Viral metagenomics performed in patients with acute febrile syndrome during Toxoplasma gondii outbreak in south Brazil

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Viral

metagenomics

performed

in

patients

with

acute

febrile

syndrome

during

Toxoplasma

gondii

outbreak

in

south

Brazil

Rafael

dos

Santos

Bezerra

_,

_Cristiane

_Frac¸ão

_Diefenbach

_,

_Dalnei

_Veiga

_Pereira

_,

Simone

Kashima

_,

_Svetoslav

_Nanev

_Slavov

a_{UniversidadedeSãoPaulo,FaculdadedeMedicinadeRibeirãoPreto,CiênciasBiológicasedaSaúde,RiberãoPreto,SP,Brazil} b_{UniversidadedeSãoPaulo,FaculdadedeMedicinadeRibeirãoPreto,Hemocentro,RibeirãoPreto,SP,Brazil}

c_{HospitalDeCaridadeDr.AstrogildoDeAzevedo,DepartamentodeHematologiaeOncologia,RioGrandedoSul,RS,Brazil} d_{UniversidadedeSãoPaulo,FaculdadedeMedicinadeRibeirãoPreto,DepartamentodeMedicinaInterna,RibeirãoPreto,SP,Brazil}

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Articlehistory:

Received8January2020

Accepted17April2020

Availableonline15May2020

Keywords: Toxoplasmagondii Viralmetagenomics Epstein–Barrvirus HumanParvovirusB19

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ToxoplasmosisisazoonoticinfectioncausedbytheprotozoanparasiteToxoplasmagondii.

Theinfectioniswidelydisseminatedinthehumanpopulationandisusuallybenignor

asymptomatic.SystemicT.gondiiinfectionpresentsrisksforpregnantwomenandAIDS

patients.Althoughrare,T.gondiicancauseoutbreaksinurbancenters.Theoriginofthese

outbreaksisnotcompletelyunderstoodbutprobablyresultsfromintroductionofzoonotic

T.gondiistrainsinthepopulation.Duringsuchoutbreaksotherpathogenswhichmimic

T.gondiiacutefebrilesyndromemayalsocirculate;therefore,detailedinvestigationofthe

outbreakisofextremeimportance.Inthisstudyweperformedviralmetagenomics

next-generationsequencing(mNGS)inpatientsamplesobtainedduringT.gondiioutbreakin

SantaMariacity,SouthBrazil.Specificbioinformaticspipelinesspecializedinvirus

dis-coverywereappliedinordertoidentifyco-circulatingvialagents.EpsteinBarrvirusand

ParvovirusB19contigswereassembledandthesevirusescancausesymptomssimilarto

toxoplasmosis.Inconclusion,ourfindingsshowtheimportanceofMetagenomicsnext

gen-erationsequencing(mNGS)usetohelpcharacterizetheoutbreakmorecompletelyandin

themanagementoftheaffectedpatients.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis

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

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

∗ _{Correspondingauthor.}

E-mailaddress:svetoslav.slavov@hemocentro.fmrp.usp.br(S.N.Slavov).

https://doi.org/10.1016/j.bjid.2020.04.011

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC

Introduction

Toxoplasmagondiiisaprotozoanparasitethatcauses

toxoplas-mosis,aworldwidespreadzoonoticinfection,whichisalso

highlyprevalentinhumans.Theinfection,inmostcasesis

asymptomaticorhasbenignclinicalcourse.However,T.gondii

cancauselife-threateningconditionswhenitistransmitted

congenitallyfrommothertofetusorwhentheparasiteinfects

patientswithimmunedeficiency,especiallyAIDS.1

Afterbirth,humantoxoplasmosisisacquiredbyingesting

waterorundercookedmeatcontaminatedwithoocysts.2–4_For

thatreason,sporadicoutbreaksofT.gondiicanoccur,andare

concentratedinsmallgroups ofindividuals whohave

con-sumedcontaminated food or inhaled oocystsfrom animal

feces.5–8 _{InBrazil,T.gondiioutbreakshavebeen reportedin}

differentregionsofthecountry(Northeast,Southeast,South),

bothingroupsofindividualsorurbanareas.9–11_The

investiga-tionoftheoriginofaT.gondiioutbreakisdifficultmainlydue

tothesimilarityofthesymptomswithotherinfectiousagents,

especiallyviruses,thecirculationofwildT.gondiistrains,and

thewidedisseminationofthisinfection.Towardthatend,a

suitablemethodforidentifyingcirculatinginfectiousagents

duringanoutbreakistheapplicationofmetagenomicsnext

generationsequencing(mNGS), whichisanunbiased

tech-niqueprovidinginformationonthegenomiccompositionof

clinicalsamplesandconsequentlytheetiologicnatureofthe

outbreak.

InApril2018,the largestT.gondiioutbreakinBrazilwas

registered in the city of Santa Maria, Rio Grande do Sul

State, SouthBrazil. By this period, there was a significant

increasein patientswho represented symptoms like fever,

myalgia,headache,lymphadenopathy,andacutefebrile

syn-drome(mononucleosislike disease). Until,June, 2018there

were510confirmedcasesoftoxoplasmosisinthislocationin

almostallresidentialareas,asreportedbytheMunicipal

San-itary Agency (report available athttp://www.santamaria.rs.

gov.br/docs/noticia/2018/06/D08-1465.pdf).Inordertoobserve

ifviralagentsalsocirculated duringthat outbreak,we

per-formed a viral metagenomic survey aiming to investigate

possibleviralcausativeagentsfortheobservedsymptoms.

Materials

and

methods

Atotalof14plasmasamplescollectedfrompatientstesting

positiveforanti-T.gondiiIgMandIgG(exceptone)duringthe

outbreakwerecollected.Allpatientspresentedsymptomsof

acutefebrilesystemicdisease.Onaverage,thesampleswere

collectedtwoweeksafterappearanceoftheacutesymptoms.

Peripheralbloodsampleswerecollectedbybrachialvein

punc-ture from each patient using sterile EDTAK3 tubes (INJEX

VÁCUO,Ourinhos,SãoPaulo,Brazil).Forplasmaseparation

frombloodcellcomponents,thesampleswerecentrifugedat

lowspeed(1455×g,10min).Allofthepatientswere

individu-allyinformedaboutthestudyandsignedawritteninformed

consent.SantaMariacityisamedium-sizedcity,locatedin

thecentralpartoftheRioGrandedoSulState(29◦ 41’02”S

53◦48’25”W).

Themeanageofthepatientswas31.85years(range5–51

years),57.1% werefemaleand42.9%male.All patientshad

aclinicalconditioncompatiblewithsystemictoxoplasmosis,

whichincludedmyalgia,asthenia,prolongedhighfever,and

lymphadenopathy.Noocularmanifestationswere reported.

Oneofthepatientswaspregnantandpresentedspontaneous

abortion.

Beforenucleicacidextraction,800␮Lofeachsamplewas

centrifuged(17,000×g,5min)forremovalofplasmaproteins

andcelldebris.Consequently,avolumeof600␮Lofplasma

was separated and treated with Turbo DNase (Turbo

DNA-freekit,ThermoFisherScientific,SãoPaulo,Brazil)toremove

free host/bacterial/protozoan DNA (incubation at 37◦C for

30min).AfterDNAseinactivation,all14samplesweremixed

inordertoformasamplepool.Thetotalvolumeofthepool

was usedtoextractDNA usingtheHighPure ViralNucleic

AcidLargeVolumeKit(Roche,SãoPaulo,Brazil)followingthe

manufacturer’s instructions. Reversetranscription was

per-formed using Superscript III First-StrandSynthesis System

(ThermoFisherScientific,SãoPauloBrazil)andamplification

ofcDNAwascarriedoutbytheQuantiTectWhole

Transcrip-tomeKit(QIAGEN,SãoPaulo,Brazil).Thelibrarypreparation

wasperformedusingtheNexteraDNAFlexLibrary

Prepara-tionKit(Illumina,SanDiego,CA,USA)andtheNexteraDNA

CDIndexeskitfollowingthemanufacturer’sinstructions.The

sequencingofthelibrarieswascarriedoutinIlluminaNextSeq

500sequencerusingtheNextSeqHighOutputKitv2.5,300

cycles(Illumina,SanDiego,CA,USA)followingmanufacturer’s

instructions.TocontrolthemNGSreactionsweusedthePhiX

SequencingControlv.3(Illumina,SanDiego,CA,USA);

how-ever,duetothehighcostofthesequencinginternalpositive

and/orblankcontrolswerenotused.

Therawsequencedatawas subjectedtoqualitycontrol

analysisusingFastQCv.0.11.8software.Afterevaluationthe

quality oftheobtainedreads, trimmingwasperformedfor

selectionofthesequenceswithbestquality.Trimmingwas

doneintwodistinctways:(i)bytheuseofTrimmomaticv.0.3.9

forremovinglowqualitysequences,duplicatesand

ambigu-ous bases; and (ii) Cutadapt v. 2.4 forremoval ofIllumina

adapters. For subsequent analysisweused onlysequences

withanoverall qualityofabove30scores.Additionally, we

performedasecondtrimmingstepusingtheAfterQCv.0.9.7

softwaretoremovepoliAtailsandmoreambiguousbases.

Abioinformaticpipelinewasdevelopedfocusingonviral

metagenomicsanalysis,butwewerealsoabletoidentifysome

protozoabecauseourcustomdatabasecontainedprotozoan

taxonomicinformation.Toinferthetaxonomicclassification,

weusedKraken2program.The“denovo”assemblywas

per-formedusingSPAdesv.3.13.0.Finally,inordertoidentifythe

nucleotideandaminoacidsimilarityweperformedaBlastn

andafteraBlastxapproachuseingDiamondv0.9.29.

Thephylogeneticanalysiswasperformedusingadataset

of45completegenomesequencesforParvovirusB19(B19V)

and20completegenomesfromEpsteinBarrvirus(EBV).

Thealignment wasperformed usingMAFFT v.7.429 and

the phylogeneticsignalwasevaluated theTreePuzzlev.5.2

program. A phylogenetic tree was built withIQtree v.16.12

applying the maximum likelihood algorithm with statistic

support of ultrafast bootstrap with 10,000 replicates. The

K3Pu+F+R2forEBVchosenaccordingtoBIC(Bayesian

Infor-mation Criterion)statistic model. The finalformatting and

visualizationofthephylogenetictreewasperformedusingthe

FigTreev.1.4.4software.

Finally,weperformeddirectconfirmationofthepresence

ofB19VDNAintheplasmasamplespresentinthetestedpool.

For B19VDNA detection TaqMan® real-timePCRwas used

withprimersandprobesalreadydescribedintheliterature.12

The amplification was performed in Applied Biosystems®

7500real-timePCRsystemandallmeasurestoprevent

con-taminationwerestrictlyadheredto.

Results

Theperformedbioinformaticanalysisontheobtained

sam-ples, revealed in first place the presence of reads, which

weretaxonomicallyclassifiedasT.gondii(totalnumberof240

obtainedfromthewholepool).However,afterBlastn

screen-ing,onlythemostrepresentativesequenceswereconsidered

foranalysis(88readswithhighgeneticsimilarityforT.gondii).

Despite efforts tobettercharacterize the infectingT. gondii

strain,wewerenotabletoperformtheassemblyofalarger

contig.ThiswasprobablyduetothemethodappliedforDNA

extraction,thatincludestreatmentwithhighDNAse

concen-tration,whichmayhavedegradedT.gondiigenome.Moreover,

we worked with plasma samples possibly with low

para-sitemiasinceT.gondiiisanobligatoryintracellularparasite.

TheBLASTnanalysisdemonstratedthattheinfectingT.gondii

strainwasclassifiedasVEGstrain(typeIII);however,onemust

takeintoaccountthesmallsizeoftheanalyzedsequences

(averagesizeof150bp)whichmaygenerateinaccurate

classi-ficationcomparedtorobustphylogeneticapproaches.

Regardingthe viralabundanceoftheanalyzedsamples,

humancommensalviruseslikeHumanPegivirus-1(HPgV-1)

andTorquetenovirus(TTV)typeswereidentified.Thisisa

normalfindingasthesevirusesare highlyprevalentinthe

humanpopulation.TheidentifiedHPgV-1belongedto

geno-type2(Subgenotype2A).FromtheTTVwereabletoidentify

thetypesTTV-11,19and20.

Ouranalysiswasalsoabletodetectknownviralpathogens

whichwerecirculatingconcomitantlywithT.gondiiduringthe

outbreakandcouldberesponsibleforaclinicalpicturesimilar

totoxoplasmosis.Inthetestedpoolswewereabletoassemble

halfcompletegenomeofB19V(2619bp) andsmallercontig

(381bp)belongingtoEBV.

Phylogeneticclassificationwasperformedonthecontigsof

bothviruses.B19Vtaxonomywascarriedoutusingthe

max-imum likelihood methodwithstatistic support ofultrafast

bootstrapwith10,000replicates.Thegeneratedphylogenetic

treedemonstratedtheexpectedforB19Vtopology,with

divi-sionofthestrainsintothreegenotypes.TheidentifedB19V

strain(GenBankacessionnumberMN556782)was

character-izedasbelongingtosubgenotype1AoftheB19Vgenotype1.

ThisstrainwasgroupedwiththeKU5isolatewhichhas

cir-culatedintheUSAduring2013(formoreinformationonthe

B19VobserveFig.1).

We also perfomed a phylogenetic analysis of the

obtainedEBVcontigdepositedasGenBankaccessionnumber

MN551095..ThepresenceofEBVDNAsequencesisjustified

bythefinding thatfourofthepositiveforT.gondiishowed

also anti-EBV IgM seroreactivity. The performed EBV

phy-logenetic analysisdemonstratedthatoursequence belongs

to the BBRF1 region of EBV genome, responsible for

cap-sid portal proteinsynthesis (positions101,916bp and ends

at 103,757bp). The location of the detected sequence was

betweennucleotidepositions103,040bpand103,338bpwithin

the reference genome Human gammaherpesvirus4isolate

UPN252(AP019097).Thephylogeneticanalysisdemonstrated

thedivisionoftheevolutionarytreeintothetwoclassicEBV

types withourstrain belongingto EBVtype1 (Fig. 2).The

sequencedatafromtheNGSwasdepositedintheGenBank

underthefollowingnumberSRR7245009.

ThedirectconfirmationofB19VDNAdemonstrateda

pos-itiveresultinoneofthesamplesofa35-yearoldmalepatient

whopresentedacutefebrilesyndromecharacterizedbyhigh

feverandintenseasthenia.Thereactioncyclethreshold(Ct)

was30.5whichprobablywasrelatedtoaviralloaddecrease.

Alimitationofourstudywasthatwewerenotabletoconfirm

the presenceofEBV DNAbymolecularmethods.

Nonethe-less, we observed that four patients whose samples were

includedinthetestedpoolhadpositiveresultsforanti-EBV

IgMwithdifferentopticaldensity(range1.11and12U,

refer-ence values:0.5U,notreactive;0.5–0.99borderlineand>1.0

positive).Althoughtheopticaldensityisanindirectmeasure

forconfirmationofacuteinfection,furthermoleculartesting

iswarrantedinordertodirectlyshowthepresenceofviral

DNA.TheworkflowoftheperformedstudyisshowninFig.3.

Discussion

Inthisstudyweanalyzedtheviromeofplasmasamplesfrom

patientswhopresentedacutefebrilesyndrome

(mononucleo-sislikedisease)duringaT.gondiioutbreakinSouthBrazil.The

similaritiesoftheclinicalsymptomsoftoxoplasmosistoacute

viraldiseaseswarrantapplicationofmNGSinordertoidentify

otherinfectiousagentswhichmightco-circulateduringthe

outbreak.Inthisrespectwewereabletoidentifyco-circulating

B19VandEBV,bothcausingacutefebriledisease.

Theperformedmetagenomicanalysisdetectedthe

pres-enceofsequencereadscompatiblewithT.gondiiVEGstrain.

Almost all patients presented anti-T. gondii IgM and IgG,

whichcanberegardedassystemictoxoplasmosis.7_T.gondii

outbreaks have been described in different Brazilian areas

includingNorth,Southeast,andSouthregions.9,10,13_Wewere

not abletodetermine theorigin ofthe T. gondiistraindue

tothelownumberofspecificT.gondiireadsobtainedduring

themNGS.However,thiscouldbeanatypicalstrainofwild

T.gondiiwhichhasenteredtheurbanpopulation.Such

intro-ductionsofatypicalT.gondiistrainshavebeenfoundinFrench

GuianaanddifferentBrazilianregions.9,14,15_{Thisisreinforced}

bythefactthatinSouthAmerica,atypicalT.gondiigenotypes

havebeenshowntocirculateandcanberesponsibleforthe

emergenceofoutbreakswithsevereclinicalpresentation.16

We identified possible circulation of well-described

pathogenic viruses during this outbreak. Larger contigs

of B19V and EBV were assembled and primary infections

withtheseagentsmightresultinacutefebriledisease.EBV,

Fig.1–PhylogeneticanalysisofParvovirusB19(B19V).Onlycompletegenomeswereusedfortracingthephylogenetic historyofB19V.ThenucleotidesubstitutionmodelusedwasTN+F+R2fortreereconstruction,whichwaschosenbyBIC (BayesianInformationCriterion)statisticmodel,utilizing10,000ultrafastbootstrapreplicatesforstatisticalsignificance. Onlyvaluesofabove75%weredemonstratedonimportanttreebranches.Thephylogenetictreewasconstructedusingthe IQtreesoftwarev.16.12,applyingthemaximumlikelihoodapproach.

Fig.2–PhylogeneticanalysisofEpsteinBarrvirus(EBV).Onlycompletegenomeswereusedforphylogeneticsanalysis.The nucleotidesubstitutionmodelusedwasK3Pu+F+R2fortreereconstructionandwaschosenaccordingtoBIC(Bayesian InformationCriterion)statisticmodel,utilizing10,000ultrafastbootstrapreplicatesforstatisticalsignificance.Onlyvalues ofabove75%aredemonstratedontheimportanttreebranches.ThephylogenetictreewasconstructedusingtheIQtree softwarev.16.12,applyingthemaximumlikelihoodapproach.

toxoplasmosis,andthedifferentiationbasedonlyonclinical

findingsisquite difficult.17 _{Therefore,applicationofmNGS}

can lead toidentification of the respective pathogens and

adoptionofthemostadequatetreatmentandmanagementof

theinfection.SimultaneouspresenceofT.gondiiandEBVhas

beendescribed inacord-bloodtransplantrecipientleading

tosevereencephalitis18_{and,therefore,establishingthemost}

adequate treatment options has a crucial importance for

patientmanagement.Fourofthepatientspresentedanti-EBV

IgM justifying detection bymNGS reads. Thephylogenetic

analysisdemonstratedthatthedetectedEBVstrainbelongsto

type1,whichistypicalintheAmericas.19_{Wewerealsoableto}

identifycontigsbelongingtoB19Vandfrequentcirculationof

thisvirushasbeendescribedinSouthBrazil20_{whichexplains}

our findings. Thephylogenetic analysisdemonstrated that

theinfectingB19Vwasofgenotype1(sub-genotype1A)which

is the most frequent genotype circulating in Brazil.20 _The

Fig.3–Workflowofthemetagenomicnext-generationsequencing(mNGS)processofthesamplesobtainedduringaT. gondiioutbreakfromsamplecollectiontothebioinformaticanalysis.

individual samples(Ct=30.5)showingthe clinicalutility of

mNGS for the diagnosis of clinically important infections.

ThepresenceofB19VandT.gondiico-infectionhasunknown

clinicaloutcome;however,fatalmyocardialcoinfectionshave

beenobservedinpatientswithHIV.21

TheappliedmNGSmethodsshowedco-circulationofviral

agentsinindividuals withacutefebrile syndromeduringT.

gondii outbreak and, therefore, mNGS methods should be

implementedtounraveltheetiologyofoutbreaksofunknown

origin.AsmNGS isan unbiasedstrategyforpathogen

dis-covery,itcanbringimportantinformationofhowtomanage

theoutbreak, detectthe geneticdiversityofthecirculating

strains(originoftheoutbreak),preventionoftheir

dissemi-nation,andimportantly,treatmentoptionsdependingonthe

circulatingpathogen.Forthatreason,mNGShasalreadybeen

successfullyappliedforinvestigatingoutbreaksofunknown

etiology.22,23_{WealsobelievethatmNGSmustbewidely}

rec-ommendedasafirstlinetoolfortestingpatientsampleswhen

decidingonthecauseofagivenoutbreakanditssubsequent

management.

Inconclusion,theperformedstudyexaminedthe

impor-tanceofmNGSforidentifyingviralinfectionsco-circulating

during anoutbreak oftoxoplasmic acutefebrile disease in

SouthBrazil.Asshortcomingofthisstudy,wewerenotable

todirectlyconfirmallidentifiedviralpathogensintheclinical

samples; nevertheless,the performed mNGS revealed that

patient’sviromemaybeusedasaviraldiagnosticmarker.The

includingthehighcostofthereactionwhichhampers

test-ing larger numbers of individual samples and performing

population-basedstudies.Nevertheless,theconductedstudy

demonstratestheutilityofmNGSnotonlyforviraldetection,

butalsoforpolicymakingdecisionsonhowtomanagethe

outbreakandconsequentlytheinfectedpatients.

Funding

ThefinancialsupportofthisstudywasprovidedbySãoPaulo

ResearchFoundation(FAPESP), grantsnumbers: 17/23205-8,

18/15826-5,19/08528-0,19/07861-8,INCTC-FAPESP2014/50947

and Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), grants numbers 465539/2014-9 and

455503/2014-1.

Ethical

approval

ThisstudywasapprovedbytheInstitutionalEthicsCommittee

oftheUniversityHospitaloftheFacultyofMedicineofRibeirão

Preto,UniversityofSãoPaulo

Informed

consent

Verbalinformedconsentwasobtainedfromallpatients

par-ticipatinginthisstudy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Wearegratefultoallpatientswhodonatedtheirsamplesfor

therealizationofthisstudy.WearealsogratefultoRoberta

MaraninchiSilveiraandKamilaChagasPeronniforthesample

preparation.WeacknowledgeSandraNavarroBrescianiforthe

artwork.

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