国产实时荧光定量PCR试剂与GeneXpert MTB/RIF检测结核分枝杆菌的对比分析

Comparative analysis of domestic real-time fluorescence quantitative PCR reagent and GeneXpert MTB/RIF for detecting Mycobacterium tuberculosis

目的评价国产实时荧光定量PCR(FQ-PCR)试剂与GeneXpert MTB/RIF(简称“GeneXpert”)检测痰标本结核分枝杆菌的临床应用价值,并比较两者的检测效能。方法收集2017年1月至2018年12月于深圳市南山区慢性病防治院就诊的210例疑似肺结核患者的痰标本,同时做涂片镜检、BACTEC MGIT 960(简称“MGIT 960”)培养、GeneXpert和FQ-PCR检测。以MGIT 960培养为参考,比较GeneXpert和FQ-PCR检测的敏感度、特异度、阳性预测值、阴性预测值,比较两者与MGIT 960培养结果的一致性及两者之间的一致性。按照涂片镜检结果分级报告标准,将痰标本分为荷菌量逐步递增的6个组,即阴性组、少量组、+组、++组、+++组、++++组,比较GeneXpert和FQ-PCR检测各个组阈值循环数(Ct值)均值的差异。结果GeneXpert检测的敏感度、特异度、阳性预测值、阴性预测值分别为83.7%(123/147)、87.9%(51/58)、94.6%(123/130)、68.0%(51/75);FQ-PCR检测的敏感度、特异度、阳性预测值、阴性预测值分别为83.7%(123/147)、89.8%(53/59)、95.3%(123/129)、68.8%(53/77)。FQ-PCR、GeneXpert分别与MGIT 960行Kappa检验,一致性分别为85.4%(176/206)和84.9%(174/205),Kappa值分别为0.70、0.66;对GeneXpert和FQ-PCR检测结果行Kappa检验,一致性为92.8%(194/209),Kappa值为0.85。FQ-PCR检测显示,阴性组与少量组的Ct值均值比较[(33.87±5.00)和(27.29±1.30)个],差异有统计学意义(t=5.56,P<0.001);GeneXpert检测分别为(33.32±6.05)和(23.99±3.36)个,差异有统计学意义(t=5.19,P<0.001)。检测涂片阴性标本时,FQ-PCR与GeneXpert检测的Ct值均值[(33.87±5.00)和(33.32±6.05)个]差异无统计学意义(t=0.32,P=0.750);检测涂片阳性(少量组、+组、++组、+++组、++++组)标本时,FQ-PCR检测的Ct值均值[分别为(27.29±1.30)、(27.95±2.85)、(25.88±3.62)、(24.79±2.46)、(22.38±2.72)个]均明显高于GeneXpert检测[分别为(23.99±3.36)、(23.10±4.05)、(20.22±2.81)、(20.31±4.16)、(16.48±2.78)个],差异均有统计学意义(t值分别为2.60、4.71、6.08、3.85、9.02,P值分别为0.030、<0.001、<0.001、<0.001、<0.001)。结论国产FQ-PCR试剂与GeneXpert检测结果一致性较好,对结核病大规模筛查、降低检测成本具有临床应用价值。

Objective To evaluate the clinical application of domestic real-time fluorescence quantitative PCR(FQ-PCR)reagent and GeneXpert MTB/RIF(GeneXpert)in the detection of Mycobacterium tuberculosis(MTB)in sputum specimens,and compare the detection efficacy of the two methods.Methods From January 2017 to December 2018,a total of 210 sputum specimens of suspected pulmonary tuberculosis cases were collected from Shenzhen Nanshan Center for Chronic Disease Control for smear microscopy,MGIT(Mycobacteria growth indicator tube)960 culture,GeneXpert and FQ-PCR.Using MGIT 960 result as a reference standard,the sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of GeneXpert and FQ-PCR methods were calculated and compared.The consistency of GeneXpert,FQ-PCR and MGIT 960 as well as the consistency between both techniques were compared.According to the grading reporting standard of smear microscopic examination results,the specimens were divided into six groups with progressively increasing bacterial load(negative,scanty,+,++,+++,++++),and the Ct mean value of each group detected by FQ-PCR and GeneXpert was compared.Results The sensitivity,specificity,PPV and NPV of GeneXpert were 83.7%(123/147),87.9%(51/58),94.6%(123/130),and 68.0%(51/57),respectively,while the sensitivity,specificity,PPV and NPV of FQ-PCR were 83.7%(123/147),89.8%(53/59),95.3%(123/129),and 68.8%(53/77),respectively.Kappa test was performed between FQ-PCR,GeneXpert and MGIT 960 results,and the consistency was 85.4%(176/206)between FQ-PCR and MGIT 960 results and 84.9%(174/205)between GeneXpert and MGIT 960 results,with the Kappa value of 0.70 and 0.66,respectively;and the consistency between GeneXpert and FQ-PCR was 92.8%(194/209)(Kappa=0.85).The mean Ct value of negative group and scanty group detected by FQ-PCR was(33.87±5.00)and(27.29±1.30)cycles,respectively,and the difference was statistically significant(t=5.56,P<0.001);while which by GeneXpert was(33.32±6.05)cycles and(23.99±3.36)cycles,respectively,and the difference was also statistically significant(t=5.19,P<0.001).There was no significant difference in mean Ct values between FQ-PCR and GeneXpert when smear negative samples were detected((33.87±5.00)cycles vs(33.32±6.05)cycles,t=0.32,P=0.750).However,when the smear was positive(scanty,+,++,+++,++++),the mean Ct values of FQ-PCR were significantly higher than that of GeneXpert((27.29±1.30)cycles vs(23.99±3.36)cycles,t=2.60,P=0.030;(27.95±2.85)cycles vs(23.10±4.05)cycles,t=4.71,P<0.001;(25.88±3.62)cycles vs(20.22±2.81)cycles,t=6.08,P<0.001;(24.79±2.46)cycles vs(20.31±4.16)cycles,t=3.85,P<0.001;and(22.38±2.72)cycles vs(16.48±2.78)cycles,t=9.02,P<0.001,respectively).Conclusion The domestic FQ-PCR method has good consistency with the GeneXpert results,which has high clinical application value for large-scale screening of tuberculosis and reducing the detection cost.