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A . W . F O T H E R G I L L , M A , M B AU N I V E R S I T Y O F T E X A S H E A L T H S C I E N C E C E N T E R

S A N A N T O N I O , T E X A SA S S O C I A T E P R O F E S S O R , D E P A R T M E N T O F P A T H O L O G Y

T E C H N I C A L D I R E C T O R , F U N G U S T E S T I N G L A B O R A T O R Y

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Interpreting Antifungal Susceptibility Testing:Science or Smoke and Mirrors

Antifungal Pharmacotherapy2

Current options for the treatment of systemic fungal infections are limited

Amphotericin B Nystatin

Fluconazole Itraconazole

Voriconazole Posaconazole

Caspofungin Micafungin

Anidulafungin

Combinations?

Considerable confusion/disagreement regarding appropriate use of these agents

Antifungal Agents3

Amphotericin B

“Standard Therapy” for invasive, life-threatening fungal infections.

Works by attaching to sterols in cell wall

Toxic

Infusion related: chills, fever, headache

Dose-limiting nephrotoxicity

Clinical efficacy poor in some settings

Invasive aspergillosis in immunocompromised

2

Amphotericin B Lipid Formulations4

Chemical Differences from AMB Deoxycholate NONE

Advantages Increased daily dose of parent drug

High tissue concentration

Fewer infusion related side effects

Decreased nephrotoxicity

Disadvantages Superior clinical efficacy not proven

Extremely expensive

Amphotericin B Lipid Formulation Use5

Systemic mycoses, intolerant of or refractory to conventional AMB

Renal dysfunction during treatment (Cr>2.5)

Severe/persistent infusion-related side effects

Disease progression after 500 mg total dose

Typically invasive aspergillosis in patients that are severely immunocompromised

Azoles6

Favorable side effect profile

Act by inhibition of ergosterol synthesis

Ease of administration

Limitations:

Drug interactions

Static rather than cidal activity

Emergence of resistance, even during therapy, especially Candida spp. to FLU

3

Echinocandins7

Favorable side effect profile

Inhibit fungal cell wall synthesis Glucan synthase inhibitors

Broadly active in vitro

Generally well tolerated Warning for co-administration with cyclosporine

FDA approved for aspergillosis and candidiasis, refractory or AMB/ITRA intolerant

Antifungal Susceptibility Testing Methods8

Macrobroth dilution

Microbroth dilution

Disk diffusion

Agar diffusion

Solid agar with wells

CLSI (NCCLS) Publications9

2008 – M27-A3Third edition

2008 – M38-A2Second edition

2004 – M44-AApproved Standard

4

Previous Susceptibility Cutoffs for Candida spp. (µg/ml)

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S SDD I R NS

5FC <4 -- 8-16 >32 --

FLU <8 16-32 -- >64 --

ITRA <0.125 0.25-0.5 -- >1 --

VORI <1 2 -- >4 --

ANID <2 -- -- -- >2

CAS <2 -- -- -- >2

MICA <2 -- -- -- >2

M27-A3 Approved BreakpointsCandida albicans, C. tropicalis, C. krusei

Time S SDD I R

AMB 24 H -- -- -- --

ANID 24 H <0.25 -- 0.5 >1

CAS 24 H <0.25 -- 0.5 >1

MICA 24 H <0.25 -- 0.5 >1

FLU 24 H <2* 4* -- >8*

POS 24 H -- -- -- --

VORI 24/48 H <0.125 0.25-0.5 -- >1

ITRA 24/48 H -- -- -- --

5FC 24/48 H -- -- -- --

*None for C. krusei

M27-A3 Approved BreakpointsCandida glabrata

Time S SDD I R

AMB 24 H -- -- -- --

ANID 24 H <0.125 -- 0.25 >0.5

CAS 24 H <0.125 -- 0.25 >0.5

MICA 24 H <0.06 -- 0.125 >0.25

FLU 24 H -- <32 -- >64

POS 24 H -- -- -- --

VORI 24/48 H -- -- -- --

ITRA 24/48 H -- -- -- --

5FC 24/48 H -- -- -- --

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M27-A3 Approved BreakpointsCandida parapsilosis, C. guilliermondii

Time S SDD I R

AMB 24 H -- -- -- --

ANID 24 H <2 -- 4 >8

CAS 24 H <2 -- 4 >8

MICA 24 H <2 -- 4 >8

FLU 24 H <2* 4* -- >8*

POS 24 H -- -- -- --

VORI 24/48 H <0.125* 0.25-0.5* -- >1*

ITRA 24/48 H -- -- -- --

5FC 24/48 H -- -- -- --

*None for C. guilliermondii

Reading MICs14

AMB

No visible growth

Azoles/5FC

80% reduction in turbidity (macro)

50% reduction in turbidity (micro)

100% reduction in turbidity (moulds – ITRA, POSA, VORI)

Echinocandins

50% reduction in turbidity (yeast)

Minimum Effective Concentration (mould)

Macrobroth Procedure15

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Microtiter Method16

Caspofungin: Minimum Effective Concentration (MEC)

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Growth Control Increasing Concentration of Echinocandin

Healthy

Stubby

Aberrant

Sick

Point of Transition:

MEC

Photomicrographs and definition of MEC: Kurtz et al., AAC 38:1480, 1994

Slide courtesy of John Rex, MD. www.doctorfungus.org.

Disk Diffusion18

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NCCLS M44-A19

Provides

Zone interpretive criteria for FLU & VORI

QC ranges for FLU & VORI

R SDD S

FLU 25µg <14mm 15-18mm >19mm

VORI1µg <13mm 14-16mm >17mm

Sensititre Yeast One Panel20

Etest – Technical Manual21

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Etest – Technical Manual22

Is Susceptibility Testing a Predictor of Clinical Response?

23

MIC is not a physical or chemical measurement

Dependant upon testing variables

Host factors are more predictive:

Immune response, underlying disease

Drainage/removal of infected foci

Drug level to site of infection

Susceptibility Testing Clinical Utility24

Low MIC does not predict success

High MIC does not predict failure

In vitro resistance may select a population less likely to respond to a particular agent

Other factors (host, drug) are more important as predictors of outcome

Both antibacterial AND antifungal susceptibility

testing share these limitations.

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Antimicrobial Susceptibility Testing25

The “90-60 Rule” (expected correlation)

General rules to guide interpretation of results

Infections due to susceptible isolates respond to appropriate therapy ~ 90% of the time

Infections due resistant isolates (or infections treated with inappropriate therapy) respond ~ 60% of the time

Rex and Pfaller CID 2002; 35:982-9.

Trends Toward Resistance?26

Reported trends toward resistance for azoles

Questionable trends toward resistance for candins

Are moulds acquiring resistance?

Resistance and Breakthrough Infections

Antifungal Description

Micafungin 12 breakthrough infections among 649 BMT or SOT

recipients receiving micafungin prophylaxis (2006 - 2008)

• 19 isolates Candida isolates

• Micafungin MICs elevated:

‒ 5 of 7 C. parapsilosis (4 - 8 μg/ml)

‒ 5 of 6 C. glabrata (4 - 8 μg/ml)

‒ 2 of 3 C. tropicalis (2 μg/ml)

• C. glabrata & C. tropicalis cross-resistance to

caspofungin & anidulafungin (fks gene mutations)

Micafungin 293 C. glabrata bloodstream infections (2001 – 2010).

• Resistance increased from 4.9% to 12.3%

Risk factors for echinocandin resistance

• Solid organ transplantation

• Prior echinocandin exposure

Pfeiffer et al. J Clin Microbiol 2010; 48: 2373-80.

Alexander et al. Clin Infect Dis 2013; 56: 1724-32.

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Increasing Echinocandin Resistance in C. glabrata

Echinocandin

resistance increasing in

C. glabrata

May vary based on

region

MDR-resistance

problem

Azoles

Candida glabrata

2010 2011 2012 20130

2

4

6

8

10

12

14

Perc

ent R

esis

tance

Atlanta (4.7%)

Baltimore (4.0%)

Knoxville (3.4%)

Portland (0%)

Of 1340 isolates tested:

• 36.2% of echinocandin-resistant

isolates also resistant to fluconazole

Pfam et al. J Clin Microbiol 2014; 58: 4690-4696.

Clinical Relevance of Azole / Echinocandin Link?

Azole & echinocandin co-resistance noted in ICU

& non-ICU patients with C. glabrata bloodstream

isolates

No co-resistance detected in isolates collected from 2001 -2004

11% of fluconazole-resistant C. glabrata isolates also resistant to one or more echinocandin in those collected from 2006 - 2010

Pfaller et al. J Clin Microbiol 2012; 50: 1199-1203.

Pfaller et al. Int J Antimicrob Agents 2011; 38: 65-69.

Azole Resistance in AspergillusModification of Target Enzyme

A. fumigatus has two distinct genes encoding for 14-

demethylase (Cyp51p)

Point mutations in CYP51A observed in clinical and laboratory

isolates with azole resistance

Position of point mutation determines azole resistance

Mann et al. Antimicrob Agents Chemother 2003; 47: 557.

Mellado et al. Antimicrob Agents Chemother 2004; 48: 2747.

Howard et al. Emerg Infect Dis 2009; 15: 1068-1076.

Seyedmousavi et al. Drug Resist Updat 2014; 17: 37-50.

Azoles Affected Cyp51p amino acid changes

Pan-azole G138, Y131C, G434C

Voriconazole G448

Itraconazole & posaconazole G54, P216L, M220

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Environmental Exposure to Azoles

Azole-resistant IA identified in patients without prior

azole exposure in parts of Europe

Indoor environment in hospitals & direct proximity to medical centers

Fields where azole fungicides used

Tandem repeat in CYP51A promoter + point

mutation in gene

TR34/L98H

TR46/Y121F/T289A

Snelders et al. Appl Environ Microbiol 2009; 75: 4053-3057. Chowdhary et al. PLoS One 2012; 7: e52871.

van der Linder et al. Clin Infect Dis 2013; 57: 513-520. Chowdhary et al. J Antimicrob Chemother 2014; 69: 555-557.

Chowdhary et al. J Antimicrob Chemother 2014; 69: 69: 2979-2983.

Global Azole ResistanceTR32/L98H & TR46/Y121F/T289A

Belgium, Denmark,

France, Germany,

the Netherlands,

Norway, UK, Spain

Tanzania India

Iran China

Australia

Becoming a global issue

Kuwait

Antifungal Resistance

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Yeasts Candida krusei fluconazole intrinsic

Candida albicans fluconazole acquired

Candida glabrata fluconazole acquired

Candida albicans caspofungin acquired?

Candida glabrata caspofungin acquired?

Moulds Pseudallescheria boydii amphotericin B intrinsic

Fusarium spp. ALL intrinsic

Paecilomyces lilacinus amphotericin B intrinsic

Aspergillus terreus amphotericin B intrinsic

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Aspergillus fumigatus

AMBN=520

CASN=391

VORIN=428

POSAN=59

MIC50 0.5 0.125 0.5 0.125

MIC90 1.0 0.25 1.0 0.125

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34

Aspergillus terreus

AMBN=96

CASN=33

VORIN=87

POSAN=18

MIC50 4.0 0.125 0.5 0.06

MIC90 8.0 0.25 0.5 0.125

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35

Candida albicans

AMBN=1385

CASN=1273

FLUN=2213

VORIN=780

MIC50 0.25 0.06 0.25 <0.015

MIC90 0.25 0.125 1.0 1.0

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36

13

Candida glabrata

AMBN=1028

CASN=1138

FLUN=1535

VORIN=781

MIC50 0.25 0.125 8.0 0.5

MIC90 0.5 0.25 64 4.0

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37

Candida lusitaniae

AMBN=130

CASN=88

FLUN=197

VORIN=67

MIC50 0.25 0.125 0.5 0.03

MIC90 0.5 0.25 2.0 0.25

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Epidemiological Cutoff Values39

An endpoint described when there is a lack of clinical data

Chosen by taking into account the MIC distribution of a species and the inherent variability in MIC

testing methods

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MIC Distribution and ECV for Azoles Against Aspergillus spp.

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DrugRange µg/ml

ECVµg/ml % < ECV

Itraconazole 0.06-2.0 1.0 99.7

Voriconazole 0.06-2.0 1.0 97.8

Posaconazole 0.015-2.0 0.25 98.6

Rodriguez-Tudela et al AAC 2008;52:2468-72

ECV for Candins Against Aspergillus spp.41

Species Drug MEC Mode ECV

A. fumigatus

ANID 0.007 0.06

MICA 0.007 0.03

A. flavus

ANID 0.007 0.03

MICA 0.007 0.03

A. niger

ANID 0.007 0.015

MICA 0.007 0.015

A. terreus

ANID 0.007 0.015

MICA 0.007 0.015

Pfaller et al, JCM 2009; 47:3323-5

Antifungal Combinations

Rationale

Expand/enhance the spectrum of activity

Synergistic combinations may allow for the administration of lower doses of toxic agents such as amphotericin B

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Checkerboard Dilutions

2 2 0.06

2 0.125

2 0.25

2 0.5

2 1

2 2

1 1 0.06

1 0.125

1 0.25

1 0.5

1 1

1 2

0.5 0.5 0.06

0.5 0.125

0.5 0.25

0.5 0.5

0.5 1

0.5 2

0.25 0.25 0.06

0.25 0.125

0.25 0.25

0.25 0.5

0.25 1

0.25 2

0.125 0.125 0.06

0.125 0.125

0.125 0.25

0.125 0.5

0.125 1

0.125 2

0.06 0.06 0.06

0.06 0.125

0.06 0.25

0.06 0.5

0.06 1

0.06 2

0.06

0.125

0.25

0.5

1

2

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Fractional Inhibitory Concentration

An algebraic or geometric determination

The MIC of each drug in the combination expressed as a fraction of the MIC for each drug alone

MIC A within the CB MIC B within the CBMIC A alone + MIC B alone

FICA + FICB = FICI (index)

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Antifungal Combinations

Synergy (FICI = <0.5) A 2-tube or 4-fold decrease in the MIC of both drugs

Additivism (FICI = 1.0) A 1-tube or 2-fold decrease in the MIC of either drug

Indifference FICA + FICB = FICA OR FICB

No change in the MIC as a result of the combination

Antagonism (FICI is >1.0) An increase In the MIC of either drug as a result of the

combination

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16

Choosing the endpoint

2 - - - - - - -

1 - - - - - - -

0.5 - I I I I - -

0.25 + A A A I - -

0.125+ + S A I - -

0.06 + + + + I - -

+ 0.06

+ 0.125

+ 0.25

- 0.5

- 1

- 2

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Example of Indifference

2 - - - - - - -

1 - - - - - - -

0.5 - - - - - - -

0.25 + + + + - - -

0.125+ + + + - - -

0.06 + + + + - - -

+ 0.06

+ 0.125

+ 0.25

- 0.5

- 1

- 2

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Example of Additivism48

2 - - - - - - -

1 - - - - - - -

0.5 - - - - - - -

0.25 + + + - - - -

0.125+ + + + - - -

0.06 + + + + - - -

+ 0.06

+ 0.125

+ 0.25

- 0.5

- 1

- 2

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Example of Synergism49

2 - - - - - - -

1 - - - - - - -

0.5 - - - - - - -

0.25 + + - - - - -

0.125+ + - - - - -

0.06 + + + + - - -

+ 0.06

+ 0.125

+ 0.25

- 0.5

- 1

- 2

Synergy50

Examples of Antagonism51

2 - - - - - - -

1 - + + + - - -

0.5 - + + + - - -

0.25 + + + + - - -

0.125+ + + + - - -

0.06 + + + + - - -

+ 0.06

+ 0.125

+ 0.25

- 0.5

- 1

- 2

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Antagonism52

Antifungal Combinations

Theoretical basis for antagonism:

Azole & polyene

No theoretical basis for antagonism:

Azole & 5FC

Azole & echinocandin

Polyene & 5FC

Polyene & echinocandin

Echinocandin & 5FC

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Azole-Polyene Interaction

Several recently published studies have reported in vivo antagonism of the fungicidal activity of AMB

when administered concurrently with FLU or ITRA

Sugar et al., JID 1998;177:1660-3

AMB+ITRA against C. albicans

Louie et al., AAC 1999;43:2831-7

Louie et al., AAC 1999;43:2831-40

AMB+FLU against C. albicans

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19

Azole-Polyene Interaction

Discourage concomitant use of AMB and azole antifungals.

A more rational approach may be to initiate therapy

with AMB and step down to an intravenous or oral azole when appropriate.

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Summary56

Antifungal Susceptibility testing offers valuable information to assist with making treatment decisions

Trends in resistance remain stable over the last 20 years

New methods may make antifungal susceptibility testing routine in the microbiology laboratory

Questions?57