耐多药结核病向广泛耐药演变趋势和机制及控制策略研究

The effectiveness of treatment of tuberculosis(TB) is increasingly compromised by the global rise in drug-resistant and multidrug resistant (MDR) strains and more recently extensively drug-resistant (XDR) strains of M. tuberculosis (M.TB). Once MDR-TB evolves to XDR, it is significantly more difficult to treat and eradicate. As in many other countries, MDR/XDR-TB in China is rapidly growing and cause for severe public health concern. A recent national survey of drug-resistant TB in China estimated an astounding 6% of new cases (incident) and 26% of previously treated cases harbored MDR-TB. In 2007, there were 110,000 incident cases of MDR-TB and 8,200 cases of XDR-TB. China has the 2nd highest TB case burden and harbors the largest absolute number (a quarter) of MDR-TB cases globally and perhaps the largest reservoir of individuals latently infected with M/XDR M.TB. Therefore, it is increasingly important also to know more about system risk factors (e.g. social, clinical and microbiologic) that can predispose MDR-TB and its transition to extensive drug resistance. By now, the extent to which social, clinical and microbiologic (i.e. resistance profile, genotypes) determinants interact and contribute to the emergence of M/XDR is presently unknown in the Chinese context. In this proposal, we will firstly determine the population-based trend and distribution of the evolution from MDR to XDR in the bacillary population and determine on the population-level what prominent factors (e.g. social and patient) contribute to the emergence of XDR in strains of M.TB infecting populations of China. Secondly, we will identified the major bacteriological family clone with high possibility of evolution to XDR by genotyping and phylogenic analysis.Thirdly, we will define the molecular mechanism behind the acquisition of 2nd line drug resistance among the MDR-TB isolates by SNP and whole-genome based sequencing; Fourthly, we will attempt to deep understand the impact of biological behavior and bio-film on the development of extensive drug resistance among the MDR-TB strains; We will then establish a cohort of MDR patients and examine correlates of poor treatment outcome (including development of XDR) using social, clinical and microbiologic data. Based on this information, we will identify programmatic areas where intervention or policy changes will likely have a meaningful impact on more favorable outcomes. That is, the extent to which social, clinical and microbiologic determinants interact and contribute to the emergence of XDR among MDR-TB　M.TB strains in China is paramount for designing effective pre-emptive control strategies.

深入研究地区耐多药结核病向二线药耐药和广泛耐药演变趋势及相关机制，有助于更好地控制广泛耐药结核病在我国的流行势头。本研究旨在通过系统的研究方法，探索影响耐多药向广泛耐药演变的的社会、临床和微生物危险因素，分析社会经济和政策、医疗服务体系和疾病特征以及病原体属性间的相互作用机制和作用程度。研究首先在人群水平了解我国不同经济发展水平地区耐多药结核病向广泛耐药演变的趋势和分布特征；采用基因分型和进化分析阐明发生新现广泛耐药主要菌群家族及分子特征；应用单位点和基因组分析掌握耐药演变过程中所牵涉的分子机制；在体外应用巨噬细胞内培养和多细胞培养研究病原体个体生物学属性和群体行为在耐药积累过程中发挥的作用。建立不同耐药类型结核病患者队列，分析与发生耐多药相关的社会经济和卫生服务、疾病特征和病原体因素。上述研究结果对于制定针对性、有效的耐多药/广泛耐药结核病的控制策略具有重要科学价值和策略建议。

我国仍然是结核病和耐多药结核病高负担国家之一。根据已有的流行病学调查和微生物免疫学研究结果，我们假设在经过多年的标准化治疗之后，我国的耐多药结核病有向广泛耐药演变的趋势。本研究以我国江苏省灌云县和南通市、浙江省德清县以及四川省富顺县和自流井区为研究现场，以2014-2015年上述五地的结核病定点医疗机构所收治的肺结核病人为研究对象，采用问卷调查获取基本信息并收集痰标本。研究期间在五区/县共纳入4391例结核病人，经过比例法为基础的药敏实验，其中耐多药结核病患者共计349例，建立治疗队列，有20人因迁出失访。在完成治疗随访和药敏追踪调查的334人中，有69人（27.6%）出现二线药物的耐药，包括33例为喹诺酮类药物耐药，41例为二线注射类药物耐药，平均出现获得性耐药的时间为7±1.3月。进一步比较发生获得性耐药前后菌株的MIRU-VNTR基因型发现，有20对(29.0%)的菌株基因型没有或在一个位点发生变化，定义为获得性耐药；另有49对（71.0%）菌株发生2个及以上位点变化，定义为传播性耐药。进一步分析影响因素发现：治疗史、胸片结果及糖尿病共患分别与获得性耐药和传播性耐药有关；除此之外，肺空洞也与传播性耐药发生有关。耐药相关基因测序发现，有72.7%获得喹诺酮类药物耐药菌株中携带gyrA基因突变，其中以94位基因突变为主；另有92.7%获得二线注射类药物耐药的菌株在rrs基因发生突变。进一步比较发现，获得性耐药菌株中存在较高比例的gyrA基因第94位基因突变和rrs基因第1401位突变，且比例要明显高于传播性耐药菌株；与最低抑菌浓度做关联分析发现，gyrA基因第94位基因突变株表现为高水平耐药，且在获得性耐药菌株中表现更为明显。单培养和竞争性试验结果显示发生获得性耐药菌株的适应力值最高，接近野生株。不仅如此，分析适应力代价相关基因，提示桥螺旋位置相关的基因突变与适应力代价存在明显关系。本研究提示耐多药结核病向广泛耐药演变的机制，包括治疗过程中由于药物作用的耐药菌株的选择性生长以及患者体外再感染新的耐药菌株；而治疗过程中获得性耐药与病原体属性（耐药水平、携带补偿机制相关基因突变和较低的适应力代价）以及卫生服务体系政策的相关因素有关。