The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
Original
article
Molecular
diagnosis
of
cryptococcal
meningitis
in
cerebrospinal
fluid:
comparison
of
primer
sets
for
Cryptococcus
neoformans
and
Cryptococcus
gattii
species
complex
Marilena
dos
Anjos
Martins
a,b,
Kate
Bastos
Santos
Brighente
a,
Terezinha
Aparecida
de
Matos
c,
Jose
Ernesto
Vidal
d,
Daise
Damaris
Carnietto
de
Hipólito
b,
Vera
Lucia
Pereira-Chioccola
a,∗aLaboratóriodeBiologiaMoleculardeParasitaseFungos,InstitutoAdolfoLutz,SaoPaulo,SP,Brazil bNúcleodeMicologia,InstitutoAdolfoLutz,SaoPaulo,SP,Brazil
cLaboratóriodeLíquidoCefalorraquiano,InstitutodeInfectologiaEmílioRibas,SaoPaulo,SP,Brazil dDepartamentodeNeurologia,InstitutodeInfectologiaEmílioRibas,SaoPaulo,SP,Brazil
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t
i
c
l
e
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n
f
o
Articlehistory:Received13August2014 Accepted19September2014 Availableonline16December2014
Keywords:
Cryptococcalmeningitis Acquiredimmunodeficiency syndrome
Polymerasechainreaction Cerebrospinalfluid
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b
s
t
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c
t
Aim:Thisstudyevaluatedtheuseofpolymerasechainreactionforcryptococcalmeningitis diagnosisinclinicalsamples.
Materialsandmethods:Thesensitivityandspecificityofthemethodologywereevaluated usingeightCryptococcusneoformans/C.gattiispeciescomplexreferencestrainsand165 cere-brospinalfluidsamplesfrompatientswithneurologicaldiseasesdividedintotwogroups: 96patientswithcryptococcalmeningitisandAIDS;and69patientswithotherneurological opportunisticdiseases(CRL/AIDS).Twoprimersetsweretested(CN4–CN5andthe multi-plexCNa70S–CNa70A/CNb49S–CNb-49AthatamplifyaspecificproductforC.neoformans
andanotherforC.gattii).
Results:CN4–CN5primersetwaspositiveinallCryptococcusstandardstrainsandin94.8%in DNAsamplesfromcryptococcalmeningitisandAIDSgroup.Withthemultiplex,no448-bp productofC.gattiiwasobservedintheclinicalsamplesofeithergroup.The695bpproducts ofC.neoformanswereobservedonlyin64.6%ofthecryptococcalmeningitisandAIDSgroup. Thisprimersetwasnegativefortwostandardstrains.Thespecificitybasedonthenegative samplesfromtheCTL/AIDSgroupwas98.5%inbothprimersets.
Conclusions:ThesedatasuggestthattheCN4/CN5primersetwashighlysensitiveforthe identificationofC.neoformans/C.gattiispeciescomplexincerebrospinalfluidsamplesfrom patientswithclinicalsuspicionofcryptococcalmeningitis.
©2014ElsevierEditoraLtda.Allrightsreserved.
∗ Correspondingauthorat:LaboratóriodeBiologiaMoleculardeParasitaseFungos,InstitutoAdolfoLutz,Av.DrArnaldo,351,8oandar,
CEP01246-902,SãoPaulo,SP,Brazil.
E-mailaddress:pchioccola@gmail.com(V.L.Pereira-Chioccola).
http://dx.doi.org/10.1016/j.bjid.2014.09.004
Introduction
Cryptococcalmeningitisisoneofthemostserioussystemic mycoses, mainly affecting immunocompromised patients and causing high morbidity and mortality. The course of infectionisoftenalife-threateningdisease thatistypically observedinlaterstagesofacquiredimmunodeficiency syn-drome(AIDS).1–4
Cryptococcal meningitis affects approximately one mil-lion persons in the world each year and results in more than 400,000 deaths within three months after the onset ofthe disease.5 Latin America,as aglobal region, hasthe
thirdhighest incidenceof the disease,with around 54,400 casesannually.6Fortunately,inrecentyears,AIDS-associated
cryptococcalmeningitishasdecreaseddramaticallyinBrazil, which is a middle-income country with universal access toantiretroviraltherapy.4,7 However,untiltoday,
cryptococ-cal meningitis remains a common complication in Brazil andotherLatinAmericancountriesasitrepresentsthe pri-marycauseofopportunisticmeningitisandthesecond-most frequentneurologic opportunistic infection in HIV-infected patients.6,7
Patients with cryptococcal meningitis usually present severeneurologicalmanifestations,particularlysecondaryto intracranialhypertension.7–9Arapidlaboratorialdiagnosisis
extremelyimportanttoidentifyCryptococcusspecies.10
Rou-tinely,microscopicexamination,cryptococcalpolysaccharide antigen,andcultureofcerebrospinalfluid(CSF)sampleslead tothediagnosis.
Severalstudieshavedemonstrated theuse ofmolecular methodsfordeterminingthegeneticcharacteristicsof Cryp-tococcusspecies.11–15Nevertheless,thereisscarceinformation
about the use of molecular diagnosis in clinical samples. Molecularmethodssuchaspolymerasechainreaction(PCR) potentially identify the fungus and the genotype of the strain.
Consideringthatnomethodisentirelyeffectivein deter-miningthepresenceofthispathogen,itishelpfultodiagnose onthe basisofresultsobtainedfrom different methodolo-giestoruleoutfalse-negativeorfalse-positiveresults.Hence, molecularmethodssuchasPCRhaveproventobeusefulfor thispurpose,sincetheycanprovideadefinitivediagnosisas wellasevaluationofspecifictreatment.
Theaimofthis study is toevaluatethe use ofPCR for moleculardiagnosisofcryptococcalmeningitis.
Materials
and
methods
Thesensitivityandspecificityofthemethodologywere eval-uatedusingCryptococcusreferencestrainsinthefirstpartand CSFsamplesfrompatientswithneurologicaldiseasesinthe secondpartofthestudy.
Patientsandclinicalsamples
The assays were evaluated analyzing 165 CSF samples with commonly used, well-known laboratory and clini-cal diagnoses. Theclinical samples were divided into two
groups. Groupcryptococcalmeningitisand AIDS(CM/AIDS) consisted of 96 CSF samples from patients with CM/AIDS. Cases of cryptococcal meningitis were confirmed using positive microscopic examination by India ink and latex agglutination ofCSFsamples.16 All patients wereadmitted
to theInstituto de Infectologia Emilio Ribas,Sao Paulo,SP, Brazil. The CRL/AIDS group (control) consisted of 69 CSF samplesfrompatientswithconfirmeddiagnosis(byclinical, radiological and laboratory features) of other neurological opportunistic infections including cerebral toxoplasmosis, progressivemultifocal leukoencephalopathy,HIV-associated neurocognitive disorders, tuberculous meningitis, and neurosyphilis.
Ethicalconsiderations
All patients providedwritten informed consent.The insti-tutional review board of both Instituto Adolfo Lutz and Instituto de InfectologiaEmilio Ribascommitteesapproved thisstudy.
Laboratorydiagnosis
AttheInstitutodeInfectologiaEmilioRibastheCSFsamples werecollectedinasepticconditions,immediatelytransferred tosteriletubes,senttolaboratoryandprocessed.Eachsample was splitin two tubes. One of them was used for micro-scopic examination by India ink and latex agglutination. Theother wassent tothe Instituto AdolfoLutzfor molec-ular diagnosis.Microscopic examinationsbyIndia ink was conductedaspreviouslydescribed.17,18CSFsampleswere
cen-trifuged at3000×g for10min.One drop ofthe pellet was usedformicroscopicobservationafterIndiainkpreparation. ThepolysaccharidecapsuleantigenwasdetectedinCSF sam-plesusingalatexagglutinationKit(CryptococcusAntigenLatex Agglutination Test–Immy) according to the manufacturer’s instructions.
Yeastpreparation
TheassayswerealsodeterminedusingDNAextractedfrom standardstrainsrepresentingeachmoleculartypeincluding: WM148 (serotypeA,VNI), WM626(serotypeA, VNII),WM 628(serotypeAD,VNIII),WM629(serotypeD,VNIV),WM179 (serotypeB,VGI),WM178(serotypeB,VGII),WM175(serotype B,VGIII),andWM779(serotypeC,VGIV).12Thestrainswere
preparedaspreviouslydescribed.12 Briefly,eachisolatewas
platedonSabourauddextroseagarandincubatedat30◦Cfor 48h. Theyeastcells were transferredtoa microcentrifuge tube containing1mLof50mMEDTA(SigmaChemical), pH 8.0,mixed,andcentrifugedat13,000×gfor15min.The super-natantwasremoved,andthepelletwasresuspendedin200L of50mMEDTA,pH8.0,containing40Loflysingenzymefrom
Trichodermaharzianum(10mg/mL)(SigmaChemical),andthen incubatedat37◦Cfor2h.Subsequently,thismixturewas cen-trifugedat13,000×gfor10min,andthepelletwasdissolvedin lysisbuffercontaining10mMTris–HCl,pH8.0;10mMEDTA, pH8.0;0.5%sodiumdodecylsulfate;0.01%N-laurilsarcosyl; and100mg/mLproteinaseK.Themixturewasvortexmixed andincubatedat56◦Cfor2h.
DNApurificationandPCR
DNAmoleculesofprotoplastsorCSFsampleswereextracted byQIAampDNAMiniKit(Qiagen)accordingtothe manufac-turer’sinstructions.CSFsampleswerepreviouslycentrifuged for15minat9000×g.PackedcellswerewashedtwiceinPBS topreventtheactionofanyTaqpolymeraseinhibitor.Whole cellswere incubatedfor15minat100◦Cin200Lof ultra-purewatercontainingfourtofive1.5mmglassbeads.Next, thecellswereprocessedinaTissueLyserdisruptor(Qiagen)for twocyclesof3min.AfterDNAextraction,thepuritywas deter-minedbytheratioofO.D.at260and280nminaNanoDrop ND100(ThermoFisherScientific,Waltham,MA,US).
Theamplificationswerecarriedoutwithakitpurchased from Promega (Go Taq Green Master Mix). The PCR mix (12.5L) consisted of 1 unit of Green GoTaq® DNA
poly-merase,10mMTris–HCl,pH8.5;50mMKCl;1.5mMMgCl2;and
200mMofeach dNTP.Thereactionsincludedthe PCRmix, 10Lofeach DNAtemplateand 25pmolofeach primerto afinalvolumeof25L.Theamplificationswereperformed in an automated thermal cycler and each run contained one negative control (ultra-pure water) and one positive (DNA extracted from serotype A, VNI culture). After ther-malcycles,PCRproductswereelectrophoresedin2%agarose gelsinTBEbufferand stainedwithethidiumbromide.The sizeoffragmentwasbasedonacomparisonwitha100-bp ladder.
Primersetselection
Theexperimentswerecarriedoutbyusingtwoprimersets. ThefirstsetwastheCN4–CN5(5 ATCACCTTCCCACTAACACA-TT3 and 5GAAGGGCATGCCTGTTTGAGAG3), which ampli-fieda 136-bp sequence from a specific genecoding region for rDNA of C. neoformans as target.15 The
amplifica-tions were performed by one initial denaturation cycle for 5min at 94◦C, 30 cycles of denaturation at 94◦C for 1min, annealing at 55◦C for1min, and extensionat 72◦C for 1min. The procedure was completed by a final cycle extension for 10min. The second was a multiplex PCR. The CNa70S–CNa70A (5ATTGCGTCCACCAAGGAGCTC3 and 5ATTGCGTCCATGTTACGTGGC3)amplifieda695-bpfragment ofaregiononC.neoformanschromosome3thatincludesthe coding sequence ofa putative aminotransferase gene. The CNb49S–CNb-49A(5ATTGCGTCCATCCAAGGTGTTGTTG3and
5ATTGCGTCCATCCAACCGTTATC3)amplifieda448-bp frag-mentofaregiononC.gattiichromosome2,whichincludes thecodingsequence ofaputative polymerase.14,19 ThePCR
products were amplified by one initial denaturation cycle for 5min at 94◦C, 35 cycles of denaturation at 94◦C for 45s, annealing at65◦C for60s, and extension at72◦C for 60s. Theprocedure was completed bya final cycle exten-sion for 10min. To control the course of extraction and checkforPCRinhibitors,allsampleswereassayedusingthe primerset1–2(5ACCACCAACTTCATCCACGTTCACC3 and 5CTTCTGACACAACTGTGTTCACTAGC3),20whichamplifieda
140-bpfragmentofthehuman-globulingene.Thereactions usingthehumanprimersetwererunsimultaneouslywiththe sametemperatureprotocolfortheCN4–CN5primersetandin thesamePCRmachine.
Dataanalysisandqualityassurance
Separate rooms were usedfor: (1) DNA extraction; (2) PCR mix and primer preparation; (3) adding DNA from clinical samples and positivecontrol; and (4) post-PCRagarose gel electrophoresisanalysis.DNAclinicalsampleswereassayed induplicateand,atleast,twice.Clinicalandlaboratory (micro-scopic examination by India ink and latex agglutination) diagnoses were the gold-standard to establish sensitivity, specificity, positive(PPV)and negative(NPV)predictive val-ues,which were calculatedas: (i)sensitivity–ratio oftrue positives/(truepositives+falsenegatives)×100;(ii)specificity –ratiooftruenegatives/(truenegatives+falsepositives)×100; (iii) PPV – ratio of true positives/(true positives+false positives);and (iv)NPV–ratiooftruenegatives/(true nega-tives+falsenegatives).
Results
Two PCR primer sets to determine C. neoformans/C. gattii
species complex in clinical samples were evaluated since theyamplifieddifferentgeneticregions.Initiallytheprimer setswereevaluatedforsensitivityusingthestandardstrains. CN4–CN5 primer set amplified the 136-bp product in all standard strains tested. Thesame DNA sampleswere also testedforCNa70S–CNa70A/CNb49S–CNb-49Amultiplex.Four standard strains (serotypes A-VNI and VNII, serotype AD-VNIII,serotypeD-VNIV) amplifiedthe695-bpfragment;and
Table1–EvaluationofprimersetsCN4–CN5andCNa70S–CNa70A/CNb49S–CNb-49AinPCRforCryptococcusstandard strains.
Standardstrains Primersets
CN4–CN5 CNa70S–CNa70A/CNb49S–CNb-49A
WM148(serotypeA,VNI) Positive Positive
WM626(serotypeA,VNII) Positive Positive
WM628(serotypeADVNIII) Positive Positive
WM629(serotypeD,VNIV) Positive Positive
WM179(serotypeB,VGI) Positive Negative
WM178(serotypeB,VGII) Positive Positive
WM175(serotypeB,VGIII) Positive Negative
Table2–SummaryoftheresultsusingCN4–CN5andCNa70S–CNa70A/CNb49S–CNb-49AprimersetsinPCRusingCSF samplesfromAIDSpatients.
Primersets PPVa NPVb PCRresults
Numberofsamples(%)
Cryptococcalmeningitis Otherneurologicaldiseases
Positivec Negative Total Positive Negatived Total
CN4–CN5 0.99 0.93 91(94.8%) 5 96 1 68(98.5%) 69
CNa70S–CNa70A 0.99 0.53 62(64.6%) 34 96 1 68(98.5%) 69
a Positivepredictivevalue(PPV).
b Negativepredictivevalue(NPV).
c Sensitivity(inpercent)wascalculatedanddeterminedconsideringtheclinicalandlaboratorydiagnosisdescribedinMaterialsandmethods
section.
d Specificity(inpercent)wascalculatedanddeterminedconsideringtheclinicalandlaboratorydiagnosisdescribedinMaterialsandmethods
section. MM 1 2 3 4 –136 bp –448 bp 100– –695 bp 600–
Fig.1–AmplifiedPCRproductsoftwodifferenttarget regionsofCryptococcusneoformans/C.gattiispecies complex.CNa70S–CNa70A/CNb49S–CNb-49Amultiplex amplifieda695-bpproductofC.neoformans(line1)and 448-bpofC.gattii(line2).CN4–CN5primerset,amplifieda 136-bpproduct(lines3and4).TheDNAfragmentswere resolvedin2%agarosegelsstainedwithethidiumbromide. LaneMM,100-bpladder.
two(serotypeB-VGIIandserotypeC-VGIV)amplifiedthe 448-bpfragment.Noamplifiedproductwasshownintwostandard strains(serotypes B-VGI and VGIII)(Table 1). These experi-ments were conductedthree times and,as anillustration,
Fig.1showstheamplifiedPCRproductsofbothprimersets usedinthisstudy.Bothdidnotamplifyotheryeastssuchas
Candidaalbicans,C.parapsilosis,C.tropicalis,andC.glabrata.
Thegoodqualityofall165DNAsampleswasconfirmed bytheamplificationof1–2primersetthatamplifiedaPCR fragmentofhuman-globulingeneof140-bp.Positive ampli-ficationsshowedthatnosubstancepresentinDNAsamples inhibitedthereactions.
Next,the sensitivityand specificityofthe CN4/CN5and theCNa70S–CNa70A/CNb49S–CNb-49Amultiplexwere deter-minedinDNAsamplesextractedfromCSFsamplesofboth groupsofpatients.Table2showstheresultsofeachgroupin
detail.TheanalysisoftheclinicalsamplesfromtheCM/AIDS grouprevealedthat94.8%ofthesesampleswerepositive(91 outof96)usingtheCN4/CN5primerset.Thespecificityofthe assay,basedontheanalysisofall69negativesamplesfrom CTL/AIDSgroupwas98.5%.
Accordingtothemultiplex CNa70S–CNa70A/CNb49S–CNb-49A, no 448-bp product of C. gattii (CNb49S-CNb-49A) was shownintheclinicalsamplesofeithergroup.The695-bp prod-uctsofC.neoformanswereshowninonly64.6%(62outof96) oftheCM/AIDSgroup.InCTL/AIDS,thespecificitywasalso 98.5%.Onlyonesampleoutof69hadafalse-positiveresult. Althoughthespecificitywasgood,thesensitivitywasverylow aswellastheNPV(0.53).
Discussion
Despite the observed declineofcryptococcalmeningitis in AIDSpatients, itsoccurrencestillrepresentsadeterminant ofpoorprognosisinHIV-infectedpatients.6Additionally,this
fungalinfectioninthecentralnervoussystemleadstosevere neurologicalcomplications.Inthisway,itssuccessful treat-ment depends on rapid and accurate identification of the causativeagent.16Consequently,accurateandrapiddiagnosis
iscritical.Inrecentyears,greatadvanceshavebeenmadein developinglaboratorytechniquestodiagnoseinfectious dis-eases.Nevertheless,despitethewell-establisheddiagnosisof cryptococcalmeningitissomeaspectsofthediagnosisremain challengingandtherearestillgapsthatneedtobeaddressed. Forinstance,anegativeresultfromaninvitroculturemight beduetothepresenceofnonviableorfastidiousyeastinthe sample.Thus,theuseofPCRinCSFsamplesassociatedwith methods alreadyused atthe laboratorycould increase the diagnosticefficacy.
Severalstudiesbasedonmolecularmethodshaveshown thatC.neoformansisolatesexhibitdifferentdegreesofgenetic heterogeneityamongclinicalandenvironmentalisolates.The primersetsusedintheseCryptococcusstudieswerenormally used forgenotypingcharacterization.11,21–25 However,there
havebeenfewstudiesevaluatingtheefficacyofsuch method-ologyinmoleculardiagnosis.Here,wechosetwopreviously describedprimers’setwidelyusedforCryptococcus genotyp-ing. Two primersets were tested as theyamplify different
geneticregions.TheCN4–CN5primersetamplifieda136-bp sequencefromaspecificregionofgenecodingforrDNAas target.15
The sensitivity, accuracyand specificity of a diagnostic test depend also on optimal working conditions. In order toimprove the PCRsensitivity, theCSF samplesshould be processedrapidlywithin48hofcollectiontopreventTaq poly-meraseinhibitionthatcouldmodifyPCRresults.26Inaddition,
theDNAextractionandPCRinhibitorsmaybeevaluatedbya primersetthatamplifiedhumanPCRfragments.Inthisstudy, positiveamplificationsshowedthatnosubstancepresentin DNAsamplesinhibitedthereactions.
Firstly,theefficacyofbothprimersetswasanalyzedusing
Cryptococcusstandardstrainsandthentheclinicalsamples. TheCN4–CN5primersetwashighlysensitivesinceit ampli-fiedallCryptococcusstandardstrainsusedinthisstudy.These datawere furtherconfirmedusing96clinicalsamplesfrom theCM/AIDSgroupthatrevealedgoodsensitivity(94.8%)and negativepredictivevalueof0.93.FivecasesoftheCM/AIDS groupwerefalsenegatives.ThePCRresultscouldbedueto lowornon-uniformdistributionofspecimensfromwhichthe DNAwasextracted.Furthermore,thespecificitywasalsogood (98.5%)with high positivepredictive value (0.99). Onlyone DNAsamplefromCLR/AIDShadafalse-positiveresult.Similar resultshavebeenreportedpreviously.27
Theuseinmoleculardiagnosisofamultiplexprimerset could be an excellent alternative,since in the same reac-tion it could be possible to detect the organism and, at the same time,genotype the infectingstrain. For this pur-pose, the CNa70S–CNa70A/CNb49S–CNb-49A multiplexwas tested in this study. According toprevious studies, 695-bp PCRproductswereshowninC.neoformansstrainsanda 448-bpfragmentofC.gattii.14,19Inthisstudy,thePCRusingthe
(CNa70S–CNa70A/CNb49S–CNb-49A)multiplexwasunableto amplifytwoCryptococcusstandardstrains(serotypesB-VGIand VGIII).Inaddition,no448-bpproductofC.gattiiwasshownin theclinicalsamplesofeithergroup.Thesedatacouldsuggest thatthemajorityoftheDNAsamplesfromCM/AIDShadyeast belongingtoC.neoformans.However,the695-bpproductsofC. neoformanswereshownonlyin64.6%oftheCM/AIDSgroup resultinginlowsensitivitywithverylowNPV(0.53).Inthe CM/AIDSgroup,34DNAsampleshadfalse-negativeresults.
Collectivelythesedata suggestthat althoughthe multi-plexprimersetcoulddetecttheorganismand,atthesame time,genotype the infectingstrain, the sensitivitywas too low.Ontheotherhand,theprimersetCN4/CN5washighly sensitiveforC.neoformans/C.gattiispeciescomplex determi-nationinCSFsamplesfrompatientswithclinicalsuspicionof cryptococcalmeningitisorthetreatmentcontrol.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest
Acknowledgments
This study was supported by grants from: (i) FAPESP (Fundac¸ao de Amparo à Pesquisa do Estado de São Paulo, Brazil), Proc: 2011/13939-8,(ii) CNPq (Conselho Nacionalde
Desenvolvimento Científico e Tecnológico, Brazil), Proc: 303489/2012-0.JimHessonofAcademicEnglishSolutions.com revisedtheEnglish.
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