Evaluation of cytomegalovirus cell-mediated immunity assays in the healthy Singapore cohort and challenges of test validation

Abstract

Background

Cytomegalovirus (CMV) is a major cause of morbidity and mortality for transplant and immunocompromised patients. While cell-mediated immunity (CMI) is crucial for control of CMV and can influence the management of patients, commercial kits to measure CMI responses have only recently become available. In this study, we evaluated 2 different test kit platforms to determine their performance with the aim of implementing CMV-CMI testing to serve local needs.

Materials

Fresh blood samples from healthy volunteers (27 CMV-IgG positives and 10 CMV-IgG negatives) were used to evaluate the performance of CMV Interferon-gamma assays, an ELISA and an ELISpot-assay (ES-a).

Results

Specificity was 100 % for both assays, while sensitivity was 66.67 % and 88.89 % respectively for ELISA and ES-a. For the ELISA, the mean coefficient of variations (CV) for within-run and between-run precisions were 3.8 % (1.4–7.3 %) and 15.5 % (5.6–24.7 %), respectively. The mean CV for ES-a’s within-run precisions was 14 % (7.9–21.8 %), though it was not feasible to evaluate between-run precision as blood samples collected on different days from healthy volunteers may have variable results. For ES-a, both delayed blood processing and seeding of peripheral blood mononuclear cells (PBMCs) at lower densities resulted in reduced spot counts but did not affect the qualitative interpretations.

Conclusions

ES-a had better sensitivity compared to ELISA in our healthy cohort. Challenges faced in evaluating these assays comprised of the need for fresh blood sample and large blood volume, particularly for ES-a. Such challenges need to be considered during the implementation of similar tests for diagnostic use.