Fluconazole levels in serum and cerebrospinal fluid according to daily dosage in patients with cryptococcosis and other fungal infections

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

Fluconazole

levels

in

serum

and

cerebrospinal

fluid

according

to

daily

dosage

in

patients

with

cryptococcosis

and

other

fungal

infections

Letícia

Aparecida

Schiave

_,

_Erika

_Nascimento

_,

_Fernando

_Crivelenti

_Vilar

_,

Tissiana

Marques

de

Haes

_,

_Osvaldo

_Massaiti

_Takayanagui

_,

Cristiane

Masetto

de

Gaitani

_,

_Roberto

_Martinez

a_{UniversidadedeSãoPaulo(USP),FaculdadedeMedicinadeRibeirãoPreto,DepartamentodeClínicaMédica,RibeirãoPreto,SP,Brazil} b_{UniversidadedeSãoPaulo(USP),FaculdadedeMedicinadeRibeirãoPreto,DepartamentodeNeurociênciaseCiênciasdo}

Comportamento,RibeirãoPreto,SP,Brazil

c_{UniversidadedeSãoPaulo(USP),FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,RibeirãoPreto,SP,Brazil}

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t

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Received21July2017 Accepted20October2017

Availableonline13November2017

Keywords: Fluconazole Cryptococcosis Cerebrospinalfluid

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Fluconazoleisextensivelyusedforthetreatmentofcandidiasisandcryptococcosis.Among otherfactors,successfultreatmentisrelatedtoappropriatefluconazolelevelsinbloodand cerebrospinalfluid.Inthepresentstudy,fluconazolelevelsweredeterminedin15patients, 14ofwhomhadAIDSand13hadneurocryptococcosis.Theonlyselectioncriterionwas treatmentwithfluconazole,whichwasperformedwithagenericorsimilarformofthe drug.Fluconazolelevelwasdeterminedbyhighperformanceliquidchromatographyand thesusceptibilityprofileofCryptococcusspp.isolatedfromthepatientswasassessedbybroth microdilution.Bloodandcerebrospinalfluidfluconazolelevelswerefoundtoberelatedto thefluconazoledailydose,andexceededtheminimuminhibitoryconcentrationofthis anti-fungalfortheCryptococcusspp.isolates.Agoodcorrelationwasobservedbetweenserumand cerebrospinalfluiddrugconcentration.Inconclusion,treatmentwithnon-original flucona-zoleunderusualmedicalpracticeconditionsresultsinappropriatebloodandcerebrospinal fluidlevelsofthedrugforinhibitingCryptococcusspp.susceptibletothisantifungaldrug. Therelativelycommonfailuresofneurocryptococcosistreatmentappearnottobedueto insufficientfluconazolelevelsinthecerebrospinalfluid,especiallywiththeuseofdaily dosesof400–800mg.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Fluconazoleisextensivelyusedasasystemicantifungalagent becauseofitsfavorablepharmacologicalcharacteristicssuch

∗ _{Correspondingauthor.}

E-mailaddress:erika.nascimento@gmail.com(E.Nascimento).

aslowtoxicity,goodbioavailabilityandappropriatelevelsin bloodandininfectedtissues,includingthecentralnervous system (CNS).In addition, fluconazoleactsagainst various agentsthatinduceendemicandopportunisticmycoses,with emphasisonitsclinicaluseforthetreatmentofCandidaspp. andCryptococcusspp.infections.1,2

TogetherwithamphotericinBand5-flucytosine, flucona-zole is a component of the therapeuticregimen currently https://doi.org/10.1016/j.bjid.2017.10.003

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

recommended for the control of neurocryptococcosis, par-ticularly during treatment consolidation and maintenance phases.3 _{However, some cases are refractory to antifungal}

therapyandearlyorlatedeathsmayoccurinmorethan30% ofallpatients.4,5_{Poorresponsetotreatmenthasbeenrelated}

tomoreextensivedisseminationoftheinfectionandtohost factors.6_{However,treatmentfailuremayalsobedueto}

insuf-ficientantifungal dosageand tissue level or tothe lack of Cryptococcusspp.susceptibilitytofluconazole.7

Early studies have shown that fluconazolereaches suf-ficientlevels in cerebrospinal fluid (CSF) forthe treatment offungal infections inthe CNS.8 In contrast to controlled studies,incurrentclinical practicethe patientsreceive flu-conazoleofvarious originsand theclinicalconditionsmay differfrom those standardizedinstudies with theoriginal drug.Scarcedataaboutgenericantifungalsmotivateda com-parisonbetweeninnovatorfluconazoleandsimilardrugsinan experimentalmodel.9_{Forthesamereason,inthisstudy}

flu-conazolelevelswerereevaluatedinbloodandCSFofpatients undertreatmentforcryptococcosisand other fungal infec-tions.

Materials

and

methods

Fifteenadultpatientswereincludedatrandominthepresent studywhilereceivingfluconazoletreatmentbetween Septem-ber 2012 and May 2014 at the University Hospital of the RibeirãoPretoMedicalSchool,UniversityofSãoPaulo,Brazil. AllbutoneofthesepatientswereHIV-infectedand flucona-zole was being used to treat neurocryptococcosis (n=13), histoplasmosis(n=1) orcandidiasis (n=1). Neurocryptococ-cosis cases were preferably included because of periodic CSFsampling and the useofdifferent fluconazoledosages during treatment, permitting the comparisonof the levels of this drug according to daily dose received. Cryptococcus spp.was isolatedinSabouraudagar towhich theCSFwas addedandidentifiedbystandardmedicalmycologymethods. ThisidentificationwasconfirmedbyVitek2automated sys-tem(Biomeriéux,France).Histoplasmosiswasdiagnosedby histopathogicalexaminationandapositiveserologyfor Histo-plasmacapsulatumantibodies.Oralcandidiasiswasdiagnosed byoralexaminationofanAIDSpatient.Thefollowingbrands of fluconazole were administered to the patients: generic Fluconazol(Sanobiol, Brazil) for intravenous infusion; cap-sulesfororaluse –Fluxilase®,(Laboris, Brazil)orFlucazol® (Cristália,Brazil).Thepatientsreceivedfluconazoledosesof 200–800mg/dayintravenously(n=4),orally(n=5)orby intra-venousroutefollowedbyoralroute(n=6).Ninepatientswere includedinthestudymorethanonce,fiveofthemfor receiv-ingadifferentdailydoseand/orforusing anotherrouteof administration.AmphotericinBandantiretroviralswereused simultaneouslytofluconazolein8/13and10/13patientswith cryptococcosis,respectively. Amongantiretroviraldrugs, all 10patientsusedlamivudine,andotheraccompanyingdrugs oftheregimenwerecombinationsoftenofovirorzidovudine withefavirenzorlopinavir/ritonavir.

Blood and CSF samples were collected between 24h and80daysafterthebeginningfluconazoletreatment. CSF was sampled only when requested by medical staff for

laboratory control ofcryptococcosis treatment. Drug levels weremeasuredinbloodsamplescollectedoneortwohours afteradministeringafluconazoledose.CSFwascollectedby lumbarpuncturethreetofourhoursafteringestionor infu-sionofafluconazoledose.Fluconazolelevelsweremeasured intwotosevenbloodsamplesperpatientforatotalof53 sam-ples,andinonetofourCSFsamplesforatotalof22samples. ThecorrelationbetweenserumandCSFconcentrationsofthe drugwas determinedin10patientsfrom whom15sample pairswerecollectedonthesameday.

Fluconazolelevelsweremeasuredbyhighperformance liq-uidchromatography(HPLC).10_{Fluconazolewasextractedfrom}

serumandCSFbyliquid–liquidextractionusingethylacetate asanorganicsolvent.HPLCwascarriedoutusingaShimadzu chromatograph (Shimadzu Corporation, Kyoto, Japan),with a SupelcoAnalytical Ascentis4.6mm×25cm C18® column with5␮mparticles,anAscentisC18Supelguard®guard col-umn(4mm×2cm,with5␮mparticles)andamobilephase consisting of10mMsodium phosphate buffer, pH5.7, and acetonitrile(75:25,v/v)(MerckDarmstad,Germany),ataflow rateof1mL/min,withdetectionat210nm.TheHPLCmethod was validated for fluconazole (Sigma–Aldrich, USA) quan-tification in serum andCSF. Inthe rangeof 0.5–62.5␮g/mL fluconazoleconcentration, the correlationcoefficients were 1.0000and0.9998forserumand CSF,respectively.The pre-cision expressedasvariation coefficientpercentageranged 0.5–8.1forserumand0.7–4.5forCSFinintra-runandinter-run analysis. Cryptococcusspp. susceptibility tofluconazolewas determinedinisolatesfrom11patientsbybroth microdilu-tionmethodasproposedbytheClinicalLaboratoryStandards Institute.11

Fluconazolelevelswereanalyzedaccordingto neurocryp-tococcosispatients’outcomeafteroneyearofdiagnosis.Two groupsofpatientswerecompared:survivors–9/13casesvs. deathsplusonecaseofrefractorycryptoccocosis–4/13cases. In the survivalgroupthe medianage was43 years (range: 21–63), 8/9 were men and 5/9 and 6/9were also receiving amphotericinBandantiretrovirals,respectively.Fivepatients were in the consolidationphase ofantifungaltherapy and fourofthemhadbeenpreviouslytreatedwithamphotericin B.Inthedeath/refractorygroupthemedianagewas49years (range:32–65),allweremenand3/4and2/4werealsoreceiving amphotericinBandantiretrovirals,respectively.Onepatient wasintheconsolidationphaseoftreatmentandhadreceived amphotericinBpreviously.

TheMann–Whitneytestwasusedtocomparebloodand CSFfluconazolelevelsaccordingtothedailydoseofthedrug receivedbythepatients,withthelevelofsignificancesetat p<0.05.ThecorrelationbetweenbloodandCSFlevelsofthe drugwasdetermined usingExcel® software(Microsoft Cor-poration). The study was approved by the Research Ethics CommitteeoftheUniversityHospital,RibeirãoPretoMedical School(protocolno.4096/2012)andallpatientsgavewritten informedconsenttoparticipate.

Results

Table1showsthat themedianserum levelsoffluconazole increasedwithincreasingdailydoses ofthe drug,reaching

Table1–Serumandcerebrospinalfluid(CSF)fluconazolelevelsaccordingtodailydoseadministeredto15patients.

Dose 200mg/day 400mg/day 600mg/day 800mg/mg

Serumlevels(g/mL) N◦_{samples 4 15 14 20} Median 20.2a 27.7b 33.8c 34.9d Range 7.5–33.4 8.5–42.1 26.9–50.1 17.0–60.5 CSFlevels(g/mL) N◦samples 2 3 8 8 Median 10.5e 32.5f 28.7g 23.3h Range 6.2–14.7 26.8–42.3 20.9–60.3 7.3–39.7

SignificantdifferencesbytheMannWhitneytest:bvs.c–p<0.05;bvs.d–p<0.01;evs.f–p<0.05;evs.g–p<0.05;evs.h–p<0.05

Table2–SerumandCSFfluconazolelevels(␮g/mL)incryptococcalmeningitiscasesandminimuminhibitory concentration(MIC)offluconazoleforCryptococcusspp.isolatesaccordingtooutcomeofthepatients.

Patient MIC(␮g/mL) Serumlevel-median(Range)(N◦samples) CSFa_{level-median(Range)(N}◦_{samples) Fluconazoledose-g/d(Route)}

Survival(n=9) 1 (ND)b _{26.2(25.4–26.9)(3) 17.0(17.0–17.0)(1) 800(IV)} 2 0.5 40.4(35.7–44.5)(5) 39.7(37.0–42.3(2) 400–800(PO) 5 1.0 34.5(23.1–40.6)(4) 26.4(25.2–27.5(2) 800(IV)–600(PO) 7 0.5 33.0(29.7–35.9)(3) (ND) 400(PO) 8 1.0 28.6(27.2–30.0)(2) 26.1(25.3–26.8)(2) 400–600(PO) 11 0.5 34.7(32.3–37.1)(2) 14.7(14.7–14.7)(1) 200(PO) 13 1.0 33.7(27.4–39.9)(4) 11.1(7.3–14.8(2) 800(PO) 14 2.0 33.8(32.1–36.4)(4) 13.8(6.2–21.4)(2) 200–800(PO) 15 (ND) 28.3(27.5–29.1)(2) 19.1(17.2–20.9(2) 600(PO) Median 1.0 33.7(26.2–40.4) 18.1(11.1–39.7)

Refractoryanddeath(n=4)

3 0.5 32.7(29.5–36.0)(4) 32.5(22.7–42.4)(3) 400(OR)–600(IV) 4 0.25 51.2(50.1–60.5)(4) 50.0(39.7–60.3)(2) 600–800(IV) 6 0.13 27.7(25.9–29.3)(3) 29.8(29.8–29.8)(1) 600(IV) 9 0.25 28.1(26.3–31.1)(4) 28.8(27.4–30.2)(2) 600–800(PO) Median 0.25 30.4(27.7–51.2) 31.2(28.8–50.0) a _{Cerebrospinalfluid.} b _Notdone

IV,intravenous;PO,oralroute.

a significant difference between the daily dose of 600 or 800mg/dandthedoseof400mg/d.Thelowestmedianserum levelwasobservedatthedoseof200mg/d,althoughthesmall numberofsamplespreventedreachingsignificancecompared tothehigherlevelsprovidedbydailydosesbetween400and 800mg.

IntheCSF,themedianlevelsoffluconazoleweresimilar withdailydosesbetween400and800mg/d,butexceededthe levels obtainedwith200mg/d. A good correlation between serumandCSFlevelswasdetectedin15blood-CSFpairsin patientsreceivingdailydosesbetween400and800mg(Fig.1). Cryptococcusisolatedfromthepatientsshowed susceptibil-itytofluconazole,asdemonstratedbyaMICbetween0.125 and2.0␮g/mL,MIC50=0.5␮g/mLandMIC90=2.0␮g/mL.

Nodifferences were verified in MIC distribution and of serumfluconazolelevelsinpatientswithcryptococcosiswho survivedcomparedtothosewithanon-favorableoutcome. CSFdruglevelsofthedeath/refractorygroupwerehigherthan levelsofthesurvivalgroup(Table2).

Discussion

Themostrelevantresultofthepresentstudywasthat flucona-zoleconcentrationsreachedinbloodandCSFaresufficient

70 60 50 40 30 20 10 0 0 10 20 30 40 R2 _{= 0,4423} Serum (µg/mL) CSF ( µ g/mL) 50 60 70

Fig.1–Correlationbetweenserumandcerebrospinalfluid fluconazolelevelsin10patients(15pairsofsamples)with cryptococcalmeningitisandAIDS.

toinhibitCryptococcusspp.isolatedfromthepatientsofthis series.Fluconazoledoseshigherthan200mg/dledtohigher levels in CSF, which were safer regarding their antifungal actionincasesofmeningealcryptococcosis.

Thepharmacokineticsoffluconazoleislinearand dose-dependent, and higher daily doses of the drug have

been shown to result in longer survival of patients with candidiasis.12 _{Thebetterpharmacokineticsparameter}

asso-ciatedwithfluconazoleefficacyistheareaunderthecurve of fluconazole blood concentration (AUC) divided by MIC (AUC/MIC)which must be≥100 forCandidaspecies.13 _AUC

near 386/L/h can be obtained with 400mg/day of flucona-zoleandthisdoseissufficienttocontrol99%ofCandidaspp. infections whose MIC is ≤2␮g/mL.14 _{In respect to}

crypto-coccosis,it ismorecomplicated todefine pharmacokinetic parameters for better outcomes because of several factors including acommonlymeningeal infection and anon-rare cerebralinvolvement.Anexperimentalstudywithfour Cryp-tococcusisolatesestimatedfluconazolemean AUC/MIC=389 forafungistaticeffect.15_{Inamurinemodelofcryptococcosis,}

fluconazolecerebrallevelswerelowerthanbloodlevelswhich couldbelowerthantheMICforCryptococcusspp.15,16

Flucona-zole doses and respective blood and CSF levels have been associatedwithneurocryptococcosiscasesoutcome.Adose ≥1200mg/daywasrecommendedifthistriazoleisemployed inmonotherapy.3

Theserumlevelsoffluconazolemeasuredinthepresent study are comparable to those reported in other studies using daily doses of 200–400mg13,17 _{or of 800mg.}18,19 _In

patientswithcryptococcosistreatedwithdailydosesof800 and1000mg/daythemeanlevelsofserumfluconazolewere 37.0␮g/mL and 42.5␮g/mL,20 _{respectively. A mean trough}

serum concentration of 5.6 (range: 0.11–18.0) ␮g/mL was verified in hematologic patients using 200mg/day of this triazole.21 _{There was nosignificant difference betweenthe}

levelsobtainedwhenthedrugwasadministeredbyoralor intravenousroute(datanotshown).

Thelevelsoffluconazoledetectedhereinthe CSFwere closetothoseobservedinotherstudiesonpatientswith cryp-tococcosisand HIVco-infection,whose meanCSF levelsof thedrugreached25.1␮g/mL,32.7␮g/mL,and36.3␮g/mLwith dailydosesof400mg,800mg,and1000mg,respectively.19,20

ThelevelsoffluconazoleintheCSFandinbloodshoweda goodcorrelation,inagreementwiththeconceptthatthisdrug hassimilarconcentrationsinthetwofluids.22_BloodandCSF

fluconazoleconcentrationsobservedinthisstudyare appar-entlynotrelatedtothebadoutcomeofsomepatients.

All Cryptococcus spp. isolates were susceptible to flu-conazolebasedonthe epidemiologicalcut-offestimatedat 8␮g/mL.23_{Resistanceornon-susceptibilityofCryptococcusspp.}

tofluconazolemaybethereasonfortreatmentfailurein cryp-tococcosis,especiallyifonlythisantifungalagentisusedfor treatment.2 _{Refractory/relapsing cryptococcal infection has}

beenassociatedwithCryptococcusneoformansnotbeing sus-ceptibletofluconazole.24_{Fluconazoleatthedoseof800mg/d}

oralternativetreatmentwithvoriconazolewasnecessaryto maintainsurvivalofpatientsinfectedwithCryptococcusspp. withaMICabove8␮g/mLforthisdrug.7 _{Aslowordelayed}

recoveryofpatientswithneurocryptococcosisshouldindicate aCryptococcusspp.susceptibilitytestandthemonitoringof fluconazolelevelsintheCSF.25,26

Inthisstudyitwasnotpossibletoestimateseveral phar-macokineticparametersoffluconazole.However,itconfirmed thatlevelsofthistriazolicweredirectlyrelatedtothedaily dosereceivedbypatients,whichmustbecarefullymanaged, particularlyinneurocryptococcosiscases.Anotherlimitation

ofthestudyreferstothehigherpercentageofpatientsalready inconsolidationphaseoftreatmentinthegroupofsurvivors incomparisontothedeath/refractorycryptococcosisgroup, impairingtheanalysisofthefluconazolelevelsaccordingto outcome. Interaction of fluconazole with other drugs pre-scribed to meningeal cryptococcosis cases may occur, but amphotericinBandantiretroviralsinparticulardonotmodify fluconazolelevels.27

Inconclusion,fluconazolereachedsufficientbloodandCSF levelstoactagainstmostCryptococcusspp.thatcause menin-gitisinAIDSpatients,eventhoughitwasadministeredunder non-standardizedclinicalconditionsandthefluconazoleused wasnottheoriginalbrandusedtoevaluate pharmacokine-ticsininitialstudies.Dailydosesof400mgorhigherprovided moreappropriateCSFconcentrationsfortreatingCryptococcus spp.,particularlyisolateswithlowersusceptibilityto flucona-zole.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

LASchiavereceivedascholarshipfromtheSãoPauloResearch Foundation(FAPESP)relatedtoaresearchprojectonthe deter-mination of fluconazole levels (grant 2010/51030-4). The FoundationofSupporttoTeaching,ResearchandAssistance ofHospitaldasClínicas,RibeirãoPretoMedicalSchool(FAEPA) providedfinancialsupportforthisstudy.WethankJohn Car-penter,RibeirãoPreto,SP,Brazil,fortheEnglishrevision.

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1.Lass-FlörlC.Triazoleantifungalagentsininvasivefungal infections:acomparativereview.Drugs.2011;71:2405–19. 2.BicanicT,HarrisonT,NiepiekloA,DyakopuW,MeintjesG.

SymptomaticrelapseofHIV–associatedcryptococcal meningitisafterinitialfluconazolemonotherapy.Theroleof fluconazoleresistanceandimmunereconstitution.Clin InfectDis.2006;43:1069–73.

3.PerfectJR,DismukesWE,DromerF,etal.Clinicalpractice guidelinesforthemanagementofcryptococcaldisease:2010 updatebytheInfectiousDiseasesSocietyofAmerica.Clin InfectDis.2010;50:291–322.

4.JarvisJN,BicanicT,LoyseA,etal.Determinantsofmortality inacombinedcohortof501patientswithHIV–associated cryptococcalmeningitis:implicationsforimproving outcomes.ClinInfectDis.2014;58:736–45.

5.RubioFG,ZananJR,deAlmeidaMTG,deGongoraDVN. EfficacyofamphotericinBinafatemulsionforthetreatment ofcryptococcalmeningitisinAIDSpatients.BrazJInfectDis. 2007;11:203–7.

6.NascimentoE,VitaliLH,TonaniL,KressMR,TakayanaguiOM, MartinezR.Refractoryand/orrelapsingcryptococcosis associatedwithAIDS:clinicalfeatures,genotypeand virulencefactorsofCryptococcusspp.isolates.AmJTropMed Hyg.2016;94:975–81.

7.NasriH,KabbaniS,BouAlwanM,etal.Retrospectivestudyof cryptococcalmeningitiswithelevatedminimuminibitory

concentrationtofluconazoleinimmunocompromised patients.OpenForumInfectDis.2016;3,ofw076.

8. TuckerRM,WilliamPL,ArathoonEG,etal.Pharmacokinetics offluconazoleincerebrospinalfluidandseruminhuman coccidioidalmeningitis.AntimicrobAgentsChemother. 1988;32:369–73.

9. GonzalezJM,RodriguezCA,ZuluagaAF,AgudeloM,VesgaO. Demonstrationoftherapeuticequivalenceoffluconazole genericproductsintheneutropenicmousemodelof disseminatedcandidiasis.PLOSONE.2015;10:e0141872. 10.MisoguchiT,HirataK,KobayashiS,ChikumaT.Monitoringof

fluconazoleinserumofpatientsundergoing hemodiafiltrationbysolid-phaseextractionand

high-performanceliquidchromatographywithultraviolet detection.Pharmazie.2012;67:765–7.

11.ClinicalandLaboratoryStandardsInstitute(CLSI).M27-A2. Referencemethodforbrothdilutionantifungalsusceptibility testingofyeasts.Thirded.Wayne,PA,USA:CSLI;2008. 12.PaiMP,TurpinRS,GareyKW.Associationoffluconazolearea

undertheconcentrationtimecurve/MICanddose/MICratios withmortalityinnonneutropenicpatientswithcandidemia. AntimicrobAgentsChemother.2007;51:35–9.

13.SinnollaredyMG,RobertsJA,LipmanJ,etal.Pharmacokinetic variabilityandexposuresoffluconazole,anidulafungin,and caspofungininintensivecareunitpatients:datafrom multinationalDefiningAntibioticLevelsinIntensivecareunit (DALI)patientsstudy.CritCare.2015;19:33.

14.Rodríguez-TudelaJL,AlmiranteB,Rodríguez-PardoD,etal. CorrelationoftheMICanddose/MICratiooffluconazoleto thetherapeuticresponseofpatientswithmucosal

candidiasisandcandidemia.AntimicrobAgentsChemother. 2007:3599–604.

15.SudanA,LivermoreJ,HowardSJ,etal.Pharmacokineticsand pharmacodynamicsoffluconazoleforcryptococcal

meningoencephalitis:implicationsforantifungaltherapyand invitrosusceptibilitybreakpoints.AntimicrobAgents Chemother.2013;57:2793–800.

16.SantosJR,CésarIC,CostaMC,etal.

Pharmacokinetics/pharmacodynamiccorrelationsof fluconazoleinmurinemodelofcryptococcosis.EurJPharm Sci.2016;92:235–43.

17.BuijkSLCE,GyssensIC,MoutonJW,VerbrughHA,TouwDJ, BruiningHA.Pharmacokineticsofsequentialintravenousand

enteralfluconazoleincriticallyillsurgicalpatientswith invasivemycosesandcompromisedgastro-intestinal function.IntensiveCareMed.2001;27:115–21.

18.SobueS,TanK,LaytonG,LeclercV,WeilA.Pharmacokinetics offosfluconazoleandfluconazolefollowingmultiple intravenousadministrationoffosfluconazoleinhealthymale volunteers.BrJClinPharmacol.2004;57:773–84.

19.ManosuthiW,ChetchotisakdP,NolenTL,etal.Monitoring andimpactoffluconazoleserumandcerebrospinalfluid concentrationinHIVassociatedcryptococcalmeningitis infectedpatients.HIVMed.2010;11:276–81.

20.MenichettiF,FiorioM,TortiA,etal.High-dosefluconazole therapyforcryptococcalmeningitisinpatientswithAIDS. ClinInfectDis.1996;22:838–40.

21.CeesayMM,CouchmanL,SmithM,WadeJ,FlanaganRJ, PagliucaA.Triazoleantifungalsusedforprophylaxisand treatmentofinvasivefungaldiseaseinadulthaematology patients:troughserumconcentrationsinrelationtooutcome. MedMycol.2016;54:691–8.

22.NauR,SörgelF,ElfertH.Penetrationofdrugsthroughthe blood-cerebrospinalfluid/blood–brainbarrierfortreatmentof centralnervousinfections.ClinMicrobiolRev.2010;23:858–83. 23.Espinel-IngroffA,AllerAI,CantonE,etal.Cryptococcus

neoformans–Cryptococcusgattiispeciescomplex:an internationalstudyofwild-typesusceptibilityendpoint distributionsandepidemiologicalcutoffvaluesfor fluconazole,itraconazole,posaconazole,andvoriconazole. AntimicrobAgChemother.2012;56:5898–906.

24.deCarvalhoSantanaR,SchiaveLA,DosSantosQuaglioAS,de GaitaniCM,MartinezR.Fluconazolenon-susceptible Cryptococcusneoformans,relapsing/refractorycryptococcosis andlong-termuseofliposomalamphotericinBinanAIDS patient.Mycopathologia.2017,

http://dx.doi.org/10.1007/s11046-017-0165-1.

25.PeaF.Plasmapharmacokineticsofantimicrobialagentsin criticallyillpatients.CurrClinPharmacol.2013;8:5–12. 26.ShojiH,TakumaT,OhbayashiH,YoshidaK,YamamotoT,

NikiY.Measurementofantifungaldruglevelsin

cerebrospinalfluidforcryptococcalmeningoencephalitis.J InfectChemother.2012;18:775–9.

27.VadlapatlaRK,PatelM,PaturiDK,PalD,MitraAK.Clinically relevantdrug-druginteractionsbetweenantiretroviralsand antifungals.ExpertOpinDrugMetabToxicol.2014;10:561–80.