Emergence(( d’Aspergillus fumigatus*splf.fr/wp-content/uploads/2015/01/VM-At3-RissoK.pdf · Aspergillus sp. • Champignon’ﬁlamenteux’de’l’environnement ’ • Responsable’en’pathologie’humaine’:’

Emergence d’Aspergillus fumigatus résistants aux Azolés en pathologie humaine

Karine RISSO, Pneumo-‐infec4ologue PhC en Unité protégée d’Hématologie

CHU Nice

•  Pas de conflit d’intérêt en rapport avec le sujet

2

Aspergillus sp.

•  Champignon filamenteux de l’environnement •  Responsable en pathologie humaine :

Chez l’immunodéprimés §  formes invasives

Chez l’immunocompétent §  formes «semi-‐invasives »: aspergillome, aspergillose

chronique nécrosante, bronchite aspergillaire §  formes immuno-‐allergiques : ABPA (muco+++), granulome bronchocentrique, asthme à aspergillus

3

En Europe

4

•  Sur les 733 millions personnes vivant sur le con4nent Européen

§  2 100 000 souffrent d’aspergillose allergique §  240 000 d’aspergillose chronique §  63 250 cas d’aspergilloses invasives par an

Rapport 2013 ECDC

Traitement

•  Les Azolés (itraconazole, voriconazole et posaconazole) §  Pierre angulaire

-‐  préven4on -‐  traitement de première inten4on

§  U4lisa4on PO à seul traitement envisageable dans les formes chroniques

§  Seul traitement efficace dans les aaeinte SNC

•  Amphotéricine B et Echinocandines

§  IV §  place limitée dans les formes chroniques…

5

ECIL 2013 CID 2008

Objec8fs de l’exposé

ü  Etat des lieux en 2014 -‐ dans le monde -‐ en France

ü  Prévision: et demain ?

ü  Les moyens à développer pour luAer contre ceAe fatalité –  contre l’émergence et propaga4on des résistances ? –  comment adapter notre prise en charge: améliorer le rendement/rapidité d’iden4fica4on des résistances

6

ü Percevoir: -‐ les origines -‐ les mécanismes

de la résistance d’Aspergillus aux Azolés

Pas de cas clinique niçois……..

7

Mécanismes de résistances

8 Verweij, PE., et al. Lancet Infect Dis 2009; 9: 789–95

•  Cible des azolés §  Enzyme = lanostérol 14 alpha déméthylase §  Catalyse une étape de la synthèse de l’ergosterol

•  Codée par gène CYP51A

•  Résistance = muta4on empêchant la fixa4on des azolés

Modalités d’acquisi8on de résistance en clinique

9

Pression de sélec;on


Environementale = acquisi4on depuis l’environnement

Emergence de mutants R in situ = prise prolongée d’azolés

Sélec4on depuis l’environnement

Modalités d’acquisiFon de résistance en clinique

10



Environementale = acquisi4on depuis l’environnement

Emergence de mutants R in situ = prise prolongée d’azolés

I -‐ Résistance secondaire à l’ exposi8on prolongée aux azolés

•  Les 1 ères descrip4ons d’aspergillus résistants aux Azolés –  3 isolats cliniques datant de la fin des années 1980 –  1997 en Suède chez un pa4ent exposé au posaconazole suggérant un mécanisme de sélec4on

–  puis en 1999 à Manchester

•  Dite « Peu fréquente » Pfaller M. J Clin Microbiol. févr 2011;49(2):586‑90

Guinea J, An4microb Agents Chemother. 9 janv 2008

•  S’accélère ces dernières années •  Pas de documenta4on claire des facteurs prédisposant •  De très nombreuses muta4ons, principalement de CYP51A

11 ECDC 2013

Howard SJ Emerging Inf Dis 2009

12

§  Manchester §  400 isolats d’Af (1997 à 2007) §  5% R Itraconazole §  Augmenta4on significa4ve

fréquence de la résistance à l’itraconazole depuis 2004

8% vs 1% §  Af R: 14 pt analysables

§  13 formes chroniques §  13 exposés aux Azolés §  Tous en échec sous Az

�F;81?� ->1� @41�9-5:?@-E� ;2� ;>-8� @41>-<E� 2;>� -?<1>358-8;?5?��F;81�>1?5?@-:/1�5:�Aspergillus has been reported in-2>1=A1:@8E��)41�L>?@�>1?5?@-:@�5?;8-@1�5:�"-:/41?@1>��* ��C-?�01@1/@10�5:��:�-�/85:5/-8�/;881/@5;:�;2��A. fumiga-tus� 5?;8-@1?�� @41� 2>1=A1:/E�;2� 5@>-/;:-F;81� >1?5?@-:/1�C-?��-�?53:5L/-:@�5:/>1-?1�?5:/1�� <��$2�@41�� 5@>-/;:-F;81�>1?5?@-:@� 5?;8-@1?�C1� ?@A0510�� C1>1�/>;??�>1?5?@-:@� @;�B;>5/;:-F;81�-:0�� C1>1�/>;??�resistant to posaconazole. Thirteen of 14 evaluable patients 5:� ;A>� ?@A0E� 4-0� <>5;>� -F;81� 1D<;?A>1�� 5:21/@5;:?� 2-5810�@41>-<E� �<>;3>1??10�� -:0� �� 2-5810� @;� 59<>;B1� �>19-5:10�[email protected]��534@11:�-95:;�-/50�-8@1>-@5;:?�C1>1�2;A:0�5:�@41�@->31@�1:FE91��E<�� ;2�C45/4�C1>1�:;B18��<;<A8-@5;:�genetic analysis of microsatellites showed the existence of resistant mutants that evolved from originally susceptible strains, different cyp51A mutations in the same strain, and 95/>;-8@1>-@5;:?� 5:�95/>;?-@1885@1� >1<1-@�:A9.1>��F;81� >1-sistance in A. fumigatus is an emerging problem and may develop during azole therapy.

Invasive aspergillosis in immunosuppressed patients is ��

in a high mortality rate (1). Chronic and allergic pulmo-nary and sinus aspergillosis are increasingly recognized in

numerous clinical settings. Treatment with itraconazole, voriconazole, and, recently, posaconazole is the backbone of therapy for these conditions because azoles are the only licensed class of oral drugs for treatment of aspergillosis (2,3). Amphotericin B and caspofungin are licensed intra-venous agents for invasive aspergillosis but have limited utility for chronic and allergic aspergillosis.

Itraconazole resistance in Aspergillus� �� reported in 1997 in 3 clinical isolates obtained from Cali-fornia in the late 1980s (4); since then, only a few clinical cases have been published (5–9). The emergence of itra-conazole resistance alone is of concern, but widespread azole cross-resistance would be devastating.

The primary mechanism of resistance described for A. fumigatus clinical isolates is mutation in the target protein. The cyp51A gene encodes the target of azoles, lanosterol �� biosynthetic pathway of ergosterol (an essential cell mem-�� open reading frame of the cyp51A gene can result in struc-tural alterations to the enzyme, which in turn may inhibit �� resistance have been characterized in the gene at codons 54 (6,10–13), 220 (6,14,15), and 98 (16–18). Other mutations in the cyp51A gene have been reported, and additional re-sistance mechanisms have been postulated (11,19,20). The environmental or antifungal pressures driving azole resis-tance are unclear because few clinical azole-resistant Asper-gillus strains have been studied in any detail; many reports simply describe individual patient cases. In this study, we

�� Resistance in Aspergillus fumigatus Associated with Treatment Failure1

Susan J. Howard, Dasa Cerar, Michael J. Anderson, Ahmed Albarrag, Matthew C. Fisher, Alessandro C. Pasqualotto, Michel Laverdiere, Maiken C. Arendrup, David S. Perlin,

and David W. Denning

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§  Aucun Af R. au Vorico ou Posaco n’était S Itraco §  65% R voriconazole §  74% R posaconazole

…. une grande diversité de muta8ons et de profils de résistance

Pression de sélec8on: exposi8on prolongée aux azolés

14

§  Comment déterminer l’origine de la résistance ? -‐ acquisi4on depuis l’environnement? -‐ de novo sous l’effet de l’exposi4on prolongée aux Azolés ?

§  Arbre phylogénéFque:

L’existence chez un même pa4ent de souches

géné4quement proches résistante et sensibles

suggère une évolu4on in situ à par4r d’un même clone S

Verweij PE et al. NEJM 2007 Mellado E etal. AAC 2007

II -‐ Emergence d’Af résistants dans l’environnement

15

Aspergillus 14 α demethylase (gène CYP51A)

DIMs ATF agricoles Azolés

Muta4on TR34/L98H

Pan Résistance Azolés 100% R Itraco

90% R ou I Voriconazole 80% R ou I Posaconazole

Pays-‐bas 2002

16

Emergence of Azole Resistance in Aspergillusfumigatus and Spread of a Single ResistanceMechanismEveline Snelders1,2, Henrich A. L. van der Lee1,2, Judith Kuijpers1,2, Anthonius J. M. M. Rijs1,2, Janos Varga3,4,

Robert A. Samson3, Emilia Mellado5, A. Rogier T. Donders6, Willem J. G. Melchers1,2, Paul E. Verweij1,2*

1 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 2 Nijmegen Institute for Infectious Diseases, Inflammation

and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 3 Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Centre,

Utrecht, The Netherlands, 4 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Hungary, 5 Servicio de Micologia, Centro Nacional de

Microbiologia, Instituto de Salud Carlos III, Madrid, Spain, 6 Department of Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, Nijmegen, The

Netherlands

Funding: This work was funded inpart by The NetherlandsOrganisation for Health Researchand Development (ZonMw; grant:50-50800-98–030). The funders hadno role in study design, datacollection and analysis, decision topublish, or preparation of themanuscript.

Competing Interests: PV:Consultant, Merck, Pfizer; researchgrant, Cephalon, Schering-Plough,Pfizer, Merck, Basilea; speaker’sbureau: Gilead, Merck, Schering-Plough. Other authors: nocompeting interests declared.

Academic Editor: Chris Kibbler,Royal Free Hospital London, UnitedKingdom

Citation: Snelders E, van der LeeHAL, Kuijpers J, Rijs AJMM, Varga J,et al. (2008) Emergence of azoleresistance in Aspergillus fumigatusand spread of a single resistancemechanism. PLoS Med 5(11): e219.doi:10.1371/journal.pmed.0050219

Received: November 29, 2007Accepted: September 25, 2008Published: November 11, 2008

Copyright: ! 2008 Snelders et al.This is an open-access articledistributed under the terms of theCreative Commons AttributionLicense, which permits unrestricteduse, distribution, and reproductionin any medium, provided theoriginal author and source arecredited.

Abbreviations: ITZ, itraconazole;MIC, minimum inhibitoryconcentration; MTR, multiple-triazole-resistance

* To whom correspondence shouldbe addressed. E-mail: [email protected]

A B S T R A C T

Background

Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured frompatients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus isunknown. We investigated the prevalence and spread of azole resistance using our culturecollection that contained A. fumigatus isolates collected between 1994 and 2007.

Methods and Findings

We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatusisolates collected from 1,219 patients in our University Medical Centre over a 14-y period. Thespread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients,from 28 other medical centres in The Netherlands and 317 isolates from six other countries. Theisolates were characterized using phenotypic and molecular methods. The electronic patientfiles were used to determine the underlying conditions of the patients and the presence ofinvasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases wereobserved after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates alsoshowed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, andposaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with twocopies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be thedominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolateswere genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to beresponsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found inisolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which69% harboured the TR/L98H substitution, and in six isolates originating from four othercountries.

Conclusions

Azole resistance has emerged in A. fumigatus and might be more prevalent than currentlyacknowledged. The presence of a dominant resistance mechanism in clinical isolates suggeststhat isolates with this mechanism are spreading in our environment.

The Editors’ Summary of this article follows the references.

PLoS Medicine | www.plosmedicine.org November 2008 | Volume 5 | Issue 11 | e2191629

PLoSMEDICINE

Emergence of Azole Resistance in Aspergillusfumigatus and Spread of a Single ResistanceMechanismEveline Snelders1,2, Henrich A. L. van der Lee1,2, Judith Kuijpers1,2, Anthonius J. M. M. Rijs1,2, Janos Varga3,4,

Robert A. Samson3, Emilia Mellado5, A. Rogier T. Donders6, Willem J. G. Melchers1,2, Paul E. Verweij1,2*

1 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 2 Nijmegen Institute for Infectious Diseases, Inflammation

and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 3 Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Centre,

Utrecht, The Netherlands, 4 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Hungary, 5 Servicio de Micologia, Centro Nacional de

Microbiologia, Instituto de Salud Carlos III, Madrid, Spain, 6 Department of Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, Nijmegen, The

Netherlands

Funding: This work was funded inpart by The NetherlandsOrganisation for Health Researchand Development (ZonMw; grant:50-50800-98–030). The funders hadno role in study design, datacollection and analysis, decision topublish, or preparation of themanuscript.

Competing Interests: PV:Consultant, Merck, Pfizer; researchgrant, Cephalon, Schering-Plough,Pfizer, Merck, Basilea; speaker’sbureau: Gilead, Merck, Schering-Plough. Other authors: nocompeting interests declared.

Academic Editor: Chris Kibbler,Royal Free Hospital London, UnitedKingdom

Citation: Snelders E, van der LeeHAL, Kuijpers J, Rijs AJMM, Varga J,et al. (2008) Emergence of azoleresistance in Aspergillus fumigatusand spread of a single resistancemechanism. PLoS Med 5(11): e219.doi:10.1371/journal.pmed.0050219

Received: November 29, 2007Accepted: September 25, 2008Published: November 11, 2008

Copyright: ! 2008 Snelders et al.This is an open-access articledistributed under the terms of theCreative Commons AttributionLicense, which permits unrestricteduse, distribution, and reproductionin any medium, provided theoriginal author and source arecredited.

Abbreviations: ITZ, itraconazole;MIC, minimum inhibitoryconcentration; MTR, multiple-triazole-resistance

* To whom correspondence shouldbe addressed. E-mail: [email protected]

A B S T R A C T

Background

Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured frompatients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus isunknown. We investigated the prevalence and spread of azole resistance using our culturecollection that contained A. fumigatus isolates collected between 1994 and 2007.

Methods and Findings

We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatusisolates collected from 1,219 patients in our University Medical Centre over a 14-y period. Thespread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients,from 28 other medical centres in The Netherlands and 317 isolates from six other countries. Theisolates were characterized using phenotypic and molecular methods. The electronic patientfiles were used to determine the underlying conditions of the patients and the presence ofinvasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases wereobserved after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates alsoshowed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, andposaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with twocopies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be thedominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolateswere genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to beresponsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found inisolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which69% harboured the TR/L98H substitution, and in six isolates originating from four othercountries.

Conclusions

Azole resistance has emerged in A. fumigatus and might be more prevalent than currentlyacknowledged. The presence of a dominant resistance mechanism in clinical isolates suggeststhat isolates with this mechanism are spreading in our environment.

The Editors’ Summary of this article follows the references.

PLoS Medicine | www.plosmedicine.org November 2008 | Volume 5 | Issue 11 | e2191629

PLoSMEDICINE

1912 Isolats cliniques d’Af 1219 pa4ents Période de 14 ans 1 CHU Pays-‐Bas ì Prévalence Résistance

-‐ 0.6% en 1999 -‐ 6% en 2007

-‐-‐> 94% TR34/L98H

17

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

CURRENTOPINION Azole resistance in Aspergillus fumigatus:a growing public health concern

Edith Vermeulena, Katrien Lagroua,b, and Paul E. Verweijc

Purpose of reviewReports from the end of the 2000s forced the medical community to take azole resistance in Aspergillusfumigatus into account. Not only patients with chronic aspergillus disease, who develop resistance duringlong-term azole treatment, but also azole-naive patients are at risk, owing to the presence of resistantstrains in the environment. The purpose of this review is to overview the latest findings concerning theorigin, evolution, and implications of azole resistance in A. fumigatus.

Recent findingsTR34/L98H is the predominant resistance mechanism of environmental origin in A. fumigatus. Recentepidemiological data show that this mechanism is an expanding problem, with reports from China, Iran,and India. However, the TR34/L98H strains from the Middle East are genotypically different from theEuropean isolates; their emergence is, therefore, not due to simple geographical spread of the ‘European’isolates. A new environmental resistance mechanism, TR46/Y121F/T289A, was detected in theNetherlands, conferring voriconazole resistance. In patients chronically treated with triazoles, the spectrumof resistance has become more diverse, with the emergence of non-CYP51A-mediated mechanisms. Centralregistration of treatment and outcome data of patients with resistant aspergillus disease are needed.

SummaryAzole resistance in A. fumigatus is evolving to a global health problem.

Keywordsaspergillosis, Aspergillus fumigatus, CYP51A, drug resistance, fungal

INTRODUCTIONTriazoles are the mainstay of therapy in infectionswith the opportunistic fungus Aspergillus fumigatus.The emergence of resistance is, therefore, of clinicalconcern. The first reports of patients with azole-resistant A. fumigatus isolates date from 1997, frompatients receiving itraconazole therapy fromSweden [1] and California (isolates obtained in1989) [2]. The characterization of two genes(CYP51A and CYP51B) encoding the azole targetenzyme in A. fumigatus, sterol 14-a-demethylase,greatly contributed to the understanding of azoleresistance mechanisms [3]. In the first decade afterthe discovery of azole resistance inA. fumigatus, onlysporadic cases of resistance were published andresistance was considered an infrequent event.Two reports since the late 2000s changed this per-ception. First, in 2007, a series of Dutch patients –including azole-naive patients – were describedwithinvasive aspergillosis due to pan-azole-resistantstrains and resistance was attributable to one pre-dominant resistance mechanism, TR34/L98H [4].

This mechanism consists of a tandem repeat of 34bases (TR34) in the promotor of the CYP51A gene,leading to enhanced expression, combined with aleucine to histidine amino acid substitution (L98H)[4,5]. In 2009 a second report, from a specializedreferral center for patients with chronic and allergicaspergillosis in Manchester, described resistance tohave increased dramatically [6]. This situation dif-fered from the TR34/L98H-resistance problem in theNetherlands, as a variety of differentCYP51A-related

aDepartment of Microbiology and Immunology, Catholic University ofLeuven, bDepartment of Laboratory Medicine, University HospitalsLeuven, Leuven, Belgium and cDepartment of Medical Microbiology,Radboud University Nijmegen Medical Centre, Nijmegen, the Nether-lands

Correspondence to Paul E. Verweij, MD, PhD, UMC St RadboudMedicalMicrobiology, PO Box 9101, 6500 HB Nijmegen, the Netherlands.Tel: +31 24 361 43 56; fax: +31 24 354 02 16; e-mail: [email protected]

Curr Opin Infect Dis 2013, 26:493–500

DOI:10.1097/QCO.0000000000000005

0951-7375 ! 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins www.co-infectiousdiseases.com

REVIEW

MutaFon TR34/L98H chez Aspergillus fumigatus dans le monde

Dans 3 villes des Pays-‐Bas -‐ dec 2009

-‐ janv 2010 -‐ février 2010 3 isolats Af poussant en présence de voriconazole (CMI> 16

mg/ml) diminu;on de l’ac;vité de l’itraconazole et posaconazole

TR46/Y121F/T289A

van der Linden JW et al. CID 2013

Décembre 2009 à janvier 2011

à1315 isolats (921 pt) à 21 isolats/ 15 pt Haut niv de R Vorico à TR46/Y121F/T289A à 8 pt: essai Vorico = échec clinique à  Tous les pt sauf 1 étaient naifs d’Azolé à 6/10 prélèvements domiciles = +

Au total: 6.8% de résistance aux Azolés -‐ 47/63 (74.6%) TR34/L98H -‐ 13/63 (20.6%) TR46/Y121F/T289A -‐ 4.7% sans muta4on de Cyp51A

20

Tous les pa4ents avec des formes invasives traités par voriconazole sont décédés

Ceae muta4on a depuis été iden4fiée en Belgique, Allemagne, Danemark, Inde, Tanzanie et en 2014 un cas CHU Rouen !

21

Pearls

Emergence of Azole-Resistant Aspergillus fumigatusStrains due to Agricultural Azole Use Creates anIncreasing Threat to Human HealthAnuradha Chowdhary1*, Shallu Kathuria1, Jianping Xu2, Jacques F. Meis3,4

1Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, 2Department of Biology, McMaster University, Hamilton, Ontario,

Canada, 3Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands, 4Department of Medical Microbiology,

Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Aspergillus fumigatus, a ubiquitously distributed opportunisticpathogen, is the global leading cause of aspergillosis and causesone of the highest numbers of deaths among patients with fungalinfections [1]. Invasive aspergillosis is the most severe manifesta-tion with an overall annual incidence up to 10% in immunosup-pressed patients, whereas chronic pulmonary aspergillosis affectsabout 3 million, primarily immunocompetent, individuals eachyear [2]. Three triazole antifungals, namely itraconazole, vor-iconazole, and posaconazole, are recommended first-line drugs inthe treatment and prophylaxis of aspergillosis [3]. However, azoleresistance in A. fumigatus isolates is increasingly reported withvariable prevalence in Europe, the United States, South America,China, Japan, Iran, and India [4–9]. For example, about 10% ofstrains of A. fumigatus from the Netherlands are itraconazoleresistant, and in the United Kingdom, the frequency increasedfrom 0%–5% during 2002–2004 to 17%–20% in 2007–2009 [10–13]. In the ARTEMIS global surveillance program involving 62medical centers, 5.8% of A. fumigatus strains showed elevated MICsto one or more triazoles [5]. Similarly, the prospective SCARE(Surveillance Collaboration on Aspergillus Resistance in Europe)study involving 22 medical centers in 19 countries identified anoverall prevalence of 3.4% azole resistance. Azole-resistant A.fumigatus (ARAF) ranged from 0% to 26% among the 22 centresand was detected in 11 (57.9%) of the 19 participating Europeancountries [4 and P.E. Verweij, personal communication]. Inter-estingly, almost half (48.9%) of the ARAF isolates from theSCARE network in European countries were resistant to multipleazoles and harbored the TR34/L98H mutation in the cyp51A gene[4 and P.E. Verweij, personal communication]. Indeed, multi-azole resistance in A. fumigatus due to the TR34/L98H mutationshas become an emerging problem in both Europe and Asia andhas been associated with high rates of treatment failures [12–14].Azole antifungal drugs inhibit the ergosterol biosynthesis

pathway, specifically the cytochrome p450 sterol 14-a-demethy-lase encoded by the cyp51A gene, which leads to depletion ofergosterol and accumulation of toxic sterols. The majority ofARAF isolates contain alterations in the target enzyme and themutated target showed reduced or no binding to the drugs [15].While most mutations in ARAF isolates were single nucleotidesubstitutions in the target gene (cyp51A), mutations at other genessuch as the cdr1B have also been reported. For example, in theUnited Kingdom the frequency of ARAF isolates without cyp51Amutations has been reported to be more than 50% [16].

Routes of Azole Resistance Development

The epidemiologic data on azole resistance is mainly from twoclinical entities. One group comprises noninvasive diseasesincluding patients with allergic bronchopulmonary aspergillosis(ABPA), aspergilloma, and chronic pulmonary aspergillosis (CPA)

who were treated with long-term azole therapy (mainly itracon-azole) and developed acquired resistance after 1–30 months oftreatment [13]. In these patients, the ARAF isolates may beresistant to only itraconazole or exhibit a multi-azole-resistantphenotype. The underlying resistance mechanism commonlyinvolves point mutations in the cyp51A gene, indicating that inpatients exposed to long-term azole therapy, the fungus is capableof rapidly adapting to azole drug(s) [11–14]. The genotypicanalysis of serial isolates of A. fumigatus from patients with chronicaspergillosis revealed that the initial susceptible and later resistantisolates had the same genotype. The only changes were the specificmutations conferring azole resistance, consistent with the devel-opment of resistance arising from azole therapy [13].The second group of patients with ARAF are those with acute

aspergillosis but with no known prior exposure to azole drugs [12].In contrast to the first group in which de novo mutation of thefungus in cavitary lesions is the primary mechanism for thedevelopment of azole resistance, those of the second group likelyacquired ARAF strains from external environments. In fact 50%of the patients with invasive aspergillosis due to ARAF are knownto be azole naıve and the outcome of patients with azole-resistantinvasive aspergillosis has been dismal, with a mortality rate of 88%[12]. Eighty percent of the ARAF strains from patients withinvasive aspergillosis described in the SCARE network had theTR34/L98H mutations, which consist of a substitution of leucineto histidine at codon 98 of the cyp51A gene in combination with a34-bp tandem repeat in the promoter region. These mutationsenabled resistance to itraconazole and intermediate susceptibilityor resistance to voriconazole, posaconazole, or both [4,17,18]. Asdescribed above, although the environmentally derived azole-resistant strains are predominately associated with acute invasiveinfections [12,19], the same mechanism has also been reported in

Citation: Chowdhary A, Kathuria S, Xu J, Meis JF (2013) Emergence of Azole-Resistant Aspergillus fumigatus Strains due to Agricultural Azole Use Creates anIncreasing Threat to Human Health. PLoS Pathog 9(10): e1003633. doi:10.1371/journal.ppat.1003633

Editor: Joseph Heitman, Duke University Medical Center, United States ofAmerica

Published October 24, 2013

Copyright: ! 2013 Chowdhary et al. This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

Funding: The authors received no specific funding for this study.

Competing Interests: JFM received grants from Astellas, Basilea, and Merck. Hehas been a consultant to Astellas, Basilea, and Merck and received speaker’s feesfrom Merck and Gilead. All other authors have declared that no competinginterests exist. This does not alter our adherence to all PLOS Pathogens policieson sharing data and materials.

* E-mail: [email protected]

PLOS Pathogens | www.plospathogens.org 1 October 2013 | Volume 9 | Issue 10 | e1003633

Tanzanie

Avant d’aborder l’épidémiologie de la Résistance d’Af aux Azolés dans le monde et en France……………

22

23

M A J O R A R T I C L E

High-frequency Triazole Resistance Found InNonculturable Aspergillus fumigatus from Lungsof Patients with Chronic Fungal Disease

David W. Denning,1,2,3 Steven Park,4 Cornelia Lass-Florl,5 Marcin G. Fraczek,2,3 Marie Kirwan,1,2 Robin Gore,2Jaclyn Smith,2 Ahmed Bueid,2 Caroline B. Moore,3 Paul Bowyer,2 and David S. Perlin2,4

1National Aspergillosis Centre, 2School of Translational Medicine, University of Manchester, Manchester, UK, 3Mycology Reference Centre,Manchester Academic Health Science Centre, University Hospital of South Manchester, Manchester, UK, 4Public Health Research Institute, New JerseyMedical School-UMDNJ, Newark, New Jersey, and 5Department fur Hygiene, Mikrobiologie und Sozalmedizin, Medizinische Universitat Innsbruck,Innsbruck, Austria

Background. Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), andchronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally.Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequencyof triazole resistance.

Methods. Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., weassessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotidepolymorphisms (SNPs) associated with triazole resistance.

Results. Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with cultureor microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] andM220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%)with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequateplasma drug concentrations around the time of sampling.

Conclusions. The very low organism burdens of fungi causing infection have previously prevented directculture and detection of antifungal resistance in clinical samples. These findings have major implications for thesustainability of triazoles for human antifungal therapy.

Aspergillus spp. cause diseases ranging from invasive

pulmonary aspergillosis (IPA) in immunocompromised

patients to chronic pulmonary aspergillosis (CPA) and

fungal allergic diseases, including allergic broncho-

pulmonary aspergillosis (ABPA) and increased severity

of asthma (severe asthma with fungal sensitization

[SAFS]) [1, 2]. Millions of individuals worldwide are

affected or at risk; recent estimates indicate approxi-

mately 3 million patients with CPA, 3 million with

ABPA and over 10 million with SAFS [3]. Exposure to

hundreds of Aspergillus fumigatus conidia is a universal,

daily occurrence. Conidia shift from being anergic to the

human immune system [4] to producing the largest

number of documented allergens of any other living

organism on germination [5].

Antifungal therapy with triazoles is recommended

for patients with ABPA, CPA, and IPA [6]. There are

three licensed triazole compounds highly active against

Aspergillus spp.—itraconazole, voriconazole, and pos-

aconazole [7]. However, triazole resistance has emerged

Received 9 September 2010; accepted 22 February 2011.Correspondence: David W. Denning, MD, FRCP, 2nd Floor Education and

Research Centre, Wythenshawe Hospital, Southmoor Road, Manchester M23 9LT,UK ([email protected]).

Clinical Infectious Diseases 2011;52(9):1123–1129! The Author 2011. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]/2011/529-0001$37.00DOI: 10.1093/cid/cir179

Triazole Resistance in Human Samples d CID 2011:52 (1 May) d 1123

M A J O R A R T I C L E

High-frequency Triazole Resistance Found InNonculturable Aspergillus fumigatus from Lungsof Patients with Chronic Fungal Disease

David W. Denning,1,2,3 Steven Park,4 Cornelia Lass-Florl,5 Marcin G. Fraczek,2,3 Marie Kirwan,1,2 Robin Gore,2Jaclyn Smith,2 Ahmed Bueid,2 Caroline B. Moore,3 Paul Bowyer,2 and David S. Perlin2,4

1National Aspergillosis Centre, 2School of Translational Medicine, University of Manchester, Manchester, UK, 3Mycology Reference Centre,Manchester Academic Health Science Centre, University Hospital of South Manchester, Manchester, UK, 4Public Health Research Institute, New JerseyMedical School-UMDNJ, Newark, New Jersey, and 5Department fur Hygiene, Mikrobiologie und Sozalmedizin, Medizinische Universitat Innsbruck,Innsbruck, Austria

Background. Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), andchronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally.Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequencyof triazole resistance.

Methods. Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., weassessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotidepolymorphisms (SNPs) associated with triazole resistance.

Results. Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with cultureor microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] andM220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%)with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequateplasma drug concentrations around the time of sampling.

Conclusions. The very low organism burdens of fungi causing infection have previously prevented directculture and detection of antifungal resistance in clinical samples. These findings have major implications for thesustainability of triazoles for human antifungal therapy.

Aspergillus spp. cause diseases ranging from invasive

pulmonary aspergillosis (IPA) in immunocompromised

patients to chronic pulmonary aspergillosis (CPA) and

fungal allergic diseases, including allergic broncho-

pulmonary aspergillosis (ABPA) and increased severity

of asthma (severe asthma with fungal sensitization

[SAFS]) [1, 2]. Millions of individuals worldwide are

affected or at risk; recent estimates indicate approxi-

mately 3 million patients with CPA, 3 million with

ABPA and over 10 million with SAFS [3]. Exposure to

hundreds of Aspergillus fumigatus conidia is a universal,

daily occurrence. Conidia shift from being anergic to the

human immune system [4] to producing the largest

number of documented allergens of any other living

organism on germination [5].

Antifungal therapy with triazoles is recommended

for patients with ABPA, CPA, and IPA [6]. There are

three licensed triazole compounds highly active against

Aspergillus spp.—itraconazole, voriconazole, and pos-

aconazole [7]. However, triazole resistance has emerged

Received 9 September 2010; accepted 22 February 2011.Correspondence: David W. Denning, MD, FRCP, 2nd Floor Education and

Research Centre, Wythenshawe Hospital, Southmoor Road, Manchester M23 9LT,UK ([email protected]).

Clinical Infectious Diseases 2011;52(9):1123–1129! The Author 2011. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]/2011/529-0001$37.00DOI: 10.1093/cid/cir179


§  Cultures et RT-‐PCR pour Aspergillus sp. sur LBA et ECBC §  Si PCR+ et culture néga4ve à séquençage du gène CYP51A

à la recherche de SNPs associés à des résistances connues

CYP51A gene in two !900 bp fragments. Fragment 1 (876 bp)

covered the promoter tandem repeat region to codon 98. The

second amplicon (748 bp) covered codons 54 to 266. The

amplified products were evaluated in a real-time assay with

allele-specific molecular beacon directed at key single-nucleotide

polymorphisms (SNPs) linked with azole resistance (G54, L981

promoter tandem repeat [TR], G138, and M220). All results

were confirmed by DNA sequencing. Patients’ notes were re-

viewed for their antifungal treatment. Resistance data were not

used for clinical decision making.

RESULTS

Extraction of Aspergillus DNA from Respiratory SamplesExtraction of sufficient fungal DNA for molecular detection

is the most challenging technical aspect of PCR for fungi.

The combination of very few fungal cells in a clinical sample

and a sturdy cell wall requiring fracture for DNA release is

problematic. We utilized an optimized bead-beating approach

to break open cells, preceded by a digestion step. Overall,

10% efficiency from unswollen conidia was demonstrated

(Supplementary Figure S1).

Detection of Aspergillus DNA in Volunteers with PCRTo better understand Aspergillus burdens in the lungs of healthy

individuals, we tested BAL from 11 normal adults who un-

derwent bronchoscopy. Of these, 4 culture-negative samples

(36.4%) had detectable signals in the PCR assay (Table 1). No

signal was detected in 7 samples (63.6%), of which one grew

Penicillium spp. (3 morphologies) and 1 Paecilomyces spp. The

positive Ct values ranged from 36.2 to 34.3 (Figure 1), consistent

with Aspergillus spp. being present in normal lungs.

PCR in Invasive Pulmonary AspergillosisWe analyzed 22 samples from patients with IPA with myco-

logical confirmation. Of the 22 samples, 20 (90.9%) had hyphae

consistent with Aspergillus spp. visible on microscopy. All

22 (100%) were culture-positive for a filamentous fungus, 10

for A fumigatus, 9 for A terreus, and 2 for Penicillium spp., and 1

grew A niger, Rhizopus oryzae, and Lichtheimia corymbifera

(PCR-negative). Five of the patients had proven and 17 probable

IPA in the context of typical immunocompromising conditions,

including organ transplant (n 5 10) and acute leukemia. Using

the normal volunteer data as negative controls and a Ct cut-off

of 36, the sensitivity was 94%, specificity 91%, positive pre-

dictive value 97%, and negative predictive value 83%. Seventeen

patients (77.3%) had received some antifungal prophylaxis

or therapy. Aspergillus DNA was detected by PCR in 21

(95.5%) samples (Table 1) with Ct values ranging from 20.5 to

33.7 (Figure 1). Both samples that grew Penicillium were PCR-

positive. Furthermore, in these 22 samples, the signal strength

was generally much stronger than that in the normal volunteers,

indicative of a greater load of Aspergillus in IPA than in normal

people.

PCR in Chronic and Allergic AspergillosisIn spontaneously produced sputum from patients with ABPA,

SAFS, and CPA, we detected Aspergillus DNA much more fre-

quently than cultures were positive. In the ABPA patients, all

cultures were negative despite strongly positive immunoglobin

E (IgE) serology for A fumigatus. AspergillusDNA was detectable

by PCR in 15 of 19 (78.9%) ABPA patients (Table 1), 11 of these

samples having strong PCR signals (Figure 1). Among the 42

patients with CPA, all of whom had detectable Aspergillus IgG

antibodies and grossly abnormal chest radiographs, 7 (16.7%)

had a positive culture for A fumigatus and 30 (71.4%) had

Aspergillus DNA detectable by PCR (Table 1). In patients with

CPA, stronger PCR signals were generally seen in those with

positive cultures.

Direct Detection of Azole ResistanceWe selected DNA from the first 25 sputum samples obtained from

ABPA and CPA patients that were PCR-positive, culture-negative,

as well as 4 culture-positive, PCR-positive samples patients with

CPA (Supplementary Table S1). No G54 or M138 mutations were

found. Four samples had M220 mutations: 2 were M220K and 2

M220R cyp51A substitutions on sequencing. Twenty-seven of 29

(93.1%) had an L98H mutation, and 16 (55.2%) also had an

upstream 34 bp TR, the combination conferring itraconazole and

voriconazole resistance [10]. The TR was found without the L98H

mutation in 2 samples. Two samples had an M220R mutation

with both the TR and L98H mutation. Of the 4 culture-positive

Table 1. Aspergillus Culture, qPCR, and A fumigatus Resistance Mutation Detection in 4 Study Populations

Laboratory result ABPA CPA IPA Normals

Culture positive for Aspergillus spp. 0/19 7/42 (16.7%) 20/22 (90.9%) 0/11

Culture positive for A fumigatus 0/19 7/42 (16.7%) 10/22 (45.5%) 0/11

qPCR positive for Aspergillus spp 15/19 (78.9%) 30/42 (71.4%) 21/22 (95.5%) 4/11 (36.4%)

A. fumigatus CYP51A mutation detecteddirectly from qPCR-positive sample

6/8 (75%) 12/24 (50%) NTa NTa

NOTE. qPCR indicates quantitative polymerase chain reaction; ABPA, allergic bronchopulmonary aspergillosis; CPA, chronic pulmonary aspergillosis; IPA,invasive pulmonary aspergillosis.

a NT indicates not tested (insufficient sample remaining).


•  Des échecs cliniques : -‐ 14 pt TR34/L98H

§  3 perdus de vue §  6 en échec sous voriconazole ou itraconazole

-‐ 3 des 4 pt M220: échec thérapeu4que (1 itraco,3 posaconale)

24

La présence de résistances pourrait expliquer le faible taux de réponse au traitement dans les ABPA et CPA

Le point épidémiologique

Aspergillus Résistant aux Azolés

Où en somme-‐nous dans le monde et en France ?

25

Une franche hétérogénéité du problème

•  Au Etats-‐Unis

-‐ les CMI à l’itraconazole sont généralement basses à 5% des Af avec des CMI augmentées: parfois aucune muta4on de CYP51A mécanismes ? non clairement élucidés, sembleraient faire intervenir des changement au niveau des pompes de transport transmembranaires

-‐ la muta4on TR34/L98H n’a jamais été iden4fiée à les agriculteurs américains u4lise une quan4té moindre de DMI dans leurs cultures que leurs homologues européens

Pham CD. Emerg Infect Dis. sept 2014;20(9):1498‑503.

26

Une franche hétérogénéité du problème

•  En Espagne données rassurantes en 2008 –  CMI au voriconazole de 400 souches cliniques d’Aspergillus sp. avant et après (2002) l’introduc4on de ce traitement.

–  281 pt dont 51 (18.1%) ont une AI probable ou prouvée.

27

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2008, p. 3444–3446 Vol. 52, No. 90066-4804/08/$08.00!0 doi:10.1128/AAC.00629-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Clinical Isolates of Aspergillus Species Remain Fully Susceptible toVoriconazole in the Post-Voriconazole Era!

Jesus Guinea,1,2* Sandra Recio,1 Teresa Pelaez,1,2 Marta Torres-Narbona,1 and Emilio Bouza1,2

Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Maranon, Universidad Complutense deMadrid, Madrid, Spain,1 and CIBER de Enfermedades Respiratorias (CIBER RES CD06/06/0058), Palma de Mallorca, Spain2

Received 14 May 2008/Returned for modification 3 June 2008/Accepted 28 June 2008

We studied the activity of voriconazole against 400 clinical strains of Aspergillus from the pre-voriconazole(1999 to 2002) and post-voriconazole (2003 to 2007) periods. Although the mean MICs of strains from thepost-voriconazole period were slightly higher (0.39 versus 0.57 !g/ml; P < 0.001), all strains were susceptibleto voriconazole and presented an MIC of <2 !g/ml.

Based on both in vitro and clinical data, voriconazole hasbecome the drug of choice for the treatment of invasive as-pergillosis (1, 8, 10, 12).

The results of different in vitro studies showed that the vastmajority of Aspergillus clinical strains are fully susceptible tothe new triazoles, including voriconazole (1, 2, 8, 9, 11–13).However, the antifungal activity of voriconazole may havechanged since it began to be used in the clinical setting.

We analyzed the in vitro antifungal activity of voricon-azole against 400 clinical Aspergillus strains collected beforeand after its introduction in our institution (November2002). We also examined the role of previous treatment withitraconazole and/or voriconazole in the appearance ofstrains of Aspergillus with diminished antifungal susceptibil-ity to voriconazole.

Part of this work was presented at the 18th ECCMID (Eu-ropean Congress on Clinical Microbiology and Infectious Dis-eases), Barcelona, Spain, 2008 (poster P 1360).

Organisms, source of samples, and period of study. Thestrains were from 281 patients, of whom 51 (18.1%) hadproven or probable invasive aspergillosis according to the Eu-ropean Organization for Research and Treatment of Cancer

(EORTC) criteria. The species distribution of the strains an-alyzed was as follows: Aspergillus fumigatus (n " 374), Aspergil-lus terreus (n " 20), Aspergillus niger (n " 3), and Aspergillusflavus (n " 3). As for the source of the strains, 308 were fromrespiratory samples and 98 were from patients with invasiveaspergillosis.

The isolates were grouped by period: those isolated duringthe period before the introduction of voriconazole (pre-vori-conazole, 1999 to 2002) and those isolated after its introduc-tion (post-voriconazole, 2003 to 2007). Both periods were com-parable in terms of number of patients (143 versus 138),number of cases of proven/probable invasive aspergillosis (27versus 24), and number of isolates (197 versus 203). Somepatients had never received voriconazole or itraconazole, somehad received it recently, and some were even taking it when thestrains were isolated.

Analysis of the antifungal susceptibility of the strains. Theantifungal activity of voriconazole (Pfizer PharmaceuticalGroup, New York, NY) was determined by using the CLSI(formerly NCCLS) M38-A standard (4). All trays used in theassay were prepared at the same time, and all strains from bothperiods were tested using the same batch of trays.

* Corresponding author. Mailing address: Servicio de MicrobiologıaClınica y Enfermedades Infecciosas, Hospital General UniversitarioGregorio Maranon, C/Dr. Esquerdo 46, 28007 Madrid (Spain). Phone:34915867163. Fax: 34915044906. E-mail: [email protected].

! Published ahead of print on 7 July 2008.

TABLE 1. In vitro activity of voriconazole against clinical isolates of Aspergillus speciesa

Period

Activity of voriconazoleb (#g/ml) against:

All strains per period Strains per patient groupc

No. ofstrains MIC90 MIC50

GM ofMIC Range No. of

patients MIC90 MIC50GM ofMIC Range

Pre-voriconazole 197 0.5 0.25 0.39 0.125–1 143 0.5 0.25 0.40 0.125–1Post-voriconazole 203 1 0.5 0.57 0.125–2 138 1 0.5 0.61 0.125–2

Overall 400 1 0.5 0.48 0.125–2 281 1 0.5 0.51 0.125–2a Four hundred isolates were tested overall and for each study period using the CLSI M-38A procedure.b MIC endpoint for voriconazole and Aspergillus spp. was defined as the lowest concentration that produced complete inhibition of growth after 48 h of incubation.

GM, geometric mean.c In patients with multiple isolates, only the highest MIC was chosen for analysis.

3444

on Novem

ber 8, 2014 by guesthttp://aac.asm

.org/D

ownloaded from

Ø  Pas de souche résistante au vorico Ø  Augmenta4on des CMI depuis vorico Ø  Pas de corréla4on entre échec et CMI > 1

Guinea j. Agents Chemother 2008,52(9):3444

•  Dans les pays du nord de l’Europe

Pays-‐Bas Allemagne Danemark

4 – 5 – 10% selon les séries ! avec une large prédominance de TR34/L98H et une mortalité rapportée dans les formes invasives > 90% !

28

Une franche hétérogénicité du problème

Vermeulen E. Cur op in infect dis 2013 Van der Linden JW et al. CID 2013

ECDC 2013

En France 6 études (dont 2 pas encore publiées)

29

Prospectif,,12,centres,

1,an,Sept,2013,E.Dannaoui)et)al.)ICAAC)2014)

En,février:,1029,isolats,,847,exploitables,!,,9,Résistants,(7pt*),(1%):,8x,

TR34/L98H,,1x,7121F,

Et.,prospective,1,an,

2012,Mondor/Créteil,Choukri)F.)et)al.)J)of)Mycol,)Mars)2014)

165,isolats,d’A.fumigatus,(130,pt),

3,pt,avec,A.fumigatus,panTR,(aspergillome,,colonization,BPCO,et,ABPA),

dont,2,TR34/L98H,et,1,sans,mutation,identifiée,sur,cyp51A.,,Prévalence,1.9%,

Patients,d’Hématologie,Paris,Alanio a. et al. JAC 2011)

118,pt,!117,S,azolés,,1,R,chez,un,patient,jamais,exposé,aux,azolés,,Pas,de,différence,de,S,aux,azolés,en,fonction,de,l’exposition,antérieure,aux,

Azolés,,

Prévalence0.85%,

Mucoviscidose,F.)Morio)et)al.)JAC 2012,

8%,(4/50,pts,),Af.,R.,Azolés,!,Toujours,mutation,CYP51A,,,

¾,soit,6%,pop,de,muco,TR34/L98H,

Mucoviscidose,

Juin,2010,à,avril,2011,Burgel)et)al.)AAC)2012)

ECBC,de,patients,muco:,249,patients,!,285/570,(50%),ECBC,+,à,A.f.)soit,131pt/249,(52.6%),,

!,6/131,pt,avaient,un,isolat,R,itraco,et,R,Posaco(4.6%),,vorico,3x,touché,!,2,étaient,TR34/L98H,

T,20%,Af,R,chez,patients,récemment,exposés,aux,azolés,!!!,

Transpl.,pulmonaires,

APHP,T,12,ans,,,,F.Choukri)et)al.))

Résultats,préliminaires:,3,pt,Af,R,azolés,dt,2,TR34/L98H,

Case,report,Hématologie,Rocchi)S)et)al.)JCM.)Besançon,

1,fermier,Français,Allogreffé,CSH,,a,développé,une,AI,TR34/L98H,,,

,

CONCLUSION

•  2 phénomènes responsables de l’émergence de résistances aux Azolés –  Exposi4on au long court à aspergilloses chroniques –  Contamina4on depuis l’environnement à AI/aspergilloses chroniques TR34/L98H , TR46/Y121F/T289A

•  Epidémiologie –  Un phénomène sous-‐esFmé –  Des inégalités de réparFFon avec une menace en provenance de

l’environnement •  qui nous vient du nord…forte prévalence dans les pays du nord (Pays bas, Danemark, Allemagne) avec extension à tous les pays d’europe, Inde et Afrique

•  Liée à l’uFlisaFon des DMI agriculture: cross résistance prouvée •  Résistance par muta4on TR34/L98H, TR46/Y121F/T289A •  Menace galopante

–  Echec cliniques ! –  Rapport ECDC 2013 ALARMANT

30

Perspec8ves

•  Veille sanitaire recommandée par ECDC

•  Améliora4on de nos méthodes de détec4on des Af Résistants –  PCR –  Séquençage

•  Vers une évolu4on de nos stratégies thérapeu4ques ?

•  Pression sur la poli4que d’u4lisa4on des ATF en agriculture….

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Merci de votre attention

Documents

Emergence(( d’Aspergillus fumigatus*splf.fr/wp-content/uploads/2015/01/VM-At3-RissoK.pdf · Aspergillus sp. • Champignon’ﬁlamenteux’de’l’environnement ’ • Responsable’en’pathologie’humaine’:’