Antifungal Drug Resistance

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Antifungal Drug Resistance:

Antifungal Drug Resistance Ira Praharaj

Introduction:

Introduction Increasing prevalence of infections caused by fungi in past few decades Antifungal therapy has undergone a remarkable transformation in recent years Emerging trend of antifungal drug resistance

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Study of resistance to antifungal agents has lagged behind that of antibacterial resistance

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“Resistance” vs Clinical Failure Relative insensitivity of a microbe to an antimicrobial drug as tested in vitro and compared with other isolates of the same species Failure of an appropriate therapy for a certain indication to result in a clinical response Loeffler J et al CID 2003

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Katzung 11 th ed

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Primary Resistance Secondary Resistance Intrinsic Resistance Clinical Resistance

Factors for Clinical Antifungal Drug Resistance:

Factors for Clinical Antifungal Drug Resistance Manual of Clinical Microbiology,9 th ed

meChanisms of resistance:

meChanisms of resistance

Antifungal Resistance vs Antibacterial Drug Resistance:

Antifungal Resistance vs Antibacterial Drug Resistance No data to suggest that destruction/modification of antifungal agents is an important component of antifungal resistance Genetic exchange mechanisms that allow rapid transmission of antimicrobial resistance in bacteria not seen in fungi

Resistance to azoles:

Resistance to azoles Extensive utilisation of fluconazole worldwide Primary resistance to fluconazole unusual,especially among isolates from blood Intrinsic resistance to fluconazole for C.krusei,C.norvegensis ,C.inconspicua Newer triazoles exhibit more potent activity against Candida species than fluconazole

Resistance to azoles:

Resistance to azoles Alteration of drug efflux Alteration of target enzyme(lanosterol demethylase) Alteration of drug influx

Alterations in Ergosterol biosynthesis:

Alterations in Ergosterol biosynthesis

Alteration of target enzymes:

Alteration of target enzymes

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Altered target(14 α demethylase)-point mutations in ERG11 gene(Y132H,D278E) Overexpression of ERG11 gene-high concentrations of the target enzyme Resistant clinical isolates contain mutated but functional ERG11 genes ERG11 gene important for intrinsic resistance to fluconazole seen in C.krusei (reduced enzyme susceptibility)

Drug Accumulation:

Drug Accumulation

Efflux Pumps:

Efflux Pumps ATP binding cassette transporters(ABCT) Major Facilitators(MF)

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ABC proteins in S.cerevisiae 3 superfamilies-PDR(Pleiotropic Drug Resistance) -MDR -MRP Azole drug resistance associated with the (Candida Drug Resistance)CDR gene family 10 CDR genes identified(4 characterised) ABCT pumps identified in C.krusei,C.glabrata,C.tropicalis,C.neoformans,A.nidulans,A.flavus

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MF pumps-no nucleotide binding domains Proton gradient of membrane used as energy source(Antiport) 2 MF pump genes characterised in C.albicans First MFS transporter gene to be characterised from a pathogenic fungus CaMDR1(BEN’) MDR1 gene found to be azole specific

Overcoming efflux mediated drug resistance:

Overcoming efflux mediated drug resistance

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Clin Microbiol Rev 1998; 11: 382

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Mechanism of azole resistance Organism Upregulation of CDR1 genes C.glabrata ↓ susceptibility of target enzyme C.krusei to inhibition ↑ drug efflux Aspergillus spp Overexpression of MDR efflux pumps C.neoformans + Altered target enzymes

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Resistance is a composite

Mechanisms of azole resistance may act individually,sequentially and in concert:

Mechanisms of azole resistance may act individually,sequentially and in concert Redding et al,1994,CID

Cellular Phenotypes Associated With Resistance:

Cellular Phenotypes Associated With Resistance Homozygosity at MTL locus(Mating Types) Trailing Heterogenous resistance Inducible resistance Transient resistance

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Trailing- MIC-defined as the concentration of drug that inhibits growth by 80% Some fungal isolates appear susceptible at 24 hrs of growth but resistant at 48 hrs. Clinically infections with these trailing isolates usually treatable with azole drugs at standard drug concentrations Trailing reduced in acidic media

Polyene Resistance:

Polyene Resistance Uncommon Significant alteration of the lipid composition in the plasma membrane Most reports of amphotericin B resistance secondary to amphotericin B exposure during treatment

Amphotericin B resistance:

Amphotericin B resistance Reduction in the amount of plasma membrane ergosterol Primary resistance in C.lusitaniae,C.lipolytica,C.guillermondii In vivo and invitro resistance in A.terreus,A.nidulans

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Reduced total ergosterol content Replacement of ergosterol with sterols(fecosterol) that bind amphotericin B less well Masking of ergosterol in the cell membrane

Echinocandins—Spectrum of Activity:

Echinocandins—Spectrum of Activity Candida Aspergillus Cryptococcus Coccidioides Blastomyces Histoplasma Fusarium Scedosporidium Zygomycetes albicans glabrata krusei tropicalis parapsilosis lusitanae guilliermondii +++ +++ +++ +++ + +++ + +++ -- ++ ++ -- - - - Gallagher JC, et al. Expert Rev Anti-Infect Ther 2004;2:253-268

Echinocandin resistance:

Echinocandin resistance Isolates for which caspofungin MICs exceed 1ug/ml rarely occur in clinical infections Efforts to produce laboratory mutants of Candida species with reduced susceptibility to caspofungin-extremely low frequency of mutants

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Study of lab-derived mutants of Candida albicans with ↓ invivo and in-vitro susceptibility to caspofungins Point mutations in FKS1 gene encoding for FKS1 integral membrane protein

Resistance to 5-FC:

Resistance to 5-FC High rates of resistance noted Primary resistance-non-albicans Candida C.neoformans Aspergillus Secondary resistance- C.albicans Secondary resistance following Flucytosine monotherapy

Mechanisms of Resistance to Flucytosine:

Mechanisms of Resistance to Flucytosine Loss of permease activity Loss of cytosine deaminase activity Decrease in the activity of UPRTase

Fungal Biofilms and Drug Resistance:

Fungal Biofilms and Drug Resistance

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Biofilms-nidus for disease often associated with high-level antimicrobial resistance Increased resistance of Candida biofilms to fluconazole and amphotericin B has been demonstrated Efflux pumps found to be upregulated in cells within a biofilm “Antibiofilm activity” seen with echinocandins and some newer formulations of amphotericin B(mechanisms unclear)

Antifungal susceptibility testing:

Antifungal susceptibility testing

Rationale behind AFST:

Rationale behind AFST Provide a reliable measure of the relative activities of two or more antifungal agents Correlate with in vivo activity and predict outcome of therapy Monitor the development of resistance among a normally susceptible population of organisms Predict the therapeutic potential of newly discovered agents

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The 90-60 Rule

The CLSI Subcommittee on Antifungal Susceptibility Testing:

The CLSI Subcommittee on Antifungal Susceptibility Testing CLSI M27-A3 document (2009)- Broth macrodilution and microdilution susceptibility of yeasts CLSI M38-A2 document- molds(Broth macro and microdilution) CLSI M44-A2 document- disk-diffusion method for yeasts

Methods of antifungal susceptibility testing:

Methods of antifungal susceptibility testing TEST METHOD ENDPOINT DETERMINATION -Broth macrodilution(yeasts)-visual comparision of turbidity with turbidity of 80% inhibition standard -Broth microdilution-visual comparision of growth( ≥ 50% inhibit) -Spectrophotometric microdilution(yeasts)-Turbidometric MIC -Agar macrodilution(yeasts ,molds)- visual -Agar diffusion -Disk(yeasts)- zone diameter(visual) -E-Test Ellipse of inhibition(visual)

Invitro susceptibility testing for yeasts:

Invitro susceptibility testing for yeasts Broth Dilution Method Broth medium-RPMI 1640 broth buffered with MOPS buffer and 0.5% to pH 7 at 25˚C Test inoculum-0.5 x 10 3 CFU/ml Drug dilution ranges-5FC-0.12-64ug/ml others-0.03-16ug/ml MIC(visual examination) Microdilution(50% inhibition) Macrodilution(matching 80% inhibition standard)

Antifungal disk diffusion testing of Candida species:

Antifungal disk diffusion testing of Candida species Agar medium-MHA+2%dextrose+ 0.5 ug methylene blue dye/ml Inoculum -0.5 MacFarland turbidity CLSI guidelines for fluconazole,voriconazole and posaconazole

Alternative approaches:

Alternative approaches Broth based Use of colorimetric indicators-Sensititre Yeast one(Alamar Blue as redox indicator) Spectrophotometric methods Flow Cytometry Introduction of DNA binding vital dyes into the culture to detect fungal cell damage after exposure to antifungal agent

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Fluconazole,itraconazole and 5-FC strips approved by FDA RPMI medium with 2% Dextrose MIC= zone of inhibition

Methods for susceptibility testing :

Methods for susceptibility testing M38-A reference method for filamentous fungi, published by the Clinical Laboratory Standard Institute (CLSI ) E test MTT XTT Flow cytometry ……

CLSI M38-A:

CLSI M38-A Characteristics Suitable Inoculum Inoculum Standardization Test medium Format Temperature Duration of incubation Endpoint CLSI M38A Conidium-and spore forming fungi 0.4x10 4 -5x10 4 CFU/ml Spectrophotometrically RPMI 1640 Microdilution 35°C 48h No growth

Limitations of susceptibility testing methods (M38-A, …):

Limitations of susceptibility testing methods (M38-A, …) size of inoculum the use of growth medium the time of incubation the inoculum preparation method the use of Tween concentration Lack of detection of amphotericin B resistance No breakpoints Rodriguez-Tudela, 2003; Denning, 1997; Gehrt 1995; Gomez-Lopez 2005

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