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SeedingofoutbreaksofCOVID-19bycontaminatedfreshandfrozenfood*DaleFisher1,2,3,AlanReilly4,AdrianKangEngZheng5,AlexRCook2,6,DanielleE.
Anderson51.
YongLooLinSchoolofMedicine,NationalUniversityofSingapore,Singapore2.
NationalUniversityHealthSystem,Singapore3.
DivisionofInfectiousDiseases,NationalUniversityHospital,Singapore4.
CentreforFoodSafety,UniversityCollegeDublin,Ireland5.
ProgrammeinEmergingInfectiousDiseases,Duke-NUSMedicalSchool,Singapore6.
SawSweeHockSchoolofPublicHealth,NationalUniversityofSingapore*CorrespondingAuthorAuthorContributionsDFandDAconceptualisedthework.
DFcoordinatedthecontributionsanddraftedthemanuscriptARprovidedinsightsintothefoodsupplychainandprocessing,draftingrelevantsectionsofthemanuscriptAKandDEundertookallthemicrobiologyworkARprovidedthestatisticalinputsAllauthorsmadesignificantcontributionstothepaperandapprovedthefinalversion.
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255166doi:bioRxivpreprintAbstractAnexplanationisrequiredforthere-emergenceofCOVID-19outbreaksinregionswithapparentlocaleradication.
RecentoutbreakshaveemergedinVietnam,NewZealandandpartsofChinawheretherehadbeennocasesforsomemonths.
Importationofcontaminatedfoodandfoodpackagingisafeasiblesourceforsuchoutbreaksandasourceofclusterswithinexistingoutbreaks.
Sucheventscanbepreventediftheriskisbetterappreciated.
MaintextAnoutbreakofSARS-CoV-2intheXinfadiwholesalemarketinBeijinghasbeenaccreditedbyChineseauthoritiestoimportedcontaminatedfood(1).
Itoccurred55daysafterthelastidentifiedlocallyacquiredcaseinBeijing.
StepstomitigatetheriskofsimilaroutbreakshavebeentakenwithinChina,yetelsewherethisriskisunderappreciated.
VietnamandNewZealandhavenewunexplainedoutbreaks99and102days,respectively,sincetheirlastidentifiedlocaltransmissions(2,3).
Itispossibleintheseregionsthateradicationwasnevertrulyachievedandthattherehadbeenongoingunidentifiedtransmission.
Alternatively,occulttransmissionchainscanbeseededthroughtravelersbutthismechanismwouldrequireafalsenegativeswabinanasymptomaticindividualorfailuretoensurequarantine.
Forocculttransmissiontoleadtorecrudescenceofanoutbreakmonthssincethelastdetectedcasewouldrequireanascertainmentratethatformostsettingsisimplausiblysmall,asdemonstratedthroughsimulations(supplementarymaterial).
Evenforareproductionnumberof1,atransmissionchainthatavoidedstochasticextinction,andonlyoneintwentyinfectionsbeingdetected,thechanceofreaching20generationswithoutdetectionisatmostoneinathousand.
Anotherpossibilitytoexplainreemergenceistransportofcontaminatedproductssuchasfoodstuffs.
AwellrecognisedfeatureoftheCOVID-19pandemicisthenumberofclusterswithinmeatandseafoodprocessingfacilities.
IntheUK,outbreaksofCOVID-19causeddisruptioninapoultryprocessingplantandinanestablishmentproducingreadymealsforsupermarkets(4).
FishprocessingintunacanneriesinPortugalandGhanawassuspendedafterworkersinbothcountriestestedpositiveforCOVID-19(5,6).
AbattoirsinAustraliahaveclosedfollowinglargeclustersamongstitsworkers(7,8).
InGermany,morethan1,500workerstestedpositiveforCOVID-19atoneofthelargestslaughterhousesinGüterslohculminatinginalockdownoftwodistrictsandover600,000people(9).
TheBeijingXinfadiwholesalefoodmarketeventhasleadtoconcernsthatimportedcontaminatedfoodcouldseednewclusters.
SARS-CoV-2wasdetectedonworkersandenvironmentalsamples,includingacuttingboardusedtosliceimportedsalmon.
AswabbingcampaigndirectedatmillionsofnearbyresidentsaswellasallworkersatthemarketandthebroaderBeijingfoodchainrevealed335COVID-19cases(10).
ChineseauthoritiesactedquicklytosuspendimportofsalmonfromEurope(11,12),andlaterimportsfromfoodpremiseswhereoutbreaksofCOVID-19haveoccurredamongworkers,affectingtheUS,GermanyandBrazilamongothers(13).
InJuly,ChinaalsosuspendedimportsofshrimpfromthreeEcuadoreanprocessingplantsafterdetectingSARS-CoV-2onshipments(14).
Thefeasibilityofthis"nontraditional"transmissionmechanismiscurrentlydebated.
At21–23°CnoviableSARS-CoV-2wasfoundafter4hoursoncoppersurfaces,24hoursoncardboardandafter3daysonstainlesssteelandplasticsurfaces(15).
WehaveassessedthesurvivalofSARS-CoV-2on.
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255166doi:bioRxivpreprintrefrigeratedandfrozenmeatandsalmonover3weekstoassessthepotentialofoutbreaksbeingseededbyimportedcontaminatedfood.
Vero-E6cells(ATCC#CRL-1586)weremaintainedinDulbecco'smodifiedEagleMedium(DMEM)supplementedwith5%fetalbovineserum(FBS)and1%penicillin/streptomycin.
SARS-CoV-2,isolateBetaCoV/Singapore/2/2020(AccessionIDEPI_ISL_406973),wasusedasvirusinoculum.
AllworkwithSARS-CoV-2wasperformedunderBSL3containmentattheDuke-NUSMedicalSchoolABSL3laboratory.
Individualpiecesofsalmon,chickenandporksourcedfromsupermarketsinSingaporewereslicedinto500mm3cubesand200lof3x106TCID50/mlSARS-CoV-2wasaddedtoeachcube.
Thesampleswerestoredat3differenttemperatures(4C,–20Cand–80C)andharvestedatspecifiedtimepoints(1,2,5,7,14-and21-dayspost-inoculation).
Followingincubation,150lofthevirusinoculumwastransferredtoanewtubeandfrozenat–80°Cuntiltitration.
Threereplicateswereperformedforeachcondition.
Thecell-freevirustitre(TCID50/mL)foreachsampleweredeterminedbylimiteddilution.
Thelimitofdetection(LOD)was5x101TCID50/mL.
GraphPadPrism(Version5.
04)wasusedtoperformone-wayANOVA,followedbyDunnett'sMultipleComparisonTest.
ThetitreofSARS-CoV-2remainedconstantat4°C,–20°Cand–80°Cforthedurationoftheexperiment(Fig1).
Infectivitywasmaintainedfor3weeksinboththerefrigerated(4°C)andfrozen(–20°Cand–80°C)samples.
NosignificantdifferencewasobservedbetweenSARS-CoV-2recoveredafterincubationwithorwithoutthepresenceoffood.
TheWHOadvisesthatitisveryunlikelythatpeoplecancontractCOVID-19fromfoodorfoodpackaging(16).
Whileitcanbeconfidentlyarguedthattransmissionviacontaminatedfoodisnotamajorinfectionroute,thepotentialformovementofcontaminateditemstoaregionwithnoCOVID-19andinitiateanoutbreakisanimportanthypothesis.
Itisnecessarytounderstandtheriskofanitembecomingcontaminatedandremainingsoatthetimeofexport,andofthevirussurvivingthetransportandstorageconditions.
TheclustersofinfectionofCOVID-19amongworkersinslaughterhousesandmeatprocessingfacilitiesinmanycountriescanbeattributedtofactorsthatpromotetransmissionofvirusdirectlybetweenworkers,suchascrowding,poorventilation,andshoutingincloseproximityduetohighambientnoiselevels.
Workersmaygotoworkwheninfected,theymayliveincrowdedhousing,andtraveloncrowdedtransport.
Environmentalcontaminationattheworksiteislikelytobeprolongedduetolowtemperatures,metalsurfacesandlackofUVlight(17,18).
WithasignificantburdenofviruspresentininfectedworkersandtheenvironmentthencontaminationofmeatwithSARS-CoV-2ispossibleduringbutcheringandprocessing.
Thekillinglinesinabattoirsgenerallyrunatambienttemperaturebuttheprocesslatermovesintoacontrolledenvironmentaltemperatureofnotgreaterthan12°Cforthebreakdownofcarcassesandmeatismaintainedat3–7°Caslegislatedbyfoodregulations.
Theprocessingofmeatandpoultryisgenerallycarriedoutmanuallyincrowdedconditions.
Salmonprocessingis,incontrast,highlyautomatedwithfilletingandcuttingperformedbymachines,andminimalhandlingbyworkers.
However,wheresuchprocessingiscarriedoutmanuallyincrowdedconditionstheriskofcontaminationincreases.
OurlaboratoryworkhasshownthatSARS-CoV-2cansurvivethetimeandtemperaturesassociatedwithtransportationandstorageconditionsassociatedwithinternationalfoodtrade.
WhenaddingSARS-CoV-2tochicken,salmonandporkpiecestherewasnodeclineininfectiousvirusafter21daysat4°C(standardrefrigeration)and–20°C(standardfreezing).
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ItisThecopyrightholderforthispreprintthisversionpostedAugust18,2020.
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255166doi:bioRxivpreprintContaminationoffoodispossible,andvirussurvivalduringtransportandstorageislikely.
Foodtransportationandstorageoccursinacontrolledsettingakintoalaboratory.
Temperatureandrelativehumidityisconsistentandmaintainedandadverseconditionssuchasdryingoutisnotpermittedfortheintegrityofthefood.
Inquantifyingtheviraltitrewecanreasonablyassessarateofdeclineininfectivity,whichdidnotoccurinanyoftheconditionsweassessed.
Webelieveitispossiblethatcontaminatedimportedfoodcantransfervirustoworkersaswellastheenvironment.
Aninfectedfoodhandlerhasthepotentialtobecomeanindexcaseofanewoutbreak.
Theinternationalfoodmarketismassiveandevenaveryunlikelyeventcouldbeexpectedtooccurfromtimetotime.
EffortstoaverttheriskofCOVID-19outbreaksseededbycontaminatedfoodmustbeginatthesource;thatisfoodprocessingpremises.
Theseincludefrequenthandwashing,cleaningoffoodcontactsurfaces,materialsandutensils.
Fitnesstoworkprotocolsshouldbeinplaceandunwellstaffshouldbeexcluded.
Furthermore,theconditionsunderwhichworkersinourfoodchainmustbereviewedtoensurethatourfoodissafe.
Financialsupportneedstobegiventounwellworkerstoensurenodisincentivestoself-isolationorpresentingforatest.
PPEusageneedstobeoverseenandsocialdistancinginandoutoftheworkplaceneedstobesupported.
Inreceivingmarketsattheotherendofthesupplychain,foodcannotbedecontaminated,however,addedprecautionstoensuregoodhandhygieneandregularcleaningofsurfacesandutensilsisimportant.
Consumersshouldwashtheirhandsaftertouchinguncookedproductsandensurethatfoodiswellcooked.
Ourfindings,coupledwiththereportsfromChinaofSARS-CoV-2beingdetectedonimportedfrozenchickenandfrozenshrimppackagingmaterial,shouldalertfoodsafetycompetentauthoritiesandthefoodindustryofa"newnormal"environmentwherethisvirusisposinganon-traditionalfoodsafetyrisk.
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ItisThecopyrightholderforthispreprintthisversionpostedAugust18,2020.
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255166doi:bioRxivpreprintFigure1:Quantificationofinfectiousvirusover21days.
Viraltitresweredeterminedbylimitingdilution.
Titresareexpressedasmean±SDlog10TCID50/mL.
SARS-CoV-2wasstoredaloneorinthepresenceoffish,chickenorporkandtestedunderrefrigeration(4°C);andfrozen(-20°Cand-80°C).
References1.
https://www.
globaltimes.
cn/content/1192146.
shtml(accessedAugust15,2020)2.
https://www.
worldometers.
info/coronavirus/country/viet-nam/(accessedAugust15,2020)3.
https://www.
health.
govt.
nz/our-work/diseases-and-conditions/covid-19-novel-coronavirus/covid-19-current-situation/covid-19-current-cases/covid-19-current-cases-details#confirmed(accessedAugust15,2020)4.
https://www.
bbc.
com/news/uk-wales-53091149(accessedAugust15,2020)5.
https://www.
intrafish.
com/processing/thai-union-sends-home-200-portuguese-seafood-processing-workers-after-positive-covid-19-case/2-1-815103(accessedAugust15,2020)6.
https://www.
seafoodsource.
com/news/processing-equipment/covid-19-infections-force-thai-union-to-close-fish-factory-in-ghana(accessedAugust15,2020)7.
https://www.
abc.
net.
au/news/2020-05-27/coronavirus-shutdown-melbourne-cedar-meats-workers-return/12289970(accessedAugust15,2020)8.
https://www.
abc.
net.
au/news/2020-08-06/covid-19-abattoir-outbreak-raises-questions-about-dhhs-response/12526816(accessedAugust15,2020)9.
https://www.
dw.
com/en/coronavirus-g%C3%BCtersloh-mayor-henning-schulz-slams-t%C3%B6nnies-meat-producer-after-massive-outbreak-forces-lockdown/a-53932101(accessedAugust15,2020)10.
https://www.
caixinglobal.
com/2020-08-14/beijing-to-reopen-market-at-the-center-of-citys-biggest-covid-19-outbreak-101592926.
html(accessedAugust15,2020)11.
https://www.
seafoodsource.
com/news/supply-trade/seafood-industry-moves-to-quash-misinformation-of-beijing-s-covid-19-spike-being-linked-to-salmon(accessedAugust15,2020)12.
https://www.
youtube.
com/watchv=7w4z0eK0dvc(accessedAugust15,2020)13.
http://www.
cfda.
com.
cn/newsdetail.
aspxid=132934(Chinese)(accessedAugust15,2020)14.
https://in.
reuters.
com/article/health-coronavirus-china-shrimp-idINKBN24B11E(accessedAugust15,2020).
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ItisThecopyrightholderforthispreprintthisversionpostedAugust18,2020.
;https://doi.
org/10.
1101/2020.
08.
17.
255166doi:bioRxivpreprint15.
VanDoremalenN,BushmakerT,MorrisDH,etal.
AerosolandSurfaceStabilityofSARS-CoV-2asComparedwithSARS-CoV-1.
NEnglJMed.
2020;382(16):1564-1567.
doi:10.
1056/NEJMc200497316.
https://www.
who.
int/publications/i/item/covid-19-and-food-safety-guidance-for-food-businesses(accessedAugust15,2020)17.
WaltenburgMA,VictoroffT,RoseCE,etal.
Update:COVID-19AmongWorkersinMeatandPoultryProcessingFacilities-UnitedStates,April-May2020.
MMWRMorbMortalWklyRep.
2020;69(27):887-892.
Published2020Jul10.
doi:10.
15585/mmwr.
mm6927e218.
https://www.
spiegel.
de/international/why-meatpacking-plants-are-perfect-for-spreading-covid-19-a-6e122821-62af-4399-a549-fd01f0abef73(accessedAugust15,2020).
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ItisThecopyrightholderforthispreprintthisversionpostedAugust18,2020.
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255166doi:bioRxivpreprintAppendixtoFisheretalToassessthefeasibilityoflongchainsofoccultinfectionleadingtoclusterssometimeafteracommunitywasthoughttohavebeenclearofinfection,webuiltabranchingprocesssimulationmodel.
EachcaseisassumedtogenerateaPoisson-distributednumberofsecondarycaseswithmean.
Weseedthechainwithasingleunidentifiedcaseingeneration0,theuntracedandunobserveddaughterinfectionofacase.
Thenumberofinfectionsingenerationofthechainisdenoted,whichhasdistribution().
Eachcaseaftertheinitialinfectionthatseedsthechainisascertainedwithprobability.
Thechainissimulatedforwardfor30generationsoruntil1000000casesoruntilthefirstcaseisascertained.
Wethencalculatethenumberofgenerationspasseduntilthechainisuncoveredorbecomesextinct,andtheprobabilitythereison-goingocculttransmissionineachgeneration.
TheprobabilitythechainofinfectionisdetectedandtheexpectednumberofinfectionsarederivedthroughMonteCarlosimulationwith100000draws.
FigureS1:Probabilityofongoingundetectedtransmissionforthefirst20generationsforfourvaluesoftheascertainmentrate(=5%,10%,25%and50%)andreproductionnumber(=1,1.
5,2,2.
5).
Probabilitieshttps://doi.
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