Aspergillus: still the same as before?

(1)

Aspergillus

Still

the

_the

same

_same

as

_as

before

_before

?

_?

Marie

Marie--Pierre HayettePierre Hayette Service de Microbiologie m

Service de Microbiologie méédicaledicale CHU Sart

(2)

Description of the

genus

_genus

Ubiquitous

filamentous

fungi

largely

spread

in the environment

in the

environment

Many

species

described

The species Aspergillus fumigatus was described in 1863 by Johann Baptist Georg Wolfgang Fresenius

1965.

1965. Rapper

Rapper

and

Fennel

. Morphological

.

Morphological.

.

132

132 speciesspecies, 18 groups , 18 groups

1996

(3)

Identification

at

_at

the

_the

species

_species

level

_level

MACROSCOPIE + MICROSCOPIE

based

on

colour

,

shape

, ornementation

sexual

or

asexual

stages recognition

Genus level: ok Section level: ok Species level

•

•The The extendextend of of speciesspecies responsibleresponsible of invasive of invasive aspergillosis

aspergillosis isis underestimatedunderestimated •

•New New speciesspecies, , lessless susceptible susceptible isis responsibleresponsible for for invasive

invasive aspergillosisaspergillosis

(4)

Study

.

Genetic

diversity

of 50

isolates

of

A.

A. fumigatus

fumigatus

+ in vitro

sensitivity

to

antifugnal

drugs

used

in IA

Method

50 50 isolatesisolates of of A. A. fumigatusfumigatus ((phenotypicphenotypic identification)identification)

RFLPRFLP

Results

34 34 A. A. fumigatusfumigatus, , 16 16 A . non A . non fumigatusfumigatus

3 distinct 3 distinct speciesspecies: : A. A. fumigatusfumigatus + A. + A. lentuluslentulus (T(T°°<50<50°°C), A. C), A. udagawaeudagawae

In vitro In vitro susceptibilitiessusceptibilities

A. A. lentuluslentulus: : decreaseddecreased sensitivitysensitivity to to AmBAmB, VOR, , VOR, CaspoCaspo

A. A. udagawaeudagawae: : decreaseddecreased sensitivitysensitivity to to AmBAmB, VOR, VOR

Conclusion

differentialdifferential antifungalantifungal susceptibilitiessusceptibilities maymay accountaccount for for somesome of of

the

the reportedreported poorpoor outcomeoutcome of of therapytherapy in in clinicalclinical studiesstudies

Molecular studies reveal frequent misidentification of Aspergillus

fumigatus by microscopy.

Balajee SA, Nickle D, Varga J, Marr KA.

Eukaryot Cell. 2006 Oct;5(10):1705-12

(5)

Clinically

relevant

Aspergilli

7

7 «

«

subgenera

»

divided

into

sections (

species

)

Aspergillus

_

sectionsection AspergillusAspergillus (21), (21), RestrictusRestrictus (3)(3)

Fumigati

__

section section FumigatiFumigati (16(16): ): A. A. fumigatusfumigatus, , A. A. lentuluslentulus, , N. N. fischerifischeri, , N.

N. pseudofischeripseudofischeri, N. , N. spinosaspinosa

Section Section CerviniCervini (5)(5)

Ornati

_section_section OrnatiOrnati (7)(7)

Clavati

_section_section ClavatiClavati (4)(4)

Nidulantes

_section_section NidulantesNidulantes (35)(35)__VersicoloresVersicolores (24)(24)--, , UstiUsti (5), (5), TerreiTerrei

(1

(1--3), 3), FlavipesFlavipes (4)(4)

Circumdati

7 sections7 sections

Stilbothamnium

5 sections5 sections

(6)

Fumigati

section:

other

pathogenic

species

Osteomyelitis

Osteomyelitis causedcaused by by NeosartoryaNeosartorya pseudofischeripseudofischeri Padhye

Padhye AAAA, , GodfreyGodfrey JHJH, , Chandler FWChandler FW, , Peterson SWPeterson SW.... J Clin

J Clin MicrobiolMicrobiol.. 1994 Nov1994 Nov;32(11):2832;32(11):2832--6.6.

Isolation of

Isolation of NeosartoryaNeosartorya pseudofischeripseudofischeri fromfrom bloodblood: first : first hinthint of of pulmonarypulmonary aspergillosisaspergillosis

Järv H, Lehtmaa J, Summerbell RC, Hoekstra ES, Samson RA, Naaber P.

J Clin Microbiol. 2004 Feb;42(2):925-8.

First description of the new species. Optical and electronic microscopy and DNA hybridization methods were used as identificaiton methods.

17-year-old male patient with Hodgkin’s disease, Fever of 38.6°C, and a nonproductive cough.

(7)

Alternative: MASS

-

_-

SPECTROMETRY

_SPECTROMETRY

MethodMethod. .

124 124 clinicalclinical

16 16 environmentalenvironmentalisolatesisolates

CharacterisedCharacterised by partial by partial sequencingsequencing of the betaof the beta--tubulintubulin and and calmodulincalmodulin genesgenes

MethodologyMethodology: engineering of : engineering of MaldiMaldi--toftof MS data base MS data base withwithreferencereference strainsstrains

No extraction: water+ DHB No extraction: water+ DHB matrixmatrix solution (disolution (di--hydroxybenzoiquehydroxybenzoiqueacidacid))

ResultsResults

Identification Identification performedperformed in 10 minutesin 10 minutes

98.6% 98.6% correctlycorrectly identifiedidentified (2 (2 couldcould not not bebe identifiedidentified))

100% 100% specificityspecificity

ConclusionConclusion: : rapidrapid methodologymethodology thatthat replaces replaces avantageouslyavantageously phenotypicphenotypic identification. It identification. It isis lesslesstime time

consuming

consuming and must and must cheapercheaperthanthan sequencingsequencing..

MALDI

MALDI--TOF Mass TOF Mass SpectrometrySpectrometry for fastfor fast and accurateand accurate identification of identification of clinically

clinically relevant Aspergillus speciesrelevant Aspergillus species..

Alanio A. Peretti JL, Dauphin B., Mellado E., Ouesne G., Lacroix C., Amara A., Berche P., Nassit X., Bougnoux ME.

Department

Department of of MicrobiologyMicrobiology, Hôpital Necker, Hôpital Necker--Enfants Malades, Paris, France; UniversitEnfants Malades, Paris, France; Universitéé Paris Paris Descartes, Paris, France.

Descartes, Paris, France.

Clin

(8)

Neosartorya

fumigata

Anamorphic

filamentous

organisms

which

reproduce

by means

by

means

of

asexual

spores

Sexual

forms

of many

of

many

aspergillus have been described

aspergillus have been

described

«

« DiscoveryDiscovery of a sexualof a sexual cycle in the opportunisticcycle in the opportunistic fungalfungal pathogenpathogen

Aspergillus

Aspergillus fumigatusfumigatus »»

O

O’’GormanGormanC. et al. C. et al. Nature.Nature.2009 Jan 22;457(7228):4712009 Jan 22;457(7228):471--44

Nature. 2005 Dec 22;438(7071):1151-6.

Genomic sequence of the pathogenic and allergenic filamentous

fungus Aspergillus fumigatus.

Nierman WC et al. , Nature. 2005 Dec 22;438(7071):1151-6.

(9)

Clinical

aspects

_aspects

Main portal of entry:

lungs

and

sinusis

Disease

AllergicAllergic (ABPA)+ (ABPA)+ allergicallergic pulmonarypulmonary alveolaralveolar diseasesdiseases

Colonisation of air Colonisation of air spacesspaces suchsuch as as fungusfungus ballsballs of the of the lungslungs and and sinusissinusis, ,

endobronchial

endobronchial colonisation or association colonisation or association withwith EAR EAR pathologypathology..

Invasive Invasive formsforms↔↔ immunosuppressionimmunosuppression

PredisposingPredisposing factorsfactors: : neutropenianeutropenia, long, long--termterm steroidsteroid therapytherapy, , lymphomalymphoma, ,

diabetes

diabetes, , burnsburns, , alcoholismalcoholism, the , the neonatalneonatal state, state, priorprior tuberculosistuberculosis, , immunosuppressive

immunosuppressive therapytherapy, trauma, , trauma, liverliver failurefailure, , operativeoperative proceduresprocedures

RecentlyRecently: NON CLASSICAL: NON CLASSICAL TYPE of TYPE of atat riskrisk patientspatients::

ParticularlyParticularly in intensive care in intensive care unitsunits , ,

patients patients withwith chronicchroniclunglungdiseasesdiseasesor influenzaor influenza

Patients Patients withwith short courses of steroidsshort courses of steroids

AIDS patientsAIDS patients: up to 9% of patients : up to 9% of patients withwith progressingprogressingdiesasediesase maymay developdevelop thisthis

complicaton

(10)

Aspergillosis

in the ICU: the new 21st

century

problem

?

Koenraad

Koenraad H. et al. , H. et al. , MedicalMedical mycology, mycology, septemberseptember 2006, 44:S71-2006, 44:S71-S76S76

OtherOther categorycategory of population of population withoutwithout apparent apparent severesevere immunodeficiencyimmunodeficiency: :

patients patients withwith criticalcritical illnessillness

Data are Data are scarcescarce

Incidence of IA ranges Incidence of IA ranges fromfrom 0.3 to 5.8%, 0.3 to 5.8%, mortalitymortality rate 80%.rate 80%.

PredisposingPredisposing factorsfactors: Malnutrition, : Malnutrition, diabetesdiabetes mellitusmellitus, , pulmonarypulmonary disorderdisorder, , liverliver

cirrhosis

cirrhosis and and corticoidcorticoid use use

CorticoidsCorticoids are are usedusedin persistent in persistent septicseptic shockshock ((beneficbeneficeffecteffect))

Patients Patients withwith underlyingunderlyingdiseasedisease: : corticoidscorticoids are a are a riskrisk factor for IAfactor for IA

DifficultyDifficulty in in establishingestablishing diagnostic: diagnostic: whichwhich tests tests cancan bebe usedused??

Culture: first clue: Culture: first clue: diseasedisease/colonisation/ marker for /colonisation/ marker for diseasedisease??

IMPORTANT: direct positive IMPORTANT: direct positive examinationexamination

Tissue Tissue samplingsampling: not : not easyeasy in ICU patientsin ICU patients

SerologicalSerological markers: markers:

GM, beta GM, beta glucanglucan: few data: few data

AntibodiesAntibodies: no data : no data

PCR: PCR: CouldCould bebe appliedappliedin the in the bloodblood. No data in ICU. No data in ICU

RadiologicalRadiologicalfindingsfindings: not : not specificspecific: : atypicalatypical infiltratesinfiltrates++++

(11)

Still

diagnostic

problems

for

invasive

aspergillosis

?

ClinicalClinical RadiologicalRadiological

Mycological

Culture +

calcofluor

(

hyphae

)

Antigens

detection

:

Galactomannan

:

released

during

hyphae

growth

EIA:

Platelia

®

Aspergillus,

Biorad

Index: 1.5

⇒

0.5

((MaertensMaertens J.) J.)

sensitivitysensitivity 92% 92% specificityspecificity 95%95%

MetaMeta--analysisanalysis: reports : reports lessless good good resultsresults 64% 64% sensitivitysensitivity/93% /93% spcecificityspcecificity

BetterBetter resultsresults in in neutropenicneutropenic patientspatients

GM: GM: «« thethe »» IA diagnostic testIA diagnostic test

Where

?

serum

,

bronchoalveolar

lavage (

Meersseman

et al.),

CSF

(12)

Galactomannan

testing

_testing

on BAL

_{on BAL}

Galactomannan

in bronchoalveolar

lavage fluid:

a tool

for diagnosing

aspergillosis

in intensive care unit patients.

Meersseman

Meersseman WW, , LagrouLagrou KK, , MaertensMaertensJJ, , WilmerWilmer AA, , Hermans GHermans G, , VanderschuerenVanderschueren SS, , Spriet

Spriet II, , VerbekenVerbeken EE, , Van Van WijngaerdenWijngaerden EE..

Am J Respir Crit Care Med. 2008 Jan 1;177(1):27-34. Epub 2007 Sep 20.

PopulationPopulation: : host host factorsfactors: : HaematologicHaematologic malignanciesmalignancies++lessless immunosuppressedimmunosuppressedpatients patients

(short course of

(short course of steroidssteroids duringduringor or beforebefore admission), HIV, admission), HIV, solidsolid organorgan transplant transplant recipientsrecipients, , immunosuppressive

immunosuppressive treatmenttreatment. 78% non . 78% non neutropenicneutropenicpatients.patients.

MethodMethod: 18 : 18 monthsmonthsstudystudy++clinicalclinical featuresfeatures sugestivesugestive of of fungalfungal infectioninfection

Comparison

Comparison of GM in BAL (0.5 index), GM in of GM in BAL (0.5 index), GM in serumserum, CT, CT--scan.scan.

ResultsResults: 26 : 26 provenproven IA: IA:

GM in GM in serumserum

SensitivitySensitivity: 42%: 42%

MedianMedianvalue of GM in value of GM in serumserum : 0,3: 0,3

14/26 14/26 negativenegativewhenwhenGM positive in BAL(>50%)GM positive in BAL(>50%)

GM in BALGM in BAL SensitivitySensitivity: 88%: 88% SpecificitySpecificity: 87%: 87%

11/26: GM in BAL unique positive test (culture+GM in 11/26: GM in BAL unique positive test (culture+GM in serumserum: NEGATIVE): NEGATIVE)

False positive tests?False positive tests?

13% of the patients in the 13% of the patients in the trulytrulynegativenegativegroupgroup

AnyAny differencedifference betweenbetween patients patients withwith or or withoutwithout neutropenianeutropenia??

GM on BAL GM on BAL performedperformedequallyequallyin in patietnspatietnswithwithor or withoutwithoutneutropenianeutropenia

GM in GM in serumserumperformedperformedbetterbetterin patients in patients withwithneutropenianeutropenia

(13)

Galactomannan

on BAL for ICU patients

GM on BAL

performs

better

than

on

serum

GM

performs

better

than

β

-

D

-

glucan

on BAL

and

serum

Conclusion: GM in BAL

improved

the

diagnosis

of IA in

critical

ill

patients

A prospective comparison of galactomannan in bronchoalveolar lavage fluid for the diagnosis of pulmonary invasive aspergillosis in medical patients under intensive care: comparions with the diagnostic performance of galactomannan and of

(1→3)β-D-glucan chromogenic assay in serum samples

Acosta J, Catalan M, Del Palacio-Peréz-Medel A, Lora D, Montejo JC, Cuetara MS, Moragues MD, Ponton J, Del Palacio A.

Clin Microbiol Infect. 2010 Sep 3. [Epub ahead of print

BAL fluid GM for the diagnosis of invasive pulmonary aspergillosis inpatients with haematologic diseases Maertens J. et al., CID, 2009, 49/1688-1694 Index ≥1 sensitivity 96% specificity 87%

(14)

β

(1

-

3)

–

D

glucan

detection

Do

we

have to use

this

test?

Utility of GalactomannanUtility of Galactomannan Enzyme ImmunoassayEnzyme Immunoassay and (1,3) βand (1,3) β--DD-

-Glucan

Glucan in Diagnosisin Diagnosis of Invasive Fungalof Invasive Fungal Infections: LowInfections: Low SensitivitySensitivity for

for Aspergillus fumigatusAspergillus fumigatus Infection in HematologicInfection in Hematologic MalignancyMalignancy Patients

Patients

R. Y.

R. Y. HachemHachem,,**D. P. D. P. KontoyiannisKontoyiannis, R. F. , R. F. ChemalyChemaly, Y. Jiang, R. , Y. Jiang, R. ReitzelReitzel, and I. , and I. RaadRaad

Prospective study in neutropenic patients

Beter sensitivity for BG (67%) vs GM (38) (sampling only once a week)

Better sensitivity for GM for detecting non-A. fumigatus infection. No difference for BG between A. fumigatus and non-fumigatus infection.

Conclusion: BG greater sensitivity than GM in detecting IA??

different reactivity in GM production depending on the species suggests the interest of using both tests for IFI diagnosis?

(15)

EORTC/MSG

RevisedRevised definitionsdefinitions of invasive of invasive fungalfungal diseasedisease fromfrom the the EuropeanEuropean Organisation for Organisation for

Research

Research and and TreatmentTreatment of Cancer/invasive of Cancer/invasive fungalfungal infections infections CooperativeCooperative group and group and the national institue of

the national institue of allergyallergy and and infectiousinfectious diseasesdiseases mycoses mycoses studystudy group group (EORTC/MSG) Consensus Group

(EORTC/MSG) Consensus Group

Ben De

Ben De PauwPauwet al., EORTC/MSG consensus group, Clin Infect Dis 2008 46 (12)et al., EORTC/MSG consensus group, Clin Infect Dis 2008 46 (12):1813:1813--2121

2002. EORTC/MSG :standard 2002. EORTC/MSG :standard definitionsdefinitions for invasive for invasive fungalfungal infections for infections for clinicalclinical and and

epidemiological

epidemiological researchresearch..

3 3 levellevel of of probabilityprobabilityto the to the diagnosisdiagnosisof IFA in of IFA in immunocompromisedimmunocompromisedpatients patients withwith cancer cancer

and in

and in hematopoietichematopoietic stem stem cellcell transplant patients: possible/probable/transplant patients: possible/probable/provenproven invasive invasive infection.

infection.

RevisionRevision processprocess startedstarted in 2003, in 2003, approvedapproved in 2005in 2005

WhatWhat has been has been changedchanged??

DefinitionsDefinitions possible/probable/possible/probable/provenproven

Probable Probable expandedexpanded

Possible Possible diminisheddiminished

(16)

« Invasive Invasive fungalfungal infectioninfection »» diseasedisease waswas adoptedadopted to to reflectreflect an an infectiousinfectious diseasedisease processprocess

«« Possible invasive Possible invasive fungalfungalinfectioninfection»» tootoo manymanydubiousdubious cases cases werewere includedincluded

OnlyOnlycases cases thatthatare are highlyhighlylikelylikelyto to bebecausedcausedby a by a fungalfungal etiology evenevenwithoutwithout evidenceevidenceof of mycologicalmycological

evidence

Populations:Populations:tootoo restrictiverestrictive→→extendedextended to:to:

SolidSolid organorgan transplant, transplant, primaryprimary immunodeficienciesimmunodeficiencies, connective tissue , connective tissue disordersdisorders, , receiptreceipt of of

immunosuppressive agents (

immunosuppressive agents (corticosteroidscorticosteroids, T, T--CellCell immunosuppressantsimmunosuppressants))

Host Host factorsfactors: : MinorMinoror major or major clinicalclinicalcriteriacriteria: ABANDONNED in : ABANDONNED in favorfavor of more of more characteristiccharacteristic and and

objectively

objectively verifiableverifiable evidenceevidence

findingsfindingsof of medicalmedicalimagingimagingwithwithstandardizedstandardizedglossaryglossaryof of definitionsdefinitions

Ex. Patients Ex. Patients withwithIPA: focal IPA: focal ratherratherthanthandiffuse diffuse pulmonarypulmonaryinfiltratesinfiltrates

EORTC/MSG:

which

_which

changes?

_changes?

dense

dense wedgewedge--shapedshaped

infiltratein the right upper lobe.

Diffuse

Diffuse pulmonarypulmonary infiltrates

infiltratesin a bone marrowtransplant recipient due to IPA

CT scan of the chest showing multiple multiple peripheral

(17)

EORTC/MSG:

which

_which

changes?

_changes?

«

Probable and

proven

»

integration

of more indirects

tests in the

definitions

Thresholds

by the manufacturer

Platelia

Aspergillus

galactomannan

EIA

can

be

applied

to the

CSF,

serum

or plasma and BAL.

β

-

D

-

glucan

(

Fungitell

®®

)

included

in the

mycological

tests for

diagnosing

IFD

except

cryptococcosis

and

zygomycosis

(18)

PCR or not?

DNA

comes

from

breakdown of

hyphae

Specific

or

panfungal

?

First on BAL: colonisation or infection?

Quantification

can

not

resolve

the

problem

In

serum

or plasma or

blood

?

Previously

no

consensus. Short

series

. Case reports.

No commercial

valuable

Aspergillus

PCR

Many

scientists

do not

rely

on PCR for

Aspergillus

(19)

PCR: international group of

standardisation

Aspergillus PCR: one

Aspergillus PCR: one stepstep closercloser to to standardizationstandardization..

White PL

White PL, , Bretagne SBretagne S, , KlingsporKlingspor LL, , MelchersMelchers WJWJ, , McCullochMcCulloch EE, , Schulz BSchulz B, , FinnstromFinnstrom NN, , Mengoli

Mengoli CC, , Barnes RABarnes RA, , Donnelly JPDonnelly JP, , LoefflerLoeffler JJ; ; EuropeanEuropean Aspergillus PCR InitiativeAspergillus PCR Initiative..

J Clin

J Clin MicrobiolMicrobiol.. 2010 2010 AprApr;48(4):1231;48(4):1231--40. 40. EpubEpub 2010 2010 FebFeb 10.10.

Sampling: 3 ml

Standardisation of DNA Extraction method Validation of the different target used

Creation of a « quality control » for laboratories resecting the desigh of the study.

(20)

Treatment

:

_:

evolution

_evolution

…

_…

AmB

⇒

lipid

formulations

⇒

new

triazole

=

Voriconazole

: first line

therapy

with

AmB

or

Caspofungin

as 2d line

treatment

⇒

Combination

therapy

??

Still

controversial

, no

consensus

Posaconazole

:

treatment

and

prophylaxy

Comparative

Comparative survivalsurvivaland and costcostof of antifungalantifungaltherapytherapy: : posaconazoleposaconazoleversus standard versus standard antifungalsantifungalsin the in the treatment

treatmentof of refractoryrefractoryinvasive invasive aspergillosisaspergillosis..

Herbrecht

HerbrechtR, R, RajagopalanRajagopalanS, S, DannaDannaRR, , PapadopoulosPapadopoulosGG..

--CurrCurrMed Med ResResOpinOpin..2010 2010 OctOct;26(10):2457;26(10):2457--64.64.