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.

FIG. 1.

Scatter diagrams (log₁₀ scale) show the correlation between plasma CMV DNA levels and pp65 antigenemia values (number of pp65-positive cells/200,000 leukocytes examined) among all samples testing positive by the real-time PCR assay and the antigenemia assay during the study period (r = 0.501, P < 0.001, by the Pearson test) (A) and among samples drawn prior to the initiation of preemptive therapy testing positive by both methods (r = 0.789, P < 0.001, by the Pearson test) (B).

FIG. 2.

ROC curves for an antigenemia value of ≥1 pp65-positive cells/200,000 PMNLs for establishing the optimal DNAemia level cutoff (A) and DNAemia level increase threshold between two consecutive PCR-positive samples obtained a median of 1 week apart (B) for triggering the initiation of anti-CMV preemptive therapy. Arrows in panel A point to the proposed DNAemia level thresholds (the left arrow corresponds to a value of 550 CMV DNA copies/ml, and the right arrow corresponds to a value of 288 CMV DNA copies/ml). The arrow in panel B points to a value of 2.42 log₁₀ CMV DNA copies/ml.

FIG. 3.

(A) Kinetics of plasma CMV DNA load in self-clearing (PCR-positive/antigenemia-negative) episodes of active CMV infection defined by two or more PCR positive results (n = 16). (B) Kinetics of plasma CMV DNA load in 18 representative PCR-positive/antigenemia-positive episodes in which PCR-positive results preceded those of the antigenemia assay, until positive conversion of the antigenemia assay.