American Journal of Transplantation 2010; 10: 157–161Wiley Periodicals Inc.

C© 2009 The AuthorsJournal compilation C© 2009 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2009.02861.x

Clinical Predictors of Relapse after Treatmentof Primary Gastrointestinal Cytomegalovirus Diseasein Solid Organ Transplant Recipients

A. J. Eida, S. K. Arthursa, P. J. Dezielb,

M. P. Wilhelma,b and R. R. Razonablea,b,*

aDivision of Infectious Diseases and bThe William J vonLiebig Transplant Center, College of Medicine, MayoClinic, Rochester, Minnesota, MN∗Corresponding author: Raymund R. Razonable,razonable.raymund@mayo.edu

Primary gastrointestinal cytomegalovirus (CMV) dis-ease after solid organ transplantation (SOT) is difficultto treat and may relapse. Herein, we reviewed the clini-cal records of CMV D+/R− SOT recipients with biopsy-proven gastrointestinal CMV disease to determinepredictors of relapse. The population consisted of 26kidney (13 [50%]), liver (10 [38%]) and heart (3 [12%])transplant recipients who developed gastrointestinalCMV disease at a median of 54 (interquartile range[IQR]: 40–70) days after stopping antiviral prophylaxis.Except for one patient, all received induction intra-venous ganciclovir (mean ± SD, 33.8 ± 19.3 days)followed by valganciclovir (27.5 ± 13.3 days) in 18patients. Ten patients further received valganciclovirmaintenance therapy (41.6 ± 28.6 days). The mediantimes to CMV PCR negativity in blood was 22.5 days(IQR: 16.5–30.7) and to normal endoscopic findingswas 27.0 days (IQR: 21.0–33.5). CMV relapse, whichoccurred in seven (27%) patients, was significantly as-sociated with extensive disease (p = 0.03). CMV se-roconversion, viral load, treatment duration, mainte-nance therapy and endoscopic findings at the end oftherapy were not significantly associated with CMV re-lapse. In conclusion, an extensive involvement of thegastrointestinal tract was significantly associated withCMV relapse. However, endoscopic evidence of reso-lution of gastrointestinal disease did not necessarilytranslate into a lower risk of CMV relapse.

Key words: CMV disease, gastroenteritis, rejection,viral decline, viral load

Received 15 July 2009, revised 18 August 2009 andaccepted for publication 02 September 2009

Introduction

In the contemporary era when antiviral prophylaxisis widely used, delayed-onset primary cytomegalovirus(CMV) disease has emerged as one of the most im-portant clinical challenges in the management of CMV-seronegative recipients of solid organ allograft from CMV-seropositive donors (termed D+/R− patient) (1). A largenumber of CMV D+/R− solid organ transplant (SOT) recip-ients present with organ-invasive CMV disease at a delayedonset, and this most commonly involves the gastrointesti-nal (GI) tract (2). It is estimated that GI CMV disease ac-counts for 80% of all cases of organ-invasive disease inSOT recipients (3–5). Intravenous (IV) ganciclovir has beenthe cornerstone of therapy for CMV disease in general(6), and more specifically, for GI CMV disease, where ab-sorption of an orally administered antiviral drug is a poten-tial concern (6). Recently, oral valganciclovir was shownto be effective in the treatment of non life-threateningCMV diseases, including some cases of GI CMV disease,and the long-term outcome of this treatment approachwas not significantly different from standard IV ganciclovirtherapy (7).

The optimal duration of antiviral treatment of CMV diseaseis not known. Current guidelines recommend the use ofCMV load in the blood as a guide to help define the du-ration of therapy (6,8). However, while CMV polymerasechain reaction (PCR) assay in the blood is very helpful inguiding the duration of treatment for CMV syndrome (9),its utility in organ-invasive CMV diseases has been lessthan satisfactory, particularly in cases of localized or com-partmentalized diseases wherein the level of viremia doesnot necessarily reflect the severity of organ involvement.In this regard, our anecdotal clinical experience suggeststhat relapse of infection in cases of GI CMV disease is notuncommon even among SOT recipients with documentedclearance of virus from the blood by CMV PCR. Becausefactors associated with success or failure in the treatmentof GI CMV diseases are not well characterized, we adopteda clinical practice wherein follow-up endoscopic examina-tion of the GI tract was performed to document resolutionof GI CMV disease before discontinuing antiviral therapy.However, whether this practice translates into a lower riskof CMV relapse has not been systematically investigated.To address these issues, we performed a retrospective

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study investigating the outcomes of treatment of GI CMVdisease among CMV D+/R− SOT recipients.

Patients and Methods

Patient population

This retrospective study was conducted during a 5-year period from May1, 2000 to April 30, 2005. During this period, a total of 274 CMV D+/R−patients received kidney, kidney–pancreas, liver or heart transplants at theMayo Clinic in Rochester, Minnesota. This group of patients was chosenbased on known increased risk for the development of delayed-onset pri-mary GI CMV disease. All patients received antiviral prophylaxis with oralganciclovir or valganciclovir for 90–100 days as per our clinical practice proto-cols. Thirty-five (12.7%) of these patients developed symptoms suggestiveof primary CMV disease involving the GI tract; however, only 26 (9.5%)patients had biopsy-confirmed tissue-invasive GI CMV disease. All patientsconsented to review of their medical records. The study was approved bythe Institutional Review Board of the Mayo Foundation.

Definition of GI CMV disease

Tissue-invasive GI CMV disease was defined as the presence of character-istic CMV inclusions, a positive CMV-specific immunoperoxidase stain, orpositive in situ hybridization for CMV in tissue specimens obtained fromSOT recipients presenting with signs and symptoms consistent with GItissue-invasive disease (10). The occurrence of CMV viremia alone was notsufficient to establish the diagnosis even in a patient with GI symptoms ifthe virus was not detected in tissue specimens. Tissue-invasive GI CMVdisease was classified as upper GI disease if it involved the esophagus,stomach or duodenum, and lower GI disease if it involved the terminalileum or colon. Simultaneous involvement of both upper and lower GI tractwas termed extensive GI CMV disease.

Clinical follow-up

The medical records of all 26 patients with biopsy-proven GI CMV diseasewere reviewed. Data collected included demographic characteristics, clini-cal presentation, endoscopic and histological findings, CMV seroconversionafter diagnosis, development of acute rejection and its treatment, CMV loadand decline during therapy, induction and maintenance antiviral therapy andoutcome. The primary outcome of interest in this study was CMV relapse,which was defined as recurrence of CMV viremia and/or disease during orearly (<3 months) after induction antiviral therapy. CMV viremia and dis-ease were considered together as a composite outcome because bothconditions were retreated with induction doses of antiviral therapy.

All patients with CMV disease were followed-up clinically and virologicallyusing CMV PCR on a weekly basis. Induction anti-CMV therapy was admin-istered at least until resolution of clinical symptoms and CMV viremia. Inaddition, it was also a routine clinical practice in our institution to performfollow-up endoscopy to document endoscopic and histopathologic resolu-tion of GI CMV disease before stopping induction antiviral therapy. Biopsyspecimens were obtained from the involved site, usually after 3–4 weeksof induction anti-CMV therapy. In cases of persistent GI CMV disease, in-duction therapy was extended until another repeat endoscopy confirmedclearance of the virus from infected tissue.

Statistical analysis

The study cohort was characterized using descriptive statistics. Continu-ous parameters were expressed by mean and standard deviation (SD) ormedian and interquartile range (IQR) as appropriate, while categorical datawere represented by the number of patients and percentages. Univariateanalysis using logistic regression model was performed to evaluate vari-

ables associated with the primary outcome of relapse of viremia or diseaseduring or early (<3 months) after finishing treatment for GI CMV disease.JMP 7 (Cary, NC) was used to perform logistic regression analysis. Hazardratios (HRs) with 95% confidence intervals (95% CI) were computed forvariables significantly associated with outcome. A p value of <0.05 wasconsidered statistically significant.

Results

GI CMV disease and its treatment

Twenty-six of 274 (9.5%) CMV D+/R− patients developedbiopsy-proven GI CMV disease after kidney (13 [50%]), liver(10 [38%]) or heart (3 [12%]) transplantation. The clinicaland demographic characteristics of all 26 patients are sum-marized in Table 1. The mean (±SD) age of the patients atthe time of GI CMV disease diagnosis was 47.5 (±15.0)years. The majority (73%) of patients were male.

Table 1: Clinical and demographic characteristics of 26 CMVD+/R− solid organ transplant recipients who developed CMV gas-trointestinal disease

Variables n (%)1

Age at transplant in years, mean (±SD) 47.5 ± 15Male gender 19 (77)Caucasian 24 (92)Transplanted organ

Kidney 13 (50)Liver 10 (38)Heart 3 (12)

Maintenance immunosuppressive therapyPrednisone 24 (92)Tacrolimus 23 (88)Mycophenolate mofetil 20 (77)Sirolimus 3 (12)Cyclosporine 2 (8)Azathioprine 2 (8)

Median duration of CMV primary 92 (91–92)prophylaxis in days (IQR)

Median time-to-CMV disease after 54 (40–70)prophylaxis in days (IQR)

Median viral load at the 154 700time of diagnosis (copies/mL)

Extent of GI diseaseUpper GI disease 7 (27)Lower GI disease 16 (62)Upper and lower GI disease 3 (12)

Treatment for acute rejection 4 (15)Initial induction therapy

Intravenous ganciclovir 25 (96)Valganciclovir 1 (4)

Maintenance therapy 10 (38)Median time to negative CMV PCR in days (IQR) 23 (16–31)Follow-up endoscopy 21 (81)Relapse during or early after 6 (23)

finishing therapy for GI CMV diseaseDeath 1 (4)1Values are presented as number of patients (percentage), unlessotherwise specified. CMV = cytomegalovirus; GI = gastrointesti-nal; IQR = interquartile range; PCR = polymerase chain reaction.

158 American Journal of Transplantation 2010; 10: 157–161

Predictors of Relapse of GI CMV Disease after SOT

The diagnosis of GI CMV disease was established at amedian of 54 days (IQR: 40–70) after cessation of antiviralprophylaxis. CMV disease involved the upper GI tract inseven (27%) patients, the lower GI tract in 16 (62%) andboth upper and lower GI tracts in three (12%) patients.Mucosal erosions and ulcerations were the most commonendoscopic findings at the time of diagnosis (66.7%), al-though some patients had grossly normal mucosa (12.5%)or mucosal hyperemia only (20.8%). Histopathologic exam-ination demonstrated cytomegalic cells with characteristicinclusions in the majority of patients (80%); however, apositive immunoperoxidase stain or in situ hybridizationwas required to demonstrate tissue-invasive CMV diseasein 20% of patients. The median CMV load at the time ofdiagnosis was 154 700 copies/mL (IQR: 55 375–288 850).

All patients except one received induction therapy with IVganciclovir for a mean (±SD) duration of 33.8 (±19.3) days;the remaining patient received oral valganciclovir for 30days. Eighteen of the 25 patients who received IV ganci-clovir were eventually transitioned to renally adjusted in-duction dose of valganciclovir (equivalent of 900 mg twicedaily) for a mean (±SD) duration of 27.5 (±13.3) days. Tenpatients subsequently received renally adjusted mainte-nance dose of valganciclovir (equivalent of 900 mg oncedaily) for a mean (±SD) duration of 41.6 (±28.6) days.

Clearance of CMV from blood and gastrointestinal

tract

The median half-life of viral load decline during antiviraltherapy was 4.7 days (IQR: 3.5–9.1). The median time-to-CMV PCR negativity in the blood was 22.5 days (IQR:16.5–30.7; mean ± SD, 23.8 ± 9.45) after the start of in-duction therapy. Follow-up endoscopy was performed in 20(77%) of the 26 patients. Clearance of CMV from tissuespecimens was documented in 17 (85%) of 20 patients.Among 17 patients with negative follow-up endoscopy, themedian time-to-normal endoscopic finding was 27.0 days(IQR: 21.0–33.5; mean ± SD, 28.6 ± 10.6) after start ofinduction therapy. Three of 20 patients had evidence ofpersistent GI CMV disease on follow-up endoscopy per-formed at a median of 32 days after the start of inductionantiviral therapy.

Outcomes and predictors of CMV relapse

During the mean (±SD) follow-up period of 979 (±550)days, relapse of CMV viremia or disease was observedin seven (27%) patients. In one of these seven patients,CMV viremia relapsed while the patient was still receiv-ing induction IV ganciclovir therapy. In this patient, CMVviremia was low with viral load copies that ranged from2500 to 5000 copies/mL. Ganciclovir resistance was sus-pected but genotype testing could not be performed be-cause viral DNA could not be amplified. Because of lowviral load, the patient was continued on therapy with in-travenous ganciclovir with eventual resolution of viremia 1month later. Five of the seven patients had relapse of CMV

Table 2: Univariate analysis of predictors of CMV viremia or dis-ease relapse during or early after finishing treatment for GI CMVdisease

Variable p-Value

Peak CMV viral load 0.33Half-life of viral decline 0.43Time-to-negative CMV PCR 0.18Endoscopic findings1 0.39Histopathologic findings2 0.11Negative pathology at the end of therapy 0.10Positive CMV serology or seroconversion 0.97

after diagnosisTreatment of acute rejection 0.94Duration of induction therapy 0.52Maintenance valganciclovir therapy 1.00Duration of maintenance therapy 0.34Extent of GI disease3 0.031Endoscopic findings were stratified based on the presence ofmucosal ulcerations and erosions, mucosal erythema or grosslynormal mucosa.2Histopathologic findings were stratified based on whether the di-agnosis of CMV disease was made based on characteristic CMVinclusions alone whether immunoperoxidase stain or in situ hy-bridization was necessary for diagnosis.3Upper and lower gastrointestinal tract disease versus upperor lower gastrointestinal tract disease; CMV = cytomegalovirus;PCR = polymerase chain reaction.

viremia (n = 3) or disease (n = 2) within 3 months aftercompleting antiviral treatment. Finally, the seventh patienthad relapse of asymptomatic CMV viremia >3 months af-ter finishing antiviral therapy. All patients with CMV relapsewere retreated with induction doses of valganciclovir or IVganciclovir.

Table 2 shows the results of univariate analysis of potentialpredictors of the primary outcome of CMV relapse. Of allfactors evaluated, only the extent of GI disease at the timeof diagnosis was significantly associated with a higher riskof CMV relapse. Transplant recipients with extensive CMVdisease (with involvement of both the upper and lower GItracts) at the time of diagnosis had a significantly higherrisk of recurrent CMV relapse after completion of antivi-ral therapy (hazard ratio [confidence interval], 4.8 [1.14–20.7]; p = 0.031). In contrast, CMV seroconversion at thetime of diagnosis, the peak viral load, the rate of viral loaddecline and treatment for acute cellular rejection duringthe 2 months prior to and 1 month following the diagno-sis of CMV disease were not significantly associated withthe primary outcome. An initial endoscopic finding of agrossly normal mucosa or mucosal erythema, as opposedto mucosal erosions and ulcerations, were not significantlyassociated with a lower risk of CMV relapse. Transplantrecipients whose GI CMV disease diagnosis was basedon a positive CMV-specific immunoperoxidase staining orin situ hybridization only were not significantly less likelyto experience CMV relapse compared to those who hadclearly visible CMV inclusions on histopathology. A longer

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duration of induction antiviral therapy was not associatedwith a lower risk of CMV relapse. Likewise, administrationof valganciclovir maintenance therapy was not significantlyassociated with protection from CMV relapse. The inci-dence of CMV relapse was 25% among patients who re-ceived maintenance valganciclovir and 25% among thosewho did not receive maintenance valganciclovir therapy.Finally, endoscopic evidence of resolution of GI diseasebefore stopping induction antiviral therapy was not signifi-cantly associated with protection from CMV relapse.

Discussion

This study highlights the difficulty and complexity of treat-ing primary GI CMV disease in CMV D+/R− SOT recip-ients. Even with a very prolonged course of treatment,recurrence of CMV infection and clinical disease is notuncommon. This study identified extensive CMV disease,as indicated by the involvement of both the upper andlower GI tracts, as a significant predictor of relapsing CMVviremia or disease. In contrast, the degree of viral repli-cation (as measured by the peak viral load in the blood),the rate of viral decline during antiviral therapy, treatmentfor acute cellular rejection and CMV seroconversion at thetime of diagnosis were not significantly associated withCMV relapse. Likewise, endoscopic and histopathologicfindings at the time of diagnosis, endoscopic evidence ofresolution of GI CMV disease at the end of induction antivi-ral therapy, a longer duration of induction antiviral therapyand the administration of maintenance therapy were notsignificantly associated with CMV relapse.

Previous studies have indicated that 23–33% of all casesof primary CMV disease relapse after a defined courseof treatment (9,11). The propensity of CMV infection ordisease to relapse led to the search for and adoption ofstrategies aimed at minimizing this risk. Foremost amongthese strategies is the use of viral quantification as a guideto assess the duration of treatment (6,9,12,13). This stan-dard practice was based on studies demonstrating thatpersistent CMV viremia at the end of treatment, the rateof viral load decline and peak viral load were significantlyassociated with the risk of CMV relapse (9,11). However,this approach may not be optimal in some cases of tissue-invasive CMV disease, because it is not uncommon for thevirus to be cleared from the systemic circulation while itpersists in tissue. Indeed, this study of biopsy-proven GICMV disease did not find significant associations betweenthe degree of viral replication and decay and the risk ofCMV relapse after treatment. Furthermore, some cases ofGI CMV disease could be compartmentalized and may notbe accompanied by detectable viremia, especially amongCMV seropositive recipients with reactivation disease. Inthese situations, viral load surveillance in the blood is notuseful for clinical follow-up.

We had initially hypothesized that repeat endoscopic ex-amination of the GI tract with collection of tissue speci-mens to confirm the resolution of invasive CMV diseasewould be useful to define the duration of treatment andto prevent CMV relapse. With this clinical approach, in-duction antiviral therapy was continued until there was noendoscopic or histopathologic evidence of persistent CMVdisease in the GI tract. However, this study demonstratesthat viral clearance from the GI tract per se, as indicated byrepeat endoscopy and histopathologic examination, doesnot necessarily translate into protection from CMV relapse.Indeed, the rate of relapse of CMV viremia or disease re-mains high (27%) and comparable to previous reports (23–33%) (9,11). Accordingly, if a repeat invasive endoscopicprocedure, with the risk and cost that it entails, does notserve as a useful guide to assess the risk of CMV re-lapse, this practice should not be recommended as a stan-dard of care for all patients with GI CMV disease. More-over, because GI CMV disease is often patchy and mul-tifocal, a false-negative histopathologic examination mayoccur as a result of sampling error when tissue biopsiesare obtained from normal-looking mucosa during follow-upendoscopy.

Follow-up endoscopic examination in the majority of ourpatients provided unique insights into the kinetics of viraleradication from the GI tract. This study demonstrates anearlier clearance of CMV from blood compared to the GItract. While this observation is limited by the infrequentuse of follow-up endoscopy (i.e. performed once at 3–4weeks, and thereafter as indicated), it was evident thatsome patients had cleared their peripheral viremia but re-mained with active gastrointestinal disease. Hence, clear-ance of circulating virus should not be used as a surrogatemarker for CMV eradication from infected GI tissue. Thepotential clinical implication of this finding is the need totreat patients with GI CMV disease for a duration whichextends beyond the time of documented viral eradica-tion in the blood. Indeed, patients in this study receivedprolonged courses (4–8 weeks) of induction therapy withIV ganciclovir or oral valganciclovir. The prolonged dura-tion of therapy correlates with the mean time to nega-tive endoscopic findings of 4 weeks. However, despite theprolonged course of antiviral treatment, relapse of CMVdisease or viremia was not uncommon. Consequently,this raises the question as to whether maintenance treat-ment may be beneficial (7). This study observed similarrates of CMV relapse between patients who receivedor those who did not receive maintenance valganciclovirtherapy. One likely reason for relapse despite the use ofmaintenance valganciclovir therapy (and even after docu-mented clearance of CMV from the GI tract) is a persis-tent defect in CMV-specific immunity (14,15). Host immu-nity, as measured by CMV seroconversion, has not beenshown to be significantly associated with CMV relapse(13). Whether measurement of CMV-specific T cells wouldbe a better marker will need to be addressed in futurestudies (14,15).

160 American Journal of Transplantation 2010; 10: 157–161

Predictors of Relapse of GI CMV Disease after SOT

This study has limitations related to its retrospective andsingle-center design. The number of patients was limitedby the requirement for biopsy-proven cases, because partof our aim was to assess the utility of follow-up endoscopy.Nonetheless, we believe that this is the largest collectionof cases of biopsy-proven primary GI CMV disease withfollow-up endoscopy to monitor treatment response. Be-cause of our requirement for tissue diagnosis, this inclu-sion criterion may have biased our population by select-ing patients with severe disease, and may have excludedmild cases of GI CMV diseases that did not require en-doscopy and biopsy. We emphasize, however, that evenpatients with grossly normal endoscopic findings but withCMV detected only histopathologically were included inour cohort. Hence, it is likely that the spectrum of GI CMVdisease is well represented in this study. Our study pop-ulation consisted of only high-risk CMV D+/R− patients,and therefore, the extrapolation of the results to CMV R+patients with GI CMV disease may not be appropriate.Conversely, one of the strengths of this study is that itpresents a large cohort of biopsy-proven cases of primaryGI CMV disease with follow-up endoscopy. The availabilityof follow-up endoscopy in this study sheds unique insightsinto the kinetics of viral clearance from the GI tract. In thisregard, we believe that this is the first and only study thathas addressed this issue in the clinical setting.

In conclusion, our study illustrates the difficulties of treat-ing primary CMV GI disease in SOT recipients. This study,which we believe is the first to simultaneously evaluate thekinetics of CMV clearance in the blood and GI tissue, pro-vides solid evidence of CMV persistence in GI tissues longafter the virus has been eliminated from peripheral blood.This study therefore has direct implications in clinical prac-tice, as our data would support more prolonged antiviraltherapy in patients with GI CMV disease. However, theoptimal duration is not defined. Despite prolonged antivi-ral therapy, relapse of CMV viremia or disease occurred inseveral of our patients. In this regard, this study shows thatsimultaneous involvement of both the upper and lower GItract is significantly associated with CMV relapse. How-ever, follow-up endoscopy with tissue biopsies to docu-ment clearance of virus from GI tissue does not neces-sarily translate clinically into protection from CMV relapse.Therefore, considering the expense and invasive nature ofendoscopy, such a clinical practice should not be routinelyrecommended in SOT recipients with limited primary GICMV disease.

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