NEJM:DNA测序预测结核菌耐药

2018-10-19 范齐文,吴炯,殷杏,侯琦 NEJM医学前沿

WHO最新全球结核报告中显示因结核分枝杆菌复合群(下文简称结核分枝杆菌)造成的死亡人数约170万,新发病630万人。新发60万耐利福平结核中有49万为耐多药结核(MDR-TB),其中印度、中国、俄罗斯金砖三国便占据了半壁江山[1]。 来源:WHO Global Tuberculosis Report 2017. 在结核病高负担国家和地区,能够获得及时的诊断和药敏检测尤为不易

WHO最新全球结核报告中显示因结核分枝杆菌复合群(下文简称结核分枝杆菌)造成的死亡人数约170万,新发病630万人。新发60万耐利福平结核中有49万为耐多药结核(MDR-TB),其中印度、中国、俄罗斯金砖三国便占据了半壁江山[1]。

来源:WHO Global Tuberculosis Report 2017.

在结核病高负担国家和地区,能够获得及时的诊断和药敏检测尤为不易。2016年全球需要治疗的约600,000名MDR-TB患者中,仅22%得到了诊断并接受了治疗。这促进了耐多药菌株的传播,使得一部分新发病例直接成为MDR-TB。结核病的快速诊断及药敏检测便成为有效控制疫情的关键环节。

结核病防治最主要的难题之一是耐药性问题。耐药结核病,尤其是耐多药结核病(MDR)和广泛耐药结核病(XDR)的治疗非常困难,治疗周期长,治愈率低[1]。而且,耐药结核病的精确诊断,即对耐药结核菌耐药谱的快速、准确的检测非常困难。耐药结核菌的耐药谱是确定耐药结核病临床治疗方案/用药组合的重要依据,也是关系耐药结核病治疗成败的第一步。

2010年,世界卫生组织批准并广泛推广Xpert MTB/RIF检测技术。该技术能够同时检测结核分枝杆菌(Mycobacterium tuberculosis,MTB)和MTB对利福平(Rifampicin,RIF,是耐多药结核的重要定义药物之一)的耐药性,具有自动化程度高,敏感性和特异性高,时间短(2小时就可以有结果),安全性高等显著优点[2]。但是其仅限于利福平耐药的检测,因此临床上非常需要广谱、快速的高敏感性检测技术。

分子诊断技术是否能为“消灭结核病”助一臂之力?让我们先了解一下最近几年《新英格兰医学杂志》(NEJM)发表的该领域的重磅研究。

首先是诊断:2010年9月NEJM刊出了题为《结核及利福平的快速分子检测》(Rapid Molecular detection of tuberculosis and rifampin resistance)一文,使用分子诊断技术(XpertMTB/RIF)对结核进行快速检测并同时报告利福平耐药情况,将结核病诊断的漫长周期(2个月)缩短至2小时内[2]。

其次是药敏:2017年9月NEJM又刊出题为《结核病快速分子药物敏感性检测方法学评估》(Evaluation of a rapid molecular drug-susceptibility test for tuberculosis)一文,在前文的基础上将结核的快速药敏检测从单一利福平扩展到了异烟肼、氟喹诺酮类及氨基糖苷类药物[3]。

可见,分子诊断技术正在撼动传统结核培养及药敏检测的地位,但支持和反对该技术的观点仍然胶着不下。

支持者认为:快捷的分子诊断大幅降低诊断窗口时间;且结核分枝杆菌自身发生突变的频率极低,耐药机制相对保守,分子诊断方法对耐药筛查存在合理性;2008年起WHO已逐步推荐分子诊断方法应用于结核诊断及药敏[4]。从生物安全角度考虑,分子诊断技术比传统培养及药物敏感性检测要相对安全,无需因操作大量活菌而增加检测人员感染风险。

反对者认为:仅通过特异的分析一个或几个基因片段做出判断,可能会漏掉无探针覆盖区域的突变位点。耐药基因突变并非一定导致表型耐药,测试准确性方面与表型相比还有待提高。分子方法检测的是核酸,无关结核分枝杆菌的死活,结核治疗转阴后一段时间内尚能检测到死菌的核酸,故而无法有效进行疗效评估。此外,分子诊断技术虽可以快速、准确、全面地预测结核耐药情况,但仪器价格昂贵,操作复杂,需要检测人员受过专门的培训,这些都限制了该方法的推广。 

2018年9月我国发布的《结核病病原学分子诊断专家共识》中平衡了双方观点:推荐使用分子诊断技术,提出应重视分子与传统方法不一致的结果,结果解释应结合传统培养及药敏实验,尤其在患者体内同时存在敏感和耐药结核分枝杆菌的情况[5]。

时隔1月,今年10月11日出版的NEJM又放大招:结核病综合耐药性预测国际联合会(Comprehensive Resistance Prediction for Tuberculosis: an International Consortium,CRyPTIC)和100,000基因组(100,000 Genomes Project)计划联合发表了题为《通过DNA测序预测一线抗结核药物敏感性》(Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing)一文[6]。

在去年和今年的这两篇NEJM论文中,来自中国疾病预防控制中心、复旦大学基础医学院、河南胸科医院和中国科学院生物物理所等多家中国机构的科研人员做出了重要贡献。

该研究使用全基因组测序的方法检测了六大洲16个国家的10209个结核菌株,分析了涉及四个一线抗结核药物的9个耐药基因及其相关突变:异烟肼(ahpC、inhA、fabG1katG)、利福平(rpoB)、乙胺丁醇(embA、mbBmbC)和吡嗪酰胺(pncA),通过基因测序结果来预测这四个药物的药敏试验表型。结果表明,基因型预测的灵敏度(基因预测耐药结果与表型耐药结果百分比)分别为97.1%、97.5%、94.6%和91.3%,特异性(基因预测敏感结果与表型表型敏感结果百分比)分别为99.0%、98.8%、93.6%和96.8%,且大部分的预测数据符合了WHO对于结核分枝杆菌新型分子检测方法灵敏度>90%、特异性>95%的要求。

全基因组测序是目前分子诊断技术中最为先进的方法。该方法可以探得结核分枝杆菌基因组的全貌,从中选择目标基因片段分析突变位点。与市面上现有的线性探针杂交方法相比,全基因组测序更能准确预测抗结核药物敏感性,特别是预测敏感菌株。

全基因组测序检测药敏性的另一亮点是为吡嗪酰胺敏感性的检测开辟了一条全新的道路。一线药物中的吡嗪酰胺在抗结核治疗中占有重要的分量,联合使用吡嗪酰胺可以将治疗周期从9-12个月缩短至6个月。此前,由于其检测条件较其他药物特殊,结核实验室并不常规开展,且尚无商品化的分子检测试剂盒。

当然,我们也不能忽略全基因组测序预测结核耐药的不足之处。高昂的测序成本,结果分析需要掌握生物信息学的专业知识等等,这些对于“贫穷病”可谓是硬伤。单看中国国内目前状况,Xpert MTB/RIF上市已有时日,但如今也没有遍地开花,全基因组测序想要落地,恐怕还有很长的路要走。另外,对于小部分预测为耐药而表型为敏感的菌株,最终仍需要排除相关影响因素,根据表型结果联合临床治疗效果才能做出最终定论。

参考文献:

[1] World Health Organization. Global tuberculosis report 2017. Geneva: World Health Organization, 2017.

[2] Boehme CC, Nabeta P, Hillemann D, et al. Rapid Molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005-15.

[3] Xie YL, Chakravorty S., Armstrong DT, et al. Evaluation of a rapid molecular drug-susceptibility test for tuberculosis. N Engl J Med 2017;377:1043-54.

[4] World Health Organization. Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB). Geneva: World Health Organization, 2008. 

[5] 中华医学会结核病学分会临床检验专业委员会. 结核病病原学分子诊断专家共识. 中华结核和呼吸杂志 2018;41:688-95.

[6] The CRyPTIC Consortium and the 100,000 Genomes Project. Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing. N Engl J Med 2018;379:1403-15

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