液体培养法联合菌种鉴定技术诊断HIV与分枝杆菌双重感染

目的 利用分枝杆菌液体培养法联合菌种鉴定技术诊断HIV与分枝杆菌双重感染,以了解目前华北地区HIV与分枝杆菌双重感染的情况。 方法 对2009-2011年收治的53例疑似HIV与分枝杆菌双重感染患者的可疑感染部位标本进行分枝杆菌液体培养及菌种鉴定。收集所有经确诊为HIV与分枝杆菌双重感染患者的临床资料、影像学及相关实验室检查结果进行分析与总结。 结果 53例疑似HIV与分枝杆菌双重感染的患者中,病原学证实存在分枝杆菌感染的患者为19例,11例(57.9%)为单纯肺脏累及,2例(10.5%)为肺外累及,4例(21.1%)为分枝杆菌血症, 2例(10.5%)为肺脏累及和分枝杆菌血症并存。16例患者为结核分枝杆菌感染,3例患者为鸟胞内分枝杆菌复合体感染。15例（78.9%,15/19）患者CD4⁺ T淋巴细胞计数<100个/μl。 结论 联合应用液体培养法与菌种鉴定技术对于诊断HIV与分枝杆菌双重感染是有效的,值得在临床推广应用。     

114例HIV/MTB双重感染病例结核分枝杆菌Spoligotyping基因分型研究

目的 了解HIV/MTB双重感染病例中结核分枝杆菌基因型构成情况以及北京家族基因型菌株所占的比例,为HIV/MTB双重感染病例结核病预防控制提供分子流行病学依据。方法 收集HIV/MTB双重感染病例结核分枝杆菌临床分离株114株,利用间隔寡核苷酸分型(Spoligotyping)技术对所收集的临床分离株进行分型研究,分析HIV/MTB双重感染病例中结核分枝杆菌基因型构成情况及主要流行型。结果 114例HIV/MTB双重感染病例结核分枝杆菌成2个大的基因家族,即北京家族(Beijing Family)和非北京家族(non-Beijing Family),分别占66.7%(76/114)和33.3%(38/114),共30种基因型。其中,非北京家族包括T家族共18株,H家族共5株,EAI5基因家族1株,CAS1_DEHLI基因家族1株及12种在数据库中没有对应基因家族或没有匹配的结果的新基因型13株。结论 HIV/MTB双重感染病例中,结核分枝杆菌基因型呈明显的多态性,北京家族基因型菌株仍为HIV/MTB双重感染病例中结核分枝杆菌主要流行菌株。     

结核分枝杆菌实验室检测产品和技术应用进展

结核病是全球面临的重大公共卫生问题,结核病患者的早期快速检测对提高患者的疗效,并有效阻断结核人际传播至关重要.结核病诊断能力不足是导致发现率和报告率较低的根本原因,尤其在患者面对交通、经济负担等一系列问题时,进一步加剧患者的漏诊.然而传统的结核分枝杆菌实验室检测方法已无法满足临床的实际需求,亟待在结核病高负担国家使用和...     

2株胞内分枝杆菌临床分离株的鉴定

目的联合应用多种分子生物学技术进行分枝杆菌菌种鉴定。方法从经PNB、TCH鉴别培养基鉴定为结核分枝杆菌的临床分离菌株285株中选取6株,其中包括oxyR-ahpC基因间隔区检测时PCR产物分子量异常的2株和另外随机选择的PCR产物分子量正常者4株,采用H37Rv、鸟分枝杆菌95001和胞内分支杆菌95002做对照,PCR扩增oxyR-ahpC基因间隔区,对其产物进行测序和网上同源性(H37Rv、U18263、U71061)比对。同时,采用多位点PCR扩增和hsp65 PCR-限制性酶切片段长度多态性分析,从而判定分枝杆菌菌种类型。结果经PNB、TCH鉴别培养基鉴定初步判定为结核分枝杆菌的285株临床分离株中2株临床菌株oxyR-ahpC基因间隔区PCR产物大小约为1 000bp,DNA序列与胞内分枝杆菌同源性极高,达99%,而与同片段序列结核分枝杆菌H37Rv的差异大,同源性为84%。多位点PCR指纹显示为非分枝结核杆菌,hsp65 PCR-RLFP/RFLP指纹与95002一致。结论2株oxyR-ahpC基因间隔区PCR产物分子量异常的经PNB、TCH鉴别培养基鉴定为结核分枝杆菌的临床分离株确定为胞内分枝杆菌。多种分子生物学技术联合应用能够快速、简便、更准确地鉴定分枝杆菌菌种。     

多位点PCR用于安徽391株分枝杆菌分离株的快速鉴定

摘要：目的 评价多位点PCR用于分枝杆菌临床分离株快速鉴定效果。方法 收集从临床结核病患者分离到的抗酸染色阳性的培养物,经PNB/TCH鉴别培养基进行培养鉴定后,采用聚合酶链反应(PCR)对16SrRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8和Rv3120 基因位点进行扩增,鉴定至种,再经rpoB-PRA、hsp65和rpoB基因测序进行验证。结果 共对391株分枝杆菌临床分离株应用多位点PCR进行了鉴定,结果显示结核分枝杆菌378株,非洲分枝杆菌I型6株,非结核分枝杆菌7株。7株非结核分枝杆菌分别为鸟分枝杆菌1株,马赛分枝杆菌2株,胞内分枝杆菌4株。而PNB/TCH鉴别培养基培养鉴定结果为结核分枝杆菌复合群385株,非结核分枝杆菌6株。多位点PCR结果与rpoB-PRA、hsp65和rpoB基因测序结果一致。结论 多位点PCR技术鉴定分枝杆菌菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值,且对于临床诊断和治疗都具有重要意义。     

Detection of mycobacteria in joint samples from patients with arthritis using a genus-specific polymerase chain reaction and sequence analysis.

OBJECTIVE: Mycobacteria have been implicated in the pathogenesis of various forms of arthritis. The aim of this study was to examine the diagnostic potential of molecular biological techniques as well as to investigate the pathogenetic role of mycobacteria in chronic arthritis. PATIENTS AND METHODS: DNA, extracted from synovial fluid and synovial tissue samples from patients with mycobacterial septic arthritis (n = 2), seronegative spondyloarthropathies (SpA) (n = 18), undifferentiated arthritis (UA) (n = 21) and rheumatoid arthritis (RA) (n = 40), was analysed using a mycobacterial genus-specific polymerase chain reaction (PCR) applied to amplify mycobacterial DNA. Subsequently, automated sequencing was performed for speciation. Samples from patients with either non-mycobacterial septic arthritis, osteoarthritis (OA), crystal arthritis or joint trauma served as negative controls (n = 19). RESULTS: Mycobacterium tuberculosis complex and Mycobacterium marinum were detected in the two patients with mycobacterial septic arthritis. The other species identified were Mycobacterium hodleri (in one RA patient), Mycobacterium smegmatis (in one OA patient and two RA patients) and Mycobacterium austroafricanum (in one crystal arthritis patient). All other samples were negative. CONCLUSIONS: The results suggest that the mycobacterial genus-specific PCR applied on DNA extracts isolated directly from joint samples may be employed as an additional diagnostic tool in the case of clinical suspicion of a mycobacterial infection. No evidence was obtained for a pathogenetic role of mycobacteria in SpA, UA or RA.     

Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium kansasii, and other nontuberculous mycobacteria in an area of endemicity for AIDS.

Between 1981 and 1990, cultures of specimens from 86 patients at State University of New York-Health Sciences Center at Brooklyn were positive for nontuberculous mycobacteria other than Mycobacterium avium/Mycobacterium intracellulare complex or Mycobacterium gordonae. The most common species isolated were Mycobacterium xenopi (33), Mycobacterium fortuitum (28), Mycobacterium kansasii (7), and Mycobacterium chelonae (6). Thirty-five patients (41%) had clinical and/or serological evidence of human immunodeficiency virus (HIV) infection. Patients from whom M. xenopi and M. kansasii were isolated were significantly more likely to be infected with HIV than were the remaining patients in this series. Most of the mycobacterial isolates were cultured from respiratory secretions. However, extrapulmonary infections with M. fortuitum, M. xenopi, M. kansasii, Mycobacterium terrae, and Mycobacterium scrofulaceum did occur among the HIV-infected patients.     

L型结核菌rpoB基因突变与利福平耐药性的关系研究

目的探讨L型结核分枝杆菌rpoB基因突变与利福平耐药性的关系。方法对76株复制肺结核患者L型结核分枝杆菌临床分离株进行药敏试验,同时采用PCR和PCR-DS技术对L型结核菌株进行rpoB基因检测和序列分析。结果药敏结果提示28株L型结核分枝杆菌对利福平耐药,其中20株(71.4%)L型结核分枝杆菌rpoB基因发生突变。结论 L型结核分枝杆菌rpoB基因突变是造成结核分枝杆菌形成利福平耐药性的主要机制。     

Molecular genetic approaches to Mycobacterium tuberculosis

Recent progress of molecular genetics has been providing tools for new approaches to disease treatment and diagnosis of Mycobacterium tuberculosis. In 1998, Cole et al. reported the complete genome sequence of Mycobacterium tuberculosis. The new information will provide us the knowledge and understanding of the biology of Mycobacterium tuberculosis. Further, it will provide us new conception of diagnosis and treatment of the disease. Four topics were selected in this symposium. Dr. Iinuma reviewed and prospected the clinical utility of nucleic acid amplification methods of Mycobacterium tuberculosis. Dr. Suzuki reviewed the molecular mechanism of acquired resistance to anti-TB drugs and reported the early detection of genetic mutation by new designed DNA tip method. Dr. Takahashi reviewed the method of molecular epidemiology and genetic elements as a tool for strain differentiation of tuberculosis. Dr. Mizuguchi interpreted the essential feature of mycobacterial genome maps, and genes and their biological activity. He also reviewed the importance and the utility of the complete genome sequence of tuberculosis in association with pathogenecity. These topics were summarized in this report, based on the symposium of "Molecular genetic approaches to Mycobacterium tuberculosis" in the 75th annual meeting of the Japanese Society for Tuberculosis.     

Mycobacteria other than Mycobacterium tuberculosis: review of microbiologic and clinical aspects

The rate of isolation of mycobacteria other than Mycobacterium tuberculosis (MOTT) has increased over the past several years; in some areas the isolation rate for Mycobacterium avium-Mycobacterium intracellulare has surpassed that for M. tuberculosis. Simultaneously, the spectrum of clinical manifestations with the various species has widened. Outbreaks of disease due to Mycobacterium chelonae have occurred in renal dialysis patients. New species have been described: Mycobacterium malmoense is primarily a pulmonary pathogen, and Mycobacterium haemophilum has been recovered from cutaneous lesions in immunosuppressed patients. In addition, reports of disease due to species generally considered saprophytic have become more numerous. In this review, the epidemiologic, pathologic, and clinical features of the individual MOTT species are discussed. A brief summary of mycobacteria isolated at the Cleveland Clinic foundation between 1982 and 1985 is also presented.     

耐药结核分枝杆菌快速检测方法的研究进展

传统的耐药结核分枝杆菌的诊断依赖药物敏感试验.近年来,随着技术的进步,出现了大量的先进检测手段,在敏感性、特异性、检测周期方面皆优于传统方法.基于核酸扩增的分子生物学技术为临床耐药结核分枝杆菌的快速检测打开了一个广阔的前景.在自动测序分析的帮助下,耐药结核的确诊越来越简便、准确,对结核菌基因中耐药突变位点的快速检测还能够为临床诊疗提供早期的帮助信息,甚至能识别潜在的感染.本文对目前已有的快速检测方法的研究进展进行综述.     

耐药结核分枝杆菌快速检测方法的研究进展

传统的耐药结核分枝杆菌的诊断依赖药物敏感试验.近年来,随着技术的进步,出现了大量的先进检测手段,在敏感性、特异性、检测周期方面皆优于传统方法.基于核酸扩增的分子生物学技术为临床耐药结核分枝杆菌的快速检测打开了一个广阔的前景.在自动测序分析的帮助下,耐药结核的确诊越来越简便、准确,对结核菌基因中耐药突变位点的快速检测还能够为临床诊疗提供早期的帮助信息,甚至能识别潜在的感染.本文对目前已有的快速检测方法的研究进展进行综述.     

耐药结核分枝杆菌快速检测方法的研究进展

传统的耐药结核分枝杆菌的诊断依赖药物敏感试验.近年来,随着技术的进步,出现了大量的先进检测手段,在敏感性、特异性、检测周期方面皆优于传统方法.基于核酸扩增的分子生物学技术为临床耐药结核分枝杆菌的快速检测打开了一个广阔的前景.在自动测序分析的帮助下,耐药结核的确诊越来越简便、准确,对结核菌基因中耐药突变位点的快速检测还能够为临床诊疗提供早期的帮助信息,甚至能识别潜在的感染.本文对目前已有的快速检测方法的研究进展进行综述.     

DRE-PCR分型方法在唐山市结核分枝杆菌研究中的应用

目的DRE-PCRDNA指纹分型方法的建立及其在分枝杆菌基因分型中的应用。方法收集结核病患者痰标本,分离培养获得分枝杆菌,进行表型鉴定及耐药性检测;提取基因组DNA,在建立DRE-PCRDNA指纹分型方法的基础上对分枝杆菌进行DNA指纹图谱分析。结果DRE-PCR指纹图谱分析将80株分枝杆菌分为13种菌型,其中A型菌26株(32.5%),B型菌14株(17.5%),C型菌16株(20.0%),其余各型均<7.5%。分组统计显示,3种主要的DRE-PCRDNA指纹在40~60岁组的分布偏高(P=0.019),而与患者的性别、职业、长期居住地、结核接触史、痰涂片、耐药性等无关。结论DRE-PCRDNA指纹分型方法是一种简单、快速的分枝杆菌的分型方法;3种主要类型的结核分枝杆菌在唐山地区流行。     

获得性免疫缺陷综合征合并肺结核临床特征与T细胞亚群的相关性分析

目的 探讨获得性免疫缺陷综合征合并肺结核(AIDS/PTB)双重感染的类型,影像特征及临床表现为PTB菌阴及菌阳时,患者T细胞亚群计数的差异及意义.方法 回顾性分析93例AIDS/PTB双重感染患者的类型,影像特征及临床表现分别为PTB菌阴及菌阳时,检测其T细胞亚群水平并进行统计学比较.结果 ①CD4+细胞计数与结核病的发病率成反比,当CD4+＜50×106/L时,结核的发病率明显上升(67.74%,63/93).②影像特征不典型的PTB病例CD4+T细胞计数为(47.79±32.17)×106/L,影像特征典型的PTB病例CD4+T细胞计数为(95.3456±64.89)×106/L.二者之间CD4+T细胞计数差异有统计学意义(P＜0.01).③不同临床表现患者CD4+T细胞计数差异有统计学意义,PTB菌阴组患者明显高于PTB菌阳组患者(P＜0.02),CD8+T细胞计数比较差异无统计学意义(P＞0.05).结论 AIDS/PTB感染患者临床特征与其T细胞亚群计数相关.     

Genotyping did not evidence any contribution of Mycobacterium bovis to human tuberculosis in Brazil

The contribution of Mycobacterium bovis to the global burden of tuberculosis (TB) in man is likely to be underestimated due to its dysgonic growth characteristics and because of the absence of pyruvate in most used media is disadvantageous for its primary isolation. In Brazil Mycobacterium culture, identification and susceptibility tests are performed only in TB reference centers, usually for selected cases. Moreover, solid, egg-based, glycerol-containing (without pyruvate supplementation) L?wenstein-Jensen (L-J) or Ogawa media are routinely used, unfavouring M. bovis isolation. To determine the importance of M. bovis as a public health threat in Brazil we investigated 3046 suspected TB patients inoculating their clinical samples onto routine L-J and L-J pyruvate enriched media. A total of 1796 specimens were culture positive for Mycobacterium spp. and 702 TB cases were confirmed. Surprisingly we did not detect one single case of M. bovis in the resulting collection of 1674 isolates recovered from M. bovis favourable medium analyzed by conventional and molecular speciation methods. Also, bacillary DNA present on 454 sputum smears from 223 TB patients were OxyR genotyped and none was recognized as M. bovis. Our data indicate that M. bovis importance on the burden of human TB in Brazil is marginal.     

苏州市结核病耐多药情况分析

目的 了解苏州市地区感染结核分枝杆菌(Mycobacterium tuberculosis,MTB)的结核病患者耐多药情况.方法 对2008年9月～2011年5月期间在苏州大学附属传染病医院就诊的肺结核病患者的临床分离株进行菌型鉴定后,用药敏罗氏培养基进行药物敏感性试验,分析结核病患者的耐多药情况.结果 苏州市地区近三年期结核病耐多药发生率总体为10.5％,2010年及2011年度耐多药发生率(18.2％)显著高于2008及2009年度(x2 =19.9,P＜0.01),且复治组耐多药发生率显著高于初治组(x2 =70.5,P＜0.01).结论 苏州大学传染病医院收治的结核病耐多药率呈上升趋势,且复治组高于初治组.     

AmpliSensor—聚合酶链反应定量检测肺结核患者外周血结 …

目的 探讨ＡｍｐｌｉＳｅｎｓｏｒ－聚合酶链反应定量检测外周血中结核分支杆菌ＤＮＡ在肺结核的应用价值。方法 采用ＱｌＡａｍｐ和ＡｃｕＰｕｒｅ法提取，制备全血中模板ＴＢ－ＤＮＡ，应用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ定量检测，并与ＩＳ６１１０－单管巢式聚合酶链反应（ＳＮ－ＰＣＲ）作比较。结果２００例肺结核患者的血液标本中，两种方法测得结核分支杆菌ＤＮＡ的阳性率分别为６０．５％、６３．５％。８５例非结核肺病     

Enzyme linked immunosorbent assay of a 12-kDa protein of Mycobacterium leprae with sera from leprosy patients.

A low molecular weight protein was obtained from a sonicate of armadillo-derived Mycobacterium leprae cells and from a lambda gt11 phage lysate of Escherichia coli (specifying the M. leprae 12-kDa protein) by a single step of ultrafiltration. Both proteins had an approximate molecular weight of about 12,000 (by SDS-PAGE) and were recognized by the M. leprae 12-kDa-specific monoclonal antibody ML06 by immunoblotting. Sera from 79 leprosy patients across the clinical spectrum, 17 contacts, and 12 normal healthy individuals were screened in an enzyme-linked immunosorbent assay (ELISA) using the 12-kDa proteins as the antigens. Antibodies to the 12-kDa protein (from lysate as well as sonicate) were detected in patients' sera across the clinical spectrum (44%-100% positivity), while no detectable reactivity was observed with control or contact sera. Sera from patients who had undergone a year or more of chemotherapy exhibited no reactivity compared to those from patients with only 3-6 months of chemotherapy. The 12-kDa proteins were also recognized by rabbit hyper-immune M. leprae antiserum.     

Laboratory diagnosis of mycobacterial infections

This paper reviews the recent US history of infection with Mycobacterium tuberculosis and discusses the emergence of drug-resistant strains. The paper continues with brief discussions of the clinical presentation of tuberculosis, tuberculosis in the pediatric population, and nontuberculous pulmonary disease. We discuss laboratory techniques that will rapidly identify Mycobacterium tuberculosis and provide susceptibility test results. The advances and limitations of currently available diagnostic techniques are presented. The chapter concludes with an explanation of the current strengths and limitations of molecular diagnosis of disease and resistance.