Surveillance of active human cytomegalovirus infection in hematopoietic stem cell transplantation (HLA sibling identical donor): search for optimal cutoff value by real-time PCR

Abstract

Background: Human cytomegalovirus (CMV) infection still causes significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, it is extremely important to diagnosis and monitor active CMV infection in HSCT patients, defining the CMV DNA levels of virus replication that warrant intervention with antiviral agents in order to accurately prevent CMV disease and further related complications.

Methods: During the first 150 days after allogeneic HSTC, thirty patients were monitored weekly for active CMV infection by pp65 antigenemia, nested-PCR and real-time PCR assays. Receiver operating characteristic (ROC) plot analysis was performed to determine a threshold value of the CMV DNA load by real-time PCR.

Results: Using ROC curves, the optimal cutoff value by real-time PCR was 418.4 copies/104 PBL (sensitivity, 71.4%; specificity, 89.7%). Twenty seven (90%) of the 30 analyzed patients had active CMV infection and two (6.7%) developed CMV disease. Eleven (40.7%) of these 27 patients had acute GVHD, 18 (66.7%) had opportunistic infection, 5 (18.5%) had chronic rejection and 11 (40.7%) died - one died of CMV disease associated with GVHD and bacterial infection.

Conclusions: The low incidence of CMV disease in HSCT recipients in our study attests to the efficacy of CMV surveillance based on clinical routine assay. The quantification of CMV DNA load using real-time PCR appears to be applicable to the clinical practice and an optimal cutoff value for guiding timely preemptive therapy should be clinically validated in future studies.

Figure 1

Probability density of active CMV infection. Seasonal variation of active CMV infection over a 150 days after HSCT period. The highest incidence occurred during the second post-transplant month with maximum value of probability density of 0.010 at day 44.4 after HSCT

Figure 2

Active CMV infection stratification by diagnostic test. Absolute and relative number of patients with positive CMV samples stratified for diagnostic assays (n = 27). Active CMV infection: ≥ 1 cell pp65 positive/3 × 10⁵ PML, and/or 2 or more consecutive positive nested-PCR and/or load CMV ≥ 418.4 copies/10⁴ PBL by real-time PCR. All tests were performed weekly from aliquots of the same blood sample

Figure 3

ROC curve to determine optimal cutoff value by real-time PCR. ROC curve graphing sensitivity versus (1-specificity) for the prediction of determination of active CMV infection using pp65 antigenemia (1 positive pp65 cells/3 × 10⁵ PML) as the reference standard for establishing the optimal cutoff level for real-time PCR. The optimal cutoff value for real-time PCR in peripheral blood leukocytes was 418.4 copies/10⁴ PBL (sensitivity 71.4%; specificity 89.7%)