Cytomegalovirus (CMV) immune monitoring with ELISPOT and QuantiFERON-CMV assay in seropositive kidney transplant recipients

Abstract

Although cytomegalovirus (CMV) specific cell-mediated immunity (CMI) has been suggested as a predictive marker for CMV infection, proper CMI monitoring strategy in CMV-seropositive recipients and optimal method are not defined. The aim of this study was to evaluate two interferon gamma release assays during early post-transplant period as a predictor of the development of CMV infection in CMV-seropositive patients. A total of 124 CMV-seropositive recipients who received kidney transplantation from CMV-seropositive donor were prospectively examined. At pre-transplant and post-transplant 1 and 3 months, CMV-CMIs were tested using QuantiFERON-CMV assay (QF-CMV) and CMV specific T cell ELISPOT against CMV pp65 and IE-1 antigens (pp65-ELISPOT, IE-1-ELISPOT). CMV DNAemia occurred in 16 (12.9%) patients within 3 months after transplant. Post-transplant pp65 or IE-1 ELISPOT response, but not QF-CMV, was significantly associated with CMV DNAemia. The pp65 ELISPOT (cut-off; 30 spots/200,000 cells) and IE-1 ELISPOT (10 spots/200,000 cells) at post-transplant 1 month predicted the risk of post-transplant CMV DNAemia (P = 0.019). Negative predictive values (NPV) for protection from CMV DNAemia in case of positive ELISPOT results were 94.5% (95% CI: 86.9-97.8%) and 97.6% (95% CI: 86.3-99.6%) in pp65-ELISPOT and IE-1-ELISPOT assays, respectively. These results suggest that the variability may exist between CMV ELISPOT assays and QF-CMV, and CMV ELISPOT at post-transplant 1 month can identify the risk of CMV DNAemia in seropositive kidney transplant recipients.

Fig 1. QF-CMV results at pretransplant, post-transplant 1 month, and post-transplant 3 months in recipients with post-KT CMV DNAemia (-) and CMV DNAemia (+) by Pearson uncorrected Chi-square test.

There was no significant difference in frequencies of QF-CMV positivity between two groups. NS, not significant (P > 0.05).

Fig 2. Results of pp65- and IE-1 specific ELISPOT at pretransplant, post-transplant 1 month, and post-transplant 3 months in recipients with post-KT CMV DNAemia (-) and CMV DNAemia (+) by Mann-Whitney U test.

(A) For pp65-ELISPOT, patients with CMV DNAemia (+) had significantly lower response at post-transplant 1 month than patients with CMV DNAemia (-) [median (95% CI): 8.5 (1.4–130.5) vs. 138.0 (87.2–214.7), P = 0.016]. (B) For IE-1-ELISPOT, CMV DNAemia (+) patients showed significantly lower response at post-transplant 1 and 3 months compared to CMV DNAemia (-) patients (4.0 (1.0–8.1) vs. 18.0 (8.0–44.0), P = 0.019 and 2.0 (0.0–46.6) vs. 27.0 (13.0–56.4), P = 0.014, respectively).

Fig 3. Plot versus criterion value curves for the post-transplant 1 month pp65-ELISPOT and IE-1-ELISPOT assay for predicting CMV DNAemia.

The X-axis shows the cut-off spots/200,000 cells and the Y-axis shows the percentage of CMV DNAemia development. The sensitivity and specificity of pp65-ELISPOT at post-transplant 1 month for predicting CMV DNAemia were 70.0% (95% CI: 34.8–93.3) and 72.2% (95% CI: 60.4–82.1%) with a cut-off value of ≤ 30 spots/200,000 cells. IE-1-ELISPOT showed a sensitivity of 90.0% (95% CI: 55.5–99.7%) and a specificity of 54.7% (95% CI: 42.7–66.2%) to predict CMV DNAemia with a cut-off value of ≤ 10 spots/200,000 cells.

Fig 4. Kaplan–Meier analysis according to the CMV ELISPOT response.

Kaplan-Meier failure estimate showing that patients with high response of CMV ELISPOT at post-transplant 1 month had lower development of CMV DNAemia than patients with low response of CMV ELISPOT (P = 0.019). Patients were classified into high response (if the either pp65 or IE-1 antigens had spot counts more than the cut-off thresholds) and low response CMV (if level of both antigens were below the cut-off thresholds) post-KT 1 month CMV ELISPOT assay.