Cytomegalovirus (CMV), a member of the human herpesvirus family, remains an important infectious complication in individuals with compromised immune functions, including solid organ transplant (SOT) recipients. Primary infection or reactivation of a latent infection usually occurs within six months of SOT (Fishman et al., 2007; Razonable and Humar, 2019). It has direct effects (CMV syndrome and tissue invasive diseases) and indirect effects (allograft dysfunction, rejection, and opportunistic infections) (Fishman et al., 2007; Razonable and Humar, 2019). CMV prevention is therefore an important measure to improve patient and graft survival.

The risk of CMV infection in SOT recipients depends on various factors, such as the organ transplanted and immunosuppressive therapy, but donor and recipient serological status are the most well-known risks. The 2 main strategies currently used to prevent CMV infection in SOT recipients are antiviral prophylaxis and preemptive management (Razonable and Humar, 2019). Traditionally, antiviral prophylaxis has been used as a preferable option in CMV-seronegative recipients receiving organs from CMV-seropositive donors (D+/R-), who have the highest risk of primary CMV infection (Humar et al., 2010). Either an antiviral prophylaxis or a preemptive approach is recommended in CMV-seropositive recipients (R+), who have an intermediate risk of CMV infection after transplantation (Razonable and Humar, 2019).

In Thailand, the kidney is the most common organ transplanted in clinical practice, with most kidney transplant (KT) recipients (99%) demonstrating CMV seropositivity (Watcharananan et al., 2012; Chiasakul et al., 2015). Previous studies reported that one-quarter of Thai patients developed CMV replication within 6 months of transplantation. Of those, antithymocyte globulin (ATG) therapy was strongly associated with an increased risk of CMV reactivation (Watcharananan et al., 2012; Chiasakul et al., 2015). Thus, better risk stratification is needed for seropositive KT recipients who do not receive ATG induction therapy in order to guide the choice of preventative measures.

Over the past decade, specific cell-mediated immunity has been studied to help stratify the risk of CMV infection in this population. The methods include commercially available tests—CMV enzyme-linked immunospot (ELISPOT) and QuantiFERON-CMV (QF-CMV) and nonstandardized laboratory methods, such as intracellular cytokine staining (ICS) (Yong et al., 2018). Of the commercial methods, ELISPOT is much more sensitive than the QF-CMV test (Yong et al., 2018). Nevertheless, it is costly and more labor intensive since a peripheral blood mononuclear cell isolation is needed. In addition, a lack of defined cutoff values for positivity is an issue of concern. By contrast, QF-CMV is easily performed in many diagnostic laboratories with a shorter turnaround time. Other studies have demonstrated the benefits of using a pretransplant QF-CMV to help stratify the risk of CMV infection in patients undergoing a kidney transplant (KT) (Lochmanova et al., 2010; Cantisan et al., 2013; Abate et al., 2013). Cantisan and colleagues investigated the use of pretransplant QF-CMV with 55 kidney and heart transplant recipients (Cantisan et al., 2013). The seropositive recipients who had a negative pretransplant QF-CMV assay were more likely to develop a CMV infection after their transplant than the nonreactive group (50% versus 13%, respectively). Nevertheless, recent published studies from Asia have provided conflicting results (Kwon et al., 2017; Lee et al., 2017). Hence, we performed this study to evaluate the benefits of using a QF-CMV assay to predict CMV infections in seropositive KT recipients.

From July 2017 to May 2019, 67 adult patients underwent KTs at our institution. Sixty-five (97%) had CMV seropositivity at the time of transplantation. Ten patients were excluded from the study (four received ATG as an induction therapy; one received ganciclovir as a CMV prophylaxis; one died within one month of the transplant; one had a postponed KT; and three were excluded because of a shortage of QF-CMV test kits). The data related to the remaining 55 patients were analyzed.

The mean (SD) age of the patients was 46.5 (10.2) years, and the majority were men (32 patients; 58.2%). Diabetes was diagnosed in 7 patients (12.7%). The most common reason for the KT was end-stage renal disease of unknown etiology (32 patients; 58.2%), followed by end-stage glomerular disease (18 patients; 32.7%), autosomal dominant polycystic kidney disease (3 patients; 5.5%), and tubulointerstitial disease (1 patient; 1.8%). Two-thirds (69.1%) underwent a deceased-donor kidney transplantation (DDKT). Fifty-two patients (94.5%) received kidneys from CMV-seropositive donors. The mean (SD) age of the donors was 39 (13.4) years. All but one received basiliximab as an induction therapy. All patients received prednisolone, tacrolimus, or cyclosporin, plus mycophenolate mofetil for maintenance immunosuppression. Immunosuppressive agents were changed from calcineurin inhibitors to everolimus in only 2 patients. The mean dosage of prednisolone at 1^st and 6^th month posttransplant was 17 and 2.2 mg/day, respectively.

During the 6-month follow-up, CMV viremia occurred in 29 patients (52.7%); 14 of those patients (25.5%) had significant viremia, but none had CMV syndrome or CMV end-organ diseases. All but one developed CMV viremia within 3 months, with a mean (SD) onset of 65 (30) days. The median (range) CMV viral load for all CMV viremia and significant CMV viremia were 635 (157–81,363) and 1,800 (783–78,090) IU/ml, respectively. Infectious complications occurred in 29 patients (52.73%), of which urinary tract infections were the most common (17 patients; 58.62%), followed by BK polyomavirus infections (5 patients; 17.2%) and herpes simplex virus infections (3 patients; 10.3%). The infection incidence of the CMV-viremia and no-viremia groups did not differ significantly. A comparison of the baseline characteristics of the patients who did and did not develop CMV viremia is demonstrated in Table 1. The prednisolone and mycophenolate mofetil doses were slightly higher for the CMV viremia group; however, the tacrolimus trough level was lower for the CMV viremia group. A univariate analysis revealed that DDKT and increased donor age were the only factors associated with CMV viremia.

Our study could not demonstrate the value of pretransplant QF-CMV status as a reliable predictor for CMV infections in KT recipients with CMV seropositivity who were classified as intermediate-risk group for CMV infection. Although data from earlier studies support the benefits of obtaining a pretransplant QF-CMV in the high-risk population, especially for patients with a CMV serological mismatch (D+R-) (Kumar et al., 2009; Manuel et al., 2013), the benefits are still unclear for the intermediate-risk group. Our results support a previous study performed by Lee and colleagues (Lee et al., 2017), who compared the efficacies of QF-CMV and ELISPOT assays in KT recipients. They reported a lack of association between pretransplant QF-CMV reactivity and the incidence of CMV viremia after KTs in the intermediate-risk population. In contrast, they found that a CMV-ELISPOT assay performed 1-month posttransplant provided more reactive result and potentially be used as a predictor for CMV replication in this group. This finding was also confirmed by a recent meta-analysis undertaken by Ruan et al. in 2019 (Ruan et al., 2019), who reported a lower sensitivity with QF-CMV (38%) than CMV-ELISPOT (73%–84%).

There are 3 possible explanations for the superior performance of ELISPOT relative to QF-CMV. Firstly, even though both tests use the same principle of IFN-gamma release assay, the QF-CMV assay relies on the detection of interferon-gamma (IFN-γ) in whole blood, whereas ELISPOT directly detects the virus-specific T lymphocytes that produce IFN-γ, which demonstrates functioning T-lymphocytes in vivo. This hypothesis correlates with intracellular cytokine staining which also has the additional ability to detect multiple cytokines and cell-surface markers (Fernandez-Ruiz et al., 2019; Rogers et al., 2020). Secondly, QF-CMV can only detect the cytotoxic T-cell (CD8) response, whereas ELISPOT can detect the response from both the helper (CD4) and CD8. (Waldman and Knight, 1996; Snyder et al., 2016). Lastly, QF-CMV requires the ex vivo stimulation of CD8 T-cells with human leukocyte antigen (HLA)-restricted CMV peptides, while ELISPOT is usually performed with peptide library spanning the whole antigens pp65 and IE-1. (Giulieri and Manuel, 2011). IE-1 could be crucial in CMV seropositive patients (Lopez-Oliva et al., 2014) while only 4 peptides presented by specific HLA alleles are included in QF-CMV (Walker et al., 2007). Lack of these HLA alleles can also cause false-negative QF-CMV tests.

The data from another study showed that patients with indeterminate QF-CMV results had an increased incidence of CMV disease or serious infectious complications (Tarasewicz et al., 2016). These findings were explained by patients’ high net state of immunosuppression since patients with an indeterminate result could not stimulate the mitogen. However, our study did not support this finding. There were indeterminate results for the QF-CMV assay (1 month after the KTs) in only 9% of cases, which was much lower than previously reported in the literature (Kumar et al., 2009). This finding comes as no surprise since our patients were not highly immunosuppressive, having relatively low FK levels (Lochmanova et al., 2010; Abate et al., 2013; Kwon et al., 2017). This might explain the negative association of the indeterminate result.

The appropriate timeline and cutoff value for QF-CMV are not yet clear. Some studies have reported a better utility with cell-mediated immune monitoring 1 month after a KT, which is the period of maximal immunosuppressant levels (Lee et al., 2017). However, our study could not demonstrate any benefit at this timepoint. Regarding the appropriate cutoff value, we hypothesized that 0.2 IU/ml might be suitable only for CMV-seronegative patients; a higher cutoff value might be required for seropositive recipients exposed to a CMV infection before their KT. Some studies have employed a higher cutoff value to predict CMV disease in transplant recipients (Gliga et al., 2018; Krawczyk et al., 2018). For example, data from bone marrow transplant recipients indicated that there was a need for a higher QF-CMV cutoff value (> 8.9 IU/ml) to stratify the risk of CMV disease in that population (Tarasewicz et al., 2016). Further analysis using QF-CMV > 10 IU/ml as the cutoff value revealed that patients with a reactive pretransplant QF-CMV (15.4%) were less likely to develop significant CMV viremia than those in the nonreactive group (31%). However, the difference did not reach statistical significance. Additional investigation with a larger sample size is required to determine a better cutoff value for intermediate-risk KT recipients.

Interestingly, although a single-timepoint, pretransplant, QF-CMV assay or a 1-month, post-KT, QF-CMV assay might not predict CMV viremia in KT recipients, further analysis found that the incidence of CMV viremia tended to be lower in those patients remaining reactive 1 month after their KTs. These findings suggest that dynamic changes in QF-CMV assays could be more helpful for predicting the incidence of CMV viremia in seropositive KT recipients. As a result, multiple time points of QF-CMV and larger sample sizes are needed to characterize the utility of dynamic changes in the QF-CMV levels to predict CMV viremia after a transplant.

The strength of our study is the homogeneity of the study population in terms of the recipients’ serostatuses and degrees of immunosuppression. Our main limitation is the small sample size. Moreover, the relatively low frequency of CMV QNAT monitoring in this study may have resulted in the missing of some episodes of CMV viremia with spontaneous viral clearance, thereby causing an outcome measurement bias.

In conclusion, our study could not confirm that single-timepoint pretransplant QF-CMV assay or a 1-month post-KT QF-CMV assay are a useful marker for predicting posttransplant CMV viremia in seropositive KT patients. Further studies with larger sample size are needed to confirm these findings. Multiple time points to evaluate the changes of QF-CMV level with a defined cutoff value may be useful to predict CMV viremia.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Siriraj Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

KP: investigation, formal analysis, writing-original draft; MC: writing-review and editing, supervision; AV: resources, writing-review and editing; WK: resources, writing-review and editing; PS: resources, writing-review and editing; PP: conceptualization, methodology, writing-review and editing, visualization. All authors contributed to the article and approved the submitted version.

This study was supported by a research development grant from Siriraj Hospital (Grant number R016032034). Although the test kits were provided by the Qiagen company, it played no role in the study design, data collection, study analysis, or manuscript writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.