Comparison of Three Cellular Assays to Predict the Course of CMV Infection in Liver Transplant Recipients

Abstract

To estimate protection from cytomegalovirus (CMV) replication after solid organ transplantation, CMV serology has been considered insufficient and thus CMV immunity is increasingly assessed by cellular in vitro methods. We compared two commercially available IFN-γ ELISpot assays (T-Track CMV and T-SPOT.CMV) and an IFN-γ ELISA (QuantiFERON-CMV). Currently, there is no study comparing these three assays. The assays were performed in 56 liver transplant recipients at the end of antiviral prophylaxis and one month thereafter. In CMV high- or intermediate-risk patients the two ELISpot assays showed significant correlation (p < 0.0001, r > 0.6) but the correlation of the ELISpot assays with QuantiFERON-CMV was weaker. Results of both ELISpot assays were similarly predictive of protection from CMV-DNAemia ≥500 copies/mL [CMV pp65 T-SPOT.CMV at the end of prophylaxis: area under curve (AUC) = 0.744, cut-off 142 spot forming units (SFU), sensitivity set to 100%, specificity 46%; CMV IE-1 T-Track CMV at month 1: AUC = 0.762, cut-off 3.5 SFU, sensitivity set to 100%, specificity 59%]. The QuantiFERON-CMV assay was inferior, reaching a specificity of 23% when setting the sensitivity to 100%. In conclusion, both CMV-specific ELISpot assays appear suitable to assess protection from CMV infection/reactivation in liver transplant recipients.

Comparison of Humoral and Cellular CMV Immunity in Patients Awaiting Kidney Transplantation

“…To better assess the strength of the ELISpot responses in the current cohort, we compared it with results of kidney and liver transplant recipients from our center, whose data have been partially published [ 10 , 26 ]. Both previous studies included intermediate-risk (donor (D)−/recipient (R)+, D+/R+) and high-risk (D+/R−) transplant recipients.…”

“…In kidney transplant recipients, median CMV IE-1 specific responses of 1.5 SFU per 200,000 lymphocytes and median CMV pp65 specific responses of 40.5 SFU per 200,000 lymphocytes were detected ( n = 22) [ 10 ]. In liver transplant recipients, we found median CMV IE-1 specific responses of 4 SFU per 200,000 lymphocytes and median CMV pp65 specific responses of 41 SFU per 200,000 lymphocytes ( n = 103) [ 26 ]. When normalizing our current data on PBMC to lymphocytes and applying a conversion factor of 1.29 as calculated from raw data of the later study by Gliga et al [ 26 ], responses were still at a similar level (converted data of the current study: CMV IE-1 and pp65: 3 and 55 SFU per 200,000 lymphocytes, respectively).…”

“…Previous studies indicate that age significantly affects an individual’s immune response toward CMV [ 26 , 28 , 29 ]. The current study could confirm these findings, when performing multivariate analysis.…”

“…Peripheral blood was collected in 9 mL heparin tubes, and the PBMC were isolated and adjusted to 2 million PBMC per milliliter. In total, 200,000 PBMC were added to each well of 8-well ELISpot strips and stimulated for 19 h at 37 °C in duplicates with two T-activated ® CMV proteins, immediate early antigen-1 (IE-1) and phosphoprotein 65 (pp65), according to the manufacturer’s instructions (T-Track ® CMV, Mikrogen GmbH, Neuried, Germany; formerly Lophius Biosciences GmbH, Regensburg, Germany) [ 10 , 26 ]. In parallel, negative controls (cells with medium only) and positive controls (stimulated with the mitogen phytohemagglutinin, PHA) were cultured.…”

“…The results were generated according to an algorithm provided by the manufacturer. Using the provided algorithm, the arithmetic mean of square-root-transformed 2-replicate spot counts is calculated and squared [ 26 ]. Negative controls were subtracted from CMV-specific values, resulting in spot forming units (SFU).…”