实时定量PCR方法监测肾移植患者血浆巨细胞病毒DNA载量及其意义

Monitoring of plasma CMV viral DNA load by real-time qantitative PCR in renal transplant recipients and implication

目的采用实时定量荧光PCR方法检测巨细胞病毒（CMV）DNA拷贝数，探讨病毒载量变化在诊断CMV活动性感染中的意义和指导抗病毒治疗中的作用。方法对40例采用预排空治疗策略预防CMV病的肾移植受者同时进行CMV抗原血症（pp65）分析和病毒载量监测并进行随访，观察CMV—DNA载量和pp65在判断肾移植受者CMV活动性感染和指导抗病毒治疗中的价值。结果264份样本中逆转录一聚合酶链反应（RT—PCR）检测57份阳性（21．59％），病毒载量水平2．648×10^1-5．273×10^5copy／ml；抗原血症分析检测48份阳性（18．18％），抗原指数1～18／5万WBC；Cohen，s—Kappa检验结果表明两种检测方法一致性良好（Kappa=0．762）。25例（62．5％）患者PCR方法检测阳性，19例（47．5％）抗原血症分析阳性。病毒载量首次出现阳性时间为（13．224-14．78）d，pp65首次出现时间为（26．724-20．61）d，差异有统计学意义（P〈0．05）。40例患者中4例出现CMV病，发病时间133～179d，3例发病前pp65和CMV．DNA均为阳性，1例仅CMV—DNA阳性。结论血浆CMV载量检测与抗原血症分析一致性良好，而敏感性较高，更准确反映肾移植术后CMV活动性，更适合用于肾移植术后预防CMV感染的预排空治疗策略。

Objective By using real-time quantitative PCR to evaluate the eytomegalovirus （CMV） DNA copies,so as to analyze the implleation of changes in virus load in active CMV infections, and its effect in guiding the antiviral therapy. Methods In 40 renal transplant recipients receiving premptive therapy to prevent CMV diseases, CMV antigen （pp65） and virus load were measured simultane- ously during the follow-up, then the meanings of CMV-DNA load and pp65 in diagnosing active CMV infections and guiding the antiviral therapy were analyzed. Results Among the 264 samples, 57 （21.59%） were positive through RT-PCR, with a virus load varying between 2. 648 × 10^-5. 273 × 10^5 eopy/ml. Through antigenemia analysis ,48 of the above samples （ 18.18% ） were positive with an antigen index be- tween 1-18/50 000 white blood cells. Cohen＇ s-Kappa test approved that these two methods had good consisteney （Kappa=0.762）. In40 patients,25 （62.5%） and 19 （47.5%） were positive by PCR and antigenerals analysis respectively. The positive results could be first found through PCR on （ 13.22 ± 14.78） days,while on （26.72 ±20.61 ） days through pp65 analysis,with the difference being statistically significant （P 〈0.05）. Four patients developed CMV diseases between 113 and 179 days,3 of which had positive results in beth PCR and pp65 analysis and the other was only positive in CMV-DNA. Conclusion Quantitative analysis of plasma CMV virus load displayed good consistency with pp65 analysis, yet had higher sensitivity ,thus could better reflect the activity of CMV infection and guide the pre-emptive therapy after kidney transplantation.