Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
�
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
� 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
�
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
�
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
�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
�
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 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
�
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
�
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 …..
�
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
�
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)
�
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
�
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
�
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
�
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
� 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
� 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
�
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.
�
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
�
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
�
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
�
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
�
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
�
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
�
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-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
Dr.T.V.Rao MD 36
�
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 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.
�
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.
�
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
�
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
�� 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
Dr.T.V.Rao MD 49
Top Related