Serum markers as an aid in the diagnosis of pulmonary fungal infections in AIDS patients

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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

Serum

markers

as

an

aid

in

the

diagnosis

of

pulmonary

fungal

infections

in

AIDS

patients

Ana

Isabela

Morsch

Passos

a

_,

_Rachel

_Polo

_Dertkigil

b

_,

_Marcelo

_de

_Carvalho

_Ramos

a

_,

Ariane

Fidelis

Busso-Lopes

d

_,

_Cibele

_Tararan

a

_,

_Erivan

_Olinda

_Ribeiro

a

_,

Angélica

Zaninelli

Schreiber

c

_,

_Plinio

_Trabasso

a

_,

_Mariangela

_Ribeiro

_Resende

a

_,

Maria

Luiza

Moretti

a,∗

a_Universidade_de_Campinas,_Faculdade_de_Ciências_Médicas,_Departamento_de_Medicina_Interna,_Campinas,_SP,_Brazil b_Universidade_de_Campinas,_Faculdade_de_Ciências_Médicas,_Departamento_de_Radiologia,_Campinas,_SP,_Brazil c_Universidade_de_Campinas,_Faculdade_de_Ciências_Médicas,_Departamento_de_Patologia_Clínica,_Campinas,_SP,_Brazil d_Laboratório_Nacional_de_Biociências_(LNBio),_Campinas,_SP,_Brazil

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t

i

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Articlehistory: Received9March2017 Accepted9July2017 Availableonline29July2017

Keywords: Pulmonaryinfection HIV/AIDS (1-3)-␤-D-Glugan LDH LAMP Pneumocystosis Fungalinfection

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b

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Introduction:TheetiologyofpulmonaryinfectionsinHIVpatientsisdeterminedbyseveral variablesincludinggeographicregionandavailabilityofantiretroviraltherapy.

Materialsandmethods: Across-sectionalprospectivestudywasconductedfrom2012to2016 toevaluatetheoccurrenceofpulmonaryfungalinfectioninHIV-patientshospitalizeddueto pulmonaryinfections.Patients’serumsweretestedfor(1–3)-␤-D-Glugan,galactomannan, andlactatedehydrogenase.Theassociationamongthevariableswasanalyzedbyunivariate andmultivariateregressionanalysis.

Results:60 patients were included in the study. The patients were classiﬁed in three groups:Pneumocystisjirovecii pneumonia (19patients), community-acquired pneumonia (18patients),andotherinfections(23patients).Theoverallmortalitywas13.3%.Thetime sincediagnosisof HIVinfection wasshorterin thepneumocystosis group (4.94 years; p=0.001)than forthe other twogroups ofpatients. The multivariateanalysis showed thathigher(1-3)-␤-D-Glucanlevel(mean:241pg/mL)andlactatedehydrogenase (mean: 762U/L)wereassociatedwiththediagnosisofpneumocystosis.Pneumocystosiswasthe aids-deﬁningillnessin11outof16newlydiagnosedHIV-infectedpatients.

Conclusion:Intheeraofantiretroviraltherapy,PJPwasstillthemostprevalentpulmonary infectionand(1-3)-␤-D-Glucanandlactatedehydrogenasemaybesuitablemarkerstohelp diagnosingpneumocystosisinourHIVpopulation.

∗ _{Corresponding}_author.

E-mailaddresses:moretti.luiza@gmail.com,mlmoretti@hc.unicamp.br(M.L.Moretti). http://dx.doi.org/10.1016/j.bjid.2017.07.002

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Introduction

The epidemiology of pulmonary infections varies

accord-ing to geographicalregions and to whether the infection

occurredbeforeorafteractiveantiretroviraltherapy(ART)

availability. In some low-income countries, tuberculosis

andpneumocystosisarestillthemostfrequentpulmonary

complicationsinHIV/AIDSpatients.1_Conversely,_in_higher

resources settings, the frequency of pulmonary diseases

associatedwithAIDShasdecreasedduetothebeneﬁtsofthe

ART.2–4_{Hospitalization}_due_to_chronic_obstructive_respiratory

disease,lungcancer,andbacterialpneumoniahasbecome

more frequentthan pulmonaryinfections due to

Pneumo-cystisjirovecii,tuberculosis,andcytomegalovirus.5,6_However,

lowerrespiratorytractinfections(LRTI)arestill25-foldmore

commonin the HIV populationcompared to the general

communitycausinganestimatenumberof20–25episodes

per100hospitalizationsworldwide.6,7

FungalinfectionsinHIV-infectedpatientsareneglected

diseases,predominantlyincountrieswithlimitedresources,

andrepresentasigniﬁcantcauseofpulmonaryinfections.

TheburdenofHIV-relatedmycosisworldwideisestimated

toaccount,peryear,formorethan950,000casesof

crypto-coccosis,400,000casesofPJP,and300,000ofdisseminated

histoplasmosis.8

Themajorityofthediagnosesofpulmonarydiseasesare

basedonclinicalsymptomsandX-rayﬁndingsresultingin

patientsbeingtreatedempirically.Identiﬁcationofthe

eti-ologicagentisa complex task requiring thecombination

ofmicrobiologicexams,serologicmarkers,molecular

tech-niques,andinvasiveprocedures, such aslungbiopsyand

bronchoalveolar lavage (BAL). New molecular techniques

havebeenstudiedtoimprovethediagnosisofpulmonary

inﬁltrates in the HIV-infected population. Loop-mediated

isothermal ampliﬁcation (LAMP) has been evaluated for the

detection of Pneumocystis DNA in respiratory specimens

and could serve as a diagnostic tool.9 _Serum _(1-3)-

␤-D-Glugan(BG)hasbeenapromisingnon-culturemethodfor

the diagnosis of some fungal infections, including,

Can-didaspp.,Fusarium,andP.jirovecii.Recentdatahaveshown

that serum BG could be correlated with the diagnosis of

PJP10–12_with_a_sensitivity_range_of_90–100%_and_speciﬁcity_of

65–100%.13–15

Inlow/middleincomecountriesthereisanurgentneed

for prospective studies to clarify the infectiouscauses of

pulmonary diseasesthat lead to hospitalizations in AIDS

patients.In this scenario,the properidentiﬁcation of the

causativeagentswillindicateabettertherapeuticapproach

andimprovetheunderstandingoftheepidemiologyof

pul-monary affections responsible for hospitalizations in our

country.

Theaimofthisstudywastoevaluatetheoccurrenceof

pulmonaryfungalinfectionsandtheroleofserummarkers

inHIV-infectedpatientshospitalizedwithacuterespiratory

symptoms,inatertiarycarereferralhospitalinCampinas,

SaoPaulo,Brazil.

Materials

and

methods

Studypopulation

Weconductedaprospectivecross-sectionalstudy,from2012 to2016,attheHospitaldasClinicasoftheUniversityof Cam-pinas (UNICAMP), Sao Paulo,Brazil, whichis the reference hospitalformorethansixmillioninhabitants.

TheEthicalCommitteeapprovedthisstudy(No.8876/2012) andallpatientssignedtheinformedconsentform.The inclu-sioncriteriaincludedHIVinfection,ageover18yearsold,and hospitalization due tosymptoms oflower respiratory tract infection. Exclusion criteria includedpregnant women and patientswithnosocomialpulmonaryinfections.Atthetime ofhospitalization,aphysiotherapist(AIPM)performeda pul-monaryexaminationinallpatientsandcollectedsputumand orallavageforthemoleculardiagnosisofP.jirovecii.A radi-ologist assessed all chest radiographies and tomographies. For theassessmentofthepneumoniaseverity,thepatients wereclassiﬁedusingCURB-65score.16_At_the_day_of_admission,

thefollowingdatawereabstractedfromthepatients’records: age, gender, duration of HIV infection (in years), duration of hospitalization (in days), outcome,previous opportunis-ticinfections,hemoglobinlevel,hematocrit,totalleukocytes, serumcreatinine,serumurea,CD4Tcellcount,andHIVviral loadinthelasttwomonths.Inaddition,serumcryptococcal antigen, serologyforparacoccidioidomycosis, blood culture forbacteriaandfungi,cultureformycobacteriaandfungiin sputum,serumLDH,BGandGM,LAMPofrespiratory spec-imens(sputum,BAL)andorallavage(OL)forP.jiroveciiwere alsotested.

TheclinicaldiagnosisofpneumoniaduetoP.jiroveciiwas establishedaccordingtoWHOclinicalcriteriaofcase deﬁni-tionforHIV-relatedopportunisticdiseases,17_and_a_chest_X-ray

showingbilateral interstitialinfiltratesor achest tomogra-physhowingalterationscompatiblewithPJP(bilateralpatchy groundgrassopacitywithacentralperihilarpredominance). A definitive diagnosis was based on the identification of P. jirovecii incytology or immunofluorescent microscopy of inducedsputumorBALorhistologyoflungtissue.

Community-acquiredpneumonia(CAP)wasdeﬁnedasthe presenceofcoughtogetherwithoneormoreofthesymptoms: chestpain,dyspnea,presenceofnewpulmonaryinﬁltrateon chestradiography.18_Criteria_for_diagnosis_of_CAP_and_LRTI_due

tobacteria,fungiotherthanP.jirovecii,orparasiteswerebased ontheidentiﬁcationoftheetiologicagentinculturesofblood andrespiratorysecretions.

BGandGMinserumandinBALspecimen

Serumand BAL sampleswere tested forGMand BG. Con-centrations of GM were determined by using the Platelia Aspergillus Ag assay (Bio-Rad, Marnes-la-Coquette, France), accordingtothemanufacturer’sinstructions.AnOD≥0.5was consideredasapositiveresult.BGlevelsweredeterminedby theFungitell®assay(AssociatesofCapeCod,Inc.,CapeCod, MA,USA),accordingtothemanufacturer’srecommendations. BGlevels≥80pg/mLwereconsideredaspositiveresults.

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60 patients included

Serum markers Imaging exams LAMP for PJ Cultures

Serum beta glucan (n=55) Serum Galactomannan (n=56) Serology for paracoccidioidomycosis (n=40) Cryptococcal antigen (n=42) Chest radiography (n=60) Chest tomography (n=34) In blood (n=37) In sputum for Mycobacterium (n=42) In sputum for In sputum for fungi (n=45) fungi (n=44) 67 patients screened Patients excluded 1 endocarditis

4 hospital acquired pneumonia 2 refused to participate

Serologies Direct microbiology

LDH (n=55) In sputum (n=21) In oral lavage (n=19) In BAL (n=2) In sputum for Mycobacterium (n=48)

Fig.1–Patientdispositionbyaccordingtoresultsoftheexamsperformed.

LAMPforP.jirovecii

Sputum,orallavage,andBALsamplesweretestedforP.jirovecii by LAMP. DNA extraction procedures strictly followed the instructionsdeﬁnedbythemanufacturer(EikenChemicalCo., Tokyo, Japan)using theoligonucleotide primersspeciﬁcfor P.jirovecii.19

Statisticalanalysis

Categoricalvariableswereanalyzedusingchi-square,Fisher, and Mid-P exact tests, and for continuous variables t-test, ANOVA and Mann–Whitney/Wilcoxon test were used. A p-value<0.05wasconsideredstatisticallysigniﬁcant.The mul-tivariatestepwiselogisticregressionanalysiswasperformed todeterminethefactorsindependentlyassociatedwithfungal infection.Thefollowingprogramswereused:Epiinfov.7 (Cen-tersforDiseaseControlandPrevention,Atlanta,USA),Open SourceEpidemiologicStatisticsforPublicHealth,version3.03a updated2015/05/04,20_and_SAS_System_for_Windows_(version

9.1.3,SASInstitute,Cary,NorthCarolina).

Results

Atotalof67HIV-infectedpatientswereincluded.Fig.1shows thedistributionofpatientsaccordingtoresultsoftheexams performed(imaging,biomarkers,LAMP,serologies,and micro-biologicexams).

Thepatientswereclassifiedintothreegroups:(a)PJP(19 cases):includedpatientswithPJPandpatientswithdiagnosis ofPJPplusanotherpulmonaryinfection/disease;(b)patients withCAP(18cases);and(c)otherdiagnosis(23casesoflower respiratoryinfection:ninecasesoftuberculosis,fivecasesof histoplasmosis,twocasesofnocardiosis,onecaseof cryp-tococcosis, one case of disseminated strongyloidiasis, one caseofpulmonaryembolism,andthreeundiagnosedcases). PJPandCAPwere themostfrequentdiagnosisrepresenting 31.6%and 30%,respectively. Tuberculosiswas diagnosedin five (8.3%) patients. Seven patients with CAP had positive

blood culturesforbacteria (one Acinetobacterbaumanii, one Kokuria micrococcus,andﬁve Streptococcuspneumoniae). Sero-logyforparacoccidioidomycosisanddetectionofcryptococcal antigeninserumwereassessedin40and42patients, respec-tively.Onlythepatient,withpulmonarycryptococcosis,had a positive cryptococcal antigen. All serologies for paracoc-cidioidomycosisturnedoutnegative.Thetwopatientswith histoplasmosishadculturesofskinbiopsiespositivefor Histo-plasma.

Directsputumsmearmicroscopyandsputumculturefor mycobacteria wereexamined in48and 42patients respec-tively,andforfungiin44and45patients,respectively.Direct sputumsmearwaspositiveforfungiinsevencases(ﬁveyeasts and ﬁlamentous fungi,one Strongyloides stercoralis and one non-pathogenicyeast)andforMycobacteriuminfourpatients. Sputumcultureforfungiwaspositivein24 cases(18 non-pathogenicyeast,threeCandidaalbicans,oneActinomycesspp., oneCryptococcusneoformansandoneHistoplasmacapsulatum) andforMycobacteriuminfourcases(twoMycobacterium tuber-culosisandtwoMycobacteriumavium).

The mean values of hemoglobin and hematocrit were below the referencevalues (Table1). Forty-onepercent(25 patients) werefemale. Themedian timesinceHIV diagno-sis was 10 years; however, 22.8% of the patients had less than oneyear. HIV infection was first diagnosedat hospi-talization in16 (26.6%)patients; PJP wasthe AIDS-defining illnessin11patients;themeanageofpatientswas43.4years (female:43.8;male:43.1);42(70%)patientshadlessthan250 cell/mm3_CD4_cell_count_and_only₁₄_(23.3%)_patients_had_HIV viral load <50copies/mL.Themeans ofHIVviral load and CD4Tcellcountformales(760,261copies/mL;214cells/mm3₎ and females (291,703 copies/mL; 174 cells/mm3₎ _were _not significantly different (p=0.43 and p=0.56, respectively) (Table1).

Meantimeofhospitalizationwas19days.The length-of-stay inhospitalwas notassociated withoutcome (p=0.21; death:27.9days;survival:16.3days).Nine(15%)patientsdied duringhospitalization(4PJP,3CAP,1tuberculosis,and1 dis-seminatedstrongyloidiasis).Thefollowingvariableswerenot associatedwiththeoutcome:age(p=0.40),gender(p=0.46),

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Table1–Laboratorydataof60HIV-infectedpatientshospitalizedwithpulmonaryinfections.

Exams Mean(±standarddeviation) p-Value Median

Female(n=25) Male(n=35) Female Male

Leukocytes,cells/mL 8144(±5578) 10,144(±11,402) 6600 7210 Hemoglobin,g/dL 9.66(±2) 11.1(±6.06) 9.8 11.3 Hematocrit,% 29.54(±5.9) 33.3(±6.88) 28.9 35.7 Platelets,cells/mL 237,920(±111,476) 244,428(±134,035) 237,000 213,000 Creatinine,mg/dL 1.49(±2.45) 1.8(4.4) 0.78 0.94 Urea,mg/dL 47.25(±58) 49.6(±39) 32.5 39 Lactatedehydrogenase(LDH)U/L 457(±207) 589(±392) 0.39 440 442

Viralload,copies/mL 291,703(±591,325) 760,261(±2,139,278) 0.43 52,804 72,391

CD4+lymphocytes,cell/mm3 ₁₇₄_(±₂₀₉₎ ₂₁₄_(±₂₉₇₎ _0.56 ₁₃₉ ₆₄

Referencevalues.Creatinine:adults–men<1.20mg/dL; women<0.90mg/dL; urea:adult≤65years: 16.6–48.5mg/dLandadult>65years: <71mg/dL;LDH:adult≤65years:240–480U/L;leukocytes:adults–4000–10,000cell/mL;hemoglobin:adults–men14–18g/dL,women12–16g/dL; hematocrit:adults–men41–52%,women36–46%;platelets:adults–150,000–400,000cells/mL.

CD4Tcellcount(p=0.15),HIVviralload(0.37),LDH(0.09),BG (0.08),andtimesinceHIVdiagnosis(p=0.45).

GMwasanalyzedinserumof56patientsandinBALoftwo patients.OnepatienthadserumandBALpositives(PJP)and onepatienthadpositiveserumsample(CAP).AlltheotherGM testswerenegative.

Results of univariate analysis of the three groups of patientsaresummarizedinTable2.ThemeanvaluesofBG in the group of patients clinically diagnosed as P. jirovecci (19 patients; 240.8±185.7pg) was significantly higher than in patients with CAP(18 patients; 36.3±34.2pg;p<0.0001) and in those diagnosed withother infections (23 patients; 67.3±60.7pg;p<0.0001). Eightpatients notdiagnosed with PJPhadBG>80pg/mL(histoplasmosis:1;strongyloidiasis:1; tuberculosis: 1; cryptococcosis: 1; CAP: 2; undiagnosed: 2). ConsideringtheBGcut-offof>80pg/mL,thesensitivityand specificity of BG in the PJP patients were 0.90 and 0.80, respectively. Two patients withPJP had BG<80pg/mL.LDH wasalso significantlyhigher inthe group ofpatients with P. jirovecii (mean 762.47±433.18U/L) than in patients with CAP(379.5±99.9U/L),andotherdiagnosis (442.6±217.6U/L) (p=0.003). Patients with PJP had lower T CD4+lymphocyte count (p=0.001) and duration of time since HIV diagnosis (p=0.001).However,higherBGandLDHweretheonly indepen-dentvariablesassociatedwiththediagnosisofPJPinrelation tothediagnosisofCAPandotherinfections(Table3).

Eleven(68.7%)ofthe16patientswhowereﬁrstdiagnosed asHIVduringhospitalization hadPJP (ninepatients)orPJP associatedwithMycobacteriumnon-tuberculosis(twopatients) andofthe otherﬁve,twopatientshadhistoplasmosis,one cryptococcosis,onetuberculosis,andonepatienthadLRTI.

Tuberculosis(17cases),Pneumocystosis(16cases),Herpes zoster(13cases),cerebraltoxoplasmosis(10cases)and hepati-tisC(11cases)werethemostprevalentpreviousopportunistic infectionsreportedinourpatients.Sixpatientsincludedin thisstudyhadapreviousdiagnosisofPJP.Threepatientsoutof the17previouslydiagnosedwithtuberculosishadatreatment failureorrelapsed.

Thirty-ﬁvepatientshadatleastoneclinicalspecimen(BAL: 1,sputum:14;OL:13;sputum+OL:6;sputum+BAL:1)tested byLAMP.ThismolecularmethoddetectedP.jiroveciiDNA prod-uctinclinicalspecimenofﬁvepatients(1BAL;4sputum).All

patientswhoseLAMPturnedoutpositivehadchestX-rayor CTsuggestiveofPJPandBG≥341.55pg.LAMPwerepreformed in12patientswithPJPandonlyinﬁveitturnedoutpositive.

Discussion

Pulmonary infectionisoneofthemostfrequent complica-tion inpeople livingwithHIV/AIDS, remainingthe leading cause ofmorbidityandmortalityworldwide.7 _In_our_study,

pneumocystosis, CAP,tuberculosis,andLRTIwerethemost frequentcausesofhospitalization.Ofnote,fungalinfections represented36.6%(22patients)ofourcases.Pneumocystosis wasthemostfrequentAIDS-deﬁningopportunisticinfection representing 68.7% ofnewly diagnosed AIDS cases, under-scoringtheneedtoimproveearlydetectionofHIVinfection. Remarkably, tuberculosiswas AIDS-deﬁning illness inonly onepatient.

Systematicreviewshavedemonstratedthatopportunistic infections decreasedaftertheavailabilityofART,mostlyin high-income countries.21 _However,_there _is_a _lack_of

infor-mation on the epidemiology of opportunistic infections in low-andmiddle-incomecountries,includingBrazil.Ourstudy demonstratedthatopportunisticinfectionswerestill preva-lent. Astudy from San Francisco reported that PJP (39.1%) followedbyKaposisarcoma(20.1%)werethemostcommon AIDS-deﬁningdiseases.22_Grinsztejn_et_al.23_found

tuberculo-sisasthemostcommonAIDS-deﬁningopportunisticinfection in a Brazilian cohort compared to esophageal candidiasis amongUSHIV-infectedpatients.AnotherstudyfromRiode Janeiro,concludedthattuberculosiswasthe mostfrequent AIDS-deﬁning opportunistic illness,24 _whereas _in _a _recent

series of HIV-infected patients from a referral hospital in Brazil,pneumocystosiswasstilltheprevailingopportunistic infectionintheHIVgroup.25_The_difference_between_the_rates

ofopportunisticinfectionsfoundinourresultscomparedto otherstudiescouldbeexplainedbythecausesofpulmonary infectionswehavestudiedandnotallcausesof opportunis-ticinfections inAIDSpatients. Notably, mostinternational andnationalstudiesarefromsinglecenters.Althoughtheir resultscontributedtotheunderstandingofthelocal epidemi-ology,theconductofmulticenterstudieswillleadtobetter

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Table2–Univariateanalysisofthe(1-3)-␤-D-Glugan,LDH,HIVviralload,CD4Tcellcount,durationofHIVinfection,and outcomeofthethreegroupsofpatientsincludedinthestudywithpulmonaryinfections.

Variables Clinicaldiagnosis p-Value

PJP(n=19) CAP(n=18) Other(n=23) (1-3)-ˇ-D-Gluganinserum(pg/mL) Median 183 29.8 52.8 Mean(±SD) 240.8(±185.7) 36.3(±34.2) 67.3(±60.7) <0.0001 LDH(U/L) Median 761 419 441 Mean(±SD) 762.47(±433.18) 379.5(±99.9) 442.6(±217.6) 0.003

Viralload(copies/mL)

Median 62,609 3800 74,892

Mean(±SD) 801,171(±2,194,157) 149,525(±270,015) 482,859(±135,562) 0.30

LymphocyteTCD4+(cell/mm3₎

Median 40 230 97

Mean(±SD) 73(±107) 303(±267) 217(±312) 0.001

TimesincediagnosisofHIVinfection(years)

Median 0 15 11

Mean(±SD) 4.94(±6.83) 14(±7.67) 10.7(±8.32) 0.001

Outcome

Discharge 15 15 21 0.62

Death 4 3 2

Meantimeofhospitalization(indays) 22.9(±22.8) 13.4(±8.9) 16.1(±12.3) 0.31 PJP,Pneumocystisjiroveciipneumonia.PJPgroupincluded:P.jirovecii+communityacquiredpneumonia:4patients;P.jirovecii+Mycobacterium non-tuberculosis:2patients.CAP:communityacquiredpneumonia.Other:lowerrespiratoryinfection:9patients;tuberculosis:5patients; histo-plasmosis:2patients;cryptococcosis:1patient;disseminatedstrongyloidiasis:1patient;nocardiosis:1patient;pulmonaryembolism:1patient andundiagnosed:3patients.

Table3–Multivariatelogisticregressionanalysisa_of_variables_{independently}_associated_with_P._jirovecii_pneumonia,

community-acquiredpneumonia,andotherpulmonarydiseases.

Variable Group p-Value OR CI95%

(1-3)-␤-D-Glugan(pg/mL) PJPvs.Other 0.0267 1.015 1.002;1.028

(1-3)-␤-D-Glugan(pg/mL) CAPvs.Other 0.1028 0.986 0.970;1.003

(1-3)-␤-D-Glugan(pg/mL) PJPvs.CAP 0.0051 1.029 1.009;1.050

LDH(U/L) PJPvs.Other 0.0375 1.009 1.000;1.017

LDH(U/L) CAPvs.Other 0.3617 0.997 0.992;1.003

LDH(U/L) PJPvs.CAP 0.0154 1.011 1.002;1.021

PJP,P.jiroveciipneumonia;CAP,community-acquiredpneumonia;Other,otherpulmonarydiseases;LDH,lactatedehydrogenase;OR,OddsRatio; CI,conﬁdenceinterval.

a _As_the_analyzed_variables_were_continuous,_the_results_of_the_multivariate_logistic_regression_analysis_described_the_odds_ratio_values_as_the effectoftheincrementofeach“1”unitofBG(pg/mL)orLDL(U/L)levels,intheincreaseofthechanceofapatientbeinginthePJPgroup.

preventionand treatmentstrategies.One limitationofthis studywasthefactthatonly60AIDSpatientsmetthe inclu-sioncriteriaduringthestudyperiod.Thetimeframeofthe study,thelimitednumberofbedsintheinfectiousdiseases ward,andthelonglengthhospitalstaywereexternalfactors thatcontributedinreducingthesamplesizeofourstudy.

Clinicianssearchfornon-invasivemethods,suchasserum markers,asanaidtodiagnosepulmonaryinﬁltrates, partic-ularly in HIV/AIDS. BG values were considerably higher in patientsclinicallydiagnosedwithPJP and mightbeagood marker forpatients with AIDS and pulmonary symptoms. ThevisualizationoftheP. jiroveciiinpulmonaryspecimens isdifﬁcult,whereasthedosageofserumBGcouldbea reli-able marker in the diagnosis of PJP.10 _Considering _the _BG

cut-offvaluerecommendedbythemanufacturer(80pg/mL), we found a high sensitivity(0.90) and speciﬁcity (0.80). In two other studies, that included a higher number of HIV patientsthanourstudy,theauthorsfound,usingthesame cut-off,asensitivityandspeciﬁcityof0.91/0.64and0.98/0.39, respectively.10,26_One_potential_limitation_of_the_use_of_BG_in

clinicalpracticewithHIV-infectedpatientsmightbethe posi-tivityofthetestinotherinvasivemycosis,suchascandidiasis, fusariosis,aspergillosis,andhistoplasmosis.27,28

PreviousstudieshavefoundthathighlevelsofLDHwere associatedwithPJP,andthatLDHcouldbeaserummarker inthediagnosisofpneumocystosis.10,29,30_Our_data_showed

a signiﬁcant correlation between high levels of LDH and pneumocystosis(p=0.003)(Table2).Themultivariateanalysis

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showed that higher levels of BG and LDH were indepen-dentlyassociatedwithPJPwhencomparedwiththeresultsin patientswithCAPandotherpulmonaryconditions(Table3). Inthemultivariate logisticregression analysisdescribed in Table3theoddsratiosrepresenttheeffectoftheincrementof each“1”unitofBG(pg/mL)orLDL(U/L)levels,inthechance ofapatienthavingPJP, asthesevariables werecontinuous variables.31_Based_in_our_results,_the_combination_of_more_than

oneserummarkermaybehelpfulinthediagnosisofPJPand todiscriminatePJPfromotherpulmonarydiseases.

LAMPisarapidsimpleandcost-effectivemethodfor diag-nosingvariouspathogensincludingPneumocystis.9_LAMP_for_P.

jiroveciiwastestedintherespiratorysecretionsofourpatients andtheresultswerenotencouraging.Mostoftheclinical sam-plesfromourpatientsweresputumorOL,andperhapsifwe hadtestedBAL,wewouldhaveobtainedbetterresults. How-ever,inclinicalpractice,non-invasivediagnosticmethodsare preferredoverinvasiveprocedures,suchasBALorbiopsies.In ourseriesofcases,serumBGandLDHprovidedbetteraidfor thediagnosisofpneumocystosisthanLAMP.

Inconclusion,ourresultsshowedthatPJPstillhasahigher prevalenceoverotherpulmonaryconditionsinourgeographic area,and thatweneedtoimplementpromptandaccurate diagnostictestsforpulmonaryinfections.BGandLDHshowed to be suitable serum markers in the diagnosis of PJP and shouldbeimplementedinroutinecaretoassistourHIV/AIDS population.Moreover,mostofourpatientswere previously enrolled in a HIV/AIDS care center. Although, we have a well-establishedNationalprogramforHIV/AIDSinBrazil,the HIVcascade care isneglecting the early diagnosis,patient follow-up,inadditiontoinsufﬁcientpatientadherencetothe HIV/AIDSprogram. Thisstudy showedthat thereisaneed forbetterstrategiestoachieveearlydiagnosisofHIVinfection andthereforereducingopportunisticinfectionsintheinfected patients.

Conﬂicts

of

interest

Theauthorshavenoconﬂictsofinterest.

Acknowledgements

This project was supported by grants from the collabo-rative research project: Science and Technology Research Partnership for Sustainable Development, Japan (SATREPS) and University of Campinas, Brazil, No. 02P-29548-09 and Fundac¸ão de Amparo à Pesquisa do Estado de Sao Paulo, FAPESPNo.2012/51158-0.WearegratefultoMr.RogerTimothy RentfrowfortheEnglishproofreading.

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