Monitoring Therapeutic Efficacy by Real-Time Detection of Mycobacterium tuberculosis mRNA in Sputum
Monitoring Therapeutic Efficacy by Real-Time Detection of Mycobacterium tuberculosis mRNA in Sputum
复制标题
DOI：
10.1373/clinchem.2009.124396
复制
发表时间：
2009-09-01
影响因子：
9.3
通讯作者：
Tang, Yi-Wei
中科院分区：
文献类型：
--
作者：
Mdivani, Nino;Li, Haijing;Tang, Yi-Wei
背景：目前用于监测结核病（TB）治疗反应的实验室方法依赖于分枝杆菌培养。因此，它们的临床用途受到结核分枝杆菌缓慢生长速率的限制。快速的方法来可靠地量化抗TB药物的反应是可取的。方法：我们开发了2个实时PCR检测，使用水解探针，以靶向DNA的IS 6110插入元件和mRNA的抗原85 B。直接从用氢氧化钠和N-乙酰-L-半胱氨酸净化的浓缩痰液样品中提取核酸。我们前瞻性地比较了这些检测与痰分枝杆菌培养的患者接受抗TB therapeutic.Results：65例新诊断的结核病和接受标准化的一线抗结核药物治疗方案的患者在治疗开始后的第2周和第1，2和4个月进行了评价。DNA PCR（98.5%）和mRNA逆转录PCR（95.4%）检测M.培养阳性痰标本中的结核病。286份痰标本中培养法与mRNA RT-PCR检测结果的总符合率为87.1%，与培养法相比，mRNA RT-PCR检测的敏感性和特异性分别为85.2%和88.6%。为了监测治疗效果，mRNA RT-PCR结果与随访时间点的培养结果平行。结论：存活的M.结核菌培养结果与85 B抗原mRNA的RT-PCR分析结果与临床抗结核药物耐药相关，而DNA PCR分析结果显示假阳性率高。这种mRNA RT-PCR检测可以快速监测抗TB治疗的反应。(C)2009年美国临床化学协会
BACKGROUND: Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable.METHODS: We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-L-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy.RESULTS: Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RTPCR assay's diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points.CONCLUSIONS: The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy. (C) 2009 American Association for Clinical Chemistry