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

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-聚合酶链反应(RT-PCR)检测57份阳性(21.59%),病毒载量水平2.648×101～5.273×105copy/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(13.22±14.78)d,pp65首次出现时间为(26.72±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133～179 d,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.     

Comparison of quantitative PCR and antigenemia in cytomegalovirus infection in renal transplant recipients

Cytomegalovirus infection is a common complication of renal transplantation. Antigen pp65 levels serve as indicators of viral load, although the technique is difficult to perform and interpret. We sought to determine whether quantitative PCR had a higher sensitivity and predictive value in CMV infection. METHODS: The study included 100 renal transplant recipients who were screened for IgM and IgG at the time of admission. On days 7, 15, 30, 45, 60, 75, 90, 120, 180, and 360, antigenemia tests were performed on blood (pp65) and urine, and a quantitative PCR on blood. Among 59 patients recruited between November 2003 and August 2004 the mean age was 54.5 +/- 12.9 years. Two patients did not reach 90 days follow-up (3%); four patients have not surpassed 90 days (7%); 22, 120 days (37%); and 31, 180 days (53%). Ninety-three percent of patients showed anti-IgG CMV-positive titers with all being IgM CMV-negative at baseline. The patients at risk for infection were given valgancyclovir as prophylaxis throughout the study. RESULTS: At 474 visits, 8 samples (2.4%) were positive with urine; 5 (1.4%) with pp65, and 15 (4.7%) with PCR. Among the 15 positive samples, two (>100,000 and 3250 copies) revealed agreement of positive IgM and shellvial test on urine; two (15,100 and 5670 copies), antigen pp65 1+; one (17,400 copies) with pp65 2+ and shellvial urine; two (99,400 and 28,300 copies) with pp65 1+ and shellvial urine; and eight remaining determinations, 749, 2250, 686, 928, 2250, 26600, 777, and 2790 copies. The rest of the tests were negative. CONCLUSION: The preliminary results of this study demonstrated that quantitative PCR was a useful rapid tool for diagnosing and monitoring CMV infections.     

Quantification of cytomegalovirus (CMV) viral load by the hybrid capture assay allows for early detection of CMV disease in lung transplant recipients.

BACKGROUND: We prospectively compared the hybrid capture system (HCS) assay with conventional cell culture and shell vial assay for the detection of cytomegalovirus (CMV) infection and disease in the lung transplant population. METHODS: Between January 1999 and February 2000, 34 lung transplant patients at Loyola University Medical Center, who were considered to be at risk for CMV disease, underwent surveillance testing for CMV cell culture, shell vial assay and HCS assay according to a pre-determined schedule. In addition, bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsy were performed at regular intervals and for clinical indications. All BAL samples were sent for CMV cultures and biopsy specimens were analyzed for histopathologic evidence of CMV by immunoperoxidase staining using antibody to early immediate nuclear antigen. RESULTS: Ten patients developed CMV disease/syndrome during the course of the study. The sensitivity, specificity, positive predictive value and negative predictive value were >90% for the HCS assay. The sensitivity of the HCS assay (90%) was statistically significantly higher than the sensitivity of either the SV assay (40%) or the cell culture (50%). In addition, the HCS assay was able to detect CMV 50 +/- 67 days prior to clinical evidence of CMV disease and an average of 36 days prior to the other detection techniques. CONCLUSION: The HCS assay is a sensitive diagnostic technique able to reliably detect CMV disease earlier than other diagnostic methods in the lung transplant population. Future studies may be able to evaluate whether pre-emptive anti-viral therapy targeted to specific viral loads using the HCS assay will be beneficial in preventing morbidity associated with CMV disease.     

巨细胞病毒抗原检测对巨细胞病毒病预防的意义

目的：提高对肾移植术后巨细胞病毒（CMV）病的防治水平,方法：应用免疫组织化学方法（LSAB法）对1997年度肾移植患者进行CMV抗原检测,对检查阳性的患者采用丙氧鸟苷预防性治疗,并与1996年度未预防治疗组进行对照.结果：1997年预防性治疗后,CMV病发病率为3．1％（5／160）,病死率为20．0（1／5）,而1996年未预防治疗组,CMV病发病率为11．7％（16／136）,病死率为43．8％（7／16）,结论：对肾移植术后CMV抗原检测阳性患者预防性应用丙氧鸟式能显著降低CMV病的发生率和病死率。     

Relationship of cytomegalovirus viral load in blood to pneumonitis in lung transplant recipients.

We developed a multiplex, quantitative, real-time, polymerase chain reaction assay for cytomegalovirus (CMV) and used it to measure the CMV viral load in weekly blood specimens from 43 lung transplant recipients. The median viral load in blood samples immediately preceding bronchoscopy was 1150 copies/microg human DNA for 12 subjects with pneumonitis compared to 91 copies for 31 subjects without (P=0.02, Mann-Whitney U test). Each log10 increase in CMV viral load resulted in an increase of 1.92 in the odds ratio for CMV pneumonitis (95% confidence interval 1.03-3.56). CMV viral load was elevated (>100 copies/microg human DNA) for a median of 21 days before bronchoscopy in those subjects with pneumonitis versus 0 days in those without (P=0.004). We conclude that the risk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood. Quantitative PCR should be evaluated prospectively for the preemptive management of CMV in lung transplant recipients.     

Comparison of the efficacy and cost effectiveness of pre-emptive therapy as directed by CMV antigenemia and prophylaxis with ganciclovir in lung transplant recipients.

BACKGROUND: CMV disease remains a major complication of lung transplantation and attempts to prevent it have met with marginal success. In a previous study we documented that universal prophylaxis did not prevent CMV disease but merely delayed it, and was very costly. METHODS: We compared the efficacy and cost of pre-emptive therapy with ganciclovir, guided by CMV antigenemia, to that of historic controls that received universal prophylaxis with ganciclovir. CMV antigenemia assay was done routinely and pre-emptive therapy was initiated if greater than 25 CMV positive cells per 100,000 polymorphonuclear cells were found. RESULTS: Nineteen patients were enrolled; 6 of of whom received 12 courses of pre-emptive therapy. The incidence of CMV disease was 26% compared to 38% for the historical controls (p = 0.51). None of the patients that received pre-emptive therapy developed CMV disease following that therapy. Antigenemia failed to predict disease in 5 patients that developed it, and thus it is unknown if pre-emptive therapy could have prevented it. There was no mortality in either the study patients or historic controls directly related to CMV. The net savings with pre-emptive therapy was $2569 per patient. CONCLUSIONS: We conclude that pre-emptive therapy with ganciclovir is as safe and effective as universal prophylaxis in preventing CMV disease in lung transplant recipients, and is less expensive. The appropriate surveillance technique and timing remain to be determine to optimize the efficacy of pre-emptive therapy.     

Real-time PCR assay compared with antigenemia assay for detecting cytomegalovirus infection in kidney transplant recipients

Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality among kidney transplant recipients. The CMVpp65 antigenemia assay has been used for preemptive therapy. Real-time polymerase chain reaction (PCR) technology for CMV DNA quantification in blood has demonstrated a good correlation with the currently employed CMV antigenemia assay. In this study, 90 renal transplant recipients were prospectively enrolled from July 2004 and May 2005. Monitoring of CMV infection was routinely performed with CMV antigenemia and real-time PCR assays. Real-time plasma PCR and CMV antigenemia assays were assessed on 797 samples. CMV antigenemia correlated with a positive CMV PCR (chi(2) = 78.05; P < .0001). Not only the positive rate but also the number of positive cells correlated with the number of PCR DNA copies (F = 26.07, r(2) = .25, P < .0001). To define an optimal cutoff value of CMV DNA load to initiate treatment in kidney transplant patients, we considered a CMV antigenemia titer of >50 positive cells per 400,000 leukocytes as the gold standard in our previous study. The optimal cutoff value for the quantitative real-time PCR assay was predicted to be 86 copies/microL. Thus, we observed that CMV real-time PCR assay would not completely replace antigenemia assay in kidney transplant recipients, but can be used complementarily to screen antigenemia and monitor preemptive therapy.     

Long-term results of CMV hyperimmune globulin prophylaxis in 377 heart transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) has emerged as the most important pathogen to affect the post-operative course after heart transplantation. We performed a retrospective analysis to evaluate the efficiency of CMV hyperimmune globulin (CMVIG) prophylaxis in preventing CMV disease in aggressively immunosuppressed patients after heart transplantation. METHODS: We studied 377 heart transplant recipients who received quadruple-immunosuppressive therapy and CMVIG as sole CMV prophylaxis. The study population was categorized into 4 groups according to donor and recipient CMV serology at the time of transplantation (D+/R+, D+/R-, D-/R+, D-/R-) and was monitored for CMV immediate early antigen in peripheral blood cells, in urine sediments, and in throat washings; for the presence of serum CMV immunoglobulin M and CMV immunoglobulin G; and for clinical evidence of CMV-related symptoms. In addition, we compared the incidence of cardiac allograft vasculopathy and infection among the groups. RESULTS: During the first 5 years after transplantation, CMV disease developed in 79 patients (20.96%). Comparison among the groups showed significantly increased risk for CMV disease in allograft recipients of organs from seropositive donors (D+, 27.31%; D-, 11.33%; p = 0.0003). We observed 6 CMV-associated deaths, all in CMV-antibody-negative recipients. Additionally CMV-positive recipients had a greater incidence of cardiac allograft vasculopathy (p = 0.048), and a greater overall infection rate (p = 0.0034). CONCLUSIONS: Cytomegalovirus hyperimmune globulin administration prevents CMV disease and infection in aggressively immunosuppressed heart transplant recipients. Because fatal CMV disease in CMV-negative recipients of organs from seropositive donors could not be prevented with CMVIG alone, we recommend the additional use of prophylactic ganciclovir in this CMV-mismatched population.     

Delay of CMV infection in high-risk CMV mismatch lung transplant recipients due to prophylaxis with oral ganciclovir

Cytomegalovirus (CMV) is a common opportunistic infection in lung transplant recipients. Despite the use of early post-operative intravenous ganciclovir, most high-risk patients develop CMV infection. We conducted this retrospective study to determine the efficacy of extended CMV prophylaxis with oral ganciclovir in high-risk, donor-positive-recipient-negative, lung recipients. All patients initially received 3 months of intravenous ganciclovir and CMV hyperimmune globulin. Clinical outcomes in all CMV mismatch patients undergoing lung transplant surviving at least 3 months were included (n = 42). Since 1998, 14 patients received no oral ganciclovir prophylaxis (group 1) and 28 patients received indefinite oral ganciclovir after completion of intravenous therapy (group 2). In those patients receiving oral ganciclovir, the prevalence of post-transplant CMV infection was significantly reduced over the first 180 d post-transplant (50% in group 1 vs. 4% in group 2; p < 0.001). Although some CMV events were observed with additional follow-up in group 2, there remained a significantly greater freedom from CMV infection by Kaplan-Meier analysis in group 2 as compared with group 1, with over 30 months follow-up time in each group (log-rank, p = 0.02). A moderate rate of drug discontinuation was observed in group 2, and no severe drug-related events occurred. In high-risk lung transplant recipients, CMV prophylaxis with intravenous ganciclovir, followed by indefinite oral ganciclovir, significantly delays and reduces post-transplant CMV infections. A larger prospective randomized study is needed to confirm the benefits of oral ganciclovir on CMV prevention.     

Clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients

BACKGROUND: The early detection of cytomegalovirus (CMV) after liver transplantation may form the basis of a preemptive strategy for prevention of active CMV disease. METHODS: We prospectively analyzed the clinical use of weekly quantitative polymerase chain reaction-(PCR) based plasma viral load determinations and the antigenemia assay for predicting the development of active CMV disease in 97 consecutive liver transplant recipients. RESULTS: CMV disease occurred in 21/97 patients. Using a positive cut-off of >400 copies/ml plasma, PCR had a sensitivity of 100%, specificity 47.4%, positive predictive value 34.4% and negative predictive value 100% for prediction of CMV disease. Respective values for a positive antigenemia (>0 positive cells/slide) were 95.2, 55.3, 37.0, and 97.7%. Different cut-off points for a positive test were analyzed using receiver-operating characteristic (ROC) curves. The optimal cut-off for viral load was in the range of 2000-5000 copies/ml (sensitivity 85.7%, specificity 86.8%, PPV 64.3%, NPV 95.7% for >5000 copies/ml). The optimal cut-off for antigenemia was in the range of four to six positive cells/slide. Mean peak viral load in symptomatic patients was 73,715 copies per/ml versus 3615 copies/ml in patients with asymptomatic CMV reactivation (P<0.001). In a multivariate logistic regression analysis of risk factors for CMV disease (CMV serostatus, acute rejection, and induction immunosuppression), peak viral load and peak antigenemia emerged as the only significant independent predictors of CMV disease (for PCR, odds ratio=1.40/1000 copy/ml increase in viral load, P=0.0001; for antigenemia odds ratio=1.17/1 positive cell/slide). CONCLUSIONS: Plasma viral load by quantitative PCR is useful for predicting CMV disease and could be used in a preemptive strategy.     

A prospective study of a quantitative PCR ELISA assay for the diagnosis of CMV pneumonia in lung and heart-transplant recipients.

BACKGROUND: Qualitative polymerase chain reaction (PCR) for the identification of cytomegalovirus (CMV) infection has a low predictive value for the identification of CMV pneumonia. This study prospectively evaluated the application of a quantitative PCR Enzyme-Linked Immuno-Sorbent Assay (ELISA) assay in 9 lung- and 18 heart-transplant recipients who did not receive ganciclovir prophylaxis. METHODS: DNA was collected from peripheral blood polymorphonuclear leucocytes (PMNL) posttransplantation. Oligonucleotide primers for the glycoprotein B gene (149 bp) were used in a PCR ELISA assay using an internal standard for quantitation. CMV disease was defined as histological evidence of end organ damage. RESULTS: The median level CMV genome equivalents in patients with CMV disease was 2665/2 x 10(5) PMNL (range 1,200 to 61,606) compared to 100 x 10(5) PMNL (range 20 to 855) with infection but no CMV disease (p = 0.036). All patients with CMV disease had genome equivalents levels of >1200/2 x 10(5) PMNL. A cut-off level of 1,200 PMNL had a positive predictive value for CMV disease of 100% and a negative predictive value of 100%. The first detection of levels of CMV genome equivalents above a level of 1200/2 x 10(5) PMNL was at a median of 58 days (range 47 to 147) posttransplant. CONCLUSIONS: Quantitative PCR assays for the diagnosis of CMV infection may predict patients at risk of CMV disease and thereby direct preemptive treatment to high-risk patients.     

Monitoring of human cytomegalovirus infections in heart transplant recipients by pp65 antigenemia

BACKGROUND: Rapid diagnostic techniques offer the opportunity of early diagnosis of human cytomegalovirus (CMV) infection in immunocompromized patients at risk of developing CMV disease and syndrome. The use of CMV pp65 antigenemia as a predictor of CMV syndrome and disease in heart transplant recipient after induction therapy was studied retrospectively. METHODS: One hundred and nineteen consecutive heart transplant recipients treated with induction therapy who survived more then 14 d were monitored for CMV infection. Ninety-four recipients were seropositive for CMV. Twenty-five recipients were seronegative for CMV and received grafts from seropositive donors. Pre-emptive therapy was used in seropositive patients when CMV pp65 antigenemia was greater than 50 antigen-positive cells per 2 x 10(5) peripheral blood leukocytes (PBL); prophylactic therapy was done only in seronegative recipient matched with seropositive donor. RESULTS: High-level CMV pp65 antigenemia (50 antigen-positive cells 2 x 10(5) PBL) occurred in 34% (41 of 119) of patients at a median of 44 d following transplantation. In seropositive recipients, 16% (15 of 94) of patients developed CMV invasive disease or syndrome, and in seronegative recipients 20% (5 of 25) of patients developed CMV disease or syndrome. Sixty-six per cent (62 of 94) of CMV seropositive patients were identified as not requiring pre-emptive therapy. In seropositive and seronegative recipients, the sensibility and negative predictive value of the cut-off level of 50 antigen positive cell for CMV disease and syndrome was 100%. The specificity was 79% and positive predictive value was 49%. CONCLUSION: Because of the excellent sensibility and negative predictive value of the cut-off level of 50 antigen positive cell per 2 x 10(5) PBL, application of pre-emptive therapy guided by high level of CMV pp65 antigenemia in the context of induction therapy allow to omit antiviral therapy in many at risk patients. In the context of pre-emptive and prophylactic therapy, the cut-off level of 50 antigen positive cell do not allow to predict with accuracy the development of CMV disease or syndrome.     

Comparison of cytomegalovirus viral load measure by real-time PCR with pp65 antigenemia for the diagnosis of cytomegalovirus disease in solid organ transplant patients

Cytomegalovirus (CMV) infection is the most frequent complication in solid organ transplant recipients. Currently, the antigenemia assay is widely used to detect this infection, although its success is being questioned to a great extent nowadays. The aim of our study is to compare a quantitative real time PCR to measure CMV DNA to the antigenemia assay, for the diagnosis to CMV disease. For our research, we prospectively processed 1198 samples (plasma and peripheral blood leukocytes [PBMC]), which belonged to 158 transplant recipients. In every sample the detection of the pp65 antigen in PBMC was carried out, as well as the quantification of CMV DNA by PCR (Light Cycler, LC-PCR). For this process, FRET probes, which detect a 254-bp fragment from the CMV gB gene, were used. The dynamic range of the LC-PCR was 500 to 5.10(7) copies/mL plasma and from 62 to 6.10(6) copies/10(6) PBMC. Twenty-three episodes of cytomegalovirus (CMV) disease occurred in 22 out of 158 patients and PCR displayed levels of sensitivity and specificity of 100% and 67%, respectively. The antigenemia assay obtained values of 91% and 57%. We established a cutoff value of 10(3) copies/mL plasma and 315 copies/10(6) cells. According to these cutoff values, PCR showed levels of sensitivity, specificity, VPN and VPP of 95.6%, 81.6%, 99%, and 53% respectively. Moreover, the LC-PCR assay anticipated the antigenemia assay in 10 patients out of 22 who developed CMV disease and the appearance of any clinical symptoms in 12 out of 22 patients. In conclusion, we believe that the quantification of CMV DNA by LC-PCR is a superior assay to pp65 antigenemia test regarding the early diagnosis of CMV disease in solid organ transplant recipients.     

Impact of prophylaxis with cytogam alone on the incidence of CMV viremia in CMV-seropositive lung transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection remains a serious problem after lung transplantation. The purpose of this study was to evaluate the efficacy of CytoGam, a CMV hyperimmune globulin (CMV-IGIV), as CMV prophylaxis after lung transplantation. METHODS: This prospective, randomized, open-label study compared prophylaxis with CMV-IGIV and no prophylaxis in 44 CMV-seropositive lung transplant recipients. The primary end-point was development of CMV viremia during the first year after transplantation. RESULTS: Cytomegalovirus viremia was detected in 13 of 22 recipients without prophylaxis and in 16 of 22 recipients with CMV-IGIV prophylaxis (p = 0.19). Cytomegalovirus pneumonitis developed in 8 controls vs in 11 CMV-IGIV recipients (p = 0.54). We found no significant difference between the groups in the incidence of positive shell vial assays (6.8% +/- 6.5% without vs 11.2% +/- 10.1% with prophylaxis, p = 0.09) or in the attack rate of CMV pneumonitis (0.41 +/- 0.59 episodes/patient without vs 0.86 +/- 0.99 episodes/patient with prophylaxis, p = 0.07). Similarly, no difference was apparent in the time to onset of CMV viremia, to detection of CMV DNA in peripheral blood leukocytes by polymerase chain reaction, or to development of CMV pneumonitis. The incidence of acute rejection and bronchiolitis obliterans syndrome and the survival rate during the first post-transplant year did not differ between the groups. CONCLUSIONS: Prophylaxis with CMV-IGIV alone did not decrease CMV viremia or pneumonitis, did not decrease the incidence of acute rejection or bronchiolitis obliterans syndrome, and did not affect 1-year survival of CMV-seropositive lung transplant recipients at our center.     

A pharmacokinetic comparison of cyclosporin oral solution and cyclosporin capsules in heart and lung transplant recipients

Pharmacokinetic profiles were obtained for 16 heart or lung recipients following the administration of identical doses of cyclosporin as oral solution and capsules on consecutive days. A comparison of pharmacokinetic parameters (AUC, C_max, C_min and t_max) showed that there were no significant differences between the two formulations except for the t_max, which was significantly longer for the capsules. The mean variation in day-to-day trough levels produced by the two different forms was 25.6%. A retrospective study was carried out of consecutive cyclosporin levels in patients at steady state on oral solution. The mean variation in day-to-day trough levels was 32.3%. This was not significantly different from the variation in consecutive trough levels seen in the oral solution/capsule comparison. This study shows that cyclosporin capsules can be substituted for oral solution without causing acute changes in cyclosporin blood levels, and that the pharmacokinetics of the two formulations are similar.This work was carried out in partial fulfillment of the requirements for the Master of Science Degree in Clinical Pharmacy, University of London