antifungal sensitivity testing

ANTIFUNGAL SENSITIVITY TESTING basics:

ANTIFUNGAL SENSITIVITY TESTING basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1

Increase of Fungal Infections:

Increase of Fungal Infections Several factors have contributed to the increase in fungal infections - most notably, increasing number of immunosuppressed cases e.g AIDS, cancer or diabetes, the use of broad spectrum antibiotics, cytotoxic chemotherapy, and organ transplantations Dr.T.V.Rao MD 2

Fugal Infections have increased Morbidity and Mortality:

Fugal Infections have increased Morbidity and Mortality The increasing incidence of opportunistic severe fungal infections has greatly enhanced the interest in novel methods for in vitro antifungal susceptibility testing, the standardized methodology Dr.T.V.Rao MD 3

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Dr.T.V.Rao MD 4 A ntifungal agents Griseofulvin binds microtubule proteins, inhibit cell wall synthesis Terbinafine is an ergosterol inhibitor useful for systemic mycosis Echinocandins target their action on fungal cell wall 5FC converts to 5FU, incorporated into RNA, abnormal proteins Mode of action Amphotericin B binds to plasma membrane creating pores Azoles inhibits cytochrome P450 enzymes in the fungal cell

Antifungal agents :

Dr.T.V.Rao MD 5 Antifungal agents Griseofulvin Source Penicillium griseofulvum Produced in 1939 Not used until 1958 Spectrum Dermatophytes Gentles first used orally in guinea pigs prior to its use in humans Anti-inflammatory properties Inhibits keratolytic action

Antifungal agents :

Dr.T.V.Rao MD 6 Antifungal agents Polyenes Amphotericin B Nystatin Polyenes are produced from Streptomyces Natamycin Mepartricin Cyclic molecules Broad spectrum

Formulations of polyenes are Toxic:

F ormulations of polyenes are Toxic AmB The most widely used antifungal for systemic infections High level of toxicity Nystatin Significant nephrotoxicity Has not been developed to treat systemic fungal infections Dr.T.V.Rao MD 7

antifungal agents:

Dr.T.V.Rao MD 8 antifungal agents Amphotericin B Yellow powder, water insoluble Bile salt allows solubility (weak association) Floats free in the aqueous medium, causes toxic effects Broad spectrum, binds to sterol in the cell membrane Fungicidal activity @ 3 h with 1 µg/ml Amphotericin B and 5FC gives synergy Azole-amphotericin B is never synergistic Candida lusitaniae is usually resistant to Amphotericin B

Amphotericin B Toxicity :

Amphotericin B Toxicity E arly intolerance reaction Thrombophlebitis N ephrotoxicity Hematotoxic effects The liposomal preparation of Amphotericin B reduces the risk of nephrotoxicity Dr.T.V.Rao MD 9

Antifungal agents:

Dr.T.V.Rao MD 10 A ntifungal agents Azole Derivatives Water insoluble except fluconazole Preferentially inhibit cytochrome P450 enzymes Fungistatic, Modify cytochrome P450 enzyme A chemical pentacyclic structure with 2 nitrogen atoms Clotrimazole requires high doses – poorly tolerated First generation Imidazoles: Parenteral dosages no longer available for Miconazole Clotrimazole & Miconazole

antifungal agents:

Dr.T.V.Rao MD 11 antifungal agents CYP is vital to the formation of cholesterol & steroids NADPH + H+ + O2 + RH ==> NADP+ + H2O + R-OH CYP is a host of enzymes that use iron to oxidize things Cytochrome P 450 (CYP 450) CYP disposes harmful substances by making them water-soluble CYP is something like a hydroxyl group P450-mediated oxidation is referred to as " Phase I metabolism ” CYP in man is found in the liver, small intestine

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Dr.T.V.Rao MD 12 Fungal plasma membranes have nonpolar sterol (ergosterol ) Amphotericin B binds to ergosterol permitting rapid leakage Cytochrome P450 catalyzes synthesis of ergosterol Azole antifungal agents interfere with cytochrome P450 antifungal agents CYP 450 …..

antifungal agents:

Dr.T.V.Rao MD 13 antifungal agents Ketoconazole Orally well absorbed imidazole of second generation Hepatotoxicity restricts its use Also interacts with other molecules CSF penetration is very weak Ketoconazole is the only imidazole for systemic use

antifungal agents:

Dr.T.V.Rao MD 14 antifungal agents Itraconazole Voriconazole Fluconazole Third generation azoles Satisfactory tolerability, Suitable for systemic use Posaconazole Revuconazole Triazole derivatives (contain three nitrogen atoms)

antifungal agents:

Dr.T.V.Rao MD 15 antifungal agents Fluconazole has been extensively used for yeast infections Useful for systemic infections Itraconazole is used to treat Aspergillus infections Entirely metabolized in the liver Eliminated in the feces and urine Readily and completely absorbed by gastrointestinal tract Distributed equally in different organs and tissue Fluconazole & Itraconazole Candida krusei Intrinsically resistant to fluconazole

antifungal agents:

Dr.T.V.Rao MD 16 antifungal agents Voriconazole is a modified fluconazole A broad spectrum antifungal agent Rapid absorption after oral administration Distributes in tissues and body fluids Metabolized in the liver Eliminated in the urine in unchanged form Azoles carry some side effects Hepatotoxicity, gastrointestinal and endocrine toxicity Skin rash, pruritis and other hypersensitivity

antifungal agents:

Dr.T.V.Rao MD 17 antifungal agents Echinocandins Caspofungin Micafungin and Anidulafungin – are under investigation Caspofungin is semisynthetic, synthesized from Glarea lozyensis Whitish powder, water & methanol soluble, fungicidal Fungicidal against, Aspergilli, Candida and P. carinii No cross resistance amongst strains resistant to Ampho B or azoles No activity against Cryptococcus neoformans, Fusarium & Rhizopus Effective against Pneumocystis carinii

antifungal agents:

Dr.T.V.Rao MD 18 antifungal agents Terbinafine Terbinafine belongs to allylamines, synthetic, highly lipophilic Oral and topical (cream) formulations Terbinafine inhibits ergosterol biosynthesis Adverse reactions to terbinafine are in general transient and mild Used to treat superficial mycosis Also useful against systemic mycosis (yeast & other fungi)

Newer Methods are Emerging:

Newer Methods are Emerging The establishment of a standardized broth reference method for antifungal susceptibility testing of yeasts has opened the door to a number of interesting and useful developments. Also, the availability of reference methods provides a useful touchstone for the development of commercial products that promise to be more user friendly and to further improvement of test standardization. Dr.T.V.Rao MD 19

Antifungals can be Optimally Used:

Antifungals can be Optimally Used Incorporation of antifungal susceptibility testing methods into the clinical trials of new antifungal agents will facilitate the establishment of clinical correlates and further enhance the clinical utility of antifungal susceptibility testing Dr.T.V.Rao MD 20

Diagnosis of Invasive Fungal Infections :

Clinical signs and symptoms Rapid tests (issues: sensitivity, specificity) qPCR Galactomannan detection 1-3 β D-glucan detection PNA FISH Smear; histology stains Culture (issues: low % positive, time to positive result) Susceptibility testing Speciation: C. glabrata or another species? Diagnosis of Invasive Fungal Infections Alexander et al. CID. 2006;43:S15–27. Dr.T.V.Rao MD 21

Increased Interest in Antifungal Susceptibility Testing :

Increased Interest in Antifungal Susceptibility Testing Changing epidemiology of isolated organisms eg, non -albicans Candida on the increase Newer drugs; more choices More immunocompromised patients Antifungal susceptibility testing becoming more commonplace ? Turner et al. Expert Opin Emerg Drugs . 2006;11(2):231–250. Maertens et al. Curr Med Chem-Anti-infective Agents. 2002;1:65–81. Dr.T.V.Rao MD 22

What Are the Current Antifungal Susceptibility Tests?:

What Are the Current Antifungal Susceptibility Tests? Dr.T.V.Rao MD 23

Introduction:

Antifungal susceptibility testing Minimum inhibitory concentration (MIC) suggests the target fungal species is susceptible to antifungal drug MIC values in vitro might not necessarily correlate with the in vivo efficacy noted clinical testing in vivo must be done to confirm any finding in vitro Introduction Dr.T.V.Rao MD 24

Introduction:

The National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests has provided guidelines to increase the reproducibility of MIC testing of filamentous fungi M27-A broth dilution method Candida spp Cryptococcus neoformans Introduction Dr.T.V.Rao MD 25

Diagnosis of Invasive Fungal Infections :

Clinical signs and symptoms Rapid tests (issues: sensitivity, specificity) qPCR Galactomannan detection 1-3 β D-glucan detection PNA FISH Smear; histology stains Culture (issues: low % positive, time to positive result) Susceptibility testing Speciation: C. glabrata or another species? Diagnosis of Invasive Fungal Infections Alexander et al. CID. 2006;43:S15–27.

Susceptibility Testing Methods:

Susceptibility Testing Methods Disk diffusion Qualitative results- interpretation only: an isolate is S usceptible or I ntermediate or R esistant) MICs ( M inimum I nhibitory C oncentration) Preferable; quantitative results: a value in µg/mL and an interpretation (S,I,R) Other: echinocandin (eg, caspofungin) “susceptibility” tests (NOT for routine laboratories) Inhibition of glucan synthesis (IC50 values) Mutations in FKS gene Pfaller. Curr Drug Targets. 2005;6:929–943. Dr.T.V.Rao MD 27

Antifungal CLSI and EUCAST Guidelines:

Antifungal CLSI and EUCAST Guidelines *Yeast-M27-A2 0.5–2.5 x 10 3 cfu/mL RPMI1640, pH 7, MOPS Macro/microbroth 35 º C 48h (others)–72h ( Cryptococcus ) Interp: 100% inhibition Amphotericin B (AmB); prominent for others * CLSI Method **EUCAST Yeast Method 0.5–2.5 x 10 5 cfu/mL RPMI 1640 with 2% glucose Micro broth, 35ºC, pH 7, 24h incubatio n Dr.T.V.Rao MD 28

Antifungal CLSI and EUCAST Guidelines:

Antifungal CLSI and EUCAST Guidelines Yeast-M44-A (disk) 1-5 x 10 6 cfu/mL MHA +2% glucose, 0.5 ug/mL methylene blue Disk diffusion 35 º C 20–24h Interp: measure zone of inhibition; to date for fluconazole and VOR Dr.T.V.Rao MD 29

Reading Disk Diffusion Test:

Reading Disk Diffusion Test Disk with drug Measure diameter of zone of inhibition Lawn of yeast or mould Pfaller. Curr Drug Targets. 2005;6:929–943. Dr.T.V.Rao MD 30

Antimicrobial Gradient Testing E-test®:

Antimicrobial Gradient Testing E-test® Read plates after recommended Incubation Read MIC where elipse intersects scale Dr.T.V.Rao MD 31

Antifungal susceptibility testing in candidemia: current « guidelines »:

Antifungal susceptibility testing in candidemia: current « guidelines » Guideline Recommandation Comment on choice of therapy Germany 2003 None NA Spain 2003 AFST (not graded) None France 2004 Routine E-test (B-II) None U.S.A. 2004 NCCLS M27A & FCZ Not a standard of care Helpful in deep or hematogenous infections Helpful in case of lack of clinical response May support oral Switch to azole (long-term therapies) Not graded

Antifungal susceptibility testing (AFST):

Dr.T.V.Rao MD 33 Antifungal susceptibility testing (AFST) AFST should be performed in hematological patients on isolates from blood or normally sterile sites, in order

Antifungal Susceptible Testing Methods:

CLSI M27-A3 and M27-S3 method for yeasts: RPMI-1640 medium with MOPS buffer to pH 7.0. • CLSI M44-A and M44-S2 method for disk diffusion testing for yeasts: Mueller-Hinton Agar supplemented with glucose and 0.5 ug/ml methylene blue dye [GMB] medium. • CLSI M38-A2 method for filamentous Fungi RPMI-1640 medium with MOPS buffer to pH 7.0. Inoculum prepared by spectrophotometer with the spore suspension density adjusted for different species . Dr.T.V.Rao MD 34 Antifungal Susceptible Testing Methods

Methods for susceptibility testing :

Methods for susceptibility testing M38-A reference method for filamentous fungi, published by the Clinical Laboratory Standard Institute (CLSI) Dr.T.V.Rao MD 35

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 Dr.T.V.Rao MD 36

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

Dr.T.V.Rao MD 37 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

E-test:

E-test E-test is a commercially available method for antimicrobial susceptibility testing. This technique is based on a combination of the concepts of dilution and diffusion tests. For Aspergillus spp., good correlations with amphotericin B and Itraconazole Etest and M38-A method have been demonstrated . Dr.T.V.Rao MD 38

Different Tests:

MTT, XTT, viability testing……………………… and several other antifungal susceptibility testing methods for moulds have been developed all of these alternative methods correlate more or less with the standard method each also has its own disadvantages: XTT or MTT method is cumbersome E test is relatively expensive Disk diffusion Viability tests are suitable for MFC Different Tests Ramani 2003; Espinel-Ingroff 1997; Balajee 2002; Lass-Flörl 2001 Dr.T.V.Rao MD 39

Antifungal Drug Sensitivity Needs Special Skills:

Characteristics CLSI M38-A EUCAST Suitability Conidium forming fungi Aspergillus fumigatus Aspergillus spp. Inoculum 0.4-5x10 4 CFU/ml 1-2.5x10 5 CFU/ml Inoculum standardization Spectrophoto= metrically Haemocytometer Test medium RPMI 1640 RPMI 1640 G2% Format Microdilution Microdiluation Temperature 35°C 35°C Duration of incubation 48h 48h Endpoint No growth No growth Antifungal Drug Sensitivity Needs Special Skills

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Caspofungin Activity in Aspergillus spp . Different from Routine Tests

Caspofungin Activity in Aspergillus spp.:

Activity does not fit classic definition of fungicidal No reduction in the number of colony count In Aspergillus the 1,3- β -D-glucan synthase complex is localized in the apical tips of the growing hyphae Inhibition results in profound change in growth, morphology, and cell wall structure of hyphae Structural change decreases ability to invade blood vessels but does not decrease colony count Caspofungin shows in vitro activity against A. fumigatus, A. flavus, A. nidulans, A. niger, A. terreus, and A. candidus Caspofungin Activity in Aspergillus spp. Bowman et al. Antimicrob Agents Chemother . 2002;46(9):3001–3012.

Colony Forming Unit Quantitation: Yeast vs Aspergillus:

Colony Forming Unit Quantitation: Yeast vs Aspergillus 10 Colony Forming Units 4 Colony Forming Units Candida spp. and Other Yeasts 1 Colony Forming Unit 1 Colony Forming Unit Aspergillus spp. Bowman et al. Antimicrob Agents Chemother . 2002;46(9):3001–3012.

Caspofungin Activity in Aspergillus spp.:

Colony counts ≠ number of viable cells with filamentous fungi Traditional endpoints like MICs are not useful for interpretation MEC or M inimum E ffective C oncentration concentration of caspofungin where microscopically swollen, distorted hyphae are observed Caspofungin Activity in Aspergillus spp. Pfaller MA. Curr Drug Targets. 2005;6:929–943.

Caspofungin Inhibits Aspergillus spp. Growth in Liquid MIC Assays:

Caspofungin Inhibits Aspergillus spp. Growth in Liquid MIC Assays CLSI M38-A: Caspofungin — prominent inhibition (>50%) at 24 hours AmB — 100% Inhibition at 48 hours Caspofungin Amphotericin B µ g/mL 64 32 16 8 4 2 1 0.5 0.25 0.13 0.06 0.03 Pfaller. Curr Drug Targets. 2005;6:929–943. Caspofungin appears static in vitro but demonstrates cidal activity in in vivo studies

Can an In Vitro Susceptibility Test Predict the In Vivo Human Response?:

MICs are not an absolute measurement MICs can vary based on medium, temperature of incubation, inoculum, etc. The in vitro - in vivo correlation for antifungal drugs is poor “S”* does not predict successful treatment “R”* does not necessarily predict clinical failure Host factors (immune status/underlying disease) play a crucial role in clinical outcome Can an In Vitro Susceptibility Test Predict the In Vivo Human Response? Rex et al. Clin Microbiol Rev. 2001;14(4):643–658. Rex et al. Clin Infect Dis . 2002;35:982–989. Pfaller. Curr Drug Targets. 2005;6:929–943. * S= susceptible, R = resistant Dr.T.V.Rao MD 46

Speciation is Important in Optimal Antifungal Administration:

Speciation is Important in Optimal Antifungal Administration Speciation of the infecting fungal pathogen may be more important, ie, is the organism C. glabrata or not C. glabrata? Based on the species of the isolates, the choice of antifungal agent becomes important Rex et al. Clin Microbiol Rev. 2001;14(4):643–658. Rex et al. Clin Infect Dis. 2002;35:982–989. Dr.T.V.Rao MD 47

New antifungal agents :

Pradimicins-benanomicins bind to cell wall mannoproteins causing osmotic sensitive lysis and cell death Nikkonycins competitive inhibitors of fungal chitin-synthase enzymes Allylamines/thiocarbamates non-competitive inhibitors of squalene epoxidase Sordarins inhibit protein synthesis, i.e. elongation factor 2 Cationic peptides bind to ergosterol and cholesterol and lead to cell lysis New antifungal agents

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