Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation (original) (raw)
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Journal of Clinical Microbiology, 2000
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 ganciclov...
Journal of Clinical Microbiology, 2003
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 Х 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.
Journal of Clinical Microbiology, 2000
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.
Transplantation Proceedings, 2005
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 diagnososis 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 ocurred 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.
Journal of Medical Virology, 2004
cause of morbidity in solid organ recipients. Early markers to identify the progress of the infection and patients at high risk are required in order to apply a strategy of pre-emptive therapy. The efficacy of pre-emptive therapy relies on accurate laboratory tests to monitor CMV infection. The evaluation of CMV DNA kinetics by the polymerase chain reaction (PCR) is widely used for the management of CMV infection but markers predicting the progression of the infection and standardization of the technique are essential for the clinical interpretation of PCR results. A commercially available PCR system, the COBAS AMPLICOR Monitor (Roche Diagnostics, Brachburg, NJ), was used for the quantitation of CMV DNA in weekly blood samples (n ¼ 504) from 47 solid organ recipients in the first 6 months after transplantation. PCR results were evaluated according to the development of clinical disease in order to find a DNA threshold and time points predicting the progression of CMV infection.
Monitoring of Cytomegalovirus Reactivation in Bone Marrow Transplant Recipients by Real-time PCR
Pathology & Oncology Research, 2008
Cytomegalovirus (CMV) has been recognized as the most important viral pathogen in persons undergoing bone marrow transplantation (BMT). The aim was to develop a quantitative PCR assay to quantify CMV DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation (BMT) patients. An in-house real-time PCR assay based on TaqMan technology was developed to monitor the quantity of CMV DNA in PBLs of the BMT recipients. Sequential blood samples (415 specimens) were collected from 43 patients as weekly intervals until day 100 after transplantation. The CMV DNA was quantified in parallel with the pp65 antigenemia assay in PBL samples. Viral reactivation occurred in 51% and 41.8% of the recipients as detected by RQ-PCR and antigenemia assays respectively. There was a significant correlation between both assays (P<0.0001); however, the RQ-PCR was more sensitive than the antigenemia. CMV DNA was detected by the RQ-PCR by a median of 14 days earlier than the antigenemia. Preemptive therapy was implemented in the antigenemia positive cases. The administration of ganciclovir led to a rapid decrease in the viral load. After preemptive therapy, the antigenemia achieved a negative result earlier than the RQ-PCR assay (a median of 17.5 days). An increase of viral load in both quantitative assays and of cyclosporine serum level were identified as the most significant risk factors for CMV reactivation. The quantitative CMV PCR might be a useful tool for monitoring the CMV reactivation and guiding the efficacy of the CMV preemptive therapy in BMT recipients.
The performances of a commercially available qualitative plasma PCR assay (AMPLICOR CMV test; Roche Diagnostics) and the pp65 antigenemia assay (AG) were evaluated for the monitoring of cytomegalovirus (CMV) viremia in 43 allogeneic stem cell transplant recipients. In addition, the suitabilities of both assays for triggering the initiation of preemptive ganciclovir therapy were assessed. A total of 37 CMV viremic episodes were detected in 28 patients. Positivity of plasma PCR testing in one or more consecutive specimens was the only marker of CMV viremia in 18 of the 37 episodes (PCR positive and AG negative, n ؍ 50 specimens). Five episodes were diagnosed on the basis of a single positive AG result (AG positive and PCR negative, n ؍ 5 specimens); both assays were eventually positive (PCR positive and AG positive, n ؍ 27 specimens) for 14 viremic episodes; for these episodes, conversion of the PCR assay result to a positive result occurred an average of 1 week before conversion of the AG result. Overall, the concordance between the two methods was 90%, and the sensitivities of the plasma PCR assay and AG for the detection of CMV viremic episodes were 86.5 and 51.3%, respectively. Two patients who tested positive by both assays simultaneously progressed to CMV end-stage organ disease, despite the initiation of preemptive ganciclovir therapy. Conversion of the AG result to a negative result upon administration of preemptive ganciclovir therapy occurred a median of 7.5 days earlier than conversion of the plasma PCR assay result. Nineteen of the 28 patients with CMV viremia received AG-guided preemptive ganciclovir therapy; had the positivity of the plasma PCR assay triggered the initiation of preemptive therapy, 9 additional patients would have been unnecessarily treated since none of them developed CMV end-stage organ disease. Although the AMPLICOR CMV assay is more sensitive than AG, the latter appears to be more suitable both for guiding the initiation of preemptive therapy and for monitoring a patient's response to antiviral therapy.
Bone Marrow Transplantation, 2005
The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment. Bone Marrow Transplantation (2005) 35, 595-599.
Journal of Clinical Microbiology, 2002
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.