Cytomegalovirus Cell-mediated Immunity Assays in Pediatric Transplantation

Abstract

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in pediatric transplantation. However, currently utilized CMV prevention paradigms have limitations, leading to research aimed at novel strategies for mitigation of CMV infection. Cell-mediated immunity (CMI) is crucial in controlling CMV infection and the use of CMV-specific CMI assays to guide prevention and treatment of CMV infection in both solid organ transplant and hematopoietic cell transplant recipients shows great promise. In this article, we review the immune response to CMV infection to highlight the rationale for CMI assays, describe available commercial assays and strategies for their use, and summarize relevant literature regarding the use of CMI assays in transplant recipients.

INTRODUCTION

Cytomegalovirus (CMV) is one of the most common infectious complications after transplantation [1, 2]. CMV-related morbidity and mortality are primarily due to tissue invasive disease as well as indirect effects such as graft rejection or loss, higher rates of bacterial and fungal infections, or cardiovascular disease [3, 4]. Several CMV prevention strategies—prophylaxis, preemptive monitoring, or surveillance after prophylaxis—are commonly employed. For universal prophylaxis, a CMV-active antiviral is given for several months posttransplant to intermediate- or high-risk patients [1]. The preemptive approach consists of serial blood polymerase chain reaction (PCR) monitoring to identify presymptomatic CMV infection, with antiviral treatment initiated only if CMV DNA is detected. Each of these strategies has limitations, including late CMV infection [5], medication side effects [6], and increased healthcare costs [7] with universal prophylaxis and the logistical difficulties of consistent screening, lack of a standardized threshold to trigger therapy, and potential indirect effects of DNAemia with preemptive monitoring [8].

Current paradigms stratify CMV infection risk based on type of transplant and donor/recipient serologic testing. Given the limitations of current strategies, significant potential exists for use of CMV-specific cell-mediated immunity (CMI) assays to guide prevention and treatment of CMV infection in both solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients [9–14]. In this article, we review the immune response to CMV infection to highlight the rationale for CMI assays; describe available commercial assays and strategies for their use; and summarize relevant literature regarding the use of CMI assays in transplant recipients.

IMMUNE RESPONSE TO CMV INFECTION

The innate immune system plays an important role in the initial recognition of primary CMV infection. Viral detection by intracellular toll-like receptors leads to a potent immune response, including the release of type I interferons (IFNs) which restrict viral replication and contribute to natural killer cell activation [15]. Natural killer cells limit early CMV infection by lysing infected cells and secreting pro-inflammatory cytokines [16, 17]. These early immune recognition steps activate the adaptive immune response, which is the dominant means by which CMV infection is controlled, and reactivation is prevented [18].

CMI is crucial in controlling CMV infection. In fact, up to 10% of all peripheral blood T cells will recognize CMV [19]. CD8+ T cells act to clear primary infection and prevent CMV reactivation [20]. Following T cell receptor recognition of CMV peptides presented via major histocompatibility complexes (MHC), CD8+ T lymphocytes exert a direct cytolytic effect on infected cells and secrete IFN-γ and other cytokines. The presence of CMV-specific CD8+ T cells correlates with protection against CMV infection and disease [21]. CD4 + T lymphocytes also play an increasingly recognized role in the immune response against CMV infection by releasing chemokines that mediate the recruitment and expansion of CD8 + T cells [22]. Reconstitution of CD4 + T cell immunity has been shown to be critical to the control of CMV reactivation in HCT patients independent of CD8 + T cell recovery posttransplant [23]. There is growing evidence that CD4+ cells also play a role in the direct killing of CMV-infected cells via cytotoxic pathways [22].

The humoral immune response plays an important but less fully elucidated role in controlling CMV infection and preventing reactivation. Clinical studies and a recently developed animal model have indicated that strain-specific antibody may play a role in protection from CMV infection in the setting of transplantation [24–26].

CMV CMI ASSAYS

Several CMV CMI assays that measure IFN-γ expression by CD4+ and/or CD8+ cells after stimulation with CMV antigens are commercially available [20, 21]. These assays typically use overlapping peptide pools from either immediate early protein-1 (IE-1), phosphoprotein 65 (pp65), or both (Table 1). Though these are two of the most immunodominant CMV antigens which CD8+ T cells recognize [18], the human immune response to CMV is not limited to these proteins and CMI assays do not identify all patients with detectable CMV-specific immunity. Each assay has a positive control consisting of a mitogen such as staphylococcal enterotoxin B or phytohemagglutinin. No commercial CMV CMI assay has been approved by the Food and Drug Administration in the United States, though multiple assays are licensed for use in Europe. Available assays are briefly summarized here and in Table 1.

Table 1.

Commercial Assays for Detection of CMV CMI

CMV–CMI Assay Type	Commercial Assay(s)	Patient Sample	Required Quantity	Antigen(s)	Required Equipment	Results and Interpretation	Notes
ELISA	Quantiferon-CMV (Qiagen Inc, Hilden, Germany) [27]	Whole blood	3 mL	Peptide poola	ELISA microplate reader	Reports IFN-γ production in response to exposure to CMV antigen as a surrogate for CD8+ response Test considered reactive if IFN-γ response in CMV tube is significantly increased over IFN-γ response in nil antigen tube (≥0.20 IU/mL and ≥25% of nil antigen value)	CE-marked
ELISpot	T.Spot.CMV assay (Oxford Immunotec International, Oxfordshire, England) [28] T-Track CMV assay (Mikrogen GmBH/Lophius Biosciences GmBH, Neuried, Germany) [29]	Whole blood	12 mLb 7.5 mL	pp65 and IE-1c	ELISpot reader	Measures both CD4+ and CD8+ responses Test reports number of “spot-forming units,” regions in test wells that produce IFN-γ Positive cutoff has not been defined, but use of cutoffs ranging from 20 to 40 spot-forming units have been reported [30, 31, 32]	Both assays are CE-marked
Intracellular cytokine staining	CMV inSIGHT™ T Cell Immunity Panel (Eurofins Viracor, Lenexa, Kansas, USA) [33]	Whole blood	10 mLd	CMV peptidese	Flow cytometer	Reports both CD4+ and CD8+ responses distinctly as a percentage of cells producing IFN-γ A test is considered positive if ≥0.20% of T cells express IFN-γ	Not FDA-approved in United States f
MHC multimer staining	CMV CD8 T Cell Immune Competence (Mayo Clinic Laboratories, Rochester, Minnesota, USA) [34]	Whole blood	20 mL	CMV peptidese	Flow cytometer	Reports absolute CD3+ and CD8+ T cell counts, as well as CMV-specific CD8 + T cells Total number of CMV-specific CD8+ cells that express IFN-γ and cytotoxic cell surface markers is also reported Reference ranges provided to allow for interpretation of results	Assay is limited to a subset of Class I HLA alleles Not FDA-approved in United States f

Abbreviations: CE, Conformité Européenne; CMI, cell-mediated immunity; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunospot; FDA, Food and Drug Administration; HLA, human leukocyte antigen; IFN-γ, interferon gamma; MHC, major histocompatibility complex.

^aCMV peptide pool from IE-1, IE-2, pp65, pp50, and gB; designed to target CD8+ T cells, including HLA Class I haplotypes covering >98% of human population.

^bFor children ≥10 years old: 12 mL of whole blood; for children ≥2 to <10 years old: 6 mL of whole blood; and for children <2 years old: 2 mL of whole blood.

^cThe same peptides are utilized for both assays with readout of spot forming units reported independently for each protein.

^dMinimum volume in children is 4 mL.

^eAntigen content not otherwise specified.

^fThough not FDA-approved, the assay is commercially available.

Enzyme-linked immunosorbent assay (ELISA) based assays detect IFN-γ produced and secreted by whole blood T cells following CMV peptide stimulation [20, 21]. The QuantiFERON-CMV (Qiagen Inc, Hilden, Germany) ELISA-based assay measures CD8 + T cell immune responses to a CMV peptide pool that covers 98% of Class I HLA haplotypes in the human population.

Enzyme-Linked Immunospot (ELISpot) assays detect IFN-γ that is bound adjacent to activated CD8 + and CD4 + T cells following T cell stimulation in microtiter plates [20, 21]. These assays are highly sensitive and quantify not only the amount of interferon that is secreted but also the number of cells that respond to the stimulus.

Flow cytometry-based assays measure IFN-γ expression following CMV stimulation using intracellular cytokine staining (ICS) [20, 21]. In contrast to ELISA- and ELISpot-based assays, ICS assays provide readout of IFN-γ expression for both CD8 + and CD4 + T cells. Other parameters such as cell surface molecules that denote activated T cells also can be assessed.

MHC multimer staining allows for assessment of CMV-specific CMI without stimulation of T cells [20, 21]. Conjugates are created between CMV peptides and MHC multimers and then fluorescently labeled allowing for direct identification of T cells. The CMV CD8 T Cell immune Competence assay (Mayo Clinic Laboratories, Rochester, MN, USA) pairs quantification of CMV-specific CD8 T cells with evaluation of IFN-γ expression and T cell degranulation (Table 1). However, this test is only available for patients with specific HLA Class I alleles which may limit the clinical utility of the test.

Notably, commercially available CMV CMI assays all utilize different CMV antigens, incubation times, and instruments used for interpretation, leading to different rates of indeterminate tests, cutoff values, sensitivity, and specificity. This makes comparing patients in varying research studies or at different medical centers difficult.

STRATEGIES FOR USING CMV CMI ASSAYS

CMV immune monitoring for transplant patients is potentially useful in a variety of clinical scenarios and can be used to augment both universal prophylaxis and preemptive therapy strategies. CMV CMI measured pretransplant could stratify risk for posttransplant CMV infection and inform the choice of CMV prevention strategy, especially in seropositive SOT recipients. Posttransplant monitoring may stratify infection risk following discontinuation of prophylaxis. Similarly, CMI assays could be used to guide monitoring frequency or identify the need for prophylaxis during high-risk periods. Lastly, CMI assays can be used in a preemptive fashion to identify patients at higher risk for symptomatic or severe CMV disease in with the setting of low-level CMV DNAemia. A summary of these clinical scenarios and potential management decisions based on the results of CMV CMI testing is shown in Table 2.

Table 2.

Strategies for Use of CMV CMI Assays in Pediatric Transplantation

Clinical Scenario	CMV CMI Result	Potential Impact on Clinical Management Decisionsa	Key Referencesb
CMV seropositivity in infants or patients with prior blood product administration	Positivec	Indicates presence or absence of prior CMV infection and allows for more accurate posttransplant prophylaxis and monitoring	[35, 36]
	Negatived
Pretransplant risk stratification in CMV seropositive transplant candidates	Positive	Use of preemptive strategy; use of shorter duration of prophylaxis; decreased CMV PCR monitoring frequency	[30, 37, 38, 39–41]
	Negative	Use of prophylaxis strategy; lLonger duration of prophylaxis
Risk stratification posttransplant at cessation of prophylaxis	Positive	Early prophylaxis discontinuation; less frequent or no post-prophylaxis CMV PCR monitoring	SOT: [30, 31, 42, 43, 44, 45, 46, 47, ] HCT: [48, 49–53]
	Negative	Prolonged prophylaxis duration; more frequent surveillance after prophylaxis discontinuation
Preemptive monitoring with low-level CMV DNAemia	Positive	Defer initiating treatment; continue CMV PCR monitoring	SOT [12, 54] HCT [55, 56]
	Negative	Initiate treatment
Posttherapy for CMV infection or disease	Positive	No secondary prophylaxis and/or ongoing surveillance	[57, 58]
	Negative	Posttreatment surveillance or initiating secondary prophylaxis
Periods of heightened immunosuppression (treatment of rejection or GVHD)e	Positive	No prophylaxis; less frequent or no ongoing surveillance	--
	Negative	Prophylaxis or frequent surveillance

Abbreviations: CMI, cell-mediated immunity; CMV, cytomegalovirus; HCT, hematopoietic stem cell transplantation.

^aClinical management decisions are given as examples of potential utility in these scenarios. Given the paucity of data in pediatric transplant recipients, further clinical research is needed to define the utility and safety of these approaches.

^bReferences include studies in these scenarios for HCT and SOT recipients.

^cPositive result indicates presence of detectable CMV CMI by assay performed.

^dNegative result indicates absence of detectable CMV CMI by assay performed.

^eAs no studies of CMV CMI have been performed in this scenario, and with the changes in immunosuppression that occur with treatment of GVHD or rejection, caution is warranted using CMV CMI in this setting.

FACTORS THAT MAY IMPACT CMV CMI

CMV CMI is impacted by T cell impairment and reconstitution timing after transplant. For all SOT patients, the CMV-specific T cell response declines after transplantation. The CD8 + T cell response appears to decline early posttransplant before rebounding to pretransplant levels by 2 months after transplant [59]. The CD4 + T cell response reaches its nadir approximately 2 months after transplantation and does not reach pretransplant levels until approximately 12 months posttransplant. For HCT patients, T cell reconstitution lags for several months [60]. CD8 + T cell reconstitution occurs about 2–8 months after transplant, while CD4 + T cell response occurs 4–12 months after transplant. This prolonged lymphopenia may result in either indeterminate or negative CMV CMI assays.

T lymphocyte reconstitution in SOT is significantly impacted by the chosen induction immunosuppression regimen. Two recent studies in adult kidney transplant recipients demonstrated that use of T cell-depleting induction immunosuppression (either anti-thymocyte globulin or alemtuzumab) was associated with decreased CMV CMI when compared to non-T cell-depleting regimens that incorporate basiliximab or other drugs [30, 42]. A similar phenomenon is seen in HCT recipients, as T cell depletion greatly slows reconstitution of the adaptive immune system [60].

Lastly, potent suppression of viral replication by valganciclovir may limit the ability to develop CMV CMI in seronegative recipients [30, 61]. One recent clinical trial in D+/R− adult liver transplant recipients found that median CD4 + and CD8 + T cell responses measured using intracellular cytokine staining were significantly higher in patients that received preemptive therapy versus those that received valganciclovir prophylaxis [62]. It is not known if this phenomenon will hold for letermovir prophylaxis.

CMV CMI ASSAYS IN SOT

Pretransplant Measurement of CMV Immunity

An array of prospective studies measuring CMV CMI with ELISpot [63], IGRA [37], or ICS assays [64–66], as well as one interventional trial of adult SOT recipients [38] demonstrate that pretransplant CMV CMI measurement can facilitate risk stratification for posttransplant CMV infection, regardless of serostatus. Study results differ regarding the impact of anti-thymocyte globulin (ATG) induction and CMV prevention strategy on the predictive utility of pretransplant CMV CMI.

The most illustrative study is an interventional trial that enrolled CMV D+/R+ kidney transplant recipients and randomized them to either preemptive monitoring or 3 months of prophylaxis [38]. Randomization was performed 1:1 based on CMV CMI measured by the T-SPOT.CMV assay (low vs high CMV CMI). All participants received either ATG or basiliximab for immunosuppression induction. Low CMV CMI was associated with CMV infection within both the preemptive (odds ratio [OR] 3.44 [95% confidence interval (CI), 1.03–9.08]) and prophylaxis (OR 11.75 [95% CI 2.31–59.71]) groups. Pretransplant CMV CMI did not predict CMV infection in patients who received ATG induction followed by preemptive monitoring but was predictive in ATG recipients who received prophylaxis [38]. In contrast, a prior study of CMV D+/R− and R + SOT recipients showed that the predictive utility of pretransplant CMV CMI remained significant for both preemptive and prophylactic CMV prevention strategies and with ATG induction [63].

Pretransplant CMV CMI monitoring successfully predicts CMV seropositive adult SOT recipients at low risk of posttransplant CMV infection and may identify patients who would do well with a preemptive monitoring strategy. Caution is warranted when considering this approach in the setting of T cell-depleting induction therapy. To our knowledge, no study has characterized pretransplant CMV CMI in CMV seropositive pediatric SOT candidates/recipients. Applicability of this approach to children is limited by the lower likelihood of prior CMV infection in pediatric SOT recipients and lack of data. CMV CMI is not useful for pretransplant risk stratification in seronegative children [67].

An additional scenario in which pretransplant CMV CMI may be helpful is in the setting of discordant serologic testing or when differentiating between passive antibody and true prior infection, particularly in infants <12 months of age or those who have received blood products [35, 36]. Though little data has been published using this approach in children, potential exists for more accurate assignment of CMV prevention strategies in infants by using CMV CMI assays (Table 2).

Predicting Post-Prophylaxis CMV Infection

Following early studies demonstrating a correlation of CMV CMI to protection from CMV infection in SOT recipients [68, 43], four large studies have demonstrated the ability of available CMI assays to predict occurrence of post-prophylaxis CMV infection [11, 30, 44, 47]. While these studies have varied in the assay used, patient population, prevention strategy, primary endpoints, and frequency of T cell-depleting immunosuppression induction, several key conclusions can be drawn (Table 3).

Table 3.

Studies Evaluating the Ability of CMI Assays to Predict Post-Prophylaxis CMV Infection in Adult Solid Organ Transplant Recipients

	Organ Participants Age (Mean or Median)	Assay Type	CMV Serostatus Prophylaxis Duration	Study Follow-Up CMV Incidence	Subjects Receiving T Cell-Depleting Induction	Predictive Value of Undetectable CMV CMI for Development of Post-Prophylaxis CMV Infection	Predictive Value of Detectable CMV–CMI for Absence of Post-Prophylaxis CMV Infection
Kumar et al [11]	Mixed N = 108 49.7 years	IGRA	D+/R−: 32.4% R+: 67.6% 94 days (median)	6 months 16.7% (disease)	36.1% (ATG)	22.9% (95% CI NR)	94.7% (95% CI NR)
Manuel et al [44]	Mixed N = 124 50.0 years	IGRA	D+/R−: 100% 98 days (median)	12 months 22.0% (disease)	39.4% (ATG or Alemtuzumab)	24% (95% CI 16–33)	93% (95% CI 68–99)
Fernández‐Ruiz et al [47]	Kidney N = 120 53.4 years	IGRA	R+: 100% 92 days (median)	12 months 41.7% (infection)	100% (ATG)	50% (95% CI 40.3–59.6)a	71.4% (95% CI 59.9–80.7)a
Kumar et al [30]	Kidney N = 368 51 years	ELISpot	D+/R−: 45% R+: 55% 3 months: 55% 6 months: 45%	12 months 11.9% (csCMVi)	68% (ATG or Alemtuzumab)	19.5% (95% CI NR)	97.1% (95% CI NR)

Abbreviations: ATG, anti-thymocyte globulin; CI, confidence interval; CMV, cytomegalovirus; csCMVi, clinically significant CMV infection; D, donor; ELISpot, enzyme-linked immunospot; IGRA, interferon gamma release assay; NR, not reported; R, recipient.

^aUsing assay cutoff threshold identified using study data that was different than manufacturer recommended cutoff.

First, detectable CMV CMI has a high predictive value for protection from post-prophylaxis CMV infection. In three studies, the predictive value ranged from 93% to 97%, indicating that if a patient has detectable CMV CMI, they will be unlikely to go on to have post-prophylaxis CMV infection or disease [11, 30, 44]. Impact of T cell depletion on CMV CMI predictive performance was variable. In one study of adult CMV R + kidney recipients, all of whom received ATG induction immunosuppression, the predictive value of detectable CMV CMI by Quantiferon-CMV was only 71.4% [69]. In contrast, CMV CMI measurement by T-SPOT.CMV in both CMV D+/R− and R + adult kidney recipients had a high predictive value for protection from CMV (96.7%) in patients receiving T cell depletion [30].

One concern regarding the use of CMV CMI assays is their generally worse predictive performance in the setting of CMV D+/R− serostatus. In a study that included both CMV D+/R− and R + kidney recipients, predictive value of a positive T-SPOT.CMV assay for protection from CMV infection was 98.1% in the R + cohort but only 87% in the CMV D+/R− cohort using the same assay cutoff [30]. Similarly, in an earlier study, predictive value of a positive Quantiferon-CMV assay was only 85.7% for protection from CMV disease in a cohort of CMV D+/R− recipients of predominantly lung, kidney, or liver transplants [11]. In contrast, in a study of mixed organ recipients all of whom were CMV D+/R-, Quantiferon-CMV performance for predicting protection from CMV infection was excellent with predictive value of 93% [44]. Some of this difference may be due to differences in CMV prophylaxis duration in CMV D+/R− recipients (6 months in Kumar et al [30] vs 3 months in Manuel et al [44]), or potentially due to use of different assays (Quantiferon-CMV vs T-SPOT.CMV).

A last observation from these studies is the poor predictive value of negative CMV CMI for the development of post-prophylaxis CMV infection. In patients with below threshold or undetectable CMV CMI as measured by a variety of assays, the frequency of CMV endpoint occurrence ranged from 22% to 50% (Table 3). Even when stratified into a higher risk group, less than half of these patients had CMV infection or disease following completion of prophylaxis. CMV CMI measurement is likely more useful in identifying SOT patients who can safely stop CMV prophylaxis versus defining those for whom prolonged CMV prophylaxis is definitively needed.

Data on the predictive and practical utility of CMV CMI measurement in pediatric SOT recipients are severely lacking. Pediatric data using CMV CMI assays to predict post-prophylaxis CMV infection are limited to small single-center cohort studies [70–72], only one of which enrolled enough patients to assess CMV CMI predictive performance. In this single-center cohort of liver or intestinal transplant recipients (n = 109), above threshold T cell CD154 expression measured using flow cytometry posttransplant was significantly associated with absence of CMV DNAemia [70]. In a validation subset of the total cohort, CD154 expression on more than 1.7% of T cells had a 98% predictive value for absence of CMV DNAemia on the subsequent CMV PCR [70]. While these are encouraging results and this study had a robust number of participants enrolled, the lack of standardized collection timing posttransplant limits the application of these results. Additionally, currently published studies have not used commercial assays. No large prospective study in pediatric SOT recipients has been published to determine whether CMV CMI can predict posttransplant CMV infection and thereby inform CMV prevention strategies.

Predicting the Need for Treatment or Response to Therapy

Two studies employing the Quantiferon-CMV assay have evaluated whether CMV CMI measurement can stratify which patients need treatment with low-level CMV DNAemia. In a prospective cohort of 37 adult CMV D+/R− or R + SOT recipients, detectable CMV CMI using the Quantiferon-CMV assay shortly after onset of CMV DNAemia was associated with infection resolution without treatment [12]. CMV CMI was measured at a median of 7 days (IQR 5–8 days) after first positive CMV PCR. Of patients with a positive assay, 92.3% had spontaneous resolution whereas 45.5% of patients with a negative assay had resolution. A second prospective cohort included 104 adult SOT patients with detectable pretransplant CMV CMI by Quantiferon-CMV for whom preemptive monitoring was planned [73]. This study was limited by many patients with asymptomatic CMV infection receiving antiviral treatment. However, 77.4% of patients with CMV DNAemia who did not receive antiviral treatment had self-resolving CMV DNAemia.

Several studies demonstrate an association between CMV CMI and protection from recurrent CMV infection in SOT recipients [57, 74–76]. One prospective feasibility study evaluated whether use of the QuantiFERON-CMV assay enables tailored secondary CMV antiviral prophylaxis [58]. Twenty-seven adult SOT recipients were enrolled at onset of CMV DNAemia. Following the completion of treatment and concurrent CMV PCR negativity, 14 patients with positive CMV CMI had antivirals discontinued while patients with negative CMV CMI received two months of secondary prophylaxis. One patient with positive CMV CMI had recurrence of CMV infection in contrast to 9/13 (69.2%) of patients with negative CMV CMI. Thus, CMV CMI measurement is likely to inform the utility of secondary CMV antiviral prophylaxis in SOT recipients and may be a viable strategy to decrease antiviral usage.

CMV CMI ASSAYS AND HEMATOPOIETIC CELL TRANSPLANTATION

Although recent advances in management have clearly improved outcomes, CMV continues to cause significant morbidity and mortality in HCT populations [2, 8, 48]. Currently, determination of risk of CMV infection for HCT patients is based on graft type and pretransplant serology. The seropositive recipient is at the greatest risk for CMV infection posttransplant, especially in the setting of a seronegative donor. Data on the use of CMV-specific CMI assays are more limited in the HCT population. The assays have been studied in several different clinical contexts, including serial monitoring of CMV CMI to predict risk of CMV infection and correlating CMV CMI and risk of progression from low-level CMV DNAemia to clinically significant disease.

Serial monitoring of IFN-γ released by CMV-responsive CD4 + and CD8 + T cells using an ELISpot assay was initially studied in HCT recipients with the goal of predicting CMV infection occurrence [77]. In the follow-up study, Chemaly et al demonstrated that serial monitoring of CMV CMI successfully stratified risk for CMV infection amongst seropositive HCT recipients, though this study was performed prior to widespread use of letermovir prophylaxis in seropositive HCT recipients [48]. In this multicenter study of 241 adult allogeneic HCT patients, CMV CMI was assessed every 2 weeks by ELISpot CMV assay from the pretransplant period until 6 months posttransplant. Seventy patients (29%) experienced CMV infection and/or disease. Of those who developed clinically significant CMV infection (csCMVi), defined as the first CMV reactivation or CMV disease after HCT necessitating the start of anti-CMV therapy, the vast majority had low levels of CMV CMI. Despite this association, the predictive value of low CMI for occurrence of csCMVi was only 36%. Predictive value of above threshold CMV CMI for absence of csCMVi was 93%. On multivariate analysis, CMV CMI along with patient sex, age, steroid use, and ATG receipt were significantly associated with csCMVi. Notably, the combination of CMV infection and low CMV CMI was associated with higher all-cause mortality.

Several publications have addressed the utility of CMV ELISpot assays in other clinical scenarios. El Haddad et al reported that low CMV CMI using T-SPOT.CMV (Oxford Immunotec International, Oxfordshire, England) was significantly associated with progression from low-level CMV DNAemia to csCMVi [55]. In contrast, a separate group showed that CD8 + T cell-specific CMV CMI measured using a research assay was not able to predict progression to csCMVi [56]. Wagner-Drouet et al used the ELISpot-based T-Track CMV assay (Mikrogen GmBH/Lophius Biosciences GmBH, Neuried, Germany) to assess the risk of late CMV reactivation following prior CMV infection in high-risk (CMV D−/R+) adult HCT patients [78, 49]. Using a clinically relevant timepoint of day 100 post-HCT, low CMV CMI had a 90% predictive value for identifying patients with subsequent recurrence of CMV infection.

CMV CMI measurement using the QuantiFERON-CMV assay to predict CMV reactivation and infection has been assessed at multiple timepoints in the HCT population [50–53]. Though these studies have enrolled fewer patients, this assay has reasonable predictive value for absence of CMV infection in the setting of detectable CMV CMI. One small study (n = 28) performed a direct comparison of ELISpot (T-SPOT.CMV) versus IGRA (QuantiFERON-CMV), with CMV CMI measured at 28- and 100-days posttransplant [79]. The assays had moderate agreement though both assays showed good ability to predict absence of CMV infection. A study to assess CMV CMI in the setting of letermovir prophylaxis post-HCT is ongoing (ClinicalTrials.gov Identifier: NCT04017962).

Only a few small studies of CMV CMI assays focused on pediatric HCT recipients have been published. In the largest of these studies (n = 46), CMV CMI measured by the T-Track CMV assay was significantly associated with protection from CMV infection on multivariate analysis [80]. Pediatric HCT-specific studies using QuantiFERON-CMV have shown similar results in assessing the ability of detectable CMV CMI to predict protection from initial CMV infection [81] and recurrent infection [82]. Thus, while limited by small sample size, studies in pediatric HCT recipients do support the clinical utility of CMV CMI measurement to predict CMV infection and potential recurrence.

CONCLUSION

Though CMV CMI measurement holds significant potential for improving the care of pediatric transplant recipients, much work remains to be done before it becomes part of routine clinical practice. CMI assays need to be routinely available and reasonably priced, with clinically useful turnaround times and standardized interpretation to be successfully utilized in clinical practice. Understanding the levels of CMV CMI associated with risk and the appropriate timing of the assays is necessary to optimize CMV management in high-risk children. Pediatric studies evaluating CMV CMI in transplant recipients are limited by small enrollment numbers, non-standardized assay timing, and/or use of noncommercial assays. One large, multicenter prospective study conducted by the Pediatric Infectious Diseases Transplant Network that will evaluate the ability of CMV-specific T cell responses in pediatric SOT recipients to predict CMV infection risk using intracellular cytokine staining has completed enrollment (NCT03924219) and should provide valuable information for pediatric SOT recipients. However, several unique challenges exist that make evaluation and implementation of CMV CMI measurement more difficult for children than adults. Children are more likely to be CMV seronegative, making pre and even posttransplant CMV CMI measurement potentially less useful. Blood volume requirements for most CMV CMI studies limit use of these assays in smaller children and infants. In addition to these pediatric-specific challenges, there are no data on the impact of standard CMV CMI measurement on antiviral utilization, healthcare costs, and long-term transplant outcomes. Further study is required prior to broad implementation of CMV CMI measurement in children.

Notes

Financial support. This work was supported by the Turner Hazinski Research Award and the Dolly Parton Pediatric Infectious Diseases Research Fund at Vanderbilt University Medical Center to D. E. D.

Supplement sponsorship. This article appears as part of the supplement “Advances in Pediatric Transplant Infectious Diseases,” sponsored by Eurofins Viracor.

Potential conflicts of interest. Otto: No reported conflict. Vora: No reported conflict. Dulek: Non-salary research assay support from Eurofins Viracor related to CMV CMI assay.