膜芯片技术对结核分枝杆菌耐药性检测的研究

目的 验证膜芯片技术在结核分枝杆菌对利福平（RFP）、异烟肼（INH）、链霉素（SM）和乙胺丁醇（EMB）耐药性检测中的应用价值.方法 收集393例临床标本（包括痰液、尿液、胸腔积液、腹水标本）,根据结核分枝杆菌耐药位点的DNA序列,设计用于检测rpoB、katG、inhA、rpsL和embB基因常见突变类型的膜芯片,对上述临床标本进行检测,并将其与传统微生物敏感性试验结果进行比较,对不相符结果用聚合酶链反应产物直接测序（PCR-DS）进行验证.结果 以常规培养法为金标准,膜芯片法扩增结核分枝杆菌的敏感性、特异性、准确性分别为90.4%、98.8%和95.7%,其对结核分枝杆菌RFP、INH、SM和EMB耐药性检测的敏感性分别为92.3%、83.3%、68.8%和83.3%;准确性分别为98.6%、97.3%、96.6%、和98.5%;特异性均达到100.0%.结论 膜芯片法检测结核分枝杆菌耐药性具有较高的敏感性、特异性和准确性,可作为常规微生物敏感性试验的补充.     

基因芯片技术检测结核杆菌耐药的临床研究

目的 评价基因芯片技术在结核病防治工作中对结核分枝杆菌异烟肼和利福平耐药性的检测效果.方法 利用基因芯片技术对38例涂阳肺结核患者的痰标本进行异烟肼和利福平耐药检测,以传统罗氏药敏试验为金标准,对基因芯片技术的检测效果进行评价.结果 在38例菌株中,用基因芯片法进行异烟肼耐药基因检测,与罗氏药敏的符合率为84.2％,对38例进行利福平耐药基因检测,符合率为89.5％.结论 基因芯片检测异烟肼和利福平的耐药性与罗氏药敏方法具有很好的一致性,具有简便快速,灵敏度高,特异性好的优点,对临床及时准确进行抗结核治疗具有重要意义.     

二代线性探针技术在结核病诊断及耐药检测中的应用研究

目的 探讨二代线性探针技术(MTBDR plus V2.0)在结核病及其耐药性诊断中的应用价值.方法 选取2018年6月至2019年6月于该院就诊的1322例疑似肺结核患者为研究对象,所有研究对象均用同一份痰标本同时进行痰涂片检测、MGIT 960液体培养和MTBDR plus V2.0基因检测.对培养阳性且鉴定为结核分枝杆菌的菌株进行MGIT 960药敏试验.采用Kappa检验比较药敏试验结果及效能.结果 以MGIT 960液体培养结果为标准,痰涂片和MTBDR plus V2.0检测结核分枝杆菌的灵敏度分别为46.74％和87.15％,特异度分别为98.73％和92.74％,Kappa值分别为0.45和0.80;MGIT 960液体培养和MTBDR plus V2.0对痰涂片阴性肺结核患者检测效能差异无统计学意义(χ2=0.071,P=0.790);MGIT 960和MTB-DR plus V2.0对利福平和异烟肼耐药性检测,Kappa值分别为0.84和0.69.结论 MTBDR plus V2.0能够快速对结核病进行诊断,且效能与MGIT 960液体培养相似,高于痰涂片检测.在利福平耐药检测方面,MTB-DR plus V2.0和MGIT 960有较好的一致性,但对异烟肼耐药性检测一致性一般.     

浙江省杭州市肺结核患者耐药监测结果分析

目的了解杭州市耐药结核病疫情现状及特点,为及时调整杭州市结核病控制策略提供依据。方法收集2011年杭州市登记所有涂阳患者痰标本,采用传统生化反应法进行菌型鉴定,采用世界卫生组织推荐的改良罗氏培养基比例法,进行异烟肼(Isoniazid,H)、利福平(Rifampicin,R)、乙胺丁醇(Ethambutol,E)、链霉素(Streptomycin,S)、氧氟沙星(Ofloxacin,O)和卡那霉素(Kanamycin,KM)的药物敏感性试验。利用结核病管理信息系统,收集患者人口学资料。结果全年共报告1845例涂阳患者,开展痰培养并培养阳性1394例,其中菌群鉴定为结核分枝杆菌并有药敏试验结果者1184例。1184例患者中对4种一线抗结核药物(H、E、R、S)的总耐药率为31.33%,总耐多药率为11.57%。广泛耐药率为0.90%,其中耐多药患者中,耐氧氟沙星者占29.93%,耐卡那霉素者占3.65%。一线药物耐药率由高到低依次为H(19.51%)、S(17.15%)、R(16.98%)和E(5.07%),复治患者耐药率明显高于初治患者。结论杭州市耐药结核病疫情相对较重,需进一步研究耐药病例合理的化疗方案,重点加强对初、复治患者的管理,从源头上减少耐多药甚至广泛耐药的发生。     

新疆阿克苏地区维吾尔族结核分枝杆菌（MTB）耐药突变基因检测的耐药性研究

目的了解新疆阿克苏地区维吾尔族结核患者对四种一线抗结核药利福平、异烟肼、链霉素、乙胺丁醇的耐药情况。方法参照结核痛诊断细菌学检验规程,用亚能生物的基因芯片检测技术对不同来源的结核茵阳性标本经处理后,进行耐药突变基因检测,来判断结核分枝杆菌的耐药情况。结果通过对100例10至70岁的维吾尔族结核患者的耐药突变基因检测,共59株耐药,利福平的耐药率为47．5％,异烟肼为37％,链霉素为8．4％,乙胺丁醇为6．7％。结论阿克苏地区维吾尔族人群的结核病人耐药率高于全国水平。初发的结核藕人对四种一线抗结核药（利福平、异熘料、链霉素、乙胺丁醇）的耐药率较低,而复发的结核病人对四种一线抗结核药的耐药率较高.     

Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis

A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.     

Molecular detection of early appearance of drug resistance during Mycobacterium tuberculosis infection.

During the early development of drug resistance in Mycobacterium tuberculosis (M. tuberculosis) infection only a small proportion of resistant bacteria are present within a milieu of sensitive bacteria. This complicates the use of molecular methods to predict the presence of a resistant phenotype and has been largely ignored in many of the newly developed molecular methods. In this study, mixtures of DNA from M. tuberculosis strains with known wild-type and mutant sequences were used to evaluate the sensitivity of three different molecular methods for detection of drug resistance. The dot-blot and amplification refractory mutation system (ARMS) methods showed sensitivities that approach those of routine phenotypic methods and are able to detect the presence of mutant sequences at a ratio of 1 in 50 (corresponding to 2% mutant sequences). This is 10-fold more sensitive than the commercial kit. The ARMS method was also used to investigate the use of molecular methods to identify mixed infections, and both drug-resistant and susceptible strain populations were identified in a single clinical isolate. These findings highlight the applicability of molecular methods to the rapid detection of drug resistance in tuberculosis patients, particularly in those who are non-compliant and in contacts of known drug-resistant tuberculosis patients, and assistance in limiting the spread of drug-resistant strains.     

结核分支杆菌链霉素耐药基因的杂交检测

目的 探讨耐链霉素（Sm）结核分支杆菌的编码基因rpsL和ms的扩增以及突变情况。方法 采用PCR扩增技术对23株敏感株，32株耐药株，25例临床结核病人痰标本进行rpsL、ms基因扩增；并利用寡核苷酸探针反相斑点杂交技术对23株敏感株，32株耐药株，25例临床结核病人痰标本进行rpsL、ms基因突变检测。结果 23株敏感株、32株耐药株rpsL、ms基因扩增均阳性，阳性率100％；25例临床结核病人痰标本中rpsL基因扩增阳性率为72％，ms基因扩增阳性率为56％；rpsL基因突变率依次分别为4．3％、65．6％，50％；ms基因突变率依次分别为0、12．5％、7．2％。结论 耐链霉素（Sm）菌株的基因突变率明显高于药物敏感株；PER．寡核苷酸探针反相斑点杂交技术以其简便、快速、灵敏的特性能够为临床检测结核分支杆菌对链霉素（Sm）的耐药性提供初步依据。     

Multiple drug resistance of Mycobacterium tuberculosis

Mycobacterium tuberculosis acquires drug resistance by chromosomal mutation resulting in alterations of target molecules of drugs.     

DNA微阵列芯片法在海南地区结核病诊断及耐药性检测中的应用

目的 探讨DNA微阵列芯片法在海南地区结核病诊断及耐药性检测中的应用.方法 采用抗酸杆菌涂片法、罗氏培养法、比例法药敏试验及DNA微阵列芯片法对海南地区的2069例疑似结核病患者痰标本进行检测,并对结核分枝杆菌检出率、耐药性及耐药基因突变特征进行分析.结果 DNA微阵列芯片法检测结核分枝杆菌检出率为明显高于罗氏培养法和...     

基因芯片技术对结核分枝杆菌耐药性检测的临床应用

目的 评价基因芯片技术对结核分枝杆菌耐药性检测的效果. 方法 选取我院结核科2014年3月至2016年3月收治的涂片阳性的肺结核住院患者238例,取其痰标本,分别用基因芯片、传统的罗氏培养和药敏试验3种方法进行结核分枝杆菌的异烟肼耐药性检测和利福平耐药性检测;把罗氏培养和药敏试验的结果作为金标准,评价基因芯片技术的临床应用价值. 结果 基因芯片技术检测涂片阳性患者痰标本异烟肼耐药的情况与金标准无显著差异(P＞0.05);利福平耐药的情况与金标准相比也无明显差异(p>0.05). 结论 基因芯片能够快速、准确地检测结核分枝杆菌对利福平和异烟肼的耐药情况,是一种值得推广的临床实验室检测方法.     

基因芯片技术在结核分支杆菌耐药突变基因检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中结核分支杆菌耐药突变情况。方法根据结核分支杆菌标准株H37Rv序列，设计了覆盖rpoB、katG，inhA基因突变区的系列寡核苷酸探针，制作膜芯片，检测临床样品中结核分支杆菌基因突变情况，以此判断耐药结果。结果在305例临床病例中，共检出阳性病例125例，其中阳性敏感病例64例，阳性突变病例61例，阳性率为40．98％，在125例阳性样品中，共发现有8种突变类型，其中10例531L，占7．94％，19例315M，占阳性样品中总数的15．08％。结论PCR与膜芯片杂交技术可临床检测结核分支杆菌对利福平和异烟肼的耐药性，并具有快速、简便、敏感的特点。     

基因芯片早期诊断脊柱结核耐药

20世纪90年代以来,全球结核疫情"死灰复燃",中国是全球22个结核高疫情国家之一,患病人数仅次于印度,80%的患者在农村.2007-2008年全国结核病耐药基线调查显示:我国肺结核患者中耐多药率为8.32%,广泛耐药率为0.68%,严重危及社会公共卫生安全.     

基因检测及MODS技术快速诊断结核分枝杆菌对左氧氟沙星的敏感性研究

目的 探讨基因检测技术和显微镜观察药物敏感度检测技术(MODS)对结核分枝杆菌(MTB)耐左氧氟沙星(LFX)快速诊断价值.方法 选取MTB标准株(H37Rv)和32株耐LFX临床分离株,行gyrA基因喹诺酮类药物耐药决定区(QRDR)序列测定,同时,用24孔细胞培养板进行MTB液体药敏检测.结果 基因检测结果显示,H37Rv未发生gyrA基因突变;与H37Rv株序列的差异比较显示,29株耐LFX临床株发生了有义突变,突变率90.6%.MODS与罗氏绝对浓度法药敏(L-J)比较,H37Rv完全相符;32株耐LFX临床分离株,相符31株,不符1株,符合率为96.9%.结论 gyrA基因突变检测可作为快速诊断结核分枝杆菌对LFX敏感性的一种方法,但不能完全区别低耐药和高耐药而使用受到限制.MODS技术检测LFX耐药性具有快速、操作简便、灵敏度和特异性高等优点,可作为结核分枝杆菌对LFX敏感性的快速检测新方法.     

Rapid alternative methods for detection of rifampicin resistance in Mycobacterium tuberculosis

OBJECTIVE: To study the performance of three rapid low cost methods for the detection of rifampicin resistance. METHODS: A panel of 20 coded Mycobacterium tuberculosis strains was tested blindly by the low cost methods: nitrate reductase, MTT and resazurin assays, and compared with the results obtained with the gold standard methods: the proportion method on L?wenstein-Jensen medium and the BACTEC TB 460 system. We have also tested two commercial tests: MGIT and INNO LiPA Rif.TB kit. RESULTS: Complete agreement was observed among all methods. CONCLUSION: These three simple methods might become inexpensive alternative procedures for rapid detection of rifampicin resistance in low-resource countries.     

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.     

Slide drug susceptibility test for the detection of multidrug-resistant tuberculosis in Bangladesh

The present study attempted to comparatively assess and establish a suitable detection method of multidrug-resistant tuberculosis (MDR-TB) from previously treated TB cases in Bangladesh. Of 130 Zeihl–Neelsen smear-positive fresh sputum specimens, 112 samples were found to contain viable bacilli as visualized under the light-emitting diode fluorescence microscope after fluorescein di-acetate staining, and 109 positive cases were detected through Löwenstein–Jensen culture. The samples were further tested to survey the drug resistance both by slide drug susceptibility test (DST) and by conventional DST: 94 MDR-TB cases were detected within 10 days through the slide DST, whereas 82 cases were observed through the conventional DST, requiring about 3 months. Because the rapidity, sensitivity and accuracy of the slide DST method were found to be comparatively satisfactory when compared to the conventional DST method; we recommend the slide DST method as the standard diagnostic tool in perspective of Bangladesh for the detection of MDR-TB.     

结核病耐药与用药史关系的研究

目的 研究结核病耐药与用药史的关系,提高医务人员,尤其是结核病防治工作者对耐药结核病的认识并指导临床治疗。方法 应用2001年河南省第二轮结核病耐药监测资料,病例入选、药敏试验及耐药标准按照世界卫生组织（WHO）／国际防痨与肺部疾病联合会（IUATLD）结核病耐药监测指南,采用整群抽样、比例法,选取30个监测县共1487例结核病患者,其中初治1222例,复治265例;详细询问患者用药史并填写临床资料调查表,对药敏结果和用药史进行分析。结果 初治失败患者耐药率和耐多药率最高,分别为90．0％和80．0％,其次为慢性排菌者,其耐药率和耐多药率均为73．3％;耐药率和耐多药率均随用药时间的增加呈升高趋势,且从未化疗及化疗〈1个月者耐药率及耐多药率均低于化疗2个月及以上各组。结论对初治失败及慢性菌患者实行个体化治疗是十分必要的,应落实直接面视下的短程督导化疗（DOTS）策略,加强强化期督导管理,防止耐药产生。     

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.