的DHPLC检测结果进行比较以验证DHPLC检测的准确性。用2mg/L的药物浓度进行氧氟沙星(OFLX)药物敏感性试验,确定101株结核分枝杆菌临床分离株对OFLX的敏感性,通过比较药物敏感性和DHPLC检测结果,建立两者之间的对应关系。结果用DHPLC检测QRDR突变与序列测定具有同样的检出率(100%),两者都能够很好地检测QRDR突变。用单纯含有Ser284突变的临床分离株代替H37Rv作为标准进行DHPLC检测,可以检测出除与耐药性无关的Ser284突变点外所有的点突变形式。在101株结核分枝杆菌临床分离株中,有49株对2mg/LOFLX敏感,52株耐受。52株耐药菌株中,有27株能够通过序列测定和DHPLC法得到准确判定。结论DHPLC法是快速检测出结核分枝杆菌gyrA基因碱基突变的好方法,但是碱基突变与结核分枝杆菌是否耐受氟喹诺酮类药物存在复杂的关系,造成检测准确性无预期中的高,因此该方法应用于临床检测尚需进一步验证。     

A novel multiplex-PCR for the rapid identification of Mycobacterium bovis in clinical isolates of both veterinary and human origin

Bovine tuberculosis is a zoonotic disease that not only causes huge economic losses but also poses an important risk for human infection. The definitive identification of a clinical isolate relies on time-consuming, highly specialized and laborious biochemical tests. We have developed a method for the rapid and reliable identification of Mycobacterium bovis and for its simultaneous differentiation from other members of the M. tuberculosis complex. Furthermore, the technique also allowed us to distinguish M. tuberculosis complex members from other Mycobacterial species. The method comprises both a single PCR and a multiplex-PCR and can be confidently applied to samples of both veterinary and human origin.