The past 5 years has seen exciting advances related to the understanding, diagnosis, and treatment of Cytomegalovirus (CMV). We currently stand on the cusp of modernizing the management of CMV infection posttransplant. Despite these advances, CMV remains one of the most common complications affecting solid organ transplant recipients (SOTR), still befitting the designation: “a transplantation troll.”1 In addition to the direct effects of CMV infection and disease, there are “indirect effects,” both general and transplant-specific, and higher rates of all types of infection, graft loss, morbidity, and mortality.2,3 A panel of experts on CMV and solid organ transplantation (SOT) was previously convened in 2008 and 2012 by The Infectious Diseases Section of The Transplantation Society to develop consensus guidelines on CMV management, subsequently published in 20104 and 2013.5 Topics included diagnostics, immunology, prevention, treatment, resistance, and pediatrics. Given numerous recent advances in the field, a third meeting of experts was convened in March 2017 to update these guidelines.
The expert panel rated the quality of evidence on which recommendations are based by following a process used in the development of other guidelines, including those by the Infectious Diseases Society of America. The Grading of Recommendations Assessment, Development and Evaluation system allows for a systematic weighting of the strength of recommendation (eg, “high, moderate, low, very low”) and quality of evidence (eg, “strong, weak”) (Table 1).6-10,13
We used the following definitions, consistent with the American Society of Transplantation and the CMV Drug Development Forum recommendations for use in clinical trials14,15:
- CMV infection: evidence of CMV replication regardless of symptoms (differs from latent CMV); “defined as virus isolation or detection of viral proteins (antigens) or nucleic acid in any body fluid or tissue specimen”15
- CMV disease: evidence of CMV infection with attributable symptoms. CMV disease can be further categorized as a viral syndrome (ie, fever, malaise, leukopenia, and/or thrombocytopenia), or as tissue invasive (“end organ”) disease.
As in our prior versions, the term deoxyribonucleic acid in blood (DNAemia) will be used instead of viremia, to reflect the detection of CMV DNA in blood or plasma (whether actively replicating virus or not). For accuracy, the phrases “viral load” or “quantitative nucleic acid amplification testing (QNAT)” are used instead of “polymerase chain reaction (PCR).”
Given that the CMV serostatus of donor and recipient (D/R) are key predictors of the risk of CMV after transplant and guide decisions on antiviral prophylaxis or preemptive treatment, serologic tests with high sensitivity and high specificity are recommended. A test measuring CMV-specific IgG should be used, as serologic tests measuring IgM or IgG and IgM combined have poorer specificity and may cause false-positive results. Neither of the latter 2 should be used for screening.16-18 Not all serologic tests are equivalent, and performance characteristics of the specific test used should be understood.19 Discordant CMV IgG serology results have been reported for 1.0% to 2.6% of samples tested by commonly used assays.16,17 A change of the serologic assay requires evaluation of its performance, including comparison with the previously used assay.
If the donor or recipient is seronegative during the pretransplant evaluation, serology should be repeated at the time of the transplantation. Interpretation of serology results can be difficult in donors and recipients with recent transfusion of IVIG and other blood products (platelets, plasma, red blood cells), given the potential for passive transfer of antibody,20 and a pretransfusion sample should be tested when possible. In seropositive children younger than 12 months, passive transfer of antibody can lead to transient false-positive serologic results.20 Cell-mediated immunity (CMI) assays may be useful in assisting in establishing true immunologic status in individuals who may have passive antibody.21,22 Testing for CMV-specific CMI appears to be less useful in deceased donors because of a high prevalence of indeterminate results.23 In infants younger than 12 months, nucleic acid amplification tests (NAT) of urine or oral samples may be helpful to identify infected patients, as children with postnatal primary infection appear to persistently shed virus for long periods,24 whereas a negative CMV NAT cannot exclude past exposure. Thus, if equivocal serologic assay results are obtained in the donor or in the recipient, we recommend assigning the highest appropriate CMV risk group for posttransplantation management decisions.
Serology has no role in the diagnosis of active CMV replication and disease posttransplantation. Serology may be used to determine ongoing susceptibility to community-acquired disease in patients seronegative before transplantation who do not develop infection or disease after transplantation.
Using CMV-antibody binding assays, seroconversion at the end of antiviral prophylaxis was not a useful predictor of future CMV disease in CMV-mismatched patients,25,26 although seroconversion at 6 months after transplant in D+/R− patients given 100 days of antiviral therapy (ie, 3 months after the end of prophylaxis) conveyed a reduced risk for late CMV disease.26 In a pilot study in CMV-mismatched patients managed using preemptive therapy, measurement of antibodies neutralizing epithelial cell infection, particularly when combined with CMI studies was identified as a possible predictor of future CMV disease risk.27
Viral culture of blood for detecting CMV has limited clinical utility due to poor sensitivity for diagnosis of disease. Conversely, there is no role for CMV culture of urine or oral secretions due to poor specificity for CMV disease (versus viral shedding).28 Quantitative nucleic acid amplification testing in blood is the cornerstone for diagnosis and monitoring for CMV infection and disease; QNAT is preferred over antigenemia because of standardization and other technical reasons, as previously described.5
Quantitative nucleic acid amplification testing is the preferred method for diagnosis of CMV infection, guiding preemptive strategies, and monitoring response to therapy.29-35 There are numerous commercial reagents and automated platforms available for quantitative CMV DNA testing, as well as increasing data on the performance characteristics of these tests. Plasma and whole-blood specimens both provide prognostic and diagnostic information regarding CMV disease.36-40 CMV DNA is generally detected earlier and in greater quantitative amounts per unit volume (ie, mL) in whole blood compared with plasma. Persistent plasma DNAemia has been shown to be a better predictor of relapse at day 21 of treatment compared with persistent whole-blood DNAemia.38 There is evidence that CMV DNA exists predominantly as small fragments in plasma,41 this may not to be true to the same extent for whole blood as both intracellular and extracellular DNA are detected. More work is needed to understand the form of DNA in whole blood samples; paired sample studies are required to establish correlation of results between the 2 sample types. Thus, when serially monitoring patients, 1 specimen type should be used.
Calibration of tests with the World Health Organization (WHO) international standard42 has led to improved agreement of viral load values,43 therefore all results should be reported as IU/mL. Important differences still exist due to a variety of factors44-46 including extraction method, amplification target, probe, nonstandardized quantification of secondary standards45,47,48 and amplicon size.41 After calibration, commutability needs to be demonstrated, meaning that patient samples and calibrators behave similarly in a given quantitative test. Commutability of current assays calibrated to the WHO international standard varies widely.49 The increased use of commercial assays that address these factors should decrease variability; a recent study showed good reproducibility in viral load values across multiple laboratories when using a commercial test calibrated to the WHO standard.50 Such agreement allows comparison of data across different transplant centers using the same test.
The QNAT results should be demonstrated to be linear in the clinically important range between the lower and upper limits of quantification (LLOQ, ULOQ). The LLOQ varies amongst assays with newer, highly sensitive assays demonstrating LLOQ less than 200 IU/mL. Assays results reported as detectable below the LLOQ represent very low levels of CMV DNA that may not be clinically significant. The precision of QNAT results is such that changes in values should be at least threefold (0.5 log10 IU/mL) to represent biologically important changes in viral replication.50-53 The QNAT variability is greatest for viral loads of 1000 IU/mL (3 log10) and below, where changes may need to be greater than fivefold (0.7 log10 IU/mL) to be considered significant. Reporting results as both integers and log10-transformed data may help clinicians avoid overinterpreting small changes in viral load.
It remains imperative that laboratories use quantitative external standard materials (independent of that provided by the manufacturer) to monitor quantification across different lots of reagents to ensure consistency in assay performance. Quantitative nucleic acid amplification testings should accurately quantify the common CMV genotypes. If the laboratory changes QNAT or extraction methods, a comparison must be performed documenting the performance characteristics of the new versus old tests. Until QNATs have been better harmonized, a single test should be used for clinical trials and for monitoring patients over time. Given the rapid replication dynamics, CMV QNAT results should be available within 24 to 48 hours for optimal clinical decision making.
Consensus viral load thresholds to initiate preemptive therapy need to be defined, but widespread use of laboratory-developed tests and paucity of assays calibrated to the WHO International Standard has limited the ability to perform such studies. Moreover, the D/R risk group and exposure to lymphocyte-depleting antibodies need to be taken into account. Early work has shown that higher viral load values correlate with increased risk for disease30,31 (see Prevention section for further discussion). It is important to note that these recent studies were done with different laboratory developed assays, specimen types (plasma, whole blood), and patient populations, so these thresholds cannot simply be adapted. This leaves individual centers with the task of determining appropriate thresholds in their patient populations, and emphasizes the urgent need to perform collaborative studies to determine consensus threshold in IU/mL, which should be increasingly practical with the availability of commercial assays.
The viral load kinetics (rapid doubling time) in high-risk groups suggests that the frequency of viral load testing will impact the effectiveness of a preemptive strategy (ie, more frequent testing will be more effective). The half-life of viral loads is such that monitoring patients on therapy should be done at least weekly.32 Dynamics of CMV loads over time may be more important in predicting disease than any absolute viral load value,30 especially when close to the LLOQ. However, the LLOQ varies among the different viral load tests; an LLOQ of greater than 1000 IU/mL (using either whole blood or plasma) may be too insensitive and hence inadequate for preemptive strategies aiming at antiviral treatment before disease.54 Conversely, a very sensitive test (LLOQ <10 IU/mL) may detect latent virus particularly if whole-blood specimens are used, which limits the clinical utility of an extremely sensitive test.
When monitoring a response to treatment, there is concern that highly sensitive tests may increase the time to reach an undetectable viral load, thus unnecessarily exposing patients to prolonged periods of antiviral therapy. A recent study showed that using a more sensitive test does increase the time to reach an undetectable viral load; however, there was a trend towards shorter duration of therapy with the more sensitive assay.55 Finally, treatment until viral load values are less than 137 IU/mL is predictive of a clinical response.56 Though there is value to using highly sensitive assays, the practice of continuing therapy until successive viral load tests are undetectable may not be necessary when using such tests (ie, with highly sensitive assays, multiple very low results might be sufficient before stopping therapy).
Diagnostics for Tissue-invasive Disease
The definitive diagnosis of tissue-invasive disease relies on detection of CMV in the tissue specimen. Identification of CMV cytopathic changes or CMV antigens by immunohistochemistry15,57,58 represents the gold standard for the diagnosis of tissue-invasive disease. Not all antibodies and staining procedures have equal sensitivity and the performance may differ between fresh and formalin-fixed, paraffin-embedded tissue.59
Gastrointestinal disease in all organ transplants and pneumonitis in lung transplant recipients may have undetectable or low DNAemia.60,61 Plasma QNAT has a high sensitivity for diagnosis of gastrointestinal disease in D+/R− patients, which decreases substantially in R+ patients.62,63
Viral culture of tissue samples, though not available in most laboratories today, may improve sensitivity compared to histopathology eg, in gastrointestinal disease.63 However, in patients with viremia the specificity of culture results of tissue for end-organ CMV disease may be limited. Similar considerations apply to the more sensitive molecular testing. Some studies have shown positive QNAT tissue results in cases with a clinical suspicion of gastrointestinal CMV disease, however, in which histopathology and DNAemia were negative.64,65 Tissue QNAT may be preferable to qualitative NAT due to higher specificity, but studies are needed to define thresholds and to provide a standardized approach for QNAT in tissue-invasive CMV disease.
In lung transplant recipients, the detection of the CMV by QNAT in bronchoalveolar lavage (BAL) reflects CMV replication in the lung rather than contamination with oropharygeal fluids.66 Qualitative NAT detecting CMV-DNA in BAL specimens in lung and nonlung transplant recipients may not be specific for a diagnosis of CMV pneumonitis.67,68 Quantitative NAT is preferred on BAL specimens and increased DNA levels may better correlate with symptomatic CMV disease.61,69–71 Further work is needed to standardize QNAT on BAL samples including development of thresholds using normalized reporting units.69,72
Central nervous system disease in SOTRs is extremely rare. In the absence of further clinical studies, the presence of CMV DNA in the cerebrospinal fluid likely represents CMV disease necessitating treatment.
The diagnosis of retinitis is based on ophthalmologic examination; CMV DNAemia is rarely useful as a predictor of CMV retinitis, although it may be positive before and at the time of diagnosis. A positive QNAT in vitreous fluid may be helpful in guiding the diagnosis of retinitis.
Consensus Statements and Recommendations
- The availability of the WHO-approved CMV international standard has led to improved agreement of plasma viral load values between various assays.43 Differences still exist due to a variety of factors,44-46 including matrix, extraction method, amplification target, amplicon size, hybridization/detection probe, nonstandardized quantification of secondary standards,45,47,48 and use of noncommutable standards.
- Global harmonization and development of universal thresholds will be facilitated by using assays where all steps are highly standardized. The increased availability of appropriately designed, affordable commercial assays should accelerate these processes.
- We recommend performing donor and recipient CMV IgG serology pretransplantation for risk stratification (strong, high).
- We do not recommend IgM testing (strong, low).
- We recommend repeat serologic testing at the time of transplant if pretransplantation serology is negative (strong, low).
- We recommend that in adults, an equivocal serologic assay result in the donor be assumed to be positive, whereas in the recipient this result be interpreted to assign the recipient to the highest appropriate CMV risk group for posttransplantation management decisions (strong, low) (for guidance on infants and children younger than 12 months, see Pediatrics section).
- We do not recommend viral culture of blood, urine, or oral secretions for the diagnosis of active CMV infection or disease (strong, high). Positive cultures of BAL samples may not always correlate with disease.
- We do not recommend QNAT on urine and oral secretions for surveillance or diagnosis of CMV disease (strong, low).
- We recommend using QNAT calibrated to the WHO standard for diagnosis, surveillance to guide preemptive antiviral treatment, and for therapeutic monitoring due to the ability to harmonize and standardize these tests (strong, high). Results must be reported as IU/mL and termed as DNAemia rather than viremia (strong, high). If QNAT is not available, antigenemia is a less desirable alternative.
- We recommend either plasma or whole blood specimens for QNAT, with an appreciation for the differences in viral load values, viral kinetics and assay performance characteristics. (strong, high). Neither the specimen type nor the assay should be changed when monitoring patients.
- Despite reporting in IU/mL, we recommend that viral load values are not directly compared across centers and/or laboratories unless identical testing reagents and procedures can be assured or equivalence has been documented (strong, high).
- We recommend that only changes in viral load exceeding 0.5 log10 IU/mL (threefold) are considered to represent clinically significant differences in DNAemia (strong, low).
- Although harmonization of QNAT has improved, universal thresholds for therapy or treatment endpoints have not been established and current published thresholds remain assay-specific. Accordingly, we recommend that centers establish their own thresholds and audit clinical outcomes to verify the thresholds used (strong, moderate).
- We do not recommend surveillance of CMV DNAemia during routine prophylaxis.
- We recommend when monitoring response to antiviral therapy, that QNAT is performed weekly (strong, moderate).
- With the use of highly sensitive QNAT (LLOQ <200 IU/mL), we suggest discontinuing therapy after 1 result is less than the LLOQ. If this approach is used, confirmatory testing should be done 1 week after discontinuing therapy. If the assay is not highly sensitive, then 2 consecutive undetectable (negative) results are needed to discontinue therapy (weak and moderate).
- We recommend histology coupled with immunohistochemistry for the diagnosis of tissue-invasive disease. Histopathologic examination of tissue should routinely include immunohistochemistry for CMV (strong, moderate).
- Directly compare QNAT monitoring in plasma, whole blood, and BAL specimens with respect to disease prediction and monitoring response to therapy with an emphasis on using commercially available testing systems.
- Determine commutability and harmonization using the WHO International Standard for whole blood and BAL.
- To improve harmonization of QNAT, determine the viral form (virions, fragmented, or genomic CMV) and viral kinetics in whole blood.
- Assess the role of digital QNAT to improve standardization of copy number assignment for secondary standards.
- Once viral load tests are harmonized, establish thresholds and kinetics for DNAemia for initiating preemptive therapy.
- Compare the performance characteristics of the different serologic tests and assess the utility of CMI assays and QNAT using a variety of sample types for the interpretation of passive immunity.
- Standardized/optimize/harmonize tests measuring neutralizing antibody in epithelial cells and fibroblasts when used to evaluate vaccine responses, characterize immunoglobulin preparations and evaluate antibody levels as a biomarker for predicting disease risk.
- Further validate nonseroconversion at 12 to 18 months after antiviral prophylaxis as a marker of nontransmission of donor-derived CMV infection in CMV mismatched patients.
- Further evaluate the usefulness of binding and neutralizing antibody (epithelial and fibroblast) measurements pretransplant in CMV seropositive patients and posttransplant in CMV mismatched patients as a predictor of disease risk.
IMMUNOLOGIC MONITORING FOR CMV
Both innate and adaptive immune mediators are necessary for control of CMV after transplantation.73,74 Innate immune factors include Toll-like receptors in which single nucleotide polymorphisms are associated with an increased risk of CMV disease.75 Polymorphisms in genes for mannose binding lectin (MBL) associated with low MBL production and ficolin-2 may also be associated with increased risk of CMV disease.76-78 A multicenter study has recently demonstrated that patients with lower complement C3 levels (<80 mg/dL) early (7 days) after transplantation have a greater risk for CMV disease.79 Transplant recipients with an IL-28B single nucleotide polymorphism were found to be at significantly less risk of CMV replication.80 Conversely those with a CCL8 promoter polymorphism showed increased risk of CMV replication.81 In addition, lower levels of natural killer (NK) cells early after transplantation are a risk factor for CMV disease in heart and liver recipients.82,83 Inhibitory NK cell KIR genotypes have been described as a predisposing factor during CMV reactivation in kidney transplantation while B-type NK cell haplotypes containing activating NK cell KIR genes were shown to be protective in organ recipients.84-86 γδ (gamma-delta) T lymphocytes that have both innate and adaptive characteristics have been reported to have a role in the immune response to CMV in transplantation.87,88 Longitudinal kinetic surveillance of Vδ2− γδ T cells in kidney recipients has been suggested to predict CMV infection resolution.89 Although the above associations are important for the pathogenesis of CMV, changes in CMV management based on these factors have not been studied.
Nonpathogen-specific Adaptive Immunity
Adaptive or acquired immune responses of B and T lymphocytes are critical in controlling CMV replication. Methods to monitor the adaptive immune response to CMV may allow for early identification of patients at increased risk of viral replication. B cells are important in the humoral response to CMV, producing neutralizing antibodies that primarily target glycoprotein B (gB) and gH90 and the pentameric gH/gL/UL128/UL130/UL131A complex.91-93 There is emerging evidence that a significant number of posttransplant patients develop hypogammaglobulinemia (26%-70% in some series94-97). A recent meta-analysis demonstrated that severe hypogammaglobulinemia during the first year after transplant significantly increased the risk of CMV disease. Moderate and severe (IgG, < 400 mg/dL) hypogammaglobulinemia have been demonstrated to be a risk factor for CMV infection in heart and lung transplant recipients in single-center studies performed in patients with various induction immunosuppressive protocols.79,98,99 A multicenter prospective study showed that heart recipients with moderate hypogammaglobulinemia at day 30 after transplantation were at higher risk of CMV disease.79 Severe IgG hypogammaglobulinemia has been associated with CMV disease refractory to antiviral therapy.100 Interventional studies in heart transplant recipients using CMV immunoglobulin (CMV Ig) or intravenous immunoglobulin replacement have shown that IgG replacement may prevent CMV disease.101-103 Retrospective studies have suggested that replacement of IgG levels by use of IVIG in heart recipients with IgG hypogammaglobulinemia detected at the time of CMV disease including refractory cases was associated with better outcomes.104,105 Despite the evidence of IgG replacement therapy in thoracic transplant recipients, the link of hypogammaglobulinemia with CMV risk remains controversial in kidney and liver recipients.96,106
The potential role of lymphopenia, a commonly used biomarker in clinical practice, as a risk factor for development of CMV infection has been evaluated. In a multivariate analysis, pretransplant lymphopenia was the strongest independent predictor of CMV disease among 276 liver transplant patients.107 Lymphocyte counts also tended to be lower in patients who have recurrent CMV infections.108
Nonpathogen-specific immune function assays are also available. The ImmuKnow (Cylex/Viracor-Eurofins, USA) assay is not specific for CMV. This assay, which is commercially available in the United States and in some European countries, measures overall immune function and serves as a marker of immunosuppression by determining the amount of ATP produced by CD4+ T cells in response to whole blood stimulation by phytohemagglutinin. However, there are no studies indicating whether this assay is predictive of CMV DNAemia or disease. Therefore, there is insufficient evidence to recommend this assay for CMV prediction. Another immune function assay under development is the QuantiFERON-Monitor (Qiagen, USA) which measures global immune function after stimulation of whole blood with a lyosphere containing R848 and anti-CD3.109 In general, however, CMV-specific immune-based assays are likely to have greater clinical utility than a nonspecific assay.
The complexity of the immunological response to control CMV infection makes it unlikely that any single marker will be highly predictive of risk. The combined use of distinct biomarkers has been suggested to be a better approach than the use of single markers including biomarker combinations and gene profiles.110 Immunological scores including distinct biomarkers have been suggested as other option to identify patients at higher risk of CMV infection.98
CMV-specific Immune Monitoring Assays
Immune monitoring of CMV-specific T-cell responses can predict individuals at increased risk of CMV disease posttransplant and may be useful in guiding prophylaxis and preemptive therapies. There is a variety of CMV-specific T-cell assays. Many assays have now moved from the experimental to the clinical setting. The majority of assays rely on the detection of IFN-γ after stimulation of whole blood or peripheral blood mononuclear cells (PBMC) with CMV-specific antigens or overlapping peptides.111,112 In addition to IFN-γ, other markers, including IL-2, TNF-α, CD107, programmed death-1 (PD-1), and CD154 have been used to correlate CMV-specific T-cell responses with the risk of CMV infection. The expression of chemokines CCL8 and CXCL10 has also been associated with CMV control in patients with CMV DNAemia whereas expression of CCR6, a chemokine receptor, predicted CMV reactivation.81,90,113
The expert panel was of the opinion that an ideal assay should provide both quantitative and functional information on CMV-specific CD4+ and CD8+ T cells. For clinical application, an assay should ideally be simple to perform, inexpensive, highly reproducible, and amenable to either widely available platforms or shipping to specialized reference laboratories. Each of the immune monitoring assays has specific advantages and limitations and has been studied in various clinical applications to predict disease or viremia (Table 2).
The QuantiFERON-CMV assay is a commercially available kit (CE marked in Europe) and is an enzyme-linked immunosorbent assay (ELISA)-based IFN-γ release assay detecting CD8 T cells after peptide stimulation. The assay has been evaluated in clinical studies of transplant patients at high risk of CMV and shown to be predictive of disease.114-117 Moreover, a negative test before transplantation may aid in predicting viremia in the posttransplant period118 or the dynamics of T-cell responses may be used as a monitoring tool in a preemptive setting.119 In a small cohort of transplant recipients with low level CMV DNAemia, a positive assay was predictive of spontaneous clearance.120 A main drawback stems from difficulties in interpretation if a patient does not respond to the mitogen control. Nonresponse to mitogen may potentially be a marker for global immunosuppression, and has been associated with a subsequent higher incidence of CMV disease.116 Test sensitivity decreases in lymphopenic patients because an adequate number of cells are required for the production of IFN-γ.
The enzyme-linked immunosorbent spot (ELISpot) assay quantifies both CD4+ and CD8+ T cells producing IFN-γ in response to CMV. Purified PBMCs are stimulated with CMV-specific peptides or whole antigen lysates; IFN-γ is then captured, detected, and quantified using a labeled antibody. As with the QuantiFERON assay, a mitogen control may indicate general T-cell responsiveness. The ELISpot assay cannot differentiate between CD4+ and CD8+ T cells. Various in-house ELISpot assays have been evaluated and shown to be predictive of disease and viremia.121-125 Studies have used arbitrary cutoffs for defining positive responses that range between 5 and 50 spot-forming cells per 200 000 PBMC. Others have suggested that kinetics of ELISpot responses are better for risk stratification.126 ELISpot assays have also been used in the pretransplant setting to predict posttransplant outcomes.126,127 One study showed that stimulation with an overlapping peptide pool of IE-1 in the pretransplant period predicted CMV in the posttransplant period in CMV seropositive patients.127 However, another smaller study found no predictive value of pretransplant CD4 and CD8 responses.128 A commercial ELISpot assay (T-Track CMV, Lophius Biosciences, Germany) has recently received CE marking in Europe.129,130 Another assay (T-SPOT.CMV, Oxford Immunotec, UK, CE marked in Europe) is used as a laboratory developed test (LDT) in the United States.
Many studies that have analyzed CMV-specific T-cell responses have used intracellular cytokine staining (ICS) for IFN-γ using flow cytometry. Whole-blood or isolated PBMCs are stimulated with CMV peptides or CMV lysate. If whole antigen lysate is used, the assay is not HLA-restricted and knowledge of patient HLA type is not required. Stimulated cells are stained with monoclonal antibodies directed against IFN-γ. This technique is fast, versatile, and can be expanded to include other cytokines and cell surface molecules. Unlike ELISpot or QuantiFERON assays, ICS can thus provide both quantitative and qualitative characteristics of CMV-specific T cells. Clinical studies have shown that this technique can predict both CMV disease and viremia. Several studies in SOTRs showed an increased risk of CMV disease in patients with low levels of specific T-cell immunity.131-134 Similarly, the absence of anti-CMV T-cell response by this technique correlates with the inability to clear viremia.131,134,135 Stable levels of CMV-specific CD4+ T cells were associated with lower risk of CMV replication.131,134 The development of T-cell immunity especially polyfunctionality has also been shown to be associated with freedom from CMV disease after lung transplantation.136-138 The predictive value for viremia may be improved when the analysis of IFN-γ is combined with other cytokines, such as IL-2, and additional markers, such as PD-1.139,140 Advances in flow cytometry, such as the ability to test for several markers at once (eg, with CyTOF technology), can increase our understanding of immune control although these are available in the research setting only.
Major histocompatibility complex (MHC)-multimer based assays directly stain peptide-specific T cells using peptide-conjugated MHC class I tetramers or pentamers. They can determine CD8 T-cell responses but are epitope-specific and require knowledge of the patients’ HLA type. Multimer assays have only been shown to predict CMV viremia when combined with analysis of surface markers, such as PD-1.141,142 Both ICS and MHC-multimer staining require a fluorescence-activated cell sorting facility, which may limit widespread use in transplant centers.
Several clinical studies have now been published that have used immune monitoring to determine risk of CMV disease and viremia. The majority of studies have measured IFN-γ alone or in combination with other cytokines or cell-surface molecules and included both seropositive and seronegative recipients. The frequency of monitoring in these studies has been variable; high-risk patients were generally monitored starting at the end of prophylaxis and those undergoing preemptive therapy were screened weekly to monthly for up to 1 year posttransplant. Therefore, data are accumulating that suggest immune monitoring may be considered in combination with viral load monitoring to improve assessment of the individual's ability to control CMV. Data are also accumulating for measuring immune function in the pretransplant setting. Donor testing for CMV has also been performed using immune monitoring platforms although indeterminate results are frequently seen depending on the assay.23 Clinical utility studies demonstrate that alteration of patient management based on the results of an immune-based assay is feasible, safe, and cost-effective are ongoing. Recently, an interventional study using the QuantiFERON-CMV assay to stratify patients in receiving secondary prophylaxis showed that CMV-specific immune function could be measured in real time and used to make clinical decisions.143 This study showed that it was safe to discontinue treatment antivirals in patients with CMV-specific immunity without the need for secondary prophylaxis. Interventional studies of primary prophylaxis are underway. Potential clinical applications for immune-based assays are summarized in Table 3.
Adoptive T-cell Therapy
Several case reports now exist demonstrating the clinical use of CMV-specific T cells for resistant/refractory CMV infection in organ transplant recipients.144-148 In general, CMV-specific T cells undergo expansion with CMV synthetic peptides or viral lysate and are then infused into the recipient. This therapy has led to reconstitution of viral immunity, reduction in CMV viral loads or resolution of CMV disease. Cells can be obtained from the transplant recipient (autologous); however, the process of generating effector cells can take several weeks. Therefore, there is increasing interest in “off the shelf” T cells using HLA-matched third-party banked cells. This method could also allow generation of cells active against multiple viruses including CMV, Epstein-Barr virus (EBV), and adenovirus. There is a larger breadth of experience using CMV-specific T cells in the hematopoietic stem cell transplant (HSCT) field, which has shown varying degrees of efficacy with no association with graft versus host disease.149,150 Commercialization of third-party CMV-specific T cells may lead to increasing use of this modality for resistant/refractory CMV in SOTRs.151,152 Safety and efficacy studies will be needed.
Several CMV vaccines are under development, but none are currently available for routine clinical use. Types of vaccines include live attenuated, recombinant/chimeric viral vectors, recombinant subunit, or gene-based vaccines.153 A live attenuated vaccine based on the Towne strain of CMV was found to be safe during clinical testing but had a suboptimal antibody response and while CMV disease was attenuated, the vaccine failed to prevent infection.154 A recombinant gB vaccine with MF59-adjuvant was shown to induce neutralizing antibodies155 and prevent infection.156 In a recent study, this vaccine was administered in a 3-dose schedule to both CMV seropositive and seronegative transplant candidates.157 During follow-up, the vaccine reduced overall days of CMV viremia and number of days of antiviral therapy. A trial with a gB/pp65-based DNA plasmid vaccine in HSCT recipients was completed. This vaccine was administered as 1 pretransplant dose and 5 posttransplant doses. It showed a significant reduction in viremia versus placebo as well as a reduction in the number of CMV episodes.158 A multicenter clinical trial for the gB/pp65-based DNA vaccine was conducted in CMV mismatched transplant recipients but did not show a significant reduction in CMV viremia needing antiviral therapy.159 Recently, a peptide-based CMV vaccine (CMVPepVax) has been used in clinical trials. This vaccine is developed using the HLA-A*201-restricted pp65 CD8 T cell peptide epitope which is fused with the P2 peptide epitope of tetanus toxin and mixed with TLR9 agonist just before administration. This vaccine was given using a 2-dose schedule to HSCT patients in a small randomized trial and showed a significant reduction in CMV viremia.160 An alphavirus replicon vector system has been used to produce viral particles expressing gB and pp65/IE-1 fusion protein; initial studies in mice and rabbits have shown the development of neutralizing antibodies161 and a phase I trial has been done. Other vaccines include canarypox gB and pp65 vaccines that produce T-cell responses and neutralizing antibodies.162,163 An adenoviral chimeric vaccine-based replication deficient adenovirus encoding gB and multiple CMV epitopes was able to produce a robust cellular response and neutralizing antibodies in mice.164 In general, CMV vaccines have reached human studies with clinical endpoints. However, because CMV vaccines are still in early stages of clinical development, no specific recommendation for vaccine use are made.
Consensus Statements and Recommendations
- Host genetic markers and immune parameters, such as low lymphocyte counts, complement levels and/or NK cell counts as well as T cell immune functional assays, have shown association with higher incidence of CMV infection. Measurement of such parameters could potentially be used to inform the risk of infection although no interventional studies using these have been performed.
- CMV vaccines are in preclinical, phase I and phase II trials. The primary goal of a CMV vaccine should be to prevent or modulate CMV replication and/or CMV disease. Surrogate endpoints (eg, reduction in viral replication, T-cell immunity) can be used to evaluate vaccine immunogenicity and efficacy.
- T-cell therapies should be further evaluated for resistant/refractory CMV.
- IgG hypogammaglobulinemia is associated with an increased risk of CMV disease after transplantation. Measurement of total immunoglobulins is suggested in situations where CMV is difficult to control (weak, low).
- CMV-specific cellular immune monitoring has been shown to predict CMV infection in the pretransplant and posttransplant settings in prospective observational, multicenter studies. Accordingly, assessment of CMI can be used to inform risk of CMV (strong, moderate). Interventional studies to determine precise clinical utility of CMI are ongoing.
The following future research directions are important for the further development of immune monitoring and CMV vaccines:
- Immune monitoring assays should continue to be standardized. Cut-off values need to be established for ELISpot and ICS assays. In addition, regulatory T cells and γδ T cells may have predictive value for DNAemia and should continue to be studied for incorporation into immune monitoring strategies.
- Clinical utility studies of immune monitoring are needed that demonstrate that alteration of patient management based on the results of an immune-based assay is feasible, safe, and cost-effective.
- Studies are also needed to determine the comparative performance of immune monitoring assays in the prediction of CMV viremia/disease.
- Further data are needed for the safety and feasibility of adaptive T-cell therapy in organ transplantation.
- For further development of CMV vaccines, the expert panel was of the opinion that (i) given the high frequency of disease in D+/R− transplant recipients, vaccines should be evaluated specifically in this group; (ii) vaccination may also reduce burden of disease or impact the course of latent CMV infection in seropositive patients; vaccination trials should therefore focus on this group also; and (iii) vaccine studies should include an evaluation of both humoral and cellular immunities where applicable as well as longevity of responses.
Given the high frequency of primary CMV infection and reactivation, prevention strategies are of paramount importance as they augment transplant clinical success and outcomes, by decreasing the risk of CMV infection and disease, as well as the associated “indirect effects.” Universal prophylaxis and preemptive therapy are the main approaches for prevention. An additional strategy that combines both of these approaches is “surveillance after prophylaxis” (termed “hybrid approach” in prior guidelines). Herein, we cover both standard prevention methods (Table 4), as well as additional tactics. Additional information is available on thresholds for initiating treatment, and we have a better understanding of the lower risk of CMV infection with mammalian target of rapamycin (mTOR) inhibitors in CMV seropositive recipients.
Universal prophylaxis entails the administration of antiviral medication to all patients or a subset of “at-risk” patients, starting within 10 days after transplant and continuing for a finite period (ie, 3-6 months). Acyclovir, valacyclovir, intravenous ganciclovir (GCV), oral GCV, and valganciclovir (VGCV) have all been studied for universal prophylaxis. Valganciclovir is currently the most commonly used drug for prophylaxis. In early studies, GCV prophylaxis was found to be more effective than acyclovir.165 Equivalent efficacy was found in a large study of D+/R− transplant patients comparing oral GCV to VGCV (PV16000); however, in small subgroup analysis, tissue invasive disease was noted at increased incidence in liver transplant patients who received VGCV.166 Oral GCV is no longer available. High-dose valacyclovir has been shown to be effective for prophylaxis in kidney transplant recipients.167
Late-onset CMV disease, or disease occurring after the discontinuation of prophylaxis, has occurred in all studies evaluating universal prophylaxis. Late CMV disease is likely related to a lack of CMV-specific CMI in the setting of ongoing immunosuppression. Risk factors for late-onset disease include D+/R− serostatus, shorter courses of prophylaxis, higher levels of immunosuppression, certain types of transplant (eg, lung), and allograft rejection.168,169 In the PV16000 study, where 3 months of prophylaxis was used, 18% had late-onset CMV disease by 12 months (~30% when including investigator treated disease).166 This has led to studies evaluating longer durations of prophylaxis (see below).25
Effective preemptive therapy (PET) involves monitoring for CMV in blood at regular intervals (ie, CMV viral load checked weekly) to detect early viral replication. Once a predetermined assay threshold is achieved (optimally before the development of symptoms), antiviral treatment is begun, which should prevent progression to clinical disease. Better availability and standardization of assays have made this approach more feasible. Nonetheless, given the variability among diagnostic specimens (whole blood versus plasma) and testing platforms,43,170 a universal threshold for starting therapy cannot be defined. It is likely that optimal thresholds are different among different at-risk groups. There was strong consensus that a lower threshold should be used with D+/R−, as the use of higher thresholds may result in insufficient time to begin treatment for CMV and higher rates of disease.171 For example, 1 study demonstrated a median viral load doubling time of 1.54 days (range, 0.55-5.5) in D+/R− compared with 2.67 days (range, 0.27-26.7) in the D+/R+ recipients (P < 0.0001).172 Although prior guidelines mentioned that some experts, given the unpredictable viral kinetics (especially in D+/R−), recommended starting treatment with any detectable DNAemia,172,173 with increasingly sensitive assays many experts felt that very low results should not always result in initiation of treatment, even in D + R− recipients (see Diagnostics).
Advantages of preemptive therapy include a reduced rate of late CMV, selective drug use, and decreased drug cost and toxicities. Preemptive therapy can be difficult to coordinate, given the logistics of weekly testing, reviewing results, initiating therapy rapidly after positive assays, and performing subsequent monitoring and management. Preferably, 1 assay and specimen type, (whole blood or plasma) should be used for an individual patient to ensure comparability of results. Preemptive therapy may not prevent the indirect effects of CMV infection, including effects on graft and patient survival; studies have demonstrated conflicting data.174-177 Second episodes of replication are observed in about 30% of those treated for CMV DNAemia,178 so the specter of late CMV remains. Patients managed with the preemptive approach should receive oral acyclovir (or similar) for the prevention of disseminated herpes simplex infections.179
Universal Prophylaxis Versus Preemptive Therapy
Four randomized trials directly compared universal prophylaxis with preemptive therapy in renal transplant recipients.174-177 In studies using weekly monitoring for 4 months posttransplant with reported high compliance rates, no difference in the incidence of CMV disease or intragraft CMV infection was found compared to universal prophylaxis.174,175,180 Comparable long-term graft survival was observed with preemptive therapy with a similar incidence of other CMV indirect effects181; PET was better when compared with valacyclovir prophylaxis.182 It should be noted that less frequent screening (ie, less than weekly) resulted in higher rates of CMV disease and inferior long-term graft survival compared to prophylaxis.176,177
There is no randomized study directly comparing preemptive approach and prophylaxis in liver transplantation. Several meta-analyses with significant numbers of liver transplant recipients have confirmed similar efficacy in prevention of CMV disease and no difference in mortality, graft loss, and acute rejection. As expected, CMV DNAemia is more common with preemptive therapy, whereas late-onset CMV DNAemia or disease and neutropenia is more common with prophylaxis.183-185 Other herpes viral infections are more common with the preemptive strategy.185 A large prospective national cohort study found an increased risk of graft loss in patients managed by preemptive strategy, with notably short follow-up (~1 year).186 In contrast, the 4-year pooled analysis of patients included in 2 randomized studies did not identify the mode of CMV prevention (preemptive vs valacyclovir prophylaxis vs VGCV prophylaxis) as an independent risk factor of graft loss. Cytomegalovirus DNAemia with viral load of more than ~2000 IU/mL in whole blood was a strong predictor of graft loss.187
Comparison of approaches among D+/R− patients is limited by a low proportion of the D+/R− group in randomized trials.174-176 Nevertheless, the results seemed to be comparable even in higher risk patients. Both a subanalysis of a cohort study186 and meta-analysis185 showed a similar incidence of CMV disease in D+/R− group. Repeated courses of preemptive therapy are sometimes required in high-risk patients.172 In summary, using a preemptive approach with once weekly CMV surveillance for 3 to 4 months and universal prophylaxis are both comparable methods of CMV disease prevention for D+/R− and/or R+ kidney and liver transplant recipients.
The preemptive approach is not well studied in nonrenal and nonliver transplant recipients, thus we suggest universal prophylaxis in D+/R− heart and lung transplant recipients, given the high rates of CMV disease and CMV indirect effects.188,189 There are no studies available to prove efficacy of preemptive approach in R+ lung transplant thus universal prophylaxis is preferred in the majority of lung transplant centers.190
Surveillance After Prophylaxis
For prompt detection of evolving CMV infection (and to decrease the risk of late CMV), the majority of the experts utilize surveillance after prophylaxis at least sometimes. Thus far, research does not support the use of a surveillance after prophylaxis approach,191-194 although studies are limited by short monitoring periods, long interassay intervals, and other methodological issues. Use of surveillance after prophylaxis may be considered in patients at increased risk for postprophylaxis CMV disease. Based on the experience with preemptive therapy, the value is probably greatest if done weekly for 8 to 12 weeks after the end of prophylaxis (weak, low).
Thresholds for Triggering Preemptive Therapy (or Surveillance After Prophylaxis)
Specific viral load thresholds for triggering therapy in asymptomatic patients have not yet been defined, in part because of the significant interassay and interinstitutional variations seen with CMV DNA testing, along with the variable impact of immunity (primary versus reactivation infection, intensity of immunosuppression, impact of various immunosuppressive agents used for induction and maintenance). Although low viral loads are likely to be more clinically significant in those at higher risk for infection, using too low of a viral load threshold can result in unnecessary treatment. Programs may wish to define their own local thresholds based on their assay, specimen type, and patient risk factors. Table 5 highlights recent thresholds used in various research studies.
Some groups have suggested that the kinetics of the CMV DNA doubling time may be a valuable diagnostic parameter for PET, given that real-time quantitative reverse transcription PCR (qRT-PCR) assays show linearity above their limit of quantification, and may allow for direct comparison of results obtained across centers using similar or different qRT-PCR assay.199-201 Viral replication kinetics have shown that viral load in the first surveillance sample and the rate of increase defines the risk of subsequent CMV disease; this risk is different in CMV-naïve patients undergoing primary CMV infection versus those with prior immunity.30,202 This approach requires more frequent testing (at least biweekly).
Optimal Duration of Prophylaxis
Seropositive recipients generally need shorter courses of prophylaxis compared with D+/R−. Risk factors for late-onset disease include D+/R− serostatus, shorter courses of prophylaxis, higher levels of immunosuppression, and allograft rejection.168,169,203 In the Improved Protection Against Cytomegalovirus in Transplantation study, a decreased risk of CMV disease was seen in patients given 200 days of prophylaxis (21.3%) in D+/R− kidney recipients compared with those given 100 days of prophylaxis (36.8%).169 Extending CMV prophylaxis from 6 to 12 months in pediatric kidney-transplant patients, however, did not prevent CMV infection or disease.204 Long courses of prophylaxis with VGCV are associated with higher rates of leukopenia and greater cost.
Longer prophylaxis may be warranted in D+R− lung recipients. In a study of 136 lung transplant recipients (including both D+/R− and R+), VGCV prophylaxis for 12 months versus 3 months was associated with a significantly lower CMV infection and disease incidence.205 In a study of 6 months of prophylaxis, almost 50% of D+/R− lung transplant patients developed late-onset CMV infection or disease.203 Many programs have moved towards 6 to 12 months of prophylaxis.188,203,205-207 Serostatus of both donor and recipient at the time of transplant may help guide the duration of prophylaxis; after lung transplant and 6 months of prophylaxis, 34% of D+/R+ and only 6% of D−/R+ developed infection or disease.203 Some programs continue prophylaxis indefinitely after lung transplant, although there is insufficient data to support this approach.208,209
High-dose valacyclovir is effective for prevention of CMV disease and CMV DNAemia in both D+/R− and D±/R+ renal transplant recipients.167,210 Apart from comparable efficacy to oral GCV prophylaxis,211,212 a recent randomized study showed similar rates of CMV DNAemia compared with VGCV prophylaxis.213 A lower incidence of acute rejection and higher rate of polyomavirus viremia was observed with VGCV.213,214 Disadvantages of valacyclovir include high pill burden and neuropsychiatric side effects, which may be decreased if valacyclovir initiation is postponed in patients with delayed graft function. Advantages of valacyclovir include less myelotoxicity and lower cost.210,213,215
New Drugs for CMV Prevention
New potent oral drugs would be useful additions to the currently available therapies for CMV prevention. Three compounds are in various stages of late development. Letermovir inhibits the viral terminase enzyme complex encoded by UL56, does not share cross-resistance with GCV,216 and has both an oral and intravenous formulations. Letermovir has demonstrated efficacy and safely in HSCT patients217,218 and is commercially available for use as prophylaxis in HSCT recipients. A phase 3 trial for letermovir prophylaxis in kidney transplant recipients is just beginning. Maribavir is an oral drug that inhibits CMV UL97 and plays a role in inhibition of viral maturation and egress. It also appears to have a good safety profile with no evidence of myelosuppression or nephrotoxicity. Early studies for use in prophylaxis after SOT failed to demonstrate efficacy219; ongoing studies are evaluating higher doses for treatment, either for DNAemia or for patients with refractory/resistant CMV. Neither letermovir nor maribavir covers HSV and VZV, so additional antiviral prophylaxis may be needed. Brincidofovir is a lipid conjugated prodrug of cidofovir (CDV) with broad antiviral efficacy, including potent antiherpesvirus activity, through inhibition of viral DNA polymerase. It has not been evaluated for prophylaxis in SOT patients. Although there was preliminary evidence of benefit in HSCT patients,220 significant gastrointestinal toxicity in the phase III trial led to the discontinuation of a planned prophylaxis trials in kidney transplant recipients. An intravenous formulation is being developed in hopes of reducing toxicity.
Prevention Strategies for CMV D−/R−
When both donor and recipient are seronegative for CMV, there is minimal risk of CMV infection, and routine prevention of CMV is not recommended. Antiviral prophylaxis against other herpes infections (especially disseminated varicella and herpes simplex) with acyclovir, famciclovir, or valacyclovir should be considered.
Prevention During (Massive) Blood Transfusion
To avoid transfusion-transmitted CMV, we recommend the use of leukoreduced or CMV-seronegative blood products (strong, moderate). The highest risk is in D−/R−, although there may be a contribution in R+. Additional clinical benefit of combining these 2 strategies is not available.221 Extensive transfusion of blood products increases the risk of CMV disease (especially if not CMV screened or leukoreduced), and transplant centers may wish to monitor such recipients with weekly viral load testing or give CMV prophylaxis (weak, very low).
Secondary Prophylaxis and Recurrent Infection
There have been no prospective randomized trials of secondary prophylaxis for prevention of recurrent CMV infection in transplant recipients following resolution of CMV infection and/or disease. Risk factors for recurrent CMV among study populations have not been adequately defined. In particular, donor and recipient serostatus, organ transplanted, choice of immunosuppression, rejection and its treatment, initial prophylactic strategies, the presence of renal insufficiency, severity of disease, and the presence of immune reconstitution have varied in those individuals experiencing recurrent infection.108,178,222-224 Notably, risk factors for recurrent infection may differ from those associated with primary infection or disease.223,224 Retrospective analyses conducted in diverse transplant populations have not demonstrated a benefit to secondary prophylaxis.108,222,223
Recurrent DNAemia upon completion of treatment and/or preemptive therapy occurs, particularly in high-risk transplant patients,173,195 although viral load replication may be slower and subsequent peak viral load significantly lower than the initial episode,172 possibly due to an evolving immune response. Although many transplant centers treat all recurrent episodes of CMV DNAemia, irrespective of a relevant threshold,173 some episodes of CMV DNAemia may resolve spontaneously.195
Prevention During Treatment of Rejection
Prophylaxis may be preferred over preemptive therapy in certain high-risk patients, including those who have received recent antilymphocyte therapy, potent immunosuppression including desensitization or ABO incompatible protocols (including those on rituximab, bortezomib, eculizumab, and plasmapheresis/immunoadsorption), and those with human immunodeficiency virus (HIV) (weak, moderate).225 Although a retrospective cohort study (2007-2012) found no significant difference in the incidence of CMV infection in patients with or without acute rejection (13% vs 10%, P = 0.37) 6 months after kidney transplant, the incidence of tissue-invasive CMV disease (8% vs 3%, P = 0.04), particularly gastrointestinal CMV disease, was significantly greater in patients with acute rejection.226 In a retrospective cohort of 15 848 adult kidney transplant recipients between 2004 and 2010, early-onset CMV disease was identified in 1.2% and delayed-onset CMV disease in 4.0% of the kidney transplant recipients; risk factors for delayed-onset CMV disease included transplant failure or rejection (HR 3.2).227
Prevention During Critical Illness
Critical illness (including mechanical ventilation and septic shock) conveys an increased risk of CMV infection in nontransplant patients, with associated increased mortality.228,229 The duration of acute illness (not severity) plays a role in CMV reactivation.230 A randomized trial of GCV prophylaxis in CMV-seropositive adults with critical illness due to sepsis or trauma did not support its routine clinical use in that population.231 Another trial in a critical illness population found that valacyclovir or low-dose VGCV prophylaxis suppressed CMV reactivation, but demonstrated higher mortality in the valacyclovir arm.232 Transplant clinicians should be aware of the increased risk of CMV infection with critical illness in normal hosts, as there may be a similar effect in transplant recipients; however at this time there is insufficient data to support routine use of antiviral prophylaxis or enhanced monitoring in critically ill transplant recipients.
Some centers, in hopes of improving tolerability and reducing costs, employ half the recommended dose of VGCV for prophylaxis (ie, 450 mg daily in patients with normal renal function, sometimes called “mini-dosing.”233 This is based on pharmacokinetic data showing comparable GCV exposure with VGCV 450 mg and oral GCV 3 g daily.234,235 Published comparisons of low-dose versus standard-dose VGCV are limited to retrospective observational studies in kidney transplant recipients.236-238 These studies suggest similar CMV outcomes in intermediate risk patients (D+R+); however, data are less clear in D+R− who are at higher risk for breakthrough disease and development of resistance.236 Whether reduced VGCV exposure was achieved in any of these studies is unclear because compliance with renal dosing protocols cannot be assumed, and immunosuppression may not have been standardized. There are no comparative studies of low versus standard-dose VGCV in nonrenal transplant recipients.
CMV Immune Globulin
Cytomegalovirus immune globulin was first licensed for use in prevention of primary CMV disease in renal transplant recipients.239,240 Its role in prophylaxis has been limited in the era of antivirals, primarily due to limited effectiveness as a single agent compared to GCV or VGCV, as well as expense and infusion related toxicity (weak, moderate). It may be useful for prevention in combination with antivirals in higher risk (CMV D+/R−) lung or small bowel transplant recipients, based on data in cohorts compared to historical controls (weak, low).206,241,242 The combination demonstrated reduced incidence of CMV disease, and bronchiolitis obliterans syndrome (chronic rejection), with improved survival in a cohort of lung transplant recipients (weak, low).243 Use of the combination has been based, in part, on the demonstration of synergy between antibody (serum) and GCV in animal models of lethal CMV infection.244 Adequately powered, randomized trials measuring the additive benefit have not yet been performed. Cytomegalovirus immune globulin has no role for CMV prophylaxis in lung transplant recipients when given alone. Cytomegalovirus immune globulin has been used in patients for prophylaxis with prolonged neutropenia who are intolerant of GCV. It has also been used in patients with refractory CMV disease and hypogammaglobinemia.101 In summary, CMV Ig is not generally recommended for use, although there may be specific circumstances, especially in thoracic organs, when used in combination with antivirals, in which some benefit has been demonstrated.
There may be subtle in vitro differences between selected immune globulin products with respect to titers of CMV antibody but clinical differences have never been evaluated or demonstrated. In vitro studies show consistently enhanced functional antibody against CMV compared to regular intravenous immune globulin.245,246 Monoclonal antibodies are in development and show potential promise for reduction of CMV DNAemia and disease.247,248
Role of mTOR Inhibitors
Two systematic reviews and meta-analyses showed that the incidence of CMV infection/disease is lower among patients receiving immunosuppressive regimens containing mTOR inhibitors, suggesting that less prevention may be needed in such patient.249,250 These findings are consistent in kidney (high, strong),251,252 and in liver,253 heart,254-256 lung,257-259 and pediatric kidney transplant recipients260 (all moderate, strong).261 For example, in a single-center, prospective study, among 288 patients randomized to receive 1 of 3 immunosuppression regimens, the lowest incidence of CMV infection and disease was observed in patients who received either of 2 everolimus-based regimens, with a proportional reduction in CMV of 90% and 75%.262
Consensus Statements and Recommendations
- We recommend either universal prophylaxis or preemptive therapy for prevention of CMV disease (Table 6) (strong, high).
- For D+/R−, we recommend the use of either prophylaxis or preemptive therapy after kidney and liver transplant (strong, high). For programs or patients unable to meet the stringent logistic requirements required with a preemptive therapy strategy, prophylaxis is preferred.
- For D+/R−, we suggest the use of prophylaxis over preemptive therapy after heart and lung transplant, based on the available data suggesting better graft survival and clinical outcomes (weak, low). Preemptive therapy has not been well studied in pancreas, islet, intestinal, and vascularized composite allotransplantation (ie, hand, face) such that prophylaxis may be preferable over preemptive therapy until more data are available (weak, very low).
- For seropositive recipients (R+) after kidney or liver transplant, we recommend either strategy (strong, high). Preemptive therapy has not been well studied in some seropositive populations including lung, heart, vascularized composite, pancreas, islet, and intestinal transplant; we suggest prophylaxis may be preferable (weak, low).
- We suggest prophylaxis may be preferred in donor and/or recipient seropositive patients whose risk for CMV may be increased, including those on recent antilymphocyte therapy, potent immunosuppression including desensitization or ABO incompatible protocols (including those on rituximab, bortezomib, eculizumab, and plasmapheresis/immunoadsorption), and those with HIV; a longer duration of prophylaxis (ie, 6 months) may be more effective (weak, moderate).
- The choice of CMV prevention method should be determined by the individual center, based on CMV disease incidence, immunosuppression, logistics of CMV surveillance, and economic aspects.
- We recommend against routinely monitoring patients for CMV DNAemia while they are receiving standard-dose prophylactic therapy based on renal function (Table 7) (strong, moderate).
- Use of surveillance after prophylaxis may be considered in patients considered at increased risk for postprophylaxis CMV disease (weak, low). The value is probably greatest if done weekly for 8 to 12 weeks. Biweekly or monthly monitoring is insufficient for preemptive interventions (low, weak).
- With preemptive therapy, we recommend monitoring at least once weekly for 3 to 4 months after transplant; longer monitoring would be indicated if they are perceived to be at ongoing increased risk for CMV disease (strong, moderate).
- Programs should develop their own thresholds for initiating treatment in preemptive therapy; this value may be different for various populations.
- Once DNAemia is at a positive threshold, for asymptomatic patients, we recommend VGCV (treatment dose) be started [strong, high] and continued until resolution of DNAemia (as per the Diagnostic section), with a minimum of 2 weeks of treatment. After resolution, discontinue antiviral and continue weekly surveillance. Intravenous GCV is a less preferred option unless concerns about absorption exist.
- If symptomatic, see Treatment section of these guidelines.
Prophylaxis Strategy D+/R−: Recommended Durations
- For D+/R− kidney recipients, prophylaxis for 6 months is preferable (strong, high).
- In D+/R− liver, heart, and pancreas recipients, prophylaxis should be 3 months (strong, moderate) to 6 months (strong, low).
- For D+/R− islet recipients, prophylaxis for 3 months is recommended (weak, low).
- Between 6 and 12 months prophylaxis is recommended for D+/R− lung transplant recipients (strong, moderate).
- A minimum of 6 months of prophylaxis is recommended for D+/R− vascularized composite (ie, hand and face) and intestinal transplant recipients (weak, low).
- When a range is given, the duration of prophylaxis may depend on degree of immunosuppression, including the use of antilymphocyte antibodies for induction.
Prophylaxis Strategy R+: Recommended Durations
- When a prophylaxis strategy is used for prevention in R+ patients (with either D+ or D−), a majority of the experts felt that 3 months of antiviral medication should be used for routine kidney, pancreas, liver, and heart transplant recipients (strong, high/moderate) and islet (weak, low).
- For those receiving more potent immunosuppression (antilymphocyte antibody therapy, desensitization protocols) or vascularized composite and intestinal transplant recipients, between 3 and 6 months of prophylaxis can be used (weak, low).
- In R+ lung transplant recipients, a minimum of 6 months prophylaxis is recommended (strong, moderate).
Additional Consensus Recommendations in Prevention
- In CMV D−/R−, antiviral prophylaxis against other herpes infections (varicella and herpes simplex) with acyclovir, famciclovir, or valacyclovir should be considered (strong, high).
- To avoid transfusion-transmitted CMV, we recommend the use of leukoreduced or CMV-seronegative blood products (strong, moderate) especially in the highest risk group, D−/R−.
- Given the potential toxicity and cost, we do not recommend the routine use of secondary prophylaxis after treatment of CMV infection or disease (low, weak). We would consider either secondary prophylaxis or preemptive therapy in certain higher risk situations, that is, potent immunosuppression, augmented risk of complications from recurrent CMV, or inability to monitor closely due to extenuating circumstances (weak, low).
- Local practices should guide management of recurrent episodes of DNAemia or utilization of relevant thresholds to initiate therapy given that there is insufficient evidence for an optimal or cutoff or threshold (weak, low).
- We recommend that treatment of rejection with antilymphocyte antibodies in at-risk recipients should result in reinitiation of prophylaxis or preemptive therapy for 1 to 3 months (weak, moderate)266-268; a similar strategy may be considered during treatment of rejection with high dose steroids or plasmapharesis (weak, very low).
- CMV Ig is not generally recommended for use, although there may be specific circumstances, especially in thoracic organs, when used in combination with antivirals, some benefit has been demonstrated (weak, low).
- We do not recommend the routine use of low-dose VGCV (weak, low).
- CMV seropositive recipients receiving mTOR inhibitors have a significantly lower incidence of CMV infection/disease. We suggest the use of mTOR inhibitors as a potential approach to decrease CMV infection and disease in CMV seropositive kidney transplant recipients (high/strong) and in liver, heart, and lung transplant recipients (moderate/strong). Cytomegalovirus risk is only one of the factors to consider when deciding on the optimal immunosuppression regimen. The impact of mTOR inhibitors on CMV in D+R− recipients is less clear.
- Despite many centers successfully deploying preemptive therapy strategies, no clear universal thresholds have been defined. In the absence of natural history studies, these may be difficult to define in the current era.
- Future studies should assess potential ways to improve the efficacy of the surveillance after prophylaxis strategy such as more stringent monitoring, optimal thresholds for initiating antiviral therapy, use of viral kinetics, and the adjunctive use of immunodiagnostic assays.
- Studies investigating the incidence of opportunistic infections, including CMV, after treatment of acute rejection are needed. While the majority of early acute rejection episodes occur during the period of CMV prevention, a growing number of patients diagnosed with late acute rejection (cellular, antibody mediated or mixed rejection) treated with various effective immunosuppressive strategies (thymoglobulin, rituximab, plasmapheresis, bortezomib, eculizumab) requires reevaluation of CMV prevention strategies.
- We await more data on the use of novel antiviral agents for CMV prevention, including maribavir, letermovir, and brincidofovir.
- Pathogen inactivation techniques may further reduce the risk of transfusion-related CMV transmission.
- More data are needed in nonkidney solid organ transplant and in high risk (D+/R−) recipients, both on the incidence of primary and reactivation CMV episodes and on the efficacy and safety of conversion to mTOR inhibitors after the first episode of CMV infection and disease. A large independent confirmatory trial is underway in kidney transplant recipients (TRANSFORM trial).
Oral VGCV and intravenous GCV treatment are associated with similar long-term outcomes in SOTRs with CMV syndrome and tissue-invasive CMV disease based on the VICTOR study conducted in adult renal, liver, heart, and lung transplant recipients.178 Although both may be used for non–life-threatening CMV disease, VGCV is preferred when feasible due to its oral formulation which may prevent or reduce hospital stays and minimize the infectious and vascular complications associated with intravenous therapy. However, intravenous GCV is preferred as initial treatment of life-threatening CMV disease when optimal drug exposure is essential. Furthermore, there are limited pharmacokinetic data to confirm adequate VGCV bioavailability in patients with gastrointestinal CMV disease.
Appropriate antiviral dosing is essential in the management of CMV disease (Table 7). Suboptimal dosing may increase the risk for clinical treatment failure and the development of resistance,269 whereas supratherapeutic doses may increase toxicity.270 Renal function should be assessed with regular monitoring of serum creatinine. Although the PV16000166 and VICTOR271 trials used the Cockcroft-Gault formula for (val)ganciclovir dosing, as does the package insert, other methods to estimate renal function are more commonly used clinically (such as the Modified Diet in Renal Disease formula and Chronic Kidney Disease Epidemiology Collaboration), and clinicians should be cognizant of this difference. Antiviral dose adjustment based on a population pharmacokinetics Bayesian prediction model may further optimize (val)ganciclovir exposure.272 In addition, complete blood counts should also be monitored in regular intervals to assess for hematologic toxicity (eg, leukopenia).
The intensity of immunosuppressive therapy can impact the treatment outcomes associated with CMV disease.273,274 Multiple studies include a reduction in immunosuppression as a component of therapy. In addition, double (versus triple) immunosuppressive therapy and lower blood concentrations of calcineurin inhibitors are significantly associated with eradication of CMV DNA at 21 days.273
CMV viral load should be monitored at weekly intervals to guide the duration of therapy. Although whole blood is more sensitive than plasma in detecting residual DNAemia, it is not a better predictor of recurrent CMV disease.38 Failure to eradicate plasma DNAemia at the end of treatment is the major significant predictor of virologic recurrence.178 Suppression of plasma CMV DNAemia measured with an assay calibrated to the WHO predicts clinical response to (val)ganciclovir.56 Reduction in antiviral dosing in the setting of persistent CMV DNAemia at day 21 is associated with a significant risk of GCV resistance by day 49.275 Therefore, treatment doses of (val)ganciclovir are recommended until eradication of CMV DNAemia below a specific threshold and resolution of all clinical signs of CMV disease. Eradication of CMV DNAemia is defined as below LLOQ on 1 highly sensitive assay (LLOQ < 200 IU/mL; see Diagnostic section), or lack of detection on 2 consecutive less sensitive assays. With use of highly sensitive assays, a completely undetectable viral load may not always be achievable. DNAemia may not accurately reflect the clinical disease status in all situations. Therefore, longer courses of treatment may be needed in the treatment of tissue-invasive gastrointestinal disease, pneumonitis in lung transplant recipients, and central nervous system or retinal disease. Secondary prophylaxis, defined as continuing prophylactic doses after discontinuing treatment dosing, is not associated with fewer relapses after suppression of CMV DNA.60,108,222,275a
Consensus Statements and Recommendations
- For initial and recurrent episodes of CMV disease, VGCV (900 mg every 12 hours) or intravenous GCV (5 mg/kg every 12 hours) are recommended as first-line treatment in adults with normal kidney function (strong, moderate).
- Valganciclovir is recommended in patients with mild to moderate CMV disease who can tolerate and adhere to oral medication (strong, moderate).
- Intravenous GCV is recommended in life-threatening and severe disease (strong, low).
- Oral GCV, acyclovir or valacyclovir are not recommended for the treatment of CMV disease (strong, moderate).
- Adjunctive immunoglobulin therapy is not routinely recommended (strong, low).
- In patients without concomitant rejection, reduction of immunosuppression is suggested in the following settings: severe CMV disease, inadequate clinical response, high viral loads, and cytopenia (weak, very low).
- During the treatment phase, weekly plasma CMV DNA testing is recommended using an assay calibrated to the WHO standard to monitor response (strong, high).
- During the treatment phase, frequent monitoring of renal function is recommended to guide dosage adjustments (strong, moderate).
- Antiviral dosage reductions are only recommended during the treatment phase to adjust for worsening renal function as suboptimal dosing may be associated with increased risk of clinical failure and/or resistance (strong, low).
- In the setting of leukopenia, changing (val)ganciclovir to another agent is not recommended before the addition of granulocyte colony stimulating factor and/or discontinuation of other myelosuppressive therapies (strong, low).
- In patients who are intolerant to (val)ganciclovir during the treatment phase, foscarnet (FOS) is the recommended second-line agent (strong, very low).
- After clinical response, intravenous GCV may be transitioned to VGCV in patients who are able to tolerate oral therapy (strong, moderate).
- Due to individual variation in virologic response, change in antiviral agent is not recommended in clinically improving patients with unchanged or rising DNAemia during the first few weeks of therapy (strong, moderate).
- Drug resistance should be suspected in patients with a prior cumulative (val)ganciclovir exposure that exceeds 6 weeks and clinical treatment failure despite at least 2 weeks of antiviral treatment or development of CMV DNAemia during prophylaxis (see Resistance section)(strong, moderate).
- Antiviral treatment dosing should be continued for a minimum of 2 weeks, until clinical resolution of disease and eradication of CMV DNAemia below a specific threshold (LLOQ < 200 IU/mL) on 1 or 2 consecutive weekly samples (strong, moderate).
- Secondary prophylaxis is not routinely recommended (low, weak).
Prospective studies in the following areas are needed to evaluate novel strategies to optimize the treatment of CMV disease:
- The application of CMV immune assays to guide duration of treatment.
- The role for measuring and repleting immunoglobulins.
- The role for switching to an mTOR inhibitor.
- The role of therapeutic drug monitoring of GCV.
ANTIVIRAL DRUG RESISTANCE
Drug resistance is defined as a viral genetic alteration that decreases susceptibility to 1 or more antiviral drugs. It may manifest as a persistent or increasing viral load or symptomatic disease after a normally effective dosage and duration of antiviral therapy. These clinical features do not in themselves imply that drug resistance is present, but are used in the context of relevant host factors to assess the need for diagnostic testing and alternative therapy.
Risk Factors, Frequency, and Clinical Consequences
Risk factors for drug resistance include prolonged antiviral drug exposure (median, 5 months for GCV) and ongoing active viral replication due to factors such as the lack of prior CMV immunity (D+/R−), strongly immunosuppressive therapy, or inadequate antiviral drug delivery.276,277 Among solid organ recipients the usual incidence of resistance after GCV therapy is 5% to 12%,278-280 but up to 18% in lung,275,281 and 31% in intestinal and multivisceral organ transplant recipients.282,283 The incidence of resistance is lower, in the 0% to 3% range, for 100 to 200 days of GCV or VGCV prophylaxis in D+/R− kidney recipients.284 A higher incidence of GCV resistance has sometimes been reported with preemptive therapy as compared with prophylaxis where no viral load is expected at baseline.171,279 The incidence of FOS and CDV resistance in transplant recipients is not well defined, but expected to be comparable to GCV based on early studies in the AIDS setting.285 The relative incidence of resistance to currently experimental drugs, such as maribavir and letermovir, is unclear from the few case reports so far.216,286,287 Drug-resistant CMV infection may range from asymptomatic and resolving without treatment change (eg, during antiviral prophylaxis284) to severe or fatal end-organ disease.288 The development of drug resistance is correlated with increased morbidity and mortality.277,289,290
Diagnosis of Drug Resistance
When to Test
Antiviral drug resistance should be suspected and tested for when there is persistent or recurrent CMV DNAemia or disease during prolonged antiviral therapy. For GCV, prolonged therapy generally means 6 or more weeks of cumulative drug exposure, including longer than 2 weeks of ongoing full dose therapy at the time of evaluation.277,278 With adverse host factors and/or high starting viral loads, drug resistance can occasionally be detected after less than 6 weeks of drug exposure, as reported in several lung recipients.277 In a large GCV and VGCV treatment trial,54 the viral load declined with a median half-life of ~11 days after an initial lag of ~6 days before any reduction was noted. The median time to viral load below 200 copies/mL was 21 days; longer with starting viral loads of more than 50 000 copies/mL. Thus, persistent viral loads in the first 2 weeks of treatment are not predictive of emerging drug resistance,173,275 but failure to achieve significant viral load reduction beyond this period may reasonably be interpreted as an unsatisfactory response. The kinetics of viral load response and risk for early emergence of resistance may differ with newer antiviral drugs.
How to Test
Testing of CMV culture isolates for antiviral susceptibility by reduction of plaque counts under increasing drug concentrations291 is technically impractical because of poor assay standardization, slow turnaround and decreasing availability of culture isolates. Instead, genotypic assays for viral drug resistance mutations are performed on viral sequences directly amplified from blood (whole blood, plasma or leukocytes), fluids (urine, cerebrospinal, lung, eye), or tissue specimens. The same blood specimens used for CMV QNAT are usually tested by amplification and sequencing of viral DNA. Results are more reliable if the CMV copy number in the specimen is at least 1000 IU/mL.292 Quality control concerns include false positive detection of mutations, especially as mixed populations from low viral load specimens,293 and false negatives due to insensitivity in detecting mutant subpopulations comprising less than 20% to 30% of the total.292,294 Evolving deep sequencing technologies offer the potential of detecting far smaller mutant subpopulations.292,294-296 These early reports have not yet established properly calibrated, adequately sensitive, reproducible and stable technical platform to serve as a base for clinical validation, including comparison with existing technology. There are reports of discordant findings of resistance mutations at different body sites (eye, cerebrospinal fluid).287,297,298 Indications of progressive disease at tissue sites despite negative findings in blood specimens may warrant the genotypic testing of tissue-specific specimens.
Gene Regions to Test
These will evolve as new mutations are better characterized and drug targets are developed. There is an increasing database of CMV mutations associated with drug resistance.299-301
In patients initially treated with (val)ganciclovir, UL97 kinase gene mutations appear first in about 90% of cases, affecting drug phosphorylation that is necessary for antiviral action.276-278,281 UL54 DNA polymerase gene mutations usually evolve later, conferring increased GCV resistance and likely cross-resistance to CDV and/or FOS, but may uncommonly be the first mutation detected. Given increasing availability, genotypic testing after GCV exposure should now routinely include both the UL97 and UL54 genes. Ideally, testing should cover the entire UL97 and UL54 coding sequence, but many diagnostic laboratories test for more limited codon ranges, such as 450 to 650 for UL97, and 300 to 1000 for UL54, which cover the most common mutations but omit some unusual loci where resistance mutations have been reported (Figure 1, Table 8).
Interpretation of Genotypic Data
Test results are reported by diagnostic laboratories as a list of mutations detected (sometimes as a mixture of mutant and wild type sequences) and usually an indication of whether the detected mutation confers resistance to particular drug(s).303 There is lack of standardization as to the level of drug resistance conferred by the listed mutations, and whether reports include sequence variants that are not in a list of resistance mutations maintained by the laboratory.
Most UL97 mutations conferring GCV resistance are strongly clustered at codons 460, 520, or 590 to 607, although atypical loci exist.276,299,301 The 7 most common (“canonical”) mutations (Table 8) account for over 80% of cases. Other UL97 mutations may confer varying degrees of GCV resistance (Table 8). UL97 mutations do not affect FOS or CDV susceptibility. UL54 drug resistance mutations tend to occur in the conserved functional domains and usually confer cross-resistance to other drugs (Figure 1), such as the GCV-CDV dual resistance of exonuclease domain mutations and the low-grade GCV-FOS cross-resistance of DNA polymerization (palm and finger) domain mutations. A few single UL54 mutations can confer resistance to all 3 drugs.276
Individual mutations are matched to their associated levels of drug resistance by recombinant phenotyping, a research technique whereby a mutation is transferred by recombination into a baseline laboratory strain (marker transfer) and the resulting virus is tested against antiviral drugs (phenotyping) by a standardized assay calibrated to baseline and drug-resistant control strains.276 The level of resistance is reported as the change in the drug concentration that reduces viral growth by 50% (EC50). For GCV, EC50 increases of twofold to fivefold may be considered low-grade, fivefold to 15-fold considered moderate (a level that may result from a single UL97 mutation), and greater than 15-fold considered a high level that suggests the combined effect of UL97 and UL54 mutations. When both UL97 and UL54 mutations are present, the combined level of GCV resistance may be approximated by multiplying the fold changes in resistance conferred by each mutation alone.304 Mutations that confer slight decreases in susceptibility may significantly increase the overall level of resistance when combined with other mutations.305 Foscarnet resistance mutations typically confer twofold to fivefold increases in EC50 and frequently attenuate viral growth.276 Some common UL54 exonuclease domain mutations confer 10- to 20-fold increases in CDV EC50.276
Uncharacterized sequence variants without a documented phenotype cannot be presumed to be resistance-related without careful analysis of such factors as sequencing quality control, evolution in serial specimens, treatment history, proximity to known gene mutations, and corroboration by recombinant phenotyping.293
Alternate Therapy for Drug-resistant CMV
No controlled trial data define a best practice for selection of alternate therapy when suspected or confirmed drug resistance is present based on clinical risk factors or genotypic testing. An updated algorithm (Figure 2) is based on consensus expert opinion but its application to individual cases must take into account host factors that strongly influence outcomes and the urgency of treatment changes. Depending on the severity of the CMV disease (whether life or sight threatening) and host risk factors (D+/R−, severe immunosuppression), empiric changes in therapy can be made when drug resistance is suspected, pending return of genotypic resistance data. Only a fraction of cases with clinical suspicion of drug resistance will be genotypically confirmed.278 If laboratory testing returns no evidence supporting drug resistance, emphasis should be given to optimization of host factors and drug delivery rather than switching antiviral medications. Therapeutic drug monitoring may be helpful in adjusting doses to maintain effective drug levels in relation to viral inhibitory concentrations and minimize toxicity,306 although timely availability of antiviral drug assays is limited. Subtherapeutic GCV levels may increase the selection of resistant mutants and risk of treatment failure.307-309 Additional genotypic testing over time, including specimens from diseased body sites or use of validated deep sequencing technology when available, may increase the sensitivity of detection of resistance mutations. Immunosuppressive therapy should be reduced to the lowest feasible amount, and adjunctive measures described in the next section can be considered.
Some UL97 mutations confer lower levels of GCV resistance by themselves (Tables 8 and 272) and may be amenable to GCV dose escalation (up to 10 mg/kg every 12 hours) combined with optimization of host factors, if severe disease is not present.310 This is double the standard dose and needs monitoring for bone marrow suppression and dose adjustment for renal function.
Switching to FOS is recommended if a mutation confers higher level GCV resistance, or UL97 and UL54 mutations combine to confer high level GCV resistance and usually CDV cross-resistance. Foscarnet salvage therapy is often successful at least initially,279,290 but metabolic and renal toxicities may impair eventual treatment outcomes.311 There is insufficient information on the efficacy of CDV as salvage therapy in SOT.312,313 Nephrotoxicity is dose-limiting. Cidofovir can be considered when GCV and FOS dual resistance is detected without CDV resistance, but the rapid development of viral load relapse and new CDV resistance mutations has been repeatedly documented in case reports,287,294,314 probably related to undetected subpopulations of cross-resistant mutants selected during prior GCV therapy.294 High-dose GCV may also be an option for situations where FOS resistance is present without associated high-level GCV resistance.
Adjunctive treatments, defined as those without a specific CMV antiviral drug target, have not been adequately evaluated. Cytomegalovirus Ig (or IVIG) and adaptive infusions of CMV-specific T cells146,315 may improve antiviral host defenses. Several drugs used for other purposes, including mTOR inhibitors (sirolimus and everolimus), leflunomide, and artesunate, have anti-CMV effects in vitro that may sometimes act synergistically with conventional antivirals.316,317 Switching immunosuppressive therapy to an mTOR inhibitor may be reasonable if otherwise tolerated, based on studies showing a lower incidence of CMV infection and disease.318-320 Leflunomide has been advocated in case reports and small series.321-324 Evidence for efficacy is limited and caution is advised when used for cases of severe disease or with high viral loads. Monitoring for drug metabolite levels and liver toxicity is recommended. Use of artesunate has been the subject of case reports,325,326 with mixed outcomes suggesting a similar degree of caution as with leflunomide.
Experimental CMV Antiviral Agents
Brincidofovir (CMX001) is an orally bioavailable derivative of CDV with improved intracellular active drug delivery and in vitro antiviral potency. Although effective in a phase 2 trial as CMV prophylaxis in HSCT recipients,220 the follow-up phase 3 trial failed primarily because of severe gastrointestinal toxicity,327 and the drug is not currently available or being developed for CMV treatment. If eventually marketed for some other indication, case reports of successful brincidofovir salvage therapy328 may prompt further study. Resistance is expected to involve similar UL54 mutations as CDV.329
Maribavir is an oral benzimidazole L-riboside inhibitor of the CMV UL97 kinase.330 After promising early phase clinical trials, phase 3 trials in HSCT and liver transplant recipients demonstrated no prophylactic efficacy of low-dose (100 mg BID) maribavir.219,331 At higher doses, it has been used as salvage therapy for drug resistant CMV infection, with mixed results.332,333 A phase 2 trial of maribavir (400, 800, or 1200 mg bid) for salvage treatment of refractory and resistant CMV infection has been completed, with indications of success in that approximately 67% achieved clearance of DNAemia within 6 weeks, but infection recurred in approximately 35% of those who cleared.334 Salvage efficacy did not vary significantly among the 3 doses tried. Resistance to maribavir involves mutations in the UL97 kinase that confer moderate to high level resistance, and assorted mutations in the gene UL27 that confer low-level resistance.335 No maribavir resistance was observed in the phase 3 prophylaxis trials,335 but UL97 mutations T409M and H411Y/N appear to be the most commonly detected after maribavir therapy.286,287,333 These mutations confer approximately 80-fold and approximately 12-fold increased maribavir EC50 without GCV cross-resistance. Although the UL97 mutations preferentially elicited by GCV or maribavir do not confer cross-resistance, there are atypical UL97 mutations (Table 8), notably the p-loop mutation F342S that confers GCV and maribavir resistance without severely impairing viral growth fitness.336
Letermovir is a CMV UL56 terminase inhibitor with high in vitro potency, which showed antiviral efficacy in placebo-controlled phase 2 and phase 3 prophylaxis trials in HSCT recipients.217,218 A case report suggests that letermovir salvage therapy and reduced immunosuppression successfully cleared a decreasing viral load after FOS therapy was limited by toxicity,314 but the efficacy of letermovir in treating active CMV infection is unknown. A concern is that high-level resistance to letermovir is readily elicited in vitro with little impact on viral growth fitness, associated with mutations clustered at codons 231 to 261, 325 and 369 of the UL56 gene.337,338 Mutations at codon 325 confer absolute resistance. No cross-resistance with current antivirals is expected. Letermovir resistance mutation UL56 V236M was encountered once in each of the phase 2 and phase 3 prophylaxis trials,216,218 along with other mutations339; additional incidence data are needed to enable comparison with the current DNA polymerase inhibitors.
Consensus Statements and Recommendations
- Interpretation of genotypic resistance testing is based on moderate- to high-quality evidence (depending on the mutation and number of times tested) where recombinant phenotyping data are available, and low-quality evidence where a phenotype is inferred from properties of mutations other than the one being detected.
- We propose a management algorithm (Figure 2); given the lack of controlled trial data to define a best practice for selection of alternate therapy when suspected or confirmed drug resistance is present, the recommendations should be considered “strong, low” for the initial approach to diagnosis, “moderate, low” for selection of alternate therapy, and “weak, low” for CDV and other nonstandard salvage therapies.
- Prospective studies are needed to define the outcomes of drug-resistant CMV under various management options.
- New therapeutic options are needed, with adequate potency, bioavailability, and lack of toxicity and cross-resistance with current drugs, including consideration of drug combinations directed at different viral targets.
- Genotypic resistance testing needs improved quality control and standardized reporting, including documentation of the level of drug resistance and cross-resistance conferred by various mutations.
- The role of next generation genotyping technology remains to be defined.
PEDIATRIC ISSUES IN CMV MANAGEMENT
Prevention and treatment of CMV infection and disease in pediatric and adolescent SOTRs present several unique issues described here, incorporating and expanding on previous guidelines.4,5,340
Burden of CMV Disease in Pediatric SOT
There are limited data on the precise disease burden in pediatric SOTRs. Nonuniform approaches to diagnosis, varying definitions of disease, and inconsistent durations of monitoring hamper data interpretation. Epidemiological studies conducted before the advent of prophylactic or preemptive therapy indicated that as many as 40% of pediatric liver and 15% of pediatric kidney transplant recipients developed CMV disease.341,342 With various antiviral prevention strategies, CMV disease initially decreased to 10% to 20% in pediatric liver transplant recipients343 with further declines to 0% to 10% in more recent reports.344-349 Late-onset CMV disease occurred in 6% of pediatric liver transplant recipients. Declines in CMV disease from 24% to 12% also have been documented in pediatric intestinal transplant recipients after the introduction of antiviral prophylaxis.350,351
The incidence of CMV DNAemia after pediatric renal transplantation is approximately 20%, with disease in 1-10%352–354 and late-onset disease in 14%.204 CMV was detected in the blood in 29-32% of pediatric lung transplant recipients in the first year, with CMV pneumonitis in 20%. Late-onset CMV data has not been reported after pediatric lung transplantation. DNAemia occurred in 38% of a small cohort of pediatric heart transplant recipients, with 8-9% developing CMV disease in the Pediatric Heart Transplant Study cohort over the first 5 years posttransplant.355,356
Primary Risk Factors for the Development of CMV Disease in Children
As with adults, the risk of CMV disease in pediatric transplant recipients varies with donor and recipient serostatus. Interpretation of donor and recipient serostatus for infants less than 12 months of age is confounded by the potential presence of transplacentally-acquired maternal CMV antibodies, and by the fact that CMV shedding in saliva or urine among infected infants is intermittent. It is known that CMV DNAemia may occur before transplantation in infants with perinatal or postnatally acquired CMV infection, but it is not known if the presence of CMV DNAemia before transplantation impacts the risk of CMV disease posttransplantation.
Adult and pediatric patients share similar risk factors for CMV disease after SOT, but there are a number of factors that specifically influence the risk of CMV exposure and infection among children. Children are more often CMV naïve at the time of transplant and are therefore more likely to acquire primary CMV infection post transplantation. CMV D−/R− pediatric SOTRs are also more likely to acquire de novo CMV infection from community exposures, such as daycare attendance. As many as 7% of pediatric CMV D−/R− recipients developed primary CMV infection in the first year after transplantation.357
Indirect Effects of CMV in Pediatrics
The nature and definition of the indirect effects of CMV may differ between children and adults. Studies performed in the adult SOT population suggest significant indirect effects including increased risk of fungal and other opportunistic infections, coronary artery vasculopathy and chronic allograft rejection.358 Evidence does seem to support an association between CMV infection and long-term graft function in pediatric kidney transplant recipients. Early studies in pediatric kidney transplant recipients suggested an association between CMV DNAemia and an increased risk of histological graft rejection.353,359 One study was potentially confounded by the presence of EBV coinfection in half of the small sample size.353 A recent multicenter cohort analysis in pediatric renal transplant patients showed a significant association between CMV DNAemia and the decline of graft function at 3 years posttransplant.354 Multivariate analysis of variance investigating the impact of various factors on eGFR revealed that, besides recipients' age at transplantation, CMV replication and acute rejection episodes were significantly associated with a lower eGFR, whereas the type or overall intensity of immunosuppressive therapy was not. The use of antiviral prophylaxis in CMV D+/R− and D+/R+ patients was accompanied by a higher CMV-free survival and a lower eGFR loss than preemptive therapy.354
Studies have not established a clear association between CMV replication and negative outcomes in nonrenal pediatric transplant recipients. In pediatric lung transplant recipients, CMV is associated with increased mortality within the first year of transplantation,357 but an association with chronic allograft rejection and opportunistic infections has not been demonstrated.360,361 In heart transplantation, CMV prophylaxis with either CMV Ig or antiviral agents was associated with decreased mortality.362 Others have reported association between CMV seropositivity and coronary artery vasculopathy,363 but data from the multicenter Pediatric Heart Transplant Study did not demonstrate this association.356 In studies of pediatric liver transplant recipients, CMV replication was not associated with immediate transplant outcome, graft loss, death or with other potential indirect CMV effects such as acute cellular rejection, sepsis, EBV infection, biliary strictures and leakages or vascular complications.344,345,348,364
In summary, available data suggest a significant negative impact of CMV replication on pediatric renal graft function, but this association has not been established in nonrenal pediatric grafts. The lack of evidence linking CMV to substantial indirect deleterious effects in nonrenal pediatric SOTRs, coupled with the potential hematological toxicities associated with antiviral therapy in children,354,365,366 provide a less compelling rationale for prolonged antiviral prophylaxis or avoidance of preemptive therapy in most pediatric SOT.
Immunologic Monitoring in Pediatrics
The potential role of monitoring for general or CMV-specific immune reconstitution has been explored only in uncontrolled and small studies in pediatric SOT.367 Children under 2 years of age with both congenital and postnatal CMV acquisition show decreased CMV-specific CD4 responses,368 although another study reported CMV-specific responses in this cohort.21 Accordingly, it is clear that assays assessing CMV cellular immunity must be validated across the spectrum of ages in the pediatric population. Pediatric-specific studies of T-cell responses and their potential role as biomarkers of risk for CMV disease are needed before introducing these assays into clinical practice (see Immunology section).
Prevention of Pediatric CMV Disease
As a first step in the prevention of CMV disease, the use of leukodepleted or CMV-negative blood products is suggested for special populations (eg, bowel, lung, and heart transplants) and in CMV D−/R− patients. Prevention strategies in pediatric SOTRs include antiviral prophylaxis, preemptive therapy, or a sequential approach of brief prophylaxis followed by viral load surveillance. Although no pediatric trial has directly compared the relative efficacies of these 3 strategies, favorable outcomes for each have been reported among pediatric SOTRs. There is broad collective evidence to support the use of antiviral prophylaxis in pediatric recipients, but its use in young children may be limited by bone marrow suppression and limited VGCV pharmacokinetic data. Conversely, preemptive therapy may avoid the toxicities of antiviral exposure but requires intensive viral load surveillance, and the threshold viral load to prompt antiviral therapy is unknown. The rate of CMV disease has been reported to be as low as 5% in relatively small studies of children managed preemptively.369,370 The sequential approach of a short course prophylaxis followed by viral load surveillance, previously designated the “hybrid approach,” limits the duration of prophylaxis to the period of most intense immunosuppression. This approach has been used successfully in pediatric liver and heart transplant recipients, with reported CMV disease rates of 8-10%.346,355 The reported duration of prophylaxis in this setting typically ranges from 2 to 4 weeks; however, the optimum duration of prophylaxis for a sequential approach has not been defined.346,355,371
Prophylactic intravenous GCV is usually dosed at 5 mg/kg per day. Some centers start with an initial dose of 10 mg/kg in 2 divided doses based on the rationale that a higher (treatment) dose may reduce viral replication within the graft,346,371 but no data support superiority of this approach. In contrast to adults, the standard duration of prophylaxis varies significantly between individual centers and among different organs. In addition to reversible bone marrow toxicity, concerns for prolonged exposure to GCV or VGCV in the very young recipient have been raised due to animal toxicity studies demonstrating carcinogenesis or an adverse effect on spermatogenesis.372 These effects have not been observed in humans, and prolonged IV GCV (12 weeks) has been used safely in pediatric transplant recipients.373
Compared with adults, less data are available to define the role of VGCV in pediatric SOTRs. Studies have addressed pharmacokinetics (PK) in older children,374,375 and emerging data address PK in younger SOT populations, including infants. Current models support body surface area-based dosing to reach targeted GCV AUC as opposed to weight-based dosing.366,376-379 Data evaluating the efficacy of VGCV for the prevention and treatment of CMV in pediatric SOTRs are needed, particularly given concerns for lower than anticipated plasma (and presumably intracellular) GCV levels and the potential subsequent risk for GCV resistance. Absorption issues might be of particular concern in small-bowel transplant recipients. The efficacy and safety of prolonged VGCV prophylaxis has not been the subject of randomized studies in children.
The choice of immunosuppressive agent choice may impact the risk for CMV disease. Subjects who received everolimus with low-dose cyclosporine in a recent multicenter study in pediatric kidney transplant recipients experienced a decreased risk of both CMV infection and disease compared to standard therapy with cyclosporine or tacrolimus with mycophenolate.260
Cytomegalovirus Ig and IVIG are sometimes used in combination with antivirals or preemptive therapy to prevent CMV. Data do not exist to enable a definitive recommendation for or against this practice. Evidence in support of this strategy has been extrapolated from data derived mostly from adult populations; however, some recent pediatric studies have been published with variable results.369,370,380,381 In adult and pediatric heart transplant recipients, Scientific Registry of Transplant Recipients data showed an improvement in recipient and graft survival for those who received CMV Ig with or without antivirals; however, this improvement was not different from that demonstrated with antivirals alone.362,382 Krampe et al369 found a low incidence of CMV disease in 28 pediatric liver transplant recipients receiving IVIG and preemptive therapy but did not have a comparison group. In a retrospective review of 329 pediatric lung transplant recipients, of whom 62 (19%) received CMV Ig in addition to at least 3 weeks of IV GCV, CMV Ig was associated with a decreased risk of CMV infection but did not impact the incidence of CMV disease, acute rejection or early morbidity.380 A beneficial effect of CMV Ig on CMV infection rates was also suggested by a combined pediatric and adult study of intestinal/multivisceral transplant recipients.383 In 1 prospective randomized pediatric study that primarily targeted EBV, CMV Ig did not appear to have a significant impact on the development of CMV disease, although there was a trend toward a higher 2-year CMV disease-free rate in R+ children.343
Treatment of Pediatric CMV Disease
Many of the principles that guide therapy in children are similar to those among adults. There is a significant lack of published data on which to base firm recommendations for the treatment of CMV disease in children, particularly regarding intravenous versus oral therapy. Valganciclovir has been shown to be effective treatment of asymptomatic CMV DNAemia in the setting of preemptive therapy, providing rationale for consideration of its use in mild to moderate CMV disease.354
GCV Resistance in Pediatric Organ Transplantation
Due to the high likelihood of CMV D+/R− status in pediatric SOTRs, GCV resistance is of significant potential concern,384 although published reports from pediatric cohorts report a low incidence of GCV resistance of only 2% to 4%.366,385,386 It is unclear if the reported incidence is due to low resistance burden, lack of generated data, or underreporting. The currently available agents for the treatment of GCV-resistant CMV in children are similar to those used in adults.
Consensus Statements and Recommendations
- In general, the principles that guide the use of prophylaxis in adults are similar in children as defined by the organ transplanted and CMV donor and recipient serostatus.
- Significant updates from prior recommendations relate to body surface area-based dosing for younger children and infants and consideration for initial oral VGCV therapy for CMV infection and mild to moderate CMV disease.
- Some experts perform surveillance during prophylaxis due to concern for breakthrough DNAemia. If surveillance during prophylaxis is performed, the frequency of surveillance should take into account the immunosuppressive regimen (including T cell–depleting induction) and the likelihood of nonadherence with the prophylactic regimen. Adherence can be a particular problem with adolescents.
- Surveillance for CMV DNAemia among patients being managed preemptively or with surveillance after prophylaxis regimens should follow adult recommendations of weekly testing and continue for at least 3 to 4 months posttransplant.
- Children with recurrent CMV DNAemia or disease (at least 2 episodes) may benefit from secondary antiviral prophylaxis. The duration of secondary prophylaxis depends on immunosuppression regimen, age, presence of other opportunistic infections, and other risk factors. Conversion to mTOR inhibitor based immunosuppression in pediatric kidney transplant recipients may provide some protection against recurrent CMV.
- Given the challenge of characterizing donor and recipient serostatus in those less than 12 months of age (due to the possible presence of maternal antibodies), we recommend that risk assessment in this age group should assume the highest risk level for purposes of CMV prevention (strong, moderate) (Table 9). It should be noted that the negative predictive value of a culture or NAT is limited by intermittent CMV shedding.
- No pediatric trials have adequately evaluated the comparative efficacy of prophylaxis, preemptive therapy or surveillance after prophylaxis strategies. Retrospective data provide equal support for each these 3 prevention strategies, and as such all 3 are recommended (strong, moderate). The decision to pursue specific strategies is dependent on organ type and risk stratification (Table 10).
- Use of the VGCV-dosing algorithm that adjusts for body surface area and renal function using the updated Schwartz formula387 provides GCV exposures similar to those established as safe and effective in adults and is recommended in infants and children for prophylaxis (strong, moderate) (Table 11). Recent data strongly supports body surface area (BSA)-based dosing algorithm over the prior suggestion of 16 mg/kg dosing for young infants (strong, moderate).
- The risk of DNAemia is greatest during the 2 to 3 months after discontinuation of antiviral agents (for prevention or treatment). Accordingly, we recommend recipients undergo frequent monitoring for CMV DNAemia for at least this time period (strong, moderate).
- For the treatment of asymptomatic DNAemia, we suggest the use of oral VGCV (strong, low). In addition to patient age, antiviral choice should be guided by early clinical assessment for subtle CMV signs/symptoms, adherence, stable creatinine clearance and oral absorption.
- We recommend the initial treatment of severe CMV disease in children with IV GCV at a dose of 5 mg/kg every 12 hours with appropriate adjustments for renal function (strong, moderate). Some experts consider switching to oral therapy towards the end of their treatment courses (strong, low). The differential effectiveness of IV GCV or oral VGCV for the initial treatment of mild to moderate CMV disease in children younger than 12 years has not been established. Treatment decisions regarding delivery method should be individualized based on age, adherence, and other modifying factors (weak, very low).
- In the management of CMV infection and disease, immunosuppression should be reduced where feasible (strong, low).
- CMV Ig therapy is not routinely recommended for CMV disease (weak, low).
- In children at risk for CMV who receive significantly intensified immunosuppression (eg, antilymphocyte therapy, intravenous steroids) for rejection, primary disease recurrence, or other complicating condition, we recommend either prophylaxis with (val)ganciclovir or preemptive therapy (strong, low). There are no data to suggest a specific duration of prophylaxis in these circumstances.
- Reporting of the epidemiology and outcomes including CMV infection and disease rates with current preventative strategies and delineation of the short- and long-term indirect effects of CMV in pediatric transplant recipients is encouraged. Such reporting should be done using uniform criteria to enable comparisons across studies.
- Additional investigation into the use of biomarkers to aid in risk stratification for purposes of prevention and diagnosis should be explored.
- Additional work is required on the utility of immunogenetic biomarkers and adjunctive immunologic monitoring to guide treatment strategies. Such work should consider age-related immune maturity issues that might influence the optimal performance of assays (eg, interferon gamma release assays).
- Investigation into optimal CMV prevention strategies should include appreciation for the impact on different age groups and the potential consequences of antiviral side effects in pediatric-aged patients.
- Pediatric data should be obtained for emerging antiviral agents to expand the opportunities to prevent and treat CMV.
We have seen major advances in CMV management in SOT (both adult and pediatric) over the past decade, with improved molecular and immunologic diagnostics, reductions in CMV infection and disease, better understanding of treatment, and enhanced knowledge of treatment of resistant virus. Nonetheless, major gaps in our ability to provide optimal care continue to exist, including viral load thresholds to trigger antiviral therapy in asymptomatic individuals, best use of immunodiagnostics and other methods to allow more personalized approaches to CMV prevention and treatment, ideal durations for prophylaxis in individual patients and how to avoid the specter of late CMV, and determination of best candidates for prophylaxis versus PET. Resistant CMV remains challenging, with among the worst outcomes of all patients afflicted by CMV infection. We look forward to more advances, and to the day when we overcome the “transplantation troll.”1
The authors thank Danielle Viau of The Transplantation Society for her administrative support.
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*Consensus Contributors (in alphabetical order).
Leaders: Camille N. Kotton (USA) and Atul Humar (Canada).
Diagnostics: Angela M. Caliendo (leader, USA), Randall Hayden (USA), Hans Hirsch (Switzerland), Tiziana Lazzarotto (Italy), Jutta Preiksaitis (Canada).
Immunology: Deepali Kumar (leader, Canada), Davide Abate (Italy), Oriol Bestard (Spain), Javier Carbone (Spain), Hannah Kaminski (France), Rajiv Khanna (Australia), Martina Sester (Germany).
Prevention: Atul Humar (coleader, Canada) and Camille N. Kotton (coleader, USA), Laura Barcan (Argentina), Emily Blumberg (USA), Jennifer Harrison (Canada), Nassim Kamar (France), Nicolas Mueller (Switzerland), Tomas Reischig (Czech Republic), Nina Singh (USA), David Snydman (USA), Helio Tedesco-Silva (Brazil), David Thomson (South Africa), Marty Zamora (USA).
Treatment: Shirish Huprikar (leader, USA), Jay Fishman (USA), Shahid Husain (Canada), Michele Morris (USA), Claudia Nagel (Argentina), William Rawlinson (Australia).
Resistance: Sunwen Chou (leader, USA), Sophie Alain (France), Guy Boivin (Canada), Klaus Hamprecht (Germany), Ajit P. Limaye (USA).
Pediatrics: Lara Danziger-Isakov (leader, USA), Upton Allen (Canada), Michael Green (USA), Britta Hoecker (Germany), Rebecca Madan (USA), Gustavo Varela-Fascinetto (Mexico).