Evaluation of the VIDAS® Cytomegalovirus (CMV)-IGRA for Assessing CMV-Specific T-Cell Responses in Immunocompetent and Immunosuppressed Individuals

We compared the performance of the VIDAS® Cytomegalovirus (CMV)-Interferon-gamma release assay (IGRA) with that of laboratory-developed flow cytometry for intracellular cytokine staining (FC-ICS) for the assessment of CMV-specific interferon-gamma (IFN-γ)-producing T-cell responses (CMV-CMI). A total of 147 blood specimens from 78 adult participants were collected: 11 healthy controls, 34 hematological patients (HP), of which 32 had undergone allogeneic hematopoietic cell transplantation (allo-HCT), and 33 Kidney transplant recipients. Of the 147 specimens, 96 tested positive, 24 negative, 25 indeterminate, and 2 were invalid by the VIDAS® CMV IGRA. A total of 137 specimens were tested by FC-ICS, of which 107 returned positive results. There were 27 discrepancies across the assays among specimens yielding interpretable results, of which 14 tested VIDAS® CMV IGRA-positive/FC-ICS-negative and 12 VIDAS® CMV IGRA-negative/FC-ICS-positive. The overall agreement between immunoassays was 78%, and the Kappa coefficient was 0.34 (0.52 for HP). Differences in identifying CMV-infected (CMV IgG-positive) and uninfected participants (CMV IgG-negative) were noticed across both assays. The overall correlation (rho values) between IFN-γ concentrations (IU/ml) measured by the VIDAS® CMV IGRA and CMV-specific IFN-γ-producing T-cell frequencies were 0.27 for CD4 + and 0.33 for CD8 + T cells ( P =0.001). In HP, the correlation was stronger (0.48 for CD4 + and 0.49 for CD8 + T cells). A trend toward a direct association between the presence of undetectable VIDAS® CMV IGRA or FC-ICS responses and subsequent occurrence of CMV DNAemia was observed. In summary, our data lend support to the potential utility of the VIDAS® CMV IGRA to assess CMV-CMI in transplant recipients. Evaluation of the VIDAS® Cytomegalovirus (CMV)-IGRA for Assessing CMV-Specific T-Cell Responses in Immunocompetent and Immunosuppressed Individuals Estela Giménez 1,2, Eliseo Albert 1, Ester Colomer 1, Ariadna Pérez 3, Marco Montomoli 4, José Luis Piñana 3, Irina Sanchis 4, José Luis Górriz 4,5, Carlos Solano 3,5, and David Navarro 1,2,6* 1 Microbiology Service, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain. 2 CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain. 3 Hematology Service, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain. 4 Nephrology Service, Hospital Clínico Universitario de Valencia, INCLIVA Health Research Institute, Valencia, Spain. RICORS 2040 RENAL, ISCIII. 5 Department of Medicine, School of Medicine, University of Valencia, Valencia, Spain. 6 Department of Microbiology School of Medicine, University of Valencia, Valencia, Spain. Correspondence : David Navarro, Microbiology Service, Hospital Clínico Universitario, Instituto de Investigación INCLIVA, Valencia, and Department of Microbiology, University of Valencia, Valencia, Spain . Av. Blasco Ibáñez 17, 46010 Valencia, Spain. Phone: 34(96)1973500; Fax: 34(96)3864173; E-mail: [email protected]. Running title : Assessment of CMV-specific T-cell response by the VIDAS® CMV-Interferon-gamma release assay

We compared the performance of the VIDAS® Cytomegalovirus (CMV)-Interferon-gamma release assay (IGRA) with that of laboratory-developed flow cytometry for intracellular cytokine staining (FC-ICS) for the assessment of CMV-specific interferon-gamma ( IFN-γ)-producing T-cell responses (CMV-CMI). A total of 147 blood specimens from 78 adult participants were collected: 11 healthy controls, 34 hematological patients (HP), of which 32 had undergone allogeneic hematopoietic cell transplantation (allo-HCT), and 33 Kidney transplant recipients. Of the 147 specimens, 96 tested positive, 24 negative, 25 indeterminate, and 2 were invalid by the VIDAS® CMV IGRA. A total of 137 specimens were tested by FC-ICS, of which 107 returned positive results. There were 27 discrepancies across the assays among specimens yielding interpretable results, of which 14 tested VIDAS® CMV IGRA-positive/FC-ICS-negative and 12 VIDAS® CMV IGRA-negative/FC-ICS-positive. The overall agreement between immunoassays was 78%, and the Kappa coefficient was 0.34 (0.52 for HP). Differences in identifying CMV-infected (CMV IgG-positive) and uninfected participants (CMV IgG-negative) were noticed across both assays. The overall correlation (rho values) between IFN-γ concentrations (IU/ml) measured by the VIDAS® CMV IGRA and CMV-specific IFN-γ-producing T-cell frequencies were 0.27 for CD4 + and 0.33 for CD8 + T cells ( P =0.001). In HP, the correlation was stronger (0.48 for CD4 + and 0.49 for CD8 + T cells). A trend toward a direct association between the presence of undetectable VIDAS® CMV IGRA or FC-ICS responses and subsequent occurrence of CMV DNAemia was observed. In summary, our data lend support to the potential utility of the VIDAS® CMV IGRA to assess CMV-CMI in transplant recipients.

Cytomegalovirus, VIDAS® Cytomegalovirus (CMV)-Interferon-gamma release assay (IGRA), flow cytometry for intracellular staining, T-cell immunity, transplant recipients.

Functional Cytomegalovirus (CMV)-specific T-cell responses are instrumental in conferring protection against high-level virus replication and development of CMV end-organ disease in transplant recipients [1,2]. A large fraction of the CMV proteome is targeted by cytokine-producing CD8 + and CD4 + T cells [3]; however, tegument protein pp65 and immediate early protein 1 (IE-1) are immunodominant in most individuals, both independently eliciting responses that effectively contribute to virus clearance in this clinical setting [1,4,5]. Sequential monitoring of CMV-specific T-cell responses (CMV-CMI) following transplantation has been advocated as a potentially useful tool to individualize preventative antiviral strategies, either prophylactic or preemptive, aimed at minimizing the risk of CMV infection-related morbidity, both in the allogeneic hematopoietic cell transplantation (allo-HCT) and kidney transplant recipients (KTR) settings [6-9]. Recent consensus guidelines recommend CMV-CMI monitoring in certain clinical scenarios in these patient populations [10-12]. Traditionally, CMV-CMI assessment in transplant recipients has relied on flow cytometry for intracellular staining (FC-ICS)-based assays, usually enumerating cytokine-producing monofunctional (i.e., interferon-gamma ( IFN-γ) or tumor necrosis factor-alpha (TNF-α)) or polyfunctional T cells targeting CMV pp65/IE-1 [4,5]. Nevertheless, FC-ICS assays, although considered the “gold-standard” method, \soutare is time-consuming, labor-intensive, and lacks robust standardization. As an alternative, commercially available IFN-γ release assays (IGRA), including enzyme-linked immunospot-based (ELISpot) tests (T-SPOT®.CMV from Oxford Immunotec, Abingdon, UK, or T-Track® CMV from Mikrogen, Neuried, Germany) and the Quantiferon® CMV assay (Qiagen, Hilden, Germany) have emerged as appealing alternatives to FC-ICS for measuring CMV-CMI in transplant recipients. Their appeal lies in that they are relatively simple to carry out, provide reproducible results, and their clinical performance regarding the prediction of CMV DNAemia and/or end-organ disease has been extensively evaluated [e.g., 13-20]. Nevertheless, both qualitative and quantitative results returned by these assays are not interchangeable, as significant discrepancies across the tests have been reported [21-29]. Recently, a fully automated ELFA (Enzyme Linked Fluorescent Assay)-based IGRA, the VIDAS® IGRA RUO kit, has been developed by bioMérieux (Marcy l’Etoile, France). Here, we compared the performance of this IGRA with that of an FC-ICS assay for the enumeration of CMV pp65/IE-1 IFN-γ-producing CD8 + and CD4 + T cells developed at our laboratory, whose usefulness in predicting the occurrence of CMV DNAemia and its resolution was shown both in allo-HCT and KTR [30-35].

Patients and specimens A total of 147 whole-blood samples, obtained from 78 nonconsecutive adult individuals (≥ 18 years old; 47 males, 30 females; median age, 55 years; interquartile range (IQR), 39-66) were prospectively collected between April and December 2024. This series included 11 specimens from unique healthy control individuals (HC) (median age, 31; IQR, 25-37) , 78 specimens from 34 hematological patients (HP) (median age, 55 years; IQR, 35-65), and 58 specimens from 33 KTRs (median age, 64 years; IQR, 53-74) . Of the 34 HP, 32 had undergone allo-HCT and 2 CAR-T cell therapy. The types of transplant in Allo-HCT recipients were HLA-matched related (n=12), haploidentical (n=10), HLA-matched unrelated (n=7), HLA-mismatched unrelated (n=2), and HLA-mismatched related (n=1). Acute graft versus host prophylaxis consisted of mycophenolate mofetil (MMF), post-transplant cyclophosphamide, and sirolimus in 30/32 patients. A total of 26/32 Allo-HCT recipients were under letermovir primary prophylaxis at the time of sampling. Immunosuppression regimens in KTR were Everolimus/MMF/prednisone in 22 patients and Everolimus/Tacrolimus/prednisone in the remaining 11 patients. Regarding CMV serostatus, among the HC, four were CMV-seropositive and seven were CMV-seronegative. Donor(D)/Recipient(R) CMV serostatus in Allo-HCT were: D+/R+ in 19 cases, D-/R+ in 10, D-/R- in two, and D+/R- in one. Both CAR-T participants and 30/33 KTR were CMV-seropositive. The study was approved by the Ethics Committees (2023/309). All patients gave their informed consent to participate in the study. VIDAS® CMV interferon-γ Release Assay (IGRA) This fully automated ELFA-based IGRA was performed on the VIDAS® 3 system using the supplied reagents: VIDAS® IGRA 16 RUO kit and VIDAS® STIMM CMV RUO kit (bioMérieux, Marcy l’Etoile, France). The assay was performed following 16-hour in-vitro stimulation (37ºC) with a CMV lysate (VIDAS STIMMTM CMV RUO reagent) according to the manufacturer’s instructions. Briefly, 4 ml of whole blood collected in a lithium heparin tube was loaded into the VIDAS3 instrument along with the required consumables (three strips and three solid-phase receptacle (SPR) devices) and three stimulation reagents: (i) Mitogen (PHA-P) as a positive control (MIT), ii) CMV antigen (AG) (VIDAS STIMMTM CMV RUO), and iii) NIL as a negative control. These reagents were reconstituted with 5 ml of distilled water before use and were ready-to-use for three months. The automated system pipetted 0.3 mL of blood with the stimulation reagents, and after 15 minutes, both samples and reagents were ready for storing. The instrument then automatically performed all subsequent steps, including anti-IFN-γ immunocomplex formation on the SPR, fluorescence reading, and IFN-γ concentration calculation (IU/mL) according to the calibration curve stored. Results were available for export after 16 hours. The assay is calibrated against the WHO International Standard for Recombinant Human Interferon-Gamma and returns results from 0.08 to 8.00 IU/ml. All samples were processed within six hours of collection. Results are available as IU/mL and fluorescence signal (rfv). VIDAS® IGRA results were classified according to the following criteria: (i) negative samples showed positive MIT results but negative results for both NIL and AG ( 8.0 IU/ml, while samples yielding MIT values < 2.0 were classified as undetermined. From sample to result, the total technical time per run is estimated to be under five minutes. Flow cytometry for intracellular cytokine staining CMV-specific IFN-γ-producing CD8 + and CD4 + T lymphocytes were enumerated using FC-ICS (BD Fastimmune, BD Biosciences, San Jose, CA, USA) as previously described [28-33]. Whole blood (0.5 mL) collected in sodium heparin tubes was incubated with two peptide libraries (15 amino acids each) spanning the complete sequences of CMV proteins pp65 and IE-1 (1 µg/peptide/mL; JPT Peptide Technologies GmbH, Berlin, Germany). The incubation included co-stimulatory antibodies (anti-CD28 and anti-CD49d; 1 µg/mL each). Control samples were prepared by incubating an equal volume of heparinized blood with dimethyl sulfoxide in phosphate-buffered saline. Samples were stimulated for six hours at 37°C, followed by a two-hour incubation with Brefeldin A (10 µg/mL). After red blood cell lysis, samples were stored at -80°C. Upon thawing at 37°C for analysis, cells were stained for 30 minutes at room temperature with a panel of antibodies: anti-CD3-APC, anti-CD4-APC-H7, anti-CD8-PerCPCy5.5, and anti-IFNγ-FITC. The stained cells were washed and fixed in 200 µL of 1% paraformaldehyde in phosphate-buffered saline. Flow cytometric analysis was performed within two hours using a BD LSRFortessa™ Cell Analyzer, and data were analyzed using FlowJo Software (BD Biosciences Immunocytometry Systems). The frequencies of CMV-specific IFN-γ-producing CD8+ and CD4 + T-cells were calculated as the percentage of IFN-γ-producing cells within their respective T-cell populations after background subtraction. A sample was classified as Positive if it showed any detectable CMV-specific IFN-γ production by either CD8 +, CD4 + T cells, or both after background subtraction. Monitoring of CMV DNAemia Quantitation of CMV DNA load in plasma was performed by the Alinity m CMV assay (Abbott Molecular Inc., Des Plaines, IL, USA) [36]. CMV serology CMV serological testing was performed using the DiaSorin Liaison CMV IgG assay (DiaSorin, Saluggia, Italy), according to the manufacturer’s recommendations. Statistical analysis We assessed the concordance (overall percentage agreement, positive percentage agreement [PPA], and negative percentage agreement [NPA]) between the two methods using a 2x2 contingency table to calculate the following metrics: true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN). For analysis purposes, patients were categorized as displaying positive FC-ICS responses if they had detectable CMV-specific CD4 +, CD8 + T cells, or both. PPA was calculated as (2 × TP) / (VIDAS positives + ICS positives), where VIDAS positives = TP + FN and ICS positives = TP + FP. Similarly, NPA was computed as (2 × TN) / (VIDAS negatives + ICS negatives), where VIDAS negatives = TN + FP and ICS negatives = TN + FN. The overall percentage agreement was determined as (TP + TN) / (TP + TN + FP + FN). Kappa statistics was also used to assess the degree of agreement between results returned by the comparison methods. The correlation between CMV-specific IFN-γ-producing CD4 + and CD8 + T-cell frequencies and antigen-specific IFN-γ levels as measured by the VIDAS® CMV IGRA was assessed using Spearman’s rank correlation coefficient (rho). For this analysis, VIDAS IGRA IFN-γ values > 8.0 IU/ml were assigned a value of 10 IU/ml, while values < 0.08 IU/ml were assigned a value of 0 IU/ml to enable statistical analysis. The strength and significance of correlations were evaluated, with P -values < 0.05 considered statistically significant. Differences in median values between groups were evaluated using the Kruskal-Wallis test, followed by pairwise comparisons with Bonferroni correction for multiple testing. Statistical significance was set at P < 0.05. All analyses were performed using GraphPad Prism 9.0.2 software. Of the 147 specimens, 134 were collected from transplant recipients (76 from Allo-HCT and 58 from KTR); of these, 33 samples were received at baseline, before transplantation. First specimens from transplant recipients were obtained at a median of 16 days after transplantation (IQR 3-42). In all, 22/34 HP had more than one specimen collected throughout the follow-up (median 2.5 samples, IQR 2-3.8). This was also the case for 18/33 of KTR (median 2 samples, IQR 2-3). In addition, we collected 11 specimens from unique HC. Due to the nature of the study, no stringent timing criteria for sampling relative to the time of transplantation were established in HP and KTR patients. Sampling times across HP and KTR participants with more than one specimen available for testing by the VIDAS® CMV IGRA, as well as the time of first detection of CMV DNAemia in these patients, are depicted in Figure 1. Qualitative agreement between CMV-specific T-cell assays Of the 147 specimens, 96 tested positive, 24 negative, 25 indeterminate, and 2 invalid (NIL positive) by the VIDAS® CMV IGRA (Table 1). All specimens yielding indeterminate results were collected from Allo-HCT (n=15) or KTR (n=10) and had absolute lymphocyte counts (ALC) < 1000/mm 3 (median 105; range, 0-280). FC-ICS results were available from 137 samples. The remaining 10 samples (seven from HP and three from KTR) could not be analyzed due to their low cell counts (ALC ≤ 500/ mm 3 ) . FC-ICS results are also displayed in Table 1. In all, 107 specimens tested positive (detectable IFN-γ-producing CD8 +, CD4 + T cells, or both), and the remaining 30 returned negative results. Of note, out of the 16 specimens with an indeterminate result by VIDAS® CMV IGRA that were assessed by FC-ICS, 12 returned positive and 4 negative results. When only considering specimens yielding interpretable results by the VIDAS® CMV IGRA (n=101)—excluding those indeterminate or invalid—that were tested by FC-ICS, there were 27 discrepancies (6, 9, and 12 specimens from HC, HP, and KTR, respectively) (Table 2). Of these, 14 tested VIDAS® CMV IGRA-positive/FC-ICS-negative and 12 VIDAS® CMV IGRA-negative/FC-ICS-positive. This pattern of discrepant results was rather comparable across study groups. Accordingly, as shown in Table 3, the overall agreement between immunoassays was 78%, PPA was 86%, NPA 48%, and the Kappa coefficient 0.34. The level of agreement was best for HP (86% overall agreement and Kappa index of 0.52) and worst for HC (55% overall agreement and Kappa index of 0.13). Concordance between CMV serology and qualitative CMV-specific T-cell immunity assay results We next evaluated the ability of the VIDAS® CMV IGRA and FC-ICS T-cell assay to identify CMV-infected or CMV-uninfected participants, as categorized by a positive or negative CMV IgG result, respectively. To conduct this analysis, we selected 44 participants: 11 HC and 33 transplant patients with whole blood collected before transplantation (Allo-HCT, n=23; KTR, n=10). FC-ICS results were not available from 10 of these patients. As shown in Table 4, in this sub-cohort, 35 patients tested CMV IgG-positive and 9 CMV IgG-negative. Overall, the VIDAS® CMV IGRA returned positive results in 30/35 (85.7%) CMV IgG-positive participants, whereas the FC-ICS did so in 27/34 (79%). Of note, two and none of the CMV-seropositive patients tested negative by FC-ICS and VIDAS® CMV IGRA, respectively. Moreover, for both CMV T-cell assays, HP exhibited the highest rate of discordant results. As for CMV IgG-negative participants, overall, the VIDAS® CMV IGRA and FC-ICS assay yielded concordant negative results in 4/9 (44%) and 6/9 (67%) participants, respectively. HC more frequently returned discordant results with both assays. Overall, except for HC, PPA was higher than NPA (Table 5). The overall qualitative agreement, as assessed by Kappa statistics, was fair for both methods (Kappa index 0.38 for VIDAS® CMV IGRA and 0.40 for FC-ICS). Quantitative correlation between results returned by the CMV-specific T-cell assays The overall correlation between IFN-γ concentrations (IU/ml) measured by the VIDAS® CMV IGRA and CMV-specific IFN-γ-producing T-cell frequencies were statistically significant and fair, with Spearman’s rho coefficients of 0.27 (95% CI: 0.10-0.42) for CD4 + and 0.33 (95% CI: 0.16-0.47) for CD8 + T cells ( P =0.001) (Figure 2). Similar correlation coefficients were observed after excluding indeterminate VIDAS® CMV IGRA results (rho=0.29 for CD4 + T cells and rho=0.37 for CD8 + T cells). In HP, the correlation was stronger, with rho coefficients of 0.48 (95% CI: 0.27-0.64) for CD4 + and 0.49 (95% CI: 0.28-0.65) for CD8 + T cells. In contrast, null or weak correlations for CD4 + T cells (rho, 0.009) and CD8 + T cells (Rho, 0.18) ( P > 0.05), respectively, were observed in KTR. The small number of specimens from HC precluded a meaningful analysis. Quantitative IFN-γ levels measured by the VIDAS® CMV IGRA were significantly higher in FC-ICS-positive specimens, particularly in HP ( P < 0.001), compared with that in FC-ICS-negative specimens (Figure 3A). Similarly, the percentages of CMV-specific IFN-γ-producing CD4 + and CD8 + T cells were significantly higher in VIDAS® CMV IGRA-positive samples compared with negative ones; this difference was most pronounced in HP ( P < 0.001; Figures 3B and 3C). Kinetics of CMV-specific T cells as assessed by comparison assays We next investigated how trajectories described by quantitative results returned by the VIDAS® CMV IGRA (in IFN-γ IU/ml) and FC-ICS assay (frequencies of CD8 + and CD4 + T cells) did compare. To perform this analysis, we selected 17 patients (10 allo-HCT and 7 KTR) from whom three or more whole-blood specimens were collected. We noticed that seven patients (four HP and three KTR) exhibited rather comparable trajectories over time (either rising, falling, or constant), whereas the remaining did not. Congruent trajectories were more frequently observed among those failing to mount detectable T-cell responses. Representative examples are depicted in Figure 4 (i.e., SCT02 and SCT022 for congruent trajectories and the remaining panels for discordant trends). CMV-specific T-cell assays for predicting the occurrence of CMV DNAemia To assess the ability of T-cell assays to predict the development of CMV DNAemia, we included 32 patients tested by the VIDAS® CMV IGRA (13 Allo-HCT and 19 KTR) with specimens collected within the first 20 days after transplantation (median, 12 days; IQR, 6-15). Patients testing positive by VIDAS® CMV IGRA had a lower incidence of CMV DNAemia within the next 30 days (1/14, a KTR patient) than patients testing negative or indeterminate (4/18; three HP and one KTR), although the difference did not reach statistical significance ( P =0.27). Data on FC-ICS were available from 25/32 patients. CMV DNAemia occurred in 3/20 patients (two HP and one KTR) testing positive and in 2/5 (one HP and one KTR) testing negative ( P =0.26).

In this study we compared the performance of the VIDAS® CMV IGRA with that of a laboratory-developed FC-ICS enumerating CMV pp65/IE-1 IFN-γ-producing CD8 + and CD4 + T cells in a mixed population, encompassing HC, HP, mostly Allo-HCT, and KTR. The most relevant findings of this study are summarized as follows. Firstly, a slightly higher rate of positive results was obtained by the FC-ICS assay compared with the VIDAS® CMV IGRA (96/147 vs. 107/137 samples). The fact that most specimens yielding VIDAS® CMV IGRA indeterminate results (15% of specimens considering the entire panel) tested positive by FC-ICS (12 out of 16 analyzed) partly accounts for this difference. Specimens yielding VIDAS® CMV IGRA indeterminate results were collected from transplant recipients who systematically had low ALC. Qualitative discrepancies across results provided by comparison assays, after excluding indeterminate and invalid samples, were evenly distributed across VIDAS® CMV IGRA-positive/FC-ICS-negative and VIDAS® CMV IGRA-negative/FC-ICS-positive specimens. This pattern of discrepancy was comparable across study groups. The agreement between qualitative results returned by the comparison assays was overall fair (Kappa, 0.34) and moderate (Kappa, 0.52) for HP. The relatively low NPA across the assays minimized the strength of the agreement. Secondly, despite the different nature of the stimulating antigen between the assays (CMV lysate in the VIDAS® CMV IGRA and overlapping 15-mer pp65/IE-1 peptides in the FC-ICS assay), the correlation between IFN-γ concentrations (IU/ml) measured by the VIDAS® CMV IGRA and CMV-specific IFN-γ-producing T-cell frequencies enumerated by FC-ICS were fair for both CD8 + and CD4 + T cells. Of note, the level of correlation was higher (moderate) for HP. Surprisingly, the correlation was weak, at best, in KTR. Unexpectedly, trends and trajectories of IFN-γ concentrations over time, as determined by the VIDAS® CMV IGRA in transplant recipients, were frequently dissimilar to that of both CMV-specific CD8 + and CD4 + T-cell frequencies enumerated by FC-ICS. Taken collectively, our data revealed differences in the qualitative and quantitative performances across comparative assays; although not gauged in the current study, these may have clinical relevance. Notable differences in the performance of another commercialized IGRA test, the QuantiFERON-CMV assay (QF), which measures IFN-γ released by CD8 + T cells activated by small-size peptides mapping within different CMV proteins (including pp65 and IE1), and the in-house FC-ICS assay were previously reported by our group in two transplant recipient cohorts [21,26]. In effect, Clari et al . [21], in a series of allo-HCT, found that the rate of concordant qualitative results by both methods was 68.8%, and the rate of positive results returned by QF was lower than that of FC-ICS; QF yielded 18% of indeterminate results. The results obtained by the QF correlated significantly with FC-ICS (rho, 0.60), a figure close to that reported herein for HP (rho, 0.52). In turn, Fernández-Ruiz et al. [26] also reported a lower rate of detectable CMV-specific T-cell responses by QF than by the FC-ICS in a cohort of KTR; a moderate correlation was found between IU/ml measured by QF and the frequencies of CMV-specific CD8 + T cells enumerated by the FC-ICS assay (rho, 0.44). These data clearly contrast with those reported herein for KTR, suggesting a poor correlation between assays. This could be partly explained by the different timing of sampling after KTR across the studies (three and four months in [26] and a median of 16 days in the current study). Detection of CMV IgG by ELISA or chemiluminescence immunoassay (CLIA) continues to be the standard approach to identify CMV-infected transplant recipients (and donors in allo-HCT). Thus, patients can be stratified according to their risk of developing clinically significant infection following transplantation, and the most appropriate preventative strategy can be applied (prophylaxis or preemptive antiviral therapy) [10,12]. This is despite the well-known fact that CMV IgG and CMV-CMI assays return discordant results rather often [37,38]. Here, we found that both methods performed rather similarly in identifying CMV-seropositive and CMV-seronegative individuals; in fact, the overall qualitative agreement as assessed by Kappa statistics was fair for both methods (Kappa index of 0.38 for VIDAS® CMV IGRA and 0.40 for FC-ICS). Nevertheless, these figures are lower than those reported by others for QF and the T-Track-CMV assay [18,37]. The different abilities of CMV-specific T-cell assays to predict the occurrence of CMV DNAemia have been demonstrated in several studies. For example, Gigla et al . [18] reported that the T-Track ELISpot assay performed substantially better than QF for this purpose in a cohort of KTR. Gabanti et al . [23] showed that, although QF and an in-house FC-ICS assay following lymphocyte stimulation by autologous CMV-infected dendritic cells showed comparable sensitivities for predicting CMV DNAemia in KTR, the former had a lower specificity. Gigla et al. [24] compared two commercially available IFN-γ ELISpot assays (T-Track CMV and T-SPOT.CMV) and QF in 56 liver transplant recipients at the end of antiviral prophylaxis and one month thereafter. While the results of both ELISpot assays were similar in predicting protection against CMV DNAemia (≥ 500 copies/mL), they performed better than QF. Likewise, in a study conducted by Callens et al . [27], the T-SPOT.CMV and QF performed at day +28 and +100 in allo-HCT recipients yielded different predictive values for the occurrence of CMV DNAemia. In this context, the current study did not aim to evaluate the predictive ability of the compared assays for the occurrence of CMV DNAemia. In fact, the limited size of our series, the small number of transplant patients developing CMV DNAemia, and the variability of testing times across participants precluded addressing this issue meaningfully. Despite this, our data seemed to suggest that the VIDAS® CMV IGRA may perform comparably to FC-ICS in assessing the risk of CMV DNAemia in transplant recipients. Our study is limited by its relatively low sample size, which did not allow statistical inference on the clinical performance of the compared assays in predicting CMV DNAemia. Likewise, the FC-ICS method employed has not been validated externally. Moreover, qualitative discrepancies across the comparison assays were not resolved due to the absence of a reference assay. In summary, our study suggests that VIDAS® CMV IGRA provides a qualitative assessment of CMV-specific responses comparable to FC-ICS. However, disagreement between the results provided across both tests was not uncommon. Nevertheless, the data presented further reinforced the idea that the correlation between quantitative values returned by different methods assessing CMV-CMI may be far from optimal. The VIDAS® CMV IGRA is relatively simple to perform and less time-consuming and labor-intensive than FC-ICS. In addition, standardization of all the steps that could lead to improved robustness across results obtained at different laboratories. Moreover, MIT values may provide additional information as the level of antigen-independent T-cell exhaustion [39]. Studies addressing the value of the VIDAS® CMV IGRA separately in Allo-HCT and KTR to predict the occurrence of CMV DNAemia are underway. ACKNOWLEDGMENTS Eliseo Albert (Juan Rodés Contract; JR20/00011) holds a contract funded by the Carlos III Health Institute (co-financed by the European Regional Development Fund, ERDF/FEDER). FUNDING This research received private financial support from Biomerieux ( Marcy l’Etoile, France). CONFLICTS OF INTEREST DN received honoraria for conferences sponsored by Biomerieux ( Marcy l’Etoile, France). AUTHOR CONTRIBUTIONS EG, EA, EC, MM, AP, JLP, IS, JLG, and CS, Methodology and Data curation. EG and DN, Data analysis and manuscript drafting. All authors approved the final version of the manuscript. DATA AVAILABILITY STATEMENT The datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.

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Boada-Pérez M, Berastegui C, Erro M, Ussetti P, Crespo E, Donadeu L, et al. Discordance between humoral and cellular immune responses to cytomegalovirus infection in CMV seropositive patients awaiting lung transplantation. Front Immunol. 2025;15:1445553. 39. Haem Rahimi M, Daniel S, Venet F, Bidar F, Cour M, Ducrot S, et al. Fully automated interferon-γ release assay to monitor antigen-independent T cell functionality: A proof of concept study in septic shock. Cytokine . 2023;169:156263. FIGURE LEGENDS Figure 1. Time of specimen collection and results returned by the VIDAS® CMV IGRA in transplant recipients with three or more whole-blood specimens collected. SCT refers to allogeneic hematopoietic stem cell transplant recipients, KT to Kidney transplant recipients, and CMV to the time of first detection of CMV DNAemia. Pos, positive result; neg, negative result; ind, indeterminate result. Figure 2 . Correlation between quantitative results provided by the VIDAS® CMV IGRA (in IU/ml) and the frequencies of CMV-specific IFNγ-producing CD8 + and CD4 + T cells, as enumerated by flow cytometry for intracellular staining. Spearman’s Rank Rho values are shown. Figure 3. Quantitative results returned by the CMV-specific T-cell assays in specimens categorized qualitatively by either assay. (A) Quantitative results returned by the VIDAS® CMV IGRA (in IU/ml) in specimens testing positive (detectable CMV-specific IFNγ-producing CD8 +, CD4 + T cells, or both) or negative by the Flow cytometry for intracellular staining (FC-ICS) assay. (B) Frequencies of CMV-specific IFNγ-producing CD8 + (B) and CD4 + (C) T cells in specimens testing positive, negative, or indeterminate by the VIDAS® CMV IGRA. HC, Healthy controls; HP, hematological patients; FC-ICS, Flow cytometry for intracellular staining; KTR, Kidney transplant recipients. Figure 4. Representative examples of trajectories of CMV-specific IFNγ-producing T-cell responses as assessed by the VIDAS® CMV IGRA and the Flow cytometry for intracellular staining method (FC-ICS). SCT refers to allogeneic hematopoietic stem cell transplant recipients, KT to Kidney transplant recipients. Information & Authors Information Version history Peer review timeline Published Journal of Medical Virology Version of Record15 Apr 2025Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection

Authors Metrics & Citations Metrics Article Usage 362views 231downloads Citations Download citation Estela Giménez, Eliseo Albert, Ester Colomer, et al. Evaluation of the VIDAS® Cytomegalovirus (CMV)-IGRA for Assessing CMV-Specific T-Cell Responses in Immunocompetent and Immunosuppressed Individuals. Authorea. 12 March 2025. DOI: https://doi.org/10.22541/au.174176116.64618536/v1 DOI: https://doi.org/10.22541/au.174176116.64618536/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.