Name: diagnosing pulmonary tuberculosis with the xpert mtb/rif test
Uploaded: 2012-04-09T12:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

作者承认温家宝仔细审阅的手稿发疯。

结核护理国际标准，所有怀疑有肺结核患者应提交至少有两个痰标本细菌学检查标准2。如果可能的话，至少有一个清晨的标本应该得到，此时痰收集具有最高产量11 Xpert山地车/ RIF检测可应用于痰标本或浓缩痰沉积物准备从诱导或咳出的痰是要么抗酸杆菌（AFB）涂片阳性或阴性6，12，13拟从患者标本，对他们来说，是临床怀疑肺结核的人使用该法是：1）不接受抗结核治疗，2 ）曾&lt;7天的治疗，或3）没有收到在过去60天的治疗。要注意，它不应该被用于监测药物治疗7的影响，因为细菌的DNA可能坚持抗生素治疗后。 <p类=“jove_content”的治疗临床标本，包括使用的墨盒，仿佛发射传染性。使用适当的预防措施，如穿着一次性防护手套，实验室外套和保护眼睛，并按照您的机构的安全程序和指引。 1。咳出痰液样本 注意：进程只作为许多标本在同一时间，因为有GeneXpert DX系统模块运行测试！ <ol><li>每个Xpert山地车/ RIF墨盒上标明相应的标本ID。 </li><li>转让1.0毫升咳出痰一个圆锥形，螺丝帽的管，用无菌移液管。此外，整个标本可在原有的防漏痰收集容器处理。 </li><li>咳出痰，用无菌移液管加入2.0毫升Xpert山地车/ RIF样品试剂（2:1 V / V）。 </li><li>更换的盖子，并大力摇晃管10-20倍。另外，混合物可涡旋（30秒）。 </li><li>允许管站直立在室温下5分钟。 </li><li>大力摇动管10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管直立站在另一个在室温10分钟。 </li><li>检查标本：样品应与液化痰不可见的团块。 </li></ol> 2。集中沉积物 注：过程中只有一次是GeneXpert DX系统的许多标本模块运行测试！ <ol><li>每个Xpert山地车/ RIF墨盒上标明相应的标本ID。 </li><li> 1.5毫升Xpert山地车/ RIF样品试剂转移到一个圆锥形，螺丝帽的管，用无菌移液管。 </li><li>新增至少0.5毫升浓缩沉积物样品试剂含有使用无菌移液管的管。 </li><li>更换盖子，摇管至五igorously 10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管站直立在室温下5分钟。 </li><li>大力摇动管10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管直立站在另一个在室温10分钟。 </li><li>检查标本：样品应与液化痰不可见的团块。 </li></ol> 3。载入Xpert山地车/ RIF墨盒 注意：样品墨盒30分钟内开始测试！ <ol><li>检查的Xpert山地车/ RIF盒和标本编号的标签。 </li><li>打开墨盒盖。 </li><li>使用无菌移液管，移液管吸进液化标本，直到半月板以上的最低标志是，转移到开放端口的Xpert山地车/ RIF墨盒样品。 </li></ol> 注意：免除慢慢气溶胶的形成，以尽量减少风险！ <ol start="4"><li>合上墨盒盖。 </li></ol> 4。开始测试 注：在开始测试之前，确保的GeneXpert DX系统配备与GX2.1软件，软件进口的Xpert山地车/ RIF检测到9！ <ol><li>打开计算机，然后打开了GeneXpert DX仪器。 </li><li>在Windows桌面上，双击的GeneXpert DX快捷图标。 </li><li>登录到的GeneXpert DX系统软件，使用您的用户名和密码。 </li><li>在GeneXpert DX系统“窗口，单击” 创建测试 。 扫描盒条码对话框。 </li><li>在样品编号 “框中，扫描或输入样品编号。 </li><li>扫描Xpert山地车/ RIF墨盒上的条形码。 创建测试窗口出现。 </li><li>点击开始测试 </strong&gt;，然后在出现的对话框中输入密码。 </li><li>绿色指示灯闪烁的仪器模块门打开，装入墨盒。 </li><li>关闭的大门。在测试开始和指示灯停止闪烁，并保持不断的绿色。当测试完成后，指示灯熄灭。 </li><li>等到系统在运行结束后释放的门锁，然后打开模块的门并取出硒鼓。 </li><li>弃置在适当的废物容器使用的墨盒，根据你的机构的标准做法。 </li></ol> 5。代表结果每个Xpert山地车/ RIF墨盒包括试剂MTB的复杂和RIF耐药的检测以及样本处理控制（SPC）控制目标细菌有足够的处理和监测PCR反应抑制剂的存在（S） 。探头检查控制（PCC）验证试剂补液，PCR管充填墨盒，探针的完整性，和染料的稳定性。9的Xpert山地车/ RIF检测的引物扩增的 rpoB基因含有81个碱基对的“核心”地区的一部分。五个不同颜色的荧光核酸杂交探针，称为分子信标，询问整个81 bp的核心。14每个分子信标被设计成具体的，它并没有绑定到它的目标，如果目标序列从野生型的rpoB基因不同如单核苷酸替代序列。因为只有当它们绑定到自己的目标，即野生型rpoB基因序列的分子信标荧光，没有任何一个在检测的五种颜色区分核心区域与野生型之间的保守序列和突变RIF的阻力。 SPC应该负样本的积极和消极或积极的，可以在一个积极的范例。测试结果将是“无效”，如果最高人民法院不以一种消极的测试检测。 PCR反应开始前，GeneXpert DX系统的措施，从探针的荧光信号的监测，反应管内充珠补液，探针的完整性和染料的稳定性。该委员会通过，如果从探针的荧光信号满足指定的验收标准。结果解释的GeneXpert DX系统测量荧光信号和嵌入式计算算法，并在9 查看结果窗口中显示，如下所示： 结核菌检测：山地车目标的DNA检测，这两个控制，SPC和PCC，满足分配的验收标准。较低的Ct值代表的DNA模板起始浓度较高;更高的Ct值代表低浓度的DNA模板。在结核菌检测结果没有检出“RIF的电阻检测”（ 图1A），“RIF的抵抗”（ <strONG&gt;图1B），或“RIF的电阻不确定”上会显示一个单独的行。 山地车未检出：山地车的目标DNA检测不到，这两个控件，SPC和PCC，满足指定的验收标准（ 图2）。 无效：不能确定存在或没有山地车的SPC不符合验收标准，即该样品不妥善处理，或PCR的抑制（ 图3）。注：重复与多余的试样测试！ 错误：一个或多个失败（FAIL）政协结果。山地车和SPC都显示没有结果。注：重复与多余的试样测试！如果该委员会通过（PASS），错误是由系统组件故障引起的。 图1：山地车目标DNA检测，这两个控制，SPC和PCC，满足分配的验收标准。较低的Ct值代表在更高的起点DNA模板的Ct值较高的浓度，低浓度的DNA模板。 “RIF的阻力在山地车检测结果没有检出”（ 图1A），“RIF的电阻检测”（ 图1B），或“RIF的电阻不确定”上会显示一个单独的行。 <img alt="图1A" src="/files/ftp_upload/3547/3547fig1A.jpg" /> 是目前的6。 <a href="https://www.jove.com/files/ftp_upload/3547/3547fig1A.jpg" target="_blank">点击这里查看大图</a> 。图1A山地车检测RIF的电阻没有检测到，即只有野生型的81-BP RIF的耐药决定区（RRDR）rpoB基因核酸序列。 <img alt="图1B" src="/files/ftp_upload/3547/3547fig1B.jpg" /> 图1B山地车检测; RIF的电阻检测。完成下降探测器B（箭头），其中包括职位513至517的R<html>共和的rpoB基因。在这个例子中，部分测序结果显示，检测rpoB基因突变D516V。8 <a href="https://www.jove.com/files/ftp_upload/3547/3547fig1Blarge.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3547/3547fig2.jpg" /> 图2山地车目标DNA检测;。这两个控件，SPC和PCC，满足分配的验收标准， <a href="https://www.jove.com/files/ftp_upload/3547/3547fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3547/3547fig3.jpg" /> 图3的测试结果无效，即存在或山地车的情况下，不能确定：最高人民法院不符合验收标准，因为样本不妥善处理，或因为PCR的抑制。3547fig3large.jpg“目标=”_blank“&gt;点击这里查看大图。

<ol>
	<li>Sudre, P., Dam, t. e. n., Chan, G., C, A., Kochi, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+in+the+present+time:+a+global+overview+of+the+tuberculosis+situation.">Tuberculosis in the present time: a global overview of the tuberculosis situation.</a> World Health Organisation Document WHO/TUB/91. 158, 1-47 (1991).</li><li>World Health Organisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+tuberculosis+control:+epidemiology,+strategy,+financing.">Global tuberculosis control: epidemiology, strategy, financing.</a> WHO report 2010. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The global plan to stop TB, 2006-2015. , (2006).</li><li>Morris, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+multiple+drug+resistance+in+clinical+isolates+of+Mycobacterium+tuberculosis.">Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis.</a> J. Infect. Dis. 171, 954-960 (1995).</li><li>Traore, H., Fissette, K., Bastian, I., Devleeschouwer, M., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin+resistance+in+Mycobacterium+tuberculosis+isolates+from+diverse+countries+by+a+commercial+line+probe+assay+as+aninitial+indicator+of+multidrug+resistance.">Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as aninitial indicator of multidrug resistance.</a> Int. J. Tuberc. Lung Dis. 4, 481-484 (2000).</li><li>Helb, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Detection+of+Mycobacterium+tuberculosis+and+Rifampin+Resistance+by+Use+of+On-Demand+Near-Patient+Technology.">Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand Near-Patient Technology.</a> J. Clin. Microbiol. 48, 229-237 (2010).</li><li>Blakemore, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Analytical+Performance+of+the+Xpert+MTB/RIF.">Evaluation of the Analytical Performance of the Xpert MTB/RIF.</a> Assay. J. Clin. Microbiol. 48, 2495-2501 (2010).</li><li>Telenti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin-resistance+in+Mycobacterium+tuberculosis.">Detection of rifampicin-resistance in Mycobacterium tuberculosis.</a> Lancet. 341, 647-650 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneXpert Dx System Operator Manual. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Standards for Tuberculosis Care (ISTC). , (2009).</li><li>Boehme, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Molecular+Detection+of+Tuberculosis+and+Rifampin+Resistance.">Rapid Molecular Detection of Tuberculosis and Rifampin Resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li><li>Marlowe, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Cepheid+Xpert+MTB/RIF+assay+for+the+Direct+Detection+of+Mycobacterium+tuberculosis+Complex+from+Respiratory+Specimens.">Evaluation of the Cepheid Xpert MTB/RIF assay for the Direct Detection of Mycobacterium tuberculosis Complex from Respiratory Specimens.</a> J. Clin. Microbiol. 49, 1621-1623 (2011).</li><li>El-Hajj, H. H., Marras, S. A. M., Tyagi, S., Kramer, F. R., Alland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampin+resistance+in+Mycobacterium+tuberculosis+in+a+single+tube+with+molecular+beacons.">Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons.</a> J. Clin. Microbiol. 39, 4131-4137 (2001).</li><li>Hillemann, D., Ruesch-Gerdes, S., Boehme, C., Richter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+extrapulmonary+tuberculosis+by+automated+GeneXpert+MTB/RIF+system.">Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system.</a> J. Clin. Microbiol. 49, 1202-1205 (2011).</li></ol>

没有利益冲突的声明。

集成的Xpert山地车/ RIF检测山地车和RIF耐药的同时检测样品净化，免提操作，板来样加工，超灵敏的半巢式PCR，无论是在咳出痰或集中沉积物，在一个单一的易于使用的系统。该法的检测分析限制被认为是131个/毫升痰液。6 Ct值与的山地车细胞的数量之间的对数线性关系，目前拥有来自10 2 10 7 CFU /毫升痰液真实。作为临床有关的山地车浓度检测的线性范围内所有的秋天，该法提供了半定量估计的细菌负荷。该法只需要温和的实验室基础设施和培训。在于检测限至少有两个幅度较低的订单，比传统的显微镜。6特异性，是优秀的。此外，同时检测对RIF-resistanţ山地车3是在耐药结核病的时代设置的医疗点测试的显着效益。 最新的只有有限公布的检测的性能数据诊断在临床上肺结核12;。13的Xpert的山地车/ RIF检测的整体检测疑似肺结核病患者的痰结核菌的临床灵敏度为97.6％，比文化作为参考。这是一项前瞻性多中心研究，涉及五个研究基地（利马，秘鲁，阿塞拜疆的巴库，南非开普敦和德班;孟买，印度）的调查结果，共1462例。预测试整体文化研究人群中证实结核病的概率为50.7％。涂片和培养阳性的情况下，灵敏度为99.8％和90.2％，涂片阴性，培养阳性的情况下。在艾滋病毒阳性肺结核患者中，山地车/ RIF的测试灵敏度为93.9％，98.4％相比，整体检测山地车的协议。艾滋病毒阴性的患者12在低结核病患病率的设置，为89％相比，文化;涂片阴性的标本涂片阳性和72％的敏感性为98％，分别与特异性为13。灵敏度测试和未经处理的痰集中沉积物之间没有显着性差异。在同一研究中12，山地车/ RIF的测试正确检测RIF的阻力，在209 211例（敏感性99.1％）和所有RIF的易感性506例（特异性100％），测序结果时，考虑到12。研究人员发现，两个案件侦破，对RIF耐药性的歧视，表现在不同地点的类似，暗示的结果有可能广泛适用12。此外，集中沉积物比其他呼吸道痰标本，如支气管肺泡灌洗和支气管升的分泌物，具有可比的灵敏度和特异性进行了测试，13日检测的性能测试数据时，肺外结核病是新兴15。 全球结核病控制的迫切需要，允许新的结核病例的快速诊断和检测耐RIF的普遍访问山地车保健点的设置。3电流的核酸扩增方法用于检测山地车是复杂的，劳动密集型的有效工具，和技术要求。 Xpert山地车/ RIF检测有可能使结核病和耐药性，他们将能够负担得起这些设置，标准化，敏感和非常具体的诊断测试。为了方便访问，基金会（FIND）创新诊断Xpert山地车/ RIF测试谈判明显的降价。 10当前查找议价，随着折扣资格的国家名单，在互联网上提供。 </suP&gt;

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Xpert山地车/ RIF的测试</td><td>造父变星</td><td> GXMTB/RIF-10 </td></tr><tr><td>转移移液器</td><td>萨尔斯塔特</td><td> 86.1172.001 </td></tr><tr><td>锥形管，15毫升，120×17毫米，PS </td><td>萨尔斯塔特</td><td> 62.553.542 </td></tr></tbody></table>

diagnosing pulmonary tuberculosis with the xpert mtb/rif test

由于结核分枝杆菌 （MTB）的结核病（TB）仍然是一个重要的公共卫生问题：感染会影响到三分之一的世界人口1，近两百万人每年结核病死亡2普及高品质。世卫组织控制结核战略，是强调以病人为中心，对所有结核病患者治疗3山地车在快速检测呼吸道标本和可靠的抗药性测试结果为基础的药物治疗是这一战略的成功实施的先决条件。然而，在世界许多地区，结核病的诊断仍依赖于敏感，不善标准化痰显微镜方法。无效的结核病检测和耐药性的结核菌株的出现和传播日益危及全球结核病控制活动。2 有效的诊断肺结核需要的可用性 - 在全球范围内的立场 -ardized，易于使用和强大的诊断工具，将使双方的山地车复杂和关键的抗生素，如利福平（RIF），阻力的直接检测。后者的结果可以作为标记为耐多药结核菌（MDR TB）的，并已在迅速提供可靠的测试结果&gt; 95％的MDR-TB菌株。4，5报道很可能直接转化成无害的病人管理决定，最终会治愈，个别病人，并打破了结核病的传播链在社区。 造父变星（桑尼维尔，加利福尼亚，美国）的Xpert山地车/ RIF检测6，7，符合上述要求的一个显着的方式。这是1 1核酸，氨基酸放大试验）山地车痰或集中痰沉积物中复杂的DNA检测;和2）的RIF的耐药相关的 rpoB基因突变检测8它是利用造父变星的GeneXpert霉素设计。系统日在集成和自动化样品处理，核酸扩增，目标序列，采用实时PCR和逆转录聚合酶链反应检测。系统由仪器，个人电脑，条码扫描器，预装软件的运行测试和查看结果。9它采用单使用一次性Xpert山地车/ RIF墨盒持有PCR试 ​​剂和主办PCR过程。由于墨盒是自足的，被淘汰样品之间的交叉污染。6当前核酸扩增方法用于检测山地车复杂，劳动密集，技术要求高。 Xpert山地车/ RIF检测有可能使结核病和耐药普遍接入点保健设置3标准化，敏感和非常具体的诊断测试，他们将能够负担得起。为了便于访问，创新诊断基金会（FIND）谈判的意义不能价格下降。 10当前查找议价，随着折扣资格的国家名单，在互联网上提供。

Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Xpert山地车/ RIF的测试样品去污，免提操作，上板来样加工，超灵敏的半巢式PCR同时检测集成<em&gt;结核分枝杆菌</em&gt;和利福平耐药，无论是在咳出痰或浓缩痰沉积物，在约两个小时。测试规范和要求只有温和的实验室基础设施和培训。

Xpert山地车/ RIF测试与诊断肺结核

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

diagnosing-pulmonary-tuberculosis-with-the-xpert-mtbrif-test

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

80.4K 次观看.  University of Bern. Xpert山地车/ RIF的测试样品去污，免提操作，上板来样加工，超灵敏的半巢式PCR同时检测集成结核分枝杆菌和利福平耐药，无论是在咳出痰或浓缩痰沉积物，在约两个小时。测试规范和要求只有温和的实验室基础设施和培训。

视频: Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

结核分枝杆菌综合肉汤稀释药敏试验微孔板格式为一线和二线药物

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

科研

免疫与感染

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

MODS的方法和诊断结核病耐药结核病

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

生物学

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

结核病分子细菌负荷测定（TB-MBLA）

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

医学

使用 Micro-CFU 方法加速结核病测定

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

作者聚焦:优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

用于结核病疫苗功效评估的微集落形成单位测定

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Xpert山地车/ RIF测试与诊断肺结核

摘要

探索更多视频

此视频中的章节

结核分枝杆菌综合肉汤稀释药敏试验微孔板格式为一线和二线药物

MODS的方法和诊断结核病耐药结核病

展现着多药耐药

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

分析¹⁸葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

报告酶荧光法检测小鼠结核分枝杆菌的影像学

结核病分子细菌负荷测定（TB-MBLA）

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

用于结核病疫苗功效评估的微集落形成单位测定