Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

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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

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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

Dr.T.V.Rao MD 4

Antifungal 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

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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

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Dr.T.V.Rao MD 6

Antifungal agents

Polyenes

Amphotericin B

Nystatin

Polyenes are produced from Streptomyces

Natamycin

Mepartricin

Cyclic molecules

Broad spectrum

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Formulations 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

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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

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Amphotericin BToxicity

• Early intolerance reaction

• Thrombophlebitis• Nephrotoxicity• Hematotoxic effects

• The liposomal preparation of Amphotericin B reduces the risk of nephrotoxicity

Dr.T.V.Rao MD 9

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Dr.T.V.Rao MD 10

Antifungal agentsAzole 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

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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

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 …..

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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

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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)

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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

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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

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Dr.T.V.Rao MD 17

antifungal agentsEchinocandins

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

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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)

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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

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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

��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

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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?

Dr.T.V.Rao MD 23

��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

��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

��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.

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Susceptibility Testing Methods

�Disk diffusion

�Qualitative results- interpretation only: an isolate is Susceptible or Intermediate or Resistant)

�MICs (Minimum Inhibitory Concentration)

� 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

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Antifungal CLSI and EUCAST Guidelines

*Yeast-M27-A20.5–2.5 x 103 cfu/mLRPMI1640, pH 7, MOPSMacro/microbroth 35ºC48h (others)–72h (Cryptococcus)

Interp: 100% inhibition Amphotericin B (AmB); prominent for others

� * CLSI Method

�**EUCAST Yeast Method

�0.5–2.5 x 105

cfu/mL�RPMI 1640 with 2% glucose

�Micro broth, 35ºC, pH 7,

24h incubation

Dr.T.V.Rao MD 28

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Antifungal CLSI and EUCAST Guidelines

�Yeast-M44-A (disk)

�1-5 x 106 cfu/mL

�MHA +2% glucose, 0.5 ug/mL methylene blue

�Disk diffusion 35ºC

�20–24h

� Interp: measure zone of inhibition; to date for fluconazole and VOR

Dr.T.V.Rao MD 29

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Reading Disk Diffusion Test

Disk with drug

Measure diameterof zone of inhibition Lawn of yeast

or mould

Pfaller. Curr Drug Targets. 2005;6:929–943.

Dr.T.V.Rao MD 30

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Antimicrobial Gradient TestingE-test®

Read platesafter

recommendedIncubation

Read MICwhere elipseintersectsscale

Dr.T.V.Rao MD 31

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

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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

��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

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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

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CLSI M38-ACharacteristics�Suitable� Inoculum� Inoculum

Standardization�Test medium� Format�Temperature�Duration of incubation� Endpoint

CLSI M38A� Conidium-and spore forming

fungi� 0.4x104-5x104 CFU/ml�Spectrophotometrically

� RPMI 1640�Microdilution� 35°C� 48h

�No growth

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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

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E-test

E-test is a commerciallyavailable method forantimicrobialsusceptibility testing. Thistechnique is based on acombination of theconcepts of dilution anddiffusion tests.

For Aspergillus spp., goodcorrelations withamphotericin B andItraconazole Etest andM38-A method have beendemonstrated.

Dr.T.V.Rao MD 38

��MTT, XTT, viability testing……………………… and several other

antifungal susceptibility testing methods for moulds have beendeveloped

�all of these alternative methods correlate more or less with thestandard 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

�Characteristics CLSI M38-A EUCASTSuitability Conidium forming fungi Aspergillus fumigatus

Aspergillus spp.

Inoculum 0.4-5x104 CFU/ml 1-2.5x105 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

Caspofungin Activity in

Aspergillus spp.Different from Routine Tests

��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.

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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.

��Colony counts ≠ number of viable cells with filamentous

fungi

� Traditional endpoints like MICs are not useful for

interpretation

�MEC or Minimum Effective Concentration

� concentration of caspofungin where microscopically

swollen, distorted hyphae are observed

Caspofungin Activity in Aspergillus spp.

Pfaller MA. Curr Drug Targets. 2005;6:929–943.

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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

µg/ml 64 32 16 8 4 2 1 0.5 0.25 0.125 0.06 0.03

Caspofungin

Amphotericin B

µg/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

��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 = resistantDr.T.V.Rao MD 46

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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.

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�� 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

��Created by Dr.T.V.Rao MD for

‘e’ learning for Medical and Paramedical Students in the

Developing World

�Email

�doctortvrao@gmail.com

Dr.T.V.Rao MD 49