Monitoring of Viral Load by Quantitative Plasma PCR during Active Cytomegalovirus Infection of Individual Liver Transplant Patients

Abstract

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 >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.Cytomegalovirus (CMV) infection is a common complication after liver transplantation. A variety of clinical manifestations of CMV, such as fever, leukopenia, thrombocytopenia, colitis, pneumonia, and hepatitis, have been described (16). Immunosuppressed organ transplant patients are usually frequently monitored for CMV and treated with antiviral agents. The antiviral treatment is based on the clinical symptoms and/or rapid laboratory diagnosis. Also, preemptive therapy, which is commonly used to prevent the development of CMV disease after transplantation, is based on monitoring the viral load of the patients (5,6,15,20).For more than 10 years, the CMV-pp65 antigenemia assay has been the most commonly used method for monitoring the appearance of CMV infection in transplant patients (2,8,22,25). The antigenemia test is quantitative and can also be used to assess viral load and to monitor the response to antiviral treatment. Although there have been attempts to standardize the assay (8, 23), the great variety of in-house and commercial modifications of the method make it difficult to compare the results and run clinical trials based on this technique. Quantitative PCR techniques, which are easy to standardize, are less laborious for laboratory personnel, and can be autom...

Validation of Clinical Application of Cytomegalovirus Plasma DNA Load Measurement and Definition of Treatment Criteria by Analysis of Correlation to Antigen Detection

“…When these quan- titative PCR results were compared with results of the pp65 antigenemia assay, the median CMV DNA copy numbers in plasma increased proportionally with the CMV antigenemia values, confirming results obtained in earlier studies (5,6,11,12,13,16,20,23,24). Nevertheless, some discrepancies were observed in this analysis of 409 plasma samples.…”

Evaluation of the COBAS Amplicor HCMV Monitor for early detection and monitoring of human cytomegalovirus infection after allogeneic stem cell transplantation

“…If CACM results were considered for clinical assessment, 42 vs 41 patients would have received antiviral therapy. Other studies that compared the CACM with pp65-antigenemia assay, 9,25-27 a quantitative real-time PCR method, 27 the Murex Hybrid Capture CMV DNA Assay 2.0, 28,29 the Quantiplex CMV bDNA 2.0 30 and the Digene CMV DNA test 31 also showed high concurrence of results for early HCMV detection.…”

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

“…The CMV pp65 antigenemia assay, a specific quantitative method developed in the early 1990s, has been widely used for the diagnosis of CMV disease and monitoring of responses to antiviral therapy (3,6,11). However, this assay sometimes shows false-negative results due to a low-level expression of the pp65 antigen in white blood cells in a small number of patients with definite CMV disease (20,22,28). The labor-intensive nature of the procedure, the requirement for immediate sample processing, and the subjective interpretation of slides place limitations on this assay as a routine diagnostic procedure (4,16,22).…”