Etude prospective multicentrique (RADAR)

de la résistance d’Aspergillus fumigatus aux

azolés en FranceJ.B. MURAT1,2, F. CHOUKRI1, J.M. COSTA3, I. ACCOCEBERRY4, C. ANGEBAULT5, M.E.BOUGNOUX5, N. BOURGEOIS6, M. CORNET7, L. DELHAES8, N. FAUCHET9, O.FAURE7, A. FEKKAR10, J. GUITARD11, C. HENNEQUIN11, L. LACHAUD12, A. PAUGAM13,R. KIM2, E. SITTERLE1, J. GUILLOT1, F. BOTTEREL1,2, E. DANNAOUI1,14.

1Dynamyc, UPEC, ENVA, 2Hosp. Mondor, Créteil, 3Lab. CERBA, 4Hosp. Pellegrin, Bordeaux, 5Hosp. Necker-Enfants malades, Paris, 6CHRU, Montpellier, 7CHU, Grenoble, 8CHU, Lille, 9CHIC, Créteil,10Hosp. Pitié, Paris, 11Hosp. Saint-Antoine, Paris, 12Hosp. Caremeau, Nimes, 13Hosp. Cochin, Paris,14Hosp. Pompidou, Paris, France.

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Aspergillus and aspergillosis

Filamentous fungus Infection - Treatment

A. fumigatus: the

most common +++

Soil-borne

microorganism

Aspergillus heads �

numerous spores

spores inhalation � pulmonary disease

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is, T

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., 2

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9. C

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iol

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v2

2:4

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-65

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Aspergillose pulmonaire invasive (API)

Aspergillome (balle fongique)

Aspergillose brocho-pulmonaire allergique (ABPA)

Aspergillose pulmonaire invasive (API)

Aspergillome (balle fongique)

Aspergillose brocho-pulmonaire allergique (ABPA)

� Voriconazole +++

Aspergillus – Azole resistance

Emergence of resistance Worldwide distribution

Snelders et al. 2008 PLoS Med. 5:e219

Detection of resistance in clinical samples (irrespective to mecanism)

Iran

India

China

Japan

Iran

India

China

Japan

AustraliaAustralia

Iran

India

China

Japan

Australia

The Netherlands

Germany

Norway

Spain

The Netherlands

Germany

Norway

Spain

Denmark

UK

France

Sweden

Denmark

UK

France

Sweden

Belgium

Greece

Italy

Austria

Belgium

Greece

Italy

Austria

The Netherlands

Germany

Norway

Spain

Denmark

UK

France

Sweden

Belgium

Greece

Italy

Austria

USAUSAUSA

Aspergillus

Azole resistance Mecanisms of

resistance

Origin of resistance

Azole resistance - France

1. Dannaoui, E., et al. 1999. JAC 44, 553-555

2. Dannaoui, E., et al. 2001. JAC 47, 333-340

3. Morio, F., 2012. JAC 67:1870-3.

4. Burgel, P. R., 2012.. AAC 56:869-74

5. Rocchi, S. 2014, JCM 52, 1724-6

6. Lavergne RA., 2015, AAC Apr 27

7. Choukri, F., et al. 2015 Med Mycol, in Press

8. Choukri, F., et al. 2013 - TIMM

Few data

� First detection in the late 1990s1,2

� Few data3-6

� Mostly in selected populations

� Retrospective analysis7 – 1 year (2012) – all patientso Prevalence 1.8% (isolates)

� Retrospective analysis8 – 12 years (2000-2012) –Lung Tx patients

o Prevalence 8.9% (isolates)

Detection of resistance in clinical samples (irrespective to mecanism)

Aspergillus

Azole resistance

Aim of the study

Aim

Risk in

Aspergillosis:

Detection of

Azole

Resistance

C

ITZ VRZ

� Surveillance study – Evaluation ofprevalence and mechanisms

� Prospective - Multicenter

� All Aspergillus spp. screened onazole-containing plates

The RADAR prospective study

Lille

Laurence Delhaes

Bordeaux

Isabelle Accoceberry

Montpellier

Nathalie Bourgeois

Nîmes

Laurence Lachaud

Grenoble

Odile Faure

Muriel Cornet

NeckerCecile Angebault

Marie-Elisabeth Bougnoux

PitiéArnaud Fekkar

CochinAndré Paugam

Saint-AntoineJuliette Guitard

Christophe Hennequin

HEGPEric Dannaoui

HMNFrançoise Botterel

CHICNathalie Fauchet

RADAR

� Screening – 1 year

� 2013 – 2014

� Standardized protocol

� Meeting pre-study

� Video

� QC exercise

� Video

� All samples – 1 colony

� Incubation 48h – 37°C

RADAR study – Study design

http://www.dynamyc.fr/en-55-dynamyc-mycology-videos.html

Methods

Methods

Screening

EUCAST

Speciesidentification

(Beta-tubulin)

Mutations

(Cyp51 sequencing)

Resistant

isolates

Resistant

isolates

12 centers

Isolates centralized in

one lab

Screening results

Total isolates A. fumigatus resistant

isolates

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Nu

mb

er

of

iso

late

s

screened expected

screened real

0

20

40

60

80

100

120

140

160

août-13 nov.-13 mars-14 juin-14 sept.-14 déc.-14

Resistant Af expected

Resistant Af real

Screening results

Number by center Distribution of species

0

100

200

300

400

500

1 2-3 4 5 6 7 8 9 10 11-

12

Nu

mb

er

of

iso

late

s

Center #

N=3049

A. fumigatus

A. niger

A. flavus

A. nidulans

A. terreus

A. versicolor

Other

Screening

results

The RADAR prospective study

142

208

3049

A. fumigatus R

Resistant

Screened

PRELIMINARY

results EUCAST

N=134

110 R/I

2 N.

pseudofisheri

108 A.

fumigatus

24 S

A. fumigatus resitant isolates - Analysis in progress

Prevalence: 4.2%

Results108 A. fumigatus resistant isolates

TR34/L98H

mutation

Cyp51 mutation n Cyp51 mutation n

TR34/L98H 66 G448S 1

Pas de mutation 19 G54W 1

G54R 5 M220V 1

TR34/L98H/H147Y 5 TR34/L98H/S297T/F495I 1

H285Y 3 TR46/Y121F/T289A 1

M220I 2 Y121F 1

The RADAR prospective study

� Various underlying diseases� CF

� SOT

� Kc

� Hematol malignancies

� …

� Various aspergillosis� Colonization

� ABPA

� IA

� Aome

� …

Conclusions � Our results provide new data about azoleresistance in France

� Azole resistant A. fumigatus isolates can berecovered in France from patients with differentunderlying diseases

� The TR34/L98H mutation is the most frequentCyp51A mutation involved

� AFST of Aspergillus spp. is recommendedand further surveillance epidemiological studiesare needed

Thank

you

Epidemiology – Unresolved issues

Problem of resistance detection – Available techniques

Culture positive samples

Screening

agar plates

/ Etest

EUCAST

Cyp51 mutation

Culture negative samples

� PCR (Mycogenie, Aspergenius)

� Not all mutations

� Pb of sensitivity

� NGS