Clinical experience with a novel assay measuring cytomegalovirus (CMV)-specific CD4+ and CD8+ T-cell immunity by flow cytometry and intracellular cytokine staining to predict clinically significant CMV events

Abstract

Background: Cytomegalovirus (CMV) infection is one of the most common opportunistic infections following organ transplantation, despite administration of CMV prophylaxis. CMV-specific T-cell immunity (TCI) has been associated with reduced rates of CMV infection. We describe for the first time clinical experience using the CMV T-Cell Immunity Panel (CMV-TCIP), a commercially available assay which measures CMV-specific CD4+ and CD8+ T-cell responses, to predict clinically significant CMV events.

Methods: Adult (> 18-year-old) patients with CMV-TCIP results and ≥ 1 subsequent assessment for CMV DNAemia were included at Brown University and the University of Maryland Medical Center-affiliated hospitals between 4/2017 and 5/2019. A clinically significant CMV event was defined as CMV DNAemia prompting initiation of treatment. We excluded indeterminate results, mostly due to background positivity, allogeneic hematopoetic cell transplant (HCT) recipients, or patients who were continued on antiviral therapy against CMV irrespective of the CMV-TCIP result, because ongoing antiviral therapy could prevent a CMV event.

Results: We analyzed 44 samples from 37 patients: 31 were solid organ transplant recipients, 4 had hematologic malignancies, 2 had autoimmune disorders. The CMV-protection receiver operating characteristic (ROC) area under the curve (AUC) was significant for %CMV-specific CD4+ (AUC: 0.78, P < 0.001) and borderline for CD8+ (AUC: 0.66, P = 0.064) T-cells. At a cut-off value of 0.22% CMV-specific CD4+ T-cells, positive predictive value (PPV) for protection against CMV was 85% (95%CI 65-96%), and negative predictive value (NPV) was 67% (95%CI 41-87%).

Conclusions: The CMV-TCIP, in particular %CMV-specific CD4+ T-cells, showed good diagnostic performance to predict CMV events. The CMV-TCIP may be a useful test in clinical practice, and merits further validation in larger prospective studies.

Keywords: (val)ganciclovir; CMV; Cytomegalovirus; Immunoassays; Transplantation.

Fig. 1

CMV-TCIP flow images for CD4+ (top) and CD8+ (bottom) populations demonstrating CMV-specific response (left) or no response (right). IFNG: Interferon-gamma

Fig. 2

%CMV-specific CD4+ (a Mann-Whitney P = 0.002) and CD8+ (b Mann-Whitney P = 0.08) T-cells in patients who subsequently had CMV events compared to those who did not (median, IQR, 1.5xIQR). Horizontal reference lines correspond to cut-off values of 0.22% (CMV-specific CD4+ T-cells), and 0.21% (CMV-specific CD8+ T-cells), as explained in the text

Fig. 3

Scatterplot diagram and linear regression line (95%CI) for the correlation between %CMV-specific CD4+ and CD8+ T-cells (b: slope coefficient). The reference lines correspond to cut-off values of 0.22 and 0.21% CMV-specific CD4+ and CD8+ T-cells, respectively, as explained in the text

Fig. 4

Receiver-operating characteristic (ROC) curves of %CMV-specific CD4+, CD8+ T-cells and the absolute lymphocyte count (ALC) as tests to predict subsequent CMV events. The diagonal line corresponds to area under the curve (AUC) of 0.5

Fig. 5

Positive (PPV) and negative (NPV) predictive values of %CMV-specific CD4+ T-cells for different frequencies (pre-test probabilities) of CMV events

Fig. 6

Significant increases in the % of CMV-specific CD4+ (left) and CD8+ (right) T-cells with time, after the initial CMV event (Wilcoxon rank sum P = 0.028). Five of six patients (black lines), who all had > 0.22% CMV-specific CD4+ T-cells on repeat testing, did not experience subsequent CMV events after discontinuation of valganciclovir (Reference lines: 0.22 and 0.21% for CD4+ and CD8+, respectively, as explained in the text)