深圳地区结核分枝杆菌耐药分离株分子特征与表型特征相关性研究

目的 研究深圳地区2007-2008年分离的结核分枝杆菌耐药株分子特征与表型特征的相关性.方法 参照WHO/IUATLD标准,使用L-J药敏培养基,1%比例法药敏试验筛选针对异烟肼、利福平、链霉素、氧氟沙星、卡那霉素5种药物耐药或敏感的临床分离株,通过PCR扩增经筛选菌株的rpoB、katG、rpsL、rrs1、gyrAB、rrs2基因的相关序列,运用DNAStar和BLASTN进行序列分析,应用二倍稀释法测定其表型最低抑菌浓度(MIC)值.结果 筛得实验菌株123株,其中耐药株73株,全敏感株50株.异烟肼耐药株katG基因突变率为84.6%,突变位点全部为S315T或S315N.利福平耐药株rpoB基因突变率为93.6%,突变位点主要集中在S531L(30/44,68.2%)和H526D(9/44,20.5%)或H526R(1/44,2.3%).链霉素耐药株以rpsl基因突变为主,突变位点为K43R(19/27,70.4%)和K88Q(6/27,22.2%);rrs1基因突变较少见,仅491C→T(2/27,7.4%)及513A→C(1/27,3.7%);2个基因突变率合计为65.9%.氧氟沙星耐药株突变率为100%,以gyrA基因突变为主,突变位点包括D94A(2/11,18.2%)、S91P(4/11,36.4%)和A90V(3/11,27.3%),3个突变位点总突变率为81.8%(9/11);S95T存在于所有氧氟沙星耐药株及部分全敏感株gyrA基因中;gyrB未发现突变.卡那霉索耐药株rrs2基因突变率为61.1%;突变位点为1400 A→C(9/11,81.8%)和1483 G→T(2/11,18.2%).其他突变位点在全敏感株中未发现.临床耐药株同一耐药组别所包含耐药类型不同,相关耐药基因的突变位点相同,其MIC值基本一致;相关耐药基因突变位点不同,其MIC值存在明显差异.结论 结核分枝杆菌耐药基因突变存在一定的地域特性,表型特征因耐药基因突变位点不同存在耐药程度差异.     

结核分枝杆菌临床分离株异烟肼耐药相关基因突变的分子特征

目的 阐明结核分枝杆菌异烟肼（INH）耐药相关基因突变特征.方法 对137株结核分枝杆菌临床分离株（耐异烟肼菌株87株,异烟肼敏感菌株90株）的9个结构基因furA、katG、inhA、kasA、Rv0340、iniB、iniA、iniC和efpA以及两个调控区oxyR-ahpC基因间隔区和mabA-inhA启动子进行DNA片段扩增及序列分析.结果 82株（94.3%）INH耐药分离株的katG基因存在突变,其中katGSer315Thr突变占优势（55.2%）.50株INH敏感的分离菌katG的463密码子没有突变.35株（40.2%）INH耐药的分离株katG的463有突变.87株INH耐药株中,20株（23.0%）的katG基因存在两重突变.13株（14.9%）分离菌inhA基因的启动子区存在突变,4.6%的分离菌有inhA结构基因突变,11.5%oxyR-ahpC基因间区存在突变.iniBAC区域和efpA中发现耐药性关联突变.结论 研究证实多个基因突变与异烟肼耐药之间的关系,并且为阐明结核分枝杆菌耐药机制提供线索.     

四川地区200例随机临床分枝杆菌分离株耐药状况的分析研究

目的 了解四川地区分枝杆菌的耐药状况,为临床用药提供参考依据.方法 采用罗氏绝对浓度法对我院2009年6月-2010年12月间的200株分枝杆菌随机临床分离株进行9种抗结核药物的敏感性试验,并同步进行微量药敏(MIC)检测.结果 200株分枝杆菌临床分离株中192株(96.0％)为结核分枝杆菌(MTB),8株(4.0％)为非结核分枝杆菌(NTM),两种分群方法结果一致.192株MTB中108株对9种抗结核药物全部敏感,对≥1种药物耐药者84株,总耐药率为43.7％ (84/192),多重耐药( MDR)23株(12.0％),广泛耐药4株(2.1％),全耐药2株(1.0％).耐9种不同抗结核药物的顺位由高到低依次为:丙硫异烟胺(PTA,33.3％)、异烟肼(INH,20.8％)、利福平(RFP,17.2％)、硫酸链霉素( SM,16.7％)、硫酸阿米卡星注射液(AMK,16.7％)、力克肺疾(PI,16.1％)、乙胺丁醇(EMB,10.9％)、左氧氟沙星( LFX,8.8％)、硫酸卷曲霉素(CPM,6.2％);单一耐1、2、3、4、5、6、7、8和9种药物耐药率分别为12.5％、7.3％、6.2％、4.7％、4.2％、3.6％、3.1％、1.0％和1.0％.对≥2种药物耐药者60株(31.2％);对≥4种药物耐药者34株(17.7％).M DR-TB对另外7种药物的罗氏药敏耐药率从高到底依次为:PI(78.3％)、EMB(69.6％)、SM(65.2％)、PTA (65.2％)、LFX (39.1％)、AMK( 30.4％)、CPM(8.7％).结论 四川地区耐药性结核病仍处于全国较高水平,特别是同时耐多种(≥4种)药物的耐药率较高,应引起重视.     

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.     

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.     

新疆南疆地区维吾尔族结核病患者结核分枝杆菌临床分离株的基因分型研究

目的 应用多位点数目可变串联重复序列(VNTR)分析技术,对新疆南疆地区维吾尔族结核病患者结核分枝杆菌临床分离株进行基因分型,探讨其数目VNTR基因型种类及其分布.方法 收集结核分枝杆菌,采用PCR和琼脂糖凝胶电泳技术,结合BioNumerics 5.0软件,对其24个VNTR位点进行结果分析.结果 分离出151株结核分枝杆菌,分为8个基因群151个基因型,其中Ⅵ群为主要基因群,占44.4%,有67个基因型,其次是Ⅷ群(23.2%)和Ⅳ群(20.5%).结论 新疆南疆地区维吾尔族结核病患者的结核分枝杆菌存在明显基因多态性,且存在主要流行菌群.     

Detection of novel mutations associated with independent resistance and cross-resistance to isoniazid and prothionamide in Mycobacterium tuberculosis clinical isolates

ObjectivesProthionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can be ineffective against isoniazid-resistant (INH^R) Mycobacterium tuberculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to INH^R and/or prothionamide-resistant (PTO^R) M. tuberculosis isolates.MethodsWe sequenced the above genes in 206 M. tuberculosis isolates with susceptibility testing against ten drugs.ResultsOf the 173 INH^R PTO^R isolates, 170 (98.3%) harboured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 INH^R PTO^S isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five INH^S PTO^R isolates, four showed mutations in ethA and two in the inhA promoter. Notably, 55 novel non-synonymous mutations were found in them and 20.2% of the PTO^R M. tuberculosis isolates harboured no known mutations.ConclusionsThis is the first report to investigate cross-resistance between INH^R and/or PTO^R isolates. Among INH^R (94.4% MDR-TB) M. tuberculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of both phenotypic susceptibility and genotypic diagnosis when using it to treat patients with INH^R-TB. The high proportion (one-fifth) of PTO^R M. tuberculosis isolates showed no known mutation related to PTO^R genes, so uncovered resistance mechanism(s) of prothionamide exist.     

Molecular characterization of varicella-zoster virus clinical isolates from 2006 to 2008 in a tertiary care hospital, Dublin, Ireland, using different genotyping methods

Varicella‐zoster virus (VZV), a herpesvirus, is a ubiquitous organism that causes considerable morbidity worldwide and can cause severe complications on reactivation. Phylogenetic analysis was performed on 19 clinical VZV isolates (16 zoster and 3 varicella) found in Ireland, between December 2006 and November 2008, in order to determine whether previously reported viral heterogeneity was still present and whether viral recombination was evident. Open reading‐frames (ORFs) from genes 1, 21, 50, and 54, were sequenced. Clades 1, 2, 3, and 5 were identified. Four putative recombinant isolates were detected (three clade 3/1 and one clade 5/3/1). Further sequencing and examination of ORF 22 and 21/50, did not elucidate the putative recombinant genotypes further. These two previously published genotyping schemes were examined in light of the new consensus genotyping scheme proposed in 2010. Remarkable VZV heterogeneity remains prevalent in Ireland. This is the first evidence of putative VZV recombination found in Ireland. J. Med. Virol. 84:1672–1679, 2012. © 2012 Wiley Periodicals, Inc.