COBAS AMPLICOR Cytomegalovirus MONITOR Test Research Articles

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log(10) copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to ≥ 200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

Preemptive valganciclovir for cytomegalovirus infection in hematological patients

To the Editor We read with interest the ¢ndings by Busca et al. (1) published in the Journal, adding to similar recent ¢ndings supporting the e⁄cacy of preemptive valganciclovir in allogeneic hematopoietic stem cell transplant (HSCT) recipients (2, 3). We add here our single-center preliminary experience in hematological patients. Before November 2005 we usually treated cytomegalovirus (CMV) reactivation (higher than 1500 copies/mL) with intravenous ganciclovir. During a 24 -month period starting in November 2005, all our patients undergoing HSCTor aggressive chemotherapy were monitored with weekly real-time quantitative polymerase chain reaction (PCR) assay (Cobas Amplicor CMV Monitor test, Roche Diagnostics, Mannheim, Germany; sensitivity5 600 CMV DNAcopies/mL) on peripheral blood during treatment and in the ¢rst 3 months after discharge. Thirty-seven patients with CMV viremia higher than 1500 copies/mL (at a median of 45 from end of chemotherapy or HSCT) were identi¢ed and treated for 3 weeks with preemptive oral valganciclovir 450 mg b.i.d. (n5 5) or 900 mg b.i.d. (n5 32) according to renal function. No patient had concurrent infections or was receiving steroids at the time of reactivation. No reduction in immunosuppression was performed in allogeneic HSCT recipients at the time of CMVreactivation. All but 2 patientswho reactivated CMVafter aggressive chemotherapy (n513: 7 myeloablative and 6 nonmyelobalative), or autologous (n5 5: 4 high-dose melphalan and 1 BEAM) or allogeneic (n519) HSCTshowed weekly drops in CMV levels and ¢nally cleared viremia at the ¢nal control on day 21 day since beginning of treatment. Both patients who failed the endpoint were allogeneic HSCT recipients with initial loads lower than 5000 copies/mL who had been treated with valganciclovir 900mgb.i.d.; they had persistent low-level viremia (4000 copies/mL) for at least 2 months despite salvage treatment with intravenous foscarnet. Only 3 patients, all allogeneic HSCTrecipients, developed neutropenia (absolute neutrophil counto500/mL) while on valganciclovir, which could be explained by concurrent immunosuppressive drugs. The median initial viral load in our cohort was 3900 copies/mL, which is a relatively low level and could have contributed to the high e⁄cacy of valganciclovir.W|th the use of such a low threshold, we did not observe any case of symptomatic CMVdisease. Recent data support that the e⁄cacy of valganciclovir 450mg b.i.d. is identical to 900mg b.i.d. in allogeneic HSCT recipients (90% clearance of antigenemia on day 121) (4), and this is likely truewhenever initial CMV load remains quite low, allowing proper treatment of patients with renal failure. In reduced-intensity conditioning HSCT recipients without gastrointestinal graft-versus-host disease (GvHD), the r 2008 Wiley Periodicals, Inc.

Evaluation of the ReSSQ Assay in Relation to the COBAS AMPLICOR CMV MONITOR Test and an In-House Nested PCR Method for Detection of Cytomegalovirus DNA

The ReSSQ CMV assay is a novel commercially available kit for quantification of cytomegalovirus (CMV), based on real-time PCR with a peptide nucleic acid probe coupled with a single dye. In combination with the LightCycler, the ReSSQ CMV assay was evaluated with respect to specificity, PCR inhibition, linearity, reproducibility, and sensitivity. All nontested CMV materials were negative, and the assay was not inhibited by the use of different anticoagulants or other factors that may influence blood samples. The dynamic range was between 10 and 5 x 10(8) copies/PCR, and intra- and interassay variabilities were below 0.10 and 0.12 log10 standard deviations, respectively. Assay sensitivity was validated by analysis of 24 samples from a proficiency panel and by comparison to a nested in-house CMV PCR and the COBAS AMPLICOR CMV MONITOR test, using 159 clinical samples. Results from the proficiency panel were well in accordance with input values over the entire range of viral concentrations tested (50 to 31,250 copies/ml). The association between the ReSSQ CMV assay and the in-house PCR was in agreement in 90% of the clinical samples, and discordant results were found for all types of sample materials analyzed. The ReSSQ CMV and COBAS AMPLICOR assays showed no significant differences for samples containing >1,000 CMV copies/ml, but results differed to a greater extent at lower viral concentrations. The results demonstrate that the ReSSQ CMV assay is a CMV-specific, robust, and reproducible method and hence is well suited for routine use in clinical virology laboratories.

FUNCTIONAL CHARACTERIZATION OF CMV-SPECIFIC CD8 T LYMPHOCYTES IN HUMAN LUNG TRANSPLANT RECIPIENTS

P1017 Aims: Human cytomegalovirus (CMV) continues to be a significant cause of morbidity in immunosuppressed lung transplant recipients (LTR), this despite the routine use of prophylactic antiviral therapies. Given the importance of an intact effector immune system to suppress CMV reactivation in normal individuals, we studied the impact of immunosuppression on the frequencies, function and phenotype of CMV-specific CD8+ T cells in a cohort of LTR. Methods: LTR, with known donor/recipient (D/R) CMV serostatus, were cross-sectionally studied before and following (1, 2, and 3 months post) lung transplantation. The frequency and functional characteristics of circulating CMV-specific CD8+ T lymphocytes was assessed by binding of specific tetrameric peptide/HLA complexes and peptide-induced interferon-γ (IFN-γ) production, respectively. HLA-A2, -B7, and –B8 CMV-specific epitopes were used as CD8+ antigenic stimulants. Phenotypic classification of CD8+ T cells into naïve, memory and effector subsets was based on the expression of the four T cell surface markers, CD27, CD28, CD45RA and CCR7. CMV viral load measurements were made in the plasma and bronchoalveolar lavage (BAL) using the COBAS Amplicor CMV monitor test. Results: The frequencies of CMV-specific CD8+ T cells varied considerably between CMV-seropositive LTR (range 0.1-8.1% of all CD8+ T cells), but remained relatively stable over time in any one individual, and were not significantly reduced compared to pre-transplant levels. In those “at-risk” LTR (CMV serostatus D+/R+, D+/R- and D-/R+) who did not demonstrate CMV reactivation during the study period, there was no evidence of any functional impairment in their CMV-specific CD8+ T cell subset (50-70% of tetramer positive CD8+ T cells produced IFN-γ following specific CMV peptide stimulation). In those LTR who were mismatched for CMV (D+/R-) the appearance of CMV-specific immunity (circulating tetramer positive CD8+ T cells) was often not associated with either a clinical CMV syndrome or measurable CMV DNA (viral load <400 copies) in the blood or BAL. Following primary CMV infection, CMV-specific CD8+ T cells were skewed predominantly towards an effector phenotype, CD45RA+/−CCR7- or CD27+/−CD28-. Conclusions: The maintenance of CMV latency in immunosuppressed CMV seropositive lung transplant recipients is associated with the persistence of functionally active circulating CMV-specific CD8+ T cells. Combining the measurement of CMV viral load with a detailed analysis of the frequency, function and phenotype of CMV-specific CD8+ T lymphocytes offers insights into the dynamic relationship that exists between host immunity and viral replication, particularly in the transplant setting.

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.

Performance characteristics of an automated PCR assay for the quantification of cytomegalovirus DNA in plasma

The COBAS Amplicor CMV Monitor test (Roche Diagnostics), an automated polymerase chain reaction (PCR) assay for the quantification of cytomegalovirus (CMV) DNA in plasma samples, was evaluated in a routine diagnostic laboratory. Using cell culture-derived CMV and CMV-negative human plasma, the linear detection range of the assay as well as its intra-and inter-assay variabilities were assessed. The study design allowed distinguishing variations in results related to amplification and detection from those caused by differences in the efficiency of DNA extraction. The assay was able to identify the majority of samples correctly as positive with CMV DNA concentrations above the limit of detection. However, the reported values were often twofold or more different from the (theoretical) input, which could be explained partly by inefficient DNA extraction. The following values were computed for the coefficients of determination R 2: inter-assay variability excluding DNA extraction, R 2=0.982; including DNA extraction, R 2=0.977; intra-assay variability excluding DNA extraction, R 2=0.992; including DNA extraction, R 2=0.992. On balance, the test has acceptable within-run and between-run reproducibility. It therefore allows the comparison of results obtained at different time-points as well as in different laboratories, e.g. in multi-centre studies.

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation.

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation.

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.