Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection

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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,∗

a_Universidade_Federal_da_Bahia_(UFBA),_Hospital_{Universitário}_Professor_Edgard_Santos_(HUPES),_Laboratório_de_Pesquisa_em

Infectologia(LAPI),Salvador,BA,Brazil

b_University_of_Hawaii_at_{M ¯anoa}_(UH_Manoa),_Department_of_Tropical_Medicine,_Medical_Microbiology_and_{Pharmacology,}_Hawaii,_USA

a

r

t

i

c

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o

Articlehistory:

Received9July2020

Accepted8August2020

Availableonline31August2020

Keywords: SARS-CoV-2 COVID-19 saliva Diagnostics

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b

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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.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mailaddresses:[email protected](S.N.Vaz),[email protected](D.S.Santana),[email protected]

(E.M.Netto),[email protected](C.Pedroso),[email protected](W.Wang),[email protected](F.D.Santos),[email protected]

(C.Brites).

https://doi.org/10.1016/j.bjid.2020.08.001

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Introduction

InDecember2019,ChinareportedtheSevereAcute

Respira-torySyndromeCoronavirus 2(SARS-CoV-2)as thecause of

CoronavirusDisease2019(COVID-19).1_Although_Severe_Acute

RespiratorySyndromeCoronavirus(SARS-CoV)2 _and_Middle

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 _At_that_time, _Brazil_conﬁrmed

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.6_Although_80%_of_cases_has

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 _also_saves _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,_and_stored_at_-80◦_C_until_nucleic_acid_extraction.

Wheneverpossible,RNAwasisolatedfromfreshsalivawithin

sixhoursaftercollection.

Sampleprocessingandviralnucleicacidisolation

Sampleswerehomogenizedbyrepeatedpipettinganddiluted

1:1withPBS1x(phosphatebufferedsaline)beforeRNA

isola-tion.RNAisolationwasperformedbyusingQIAGENQIAamp®

RNAMiniKit,accordingtomanufacturer’sinstructions.Viral

nucleic acid was extracted from 140␮l diluted saliva, and

elutedto60␮lofelutionbuffer.

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.10_This_protocol

consisted of three stages: ﬁrst (screening) to amplify the

E gene, a conﬁrmatory and eliminatory step.The last two

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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 _Concordant_results_for_SARS-CoV-2_by_RT-PCR_between_swab_and_saliva_samples.

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)

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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_(negative_antibody_detection)_had_unspeciﬁc

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.13_To_et_al._reported_a_high

con-cordancebetweensalivaandnasopharyngealaspiratesforthe

detectionofrespiratoryvirusesandmonitoringSARS-CoV-2

viralloadduringthecourseofinfection.12,16_There_are_several

advantagesofusingsalivasamplesfordetectionof

SARS-Cov-2:ﬁrst,salivacanbeprovidedeasilybythepatientwithout

any invasive procedures.Therefore, the use ofsaliva

sam-plescouldreducetheriskofvirustransmissiontohealthcare

professionals.17_Second,_the_use _of_saliva_will_allow_sample

collectionoutsidehospitalsorhealthcentersareas.Finally,it

couldbeconvenientlypooledtoscreenlargerpopulations,as

demonstrated inthiswork.Inthesesettings,wherealarge

numberofindividualsrequirescreening,salivarepresentsa

practicalandnon-invasivespecimentype.19

Inthe presentstudy salivawasself-collectedby

partici-pantsinsidethehospitalarea.Despitetherecommendation

ofsomeauthorstousesamplescollectedimmediatelyupon

wakingup,11,16_without_previous_food_consumption_or_teeth

brushing,wecollectedsalivaatanytimefrompatientswho

had atest requestedbya physician.Thiseasy toperform,

simpleprocedure,providedasimilarsensitivity/speciﬁcityto

samplescollectedbyNPswabs.11

Insomepreviousstudies,salivawasobtainedusing

spe-cialcollection devices,20 _improving_quality _and _quantity_of

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

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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.23_The_two_participants_presenting_negative_results

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,24_like_the

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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