Herpesvirus infections in hematopoietic stem cell transplant recipients seropositive for human cytomegalovirus before transplantation

Herpesvirus

infections

in

hematopoietic

stem

cell

transplant

recipients

seropositive

for

human

cytomegalovirus

before

transplantation

Jun

Fan,

Min

Jing,

Meifang

Yang,

Lichen

Xu,

Hanying

Liang,

Yaping

Huang,

Rong

Yang,

Genyong

Gui,

Huiqi

Wang,

Shengnan

Gong,

Jindong

Wang,

Xuan

Zhang,

Hong

Zhao,

Hainv

Gao,

Huihui

Dong,

Weihang

Ma,

Jianhua

Hu

*

StateKeyLaboratoryfortheDiagnosisandTreatmentofInfectiousDiseases,CollaborativeInnovationCenterfortheDiagnosisandTreatmentofInfectious Diseases,TheFirstAffiliatedHospital,CollegeofMedicine,ZhejiangUniversity,310003,Hangzhou,China

1. Introduction

Hematopoieticstemcelltransplantation(HSCT)hasproven tobe an effectivemeasureinthe treatmentof hematological malignancies.However,thisprocedureisnotwithoutsignificant risks,particularlythatofviralinfections,whichremainoneof the major causes of morbidity and mortality after HSCT.1,2 Transplantation is often accompanied by the use of potent

immunosuppressive drugs to both prevent and treat graft-versus-hostdisease(GVHD).Theuseofthesedrugsresultsina severelycompromisedimmunesystem,makingHSCTpatients morevulnerabletoprimaryviralinfectionsandreactivation.

Herpesviruses are among the most common opportunistic viral infections in HSCT recipients. Of these infections, human cytomegalovirus(HCMV)pneumonia andenteritisarethemost serious and often fatal complications, with a mortality rate exceeding 50% after HSCT.3 _{Primary Epstein–Barr virus (EBV)} infection and lymphoproliferative disorders can occur after T-cell-depleted HSCT,4 _{while human herpesvirus 6 (HHV-6),}5 _a memberofthe

b

-herpesvirusfamilyalongwithHCMV,canachieve ARTICLE INFO

Articlehistory:

Received6January2016

Receivedinrevisedform8March2016 Accepted30March2016

CorrespondingEditor:EskildPetersen, Aarhus,Denmark.

Keywords:

Hematopoieticstemcelltransplantation Herpesvirus

Epstein–Barrvirus Humancytomegalovirus Humanherpesvirustype6 Infection

SUMMARY

Background: Viralinfectionsareamajorcauseofmorbidityandmortalityafterhematopoieticstemcell transplantation (HSCT). The effect of herpesvirus infections in human cytomegalovirus (HCMV)-seropositive(IgG-positive/IgM-negative)HSCTrecipientsremainspoorlyunderstood.Theriskfactors associatedwithEpstein–Barrvirus(EBV),HCMV,andhumanherpesvirustype6(HHV-6)infectionsafter HSCT,bothaloneandincombination,wereinvestigatedinthisstudy.

Methods:Peripheralbloodspecimenswerecollectedfrom44HSCTrecipientsandexaminedforviral DNAusingquantitativefluorescencePCRassays.RiskfactorsforEBV,HCMV,andHHV-6infectionswere analyzedbybinarylogisticregression,andrelationshipsbetweentheseviruseswereanalyzedusingthe Chi-squaretest.

Results:EBV,HCMV,andHHV-6weredetectedin50%,45.45%,and25%ofHCMV-seropositive (IgG-positive/IgM-negative)HSCTrecipients,respectively.Malesex(p=0.007)andconditioningregimens includinganti-thymocyteglobulin(ATG)(p=0.034)werestronglyassociatedwithanincreasedriskof EBVinfection.Graft-versus-hostdisease(GVHD)prophylaxiswithcorticosteroidswasariskfactorfor bothEBV(p=0.013)andHCMV(p=0.040)infections,whileEBVinfection(p=0.029)wasfoundtobean independentriskfactorforHHV-6infection.Pre-existingHHV-6infectionwasassociatedwithlower ratesofHCMVinfection(p=0.002);similarly,pre-existingHCMVinfectionwasprotectiveagainst HHV-6infection(p=0.036).

Conclusions: HCMV-seropositive(IgG-positive/IgM-negative)HSCTrecipientsexhibitedahighrateof herpesvirusinfections,particularlyEBV.ATGandmalesexwerestronglyassociatedwithanincreased riskofEBVinfection.GVHDprophylaxiswithprednisonewasfoundtoaffectbothEBV andHCMV infections.PriorinfectionwithEBVwasshowntopromoteHHV-6infection.Takentogether,thesedata highlighttheneedforactivemonitoringofherpesvirusinfectionsinpatientsundergoingHSCT. ß2016TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Correspondingauthor.Tel.:+8657187236581;fax:+8657187236444.

E-mailaddress:hjianhua0825@126.com(J.Hu).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d

http://dx.doi.org/10.1016/j.ijid.2016.03.025

1201-9712/ß2016TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

lifelonglatencyinthehost.Reactivationoftheselatentinfections after HSCT has been associated with a variety of symptoms includingskinrash,fever,interstitialpneumonitis,bonemarrow suppression,encephalitis,andGVHD.6–9

The vast majority of research into herpesvirus infections in HSCT recipientshas focused on HCMV, with very little known regarding either EBV or HHV-6. Moreover, few studies have examinedtheeffectsofthesevirusesincombination,particularly inthecontextofHCMV-seropositive(IgG-positive/IgM-negative) HSCTrecipients.Inpreviousstudiesexaminingtherelationships between the herpesviruses,

b

-herpesviruses were found to transactivate each other, while HCMV infection appeared to trigger HHV-6 and/or HHV-7 co-infection and vice versa.10 However, the relationship between EBV and

b

-herpesvirus infections remains poorly understood. In this study, HSCT recipientswho were seropositive for HCMV (IgG-positive/IgM-negative) before transplantation were examined to assess the relationships between HCMV, EBV, and HHV-6 infections after HSCTandtoidentifypotentialriskfactorsforviralinfection.

2. Methods

2.1. Humansubjectsandsamples

HSCTrecipientstreatedatthestudyhospitalbetweenJanuary 2012andJune2012weretestedforseropositivity(IgGandIgM)to HCMVprior totransplantation;almostall ofthe patients were infectedwithHCMVbeforetransplantation.Followingthe exclu-sionofthefewHCMVIgG-negativepatients,44patientswith IgG-positive/IgM-negativeHCMVwereenrolledinthisstudy.Detailed demographic and clinical data for thesepatients are shown in

Table1.Plasmasampleswerecollectedonceweeklyinthefirst

month,twiceinthesecondandthirdmonthsaftertransplantation, andthenevery1–2monthsuntilDecember2012(range3months to1year).Intotal,392peripheralbloodspecimens(range5–17 samplesperpatient)werecollected,fromwhichperipheralblood leukocytes(PBLs)wereisolatedandstoredat 708CuntilDNA extraction.Forsomepatients, thenumberoffollow-upsamples waslimitedduetoearlydeathorlosstofollow-up.

2.2. Conditioningregimenandpost-transplanttreatment

HSCTrecipientsweretreatedwithorwithoutanti-thymocyte globulin(ATG)beforetransplantation.Patientsweretreatedwith mycophenolate mofetil plus cyclosporine in combination with short-termmethotrexateandintravenousganciclovir(5mg/kgper day)forHCMV,for7dayspriortotransplantation.Thiswasfollowed by long-term mycophenolate mofetil plus cyclosporine and prednisone,ormycophenolatemofetilpluscyclosporineforGVHD prophylaxis,sulfamethoxazoleforPneumocystiscariniipneumonia (4tablets,twicedaily),andintravenousganciclovirforHCMV(5mg/ kgperdayforthefirst2weeks)aftertransplantation.AcuteGVHD and chronic GVHD were diagnosed and graded according to standard criteria.11 _{Corticosteroids were used in patients with} gradeII–IVacuteGVHD,withvaryingdurations.

2.3. DNAdetectionofherpesviruses 2.3.1. Primersandprobes

Herpesvirus DNA was extracted using a commercial DNA extraction kit (Promega Biological Technology Co. Ltd, Beijing, China)in accordance withthe manufacturer’sinstructions. The primersandprobesusedtodetectEBV,HCMV,andHHV-6have beendescribedpreviously.4,12,13_{Briefly,PCRprimersandprobes} for EBV wereselected from BALF5,4 _{those for HCMV from the} immediateearly(IE)gene,13_{andthoseforHHV-6fromtheU31}

gene.12 _{All primers and probes were synthesized by ZeHeng} Technology(Shanghai,China).

2.3.2. QuantitativefluorescencePCRassay

QuantitativefluorescencePCRwasperformedusingaTaqMan PCRKit(Takara,Dalian,China)andrunonanABI7500Real-Time PCRSystem(USA),asdescribedpreviously.3_{Standardstrainswere} used aspositive controls in each amplification(B95-8 for EBV, AD169forCMV,andGSforHHV-6A).Inadditiontoablankcontrol, distilled water was used as a negative control. Real-time fluorescencewasmeasured,andcyclethreshold(Ct)valueswere calculatedforeachsample.

SpecificitywasconfirmedusingviralDNAfromstandardstrains. SensitivitywasconfirmedbyTaqManQualitativefluorescencePCR usingserialdilutionsofstandardstrains,withaminimumdetectable Ctvalue of48 relativetoundilutedsamples,whichproduced Ct valuesof16.Nopeaksweredetectedinthenegativecontrol.

2.4. Statisticalanalyses

AllstatisticalanalyseswereperformedusingSPSSversion16.0 (SPSS Inc., Chicago, IL, USA). Qualitative variables, such as the clinicalcharacteristicsoftheHSCTrecipients,wererecordedasthe percentage of the positive results, and differences in these variableswereevaluatedusingtheChi-squaretest.Quantitative variables,suchasage,wererecordedasthemedianandrange.Risk Table1

Characteristicsofthestudypatients

Characteristicsofpatients Value

Patients,n 44

Age,years,median(range) 26(16–55)

Sex,n(%)

Male 23(52.30%)

Female 21(47.70%)

Underlyingdisease

Acutemyelogenousleukemia 19(43.20%)

Acutelymphoblasticleukemia 18(40.90%)

Non-Hodgkinlymphoma 1(2.27%)

Myelodysplasticsyndrome 5(11.36%)

Lymphosarcomacellleukemia 1(2.27%)

Conditioningregimen

Ara-c+BUCY+MeCCNU+ATG 21(47.73%)

Ara-C+BUCY+MeCCNU 4(9.09%)

BUCY+ATG+MeCCNU 5(11.36%)

Ara-C+BUCY+ATG 1(2.27%)

BUCY+MeCCNU 11(25.00%)

BUCY+ATG 1(2.27%)

BUCY 1(2.27%)

Typeofdonor

HLA-identicalsibling 16(36.36%)

Mismatchedrelateddonor 14(31.82%)

Matchedunrelateddonor 11(25.0%)

Mismatchedunrelateddonor 3(6.82%)

Stemcellsource

Peripheralblood 42(95.45%)

Peripheralbloodandbonemarrow 2(4.55%)

GVHDprophylaxis

Mycophenolatemofetil+cyclosporine 10(22.73%) Mycophenolatemofetil+cyclosporine+prednisone 34(77.27%) aGVHD

Grade0–I 36(81.82%)

GradeII–IV 8(13.64%)

Deatha

5(11.36%)

Pulmonaryfungalinfection 2(4.55%)

Pulmonaryhemorrhage 1(2.27%)

Hemorrhageofdigestivetract 1(2.27%)

Pulmonaryfungalinfectionandhemorrhageofdigestivetract 1(2.27%) Ara-C, cytosine arabinoside; BU, busulfan; CY, cyclophosphamide; MeCCNU, methylcyclohexylnitrosamine;ATG,anti-thymocyteglobulin;HLA,humanleukocyte antigen;GVHD,graft-versus-hostdisease;aGVHD,acutegraft-versus-hostdisease.

a

factorsfor EBV,HCMV,and HHV-6 wereanalyzedusing binary logisticregression.Theresultswereexpressedastheoddsratio (OR)withcorresponding95%confidenceinterval(CI).Ap-valueof

<0.05wasconsideredstatisticallysignificant.

3. Results

The aim was to examine HCMV, EBV, and HHV-6 viral

infections, therisk factors forinfection, and their relationships inHSCTrecipients.TheCtvaluecanreflecttheconcentrationof virus.AstheCtvaluewasconsideredsufficientforthepurposesof this research, the quantitation of standard strains was not performed. Serial dilutions of standard strains detected in the study wereused to verify the validity of PCR and the relative detectionrange,inordertoensurethereliabilityofthePCR.The aim when obtaining multiple specimens for an objective is to observethechangeinvirusesoveraperiodoftime.Astheoverall relationship of the three virusinfections was analyzed in this study,theuseofthemedianCtvaluewasconsideredsufficientfor thispurposeandnottohaveanyinfluenceontheanalysis.Theuse of the Ct value to determine expression has been reported previouslyintheliterature.14

3.1. Herpesvirusinfections

Thefirstsamplewascollectedfromeachpatientduringthefirst weekaftertransplantation. Thesefirstpatient sampleswereall PCR-negativefor theviruses.Among the44 HCMV-seropositive (IgG-positive/IgM-negative) HSCT recipients included in this study,22(50%)testedpositiveforEBVaftertransplantationata medianCtvalueof35.42(range31.04to38.67cycles).Themedian time to EBV DNA detection was 45 days post-transplantation (range14–88days).Twentypatients(45.45%)testedpositivefor HCMVatamedianCtvalueof23.90(range17.96to27.42cycles). The median time to HCMV DNA detection was 32 days post-transplantation(range11–76days).Elevenpatients(25%)tested positiveforHHV-6atamedianCtvalueof35.00(range30.90– 37.44 cycles). The median time to HHV-6 DNA detection was 39dayspost-transplantation(range24–87days)(Table2). Co-infections were observed in 18 patients. Ten patients (22.73%) wereco-infectedwithEBVandHCMV,ofwhomfivetestedpositive forEBVfirst,fourforHCMVfirst,andoneforbothsimultaneously. Sixpatients (13.64%)wereco-infectedwithEBVand HHV-6,of whomthreetestedpositiveforEBVfirst,oneforHHV-6first,and two for both infectionsat thesame time. Finally, two patients (4.55%)were co-infected with all three viruses, both of whom testedpositiveforHCMV,thenEBV,andfinallyHHV-6(Table2).

3.2. RiskfactorsforEBV,HCMV,andHHV-6infectionsafterHSCT

PotentialriskfactorsforEBV,HCMV,andHHV-6infectionsin HSCT recipients were analyzed using binary logistic regres-sion.3,10,12,15–18_{Malepatientswere13.24timesmoresusceptible} to EBVinfection than female patients (p=0.007). Conditioning regimens that included ATG (OR 7.690, p=0.034) and GVHD

prophylaxis regimens that included prednisone (OR 23.681,

p=0.013) werealso strongly associated withan increased risk of EBV infection. Similarly, GVHD prophylaxis regimens that includedprednisone(OR13.565,p=0.040)werealsosignificantly associatedwithanincreasedriskofHCMVinfection.EBVinfection (OR6.726,p=0.029)wasidentifiedasanindependentriskfactor forHHV-6infection(Table3).Otherpotentialriskfactors,suchasa sexmismatchbetweenthedonorandrecipient,ABObloodtype mismatch,humanleukocyteantigen(HLA)mismatches,etc.were foundnottoberiskfactorsforEBV,HCMV,andHHV-6infections.

3.3. RelationshipsbetweenEBV,HCMV,andHHV-6infectionsafter HSCT

PriortoEBVdetection,15patientswerealreadyinfectedwith HCMV and six with HHV-6; seropositivity for both of these infectionsdidnotaffectEBVinfection(p=0.750,p=1.000).Among the HCMV-infectedindividuals, six werepre-infected withEBV andninewerepre-infectedwithHHV-6.EBVstatusdidnotaffect theinfectionrate,whileHHV-6pre-infectionwasassociatedwitha significantly lower rate of HCMV infection (p=0.002). Finally, amongtheHHV-6-infectedpatients,sevenwerepre-infectedwith EBVandtwowithHCMV.HCMVpre-infectionwasalsofoundtobe aprotectivefactorforHHV-6infection(p=0.036);noeffectwas seenforEBV.TherelationshipsbetweenEBV,HCMV,andHHV-6 infectionratesinHSCTrecipientsareshowninTable4.

4. Discussion

HCMV is one of the mostcommon humanpathogens, with seroprevalencerangingfrom40%to100%.19Despiteitsprevalence, little isknown ofitsco-occurrencewithothercommonviruses such as EBVand HHV-6, particularlyin the context of HCMV-seropositiveHSCTrecipients.Inthisstudy,HSCT recipientswho were seropositive for HCMV (IgG-positive/IgM-negative) before HSCTwereexamined.TherateofHCMVinfectionafterHSCTwas 45.45% (20 patients), a rate similar to that seen in previous studies.15,20 _{Immunosuppressive drug regimens put transplant} recipientsatahigherriskofEBVinfection,whichoftenmanifests intheformofpost-transplantationlymphoproliferativedisease.21 Table2

HerpesvirusinfectionsafterHSCT

Virus Patients Ctvalue Timeto

infection,days Median Range Median Range

EBV 22(50%) 35.42 31.04–38.67 45 14–88 HCMV 20(45.45%) 23.90 17.96–27.42 32 11–76 HHV-6 11(25%) 35.00 30.90–37.44 39 24–87 EBV+HCMV 10(22.73%) EBV+HHV-6 6(13.64%) HCMV+HHV-6 0(0%) EBV+HCMV+HHV-6 2(4.55%)

HSCT,hematopoieticstemcelltransplantation;Ct,cyclethreshold;EBV,Epstein– Barrvirus;HCMV,humancytomegalovirus;HHV-6,humanherpesvirustype6.

Table3

BinarylogisticanalysisforEBV,HCMV,andHHV-6riskfactors

Virus Factors Coefficient(B) OR(95%CI) p-Value

EBV Maledonor 2.583 13.240(2.006–87.387) 0.007

GVHDprophylaxisincludingcorticosteroids 3.165 23.681(1.924–291.449) 0.013

ATGincludedinconditioningregimen 2.040 7.690(1.171–50.493) 0.034

HCMV GVHDprophylaxisincludingcorticosteroids 2.607 13.565(1.125–163.496) 0.040

HHV-6 EBVinfection 1.906 6.726(1.213–37.303) 0.029

EBV,Epstein–Barrvirus;HCMV,humancytomegalovirus;HHV-6,humanherpesvirustype6;OR,oddsratio;CI,confidenceinterval;GVHD,graft-versus-hostdisease;ATG, anti-thymocyteglobulin.

Inthisstudy,EBVwasdetectedin22HSCTrecipients(50%),arate similartothoseinotherreports.16,17

HHV-6 and HCMV are closely related members of the b-herpesvirus family and share many characteristics. Zerr et al. reportedthatmorethan90%ofthepopulationareinfectedwith HHV-6withinthefirst18monthsoflife.6_{Amongthe44patients} includedinthisstudy,25%(11patients)testedpositiveforHHV-6 DNAintheirPBLs,a muchlowerratethanreportedforthetwo virusesdescribedabove.Theseobservationsaresimilartothoseofa previousreport,16_{although considerable variationis seenin the} literature.1,7,17_{Whencomparingthestudies,itisimportanttonote} that the HSCT recipientsin the present study were adults and teenagers,withmostbeingadults(81.82%),whilethepreviously reportedHSCTrecipientswithhigherHHV-6infectionrateswereall children.1,7,17_{Inaddition,differentsampletypes(e.g.,wholeblood} samples,plasma,orPBLs)andthelackofinternationally standard-izedPCRassaysarelikelytoaccountforsomeofthediscrepanciesin HHV-6infectionrates.Thethreeherpesvirusesdescribedherewere alldetectedwithinthe first3monthspost-transplantation, with HCMVinfectionoccurringfirst,followedbyEBVandthenHHV-6,a patternconsistentwiththoseseeninpreviousstudies.5,16,17

Reported riskfactorsfor EBV,HCMV,andHHV-6 afterHSCT includeage,HLAmismatch,thepresenceofacuteGVHD,ATG,and GVHDprophylaxisregimenscontainingcorticosteroids.3,10,12,15–18 Inadditiontotheseknownriskfactors,otherpotentialriskfactors includethesexoftherecipientanddonor,sexmismatchbetween thedonorandrecipient,ABObloodtypemismatch,andotherviral infections.

It was found that GVHD prophylaxis regimens including prednisone represented an independent risk factor for HCMV infection,consistent with other studies.3,15 _{Immunosuppressed} patientsexhibitdelayedorreducedimmunereconstitution,which hasbeenshowntohaveadirecteffectonviralreplicationdynamics invivo;22_{thiseffectmayinturnaffectHCMVreplication.Close} monitoringforHCMVisthereforeextremelyimportantinpatients whoseimmunosuppressiontherapyincludescorticosteroids.

Inthisstudy,theuseofATGintheconditioningregimenand GVHD prophylaxis regimens containing corticosteroids were associated with significant increases in EBV infection post-transplantation, with patients 7.7- to 23.7-times more likely to become infected; this is similar to the findings of previous studies.12,16–18 Thiseffect is likely due tothe actionof ATG on cellularimmunity.Asmorethan90%ofadultshavebeeninfected withEBV,exposuretothevirusisinevitable.Afterinfection,EBV persistswithinthebodyinrestingmemoryB-cells,withcellular immuneresponses controlling proliferating EBV-infected B-cells. High-levelimmunosuppression,asachievedwithATGplus high-dosecorticosteroids,affectsboththenumberandfunctionofT-cells tothepointwhereexistingT-cellsmaybeunabletocontrolEBV proliferation,whetherdue toprimary infectionorreactivation.17 Thismodelisalsosupportedbythewell-establishedmechanismof

actionofATGintermsofinvivopartialT-celldepletion.18,23_Thesex ofthedonorwasalsofoundtobeasignificantriskfactorforEBV infection, although the specific reasons underlying this effect remainunknown.

GVHD, the administration of steroids for GVHD, and allele mismatched donors have previously been associated with an increased risk of active HHV-6 infection,7,10 _{although these} findings were not confirmed in thepresent study. However, a surprisingfindingwasthat EBVpre-infection facilitatedHHV-6 infection thereafter (Table 3). This association may be due to impairedT-cellimmunitystemmingfromtheGVHDprophylaxis regimen,resultinginanimmunesystemunabletosuppressEBV reactivation.17 EBV proliferation would further suppress T-lymphocytefunctionand/orexhaustT-cells,enabling opportunis-ticinfectionssuchasHHV-6.Alternatively,EBVproliferationmay inducethesynthesisofproinflammatorycytokinesandsuppress HHV-6-specificlymphoproliferativeresponses,triggeringHHV-6 primaryinfectionfollowedbyproliferationandreactivation.

Differentstudiesmayidentifydifferentriskfactorsdepending onthetargetpopulation,methodsandsamplesused,etc.Therisk factors identified in the present study are just possibilities. However,aspotentialriskfactors,theyareworthyofattention.

Despite the apparent associations observed between viral infections,itisdifficulttodeterminewhethertheseco-infections represent new or reactivated infections, and what effectsthese virusesmayhaveoneachotherinvivo.Growingevidencesuggests that the

b

-herpesvirusesareable toreverselyactivate other

b

-herpesviruses due to their effectson immune regulation.10,24–29 However,Tormo etal. have arguedagainst a role forHHV-6 in promoting HCMV replication by inhibiting the reconstitution of HCMV-specificT-cellimmunity,whichisconsistentwiththepresent findings.10Here,bothHCMV andHHV-6werefoundtodecrease susceptibilitytotheothervirus.Whiletheexactmechanismremains unknown,ithasbeensuggestedthatvirus-specificIgGraisedagainst one virus may confer cross-immunity to the other, thereby minimizing further infection. Alternatively, due to the strong homologybetweenHCMVandHHV-6,priorinfectionmayconfer resistance due to competition for shared replication machinery. Furtherresearchwillbenecessarytovalidatethesehypotheses.

Few studies have examined the relationship between

b

-herpesvirusesandEBV.Aaltoetal.30_{andRazonableetal.}31_have suggested that HCMV infection may induce EBV infection and proliferation. In this study,no relationship wasfoundbetween HCMVandEBV,orbetweenHHV-6andEBV,asanalyzedby Chi-squaretest(Table4).However,EBVpre-infectionwasfoundtobe anindependentriskfactorforHHV-6infectiononbinarylogistic regressionanalysis(Table3).Theexactpathophysiologicalbasis underlyingthisinteractionislargelyunknown,althoughitremains possiblethattheimmunosuppressiveeffects oftheEBVsystem mayfacilitateotherinfectionssuchasHHV-6.Furtherresearchwill benecessarytovalidatethishypothesis.

Table4

RelationshipsofEBV,HCMV,andHHV-6infections

Total EBVinfection Total HCMVinfection Total HHV-6infection

Number p-Value Number p-Value Number p-Value

Total 44 22 44 20 44 11 EBVpre-infection 0.423 0.223 Yes 16 6 21 7 No 28 14 23 4 HCMVpre-infection 0.750 0.036 Yes 15 7 20 2 No 29 15 24 9 HHV-6pre-infection 1.000 0.002 Yes 6 3 9 0 No 41 19 35 20

Takentogether,thedatapresentedhererevealafrequent co-occurrenceofEBV,HCMV,andHHV-6inHCMV-seropositive (IgG-positive/IgM-negative)HSCTrecipients,withEBVbeingthemost common.Furthermore,theseresultsconfirmthestrong associa-tion between T-cell depletion, male sex, and EBV reactivation. GVHD prophylaxis regimens containing corticosteroids were identifiedasariskfactorforbothEBVandHCMVinfections,with each of thethreeviruses exhibiting someformof relationship. HCMVandHHV-6appeartoserveasprotectivefactorspreventing furtherinfection,whileEBVhelpsfacilitateHHV-6infection.

Some encouraging results were obtained and conclusions drawnfromthisstudy,buttherearesomeinevitablelimitations. First,theherpesvirusfamilyincludesavarietyofviruses:herpes simplexvirus(HSV)-1,HSV-2,varicellazostervirus(VZV),HCMV, EBV,HHV-6,HHV-7,andHHV-8.Unfortunatelyonlythreeviruses wereinvestigatedinthisstudy(HCMV,EBV,andHHV-6).However, this study will form part of a series of future studies on herpesviruses,which will beperformed stepby step. A second limitation was the use of Ct values; however, these were considered sufficiently accurate for the purposes of this study and not to have any influenceon theanalysis. Nevertheless it wouldbebettertoreportthenumberofcopiesofviralDNAto demonstratethechangeinvirusinfection.Third,theaimofthis studywastoinvestigatevirusinfectionsinHSCTrecipientswho were HCMV-seropositive (IgG-positive/IgM-negative) before transplantation,whileEBVandHHV-6werenotdetectedbefore transplantation.Thiswouldnot,therefore,allowustodetermine whethertheinfectionthat occurredlaterwasa consequenceof reactivation or a primary infection.This should be clarified in future studies.Finally, therate ofHHV-6 infection is higherin childrenthaninadults.1,7,17Whetheryoungeradultshaveahigher rateofinfectionshouldalsobestudiedinthefuture.

Acknowledgements

This study was supported by the Medical Science and

Technology Project of Zhejiang Province (2013KYB084), the National Natural Science Foundation of China (30872239), the Zhejiang Provincial Natural Science Foundation of China (LY14H190002), and the Independent Research Topics of State keyLaboratoryforDiagnosisandTreatmentofInfectiousDiseases.

Ethical approval: This study was approved by the Ethics CommitteeoftheFirstAffiliatedHospitalofZhejiangUniversity. Informedconsentwasobtainedfromthepatients.

Conflictofinterest:None.

Contributions:Studydesign:JianhuaHu,JunFan,WeihangMa; performanceoftheexperiments:MinJing,JunFan,YapingHuang, RongYang;provisionofpatients:MeifangYang,XuanZhang,Hong Zhao, Hainv Gao; data collection: Lichen Xu, Hanying Liang, GenyongGui,HuiqiWang,ShengnanGong,JindongWang;data analysis:JianhuaHu,MinJing,MeifangYang,HuihuiDong;writing ofthepaper:JunFan.

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