Dr.T.V.

Rao MD

Dr.T.V.Rao MD 1
 Increase of Fungal Infections
e
a 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
e
a 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
 Antifungal agents
Mode of action

Amphotericin B binds to plasma membrane creating pores

Azoles inhibits cytochrome P450 enzymes in the fungal cell

5FC converts to 5FU, incorporated into RNA, abnormal proteins

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

Dr.T.V.Rao MD 4
 Antifungal agents
Griseofulvin

e
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 5
 Antifungal agents

Poelyenes
Polyenes are produced from Streptomyces
Cyclic molecules
Nystatin

Amphotericin B

Natamycin

Mepartricin

Broad spectrum

Dr.T.V.Rao MD 6
 Formulations of polyenes are
Toxic
e
a AmB
e The most widely used
antifungal for systemic
infections
e High level of toxicity
a Nystatin
e Significant
nephrotoxicity
e Has not been developed
to treat systemic fungal
infections

Dr.T.V.Rao MD 7
 antifungal agents
Amphotericin B
Yellow powder, water insoluble

e
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

Azole-amphotericin B is never synergistic

Amphotericin B and 5FC gives synergy

Candida lusitaniae is usually resistant to Amphotericin B

Dr.T.V.Rao MD 8
 Amphotericin B
Toxicity
e
• Early intolerance
reaction
• Thrombophlebitis
• Nephrotoxicity
• Hematotoxic effects

• The liposomal
preparation of
Amphotericin B
reduces the risk of
nephrotoxicity
Dr.T.V.Rao MD 9
 Antifungal agents
Azole Derivatives

e
A chemical pentacyclic structure with 2 nitrogen atoms
Water insoluble except fluconazole
Preferentially inhibit cytochrome P450 enzymes
Fungistatic, Modify cytochrome P450 enzyme
First generation Imidazoles:

Clotrimazole & Miconazole

Clotrimazole requires high doses – poorly tolerated

Parenteral dosages no longer available for Miconazole

Dr.T.V.Rao MD 10
 antifungal agents

Cytochrome P 450 (CYP 450)

e
CYP is a host of enzymes that use iron to oxidize things

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
CYP is vital to the formation of cholesterol & steroids

NADPH + H+ + O2 + RH ==> NADP+ + H2O + R-OH

Dr.T.V.Rao MD 11
 antifungal agents

CYP 450 …..

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

Dr.T.V.Rao MD 12
 antifungal agents

Ketoconazole
e
Orally well absorbed imidazole of second generation

Ketoconazole is the only imidazole for systemic use

CSF penetration is very weak

Hepatotoxicity restricts its use

Also interacts with other molecules

Dr.T.V.Rao MD 13
 antifungal agents

Third generation azoles

Triazole derivatives (contain three nitrogen atoms)

Fluconazole
e
Itraconazole

Voriconazole

Posaconazole

Revuconazole

Satisfactory tolerability, Suitable for systemic use

Dr.T.V.Rao MD 14
 antifungal agents
Fluconazole & Itraconazole

e
Fluconazole has been extensively used for yeast infections
Useful for systemic infections
Readily and completely absorbed by gastrointestinal tract

Distributed equally in different organs and tissue

Candida krusei Intrinsically resistant to fluconazole
Itraconazole is used to treat Aspergillus infections

Entirely metabolized in the liver

Eliminated in the feces and urine

Dr.T.V.Rao MD 15
 antifungal agents

Voriconazole is a modified fluconazole

e
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 16
 antifungal agents
Echinocandins
Caspofungin

e
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

Micafungin and Anidulafungin – are underinvestigation

Dr.T.V.Rao MD 17
 antifungal agents
Terbinafine

e
Terbinafine belongs to allylamines, synthetic, highly lipophilic

Oral and topical (cream) formulations

Terbinafine inhibits ergosterol biosynthesis

Used to treat superficial mycosis

Also useful against systemic mycosis (yeast & other fungi)

Adverse reactions to terbinafine are in general transient and mild

Dr.T.V.Rao MD 18
 Newer Methods are
Emerging
a The establishment of a e
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
ea 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
e
a Clinical signs and symptoms
a Rapid tests (issues: sensitivity, specificity)
e qPCR
e Galactomannan detection
e 1-3 β D-glucan detection
e PNA FISH
e Smear; histology stains
a Culture (issues: low % positive, time to positive result)
e Susceptibility testing
e Speciation: C. glabrata or another species?

Dr.T.V.Rao MD 21

Alexander et al. CID. 2006;43:S15–27.

 Increased Interest in Antifungal
Susceptibility Testing
e
aChanging epidem iology of isolated
organisms
e e g , non-albicans Candida on the increase
aNewer drugs; more choices
aMore immunocompromised patients
aAntifungal susceptibility testing
becoming more commonplace
?
Dr.T.V.Rao MD 22
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.
What Are the Ceurrent Antifungal
Susceptibility Tests?

Dr.T.V.Rao MD 23
 Introduction
e
aAntifungal susceptibility testing
eMinimum inhibitory concentration (MIC)
asuggests the target fungal species is
susceptible to antifungal drug
e M I C values in vitro might not necessarily
correlate with the in vivo efficacy noted
aclinical testing in vivo must be done to
confirm any finding in vitro
Dr.T.V.Rao MD 24
 Introduction
e
e T h e National Committee for Clinical
Laboratory Standards (NCCLS) Subcommittee
on Antifungal Susceptibility Tests
a h a s provided guidelines to increase the
reproducibility of MIC testing of
filamentous fungi
aM27-A broth dilution method
aCandida spp
aCryptococcus neoformans
Dr.T.V.Rao MD 25
 Diagnosis of
Invasive Fungal Infections
e
aClinical signs and symptoms
aRapid tests (issues: sensitivity, specificity)
e qPCR
e Galactomannan detection
e 1-3 β D-glucan detection
e PNA FISH
e Smear; histology stains
aCulture (issues: low % positive, time to positive
result)
e Susceptibility testing
e Speciation: C. glabrata or another species?
Alexander et al. CID. 2006;43:S15–27.
 Susceptibility Testing Methods
aDisk diffusion
e
e Qualitative results- interpretation only: an isolate is Susceptible
or Intermediate or Resistant)
aMICs (Minimum Inhibitory Concentration)
e Preferable; quantitative results: a value in µg/mL and an
interpretation (S,I,R)
aOther: echinocandin (eg, caspofungin) “susceptibility”
tests (NOT for routine laboratories)
e Inhibition of glucan synthesis (IC50 values)
e Mutations in FKS gene
Dr.T.V.Rao MD 27

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

 Antifungal CLSI and EUCAST
Guidelines
*Yeast-M27-A2 e a * * E U C A S T Yeast
0.5–2.5 x 103 cfu/mL Method
RPMI1640, pH 7, MOPS a0.5–2.5 x 105
Macro/microbroth 35ºC cfu/mL
48h (others)–72h
(Cryptococcus) aRPMI 1640 with
Interp: 100% inhibition 2% glucose
Amphotericin B (AmB); aMicro broth, 35ºC,
prominent for others pH 7,
a * CLSI Method
24h incubation

Dr.T.V.Rao MD 28
Antifungal CLSI and
EUCAST Guidelines
e aYeast-M44-A (disk)
a 1-5 x 106 cfu/mL
a MHA +2% glucose, 0.5
ug/mL methylene blue
a Disk diffusion 35ºC
a 20–24h
a Interp: measure zone of
inhibition; to date for
fluconazole and VOR

Dr.T.V.Rao MD 29
 Reading Disk Diffusion Test
e
Disk with drug

Measure diameter
of zone of inhibition Lawn of yeast
or mould

Dr.T.V.Rao MD 30

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

 Antimicrobial Gradient Testing
E-test®
e
Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale

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 Helpful in case of lack of

Not a standard of care clinical response
Helpful in deep or deMdaysupport oral Switch to
hematogenous infections azole (long-term therapies)
 Antifungal susceptibility testing
(AFST)
e
AFST should be
performed in
hematological
patients on
isolates from blood
or normally sterile
sites, in order

Dr.T.V.Rao MD 33
Antifungal Susceptible Testing
Methods
e
a CLSI M27-A3 and M27-S3 method for yeasts: RPMI-1640
medium with MOPS buffer to pH 7.0.
a • 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.
a • 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
 Methods for susceptibility
testing
aM38-A reference e
method for
filamentous fungi,
published by the
Clinical Laboratory
Standard Institute
(CLSI)

Dr.T.V.Rao MD 35
 CLSI M38-A
Characteristics CLSI M38A
a Suitable
e a Conidium-and spore forming
fungi
a Inoculum
a 0.4x104-5x104 CFU/ml
a Inoculum
a Spectrophotometrically
Standardization
a Test medium a RPMI 1640
a Format a Microdilution
a Temperature a 35°C
a Duration of incubation a 48h
a Endpoint
a No growth

Dr.T.V.Rao MD 36
 Limitations of susceptibility testing
methods
(M38- A, …)
a size of inoculum
e
a the use of growth medium
a the time of incubation
a the inoculum preparation
method
a the use of Tween
concentration
Lack of detection of amphotericin B
resistance
No breakpoints

Dr.T.V.Rao MD 37
 E-test
E-test is a
e
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
e
a MTT, XTT, viability testing……………………… and several other
antifungal susceptibility testing methods for moulds have been
developed
a a l l of these alternative methods correlate more or less with the
standard method
a e a c h also has its owndisadvantages:
e XTT or MTT method is cumbersome
e E test is relatively expensive
e Disk diffusion
e Viability tests are suitable for MFC

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
e
Conidium forming fungi Aspergillus fumigatus
Aspergillus spp.

Inoculum 0. 4- 5x1 04 CFU/ml 1 - 2. 5x1 05 CFU/ml

Inoculum Spectrophoto= Haemocytometer

standardization metrically
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

Caspofungin Activity in
Aspergillus spp.
Different from Routine Tests
 Caspofungin Activity in Aspergillus
spp.
e
a Activity does not fit classic definition of fungicidal
e No reduction in the number of colony count

a In Aspergillus the 1,3-β-D-glucan synthase complex is

localized in the apical tips of the growing hyphae
e Inhibition results in profound change in growth, morphology, and cell wall
structure of hyphae
e Structural change decreases ability to invade blood vessels but does not decrease
colony count
a Caspofungin shows in vitro activity against A.
fumigatus, A. flavus, A. nidulans, A. niger, A. terreus, and
A. candidus

Bowman et al. Antimicrob Agents Chemother. 2002;46(9):3001–3012.

 Colony Forming Unit Quantitation: Yeast
vs Aspergillus
Candida spp. and Other Yeasts
e Aspergillus spp.

10 Colony Forming Units 1 Colony Forming Unit

4 Colony Forming Units 1 Colony Forming Unit

Bowman et al. Antimicrob Agents Chemother. 2002;46(9):3001–3012.
 Caspofungin Activity in
Aspergillus spp.
e
a Colony counts ≠ number of viable cells with filamentous
fungi
a Traditional endpoints like MICs are not useful for
interpretation
a MEC or Minimum Effective Concentration
e concentration of caspofungin where microscopically
swollen, distorted hyphae are observed

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

 Caspofungin Inhibits Aspergillus spp.
Growth in Liquid MIC Assays
Caspofungin appears static in vitro but demonstrates cidal activity
in in vivo studies e
µg/ml 6464 3232 1616 8
µg/mL 8 4 4 2 21 10.5 0.5
0.25 0.25
0.13 0.125 0.06 0.03
0.06 0.03

Caspofungin

Amphotericin B

CLS
Caspofungin — prominent inhibition (>50%) at 24 hours
AmB — 100% Inhibition at 48 hours

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

 Can an In Vitro Susceptibility Test Predict
the In Vivo Human Response?
e
aMICs are not an absolute measurement
e MICs can vary based on medium, temperature of
incubation, inoculum, etc.
a T h e in vitro - in vivo correlation for antifungal
drugs is poor
e “S”* does not predict successful treatment
e “R”* does not necessarily predict clinical failure
aHost factors (immune status/underlying disease)
play a crucial role in clinical outcome
* S= susceptible, R = resistant
Dr.T.V.Rao MD 46
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.
 Speciation is Important in Optimal
Antifungal Administration
e
a Speciation of the infecti ng
fungal pathogen may be
more important, ie, is th e
organism C. glabrata or
not C. glabrata?
e Based on the species o f
the isolates, the choice of
antifungal agent
becomes important

Dr.T.V.Rao MD 47
Rex et al. Clin Microbiol Rev. 2001;14(4):643–658.
Rex et al. Clin Infect Dis. 2002;35:982–989.
 New antifungal agents
e Pradimicins-benanomicins e
a bind to cell wall mannoproteins causing osmotic sensitive
lysis and cell death
e Nikkonycins
a competitive inhibitors of fungal chitin-synthase enzymes
e Allylamines/thiocarbamates
a non-competitive inhibitors of squalene epoxidase
e Sordarins
a inhibit protein synthesis, i.e. elongation factor 2
e Cationic peptides
a bind to ergosterol and cholesterol and lead to cell lysis
 e
aCreated by Dr.T.V.Rao MD for
‘e’ learning for Medical and
Paramedical Students in the
Developing World
aEmail
adoctortvrao@gmail.com
Dr.T.V.Rao MD 49