Case report of a neonate with high viral SARSCoV-2 loads and long-term virus shedding

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JournalofInfectionandPublicHealth13(2020)1878–1884

ContentslistsavailableatScienceDirect

Journal

of

Infection

and

Public

Health

jo u r n al hom ep age :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / j i p h

Case

Report

Case

report

of

a

neonate

with

high

viral

SARSCoV-2

loads

and

long-term

virus

shedding

Monique

A.L.J.

Slaats

a,∗

_,

_Maud

_Versteylen

a

_,

_Karin

_B.

_Gast

b

_,

_Bas

_B.

_Oude

_Munnink

c

_,

Suzan

D. Pas

b

_,

_Robbert

_G.

_Bentvelsen

b

_,

_Ron

_van

_Beek

a

a_Department_of_Pediatrics,_Amphia_Hospital,_Molengracht_21,₄₈₁₈_CK,_Breda,_The_Netherlands b_Microvida,_Location_Amphia,_Molengracht_21,₄₈₁₈_CK,_Breda,_The_Netherlands

c_Department_of_Viroscience,_WHO_{Collaborating}_Centre_for_Arbovirus_and_Viral_Hemorrhagic_Fever_Reference_and_Research,_Erasmus_University_Medical

Center,Molewaterplein50,3015CE,Rotterdam,TheNetherlands

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29June2020 Receivedinrevisedform 23September2020 Accepted13October2020 Keywords: COVID19 SARS-CoV-2 Novelcoronavirus Neonates

a

b

s

t

r

a

c

t

Background:SARS-CoV-2hasspreadglobally.Currently,literatureofSARS-CoV-2inneonatesisscarce. Wepresentacaseofaneonatewithahighviralloadandprolongedvirusshedding.

Methods:Epidemiology,clinicalcharacteristics,treatment,laboratorydataandfollow-upinformation andthetreatmentofaneonatewithCOVID-19wererecorded.

Results:A7-day-oldboywasadmittedtothehospitalwithfever,lethargyandapnoea.Hewasfound SARS-CoV-2RNApositivewithanexceptionallyhighviralloadinnasopharyngealswabandstool.The fatherandtwomaternitynursesathomehaddetectableSARS-CoV-2RNAaswell.Sequencingshowedall strainsbelongedtothesamecluster.Thefatherwasasymptomaticandthematernitynursesdeveloped symptomsaftervisiting.In themother,noSARS-CoV-2RNAcouldbefound.Six daysafter admis-sion,theneonatewasdischargedafterclinicalimprovementwithoralantibioticsbecauseofapossible pyelonephritis.MonitoringthecourseofthisinfectionshowedthatSARS-CoV-2RNAwasdetectablein thenasopharynxuntilday19andinstooluntilday42aftersymptomonset.

Conclusions:ThiscaseshowsthatneonatescanhaveahighviralloadofSARS-CoV-2andcanshedthe virusforoveronemonthinstool.Despitethehighviralloadintheneonate,themotherandasiblingdid notgetinfected.

Background

StartingfromDecember2019,anovelcoronavirus (SARS-CoV-2),causingcoronavirusdisease2019(COVID-19),hasspreadon a globalscale[1].Comparedtoadults,children seemtobeless affectedbySARS-CoV-2infectionastheyareusuallyasymptomatic orhavemildsymptoms[1–4].However,littleisknownabout dis-easeseverityinneonates,theneedfortreatmentandtheirrole intransmissionofSARS-CoV-2[5].Itisreportedthatchildrenor neonatescanhavehighviralloads,whileremainingasymptomatic orhavingmildsymptoms[6,7].However,thereisnoclear associa-tionbetweentheviralloadandinfectivity.

Furthermore,inchildrenagedbetween2monthsand15years, SARS-CoV-2RNAcouldstillbedetectedintheirstoolinupto70

∗ Correspondingauthor.

E-mailaddress:moniqueslaats@gmail.com(M.A.L.J.Slaats).

days[4,8,9].Dataaboutdurationofviralsheddinginneonatesis scarce[10,24,27,33].

Inthiscase reportwepresent a7-day-oldneonatewhowas hospitalizedintheNetherlandswithCOVID-19andhadahighviral loadofSARS-CoV-2inthenasopharynxandstool.Wereviewedthe literatureandinvestigatedthedurationofviralsheddingandthe sourceofinfection.OnlydetectionofviralRNAwasdescribedin thisarticle,nottheisolationoflivevirus.

Casepresentation

A7-dayoldboywasadmittedtotheemergencywardofthe AmphiahospitalinBreda,theNetherlandsbecauseoffever (rec-taltemperature38.4–39.4◦C),lethargyandapnoea.Thedaybefore thispresentation,hewastreatedwithphototherapyfor1dayfor hyperbilirubinemia.Hehadnorunnynoseandnotachypnoea, oxy-gensaturationwas99%onroomair,andauscultationofhislungs wasclear.AlllaboratorycharacteristicsarepresentedinTable1. https://doi.org/10.1016/j.jiph.2020.10.013

1876-0341/©2020TheAuthors.PublishedbyElsevierLtdonbehalfofKingSaudBinAbdulazizUniversityforHealthSciences.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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M.A.L.J.Slaatsetal. JournalofInfectionandPublicHealth13(2020)1878–1884

Table1

Laboratorycharacteristics.

DayssincepositivePCRSARS-CoV-2neonate Day-1 Day0-morning Day0-evening Day1 Day2 Day5

Haemoglobin,g/L 11.8 10.6 11.1 10.3

Whitebloodcellcount,×109_/L _7.3 _8.6 _15.1 _7.4

Lymphocytecount,×109_/L _1.47 _3.84

Plateletcount,×109_/L ₂₅₈ ₂₆₉ ₂₅₄ ₁₅₈

C-responseprotein,mg/L 1 1 5 54 23

Alanineaminotransferase,U/L 14

Aspartateaminotransferase,U/L 51

Totalbilirubin,mmol/L 362 229 203 201

Creatinekinase,U/L 101

Lactatedehydrogenase,U/L 634 499

Urine-Whitebloodcellcount,×109_/L _>800 ₂₂

Urine-erythrocytcount,×109_/L _<5 _<5

Lumbarpuncture-whitebloodcellcount,×109_/L ₁

Lumbarpuncture-erythrocytcount,×109_/L ₁₀₀₀

Bloodculture Neg

Urineculture Neg

PCREnterovirus/parechovirus(stool) Neg

PCREnterovirus(lumbarﬂuid) Neg

PCRinﬂuenza/RSV(nasopharyngealswab) Neg

(Abbreviations:COVID-19=newcoronavirus2019,PCR=polymerasechainreaction,RSV=respiratoirsyncytieelvirus,pos=positive,andneg=negative).

Onlytheﬁrstlymphocytecountwaslowandtherewasanincrease

of c-responseproteinonday1.Sincetherewerenorespiratory

symptoms, no chest radiographwas performed.Because of his

age, a total sepsis work-upwas performed includingcomplete

blood count,urinalysis,cerebrospinal ﬂuid(CSF)studies,blood,

urineandCSFculture,andnasopharyngealswabandstoolforPCR

viruses.Afterthework-up,empiricaltherapyforlate-onsetsepsis

(intravenoustherapyofamoxicillinandcefotaxime)wasinitiated.

SARS-CoV-2RNAwasdetectedinanasopharyngealswabandstool

byasemi-quantitativereal-timereversetranscriptasepolymerase

chainreaction(sqRT-PCR)withlowcyclethreshold(Ct)-valuesof

13.9and20.5respectively,suggestingahighviralload[11,12].No

SARS-CoV-2RNAwasfoundinurineandCSF.sqRT-PCRwas neg-ativeforotherpathogensonnasopharyngealswab,CSFandstool (Table1).Bloodandurinebacterialcultureremainednegative.The urine culture couldbefalse negativebecause ofthe antibiotics whichwereadministratedpriortotheurinesample.Becauseofthat factandleukocyturia,apossiblebacterialpyelonephritiscouldnot beexcluded.Fromthreedayspost-symptomonset,therewereno clinicalsymptomswithstableparameters.Sixdaysafteradmission, theneonatewasdischargedingoodconditionwithoralantibiotics (amoxicillin80mg/kg)forthreemoredays.

Courseofinfection

NoCOVID-19-relatedsymptomswererecordedduringthe14 days follow-up afterdischarge. Nasopharyngeal swabremained SARS-CoV-2RNApositiveuntilday19andbecamenegativeafter day20ofdiseaseonset.Stoolsamplesremainedpositiveuntilday 42(Fig.1).

Sourceofinfection

The neonate had been delivered vaginally at term after an uneventful pregnancy and was breastfed. We investigated the sourceof thisinfectionand Fig.1illustrates thedatesof expo-sure,illnessonsetandsamplingandreal-timeRT–PCRresultsof nasopharyngealandrectalswabsofclose-contactsoftheboy.His parentsdidnotreportfeverorupperairwaycomplaints.His two-year-oldsisterhadarunnynoseforweeksandhisgrandparents reportedafeverwithupperairwaycomplaints(runnynoseand cough)28daysbeforeunsetofillnessoftheboy.Maternitynurse1 (MN1)to.okcarefortheneonateforonedayathomewithout symp-tomsofCOVID-19.Thishappenedtwodaysbeforetheneonatehad

feverfortheﬁrsttime.Onedayafterthevisit,shedevelopedasore throat.Maternitynurse2(MN2)startedworkingwithout symp-tomsthedayafterthevisitofMN1.Shehadsymptomsoffever, headacheandchestpainonedayaftervisitaswell.Thefather,MN1, andMN2testedSARS-CoV-2RNApositive,withinitial nasopharyn-gealCtvalues33.3,18.3and26.2respectively(Fig.1).Follow-up showedthatthefatherhadanincreasingviralloadonday6 (Ct-value21.9).BothmotherandsisterremainedSARS-CoV-2

RNAnegative atday13 and day19, respectively. Expressed breastmilkwastestednegative.

Wholegenomesequencingofthevirusfromthenasopharyngeal swabspecimenswasperformedasdescribedbefore[13].MN2and theneonatehadidenticalgenomeswhilethefatherandMN1had adifferenceof1single-nucleotidepolymorphism,placingallviral genomesinonecluster.Aphylogenetictreewasconstructed con-tainingallDutchSARS-CoV-2sequencesusingIQ-TREEunderthe GTR+F+I+G4asbestpredictedmodelusingtheultrafastbootstrap option[14,15].

Toinvestigatewhethermotherandsisterhadpreviouslybeen infected and already turned negative SARS-CoV-2 PCR results, serumsamplesweretestedatday3andday19(mother)andday19 (sister)forthepresenceofIgGantibodiesagainstSARS-CoV-2using anenzymelinkedimmunosorbentassay(ELISA)kit(EUROIMMUN, Luebeck,Germany);bothtestednegative.

Reviewoftheliterature

Anelectronicsearchwasconductedonstudiespublishedfrom1 December2019to29Junebysearchingforthekeywords‘covid-19’ OR‘covid’OR‘SARS-CoV-2’AND‘neonate’onPubMed,MEDLINE, GoogleScholar,medRxiv,andbioRxivdatabase.Wealsosearched the references listed within the articles to identify additional articlesthatmayhavebeenmissedduringtheelectronicsearch. Eli-giblestudies(includingcasereports,caseseries,cohortstudiesand retrospectivestudies)publishedinEnglish-languagejournalsthat havedescribedneonatesdiagnosedwithCOVID-19wereincluded for analysis.We summarized the studiesinvolvingneonates in Tables2Aand2B[10,16–34].Mostoftheseneonateshadaninfected motherandwereasymptomatic(n=7)orbecamesymptomatic after16huntil17daysafterbirth(n=20).

They all recovered. To our knowledge, there is only one series report about neonates that showed viral loads (Ct val-ues)in neonates. Thisreport included18 neonatesand infants agedbetween10–88daysoldandshowedextremelylowvalues

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M.A.L.J. Slaats et al. Journal of Infection and Public Health 13 (2020) 1878–1884 Table2A

SummaryofstudiesofneonateswithconﬁrmedCOVID-19.

Author Hanetal [10].(n=1) Aghdametal [16].(n=1) Wangetal. [17].(n=1) Zengetal [18].(n=3) Zhangetal [19].(n=4) Coronado Munozetal [20].(n=1) Dumpaetal [21].(n=1) Saliketal [22].(n=1) Precitetal [23].(n=1) Piersigillietal [24].(n=1) Paretetal [25].(n=1) Alzamoraetal [26].(n=1) Gestational age (weeks) 38 Unknown 40 39,39 >39 36 39 37 39 26+4 Unknown 33 Dayoflife when illness started

27days 15days 36h Unknown 30h–17days 21days 22days 7days 10days 7days 25days 16h

Modeof delivery

Vaginal CS CS CS CS Unknown Vaginal Unknown Vaginal CS Unknown CS

Presentation Fever,cough, rhinorrhoea, feeding intolerance Fever, feeding intolerance, mottling Asympto-matic Fever, cyanosis, feeding intolerance, dyspnoea Fever,cough, feeding intolerance, dyspnoea Rhinorrhoea, feeding intolerance, dyspnoea Fever, feeding intolerance Fever, dyspnoea, cyanosis, feeding intolerance, lethargy Rhinorrhoea, dyspnoea No COVID-19-related symptoms Fever, irritability Respiratory difﬁculty Abnormal labs / None ElevatedCK andASAT None / Rhinovirus positive, elevatedCRP andPCT

None ElevatedIgM,

cytokine,and lymphocyte count LowMCVand MCH Leukopenia / None Chest radiog-raphy

Normal Normal Abnormal / Abnormal Bilateral

infiltration Normal Bilateral pulmonary granular opacities Bilateral groundglass opacities Non-specific bilateralstreaky infiltrates Not performed Normal SARS-CoV-2 testing PCRNP,stool, plasma,and urine PCRNP PCRNP / PCRNPand analswabs PCRNP PCRNP PCRNP PCRNP, plasma, nares,and stool PCRNP PCRNP PCRNPand plasma

Medication None Antibiotics,

antiviral agents

Antibiotics Antibiotics Antibiotics Antibiotics Antibiotics None None None Antibiotics None

Probable modeof transmis-sion

Horizontal Horizontal Vertical? Horizontal Horizontal Horizontal Horizontal Horizontal Horizontal Vertical? Horizontal Vertical?

Outcome Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered

(Abbreviations:ASAT=Aspartateaminotransferase,CK=Creatinekinase,COVID-19=coronavirusdisease2019,CRP=C-reactiveprotein,CS=sectiocaesarea,GA=gestationalage,MCH=meancellhemoglobin,MCV=meancell volume,NP=nasopharyngealswab,PCR=polymerasechainreaction,PCT=procalcitonine).

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M.A.L.J. Slaats et al. Journal of Infection and Public Health 13 (2020) 1878–1884 Table2B

SummaryofstudiesofneonateswithconﬁrmedCOVID-19.

Author Diazetal [27].(n=1) Gregorio-Hernandez etal[28].(n= 2) Whiteetal [29].(n=2) Huetal[30]. (n=1) Buonsenso etal[31].(n= 1) Pateketal[32]. (n=1) Chacon-Aguilaretal [33].(n=1) Gordonetal.[34] (n=1) Gestational age (weeks) 38+4 38+1,39 39,39 40 38+3 39 Unknown 32 Dayoflife when illness started

8days 2days,6days 16days,25

days

36h 15days 14days 26days 4days

Modeof delivery

CS Vaginal Vaginal,CS CS CS CS Unknown CS

Presentation Asymptomatic Asymptomatic

besides previous diagnosis Fever, rhinorrhoea, hypoxia, conjunctivitis

Asymptomatic Asymptomatic Fever,fussiness,

apnoea Paroxysmal episodes, fever, rhinorrhoea, vomiting Asymptomatic Abnormal labs None ElevatedCRP, PCT, lympho-cytopenia Neutropenia, elevatedCRP

Lymphocytosis / Elevatedliver

enzymes, neutropenia, monocytosis ElevatedCK andLDH / Chest radiog-raphy Groundglass opacities Consolidation lateraland posterior Hazy opacities without con-solidations

Normal / Bilateralperihilar

streaking withoutfocal consolidation Not performed Findings consistentwith surfactantlung disease SARS-CoV-2 testing PCRNP PCRNP PCRNP PCRNP PCRNP PCRNP PCRNP,stool PCRNP

Medication None None None None None Antibiotics,

acyclovir Antibiotics None Probable modeof transmis-sion

Horizontal Horizontal Horizontal Horizontal Horizontal Horizontal Horizontal Vertical?

Outcome Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered

(Abbreviations:ASAT=Aspartateaminotransferase,CK=Creatinekinase,COVID-19=coronavirusdisease2019,CRP=C-reactiveprotein,CS=sectiocaesarea,GA=gestationalage,MCH=meancellhemoglobin,MCV=meancell volume,NP=nasopharyngealswab,PCR=polymerasechainreaction,PCT=procalcitonine).

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Fig.1. Chronologyofmajorepidemiologicaleventsandmoleculartestingresultsofneonateandclose-contactsafterpositivePCRSARS-CoV-2inneonate(Abbreviations: Adm=admission,BM=breastmilk,CSF=cerebrospinalﬂuid,CT=cyclethresholdvalues,MN=maternitynurse,neg=negative,NP=nasopharyngealswab,PCR=polymerase chainreaction,ReAdm=readmission,SNP=singleNucleotidePolymorphisms,WGS=whole-genomesequencing).

between3.00–6.58[7].Addiotionally,therearesomecasereports thatreportviralsheddinginneonates.Gordonetal.reportedthe longestsheddingof28days(nasopharyngealswab)inaneonate [34].Hanetal.reportedvirussheddinginnasopharyngealswab untilday17,stooluntilday18,andurineuntilday10afteronset ofsymptomsofa27-dayoldneonate[10].

Discussion

We reporta 7-day-oldneonatewithCOVID-19conﬁrmedby PCR andwithpossiblepyelonephritis. Hehadnorespiratory or gastrointestinalsymptoms.Ahighviralloadof SARSCoV-2RNA wasfoundinnasopharynxandstoolspecimensandremained pos-itiveuntilday42.Thefather,whohadnoclinicalsymptoms,and two maternity nurseswho cared for theboy for oneday only, werebothpositiveforSARS-CoV-2RNAand(oneofthem) prob-ably infectedthisneonate.Themother hadnotpreviouslybeen infectedandwasnotinfectedbythisneonateduringtheperiod ofviralsheddingdespitethehighviralload.Asdescribedin cur-rentliterature,symptomsofCOVID-19innewbornsweremild,or evennonewithcompleterecovery;whichisconsistentinourcase report[7,10,16–34].Inmostcases,themotherwasinfectedand probablytransmittedhorizontallytotheneonate.Themotherin ourcaserepeatedlytestednegativeforSARS-CoV-2byPCR,and noIgGsero-reactivitywasseen.Therefore,weconcludethatthe motherwasnotthesourceofthisinfection[35].

Literature shows SARS-CoV-2incubationperiod arebetween 4–21days[1–3,11].Thisneonatebecamesymptomatic1.5and2.5 daysafterthevisitofthematernitynurses.Inthiscase,itishard todeterminethesourceoftheinfection.Theneonatewasbornin thehospitalandstayedfor1dayafterbirth,thefathershowedno symptomsandthematernitynursesshowedsymptoms1dayafter visitingthisfamily.Sequencingshowedallstrainsbelongedtothe samecluster.Arelativefastincubationtimecouldbepossiblydue tothehighviralloadofSARSCoV-2RNAinourneonate.TheseCt valuesarelowercomparedtochildrenandadults[35–37].Possible explanationincludethatneonatesarepresentingearlierinillness coursethanolderpeople.

Viralsheddingindifferentspecimentypessuchasstool,urine, CSF, orblood remainsuncertainin neonates.Limiteddata have shown that viralRNAcouldbedetectedinplasma orserum of neonates[10,22,25]andadultpatients[37].Justonecasereport detected viralRNA inurine of a neonate [10]. A recentreview

concludedthat3.7%of430adultpatientswerepositiveinurine [38]. We tested for SARS-CoV-2 RNA in urine, nasopharyngeal swab,stool,andCSF oftheneonate, onlynasopharyngealswab andstoolsampletested positivewithprolongedvirusshedding (Tables2Aand2B).Thisisconsistentwiththecurrentliterature ofchildren, PCR instool waspositive until70 days in children [8,9,11,39,40,41]. The duration of viral shedding in neonates is scarce.Becauseofprolongedsheddinginstool,itisnecessarytobe awareofthepossibilityoffecal-oraltransmissionofSARS-CoV-2 infection,especiallyinneonatesandchildren.

Additionally,duetotherelativelylongdurationofviral shed-ding, caution should be taken with diagnosis of COVID-19 in neonatesandchildrenpresentingwithCOVID-19likesymptoms.

Currently,thereisnoconsensusoninfectivityduringviral shed-ding[42,43].Cultureofliveviruswasnotpossibleinourhospital. Wolfeletal.demonstratedthatlivevirusSARS-CoV-2canbe cul-turedfromnasal/throatandsputumsamplesinpatients,however, noliveviruswassuccessfullyisolatedafterday8fromsymptom onsetdespiteongoinghighviralloads[43].Bullard etal. inves-tigated90 SARS-CoV-2 RT-PCR conﬁrmedpositive samples and determinedtheirabilitytoinfectVerocelllines.Inthisstudy,28.9% demonstratedviralgrowthandtheyconcludedthatinfectivityof patientswithCt>24anddurationofsymptoms>8daysmaybe low[12].Furtherresearchisneededtodeterminetheviabilityof thevirusoutsideandthedurationofinfectivity.

Conclusion

Inconclusion,dataofneonateswithCOVID-19areverylimited. Thiscasereportshowedaneonatewithahighviralloadof SARS-CoV-2andprolongedsheddingwithcompleterecovery.Thereby, onlynasopharyngealsecretionandstoolswabtestedpositive.Stool remainedpositivethelongestuntilday42.However,thiscourseof infectionislimitedbythissinglecase,additionaldataon COVID-19infectedneonatesarenecessarytoconﬁrmtheseﬁndings.Until then,isolationofchildrenorneonateswithCOVID-19symptoms shouldbeperformedassoonaspossibletopreventtransmission tohealthcareprofessionalsandothers.

Authors’contributions

MSandMVconceivedthestudy,andparticipatedinitsdesign. MSandMVDraftedandrevisedthemanuscriptforcontent

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ingmedicalwritingforcontent,analysisandinterpretationofdata. RB,SP,KG,BOconceivedthestudy,participatedinitsdesign man-agedmicrobiologylaboratoryassays,andrevisedthemanuscript. BOperformedsequencing.Allauthorsreadandapprovedtheﬁnal manuscript.

Ethicsapprovalandconsenttoparticipate Notapplicable.

Consentforpublication Included.

Availabilityofdataandmaterials Notapplicable. Competinginterests Nonedeclared. Funding Notapplicable. Sourcesofsupport None. Acknowledgements Notapplicable.

AppendixA. Supplementarydata

Supplementary materialrelated tothis article canbefound, intheonlineversion,atdoi:https://doi.org/10.1016/j.jiph.2020.10. 013.

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