Antifungal Drug Resistance: a Cause for Concern?

Transcription

1 Antifungal Drug Resistance: a Cause for Concern? Sharon Chen Centre for Infectious Diseases and Microbiology CIDM-PH, CRE in Critical Infections, June 2014

2 Vis-à-vis bacteria - lesser scale and emotive response No plasmids and transposons

3 Outline for today Definition of antifungal resistance Overview of epidemiology, burden Candida & azoles, echinocandins Aspergillus & azoles Mechanisms of resistance Does genotypic resistance = phenotypic resistance Whether MICs are useful Detection methods and derivations of breakpoints

4 Global burden: serious infections (Global Action Fund for Fungal Infections) Infection Fatality rate Estimated deaths p.a. Cryptococcal meningitis Invasive aspergillosis Chronic lung IA 15-20% USA >50% Africa 50% (on treatment) Comments 600,000 CDC estimate >100,000 Many missed diagnoses 15% >450,000 Under-diagnosed Candidemia 40% (untreated) >120,000 Pneumocystis 15% (AIDS) 40-50% (non- AIDS) >80,000 Most cases in Africa not diagnosed TOTAL >1,350,000 Significant D. Denning, UK: underestimate

5 Meningitis Exserohilum rostratum multi-state outbreak USA Fungal meningitis cases in USA linked to contaminated methyl prednisolone injections produced by the New England Compounding Center * As As of Oct of 8 23 th Nov rd CDC website Exserohilum rostratum Keratitis caused by Fusarium species MRL in discussion with MHRA * * 14,000 injected 751 total cases 384 fungal meningitis 325 paraspinal only 33 peripheral joint 64 deaths (Accessed Nov 2013). Brilliant blue, triamcinolone injections Bipolaris hawaiiensis Mikosz et al, Emerg Infect Dis Feb 2014 Slide: E.Johnson, Bristol UK

6 Definition(s) of resistance Microbiological Definition Susceptible: Growth of fungus is inhibited by drug at concentration in the range for wild-type (WT) strains (no acquired or intrinsic resistance mechanisms) I.e. = or below susceptibility breakpoint MIC Resistance: Growth of fungus is inhibited by drug at concentration that exceeds the MIC breakpoint Can be primary (intrinsic) or secondary (acquired) Kanafani and Perfect, CID 2008

7 Definition of resistance Clinical Definition Susceptible: inhibition of organism by a concentration of the drug = associated with high likelihood therapeutic success Resistant: Failure to eradicate the infecting organism by the drug despite in vitro activity against the organism = associated with high likelihood therapeutic failure Kanafani and Perfect, CID 2008

8 Susceptibility and resistance: what does it mean Composite definition: CLSI & EUCAST Susceptible: Isolate inhibited by usually achievable drug concentration when recommended dosage is used for that site of infection. Resistant: Isolate not inhibited by usually achievable drug concentrations with normal dosage schedules and/or demonstrate MICs where specific resistance mechanisms likely and clinical efficacy against isolate not been shown Turnidge and Paterson, ClIn Microbiol Rev 2007

9 1. Candida and azole resistance Fluconazole (FLU; data most robust) Voriconazole (VRC); data on other azoles Pan azole The last occurs in animals eg. Brazilian porcupine, and in soil (C. tropicalis, Japan) Azole MICs ug/ml Coendou prehensilis Castelo-Branco et al, Med Mycol 2013; Yang et al., Plos One 2012

10 Estimated resistance: Candida and fluconazole by region Country Year Main species Inc. per 10 3 adm % FLU resistant % FLU- R nonalbicans Australia 2006 C. albicans S. Africa 2013 C. albicans India 2012 C. tropicalis Denmark 2013 C. albicans Spain 2005 C. albicans England 2012 C. albicans Brazil 2013 C. albicans USA 2012 C. albicans ANZMIG, 2013; WHO, June 2013

11 Mode of action of azole antifungals Fungal Cell membrane acetyl-co-a squalenes azoles lanosterol ergosterol Cellular target cytochrome P demethylase (Erg 11p is the product of ERG 11 gene, Cryp 51A in Aspergillus) Disruption of membrane structure and function and accumulation of toxic metabolites Slide: Elizabeth Johnson, Bristol, UK

12 Mechanisms of azole resistance 4 major mechanisms: may co-exist in any 1 strain Decreased drug concentration at site of action/ target: active efflux pumps in fungal cell membrane Target site alteration (lanosterol steroldemethylase) Up-regulation of target enzyme (rel. minor, no > 5 X incr. expression in resistant isolates) Alteration in sterol composition (minor mechanism) Can occur in sequence leading to additive effects Kanafani and Perfect, CID 2008

13 Decreased susceptibility due to reduced drug concentration Induction of efflux pumps is important Encoded by 2 main families of gene transporters (CDR, MDR, CFTR, YCF) CDR genes: ATP binding cassette family (resistance to all azoles; sufficient for resistance in itself) MDR genes: major facilitator (proton motive force) (more selective for FLU) C. albicans: CDR1, CDR2, MDR1 (BENr) C. glabrata: CgCDR1, CgCDR2 C. dubliniensis: CdCDR1, CdMDR1 Gene deletion and mrrna expression experiments

14 Pump up-regulation Constituitive or transient (induced by antifungals, corticosteroids, H 2 O 2 ) Mutation of genes that up-regulate transporter genes CDR1 and CDR2 promoters: common drug responsive regulatory element (DRE) critical in resistance (C. albicans) PDRE (pleotropic drug responsive elements) lead to high constitutive expression of CgCDR1 and CgCDR2 correlates with clinical resistance

15 Target site alteration Erg11p is the azole target (C14 - -demethylase) Point mutations in ERG11 results in decrease affinity for target, cannot bind azole > 80 amino acid substitutions (matched pairs of S and R isolates) Co-existence, > 1 mutation with additive effects. Detection methods: DNA sequencing or functional expression of ERG11 alleles eg. in S. cerevisiae Paired isolates to prove resistance Note: 2 alleles diploid state

16 C. albicans Erg11p: modelling Red: impt azole affinity Gly464 to Ser Arg 467 to Lys Ser405 to Phe Tyr132 to His (Y132H) Green: haem Blue: subs. in azole-r and azole-s strains

17 CDR pumps and ERG11 mutations have role in resistance phenotype Strain CDR ERG11 VRC MIC FLU MIC 1 Basal WT/WT Increase G464S/G464S Basal G464S/G464S Basal G464S/WT Increase WT/WT Key finding: MICs were highest in strain with CDR over expression and ERG11 mutations in both alleles; C. albicans Additive nature of resistance mechanism. MICs in mg/l MacCallum et al., AAC 2010;54

18 Genetics of azole resistance Are important and relevant but.. Difficult to implement routinely since no direct relationship with clinical resistance or outcome although able to trace resistance mechanistically Exception of pan azole resistance in C. glabrata: over expression CgCDR1, CgCDR2

19 For now we still need the MIC (phenotype) Reference CLSI, EUCAST methods are harmonized for Candida (azoles, echinocandins) Both discriminate between S and R. Both provide species-specific clinical breakpoints (FLU and VRC) wrt WT MIC distribution (ECV) drug PK/PD Resistance mechanisms clinical outcomes (hence clinically relevant)

20 Fluconazole and Candida Case in point

21 Summary FLU MICs: predicting outcome in candidemia and mucosal candidiasis (CLSI) C. albicans, C. tropicalis, C. parapsilosis: Pfaller et al. Drug Resist Updates 2010; 13: Outcome at MIC MIC (µg/ml) No. of Events 24-h 48-h % Success No. of Events % Success < > Similar results by EUCAST

22 New 24-H CLSI breakpoints for azoles: C. albicans, C. tropicalis, C. parapsilosis MIC (mg/l) defining: Drug S S-DD/I R Fluconazole < 2 (8) 4 > 8 (64) Voriconazole < 0.12 (1) >1.0 (4) Pfaller et al., * Drug Resist Updates 2010; 13: ; Pfaller et al., DMID 2011;70:

23 Australia: C. albicans (n=272) MIC 90 = 2 mg/l modal MIC 1 mg/l Sensititre Yeast One Van Hal S et al, JAC 2014

24 CART analysis: MIC as dichotomous variable vs. infection related deaths P <0.05 Van Hal et al., JAC 2014

25 C. glabrata: Inspires fear New 24-H CLSI Breakpoints for azoles Similar outcome studies MIC (mg/l) defining: Drug S S-DD R/NS Fluconazole - < 32 > 64 Voriconazole * < > 1 * ECV only max. doses FLU used 12 mg/kg or minimum 800 mg/d

26 EUCAST: no breakpoint, poor target for FLU Only 80% of isolates have MICs that are less than the ECV of 32 Slide adapted from M. Arendrup, SSI, 2011

27 C. glabrata FLU resistance: (new CBPs) Country C.glabrata (%) % FLU resistance % other azole resistance ARTEMIS USA Approx. 10 SENTRY USA (VRC) SENTRY Europe (VRC) SENTRY Asia-Pacific (VRC) SENTRY Total SENTRY Latin America (VRC) POS (3.5), VRC (10.5) ACS Australia VRC Results: those > ECV Pfaller et al., JCM 2013; 51:2571

28 Voriconazole and Candida

29 Summary analysis: VRC MIC ranges predicting outcome in invasive candidiasis (CLSI) for C. albicans, C. tropicalis, C. parapsilosis: Pfaller et al. Diagn Microbiol Infect Dis 2011 MIC (µg/ml) No. of Events Outcome at MIC 24-h 48-h % No. of Success Events % Success < >

30 New 24-H CLSI breakpoints for azoles: C. albicans, C. tropicalis, C. parapsilosis MIC (mg/l) defining: Drug S S-DD/I R Fluconazole < 2 4 > 8 Voriconazole < >1.0 Pfaller et al., * Drug Resist Updates 2010; 13: ; Pfaller et al., DMID 2011;70:

31 C. glabrata: poor clinical response, lack of relationship between MIC and outcome precludes breakpoint MIC (mg/l) defining: Drug S S-DD R/NS Fluconazole - < 32 > 64 Voriconazole * < > 1 * ECV only differentiate WT vs. non-wt for resistance surveillance. Pfaller et al., Diagn Microbiol Infect Dis 2011; 70

32 2. Candida and echinocandins Inhibits glucan synthesis (Yeasts: β-1,3 & 1,6; moulds: β-1,3 & 1,4 links) Cell lysis in yeasts, aberrant growth in moulds Encoded by FKS1, FKS2, FKS3 (catalytic subunit) controlled by RHO1 (Rho1p) In Candida, resistance associated with point mutations in these genes (incr. IC 50 and K i )

33 Caspofungin resistance mutations AA Fks1p F641-P649 HSP HSP2 2 regions of Fks1p are associated with acquired resistance, but region around Ser 645 is most significant Seen in C. albicans, C. glabrata (HSP2 also), C. krusei Confers resistance to entire class (can vary with drug) Perlin, D.S Drug Resistance Updates. 10:121-

34 CLSI echinocandin breakpoints: Candida Designed to identify FKS mutants from WT Is specificity of breakpoints too low to be useful? Pfaller et al., DMID 2011; 70: 330

35 C. glabrata: CLSI breakpoints No. Drug CBP S I R strains no.(%) no.(%) no.(%) 169 ANID <= (2)* 4 (3) 30 (27) CASPO <= (1) 5 (3) 35 (26) MICA <= (1) 6 (3) 29 (26) AUS: R = 12.5% I = intermediate; R = resistant, S = susceptible *no. of strains with fks mutations in parentheses Data from Pfaller et al., DMID 2011; 14: ; Castanheira et al, AAC Jan 2014

36 Determining presence of FKS mutants is a better idea Induced by prior echinocandin exposure FKS mutants less likely to respond to echinocandins FKS mutants independent risk factor for failure of echinocandin therapy for IC due to C. glabrata For C. glabrata: Caspo MIC of 0.5 mg/l rather than 0.12 mg/l, or Mica MIC of > 0.06 mg/l has been associated with mutations hence may be proxy for FKS mutations Alexander et al, CID 2013; 56; Shields et al, AAC 2012; 56

37 Detecting echinocandin resistance Emerging consensus that FKS genotype is a better predictive of clinical response than MIC alone Real time PCR, allele-specific molecular beacons Sequenom High throughput sequencing Microarrays (C. glabrata, +++ FKS mutations) Garcia-Effron G et al., AAC 2009; 53:

38 Echinocandin Resistance in Candida glabrata Increasing echinocandin co-resistance among already fluconazole resistant C. glabrata isolates (SENTRY) no echinocandin resistance detected % echinocandin co-resistance NMRC Data Flu-R C. glabrata Isolates All C. glabrata isolates No. Isolates Caspofungin Resistance No. (%) Anidulafungin Resistance No. (%) Micafungin Resistance No. (%) (16%) 4 (2.8%) 5 (3.5%) /640 (13%) 16/492 (3.3%) 13/491 (2.6%) 1 Pfaller et al. J Clin Microbiol ; SENTRY Study: all C glabrata: 16.7 % resistance to echinocandins: 33% resistant also to fluconazole - OLDER STUDY

39 Azole resistance: Aspergillus fumigatus Increasingly recognised esp. environmental CYP51A mutations esp. M220 (all azoles), G54 (ITC and POS) amino acid substitutions Mutations in promoter region of CYP51A Predominant mechanism TR 34 /L98H Mutation in L98H coupled with duplication of fragment in the promoter (34 bp tandem repeat, TR) up-regulates expression of CYP51A > 8 fold - multi-azole resistance - simple PCR-RFLP assays Linked to treatment failure Ahmad et al., JCM, epub April 2014; Van der Linden et al., EID; 2011; 17

40 Summary I. Antifungal resistance Prevalence not yet high enough to interest public health practitioners Principal consequence is clinical resistance Elevated MICs associated with poorer outcomes Breakthrough infections Represent a reservoir of resistant organisms

41 Summary II: Candida & azole MIC C. albicans, C. tropicalis, C. parapsilosis About 8-10% FLU resistant (MIC > 2 mg/l) MICs are reliable C. glabrata 15-20% FLU resistant High dose FLU? no systematic evidence of efficacy VRC (and POS) are not the answer cross resistance likely response to VRC is suboptimal

42 Summary III: Candida & echinocandins C. albicans, C. tropicalis, C. parapsilosis Resistance is uncommon. New CLSI breakpoints are (probably) OK C. krusei MIC >=1 mg/l resistant C. glabrata Resistance % MICs less reliable in picking up strains with FKS1 mutations but can be used as a screening tool (CASPO 0.12 mg/l or 0.5 mg/l).

43 A. fumigatus: Multi-azole resistance encountered after long-term therapy of chronic infections Multi-azole resistance encountered in azole naïve patients Key recommendations: obtain good and reliable data re A.fumigatus resistance in the patient population and the environment Screening agar?

44 THANK YOU

45 Induction or selection of resistance Prolonged therapy or prophylaxis Persistent infection Sub-therapeutic drug levels Fungistatic drugs Biofilms Exposure to environmental antifungal agents

46 Position of molecular detection of resistance Drug group Species Clinical relevance Available tools Integration into practice Azoles Candida Yes Yes Possible Echinocandins Aspergillus Yes Yes Possible Candida Yes Yes Yes * Aspergillus No Yes No Although the complement of resistance mechanisms are incompletely defined, FKS sequencing is reasonable Cuenca-Estrella M et al, Clin Microbiol Infect 2013; Dec 21

47 Up-regulation of target enzyme Up-regulation of ERG11 in C. albicans and C. glabrata No > 5 X incr. expression in resistant isolates Relatively minor mechanism in Candida

48 Azole-resistance in clinical A. fumigatus isolates Manchester (519 isolates) includes azole naiive Cross-resistant: 65% voriconazole 74% posaconazole Howard et. al Emerging Infect Dis. 15:

49 CLSI Breakpoints for echinocandins vs Candida Pfaller et al Drug Resist. Updates 2011;14:164 No. Category Species Antifungal agent tested S I R C. albicans ANF CSF (1) 41 2(2) 2(2) 8(8) 9(9) MCF 52 43(2) 1(1) 8(8) C. glabrata ANF CSF (2) 129(1) 4(1) 5(3) 30(27) 35(26) MCF (1) 6(3) 29(26) C. tropicalis ANF CSF (2) 26(1) 1(1) - 4(4) 5(5) MCF 31 25(1) 3(2) 3(3) What does this mean?

50 Fluconazole MIC distribution: 10,803 invasive Candida spp ECV for most species ECV for CG Pfaller et al., Drug Res Update 2010; 13: 180 <= >128 micrograms/ml C. albicans C. glabrata C. tropicalis C. krusei C. parapsilosis Other

51 USA Argenti Brazil Chile Colom Ecuador Hondu Venez South Australia India Japan Pakistan Korea( France Finland Norway Iceland Sweden England UK Italy Spain Scotland Denmark % resistant to fluconazole (Candida spp.) global 35% 30% 25% 20% 15% 10% 5% 0%

52 No. Isolates Candida glabrata: Caspofungin Australian Data N=640 S I R R = 12.5% Old bp >2 µg/ml R= 1.6% MIC (µg/ml) Slide: S. Kidd Feb 2014

53 Caspofungin MICs Lack of reproducibility (E test is worse) Variability in preparation and quality Do we use Micafungin or Anidulfungin as a better predictor for echinocandin resistance in Candida

54 Resistant phenotype: genetic changes Survey of high level FLU resistance (MIC >64 mg/l) 20 clinical OPC C. albicans from 12 HIV+ve patients Results 75% multi-factorial, step-wise resistance 85% overexpression efflux genes 65% ERG11 mutations 35% overexpression ERG11 Perea et al., AAC 2001

55 Prospective 4 year study 89 consecutive patients - 51 proven or probable IA 118 strains Azole naive Itraconazole R 0.85% = 1 isolate Itraconazole MIC 16 mg/l Voriconazole MIC 0.38 mg/l Posaconazole MIC 0.25 mg/l G432S substitution