International Research in Korea: Clinical Studies of Drug-Resistant Tuberculosis

该项目是由LCID/NIAID结核病研究科、韩国卫生、福利和家庭部疾病控制中心以及大韩民国延世大学医学院合作开展的。这些合作者共同努力建立了国际结核病研究中心（ITRC），该中心管理实验室设施内的财务和科学活动，包括一个功能齐全的生物安全3级实验室设施。此外，还与首尔峨山医院、三星首尔医院、国立首尔医院等共同进行研究。具体方案驱动的研究正在进行中，包括：(1)NIAID 05-I-N069：韩国肺结核患者多药耐药结核染色和宿主易感基因的自然历史研究。本研究旨在确定耐多药和广泛耐药结核分离株的特征及其对人类疾病的影响，并招募了740多名受试者。该方案的一个子研究是与斯泰伦博斯大学和新泽西医学和牙科大学合作调查疾病反应的生物标志物。耐多药结核病和广泛耐药结核病的自然史研究已经允许通过全基因组测序解决关于高度耐药结核病和药物敏感结核病差异的一些基本生物学问题，这项分析正在进行中。我们还在研究各种结核病药物的作用机制，以及结核分枝杆菌基因组的突变如何赋予对这些药物的耐药性。最近，作为结核病临床诊断研究联盟（TB- cdrc）的一部分，我们正在使用根据该方案收集的200株分离株来研究基于微滴板的药物敏感性试验的性能，该试验称为SENSITITREMYCOTB。该测试具有潜在的优势，可以比传统方法更快地给出一线和二线药物的最小抑制浓度（MIC）结果。涉及新诊断工具的几项合作正在进行中，包括使用独特的马氏信标方法开展下一代RIF GeneXpert检测工作，该检测将涵盖更多突变，以及与David Alland博士合作开展用于检测Mtb中氟喹诺酮类药物耐药性和卡那霉素耐药性的XDR检测。此外，正在对GeneXpert检测进行研究，该检测增加了一个步骤，旨在抑制来自死细菌的DNA扩增，作为测量对治疗反应的检测。洛斯阿拉莫斯国家实验室正在开发另一种有前途的直接检测病原体特异性生物标志物的测试。传统的免疫检测平台灵敏度较差，但我们的合作已经证明了使用基于波导的光学生物传感器平台直接检测三种结核病特异性生物标志物，即LAM，早期分泌抗原靶标6 （ESAT6）和抗原85复合物（Ag85）。将平面光波导的倏逝场内的检测与减少非特异性相互作用的功能表面相结合，可以在短时间内对复杂患者样本（尿液、血清）中的生物标志物进行超灵敏检测。这些平台和易于获取的人体样本（如尿液和呼出气体凝析物）中的生物标志物组可用于报告治疗成功，以开发护理点测试。在分析本研究期间收集的医学数据时，我们确定，在自然史队列中，服用二线药物的患者中发生了大量的主要药物不良反应（MADRs）。最常见的madr是胃肠道、肌肉骨骼、精神、视觉和周围神经病变。与一线治疗方案（2.5%）相比，二线治疗方案中mdr的发生率更高（16%）。与MADRs相关的常见药物有阿米卡星（30%）、利奈唑胺（28%）、对氨基水杨酸（24%）、吡嗪酰胺（5.8%）、大环内酯类（4.5%）和环丝氨酸（4.4%）。氟喹诺酮类药物尽管广泛使用，但仍占单一MADR（0.003%）。在多因素分析中，多重或广泛耐药疾病感染和既往抗结核治疗史是MADR的危险因素。

The project is conducted as a collaboration between the Tuberculosis Research Section of LCID/NIAID, the Korean Ministry of Health, Welfare and Familys Center for Disease Control, and Yonsei University College of Medicine in the Republic of Korea. These collaborators have worked together to establish the International Tuberculosis Research Center (ITRC) that manages both financial and scientific activities within the laboratory facilities, including a fully functional Biosafety Level 3 laboratory facility. In addition, studies are also being conducted in collaboration with Asan Medical Center, Samsung Medical Center, and the National Medical Center, all located in Seoul. Specific protocol-driven investigations underway include: (1) NIAID 05-I-N069: A Natural History Study of Multidrug-Resistant TB Stains and Host Susceptibility Genes in Korean Patients with Pulmonary TB. This study seeks to characterize MDR and XDR tuberculosis isolates and their contribution to human disease and has over 740 subjects enrolled. A substudy of this protocol is investigating biomarkers of disease response in collaboration with University of Stellenbosch and University of Medicine and Dentistry of New Jersey. The Natural History study of MDR and XDR TB has allowed a number of basic biology questions about the differences in highly drug resistant and drug sensitive tuberculosis to be addressed by whole genome sequencing and this analysis is ongoing. We are also studying the mechanism of action of various TB drugs and how mutations in the Mtb genome confer resistance to these drugs. Most recently we as part of the TB Clinical Diagnostics Research Consortium (TB-CDRC), are using 200 isolates collected under this protocol to investigate the performance of a microtiter plate-based drug susceptibility assay called SENSITITREMYCOTB. This test has the potential advantages of giving minimal inhibitory concentration (MIC) results to both first and second line agents more rapidly than traditional methods. Several collaborations involving new diagnostic tools are underway including work on the next generation GeneXpert test for RIF that will cover more mutations using a unique sloppy beacon approach and a XDR test for detection of fluoroquinolone resistance and Kanamycin resistance in Mtb with Dr. David Alland. In addition, the GeneXpert assay is being investigated, with the addition of a step meant to inhibit DNA from dead bacteria from amplifying, as an assay for measuring response to treatment. Another promising test for the direct detection of pathogen-specific biomarkers is under development with Los Alamos National labs. Traditional immunoassay platforms suffer from poor sensitivity, but our collaboration has demonstrated direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive detection of biomarkers in complex patient samples (urine, serum) within a short time. These platforms and biomarker sets in easily accessible human samples, such as urine and exhaled breath condensate, may be adaptable to report on treatment success, for development of point of care tests. In analyzing the medical data collected during this study, we determined that significant numbers of major adverse drug reactions (MADRs) were occurring in patients taking second line agents in the Natural History cohort. The most common MADRs were gastrointestinal, musculoskeletal, psychiatric, visual and peripheral neuropathic. MADRs were more frequent in subjects being treated with second-line regimens (16%) compared to first-line regimens (2.5%). Drugs frequently associated with MADRs were amikacin (30%), linezolid (28%), para-aminosalicylic acid (24%), pyrazinamide (5.8%), macrolides (4.5%) and cycloserine (4.4%). Fluoroquinolones accounted for a single MADR (0.003%), despite widespread usage. In multivariate analysis, infection with multi- or extensively drug-resistant disease and previous history of anti-tuberculosis treatment were risk factors for MADR. (2) NIAID 07-I-N041: A Randomized, Double-blind, Placebo-controlled Pilot Study of Metronidazole Combined with Antituberculous Chemotherapy vs. Antituberculous Chemotherapy with Placebo in Subjects with Multidrug-Resistant Pulmonary Tuberculosis. The importance of anaerobic activity in candidate TB drugs is under investigation in this study. In 2009, the trial closed to enrollment after 35 patients enrolled because of concerns about side effects, but analysis of the data collected continues. (3) NIAID 08-I-N167: A Phase 2a, Randomized, 2 Arm, Open-label, Clinical Trial of the Efficacy of Linezolid Combined with Antituberculous Therapy in Subjects with Extensively Drug-Resistant (XDR) Pulmonary Tuberculosis. The major aim of this study is to evaluate the efficacy, safety and tolerability of one of the drugs of last resort for XDR TB patients, linezolid (LZD, Zyvox, Pfizer). LZD is infrequently used in TB patients because of its prohibitive cost and adverse effects but the emergence of XDR TB is spurring doctors into off-label, uncontrolled use in salvage therapy for the few patients who can afford it. This trial opened to enrollment in December 2008 and has enrolled 39 evaluable patients with XDR TB and closed to enrollment in July 2010. By four months, 15/19 (78.9%) subjects on the immediate-start arm and 7/20 (35.0%) subjects on the delayed-start arm achieved culture conversion (p=0.001). Thirty-four of 39 (87.2%) subjects achieved culture negative sputum within six months of adding LZD to their failing drug regimens. Among the 38 subjects exposed to LZD, 31 (82%) experienced clinically significant adverse events (AEs) possibly or probably related to LZD that resulted in 3 subjects discontinuing therapy. Subjects who de-escalated to 300 mg/day after the second randomization experienced fewer adverse events than subjects who continued taking 600 mg/day. Thirteen subjects successfully completed therapy and remain relapse-free. Four cases of acquired resistance to LZD have been observed thus far. LZD is highly effective at achieving culture conversion among subjects with treatment- refractory pulmonary XDR tuberculosis but adverse events must be monitored carefully. (4) NIAID 09-I-N061; Pharmacokinetics of Standard First and Second Line anti-TB Drugs in the Lung and Lesions of Subjects Elected for Resection Surgery. This is a multicenter study of the differential penetration of tuberculosis chemotherapeutics into pulmonary tubercular lesions. The study opened to enrollment in 2010 and presently 6 subjects have been enrolled by Asan Medical Center, 3 subjects have been enrolled by Pusan National University Hospital, and 2 subjects have been enrolled by the National Medical Center. The study will further define the relationship between pathology and drug penetration in the types of lesions commonly seen in TB patients and follow up the work we have conducted on lesion penetration in rabbits. Initial imaging mass spectrometry results indicate that similar drug distribution results to those in the rabbit model indicating concentration of Moxifloxacin in the livable rim of lesions. (6) NIAID 12-I-N036; Risk Stratification in Latent Tuberculosis: PET/CT Findings in TB Contacts and the Effect of Preventive Treatment. The primary purpose of this study, which is about to open to enrollment, is to determine whether or not FDG uptake by PET scanning can predict activation of latent TB infection in close contacts of active TB cases. This is a critically important question as there is currently no biomarker that can predict activation of latent TB disease.