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

该项目由LCID/NIAID结核病研究科、韩国卫生、福利和家庭疾病控制中心、国立马山结核病医院（NMTH）和大韩民国延世大学医学院合作开展。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，共有730多名受试者入选。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。(2) NIAID 07-I-N041：一项随机、双盲、安慰剂对照的甲硝唑联合抗结核化疗与安慰剂联合抗结核化疗在多重耐药肺结核患者中的初步研究。在这项研究中，厌氧活性在候选结核药物中的重要性正在进行调查。2009年，由于担心副作用，在33名患者入组后，该试验停止了入组，但对这些受试者的随访和收集到的数据的分析仍在继续。(3) NIAID 08-I-N167：一项利奈唑胺联合抗结核治疗广泛耐药（XDR）肺结核的2a期、随机、2组、开放标签临床试验。本研究的主要目的是评估广泛耐药结核病患者最后使用的药物之一利奈唑胺（LZD, Zyvox, Pfizer）的疗效、安全性和耐受性。目前，LZD很少用于结核病患者，因为它的成本和副作用令人望而却步，但广泛耐药结核病的出现正在促使医生在标签外不加控制地使用它，为少数能够负担得起的患者提供补救性治疗。该试验于2008年12月开始招募，已招募了39名可评估的广泛耐药结核病患者，并于2010年7月结束招募。两组患者痰培养转化为结核阴性的时间差异的中期分析表明LZD具有潜在的活性，但最终分析有待于最后几名受试者达到其主要终点。在该人群中观察到意想不到的毒性，并已向FDA， IRB和发起人报告，并提交了一份描述观察到的rabdomyolysis的手稿。(4) niaid 09-i-n061；标准一线和二线抗结核药物在切除手术患者肺部和病变中的药代动力学。这是一项多中心研究结核化疗药物在肺结核病变中的差异渗透，于2010年开始入组；首尔峨山医疗中心招募了4名受试者。本研究将进一步明确结核患者常见病变类型的病理与药物渗透之间的关系，并跟进我们在家兔中进行的病变渗透工作。

The project is conducted as a collaboration among the Tuberculosis Research Section of LCID/NIAID, the Korean Ministry of Health, Welfare and Families Center for Disease Control, National Masan Tuberculosis Hospital (NMTH), 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 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 730 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. (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 Multi-drug 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 33 patients enrolled because of concerns about side-effects, but follow up of these subjects and 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). For the moment 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 that 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. The interim analysis of the difference in time to sputum culture conversion to tuberculosis negative between the two arms of the study indicates LZD is potentially active, but the final analysis is pending until the last few subjects have reached their primary endpoint. An unexpected toxicity has been observed in this population and has been both reported to the FDA, the IRB and the sponsor and a manuscript describing the observed rabdomyolysis has been submitted. (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 opened to enrollment in 2010; 4 subjects have been enrolled by Asan Medical Center in Seoul. 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. 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. The mechanism of pyrazinoic acid (POA), the active form the pro-drug Pyrazinamide (PZA), was found to bind to the ribosomal protein RpsA and that the site at which it binds corresponds to the binding site of tmRNA. This RNA species is required for rescuing stalled ribosomes by trans-translation. These results not only demonstrate the novel target of this drug which has to date remained elusive, but also points to the unexpected critical role of ribosomal homeostasis in stressed cells in human pathogenesis by MTb indicating that PZA may act to poison cells by accumulation of toxic peptides released from stalled ribosomes in addition to a bottleneck in the amount of available free ribosomes in cells under in vivo stress. Further support for these studies was established by the finding that some clinical strains of MTb with PZA resistance have RpsA mutations at the POA binding site. In 2010, we reported a number of mutations in the gibB gene that encodes a putative 16S rRNA methyltransferase. Recently, by evaluating an isogenic gidB mutant strain constructed from strain H37Rv, we demonstrated the causal role of gidB in conferring a low-level SM-resistant phenotype in M. tuberculosis with a 16-fold increase in the MIC over the parent strain. Among clinical isolates, the modest increase in SM resistance conferred by a gidB mutation leads to an MIC distribution of gidB mutation-containing strains that spans the recommended SM breakpoint concentration currently used in drug susceptibility testing protocols. As such, some gidB mutation-containing isolates are found to be SM sensitive, while others are SM resistant. On the basis of a pharmacodynamic analysis and Monte Carlo simulation, those isolates that were found to be SM sensitive should still respond favorably to SM treatment, while nearly half of those found to be SM resistant would likely respond poorly. This investigation provided the first microbiological evidence for the contribution of gidB in streptomycin and demonstrates its clinical importance. Several collaborations involving new diagnostic tools are also underway including work on the next generation GenXpert 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. We are also collaborating on work on biomarkers of treatment success and to assess the presence of fragments of Mtb in easily accessible human samples, such as urine and exhaled breath condensate, for development of point of care tests. These initial studies been productive enough for us to add a substudy to the Natural History protocol and enter into a formal discovery effort.