new tools for the diagnosis of

New Tools for the Diagnosis ofPnemocystis pneumonia (PcP)

MMííriamriam J. J. ÁÁlvarezlvarez--MartMartííneznez

Hospital Hospital ClinicClinic, Barcelona (, Barcelona (SpainSpain))CRESIB (Barcelona CRESIB (Barcelona CenterCenter forfor InternationalInternational HealthHealth ResearchResearch))

UniversityUniversity ofof [email protected]@clinic.ub.es

without forgetting the old ones

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OUTLINEOUTLINE

1. 1. WhyWhy PcPPcP stillstill happeninghappening??

2. 2. PneumocystisPneumocystis changeschanges overover thethe timetime

3. 3. OldOld diagnosticdiagnostic toolstools in usein use

4. 4. NewNew diagnosticdiagnostic toolstools overviewoverview & & challengeschallenges

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AnnualAnnual IncidenceIncidence ofof firstfirst AIDSAIDS--definingdefining OIsOIs, 1994, 1994--20072007

Brooks et al, CID, 2009.

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HIV-Associated OpportunisticInfections-Going, Going,

But Not Gone.

BrooksBrooks et al, CID, 2009.et al, CID, 2009.

““OIs are here to stay...OIs are here to stay...””

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OpportunisticOpportunistic InfectionInfection

HostHost--Parasite Parasite InteractionInteraction

Banerjee et al., Ind J Med Res, 2005.

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WHY WHY OIsOIs STILL HAPPENING ?STILL HAPPENING ?

•• OIsOIs as HIV Debutas HIV Debut

•• PatientsPatients w/o w/o ProphylaxisProphylaxis //TreatmentTreatment

•• TreatmentTreatment FailureFailure

•• FailureFailure toto ReinitiateReinitiate ProphylaxisProphylaxis ( CD( CD44))

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ECDC/ WHO. AIDS/ HIV ECDC/ WHO. AIDS/ HIV SurvillanceSurvillance in in EuropeEurope, 2009. , 2009.

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82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 980

10

20

30

YEAR

NO. x

100

EXPO

SED

PATI

ETNS

xYEA

RIncidence of PcP in HIV-infected Patients

Hospital Clínic Barcelona 1984 -2008

HAART: Highly Active Antiretroviral Therapy (≥2NRTI plus ≥1PI/NNRTI)

99 00 02 04 06 08

PcPPcP PROPHYLAXISPROPHYLAXIS HAARTHAART

• PcP debut of HIV• Patients withoutprophylaxis/ treatment• Treatment failure

•• PcPPcP debut debut ofof HIVHIV•• PatientsPatients withoutwithoutprophylaxisprophylaxis/ / treatmenttreatment•• TreatmentTreatment failurefailure

??

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OUTLINEOUTLINE





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19081908

19101910

19131913--19171917

19421942

19601960

19811981

20082008

ChagasChagas, , TrypanosomaTrypanosoma cruzicruzi, , SchizotrypanumSchizotrypanum lewisilewisi

CariniCarini, , DelanDelanööee, , PneumocystisPneumocystis cariniicarinii

FirtsFirts studiesstudies in in animalsanimals

PlasmaticPlasmatic cellscells pneumonitispneumonitis

FirstFirst immunosuppressedimmunosuppressed patientspatients

PneumocystisPneumocystis discoversdiscovers AIDSAIDS

PneumocystisPneumocystis jiroveciijirovecii & XXI & XXI centurycenturyPcPPcP ((PPneumoneumoccystisystis PPneumonianeumonia))

Once Once uponupon thethe time...time...

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•• TrypanosomeTrypanosomeSchizotripanumSchizotripanum lewisilewisi

•• ProtozoanProtozoan differentdifferent toto TrypanosomesTrypanosomesTreatedTreated withwith antiprotozoanantiprotozoan drugsdrugs ((PentamidinePentamidine))

•• FungiFungidivisiondivision AscomycotaAscomycotaclassclass ArchiascomycesArchiascomycesgenusgenus PneumocystisPneumocystisspeciespecie PneumocystisPneumocystis jiroveciijirovecii-- No response No response toto antifungicalantifungical drugsdrugs-- No No growthgrowth in culturein culture

Peculiar Peculiar TaxonomyTaxonomy

•• CombinedCombined fungalfungal & & parasiticparasitic termstermsTrophozoiteTrophozoite = = TrophicTrophic formformPrecystPrecyst = = SporozoiteSporozoiteCystCyst = = AscusAscus oror bagbag ofof sporessporesIntracysticIntracystic bodiesbodies = = SporasSporas

•• AcronimAcronim PcPPcP ((PPneumoneumoccystisystis PPneumonianeumonia))•• P. P. cariniicarinii forfor speciesspecies affectingaffecting rodentsrodents•• StenoaxenicStenoaxenic organismorganism ((speciespecie--specificspecific))

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PathogenesisPathogenesis mechanismmechanism

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BB

CD 4

CD 8

LymphocytesLymphocytes

TrophozoitesTrophozoites

MacrophageMacrophage

Alveolar Alveolar cellscells typetype II

CystsCysts

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Alveolar Alveolar cellscells typetype II

•• AnatomicAnatomic--functionalfunctional changeschanges

-- PneumocytesPneumocytes typetype II II degenerationdegeneration

- Hipertrophy Pneumocytes type II

-- DecreasingDecreasing surfactantsurfactant

•• AlterationAlteration gas gas exchangeexchange

•• FoamingFoaming alveolar alveolar exudateexudate

PNEUMONIA

PPNNEEUUMMOONNIIAA

PneumocystisPneumocystis proliferationproliferation

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CLINICAL DiagnosisCLINICAL Diagnosis

•• FeverFever•• CoughCough•• DisneaDisnea•• LDHLDH•• PaoPao22•• (( PAo2PAo2 -- Pao2)Pao2)

ComplicationsComplications::•• AcuteAcute respiratoryrespiratory insuficiencyinsuficiency•• PneumotoraxPneumotorax••ChronicChronic pulmonarypulmonary diseasedisease

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OUTLINEOUTLINE





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• Type of Sample– , IS, pulmonar and transbronchial biopsy

• Optical microscopy– Stains:

ToluidineBlueGiemsa, Gram, Papanicolau.

– Inmunofluorescence• Electronic microscopy• Molecular biology techniques

– > Sensitivity & Specificity– Non invasive samples: OW– Assymptomatic carriers diagnostic

• Other techniques: plasma (S-adenosylmethionine)serum (ß-glucans)

PcPPcP LABLAB DIAGNOSISDIAGNOSIS

BALBAL

Silver Methenamine- Gomori,

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18BALBAL--SilverSilver MethenamineMethenamine stainedstained 100X100XBALBAL--SilverSilver MethenamineMethenamine stainedstained

((magnifiedmagnified) )

CourtesyCourtesy Dr. J MasDr. J Mas

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19BALBAL-- GiemsaGiemsa stainedstained 100X100XBALBAL-- GiemsaGiemsa stainedstained

((magnifiedmagnified) )

CourtesyCourtesy Dr. J MasDr. J Mas

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BALBAL-- GiemsaGiemsa stainedstained((magnifiedmagnified)) CourtesyCourtesy Dr. J MasDr. J Mas

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22ElectronicElectronic MicroscopyMicroscopy ((trophozoitestrophozoites attachedattached toto alveolar alveolar cellscells))

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23Cyst of Pneumocystis with spores inside

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OUTLINEOUTLINE





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25QuantitativeQuantitative rTrT--PCRPCR

nestednested--PCRPCR 335pb335pb

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SamplesSamples

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SensitivitySensitivity betweenbetween 62.5%62.5% andand 100%,100%, accordingaccording typetype ofofsamplesample andand storagestorage..

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QuantitativeQuantitative rTrT--PCR PCR showedshowed statisticallystatistically betterbetter specificity specificity (p=0.015 ) than nested(p=0.015 ) than nested--PCR, PCR,

reducing false positive percentage.reducing false positive percentage.

S: 94 %S: 94 %SpSp: 81%: 81%

S: 94%S: 94%SpSp: 96%: 96%

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31VPP= VPP= SensitivitySensitivity**PrevalencePrevalence//SensitivitySensitivity* * PrevalencePrevalence + (1+ (1--PrevalencePrevalence)* (1)* (1--SpecificitySpecificity))

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Para-aminobenzoic acid (PABA) Dihydropteroate synthase Inhibitory action of (DHPS) Sulfametoxazole (SMX) and Dapsone Dihydropteroate Dihydrofolate Dihydrofolate reductase Inhibitory action of (DHFR) Trimethoprim Tetrahydrofolate (TMP) Nucleic acids

MODE OF ACTIONMODE OF ACTION

OF FOLATE INHIBITORSOF FOLATE INHIBITORS

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33KovacsKovacs et al. , 2001.et al. , 2001.

DHPSDHPS PneumocystisPneumocystis jiroveciijirovecii

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1.1. IncreasedIncreased riskrisk ofof mortalitymortality in in followingfollowing threethreemonthsmonths afterafter diagnosisdiagnosis ((HelwengHelweng--LarsenLarsen et al.)et al.)

2. Sulfa2. Sulfa--drugsdrugs prophylaxisprophylaxis isis associatedassociated totopresencepresence ofof DHPS DHPS mutationsmutations butbut outcomeoutcome isisnotnot affectedaffected ((KazanjianKazanjian & & MeshnickMeshnick))

3.3. SulfaSulfa--drugsdrugs prophylaxisprophylaxis isis anan independentindependentfactorfactor forfor DHPS DHPS mutationsmutations ((NavinNavin et al.)et al.)

Role Role ofof DHPS Gene DHPS Gene MutationsMutations in in PcPPcP OutcomeOutcome

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KovacsKovacs et al. , 2001.et al. , 2001.

•• PrevalencePrevalence ofof PneumocystisPneumocystis DHPS DHPS gene in gene in SpainSpain in cin c--ART era: ART era: 3.7%3.7%

•• Global Global mortalitymortality 15%, 15%, risingrising toto 80% 80% in in patientspatients withwith mechanicalmechanicalventilationventilation..

•• PresencePresence ofof DHPS DHPS mutationsmutations werewerenotnot associatedassociated toto worseworse outcomeoutcome. .

•• TMPTMP--SMX SMX isis effectiveeffective atattherapeuticatherapeutica dosesdoses..

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••DHPS DHPS mutationsmutations more more frequentfrequent in in prepre--cARTcART (1989(1989--1995) era,1995) era,thanthan in cin c--ART era (2001ART era (2001--2004), 2004), 33% vs. 5.5%,33% vs. 5.5%, respectivelyrespectively..

•• Sulfa Sulfa ––drugs prophyllaxisdrugs prophyllaxis•• PrePre cc--ART ART periodperiod•• HMSHMS

IncreaseIncrease riskrisk ofof mutationsmutations

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

89 90 91 92 93 94 95 2001 2002 2003 2004

DHPS GENOTYPES BY YEAR

1 2 3 4DHPS genotypes

YEAR

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FutureFuture::NonNon--invasiveinvasive TestTest forfor PcPPcP Diagnosis Diagnosis

•• Oral Oral oror oropharingealoropharingeal washwash specimensspecimens+ PCR + PCR assaysassays..–– S: up S: up toto 88%; 88%; SpSp: up : up toto 90%90%

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EfficiencyEfficiency ofof Oral Oral WashesWashes & BAL in & BAL in PcPPcP Diagnosis by realDiagnosis by real--time PCRtime PCR

MMííriamriam J. J. ÁÁlvarezlvarez--MartMartííneznez et al.et al. SEIMCSEIMC--2010.2010.

BAL BAL OWOW

sensitivity(Ssensitivity(S)) 100%100% 53%53%specificityspecificity (E) (E) 78%78% 100%100%positive positive predictivepredictive valuevalue (PPV)(PPV) 88%88% 100%100%negativenegative predictivepredictive valuevalue (NPV)(NPV) 100% 100% 56%56%

rTrT--PCR in BALPCR in BALa) a) ExcelentExcelent sensitivitysensitivity accordingaccording toto microscopymicroscopy..b)b) PcPPcP diagnosis in cases diagnosis in cases ofof lowlow numbernumber ofof cystcyst indetectable by indetectable by microscopymicroscopy..c)c) AllowsAllows PneumocystisPneumocystis quantificationquantification..

rTrT--PCR in Oral PCR in Oral WashesWashesa)a) VeryVery goodgood specificityspecificity, , furtherfurther researchresearch isis neededneeded toto improveimprove sensitivitysensitivity andand

considerconsider itit as as anan aternativeaternative diagnosisticdiagnosistic methodmethod..

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FutureFuture::NonNon--invasiveinvasive TestTest forfor PcPPcP Diagnosis Diagnosis

•• Plasma SPlasma S--adenosylmethionineadenosylmethionine (SAM)(SAM)–– DeplectionDeplection ((SkellySkelly et alet al., CID, 2008)., CID, 2008)

((distinguish between persons w PcP & those w non-PcPpneumonia)

•• SerumSerum (1(1--3)3)--betabeta-- DD-- glucanglucan–– HorseshoeHorseshoe crabcrab-- G factorG factor-- glucanglucan–– FungalFungal infectioninfection–– Variable S & Variable S & SpSp

HuangHuang et et al.al.ParasiteParasite, 2010., 2010.

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CONCLUSIONSCONCLUSIONS•• OisOis ((PcPPcP) has ) has beenbeen decreasingdecreasing dramaticallydramatically afterafter

cc--ART.ART.•• HoweverHowever,... ,... PcPPcP stillstill herehere. . •• ClassicalClassical diagnosticdiagnostic techniquestechniques are in use are in use •• NewNew molecular molecular methodsmethods supportsupport

classicalclassical diagnosis: diagnosis: -- advantagesadvantages: : rapidityrapidity; ; quantificationquantification; monitor ; monitor treatmenttreatment response; response; studystudy ofof resistancesresistances; ; molecular molecular epidemiologyepidemiology. . -- disadvantagesdisadvantages: : expensiveexpensive; ; equipmentequipment,,lacklack ofof protocolsprotocols..

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ACKNOWLEDGEMENTSACKNOWLEDGEMENTS•• Dr. JosDr. Joséé MarMaríía Mira Miróó•• Dr. AsunciDr. Asuncióón Morenon Moreno

–– InfectiousInfectious DiseasesDiseases, Hospital , Hospital ClinicClinic, Barcelona, Barcelona

•• Dr. Jorge Puig de la Dr. Jorge Puig de la BellacasaBellacasa•• Dr. MarDr. Maríía Eugenia Vallsa Eugenia Valls•• Dr. Jordi MasDr. Jordi Mas

–– MicrobiologyMicrobiology, Hospital , Hospital ClinicClinic, Barcelona, Barcelona

•• Dr. Dr. StevenSteven MeshnickMeshnick–– UniversityUniversity NorthNorth Carolina,Carolina,

ChapelChapel Hill, NC, USA.Hill, NC, USA.

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Pneumocystis: round tableS. Meshnick, JM. Miró, F. Derouin,

M. Álvarez

ESGCP, Barcelona, September 6, 2010

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KovacsKovacs et al. , 2001.et al. , 2001.

•• PrevalencePrevalence ofof PneumocystisPneumocystis DHPS DHPS gene in gene in SpainSpain in cin c--ART era: ART era: 3.7%3.7%

•• Global Global mortalitymortality 15%, 15%, risingrising toto 80% 80% in in patientspatients withwith mechanicalmechanicalventilationventilation..

•• PresencePresence ofof DHPS DHPS mutationsmutations werewerenotnot associatedassociated toto worseworse outcomeoutcome. .

•• TMPTMP--SMX SMX isis effectiveeffective atattherapeuticatherapeutica dosesdoses..

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CLINICAL & DEMOGRAPHICAL CHARACTERISTICSCLINICAL & DEMOGRAPHICAL CHARACTERISTICSOF PATIENTS ACCORDING TO GENOTYPEOF PATIENTS ACCORDING TO GENOTYPE

50% 50% PcPPcP debut debut ofof VIHVIH

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ThereThere waswas notnot differencedifference in in clinicalclinical coursecourse amongamong

patientspatients withwith oror withoutwithout DHPS DHPS mutationsmutations

PatientPatient withwith DHPS DHPS mutationsmutations diddid notnot diedie,,neitherneither neededneeded ICU ICU oror mechanicalmechanical ventilationventilation

CLINICAL COURSECLINICAL COURSE

TREATMENT & OUTCOMETREATMENT & OUTCOME

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Global Global mortalitymortality 15%.15%.

CLINICAL & DEMOGRAPHICAL CHARACTERISTICSCLINICAL & DEMOGRAPHICAL CHARACTERISTICSOF PATIENTS ACCORDING TO MORTALITYOF PATIENTS ACCORDING TO MORTALITY

BaselineBaseline clinicalclinical characteristicscharacteristicssamesame in in bothboth groupsgroups

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MechanicalMechanical ventilationventilation /ICU /ICU increasedincreased mortalitymortality toto 80%80%

CLINICAL COURSECLINICAL COURSE

TREATMENT & OUTCOMETREATMENT & OUTCOME

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MortalityMortality multivariatemultivariate analysisanalysis

BASELINE SEVERITY OF RESPIRATORY INSUFICIENCYBASELINE SEVERITY OF RESPIRATORY INSUFICIENCYDETERMINED DETERMINED PcPPcP OUTCOMEOUTCOME

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0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

89 90 91 92 93 94 95 2001 2002 2003 2004

DHPS GENOTYPES BY YEAR

1 2 3 4

DHPS genotypes

YEAR

PossiblePossible transmissiontransmission personperson toto personperson

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OPEN QUESTIONS TO THE TABLEOPEN QUESTIONS TO THE TABLE

•• TrueTrue clinicalclinical role role ofof DHPS DHPS mutationsmutations•• MortalityMortality ofof PcPPcP ((patientpatient/ / microorganismmicroorganism))•• RespiratoryRespiratory baselinebaseline influenceinfluence in in outcomeoutcome•• GenotypesGenotypes ofof DHPS DHPS mutationsmutations as as markersmarkers

ofof transmissiontransmission•• ValueValue ofof classicalclassical methodsmethods•• OW OW vsvs BALBAL

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new tools for the diagnosis of

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