Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.

BACKGROUND: Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories. Objectives: The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients. STUDY DESIGN: The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test. RESULTS: The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients. CONCLUSIONS: The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.     

Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants.

BACKGROUND: Although several diagnostic methods are available for the surveillance of patients at risk of human cytomegalovirus (CMV) infection and disease, little data is available on their comparative performances in the diagnostic setting. OBJECTIVES: To compare different assays for CMV detection, especially assays based on (quantitative) DNA and mRNA detection. STUDY DESIGN: Eight allogeneic bone marrow and stem cell transplant recipients at high risk for developing CMV disease (donor CMV-negative, recipient positive) were regularly tested for 7-20 weeks post-transplant by spin-amplification rapid culture from urine (viruria), antigenemia (pp65 assay), pp67 mRNA in whole blood (NASBA), and CMV DNA both qualitatively (in-house PCR, whole blood) and quantitatively (in-house PCR, plasma; Cobas Amplicor CMV Monitor Test, plasma and whole blood; Hybrid Capture, whole blood). RESULTS: Four patients (50%) suffered CMV reactivation during follow-up. Out of 104 sample dates, 41 (39.4%) yielded a positive CMV result in at least one assay. Out of the 28 samples tested by all assays, the highest percentage of positive results was obtained with the in-house quantitative PCR (60.7%), followed by the Hybrid Capture system (39.3%), the Cobas Amplicor CMV Monitor Test, plasma version (35.7%), the Cobas Amplicor CMV Monitor Test, whole blood version (32.1%), in-house qualitative PCR (28.6%), and the mRNA assay (21.4%). Viruria was positive in one sample and pp65 antigenemia was found in two samples. CONCLUSIONS: Despite a considerable incidence of CMV reactivations, pre-emptive anti-CMV chemotherapy prevented the development of CMV disease with the exception of one case. The molecular assays had superior sensitivity to conventional ones. The antigenemia assay proved unsuitable for the surveillance of hematological transplant patients. However, none of the tests recognized all timepoints with CMV reactivation. Further comparative studies are needed to determine their respective diagnostic values.     

Monitoring of viral load by quantitative plasma PCR during active cytomegalovirus infection of individual liver transplant patients

A quantitative PCR test, the Cobas Amplicor CMV Monitor, was used for the monitoring of viral load in the peripheral blood of 27 individual liver transplant patients and correlated with cytomegalovirus (CMV) pp65 antigenemia. Altogether, 243 specimens were analyzed. During the first 3 months, 20 patients showed PCR positivity which correlated with pp65 antigenemia. Of those, 13 patients developed symptomatic CMV infection 27 to 52 days after transplantation, with a significantly higher peak viral load in PCR and in pp65 assay compared with the seven asymptomatic infections (median 10,200 versus 2,240 copies/ml, P < 0.05, and median 100 versus 30 pp65-positive cells/50,000 leukocytes, P < 0.01). Five were primary infections of D+/R- cases (donor CMV seropositive and recipient seronegative) and demonstrated, except in one case, a high peak viral load (>10,000 copies/ml; range, 10,200 to 21,600 copies, and > or =50 positive cells, range, 50 to 800 cells). The peak viral loads of the six D+/R+ patients with symptomatic infection varied widely (range, 2,290 to 126,000 copies and 50 to 300 positive cells). Two D-/R+ patients developed symptomatic infection with a lower viral load (range, 1,120 to 6,510 copies and 25 to 100 positive cells). All symptomatic infections were successfully treated with ganciclovir. The asymptomatic infections all in D+/R+ patients with low copy numbers (<5,500 copies) were monitored until CMV disappeared. One of the seven PCR-negative patients had one sample with low antigenemia, but the subsequent specimens were all negative. The time-related correlation of the two methods was also good. In summary, quantitative PCR could equally well be used as the CMV pp65 assay for the monitoring of viral load in individual transplant patients.     

Comparison of plasma polymerase chain reaction and pp65-antigenemia assay in the quantification of cytomegalovirus in liver and kidney transplant patients.

BACKGROUND: Cytomegalovirus (CMV) is a significant problem in transplantation. The antiviral treatment is based on the clinical symptoms and the rapid laboratory diagnosis. Although polymerase chain reaction (PCR) methods have already been widely used, the clinical correlation of the findings is not clear. OBJECTIVE: The objective of this study was to investigate the usefulness of a quantitative plasma PCR test and compare it with the pp65-antigenemia test in the detection of clinically significant CMV infections in liver and kidney transplant patients. STUDY DESIGN: The clinical material consisted of 253 consecutive blood samples was tested using a quantitative polymerase chain reaction test, Cobas Amplicor CMV Monitor (Roche) and pp65 antigenemia assay. Plasma was used for PCR and leucocytes were used for the antigenemia test. RESULTS: CMV was detected in 89 out of 253 blood samples by one or both methods. PCR detected 78 (range 274-165000 copies/ml) and pp65 antigenemia test 79 (range 1-1500 positive cells/50000) of the positive findings. The sensitivity and specificity of PCR test was 86 and 94%, respectively. The PCR detected all clinically significant CMV infections (>10 positive cells in pp65 test) and infections which required antiviral treatment. In addition, the correlation between the two tests was almost linear. CONCLUSIONS: The quantitative PCR appears to be a suitable alternative to diagnose and monitor CMV infections in transplant patients.     

Differences between the quantitative antigenemia assay and the cobas amplicor monitor quantitative PCR assay for detecting CMV viraemia in bone marrow and solid organ transplant patients.

The relationship between quantitative PCR (COBAS Amplicor CMV Monitor, Roche Diagnostics) and quantitative antigenemia (Monofluor pp65, Sanofi Diagnostics) was examined for monitoring CMV viraemia. A total of 469 specimens from immunocompromised haematology and solid organ transplant patients were tested by quantitative antigenemia and qualitative PCR. Quantitative PCR (QPCR) was performed on the 245 specimens in which CMV DNA was detected by qualitative PCR. To exclude any effect due to specific anti-CMV treatment, analysis of antigenemia and QPCR results was only performed on the 164 of 245 specimens collected from patients not on ganciclovir or foscarnet treatment. Forty seven specimens had <400 CMV copies/mL and a negative antigen result, four specimens were antigen positive (all between 1 to 10 positive CMV cells/2 x 10(5) leucocytes) and had <400 CMV copies/mL. Fifty-one specimens had a CMV viral load > or = 400 copies/mL and a negative antigen result and 62 specimens had a CMV viral load > or = 400 copies/mL and a positive antigen. The viral load was shown to be as high as 43,000 copies/mL in some patients with a negative antigen and occurred in non-neutropenic patients. The correlation coefficient for antigen and QPCR results for specimens from bone marrow transplant patients, was 0.69 with an average CMV viral load of 3,200 copies/mL (SEM = 800) and an average antigen of nine positive CMV cells/2 x 10(5) leucocytes (SEM = 3). In the corresponding solid organ transplant group, the correlation coefficient for antigen and QPCR results was 0.71 with an average CMV viral load of 9,900 copies/mL (SEM = 2,100) and an average antigen of 26 positive CMV cells/2 x 10(5) leucocytes (SEM = 6). Both the average viral load and the average antigen result in specimens from solid organ transplant patients, were significantly higher than the average viral load and antigen result in the corresponding group of bone marrow transplant patients (Two-Sample-for-Means z-Test, P = 0.001 and P = 0.003, respectively). The differences in the kinetics of the two assays in monitoring CMV and their ability to predict CMV disease was also assessed in a sub-group of patients. In conclusion, the two assays used in this study do not always show parallel changes in CMV viral load, but may be complementary for the diagnosis and management of CMV disease. The observation that non-neutropenic patients can have a high viral load in plasma and a negative antigenemia has implications for laboratories using antigenemia alone to monitor patients for CMV disease.     

Distinguishing cytomegalovirus (CMV) infection and disease with CMV nucleic acid assays

Human cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality among transplant recipients. Molecular assays have been developed for the detection and quantification of CMV nucleic acid. In evaluating the clinical utility of these assays, correlations with clinical outcome are essential. The Amplicor CMV Monitor and NucliSens CMV pp67 tests were compared to the CMV antigenemia assay for 45 transplant recipients and 1 patient with Wegener's granulomatosis. Twenty-three patients remained antigenemia negative throughout the monitoring period, none of whom developed CMV disease. In this patient group, both the Amplicor and NucliSens assays showed very high specificity; only 1 of the 324 specimens assayed by NucliSens and none of the 303 specimens assayed by Amplicor were positive. Twenty-three patients were antigenemia positive during the monitoring period, 12 of whom developed 13 episodes of symptomatic CMV disease. In this patient group, the NucliSens assay was positive at or before the development of symptoms in 12 of the 13 episodes of CMV disease. All eight patients with symptomatic CMV disease who were tested by the Amplicor assay were positive at or before the development of disease. For the 11 asymptomatic patients, the NucliSens assay was positive less frequently than the antigenemia or Amplicor assays. The NucliSens assay was more likely to be positive at higher antigenemia or viral load levels. Both the NucliSens and Amplicor assays appear to have clinical utility in monitoring patients for CMV disease.     

Detection of CMV-DNA in peripheral blood leukocytes of liver transplant patients after ganciclovir treatment

Summary.  Cytomegalovirus (CMV) infections are common after transplantation, but usually successfully treated with antivirals. In this study, the detection of CMV-DNA in peripheral blood leukocytes was monitored and compared with CMVpp65-antigenemia in liver transplant patients receiving ganciclovir treatment. Twenty adult liver transplant recipients were frequently monitored for CMV up to 6 months after transplantation. CMV infections were diagnosed by pp65-antigenemia and the same specimens were used for CMV-DNA in situ hybridization. Altogether 202 blood specimens were analyzed. During the first 6 months, 14/20 patients developed CMV antigenemia and 11 were treated with ganciclovir. In all patients, CMV-DNA was detected before antigenemia (mean 15 days earlier). All patients responded to ganciclovir and pp65-antigenemia disappeared. However, 8/11 demonstrated persistence of CMV-DNA for up to 6 months. Recurrences appeared in 6/11 patients. In conclusion, detection of CMV-DNA preceded pp65-antigenemia. Persistence of CMV-DNA demonstrates that the virus is not eliminated by ganciclovir and recurrences can be expected. Received December 4, 2002; accepted March 14, 2003 Published online June 2, 2003     

Evaluation of New Quantitative Assays for Diagnosis and Monitoring of Cytomegalovirus Disease in Human Immunodeficiency Virus-Positive Patients

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman rho = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97. 8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment.     

Use of Molecular Assays in Diagnosis and Monitoring of Cytomegalovirus Disease following Renal Transplantation

We compared two commercial molecular assays (the Murex Hybrid Capture CMV DNA assay [HCA], version 2, and the Roche Amplicor plasma PCR assay) with a standard shell vial assay in detecting and predicting cytomegalovirus (CMV) disease in a group of renal transplant patients and assessed the role of viral load measurements (using the HCA) in their management. The sensitivity of the HCA and Amplicor assay in terms of disease detection was 100%, compared to 71% for the shell vial assay. Both the HCA and the PCR assay detected all cases of disease, at medians of 11 and 12.5 days before the onset of symptoms, respectively. Significantly higher viral loads were detected in those patients with symptoms (7.9 x 10(5) copies/ml) than in patients without symptoms (7.9 x 10(4) copies/ml; P < 0.0001). There was also a trend towards higher viral loads in those patients with primary infections (7.8 x 10(5) copies/ml) than in those patients with reactivations of CMV disease or reinfections. Successful treatment with ganciclovir was associated with a >90% reduction in viral load. Both of these new assays are sensitive and easy to use. A comparison of accurate quantitation is also useful in monitoring responses to antiviral therapy.     

Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.

BACKGROUND: Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis. OBJECTIVES: To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag. STUDY DESIGN: Prospective study conducted in liver transplant recipients from February 2000 to February 2001. RESULTS: Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively. CONCLUSIONS: Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating preemptive therapy with high specificities and sensitivities. Diagnostic assays like CMV pp65 Ag and quantitative PCR for CMV have similar efficiency and could be recommended as methods of choice for diagnosis and monitoring of active CMV infection after transplantation.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.     

Comparison of LightCycler-based PCR,COBAS amplicor CMV monitor,and pp65 antigenemia assays for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r congruent with 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10(7) DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.     

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.     

Cytomegalovirus infection in paediatric haemopoietic stem cell transplantation

A retrospective audit of CMV infection was undertaken to determine prevalence and outcome in the national paediatric Haemopoietic Stem Cell Transplant (HSCT) unit, with particular reference to surveillance and treatment. All patients undergoing HSCT (125 allogeneic, 50 autologous) from January 1994 to December 2004 were included. Nine underwent a second transplant for graft failure or disease recurrence. Of 134 allogeneic transplants performed, 62 were unrelated. Shell vial cultures of throat swabs and urine, and blood samples for pp65 antigenemia +/- PCR were tested weekly for a mean of 147 days post transplant. CMV negative blood products and filters were used in all. 11 rec+/donor-, 12rec-/donor+ and 10rec+/donor+ transplants were performed. All received prophylactic acyclovir, IVIG was prescribed for all but CMV -/- transplants. Initial detection of CMV was urine in 5 cases, four of whom developed antigenemia. Of ten patients who developed antigenemia, nine were treated with ganciclovir +/- foscarnet and two of these patients developed CMV pneumonitis and died. The current policy of strict surveillance, matching donor and recipient CMV status, use of CMV negative blood products and filters and pre-emptive therapy appears to be effective in controlling CMV disease/infection in the peritransplant period.     

Cytomegalovirus management after allogeneic hematopoietic stem cell transplantation: A mini-review

Because of the high incidence of cytomegalovirus (CMV) seropositivity in the population, CMV infection is a common and severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Taiwan. Here we propose a CMV management strategy for patients undergoing allo-HSCT from the Taiwanese perspective, which focuses on the epidemiology, diagnosis, monitoring, prophylaxis, and treatment of CMV infection after allo-HSCT. In terms of CMV monitoring, weekly CMV monitoring with the COBAS® AmpliPrep system is the standard approach because the pp65 CMV antigenemia assay has a lower sensitivity than CMV monitoring with the COBAS® AmpliPrep system. However, pp65 CMV antigenemia assay has a better correlation with clinical symptoms in immunocompromised patients. A 14-week prophylactic course of letermovir is recommended for allo-HSCT recipients in Taiwan, especially for recipients of hematopoietic stem cells from mismatched unrelated and haploidentical donors. Preemptive ganciclovir therapy should be initiated when the CMV viral load exceeds 1000 copies/mL, and should not be discontinued until CMV DNA is no longer detected in the blood. For allo-HSCT recipients who have CMV-related diseases, ganciclovir with or without CMV-specific intravenous immunoglobulin is the standard of care. The limited availability of foscarnet, an alternative for patients who are not responsive to or cannot tolerate ganciclovir, is a crucial issue in Taiwan. For pediatric allo-HSCT recipients, more data are needed to propose a CMV management recommendation.     

Cytomegalovirus DNA concentration in plasma predicts development of cytomegalovirus disease in kidney transplant recipients

The clinical significance of cytomegalovirus (CMV) DNA detection in post-kidney transplantation infection surveillance was examined by comparing the performance of three assays for detection of CMV in blood: the test for CMV-pp65-antigen in leukocytes, which is routinely employed in our laboratory, the quantitative plasma CMV-DNA-polymerase chain reaction (PCR; Cobas Amplicor CMV Monitor test^®) and the qualitative plasma CMV-DNA-PCR (Amplicor CMV test^®). Thirteen kidney transplant recipients were monitored with serial samples taken over a period of 3 months following transplantation. The quantitative CMV-PCR was the test with highest sensitivity, 95.9%, vs. 88.9% and 76.9% for the CMV-pp65 antigen assay and qualitative CMV-PCR, respectively. The virus load in the first positive specimens, assessed as DNA-copies/mL, was significantly associated with CMV disease because five of the six patients who developed disease, but only one of the seven who did not develop disease, had more than 3000 CMV-DNA-copies/mL. The number of CMV-pp65 antigen-positive cells in the first positive specimens did not have predictive value for development of CMV disease. Assessment of CMV in plasma by the quantitative CMV-PCR is especially useful since it has a high sensitivity and the amount of CMV DNA in plasma is a good predictor of CMV disease.     

肝移植术后早期巨细胞病毒感染的先驱性治疗

目的 通过临床病例对照研究,寻求肝移植术后防治巨细胞病毒(CMV)感染的较好方法。方法 将63例原位肝移植患者分为预防性治疗组与先驱性治疗组,术后3个月内定期进行CMV-PP65定性和CMV-DNA定量检测,预防性治疗组均在术后2周时给于静脉更昔洛韦治疗,先驱性治疗组仅在检测阳性时给予更昔洛韦治疗。结果 预防性治疗组中17％(5／35)出现了CMV感染;先驱性治疗组中36％(10／28)出现了CMV感染。两组中全部病例均未发生CMV病。结论 肝移植术后早期采用先驱性治疗不增加巨细胞病毒病的发生率。     

Prognosis value of the pp65 antigenemia and semi-quantitative PCR in the detection of the CMV reactivation in bone marrow grafted patients

In this article a Cytomegalovirus (CMV) antigenemia and semiquantitative PCR retrospective evaluation of 26 bone marrow allo-grafted patients for different haematological disease is reported. Eighteen patients had a CMV reactivation despite a prophylactic treatment, seven of those patients had both positive antigenemia pp65 and positive semi-quantitative CMV PCR. During CMV reactivation, 3 patients developed a CMV disease despite a pre-emptive therapy. The follow up of the antigenemia was performed since D21 until D100 post transplantation, the antigenemia positivity occurred at D53 and was preceded about 7 days by CMV PCR positivity The CMV disease wasn't associated with a high viral load. All patients that had CMV reactivation had a positive CMV serology before the graft, whereas only 37.5% of the patients who did not reactivate had a positive CMV serology. Respectively half patients who reactivated and only 12.5% of those who didn't had a Graft versus host disease (GVHD), witch preceded the reactivation about 21 days in six of the formers. Clinical and biological signs presented by our patients in this cases report, seems to be associated more with the GVHD than with CMV reactivation.     

Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantitation assay

Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene, Argene, France), a pp65 antigenemia assay and four different "in-house" real-time PCR assays were compared to the CMV R-gene for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r=0.63, p<0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200000 polymorphonuclear leukocytes was 5.26log(10)copies/mL of whole blood. When the CMV R-gene kit was compared to the four other "in-house" real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good (r=0.82 for laboratory 1, r=0.66 for laboratory 3) to excellent (r=0.99 for laboratory 2, r=0.94 for laboratory 4) correlation between CMV R-gene and the four real-time "in-house" PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene test were constantly higher than those generated by three out of four "in-house" PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene and "in-house" PCRs was of 0.77log(10), 0.04log(10), 0.77log(10) and 0.97log(10), for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an "inter-laboratory" comparison of results. Our study showed that CMV R-gene is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.     

Quantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapy

In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan PCR >10/150,000 cells (RR: 27.6, IC95: 7.1-107.2), the CD4 cell count <75 cells/mm(3) and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml.