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EBV DNA increase in COVID-19 patients with impaired lymphocyte subpopulation count

Stefania Paolucci

^a

, Irene Cassaniti

^a

, Federica Novazzi

^a

, Loretta Fiorina

^b

, Antonio Piralla

^a

, Giuditta Comolli

^a,c

, Raffaele Bruno

^d,f

, Renato Maserati

^d

, Roberto Gulminetti

^d

,

Stefano Novati

^d

, Francesco Mojoli

^e,f

, Fausto Baldanti

^a,f,

*, for the San Matteo Pavia COVID-19 Task Force, R Bruno, M Mondelli, E Brunetti, A Di Matteo, E Seminari,

L Maiocchi, V Zuccaro, L Pagnucco, B Mariani, S Ludovisi, R Lissandrin, A Parisi, P Sacchi, SFA Patruno, G Michelone, R Gulminetti, D Zanaboni, S Novati, R Maserati, P Orsolini, M Vecchia, M Sciarra, E Asperges, M Colaneri, A Di Filippo, M Sambo, S Biscarini,

M Lupi, S Roda, TC Pieri, I Gallazzi, M Sachs, P Valsecchi, S Perlini, C Alfano, M Bonzano, F Briganti, G Crescenzi, AG Falchi, R Guarnone, B Guglielmana, E Maggi, I Martino, P Pettenazza, S Pioli di Marco, F Quaglia, A Sabena, F Salinaro, F Speciale, I Zunino, M De Lorenzo, G Secco, L Dimitry, G Cappa, I Maisak, B Chiodi, M Sciarrini, B Barcella, F Resta, L Moroni, G Vezzoni, L Scattaglia, E Boscolo, C Zattera, MF Tassi, V Capozza, D Vignaroli, M Bazzini, G Iotti, F Mojoli, M Belliato, L Perotti, S Mongodi, G Tavazzi, G Marseglia, A Licari, I Brambilla, D Barbarini, A Bruno, P Cambieri, G Campanini, C. Cavanna, G Comolli, M Corbella, R Daturi, M Furione, B Mariani, P Marone,

R Maserati, E Monzillo, S Paolucci, M Parea, E Percivalle, A Piralla, F Rovida, A Sarasini, M Zavattoni, G Adzasehoun, M Ardizzone, L Bellotti, V Brunco, E Cabano, G Casali, L Capella, D Devitis, L Dossena, G Frisco, G Garbagnoli, F Gardellini, A Girello, A Guerrizio, V Landini, C Lucchelli, V Maliardi, P Piemontese, S Pezzaia, M Premoli, C Rebuffa, C Zanello, J Bagnarino, F Bergami, A Bonetti, G Caneva, I Cassaniti, A Corcione, R Di Martino, A Di Napoli, A Ferrari, G Ferrari, L Fiorina, A Gallone, F Giardina, A Girardi, A Mercato, C Merla, F Novazzi, G Ratano, B Rossi, G Saveriaempillai, IM Sciabica,

M Tallarita, E Vecchio Nepita, J Vitali, A Cerino, S Varchetta, B Oliviero, S Mantovani, D Mele, M Calvi, M Tizzoni, C Nicora, A Triarico, V Petronella, C Marena, A Muzzi, P Lago, S Cutti, V Novelli, F Comandatore, G BatistiBif ﬁgnandi, S Gaiarsa, M Rettani, C Bandi, A Ferrari

aMolecularVirologyUnit,MicrobiologyandVirologyDepartment,FondazioneIRCCSPoliclinicoSanMatteo,Pavia,Italy

bLaboratorioGenetica-TrapiantologiaeMalattieCardiovascolari,FondazioneIRCCSPoliclinicoSanMatteo,Pavia,Italy

cExperimentalResearchLaboratories,BiotechnologyArea,FondazioneIRCCSPoliclinicoSanMatteo,Pavia,Italy

dInfectiousDiseaseDepartment,FondazioneIRCCSPoliclinicoSanMatteo,Pavia,Italy

eAnesthesiaandIntensiveCare,EmergencyDepartment,FondazioneIRCCSPoliclinicoSanMatteo,Pavia,Pavia,Italy

fDepartmentofClinical,Surgical,DiagnosticandPediatricSciences,UniversityofPavia,Pavia,Italy

*Correspondingauthorat:DepartmentofClinical,Surgical,DiagnosticandPediatricSciences,UniversityofPavia,27100Pavia,Italy.

E-mailaddresses:f.baldanti@smatteo.pv.it,fausto.baldanti@unipv.it(F.Baldanti).

https://doi.org/10.1016/j.ijid.2020.12.051

1201-9712/©2020TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International Journal of Infectious Diseases

j o u r n a l h o m ep a g e : w w w . e l s e v i e r . c o m / l o c a te / i j i d

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ARTICLE INFO

Articlehistory:

Received4November2020

Receivedinrevisedform10December2020 Accepted17December2020

Keywords:

COVID-19 EBVDNA

Lymphocytesubpopulation

ABSTRACT

Objectives:TheimmunologicproﬁleandopportunisticviralDNAincrease weremonitoredinItalian patientswithCOVID-19inordertoidentifymarkersofdiseaseseverity.

Methods:Atotalof104patientsinfectedwithSARS-CoV-2wereevaluatedinthestudy.Ofthem,42/104 (40.4%)werehospitalizedinanintensivecareunit(ICU)and62/104(59.6%)inasub-intensivecareunit (SICU). Humancytomegalovirus (HCMV) andEpstein-Barr virus (EBV),ParvovirusB19 andHuman Herpesvirus6virusreactivationsweredeterminedbyreal-timePCR,andlymphocytesubpopulation countsweredeterminedbyﬂowcytometry.

Results:Amongopportunisticviruses,onlyEBVwasconsistentlydetected.EBVDNAwasobservedin40/

42(95.2%)oftheICUpatientsandin51/61(83.6%)oftheSICUpatients.Comparingthetwogroupsof patients,theEBVDNAmedianlevelamongICUpatientswassignificantlyhigherthanthatobservedin SICUpatients.Inparallel,asignificantreductionofCD8TcellandNKcountinICUpatientsascompared withSICUpatientswasobserved(p<0.05).Incontrast,BcellcountwassignificantlyincreasedinICU patients(p=0.0172).

Conclusions:AcorrelationbetweenreducedCD8⁺TcellsandNKcounts,EBVDNAlevelsandCOVID-19 severitywasobserved.Otheropportunisticviralinfectionswerenotobserved.Therelationshipbetween EBVloadandCOVID-19severityshouldbefurtherevaluatedinlongitudinalstudies.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc- nd/4.0/).

Introduction

Coronavirusdisease2019(COVID-19),causedbysevereacute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was ﬁrst reported in China in December 2019. As of February 21,2020, more than 7,151,000 cases of COVID-19 have been reported worldwideand therearemorethan407,145 deaths.Thespread has already takenonpandemic proportions,affectingover 100 countries, including Italy that counts more than 235,270 cases (Ghinai et al., 2020; Remuzzi and Remuzzi, 2020). In February 2020,alargeCOVID-19outbreakinLombardyItalywasobserved.

The SARS-CoV-2 inﬂuence on immunological response, and its relationshipwiththereactivationofopportunisticviralinfection in the clinical course of COVID-19 is still under investigation.

Amongvariouspathogens,opportunisticviralinfectionssuchas humancytomegalovirus(HCMV)andEpstein-Barrvirus(EBV)are particularlyrelevantinimmunocompromisedpatients(Fishman, 2017;Fishman,2013)andtheirreactivationhasalsobeenreported inpatientsinintensivecareunits(ICU)withnopreviousimmune suppression (Schildermans and De Vlieger, 2020; Limaye and Boeckh,2010;Frantzeskakietal.,2015;Ongetal.,2017;Cantan etal.,2019;Coşkunetal.,2017).

Ontheotherhand,theimpactofopportunisticviralreactivation incriticallyillpatientsremainstobeelucidated.

Finally, the impact of COVID-19 infection on the immune systemisnotyetwellknown.Inthisstudy,apossiblerelationship withalterations oftheNKand T-cell subpopulationsleadingto opportunistic virus reactivationwereanalyzed inpatientswith COVID-19havingdifferentdegreesofseverity.

Materialandmethods

Patientcharacteristicsandcomorbidity

Writteninformedconsentwascollected.Atotalof104patients infected with SARS-CoV-2 referred to the Fondazione IRCCS PoliclinicoSanMatteo,PaviabetweenFebruary2020andMarch 2020wereevaluated.Ofthem,42/104(40.4%)werehospitalizedin an ICUand62/104(59.6%) werehospitalized ina sub-intensive care unit(SICU). Thedegreeof severityofCOVID-19(severe vs mild)wasdeﬁnedusingtheAmericanThoracicSocietyguidelines forcommunity-acquiredpneumonia(Metlayetal.,2019).Patient characteristicsandcomorbidityweredescribedinTable1.

CharacterizationofserologicalstatusforHCMV,EBVandparvovirus B19

Antiviralcapsidantigen(anti-VCA),anti-EBNA,anti HCMVand anti-parvovirus B19 specific IgG were quantified from serum samplesusinga chemiluminescentassayaccordingtomanufac- turers’ instructions (Liaison, Diasorin, Saluggia, Italy). Positive results were defined as IgG> 20U/mL for VCA and EBNA IgG, IgG>14U/mLforCMVIgGandIndex>1.1forParvovirusB19IgG.

ViralquantiﬁcationinCOVID-19patients

Nasalswabs(NS)orbronchoalveolarlavage(BAL)andwhole bloodwerecollectedduringthehospitalizationfromallpatients, and 200

m

^L ôf ^clinical ^specimens ^were êxtracted ûsing ^the

QIAsymphony¹ instrument with QIAsymphony¹ DSP Virus/

PathogenMidiKit(QIAGEN,Hilden,Germany).Speciﬁcreal-time reversetranscriptase-polymerasechainreaction(RT-PCR)target- ingRNA-dependent RNApolymeraseandE geneswereusedto detect the presence of SARS-CoV-2 according to the WHO guidelines (Anon, 2020) and Corman et al. protocols (Corman etal.,2020).TheCMVDNAandEBVDNAloadweredeterminedby home-madereal-timePCRinCMVandEBVseropositivesubjects using primersand probesand Applied Biosystems Instruments

Table1

Demographiccharacteristicsofthe104analyzedpatients.

Hospitalization N104

Total ICU^a

N42

SICU^b N62

P-value

no(%) 42(40.4%) 62(59.6%)

Gender

Male/Female 36/6(85.7%/14.3%) 41/21(66.1%/33.9%) 0.0391 Medianage(years;IQR) 61.5(55 71.25) 73.5(57.8 80) 0.05 Comorbidity

Heartdisease 19.0% 33% 0.25

Hypertension 40.4% 48.1% 0.62

Obesity 30.9% 18.5% 0.27

Lungdisease 4.7% 14.8% 0.20

Diabetes 14.3% 11.1% 1.00

Carcinoma 9.5% 3.7% 0.64

aICU,IntensiveCareUnit.

b Sub-IntensiveCareUnit,Inboldisthesigniﬁcantvalue.

S.Paolucci,I.Cassaniti,F.Novazzietal. InternationalJournalofInfectiousDiseases104(2021)315–319

316

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(ThermoFisher, Waltham, Massachusetts, United States) as reported(Baldantietal.,2008;Furioneetal.,2012).Atthesame time,ParvovirusB19andHumanHerpesvirus6wereanalyzedina fractionofpatients(Corcoranetal.,2010;Watzingeretal.,2004).

Lymphocytesubpopulationscountandcharacterization

Blood samples were stained with CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetra- CHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5, all from BeckmanCoulter,Milan,Italy).ThepercentageofCD4+andCD8+T lymphocytesandCD56+NKandCD19+Bcellswasdeterminedby Navios FlowCytometer System(Beckman Coulter). UsingFlow- CountFluorospheres,CD4+,CD8+TcellsubsetsandCD56+NKand CD19+Bcellswereexpressedasabsolutecounts(cells/

m

^l).^Gating

strategywassetuponCD45⁺andsidescatter(SSC).

Statisticalanalysis

Descriptivedatawerereportedorconsideredasabsoluteand relativefrequencies,medianandinterquartilerange(IQR)basedon thetypeofvariabledistribution.Forqualitativevariables,Fisher’s test was used, while the Mann-Whitney test was used for quantitative variables in ordertoperform comparisonbetween groups.Spearman’stestwasusedforthecorrelationanalysis.All testsweretwo-tailed.Ap-value<0.05wasconsideredstatistically signiﬁcant.AnalyseswereperformedusingtheGraphPadPrism5 (GraphPadSoftware,CA,USA).

Results

Clinicalcharacteristics

Male COVID-19 patients were more frequent in both units (p=0.039).Themostcommonsymptomsdetectedinpatientson admissionorduringhospitalizationinICUandSICU,respectively, were fever (80.9% and 100%; p=0.02), interstitial lung disease (100%and55.6%;p<0.001),dyspnea(100%and37%;p<0.001), and diarrhea (2.3 and 3.7%; p>0.05). Overall, all the patients exceptonewerepositiveforEBVIgG(99%),while100/104subjects (96.2%)wereCMVseropositiveattimeofhospitalization.Finally, 72/104(69.2%)werepositiveforParvovirusB19IgG.

Viraldetectionandquantiﬁcation

CMV,ParvovirusB19andHHV-640DNAwerenegativeinall patientsanalyzed.EBVpositiveDNAwasobservedin91/103EBV seropositivepatients(88.3%).Ofthem,40/42patients(95.2%)were admittedtotheICUand51/61patients(83.6%)wereadmittedto theSICU.Intheremaining2/42patients(4.8%)and10/61patients (16.4%)intheICUandSICU,respectively,noEBVDNAemiaevents werereported(p=0.1161).TheEBVDNAmedianlevelamongICU patients[4709(IQR1284–32075)IU/mL]wassigniﬁcantlyhigher than that observed in SICUpatients[median 1890 (IQR 370.1– 8820)IU/mL;p=0.0289)asshowninFigure1.

Lymphocytesubpopulationcounts

Lymphocytesubpopulationcountswereavailablefor31/42ICU patients (73.8%) and for 55/62 SICU patients (88.7%). A slight reduction of the total lymphocyte count was observed in ICU patientsascomparedwithSICUpatients[median617.5(IQR473.8– 858)cells/

m

^l^vs⁸²³^(IQR⁵³³–1044)cells/

m

^l;^p⁼^0.0502].^In^detail,

CD4Tcellcountwasnotstatisticallydifferentbetweenthetwo groupsofpatients[median249(IQR185.3–393.3)cells/

m

^l^vs²⁸⁶

(IQR 182–403) cells/

m

^l, respectively; p=0.6953]. On the other

hand,weobservedasigniﬁcantreductionofCD8⁺Tcellcountin ICUpatientsascomparedwithSICUpatients[median95(IQR58– 174) cells/

m

^l^vs ¹⁸¹⁽¹¹⁵–257) cells/

m

^l; ^p⁼^0.0011]. ^Similarly, ^a

lowermedianNKcountwasreportedinICUpatientsascompared withthegroupofSICUpatients[50(IQR36–90)cells/

m

^l^vs⁹⁸^(IQR

56–163) cells/

m

^l; ^p⁼^0.0131]. ^In ^addition, ^comparing ^the ^two

groupsofpatients,BcellcountwassigniﬁcantlyincreasedinICU patients[median81(IQR65–203)cells/

m

^l^vs⁶⁹⁽³⁶–113)cells/

m

^l,

respectively;p=0.0172)(Figure2).Nolinearcorrelationbetween EBVloadandCD8T cells,BcellsorNKcellswasobserved(Figure3).

Discussion

Thestrictrelationbetweenimmunosuppressionandreactiva- tionoflatentvirusesandanincreasedprevalenceofopportunistic viralinfectionsiswellknown,especiallyintransplantedpatients andinICUpatients(Fishman,2013;SchildermansandDeVlieger, 2020; Limayeand Boeckh, 2010; Frantzeskakiet al., 2015;Ong etal.,2017;Cantanetal.,2019;Coşkunetal.,2017).

TheimpactofCOVID-19infectionontheimmunesystemisstill largelyunknown.WeobservedasigniﬁcantlossofNKandCD8⁺T cells inCOVID-19patients, which paralleledtheseverityof the Figure1.EBVDNAwasmeasuredin42COVID19-positivepatientsinICU(black dots)andin62COVID19-positivepatientsinSICU(whitedots).MedianandIQRare shownandp-valuewasgiveninthegraph.

Figure2. Lymphocytesubpopulationcountswerecomparedinthe42patientsof theIntensiveCareUnit(greybars)andinthe62patientsoftheInfectiousDisease Department(whitebars).Thep-valuewasmeasuredbytheMannWhitneytestand wasgivenforeachgroupofcomparison;*p<0.05;**p<0.01.

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infection.Normally,inhealthysubjectslymphocyteCD3⁺counts rangefrom690to2540cells/

m

^l,^lymphocyte^CD4⁺^and^CD8⁺^counts

rangefrom410to1590andfrom190to1140cells/

m

^l,respectively;

moreover,inhealthysubjects,wereportedthatCD19⁺Bcellcounts andCD56+NKcellcountsrangefrom163to288cells/

m

^l^and^from

151to296cells/

m

^l.

Surprisingly,thisNKandCD8⁺Tcellreductionwasassociated withpresenceofEBVDNAonly.Despitethatthelargemajorityof patientswereseropositiveforbothCMVandB19,noevidenceof circulating CMV and B19 DNAwas observed. In this study, we analyzedthecorrelationbetweenCOVID-19infectionandimmu- nologicalandvirologicalfactors,classifyingpatientsaccordingto theseverityoftheCOVID-19disease.Overall,ahigherproportion ofinterstitiallungdiseaseanddyspneawasobservedamongICU patientsas comparedtoSICUpatients(p<0.001).Interestingly, positiveEBVDNAwasobservedinapproximately87%ofpatients.

TherateofpositiveEBV DNAobservedwashigherinCOVID-19

patients with severe infection (ICU patients) as compared to patients with mild symptoms (SICU patients). Moreover, the median EBV DNA levels in patientswith severe COVID-19was significantlyhigherthaninpatientswithmildCOVID-19disease, supportingtheevidenceofahigherimmuneimpairmentinthe previouspatients. However,no linear correlationbetween EBV DNAloadandlymphocytecountswasobserved.Inlinewiththese results,areducedtotallymphocytecountinpatientsreferredto theICUwasobserved,reportingasignificantreductionintermsof bothCD8⁺andNKcells ascompared toSICUpatients, whileno significantreductionofCD4⁺Tcellcountswasobserved.Recent studiesunderlinedamarkedlymphopeniainalargefractionof COVID-19patients(Huang etal., 2020;Tan etal.,2020)witha reductionoftotalTlymphocytes,CD4⁺TcellsandCD8⁺Tcellsanda moreprofoundreductioninseverecases(Chenetal.,2020).

Ofnote,thehigherincreaseofBcellcountobservedinpatients with severe symptoms as compared to patients with milder symptoms was in line with results obtained by Chen and colleagues(Chenetal.,2020).However,ourdatawasincontrast withWangandcolleagues’observation,reportingadecreaseofB cellsinpatientswithsevereCOVID-19disease(Wangetal.,2020).

SinceBlymphocytesarethesiteoflatencyforEBV,anincrease ofBcellsinpatientswithseveresymptomscouldberelatedtothe increasedEBVDNAlevelsasoccurringinpatientswithPTLD(post- transplantlymphoproliferativedisorder).Ontheotherhand,the increase of B cell counts during COVID-19 could reﬂect the antibodyresponse toSARS-CoV-2.Finally,therole of increased levelsofEBVDNAonBcellfunctions,withapotentialimpacton antibodyproduction,isunknown.Thus,wecanonlyspeculatethat thepresenceofdetectableEBVDNAmayplayaroleintheseverity ofCOVID-19diseaseonthebasisofthehigherfrequencyofEBV DNApositivepatientsandthehigherEBVDNAlevels.

Interestingly,amongallopportunistic viruses,reactivationin COVID-19 patients seems to be restricted to EBV, an event somewhatcorroboratingaroleintheseverityofthedisease.In fact,despitethelowrateofCD8⁺TcellsandNKcellsinpatients withseveresymptoms,CMVreactivation,forexample,wasnever observed.Usually,detectableCMV DNAinwholebloodismore frequent than EBVDNA in immunocompromised patientswith reducedlymphocytecounts.

In conclusion, our data support the possible correlation between lymphopenia and EBV load with COVID-19 disease severity. In order to corroborate ourhypothesis, monitoringof the immunological proﬁle and opportunistic virus reactivation overtimeiswarrantedinordertobetterunderstandtheroleinthe pathogenesisofthediseaseoritsrelationshipwiththeseverityof symptoms.

Funding

This work was supported by the Ministero della Salute, FondazioneIRCCSPoliclinicoSanMatteoRicercaCorrente(grant 80207).

Conﬂictofinterest

Theauthorsdeclarethattheyhavenocompetinginterests.

Authorcontributions

Stefania Paolucci and Irene Cassaniti: data analysis and interpretation,draftedthearticle;FedericaNovazzi,LorettaFiorina andGiudittaComolli:sampleprocessing.AntonioPiralla:software and data analysis.Raffaele Bruno, Renato Maserati, Roberto Gulminetti,StefanoNovatiandFrancescoMojoli:patientenroll- ment. Fausto Baldanti: critical revision of the article and ﬁnal Figure3.EBVDNAloadwascorrelatedtoBcellcount(a),CD8+Tcellcount(b)and

NKcount(c);r(RemuzziandRemuzzi,2020)andpvaluewerecalculatedusing Spearman’scorrelationtestandresultsweregivenineachgraph.

S.Paolucci,I.Cassaniti,F.Novazzietal. InternationalJournalofInfectiousDiseases104(2021)315–319

318

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approvaloftheversiontobepublished.Allauthorscontributed towarddataanalysis,draftingandrevising thepaper, gaveﬁnal approvaloftheversiontobepublishedandagreetobeaccountable forallaspectsofthework.

Acknowledgements

We thank Daniela Sartori for careful preparation of the manuscript.

References

https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf.

BaldantiF,GattiM,FurioneM,PaolucciS,TinelliC,ComoliP,etal.Kineticsof Epstein-Barr virusDNAloadindifferentbloodcompartmentsofpediatric recipientsofT-cell-depletedHLA-haploidenticalstemcelltransplantation.J ClinMicrobiol2008;46:3672–7.

Cantan B, LuytCE,Martin-Loeches I.InﬂuenzaInfections and EmergentViral InfectionsinIntensiveCareUnit.SeminRespirCritCareMed2019;40:488–97.

ChenG,WuD,GuoW,CaoY,HuangD,WangH,etal.Clinicalandimmunologic featuresinsevereandmoderateCoronavirusDisease2019.JClinInvest2020;

pii:137244.

CorcoranC,HardieD,YeatsJ,SmutsH.GeneticvariantsofhumanparvovirusB19in SouthAfrica:cocirculationofthreegenotypesandidentiﬁcationofanovel subtypeofgenotype1.JClinMicrobiol2010;48:137–42.

Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al.

Detection of2019novelcoronavirus(2019-nCoV)byreal-timeRT-PCR.

Euro Surveill 2020;25:2000045, doi:http://dx.doi.org/10.2807/1560- 7917.ES.2020.25.3.2000045.

CoşkunO,YaziciE,ŞahinerF,KarakaşA,KiliçS,TekinM,etal.Cytomegalovirusand Epstein-Barrvirusreactivationintheintensivecareunit.MedKlinIntensiv- medNotfmed2017;112(3):239–45.

FishmanJA.Overview:cytomegalovirusandtheherpesvirusesintransplantation.

AmJTransplant2013;13(Suppl3):1–8.

FishmanJA.InfectioninOrganTransplantation.AmJTransplant2017;17:856–79.

FrantzeskakiFG,KarampiES,KottaridiC,AlepakiM,RoutsiC,TzanelaM,etal.

Cytomegalovirusreactivationinageneral,non-immunosuppressedintensive careunitpopulation:incidence,riskfactors,associationswithorgandysfunc- tion,andinﬂammatorybiomarkers.JCritCare2015;30:276–81.

FurioneM,RognoniV,CabanoE,BaldantiF.Kineticsofhumancytomegalovirus (HCMV)DNAemiaintransplantedpatientsexpressedininternationalunitsas determinedwiththeAbbottRealTimeCMVassayandanin-houseassay.J ClinVirol2012;55:317–22.

GhinaiI,McPhersonTD,HunterJC,KirkingHL,ChristiansenD,JoshiK,etal.First knownperson-to-persontransmissionofsevereacuterespiratorysyndrome coronavirus2(SARS-CoV-2)intheUSA.Lancet2020;395(10230):1137–44.

HuangC,WangY,LiX,RenL,ZhaoJ,HuY,etal.Clinicalfeaturesofpatientsinfected with2019novelcoronavirusinWuhan.China.Lancet2020;15(395):497–506, doi:http://dx.doi.org/10.1016/S0140-6736(20)30183-185.

LimayeAP,BoeckhM.CMVincriticallyillpatients:pathogenorbystander?.Rev MedVirol2010;20:372–9.

MetlayJP,WatererGW,LongAC,AnzuetoA,BrozekJ,CrothersK,etal.Diagnosisand treatmentofadultswithcommunity-acquiredpneumonia:anofﬁcialclinical practiceguidelineoftheAmericanThoracicSocietyandInfectiousDisease SocietyofAmerica.AmJRespirCritCareMed2019;200(7):e45–67.

OngDSY,BontenMJM,SpitoniC,VerduynLunelFM,FrenckenJF,HornJ,etal.

EpidemiologyofMultipleHerpes ViremiainPreviouslyImmunocompetent PatientswithSepticShock.MolecularDiagnosisandRiskStratiﬁcationofSepsis Consortium.ClinInfectDis2017;64:1204–10.

Remuzzi A, Remuzzi G. COVID-19 and Italy: what next?. Lancet 2020;395 (10231):1225–8.

SchildermansJ,DeVliegerG.Cytomegalovirus:ATrollintheICU?Overviewofthe LiteratureandPerspectivesfortheFuture.FrontMed(Lausanne)2020;7:188, doi:http://dx.doi.org/10.3389/fmed.2020.00188.

TanL,WangQ,ZhangD,DingJ,HuangQ,TangYQ,etal.Lymphopeniapredicts disease severityof COVID-19: a descriptive and predictive study. Signal TransductTargetTher2020;5(33)eCollection2020.

WangF,NieJ,WangH,ZhaoQ,XiongY,DengL,etal.Characteristicsofperipheral lymphocytesubsetalterationinCOVID-19pneumonia.JInfectDis2020;pii:

jiaa150.

WatzingerF,SudaM,PreunerS,BaumgartingerR,EbnerK,BaskovaL,etal.Real-time quantitative PCR assaysfor detectionand monitoring of pathogenichuman viruses inimmunosuppressedpediatricpatients.JClinMicrobiol2004;42:5189–98.