w w w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Saliva
is
a
reliable,
non-invasive
specimen
for
SARS-CoV-2
detection
Sara
Nunes
Vaz
a,
Daniele
Souza
de
Santana
a,
Eduardo
Martins
Netto
a,
Celia
Pedroso
a,
Wei-Kung
Wang
b,
Felice
Deminco
Alves
Santos
a,
Carlos
Brites
a,∗aUniversidadeFederaldaBahia(UFBA),HospitalUniversitárioProfessorEdgardSantos(HUPES),LaboratóriodePesquisaem
Infectologia(LAPI),Salvador,BA,Brazil
bUniversityofHawaiiatM ¯anoa(UHManoa),DepartmentofTropicalMedicine,MedicalMicrobiologyandPharmacology,Hawaii,USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received9July2020
Accepted8August2020
Availableonline31August2020
Keywords: SARS-CoV-2 COVID-19 saliva Diagnostics
a
b
s
t
r
a
c
t
Background:SevereAcuteRespiratorySyndromeCoronavirus2(SARS-CoV-2)isthecauseof
CoronavirusDisease2019(COVID-19).AlthoughRealTimeReverseTranscriptionPolymerase
ChainReaction(qRT-PCR)ofrespiratoryspecimensisthegoldstandardtestfordetectionof
SARS-CoV-2infection,collectingnasopharyngealswabscausesdiscomforttopatientsand
mayrepresentconsiderableriskforhealthcareworkers.Theuseofsalivaasadiagnostic
samplehasseveraladvantages.
Objectives:Theaimofthisstudywastovalidatetheuseofsalivaasabiologicalsamplefor
diagnosisofCOVID-19.
Methods:ThisstudywasconductedatInfectiousDiseasesResearchLaboratory(LAPI),in
Sal-vador,Brazil.Participantspresentingwithsigns/symptomssuggestingSARS-CoV-2infection
underwentanasopharyngealswab(NPS)and/ororopharyngealswab(OPS),andsaliva
col-lection.SalivasamplesweredilutedinPBS,followedbyRNAisolationandRT-RealTimePCR
forSARS-CoV-2.Resultsofconventionalvssalivasamplestestingwerecompared.Statistical
analyseswereperformedusingStatisticalPackagefortheSocialSciencessoftware(SPSS)
version18.0.
Results:Onehundredfifty-fiveparticipantswererecruitedandsamplespairsofNPS/OPSand
salivawerecollected.ThesensitivityandspecificityofRT-PCRusingsalivasampleswere
94.4%(95%CI86.4–97.8)and97.62%(95%CI91.7–99.3),respectively.Therewasanoverall
highagreement(96.1%)betweenthetwotests.
Conclusions:Useofself-collectedsalivasamplesisaneasy,convenient,andlow-cost
alterna-tivetoconventionalNPswab-basedmoleculartests.Theseresultsmayallowabroaderuse
ofmoleculartestsformanagementofCOVID19pandemic,especiallyinresources-limited
settings.
©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddresses:saranunes02@hotmail.com(S.N.Vaz),dnssan@hotmail.com(D.S.Santana),nettoeduardom@hotmail.com
(E.M.Netto),cpedrosoj@gmail.com(C.Pedroso),wangwk@hawaii.edu(W.Wang),fdeminco@gmail.com(F.D.Santos),crbrites@gmail.com
(C.Brites).
https://doi.org/10.1016/j.bjid.2020.08.001
1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC
Introduction
InDecember2019,ChinareportedtheSevereAcute
Respira-torySyndromeCoronavirus 2(SARS-CoV-2)as thecause of
CoronavirusDisease2019(COVID-19).1AlthoughSevereAcute
RespiratorySyndromeCoronavirus(SARS-CoV)2 andMiddle
East RespiratorySyndrome Coronavirus(MERS-CoV)3
infec-tionshavehighermortalityratesthanCOVID-19,SARS-CoV-2
spreadsmuchmorerapidly thanMERS-CoVandSARS-CoV.
On March 11, 2020, the World Health Organization (WHO)
announcedthatepidemicofthenovelcoronaviruswasa
pan-demic,andasofJuly8,2020,thenumberofconfirmedcases
globallywas11,874,226,associatedtoover545,481deathsin
216countries orterritories.4 Atthattime, Brazilconfirmed
over1.7millioncases,67,964deaths,andamortalityrateof
32.3/100,000.5
Direct human-to-human transmission of SARS-CoV-2
occursthroughdropletinhalationwhilecoughing,sneezing,
oreven talking,and transmissions causedbycontact with
nasal,ocularandoralmucousmembranesarealsopossible.
SeveralclinicalsymptomsareassociatedtoCOVID-19,such
asfever,cough,shortnessofbreath,sorethroat,chestpain,
headache,anosmiaandageusia.6Although80%ofcaseshas
lightormildsymptoms,theremaining20%canpresentwith
severedisease,andaround5%willrequireintensivecare
treat-ment,especiallythosewithchronichealthconditions
includ-ingcardiopathy,arterialhypertension,diabetesandobesity.7
RealTimeReverseTranscriptionPolymeraseChain
Reac-tion (qRT-PCR) of respiratory specimens (oropharyngeal
andnasopharyngealswabs,bronchoalveolarlavage,tracheal
aspirate)isthegoldstandardtestfordetectionof
SARS-CoV-2 infection.8–10 However, collecting nasopharyngeal swabs
causesdiscomforttopatientsduetoinvasivenessofthe
pro-cedure,whichcanreducethepossibilityofpatientconsent
toretest,andmayrepresentconsiderableriskforhealthcare
workers,becauseofitspotentialtoinducepatientstosneeze
orcough,expellingvirusparticles.11
Previous studies have shown high detection rate using
salivaas specimens forlaboratory diagnosis ofrespiratory
viruses.12,13 Recent studies reported the use of oral
flu-ids/saliva for the detection of SARS-CoV-2.14–18 The use of
salivaasadiagnosticsamplehasseveraladvantages,suchas
easyself-collectionevenathome,andnoneedofspecialized
personnelforsamplecollection.Inaddition,salivacollection
ismuchmorecomfortableforthepatientthan
nasopharyn-geal/ oropharyngeal swabs procedure.17 It alsosaves time,
and is less costly, because it does not require the use of
personalprotectiveequipmentnorviraltransportation
solu-tion.
Timely,accurateandnon-invasivesamplesforSARS-CoV-2
detectionwillfacilitateeffectivelarge-scalepandemiccontrol
measurestopreventthespreadofCOVID-19.Theaimofthis
studywastovalidatetheuseofsalivaasabiologicalsample
forthediagnosisofCOVID-19.
Material
and
methods
Studypopulation
This study was conductedat Infectious Diseases Research
Laboratory (LAPI),ofComplexo HospitalarProfessor Edgard
Santos (C-HUPES),Federal University ofBahia, inSalvador,
Brazil.
C-HUPES healthcare workers presenting with signs/
symptoms suggesting SARS-CoV-2 infection, as well as
patients at the COVID-19 ward of C-HUPES, underwent a
nasopharyngealswab(NPS)and/ororopharyngealswab(OPS)
collection.Allparticipantsrecruitedtothisstudy,aftersigning
informed consent, wereasked toprovide asampleof
self-collectedsaliva.
ThisstudywasapprovedbytheResearchEthicsCommittee
ofMaternidadeClimériodeOliveira–UFBA(4.042.620).
Samplecollection
Salivasampleswere collectedinto 30mlsterileurinecups.
Participantswereinstructedtorepeatedlyspituntil
approx-imately2mlofsamplewasobtained,thusavoidingmucous
secretions from oropharynx or lower respiratory tract (i.e.,
sputum).SamplesweretransportedtoLAPIinathermalbox
at2−8◦C,andstoredat-80◦Cuntilnucleicacidextraction.
Wheneverpossible,RNAwasisolatedfromfreshsalivawithin
sixhoursaftercollection.
Sampleprocessingandviralnucleicacidisolation
Sampleswerehomogenizedbyrepeatedpipettinganddiluted
1:1withPBS1x(phosphatebufferedsaline)beforeRNA
isola-tion.RNAisolationwasperformedbyusingQIAGENQIAamp®
RNAMiniKit,accordingtomanufacturer’sinstructions.Viral
nucleic acid was extracted from 140l diluted saliva, and
elutedto60lofelutionbuffer.
Testingofsamplespoolswithonepositiveandfour
nega-tivesamplesandonepositiveandninenegativesampleswas
alsoperformed. Itincluded positivesamples(RT-RealTime
PCR)withlowviralloadandhighviralload.
RT-RealtimePCR
Nasopharyngealandoropharyngealspecimensweresentto
BahiaCentral Laboratory(LACEN),aState´sReference
Labo-ratory, forSARS-CoV-2 investigation byRT-PCR method, in
accordancetoBIOMOLOneStep/COVID-19Kit(Paraná
Molec-ularBiologyInstitute)protocol.
ForsalivasamplesRNAtemplatewassubjectedto
ampli-ficationaccordingtoCharité-Berlinprotocol.10Thisprotocol
consisted of three stages: first (screening) to amplify the
E gene, a confirmatory and eliminatory step.The last two
Table1–Characteristicsofparticipantsenrolledinthisstudy.
Overall(n=149)a COVID-19(n=67) Non-COVID19(n=82) p-value
Age(years),median(IQR) 40(33−48.5) 40(35−50) 39.5(33−48.3) 0.588
Male,n.(%) 46(30.9) 23(34.32) 23(28.04) 0.470
Onsetofsymptomsbeforethetest(days),median(IQR) 4(3−6) 4(3−6) 4.5(3−6) 0.548 Symptomsatsamplecollection
Headache,n(%) 82(61.2) 33(53.2) 49(68.1) 0.109 Cough,n(%) 60(45.1) 30(49.2) 30(41.7) 0.484 Myalgia,n(%) 54(40.3) 25(40.3) 29(40.3) 1 Sorethroat,n(%) 50(37.3) 14(22.6) 36(50) 0.001 Fever,n(%) 37(27.6) 25(40.3) 12(16.7) 0.003 Anosmia,n(%) 29(21.6) 20(32.3) 9(12.5) 0.007 Ageusia,n(%) 28(20.9) 18(29.0) 10(13.9) 0.035 Chills,n(%) 27(17.9) 10(16.1) 14(19.4) 0.658 Chestpain,n(%) 18(13.4) 9(14.5) 9(12.5) 0.802 Dyspnea,n(%) 16(11.9) 9(14.5) 7(9.7) 0.433
a ConcordantresultsforSARS-CoV-2byRT-PCRbetweenswabandsalivasamples.
wasdetectedonscreening.Amplificationreactionswere
car-riedoutonAppliedBiosystems7500RealTimePCRdetector,
andresultswereclassifiedaspositiveforSARS-CoV-2when
bothE and RdRpgenes were detectedand cycle threshold
(Ct,number ofcycles requiredforthe fluorescentsignal to
exceedbackgroundlevel)valueswere lessthan orequalto
40.
Immunologicalassay
In order to confirm the absence of SARS-CoV-2 infection
insomediscordantresults,EnzymeLinkedImmunosorbent
Assay(Euroimmun,Lüebeck,Germany)wasperformed.
Detec-tion of IgG antibodies was attempted in plasma samples
collectedafterthreeweeksofsymptoms ´onset,accordingto
manufacturer’sinstructions.
Statisticalanalysis
Descriptive statistics were presented as a percentage (%)
for categorical variables and median (interquartile range;
IQR) for continuous variables. Fisher’s and Mann-Whitney
tests were used for categorical and continuous variables,
respectively.Sensitivity,specificity,positivepredictivevalue,
negative predictive value and a 95% confidence intervals
(CI)were calculated toaccess diagnostic performance.The
kappa coefficient was used to estimate the agreement
beyondchancebetweensalivaand NPS/OPSRT-PCRresults.
All statistical analyses were performed using Statistical
Package for the Social Sciences software (SPSS) version
18.0.
Results
Between May 5thand June5th 2020,155participants were
recruitedandsamplespairsofNPS/OPSandsalivawere
col-lected.Ofthese, 149(96.1%)had concordantresultson the
detection of SARS-CoV-2 RT-PCRin bothspecimens.
Forty-sixindividuals (30.9%)were male,median(IQR)age was40
(33−48.5)years,andmedian(IQR)timefromonsetof
symp-tomswas4(3−6)days.Participants ´characteristicsareshown
inTable1.
TheprevalenceofCOVID-19diagnosedbyNPS/OPSRT-PCR
and bysalivaRT-PCRinthis study were 45.8%and 43.22%,
respectively. Most ofthe tested healthcareworkers (51.6%)
werenurses/nursingtechnicians.
Allthe67participantsdiagnosedwithCOVID-19hadmild
to moderate symptoms. No one needed hospitalization or
intubation.Nodeathwasreported.Themostcommon
symp-tomsatsamplingtimewereheadache(53.2%),cough(49.2%)
and myalgia (40.3%). Frequency of fever and anosmia was
significantlyhigher (p=0.003; 0.007),amongpatientstested
positiveforCOVID-19,while,frequencyofsorethroatwas
sig-nificantlylower(p=0.001)(Table1).
Using RT-PCRofNPS/OPSsamplesasthegoldstandard,
thesensitivityandspecificityofRT-PCRusingsalivasamples
were94.4%(95%CI86.4–97.8)and97.62%(95%CI91.7–99.3),
respectively(Table2).Positivepredictivevalueandnegative
predictivevaluewere97.1%(95%CI90.0–99.2)and95.35%(95%
CI 88.6–98.2),respectively.There wasanoverallhigh
agree-ment(96.1%)betweenthetwotests(kappacoefficient0.922,
95% CI0.765–1.00, p<0.001).Themedian(IQR)Ct valuesof
Table2–DetectionofSARS-CoV-2RT-PCRusingNPS/OPSandsalivasamples.
SWAB
Detectable Nondetectable Total
Saliva Detectable n.(%) 67(94.4) 2(2.4) 69(44.5)
Nondetectable n.(%) 4(5,6) 82(97.6) 86(55.5)
Table3–CharacteristicsoftheparticipantswhohaddiscordantresultondetectionofSARS-CoV-2RT-PCRbyNPS/OPS andsaliva.
ParticipantN Sex Age NPS/OPS Saliva Onsetof symptoms(days)
Symptoms Ct IgG
1 F 82 ND D 3 Fever,dyspnea,drycoughand
myalgia
35.72 NA
2 M 41 ND D 3 Chills,myalgia,headache,
rhinorrhea,drycough,anosmia andageusia
32.24 NA
3 F 41 D ND 6 Nasalcongestionanddrycough R
4 F 27 D ND 4 Headache,sneezing,runnynose
andsorethroat
R
5 F 39 D ND 7 Headache,rhinorrhea,drycough
andsorethroat
NR
6 F 49 D ND 3 Headache,drycoughandsore
throat
R
Abbreviations:F,female;M,male;D,detected;ND,notdetected;NPS,nasopharyngealswab;OPS,oropharyngealswab;Ct,cyclethreshold;R, reactive;NR,notreactive;NA,notavailable.
the Egene was33 (29–36.6), and ofthe RdRpgenewas 34
(29.5–37.4).
Thedetection rate ofSARS-CoV-2 in salivawas slightly
lowerthanthatofNPS/OPS,butwithoutreachingstatistical significance.Sixparticipantshaddiscordantresultsbetween salivaandNPS/OPSRT-PCRassays,includingtwoparticipants withvirus detectedinsalivabut notin NPS/OPS,and four participantswithvirusdetectedinNPS/OPSbutnotinsaliva (Table3).
After20daysofsymptomsonset,participantswith
neg-ativesalivaresults(n=4) were invitedtocollectnewblood
samplesforanti-SARS-CoV-2antibodies.IgGwasreactivein
all but one of them, confirming that at least one patient
probably had not been previously infectedby SARS-CoV-2.
Participantno5(negativeantibodydetection)hadunspecific
symptoms,commontoanyviralinfection(Table3).Thetwo
participantswithsalivapositive/NP-negativetestingdidnot
providebloodsamplesforantibodytesting.
Onepositiveandfournegative(lowdilution)andone
pos-itive andnine negative(high dilution)saliva sampleswere
pooled.LowandhighdilutionwerepositiveforlowCtsamples
(highviralload),butonlypositiveinlowdilutionwhenalow
viralloadsamplewasincluded.
Discussion
Ourfindings demonstratethattesting salivaasan
alterna-tivetoNPswabsissensitiveandspecificenoughtobeused
inaroutinepractice.ConcordancebetweenRT-PCRresultsfor
SARS-CoV-2detectioninsalivaandNPswabswas96.1%.In
addition,amongsampleswithdiscrepantresultswehadone
thatwasnegativeforSARS-CoV-2antibodiesthreeweeksafter
diagnosis,suggestingthatpatienthadadiagnosisotherthan
COVID-19.Inthiscase,thesensitivityofsalivatestingwould
increasefrom94.4to95.7%,whilethespecificityofNPswabs
testingwoulddecreaseinasimilarway.
Theuse ofNP swabshasbeen the standard ofcarefor
SARS-CoV-2diagnosisinBrazil,butitrequirestheuseoffull
personalprotectiveequipment,increasetheriskofinfection
forthehealthprofessionalwhocollectsthesample,andalso,
thecostoftesting.Salivahasbeenshowntobeareliable
spec-imenfordetectingSARScoronavirussince2004.Wangetal.
(2004)reportedahighviralloadofSARS-CoVinsalivasamples
whencomparedtothroatwash.13Toetal.reportedahigh
con-cordancebetweensalivaandnasopharyngealaspiratesforthe
detectionofrespiratoryvirusesandmonitoringSARS-CoV-2
viralloadduringthecourseofinfection.12,16Thereareseveral
advantagesofusingsalivasamplesfordetectionof
SARS-Cov-2:first,salivacanbeprovidedeasilybythepatientwithout
any invasive procedures.Therefore, the use ofsaliva
sam-plescouldreducetheriskofvirustransmissiontohealthcare
professionals.17Second,theuse ofsalivawillallowsample
collectionoutsidehospitalsorhealthcentersareas.Finally,it
couldbeconvenientlypooledtoscreenlargerpopulations,as
demonstrated inthiswork.Inthesesettings,wherealarge
numberofindividualsrequirescreening,salivarepresentsa
practicalandnon-invasivespecimentype.19
Inthe presentstudy salivawasself-collectedby
partici-pantsinsidethehospitalarea.Despitetherecommendation
ofsomeauthorstousesamplescollectedimmediatelyupon
wakingup,11,16withoutpreviousfoodconsumptionorteeth
brushing,wecollectedsalivaatanytimefrompatientswho
had atest requestedbya physician.Thiseasy toperform,
simpleprocedure,providedasimilarsensitivity/specificityto
samplescollectedbyNPswabs.11
Insomepreviousstudies,salivawasobtainedusing
spe-cialcollection devices,20 improvingquality and quantityof
obtainedsaliva.However,thesecollectiondevicesarenot
usu-ally availableingeneralmedicalcarecenters,especiallyin
low-income countries. Furthermore, saliva collection using
suchdevicesrequirestheassistanceofhealthcareworkers.In
thepresentstudy,participantswereinstructedtorepeatedly
spitintoasterileurinecupusedroutinelyinourhospital.As
nospecialdeviceisrequired,theuseofsalivacanbe
imple-mentedasaroutineinclinicalpracticewithoutcompromising
thesamplequality,aspreviouslyreported.19,21
Thehighlevelofconcordancebetweenthetwomethods
demonstrates thatthe use salivafor detectingSARS-CoV-2
byRT-PCRisasreliableastheuseofNP/OPswabs,butwith
diag-nosedsofar,whichmeansthatasimplertestingprocedure
couldpotentiallyreducetherisksandcostsinvolvedinsample
collection.Despitethesenumbers,theproportionofpatients
testedbyPCRinBrazilisverylow,andtheestimatesonthe
dynamicsof the pandemic islargely basedon flow lateral
immunochromatographictests,whichhavebeenconsidered
inaccuratetodefineserologicalstatusforSARS-CoV-2.22
Ourresultswerecomparabletosimilarstudiesthatused
salivafor SARS-CoV-2detection and were obtainedfrom a
population of patients closely followed up in a university
hospital.23Thetwoparticipantspresentingnegativeresults
in salivasamples but positive in NP swabs had very
non-specificsymptomsandcouldnotfulfillaclinicaldiagnosisof
COVID-19.ThemostpredictivesymptomsforCOVID-19inour
populationwerenotpresentinthesetwocases,increasingthe
possibilityofafalse-positiveresultbyNPswab.Theuseofa
three-stepamplificationprotocolalsoincreasesthe
sensitiv-ityofthetest.Arecentstudyshowedthatsomevariationsin
SARS-CoV-2genomecannegativelyimpactthesensitivityof
RT-PCRtestusedforitsdetection,andreinforcestheneedof
protocolsabletomaintainsensitivityandspecificity,24likethe
oneusedinthisstudy.
Ourworkpresentssomelimitationsincludingtheabsence
ofserologicalconfirmationofCOVID-19inallcases,andthe
lackof ct valuesfor the tests using NPS samples, as they
wereperformedinareferrallaboratoryandwerenotavailable
forcomparisonpurposes.However,wewereabletoclosely
evaluatehealthcareworkersandinpatientsadmittedtoour
hospitalandcomparetheuseoftwoapproachesformolecular
detectionofSARS-CoV-2,withahighdegreeofconcordance.
Inconclusion,wedemonstratedthatusingself-collected
salivasamplesisaneasy,convenient,andlow-costalternative
toconventionalNPswab-basedmoleculartests.Theseresults
canallowabroaderuseofmoleculartestsformanagementof
COVID19pandemic,especiallyinresources-limitedsettings.
Conflict
of
interest
Theauthordeclaresnoconflictsofinterest.
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s
1. WuF,ZhaoS,YuB,etal.Anewcoronavirusassociatedwith
humanrespiratorydiseaseinChina.Nature.2020;579:265–9,
http://dx.doi.org/10.1038/s41586-020-2008-3.
2. ChengVCC,LauSKP,WooPCY,KwokYY.Severeacute
respiratorysyndromecoronavirusasanagentofemerging
andreemerginginfection.ClinMicrobiolRev.2007;20:660–94,
http://dx.doi.org/10.1128/CMR.00023-07.
3. ChanJFW,LauSKP,ToKKW,ChengVCC,WooPCY,YueKY.
MiddleEastRespiratorysyndromecoronavirus:Another
zoonoticbetacoronaviruscausingSARS-likedisease.Clin
MicrobiolRev.2015;28:465–522,
http://dx.doi.org/10.1128/CMR.00102-14.
4. WorldHealthOrganization.WHOCoronavirusDisease
(COVID-19)Dashboard.Availableat:https://covid19.who.int/.
5. Ministeriodasaude.PainelCoronavirus.Availableat:
https://covid.saude.gov.br/.
6. HuangC,WangY,LiX,etal.Clinicalfeaturesofpatients
infectedwith2019novelcoronavirusinWuhan,China.
Lancet.2020;395:497–506,
http://dx.doi.org/10.1016/S0140-6736(20)30183-5.
7.RamanathanK,AntogniniD,CombesA,etal.Comparative tropism,replicationkinetics,andcelldamageprofilingof SARS-CoV-2andSARS-CoVwithimplicationsforclinical manifestations,transmissibility,andlaboratorystudiesof COVID-19:anobservationalstudy.LancetMicrobe.2020:19–21.
8.CenterofDiseaseControlandPrevention.InterimGuidelines
forCollecting,Handling,andTestingClinical
Speci-mensforCOVID-19.May22.Availableat:https://www.cdc.gov/
coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html.
9.WangY,KangH,LiuX,TongZ.CombinationofRT-qPCR
testingandclinicalfeaturesfordiagnosisofCOVID-19
facilitatesmanagementofSARS-CoV-2outbreak.JMedVirol.
2020;92:538–9,http://dx.doi.org/10.1002/jmv.25721.
10.CormanVM,LandtO,KaiserM,etal.Detectionof2019novel
coronavirus(2019-nCoV)byreal-timeRT-PCR.
Eurosurveillance.2020;25:1–8,
http://dx.doi.org/10.2807/1560-7917.ES.2020.25.3.2000045.
11.WyllieAL,FournierJ,Casanovas-MassanaA,etal.Salivais
moresensitiveforSARS-CoV-2detectioninCOVID-19
patientsthannasopharyngealswabs.MedRxiv.2020,
http://dx.doi.org/10.1101/2020.04.16.20067835, 2020.04.16.20067835.
12.ToKKW,YipCCY,LaiCYW,etal.Salivaasadiagnostic
specimenfortestingrespiratoryvirusbyapoint-of-care
molecularassay:adiagnosticvaliditystudy.ClinMicrobiol
Infect.2019;25:372–8,
http://dx.doi.org/10.1016/j.cmi.2018.06.009. 13.WangW-K,ChenS-Y,LiuI-J,etal.Detectionof
SARS-associatedCoronavirusinThroatWashandSalivain EarlyDiagnosis.EmergInfectDis.2004;10:1213–9.
14.AzziL,CarcanoG,GianfagnaF,etal.Salivaisareliabletoolto
detectSARS-CoV-2.JInfect.2020;81:e45–50,
http://dx.doi.org/10.1016/j.jinf.2020.04.005.
15.ChenL,ZhaoJ,PengJ,etal.Detectionof2019-nCoVinsaliva
andcharacterizationoforalsymptomsinCOVID-19patients.
SSRNElectronJ.2020,http://dx.doi.org/10.2139/ssrn.3557140.
16.ToKKW,TsangOTY,LeungWS,etal.Temporalprofilesofviral
loadinposteriororopharyngealsalivasamplesandserum
antibodyresponsesduringinfectionbySARS-CoV-2:an
observationalcohortstudy.LancetInfectDis.2020;20:565–74,
http://dx.doi.org/10.1016/S1473-3099(20)30196-1.
17.WilliamsE,BondK,ZhangB,PutlandM,WilliamsonDA.
Salivaasanon-invasivespecimenfordetectionof
SARS-CoV-2.JClinMicrobiol.2020;50,
http://dx.doi.org/10.1128/JCM.00776-20.
18.McCormick-BawC,MorganK,GaffneyD,etal.Salivaasan
alternatespecimensourcefordetectionofSARS-CoV-2in
symptomaticpatientsusingCepheidXpertXpress
SARS-CoV-2.JClinMicrobiol.2020,
http://dx.doi.org/10.1128/jcm.01109-20.
19.ToKKW,TsangOTY,Chik-YanYipC,etal.Consistent
detectionof2019novelcoronavirusinsaliva.ClinInfectDis.
2020:4–6,http://dx.doi.org/10.1093/cid/ciaa149.
20.SuekiA,MatsudaK,YamaguchiA,etal.Evaluationofsaliva
asdiagnosticmaterialsforinfluenzavirusinfectionby
PCR-basedassays.ClinChimActa.2016:71–4,
http://dx.doi.org/10.1016/j.cca.2015.12.006.
21.ToKK,LuL,YipCC,etal.Additionalmoleculartestingof
salivaspecimensimprovesthedetectionofrespiratory
viruses.EmergMicrobesInfect.2017;6,
http://dx.doi.org/10.1038/emi.2017.35.
22.OngDSY,ManSJDe,LindeboomFA,KoelemanJGM.
Comparisonofdiagnosticaccuraciesofrapidserologicaltests andELISAtomoleculardiagnosticsinpatientswith
suspectedcoronavirusdisease2019presentingtothe hospital.ClinInfectDis.2020.
23.PasomsubE,WatcharanananSP,BoonyawatK,etal.Saliva
sampleasanon-invasivespecimenforthediagnosisof
coronavirusdisease2019:across-sectionalstudy.Clin
MicrobiolInfect.2020,
http://dx.doi.org/10.1016/j.cmi.2020.05.001.
24.Pe ˜narrubiaL,RuizM,PorcoR,etal.Multipleassaysina
real-timeRT-PCRSARS-CoV-2panelcanmitigatetheriskof
lossofsensitivitybynewgenomicvariantsduringthe
COVID-19outbreak.IntJInfectDis.2020;97:225–9,