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

Volume 181, December 2025, 105858

Author links open overlay panel, , , , , , , , , , Highlights•

Cell-mediated immunity (CMI) is crucial for control of cytomegalovirus (CMV).

•

Two different test kit platforms were evaluated for CMV-CMI testing.

•

Specificity was 100 %, while sensitivities were between 66.67 % and 88.89 %.

•

Challenges faced in implementing these assays for diagnostic use were discussed.

AbstractBackground

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.

Introduction

Cytomegalovirus (CMV) is ubiquitous and a major cause of morbidity and mortality in transplant and immunocompromised patients. While pre-transplant donor/recipient CMV IgG status defines the risk of post-transplant CMV disease, the use of serology after transplantation has limited clinical utility [1]. Conversely, cell-mediated immunity (CMI) plays an important role in the control of CMV infection [2], [3], and immunosuppressed patients with impaired CMI have higher risk of developing CMV-related disease [1]. While humoral immunity assays for CMV immunoglobulin have been widely available for decades, only recently have assays for measuring CMV-CMI been commercialised. At present, two in vitro diagnostic (IVD) kits are available in Singapore; an enzyme-linked immunosorbent assay (ELISA) and an ELISpot-assay (ES-a). These kits measure the release of interferon-γ (IFN-γ) by T-lymphocytes against CMV to determine the level of CMI, albeit with significantly different assay designs.

The measurement of CMV-CMI can guide prevention and treatment strategies for CMV particularly in solid organ transplant patients [4], [5]. We performed this study in a healthy cohort to verify the analytical performance of these assays.

Section snippetsMaterials and methods

Healthy healthcare workers were recruited with consent and screened for past exposure to CMV using ARCHITECT CMV IgG chemiluminescent microparticle immunoassay (Abbott Laboratories). Seropositive-status was used as a proxy for reactive CMV-CMI. A total of 37 volunteers were recruited, of which 27 were seropositive and 10 were seronegative.

The ELISA and ES-a were performed according to manufacturer’s instructions. Briefly, for ELISA (QuantiFERON®-CMV, QIAGEN), whole blood was deposited into

Results and discussion

The overall agreements of CMV-CMI measured by ES-a and ELISA with CMV IgG serostatus were 91.9 % and 75.7 %, respectively. All 10 samples from CMV IgG negative individuals were non-reactive in ES-a and ELISA, indicating 100 % specificity for both assays. For samples from CMV IgG positive individuals, 24 and 18 out of 27 samples were reactive in ES-a (sensitivity: 88.9 %, 95 % CI: 70.8–97.7 %) and ELISA (sensitivity: 66.7 %, 95 % CI: 46.0–83.5 %), respectively. Notably, the 3 CMV IgG positive

CRediT authorship contribution statement

Soon Hwee Ng: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation. Shireen Yan Ling Tan: Writing – review & editing, Writing – original draft, Visualization, Validation. Su Ming Thean: Writing – review & editing, Project administration, Methodology, Investigation, Data curation, Conceptualization. Poi Wah Kwek: Writing – review & editing, Methodology, Investigation.

Declaration of Competing Interest

The authors declare no conflicts of interest.

References (10)N. Zangger et al.T cell immunity to cytomegalovirus infection

Curr. Opin. Immunol.

(2022)

P.C. Yi et al.Impact of delayed PBMC processing on functional and genomic assays

J. Immunol. Methods

(2023)

V.G. Hall et al.Utility of cytomegalovirus cell-mediated immunity assays in solid organ transplantation

J. Clin. Microbiol.

(2022)

E.Y. Lim et al.The CD4+ T cell response to human cytomegalovirus in healthy and immunocompromised people