Quantification of DNA in Plasma by an Automated Real-Time PCR Assay (Cytomegalovirus PCR Kit) for Surveillance of Active Cytomegalovirus Infection and Guidance of Preemptive Therapy for Allogeneic Hematopoietic Stem Cell Transplant Recipients

Abstract

The performance of a plasma real-time PCR (cytomegalovirus [CMV] PCR kit; Abbott Diagnostics) was compared with that of the antigenemia assay for the surveillance of active CMV infection in 42 allogeneic hematopoietic stem cell transplantation (Allo-SCT) recipients. A total of 1,156 samples were analyzed by the two assays. Concordance between the two assays was 82.2%. Plasma DNA levels correlated with the number of pp65-positive cells, particularly prior to the initiation of preemptive therapy. Fifty-seven episodes of active CMV infection were detected in 37 patients: 18 were defined solely by the PCR assay and four were defined on the basis of the antigenemia assay. Either a cutoff of 288 CMV DNA copies/ml or a 2.42-log 10 increase of DNAemia levels between two consecutive PCR positive samples was an optimal value to discriminate between patients requiring preemptive therapy and those not requiring therapy on the basis of the antigenemia results. The real-time PCR assay allowed an earlier diagnosis of active CMV infection and was a more reliable marker of successful clearance of CMV from the blood. Analysis of the kinetics of DNAemia levels at a median of 7 days posttreatment allowed the prediction of the response to CMV therapy. Two patients developed CMV colitis. The PCR assay tested positive both before the onset of symptoms and during the disease period. The plasma real-time PCR from Abbott is more suitable than the antigenemia assay for monitoring active CMV infection in Allo-SCT recipients and may be used for guiding preemptive therapy in this clinical setting.Preemptive antiviral therapy based on the detection of cytomegalovirus (CMV) in blood has been shown to significantly reduce the incidence of CMV disease in the early posttransplant period following allogeneic hematopoietic stem cell transplantation (Allo-SCT) (2, 7). The pp65 antigenemia assay has been adopted by many transplant centers as the "gold standard" for monitoring active CMV infection and guiding preemptive therapy in this clinical setting. The antigenemia assay, however, requires rapid processing of specimens, is labor-intensive, cannot be automated, is subjective in reading, and is unfeasible during periods of severe neutropenia. In addition, it does not reflect the viral load in the blood compartment accurately (12) and sometimes displays negative results in the presence of CMV disease (2,31,32,36).Quantification of CMV DNA in blood by PCR is emerging as an alternative to the antigenemia assay and may soon become the standard for the surveillance of CMV infection in Allo-SCT recipients. Viral load quantification in blood allows early detection of active CMV infection, close monitoring of the response to antivirals, prediction of the risk of viremic relapse, emergence of resistant strains, and eventual development of CMV disease (8,31). In addition, it may be safely used for guiding preemptive therapy (11,16,23,24,32,37). Nevertheless, there is some controversy as to which is the optimal clinical specimen (plasma, whole blood, or leukoc...

Pre-emptive antiviral therapy for active CMV infection in adult allo-SCT patients guided by plasma CMV DNAemia quantitation using a real-time PCR assay: clinical experience at a single center

“…Our finding was not unexpected, as we have previously shown that an AG level of 1 positive cell/200 000 PMNLs corresponded roughly to 250 CMV DNA copies/mL. 6 The above discrepancies are likely to be related to the different study design, as well as to differences in the clinical characteristics of patients, the sensitivity of the QRT-PCR employed for CMV surveillance and the CMV DNA load threshold established for initiation of antiviral therapy.…”

“…Until April 2010 (group I; n ¼ 90), CMV surveillance was performed in parallel by the AG assay and by a plasma real-time PCR assay (Abbott CMV PCR Kit, Abbott Diagnostics, Des Plaines, IL, USA). 6 In this group, pre-emptive therapy was initiated upon a positive AG assay (X1 positive cell/200 000 polymorphonuclear leukocytes) and stopped upon two consecutive negative results after a minimum of 2 weeks of treatment, as previously described. 6 Since May 2010 (group II; n ¼ 57), virological monitoring was exclusively performed by QRT-PCR, and pre-emptive therapy was initiated upon a plasma CMV DNAemia threshold level of 500 CMV DNA copies/mL and was discontinued after two consecutive PCR-negative (undetectable) results, after a minimum of 2 weeks of treatment.…”

“…6 In this group, pre-emptive therapy was initiated upon a positive AG assay (X1 positive cell/200 000 polymorphonuclear leukocytes) and stopped upon two consecutive negative results after a minimum of 2 weeks of treatment, as previously described. 6 Since May 2010 (group II; n ¼ 57), virological monitoring was exclusively performed by QRT-PCR, and pre-emptive therapy was initiated upon a plasma CMV DNAemia threshold level of 500 CMV DNA copies/mL and was discontinued after two consecutive PCR-negative (undetectable) results, after a minimum of 2 weeks of treatment. Virological monitoring was performed twice a week in the presence of active CMV infection and once a week otherwise (until day 120 after allo-SCT, and at each patient visit afterwards).…”

Cytomegalovirus‐Productive Infection Is Associated With Acute Coronary Syndrome

“…To evaluate productive viral infection, we measured HHV DNA in plasma 16, 17. Although control cord blood samples were negative for the presence of HHVs in our measurements, we set an arbitrary level of 100 copies of viral DNA per microliter of plasma as a threshold for positivity.…”

“…Rather than focusing on HHV replication in particular cells, we evaluated DNA of all HHV types in plasma, into which the productively infected cells may release viruses 17. This analysis revealed all HHVs in plasma of healthy controls and patients with SCAD and ACS.…”