Quantification of Human Cytomegalovirus DNA in Bone Marrow Transplant Recipients by Real-Time PCR (original) (raw)
Related papers
Journal of Virological Methods, 1994
A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibodypositive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture.
Journal of Clinical Microbiology, 2003
A total of 676 specimens from 63 recipients of bone marrow allografts were tested for cytomegalovirus (CMV) by the following assays: CMV pp67 NucliSens (NS), AMPLICOR CMV MONITOR (RA), and the Digene CMV DNA test (DG). In a consensus analysis, the sensitivities and specificities were 60 and 99% (NS), 96 and 98% (RA), and 90 and 76% (DG), respectively; for detection of symptomatic CMV infection, they were 60 and 97% (NS), 65 and 97% (RA), and 95 and 77% (DG), respectively. In multivariate analysis, the major risk factor for symptomatic CMV infection was an increase in the viral load in the DG assay; in univariate analyses, maximum viral loads in both DG and RA assays and a rising viral load in the RA assay were also significant. The earliest detection of CMV replication was provided by the RA assay (mean, 39 days posttransplantation), followed by the DG assay (mean, 48 days) and the NS assay (mean, 58 days).
Revista da Sociedade Brasileira de Medicina Tropical, 2004
The purpose of this work was to test a cytomegalovirus qualitative PCR and a semi-quantitative PCR on the determination of CMV load in leukocytes of bone marrow and kidney transplanted (RT) patients. Thirty three BMT and 35 RT patients participated of the study. The DNA was subjected to a qualitative PCR using primers that amplify part of CMV gB gene. CMV load of positive samples was determined by a semi-quantitative PCR using quantified plasmids inserted with part of the gB gene of CMV as controls. The sensitivity of the test was determined to be 867 plasmid copies/µg DNA. CMV loads between 2,118 and 72,443 copies/µg DNA were observed in 12.1% BMT recipients and between 1,246 and 58,613 copies/µg DNA in 22.9% RT recipients. Further studies are necessary to confirm the usefulness of this CMV semi-quantitative PCR in transplanted patients.
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.
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.
Quantitation of human cytomegalovirus DNA in bone marrow transplant recipients
British Journal of Haematology, 1995
HCMV DNA was retrospectively quantitated in 22) corresponding to a mean DNA level of 184.6 (range 20-710) GE avoided occurrence of any major HCMV-related clinical complication: (ii) clinical symptoms were associated with antigenaemia levels > 100 and DNA levels > 1000 G E (iii) the effect of antiviral treatment could be more carefully monitored by quantitation of viral DNA.
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.
In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. Methods: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The -globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. Results: The assay was linear (R ؍ 0.999) from a lower detection limit of 125 copies/mL to 5 ؋ 10 9 copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n ؍ 53; R ؍ 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R ؍ 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P ؍ 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n ؍ 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n ؍ 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. Conclusions: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.
Transplant Infectious Disease, 2007
Hassen. 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. Transpl Infect Dis 2007: 9: 16^21. All rights reserved Abstract: A pp65 antigenemia assay for polymorphonuclear leukocytes (PMNLs) (CINAkit Rapid Antigenemia), and a qualitative polymerase chain reaction (PCR) test for plasma'PCR-P qual' (Amplicor cytomegalovirus [CMV] test) were performed for 126 samples (blood and plasma) obtained from 18 bone marrow transplant patients, over a 9 -month surveillance period. Among those samples, 92 were assayed with a semi-quantitative PCR test for PMNLs 'PCR-L quant.' The number of samples with a positive CMV test for antigenemia and PCR-P qual assays was 20.63% and 12.7%, respectively, whereas the PCR-L quant assay was positive in 48 of the 92 samples assayed (52.17%). The rates of concordance of the results of PCR-P qual and antigenemia, PCR-P qual and PCR-L quant, antigenemia and PCR-L quant were 92%, 65.2% and 66.8%, respectively. The analysis of the results for the 92 specimens tested by all 3 methods showed a rate of concordance of 63% among all methods. Good agreement (k 5 0.72) was found only between pp65 Ag and PCR-P qual assays. Clinical disease correlates with an antigenemia high viral load. Three patients had CMV disease despite preemptive therapy, and all of them had graft-versus-host-disease (GVHD). PMNLs-based assays are more ef¢cient in monitoring CMV reactivation, but for high-risk patients with GVHD, more sensitive assays (real-time PCR) must be done.
Monitoring of Leukocyte Cytomegalovirus DNA in Bone Marrow Transplant Recipients by Nested PCR
2000
A nested PCR assay for the detection of human cytomegalovirus (CMV) DNA was evaluated by weekly monitoring of blood samples taken from 101 bone marrow transplant (BMT) recipients. When peripheral blood leukocytes were used as the source of CMV DNA, even a modified assay with stringent temperaturecycling conditions was as sensitive as the standard assay. The sensitivity, specificity, and positive predictive value of two consecutively positive leukocytic PCR results with this modified assay in predicting CMV disease of 101 patients submitting 1,441 peripheral blood leukocyte samples were found to be 92.1, 63.5, and 60.3%, respectively. The positive predictive value of patients' seropositivity for CMV was 40%, while that of viremia was 72%. However, viremia followed CMV disease by a median of 1.5 days, while the first leukocytic positive PCR assay preceded disease by a median of 14 days. By use of the criteria of two consecutively positive PCR results instead of recipient CMV seropositivity for starting preemptive ganciclovir treatment, 38 of the 43 recipients with isolated single positive or negative assays (groups I and II) would be spared unnecessary ganciclovir treatment. Moreover, two other findings support the use of antiviral prophylaxis before engraftment in high-risk cases and subsequent preemptive treatment of patients with two consecutively positive PCR assays. First, for 7.9% of 76 patients with positive assays (groups II and III), the first positive PCR assay occurred before engraftment, which implied the presence of viral DNA in the blood (DNAemia) soon after transplantation. Second, isolated single positive assays which were clustered around the second to sixth weeks after transplantation were found for 18 patients (group II) and could represent abortive episodes of CMV infection.