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
Original
article
Design
and
evaluation
of
an
AFLP
molecular
marker
for
the
detection
of
Coccidioides
spp.
in
biological
samples
María
del
Rocío
Reyes-Montes
a,
María
Guadalupe
Frías-De-León
b,
Isai
Victoriano-Pastelín
a,
Gustavo
Acosta-Altamirano
b,
Esperanza
Duarte-Escalante
a,∗aFacultaddeMedicina(UNAM),DepartamentodeMicrobiologíayParasitología,LaboratoriodeMicologíaMolecular,CiudaddeMéxico,
Mexico
bHospitalRegionaldeAltaEspecialidaddeIxtapaluca,DireccióndeInvestigación,Ixtapaluca,Mexico
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10April2019 Accepted16August2019
Availableonline17September2019
Keywords: Molecularmarkers SCAR Coccidioidomycosis Coccidioidesspp.
a
b
s
t
r
a
c
t
Atpresent,thereisnostandardizedmarkerthatisroutinelyusedinclinicallaboratoriesto diagnosecoccidioidomycosis.Thus,thegoalsofthisstudyweretoobtainasequence charac-terizedamplifiedregion(SCAR)markerfortheidentificationofCoccidioidesspp.,evaluateits specificityandsensitivityinfungalDNA-spikedbloodandsputumsamples,andcompare itwithpreviouslydescribedmolecularmarkers.Specificamplifiedfragmentlength poly-morphism(AFLP)ampliconsforCoccidioidesimmitisandCoccidioidesposadasiiwerecloned intothevectorpGEM® -TEasyvectorandsequencedtodevelopaSCARmarker. Oligonu-cleotidesweredesignedtoidentifyCoccidioidesspp.bypolymerasechainreaction(PCR), andthespecificityandsensitivityoftheseoligonucleotidesweretestedwiththeDNAfrom relatedpathogens.ThespecificityandsensitivityoftheSCARmarkerwasevaluatedwith bloodandsputumsamplesspikedwithCoccidioidesDNAandcomparedwithother previ-ouslydescribedmarkers(621,GAC2,andAg2/PRA).Inaddition,theconditionsforitsuse wereestablishedusingbiologicalsamples.AspecificmarkernamedSCAR300wasobtainedto
identifyCoccidioidesspp.thatexhibitedgoodsensitivityandspecificity.Theresultsshowed thatallofthemarkerstestedinthisstudycanidentifyCoccidioidesspp.However,theSCAR300
and621markerswerethemostsensitive,whereastheSCAR300markerwasthemostspecific.
Thus,theSCAR300markerisausefultooltoidentifyCoccidioidesspp.
©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddress:[email protected](E.Duarte-Escalante).
Introduction
ThefungiCoccidioidesimmitisandCoccidioidesposadasiiarethe causativeagentsofcoccidioidomycosis,1apredominant
dis-easeintheAmericas.Theareasmostaffectedbythismycosis
https://doi.org/10.1016/j.bjid.2019.08.002
1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
areendemicareasoftheSouthwesternUnitedStatesof Amer-ica(USA),2northernMexico,someCentralAmericaregions3
and SouthAmerica.4,5 Althoughmostinfections caused by
thesefungiareasymptomatic,somecandevelopinto symp-tomaticclinical infections withmild respiratorysymptoms ormaydevelopintoseveredisseminatedinfectionsthatare typicallyassociatedwithAidsorHIV-infectedpatients, indi-viduals havingreceived transplants,hemodialysis patients, cancer patients undergoingtreatment (primarily Hodgkin’s lymphoma),pregnantwomen,orindividualswithdiabetesor tuberculosis.6,7
ThenumberofcoccidioidomycosiscasesintheUSAhas increasedinrecentyears.8However,thenumberofcasesin
Mexicoisunknown,ascoccidioidomycosisceasedtobea noti-fiablediseasein1994,althoughithasbeensuggestedthatthe sametrendisoccurring.9
Thediagnosisofthisdiseasehastraditionallybeenbased on the resultsofa combination ofclinical data, the isola-tionofthecausativeagentinclinicalsamples,andimaging studies.However,sincethecausativefungigrowslowly,rapid methodsforitsidentificationarerequired,suchas conven-tionalserological techniques, althoughthesemethods also havelimitations.10Forthisreason,moleculartechniquesthat
usedifferentpolymerasechainreaction(PCR)markershave been developed in recent years.11–18 However, these
tech-niqueshavenotyethadasignificantimpactonthemajority ofclinicallaboratoriesinthe diagnosisof coccidioidomyco-sis.Thus,otheridentificationstrategieshavebeendeveloped, oneofwhichistheuseoftheso-calledSCAR(sequence char-acterizedamplifiedregion)markers,whichhavebeenuseful forstudyingvariationamongorganisms,identifyingstrains ofinterest,determiningtheoriginofisolates,studying popu-lationstructure,anddetectingpestresistancegenes.19SCAR
markershavealsobeen usedfordiagnostic and epidemio-logical application inother humanpathogenic fungi, such asHistoplasmacapsulatum,20demonstratingitsusefulnessfor
theidentificationofspecificmicroorganisms.Therefore,the aimofthis study wastoobtain aSCARmarkerfrom poly-morphicpatterns obtainedfromamplifiedfragment length polymorphism(AFLP)toidentifyCoccidioidesspp.,inaddition toevaluateitsspecificityandsensitivityinfungalDNA-spiked bloodandsputumsamples.Furthermore,wecomparedthe SCARmarkertothreepreviouslydescribedmolecular mark-ersand established the conditionsfor its use inbiological samples.
Material
and
methods
Isolates
Atotalof40isolateswereincludedinthisstudy(TableS1), includingfourisolatesfromMexicothatwerepreviously iden-tifiedasC.immitisand36identifiedasC.posadasii,ofwhich25 wereisolatedinMexicoand11inArgentina.21Allofthe
iso-lateswereculturedintubescontainingMycobioticagar® (BD Bioxon,EstadodeMéxico,México)andwereincubatedat28◦C forfivedaysoruntilgoodgrowthwasobservedforsubsequent trials.
Biologicalsamples
Whole blood and sputumsamples usedinthis studywere obtainedfromahealthyhumanvolunteer.
DNAextractionfromC.immitisandC.posadasiiisolates
AlloftheC.immitisandC.posadasiiisolatesincludedinthe study were grown inYPDmedium(10% yeastextract, 10% peptone,and20%dextrose)for7–10daysat28◦C.DNA extrac-tionwasperformedasdescribedbyDuarte-Escalanteetal.21
TheDNAconcentrationwasdeterminedvia spectrophotome-try(SpectrophotometerDS-11,DeNovix,Delawere,USA),and theDNAsampleswerestoredat4◦Cuntiluse.
TheDNAfromSporohtrixschenckiiwaskindlyprovidedby ConchitaToriello(FacultaddeMedicina,UNAM,Mexico), Can-didaglabrata,waskindlyprovidedbyMaríaGuadalupeFrías DeLeón(HospitalRegionaldeAltaEspecialidaddeIxtapaluca, México),HistoplasmacapsulatumwaskindlyprovidedbyMaria LuciaTaylor(FacultaddeMedicina,UNAM,Mexico),Aspergillus fumigatus and A. niger were kindly provided by María del RocíoReyesMontes (FacultaddeMedicina,UNAM,Mexico), andMycobacteriumtuberculosiswaskindlyprovidedbyMiriam BobadilladelValle(InstitutoNacionaldeCienciasMédicasy NutriciónSalvadorZubirán,Mexico).DNAfromallofthefungi tested,aswellasfromM.tuberculosis,wasusedtocheckthe specificityoftheCoccidioidesspp.molecularmarkersstudied.
ObtainingoftheSCARmarker:AFLPandSCARmarker selection
The AFLP assays were performed according to Duarte-Escalante et al.,21 using six selective primer
com-binations: E+AA:M+CAC, E+AA:M+CAT, E+AA:M+CTG, E+AA:M+CTC,E+AC:M+CATandE+AC:M+CTC.The anal-ysis of the polymorphic patterns obtained through AFLP with the six combinations of oligonucleotides allowed the identificationofabandcommoninallisolates,of300bpwith thecombinationE+AC/M+CAT.ThespecificDNAfragmentof
Coccidioidesspp.werepurifiedusingaQIAquickgelextraction kit(Qiagen,Inc.,Valencia,California,USA)andclonedintothe pGEM-TEasyvector(Promega,Madison,WI,USA),according to FríasDe León etal.20 TheSCARmarkerwas sequenced
atthe Unidadde Biología Molecular,Instituto de Fisiología Celular, UNAM, using an ABI Prism 3100 automated DNA sequencer (Applied Biosystems,Inc., FosterCity, CA, USA). TheSCARmarker sequenceswere analyzed usingBLAST22
to verify similarities between all of the fungal sequences depositedinthedatabase.Aspecificsequenceofthefungus whichhadnocoincidencewhatsoeverwiththesequencesof relatedfungidepositedintheGenBankwasselectedtodesign thespecificnucleotidesforCoccidioidesspp.Oligonucleotides were designedbased on theSCARsequence using Primer3
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3www.cgi)
and were synthesized bySigma-Genosys (The Woodlands, Texas,USA).ThePCRwiththeSCARmarkerconditionswere establishedwiththe40DNApreparationsobtainedfrom C. immitisandC.posadasii.
300 10 2 ng/ µ L 10 1 ng/ µ L 10 0 ng/ µ L 10 -1 ng/ µ L 10 -2 ng/ µ L 10 -3 ng/ µ L 10 -4 ng/ µ L 10 -5 ng/ µ L 10 -6 ng/ µL (-)
Fig.1–SensitivityoftheSCAR300marker.PCRwasperformedwithdifferentDNAconcentrationsoftheC.posadasii(HU-1)
referencestrain,asdescribedintheMaterialsandMethodssection.Positivecontrol(+);negativecontrol(−);bp(molecular sizemarker).
EvaluationofthesensitivityandspecificityoftheSCAR, Ag2/PRA,andmicrosatellite621andGAC2markers
ThesensitivityoftheSCARmarkerandthatoftheAg2/PRA
and microsatellite 621and GAC2markers was determined usingdifferentconcentrationsofDNAfrom theC.posadasii
referencestrain(HU-1).
ThespecificitiesoftheSCAR,Ag2/PRA,11GAC2,1and6211
markerswereevaluatedusingDNAfromtheC.posadasii ref-erencestrain(HU-1)andthatofotherpathogensthatcause clinicalsymptomssimilartoCoccidioidesspp.(A.niger,A. fumi-gatus,H.capsulatum,S.schenckii,C.glabrata,andM.tuberculosis).
EvaluationoftheSCAR,Ag2/PRA,andmicrosatellite GAC2and621markersinbloodandsputumsamples spikedwithC.posadasiiDNA(HU-1)
Five hundred microliters of blood or sputum was spiked with30LofC.posadasiiDNA(HU-1)atdifferent concentra-tions(2.83×102,2.83×101,2.83×100,2.83×10−1,2.83×10−2,
2.83×10−3, 2.83×10−4, 2.83×10−5, and 2.83×10−6 ng/L). EachtubewasprocessedtoobtaintotalDNAusingaDNeasy Blood&TissueKit(Qiagen).
AllPCRassayswereperformedinatotalvolumeof50L with5,10,15,and 20LoftotalDNAobtainedfrom blood orsputumsamplesspikedwithC.posadasiiDNA(HU-1).The PCRconditionsusedforeachmarkerwerethesameasthose describedabove.
Results
ObtainingoftheSCARmarker:selectionofAFLPbandsfor theSCARmarker
Analysis of the polymorphic patterns obtained by AFLP withthesixcombinationsofselectiveoligonucleotidesused resultedin the identificationof differential bands between
C. posadasii and C. immitis. Four differential bands were obtainedfor C. posadasii:a 250-bp band obtained withthe E+AA/M+CTC combination;twobands,a150-bp bandand a 300-bp band, obtained with the E+AC/M+CAT combina-tion; and a 180-bp band obtained with the E+AA/M+CAT
combination,aswell asa200-bpdifferentialbandobtained withtheE+AA/M+CATcombination.Allofthebands were reamplified underthesame conditionsinwhich theywere generated.Thefragmentswereclonedintothevector pGEM®-TEasyandthepresenceoftheinsertswascorroboratedby colonyPCRwiththeleftandrightuniversaloligonucleotides pUC/M13 and subsequently by restriction digest analysis, whichrevealedtheexpectedsizeforeachfragment.Onlythe 300-bpsequenceshowed100%identitywithC.immitisandC. posadasiiwiththesequencesdepositedinGenBank,whereas thesequencesofthe250-,180-,and150-bpbandsshowedno identitywithCoccidioidesspp.sequences.The300-bpsequence wassubsequentlynamedSCAR300.
OligonucleotidedesignfortheidentificationofCoccidioides spp.
Using the SCAR300 marker, the specific oligonucleotides
SCAR300 (F) (5-AATGGCGTTAAGTGGGTC-3) and SCAR300
(R) (5-AAGCCACTTACACAATCCAG-3) were designed. PCR was performed in a 25-L reaction containing 10ng of DNA,2.0mMMgCl2,200Mdeoxynucleotidetriphosphates
(Applied Biosystems,Foster City, California, USA), 0.1nmol of each oligonucleotide (SCAR300-F and SCAR300-R), and 1
U ofTaq DNA polymerase (Applied Biosystems) in 1×PCR buffer (Applied Biosystems). The amplification conditions were as follows: one cycle at 94◦C for 5min; 30 cycles at 94◦C for 30s, 53◦C for 30s, 72◦C for 1min; and a final extension at 72◦C for5 min. Gel electrophoresis was per-formed in a 1%agarose gel with 0.5× Tris-borate-EDTA at 100V.
EvaluationofthesensitivityandspecificityoftheSCAR, Ag2/PRA,andmicrosatellite621andGAC2markers
The minimum amount of DNA detectedby PCR using the SCAR300markerwas1ng/L(Fig.1).
TheSCAR300markerwasamplifiedusingtheC.posadasii
(HU-1) DNA and exhibited the expected 300-bp product, whereasnoamplificationwasobservedusingtheDNAfrom theotherassayedpathogenicfungiandM.tuberculosis(Fig.2A). The expected 400-bp product was amplified for the 621 microsatellite using the C. posadasii (HU-1) DNA, whereas
SCAR300 621 Ag2/PRA GAC2 300 400 300 200 bp S . schenc kii S . schenc kii S . schenc kii S . schenc kii C . glabr ata C . glabr ata C . glabr ata C . glabr ata H. capsulatum H. capsulatum H. capsulatum H. capsulatum A. fumigatus A. fumigatus A. fumigatus A. fumigatus A. fumigatus A. fumigatus A. fumigatus A. fumigatus A. niger A. niger A. niger A. niger C . posadasii C . posadasii C . posadasii C . posadasii M. tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis (-) (-) (-) (-)
Fig.2–SpecificityofthemolecularmarkersforthedetectionofCoccidioidesspp.(A)SCAR300,(B)621,GAC2and(C)Ag2/PRA.
ThespecificitiesofthemolecularmarkersweredeterminedasdescribedintheMaterialsandMethodssectionusingDNA fromotherpathogenicfungiandM.tuberculosis.Positivecontrol(+);negativecontrol(−);bp(molecularsizemarker).
no amplification was observed using DNA from the other pathogenicfungi(Fig.2B).Theexpected200-bpfragmentwas amplifiedforthe GAC2microsatelliteusingthe C.posadasii
(HU-1)DNA,althoughadditionalproductsofdifferentsizes wereamplifiedwhenDNAfromA.fumigatus,H.capsulatum,
A.niger,S.schenckii,andM.tuberculosiswasused(Fig.2B).In addition,theexpected300-bpampliconwasobtainedforthe Ag2/PRAmarkerusingC.posadasii(HU-1)DNA,althoughita 200-300-bpproductwasalsoamplifiedusingS.schenckiiDNA (Fig.2C).
EvaluationoftheSCAR,Ag2/PRA,andmicrosatellite GAC2and621markersinbloodandsputumsamples spikedwithC.posadasiiDNA(HU-1)
EvaluationoftheSCAR300markerusingbloodandsputum
samples
TheconcentrationsoftotalDNAobtainedfrombloodand spu-tumsamplesspikedwithC.posadasii(HU-1)DNAwere10–100 and50–100ng/L,respectively.Theexpected300-bpbandwas observedwhen10,15,and20LoftotalDNAwasusedfrom thebloodspikedwith2.83×101to2.83×10−1ng/Lof
Coc-cidioidesDNA,whilethebandwasdetectedwhen5Loftotal DNAwasusedfrombloodspikedwith2.83×101ng/Lof
Coc-cidioidesDNA(Fig.3).Incontrast,forthesputumsamples,the 300-bpbandwasobservedwhen5and15LoftotalDNAwas usedfromsputumspikedwith2.83×101to2.83×100ng/L
ofCoccidioidesDNA,whiletheampliconwasobservedwhen 10and20LoftotalDNAwasusedfromsputumspikedwith 2.83×101to2.83×10−1ng/LofCoccidioidesDNA(Fig.4).
EvaluationoftheGAC2and621microsatellitesinbloodand sputumsamples
Whenbloodorsputumthatwasspikedwithdifferent concen-trationsofCoccidioidesDNAharboringtheGAC2microsatellite wasassayedforthismarkerbyPCR,theexpected200-bpband wasobservedwhenfiveand10LoftotalDNAwasusedfrom bloodspikedwith2.83×101 to2.83×10−6and 2.83×101 to
2.83×10−1 ng/L ofCoccidioides DNA, respectively. In addi-tion,theexpectedampliconwasobservedwhen15and20L oftotalDNAwasusedfrombloodspikedwith2.83×101 to
2.83×10−2ng/LofCoccidioidesDNA(Fig.3).Forthesputum, a200-bpampliconwasobservedwhen20LoftotalDNAwas usedfromsputumspikedwith2.83×101to2.83×100of
Coc-cidioidesDNA,whiletheexpectedbandwasobservedwhen5, 10,and15LoftotalDNAwasusedfromsputumspikedwith 2.83×101to2.83×10−1ng/LofCoccidioidesDNA(Fig.4).
The 400-bp 621 microsatellite amplicon was observed when 5L oftotal DNA was usedfrom blood spiked with 2.83×101to2.83×10−1 ng/LofCoccidioidesDNA,whilethe
expectedampliconwasobservedwhen10LoftotalDNAwas usedfrom blood spikedwith 2.83×101 to2.83×10−7ng/L
of Coccidioides DNA. In contrast, the 400-bp amplicon was observed when 15 and 20L of total DNA was used from bloodspikedwith2.83×101 to2.83×10−3and 2.83×101 to
2.83×10−2ng/LofCoccidioidesDNA,respectively(Fig.3).For thesputumsamples,the400-bpampliconwasobservedwhen 5and10LoftotalDNAwasusedfromsputumspikedwith 2.83×101to2.83×10−7and2.83×101to2.83×10−6ng/Lof
CoccidioidesDNA,respectively.Incontrasttheexpected ampli-conwasobservedwhen15and20LoftotalDNAwasused fromsputumspikedwith2.83×101to2.83×10−3ng/Land
2.83×101to2.83×10−2ng/LofCoccidioidesDNA,respectively
(Fig.4).
EvaluationoftheAg2/PRAmarkerwithbloodandsputum samples
For the Ag2/PRA marker, the 300-bp band was observed when 5, 10, and 15L of total DNA was used from blood spiked with 2.83×101 to 2.83×100 ng/L of
Coc-cidioides DNA. In contrast, the expected amplicon was
observed when 20L of total DNA was used from blood spiked with 2.83×101 to 2.83×10−1ng/L of Coccidioides
DNA (Fig. 3). For the sputum samples, the 300-bp band was observed when 5, 10, 15,and 20Lof total DNA was used from sputum spiked with 2.83×101 to 2.83×10−5,
2.83×101to2.83×10−3ng/L,2.83×101to2.83×10−7ng/L,
and2.83×101to2.83×10−2ng/LofCoccidioidesDNA,
respec-tively(Fig.4).
Discussion
Variousmolecularmarkershavebeendescribedforthe identi-ficationofCoccidioidesspp.fordiagnosticandepidemiological purposes.1,11,13,15,16,23However,manyofthesemarkershave
low sensitivity, specificity, and reproducibility as well as limitationsassociatedwithcomplicatedmethodologiesthat involve high costs. A small number ofmarkers have been obtainedfromribosomalgenes,whicharenaturallyconserved withinthe fungal kingdom; however, their usecan leadto nonspecificresultsamongseveralfungalspecies.24,25In
addi-tion,commerciallyavailableprobesusedfordiagnostictests alsoyieldnonspecificresultsinsomecases.26Due tothese
drawbacks,designingmorespecificandsensitivemarkersfor theidentificationofCoccidioideswasnecessary.SCARmarkers havebeendesignedforotherpathogenicfungiandare excel-lentcandidatesforthispurpose,asdescribedbyFríasDeLeón etal.20Duetotheirhighspecificityandsensitivity,molecular
methodsaregraduallybeingimplementedasroutinemethods inclinical laboratoriestoconfirmthe informationobtained through conventional methods. Furthermore, these newer techniquesareusedasauxiliarymethodsinthediagnosisof questionablecasesofsomemycosesaswellastofacilitatethe characterizationofinfectionsourcesandtoconsolidate epi-demiologicalinformationofsuchmycoses,especiallyinLatin Americancountries.
SCAR markers have proven to be very useful, with the developmentofaSCARmarkerrequiringtheuseoftwo spe-cificprimersthataredesignedfromthenucleotidesequences of amplicons generated using techniques such as random amplificationofpolymorphicDNA(RAPD)orAFLP,afterwhich theyareclonedandassociatedwithafeatureofinterest.Once developed,aSCARmarkercanbeappliedtoalargenumberof samplesthatcanbesimultaneouslyexamined,reducingthe necessarytimeandincreasingreliability.27
Itisimportanttomentionthatthedevelopmentof molec-ularmarkersfromnativeisolates,asisthecaseoftheSCAR300
marker,isveryimportant,sincegreatgeneticvariabilityhas beenobservedinisolatesofC.immitisandC.posadasiifrom differentgeographicorigins.24,28–30Furthermore,ithasbeen
suggestedthatmolecularmarkersusedtodetectpathogens fromclinicalsamplesshouldbedesignedfromnativeisolates intheregioninwhichtheyaretobeused.Thus,itis impor-tant thatthe SCAR300 markerbevalidated foruseinthese
countries.
Althoughoneoftheobjectives ofthepresent studywas todesignspecificSCARmarkersthatwereforC.immitisand
C.posadasii,thiswasnotpossibleduetothesmall number ofisolatesbelongingtotheC.immitisspeciesandbecauseof thehighdiversityfoundinallisolates,whichmadeitdifficult
5µL 5µL bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) bp 400 200 200 300 300 400 300 300 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) 10µL 5µL 10µL 5µL 10µL 10µL 15µL 15µL 20µL 15µL 20µL 15µL 20µL 20µL 621 GAC Ag2/PRA SCAR 300
Fig.3–SensitivityoftheSCAR300,621,GAC2,andAg2/PRAmarkers.Differentvolumes(5,10,15,and20L)oftotalDNA
obtainedfrombloodspikedwithdifferentconcentrationsofC.posadasiireferencestrain(HU-1)DNAwereused.Positive control(+);negativecontrol(−);bp(molecularsizemarker).
toidentifyspecies-specificbandsinthepolymorphicpatterns obtainedbyAFLP.Thus,thedesignedmarkerwasspecificonly atthegenuslevel.However,becausetheSCAR300markerwas
testedusingisolatesfromMexicoandArgentinaandshowed goodspecificityandsensitivity,itmaybeagoodcandidatefor theidentificationoffungiofthegenusCoccidioides.However,
5µL 5µL 5µL 5µL 10µL 10µL 10µL 10µL 15µL 20µL 15µL 15µL 15µL 20µL 20µL 20µL SCAR 300 621 GAC Ag2/PRA bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L 2.83x10 7 ng/ µ L Control (+) Control (-) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) bp 2.83x10 1 ng/ µ L 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (+) 2.83x10 0 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 1 ng/ µ L 2.83x10 2 ng/ µ L 2.83x10 3 ng/ µ L 2.83x10 4 ng/ µ L 2.83x10 5 ng/ µ L 2.83x10 6 ng/ µ L 2.83x10 7 ng/ µ L Control (-) 400 200 200 300 300 400 300 300
Fig.4–SensitivityoftheSCAR300,621,GAC2,andAg2/PRAmarkers.Differentvolumes(5,10,15,and20L)oftotalDNA
obtainedfromsputumspikedwithdifferentconcentrationsofC.posadasiireferencestrain(HU-1)DNAwereused.Positive control(+);negativecontrol(−);bp(molecularsizemarker).
itisnecessarythatthemarkerbeevaluatedwithclinical sam-plesandsamplesobtainedfromdifferentsourcesofinfection. TheresultsofthisstudyshowedthattheSCAR300marker
wasefficientatamplifyingCoccidioidesDNA frombiological
samples(bloodorsputum)experimentallyspikedwith differ-entconcentrationsofHU-1strainDNA,eventhoughitshowed lowersensitivitythantheGAC2and621microsatellites.1The
is highly similar to that of the marker Ag2/PRA, confirm-ingitsusefulnessasadiagnostictoolforcoccidioidomycosis (Fig.3and4).
Regarding the specificity ofthe SCAR300 marker, it was
shown to be as specific as the 621 microsatellite marker, sincebothwereonlyamplifiedusingDNAfromC.posadasii.
Incontrast,inaddition toDNA fromC.posadasii,theGAC2 microsatellitewasamplifiedusingDNAfromA.fumigatus,A. niger,H.capsulatum,S.schenckii,andM.tuberculosis,whereas theAg2/PRAmarkerwasamplifiedusingDNAfromC.posadasii
andS.schenckii,makingbothofthesemarkerslessefficientfor useinthediagnosisofcoccidioidomycosis.
Accordingtotheresultsobtainedinthepresentstudywith respecttotheuseofdifferentvolumesoftotalDNA (biolog-icalsamplespikedwithDNAfromtheHU-1isolate),theuse of10and15LoftotalDNAfrombloodandsputumis recom-mendedforthePCRwithSCAR300 marker.Thisinformation
isusefulasaguideforlaboratorypersonnelinchargeof per-formingmoleculardiagnosis,sincetheseresultsindicatethe minimumvolumeoftotalsampleDNAneededtoperformPCR andachieveapositiveresult.
Conclusions
Althoughthe621microsatellitemarkershowedgreater sensi-tivitythantheSCAR300marker,itisimportanttonotethatthe
latterpresentstheadvantageofhavingbeenobtainedfrom isolatesfromMexicoandArgentina,whichensuresthe detec-tionofthefungusinthesecountries.
However,itisessentialthattheSCAR300marker,whichwas
testedinthisstudyusingbiologicalsamplesspikedwithDNA fromaC.posadasiiisolate(HU-1),betestedwiththelargest possiblenumberofclinicalsamplesfrompatientswith sus-pectedcoccidioidomycosistodefinitivelyvalidatethemethod andcorroborateitsdiagnosticutility.
Conflict
of
interest
Alltheauthorsofthisstudydeclaretheabsenceofany poten-tialconflictsofinterest.
Acknowledgements
ThisworkwasfinanciallybyPAPIIT-DGAPA(IN215509-3).
Appendix
A.
Supplementary
data
Supplementary material related to this article can be found,intheonlineversion,atdoi:https://doi.org/10.1016/j.
bjid.2019.08.002.
r
e
f
e
r
e
n
c
e
s
1. FisherMC,KoenigGL,WhiteTJ,TaylorJW.Molecularand
phenotypicdescriptionofCoccidioidesposadasiisp.nov.
previouslyrecognizedasthenon-Californiapopulationof
Coccidioidesimmitis.Mycologia.2002;94:73–84.
2.FisherFS,BultmanMW,JohnsonSM,PappagianisD,Zaborsky
E.CoccidioidesnichesandhabitatintheSouthwesternUnited
States:amatterofscale.AnnNYAcadSci.2007;1111:47–72.
3.Laniado-LaborínR.Expandingunderstandingof
epidemiologyofcoccidioidomycosisintheWestern
hemisphere.AnnNYAcadSci.2007;1111:19–34.
4.CanterosCE,ToranzoA,Ibarra-CamouB,etal.La
coccidioidomicosisenArgentina,1892-2009.RevArg
Microbiol.2010;42:261–8.
5.deAguiarCordeiroR,Nogueira-BrilhanteRS,MarcosFábio
GadelhaRochaMF,etal.Twelveyearsofcoccidioidomycosis
inCearáState,NortheastBrazil:epidemiologicanddiagnostic
aspects.DiagnMicrobiolInfectDis.2010;6:65–72.
6.Laniado-LaborínR,Alcantar-SchrammJM,Cazares-AdameR.
Coccidioidomycosis:anupdate.CurrFungalInfectRep.
2012;6:113–20.
7.Laniado-LaborínR,ArathoonEG,CanterosC,Mu ˜niz-Salazar
R,RendonA.CoccidioidomycosisinLatinAmerica.Med
Mycol.2019;57:S46–55.
8.SondermeyerGL,LeeLA,GillissD,VugiaDJ.
Coccidioidomycosis-associateddeathsinCalifornia,
2000–2013.PublicHealthRep.2016;131:531–5.
9.Baptista-RosasRC,RiquelmeM.Epidemiologíadela
coccidioidomicosisenMéxico.RevIberoamMicol.
2007;24:100–6.
10.NegroniR,ArechavalaA,MaioloE.Coccidioidomicosis.Med
CutanIberLatAm.2010;38:179–88.
11.BialekR,KernJ,HerrmannT,etal.PCRassaysfor
identificationofCoccidioidesposadasiibasedonthenucleotide
sequenceoftheantigen2/proline-richantigen.JClin
Microbiol.2004;42:778–83.
12.AssiMA,BinnickerMJ,WengenackNL,DezielPJ,BadleyAD.
Disseminatedcoccidioidomycosisinalivertransplant
recipientwithnegativeserology:useofpolymerasechain
reaction.LiverTranspl.2006;12:1290–2.
13.UmeyamaT,SanoA,KameiK,NiimiM,NishimuraK,Uehara
Y.Novelapproachtodesignprimersforidentificationand
distinctionofthehumanpathogenicfungiCoccidioidesimmitis
andCoccidioidesposadasiibyPCRamplification.JClin
Microbiol.2006;44:1859–62.
14.PounderJI,HansenD,WoodsGL.IdentificationofHistoplasma
capsulatum,Blastomycesdermatitidis,andCoccidioidesspeciesby
repetitivesequencebasedPCR.JClinMicrobiol.
2006;44:2977–82.
15.BinnickerMJ,BuckwalterSP,EisbernerJJ,etal.Detectionof
Coccidioidesspeciesinclinicalspecimensbyreal-timePCR.J
ClinMicrobiol.2007;45:173–8.
16.TintelnotK,DeHoogGS,AntweilerE,etal.Taxonomicand
diagnosticmarkersforidentificationofCoccidioidesimmitis
andCoccidioidesposadasii.MedMycol.2007;45:385–93.
17.Duarte-EscalanteE,Frías-De-LeónMG,Zú ˜nigaG,etal.
Molecularmarkersintheepidemiologyanddiagnosisof
coccidioidomycosis.RevIberoamMicol.2014;31:49–53.
18.JohnsonL,GaabME,SanchezJ,etal.ValleyFever:Danger
lurkinginadustcloud.MicrobesInfect.2014;16:591–600.
19.XuM,KorbanSS.AFLP-derivedSCARsfacilitateconstruction
ofa1.1Mbsequence-readymapofaregionthatspanstheVf
locusinapplegenome.PlantMolBiol.2003;50:803–18.
20.Frías-De-LeónMG,Arenas-LópezG,TaylorML,
Acosta-AltamiranoG,Reyes-MontesMR.Developmentof
specificSCARmarkersfordetectingHistoplasmacapsulatumin
clinicalandenvironmentalsamples.JClinMicrobiol.
2012;50:673–9.
21.Duarte-EscalanteE,Zú ˜nigaG,Frías-De-LeónMG,CanterosC,
Casta ˜nón-OlivaresLR,Reyes-MontesMR.AFLPanalysis
revealshighgeneticdiversitybutlowpopulationstructurein
CoccidioidesposadasiiisolatesfromMexicoandArgentina.
22.AltschulS,MaddenT,ShäfferA,ZhangJ,MillerW,LipmanA.
GappedBLASTandPSI-BLAST:anewgenerationofprotein
databasesearchprograms.NucleicAcidsRes.
1997;25:3389–402.
23.GreeneDR,KoenigG,FisherMC,TaylorJW.Soilisolationand
molecularidentificationofCoccidioidesimmitis.Mycologia.
2000;92:406–10.
24.LindsleyMD,HurstSF,NaurenJI,MorrisonCJ.Rapid
identificationofdimorphicandyeast-likefungalpathogens
usingspecificDNAprobes.JClinMicrobiol.2001;39:505–11.
25.MartagonVillamilJ,ShresthaN,SholtisM,etal.Identification
ofHistoplasmacapsulatumfromcultureextractsbyreal-time
PCR.JClinMicrobiol.2003;41:1295–8.
26.BrandtME,GauntD,IqbalN,McClintonS,HambletonS,Sigler
L.False-positiveHistoplasmacapsulatumGen-Probe
chemiluminescenttestresultcausedbyaChrysosporium
species.JClinMicrobiol.2005;43:1456–8.
27.BhagyawantSS.RAPD-SCARmarkers:aninterfacetoolfor
authenticationoftraits.JBiosciMed.2016;4:1–9.
28.KoufopanouV,BurtA,TaylorJW.Concordanceofgene
genealogiesrevealsreproductiveisolationinthepathogenic
fungusCoccidioidesimmitis.ProcNatlAcadSciUSA.
1997;94:5478–82.
29.FisherMC,KoenigGL,WhiteTJ,TaylorJW.Atestfor
concordancebetweenthemultilocusgenealogiesofgenes
andmicrosatellitesinthepathogenicfungusCoccidioides
immitis.MolBiolEvol.2000;17:1164–74.
30.JewellK,CheshierR,CageGD.Geneticdiversityamong
clinicalCoccidioidesspp.isolatesinArizona.MedMycol.
