MICROBIOLOGY AND DERMATOLOPHYTE RESISTANCE RELATED TO THE TREATMENT OF TINEA PEDIS

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MICROBIOLOGY AND DERMATOLOPHYTE RESISTANCE
RELATED TO THE TREATMENT OF TINEA PEDIS

• M. Ghannoum, Ph.D.
• Professor, Department of Dermatology
• University Hospitals of Cleveland
• Case Western Reserve University
• Director, Center for Medical Mycology, Cleveland,
  Ohio, USA

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Conflict of Interest

• Funding from various pharmaceutical and biotech
  companies in the form of grants and contracts
• Act as consultant and speakers bureau member for
  different companies
• Companies relevant to this presentation include
• Pfizer
• Novartis Consumer Health and Pharma
• Schering-Plough
• Aventis
• Merck

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Antifungal Agents And Resistance

• A number of safe and highly efficacious
  antifungal agents (e.g. allylamines and azoles)
  have been introduced for the treatment of
  superficial fungal infections.
• Although the new drugs are welcome additions,
  like other antimicrobials they pose the potential
  threat of resistance.
• This is clearly illustrated by the significant
  increase in the occurrence of resistance to
  systemic antifungals (e.g., fluconazole).

• Ghannoum and Rice (1999) Clin. Microbiol. Rev.
• Hossain et al. (2001) Expert Opin. Invest. Drugs.

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Annual Number of Reports on Medline Search for
Antifungal Resistance

• Resistance is reported two years after the
  introduction of a new antifungal agent
• Most studies are limited to systemic antifungals

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Susceptibility Profile of Topical Antifungals

• In spite of the wide clinical use of topical
  antifungals, information about their
  susceptibility patterns is limited.
• Scarcity of information could be attributed to
  lack of a suitable method for determining the
  antifungal susceptibility of dermatophytes to
  clinically used topical antifungals.

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Assessment of Susceptibility of Dermatophytes

• A research program to develop a method for the
  antifungal susceptibility of dermatophytes
  against antifungals was initiated (1998) at the
  Center for Medical Mycology, Cleveland
• Antifungals tested
• Terbinafine
• Griseofulvin
• Itraconazole
• Fluconazole

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Optimal Conditions for Determination of
Antifungal Susceptibility of Dermatophytes

• Microdilution method
• Medium RPMI 1640
• Inoculum size - 2-5 x 103 conidia/mL
• Incubation time 4 days
• Incubation temperature - 35C

Norris et al. (1999) J.Amer.Acad. Derm.
40S9-S13 Jessup et al. (2000) J. Clin.
Microbiol. 38341-344
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Current Status of Susceptibility Test Methods for
Dermatophytes (NCCLS)

• Under the auspices of the NCCLS, and in
  preparation for approval of the developed assay
  as a Reference Method
• The NCCLS conducted an intra- and
  inter-laboratory multi-center study of this
  method for the testing of dermatophytes (Ghannoum
  et al., 2004, in press).
• The NCCLS is in the process of conducting a
  quality control study
• Based on the results of this mutli-center study,
  adoption of this method as an amendment to the
  NCCLS M38-A standard for the testing of
  dermatophytes will be undertaken (January, 2005)

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Use of the Developed Method to Monitor Resistance
in Dermatophytes

• Is there a resistance issue?
• At the Center for Medical Mycology, we have been
  actively monitoring susceptibnility patterns of
  various antifungals against a wide range of
  dermatophyte isolates
• Sources of dermatophyte isolates tested
• Sequential isolates obtained from an
  onychomycosis clinical trial
• Routine clinical specimens received at the Center
  
• Clinical trials of topical agents in use use, and
• Two epidemiological studies in onychomycosis and
  tinea capitis.
• In total 2,189 isolates were tested

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Dermatophytes from Epidemiological Study
Terbinafine Susceptibility
Organism Sample size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample size (n) lt0.001 0.001 0.002 0.004
T. rubrum 82 16 38 24 1
T. mentagrophytes 33 14 16 3 -
T. tonsurans 1 1 - - -
M. canis 1 - 1 - -
All derm isolates 117 31 55 27 4
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Dermatophytes from Epidemiological Study
Fluconazole Susceptibility
Organism Sample size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample size (n) 0.125 0.25 0.5 1 2 4 8 16 32
T. rubrum 82 - 6 20 41 8 6 - 1 -
T. mentagrophytes 33 - 1 5 8 6 5 3 1 4
T. tonsurans 1 - - - - - - 1 - -
M. canis 1 1 - - - - - - - -
All derm isolates 117 1 7 25 49 14 11 4 2 4
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Dermatophytes from Epidemiological Study
Itraconazole Susceptibility
Organism Sample size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample size (n) lt0.06 0.06 0.125 0.25 0.5 1
T. rubrum 82 20 29 22 4 6 1
T. mentagrophytes 33 19 11 2 - 1 -
T. tonsurans 1 1 - - - - -
M. canis 1 1 - - - - -
All derm isolates 117 41 40 24 4 7 1
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Dermatophytes from Epidemiological Study
Griseofulvin Susceptibility
Organism Sample size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample size (n) lt0.125 0.125 0.25 0.5 1 2
T. rubrum 82 1 2 16 49 9 5
T. mentagrophytes 33 12 16 5 - - -
T. tonsurans 1 - 51 - - - -
M. canis 1 - 1 - - - -
All derm isolates 117 13 20 21 49 9 5
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Susceptibility of Clinical Trial Isolates against
Terbinafine
Organism Sample Size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample Size (n) lt0.001 0.001 0.002 0.004 0.008 0.015 0.03
T. rubrum 132 40 65 23 4 - - -
T. mentagrophytes 32 14 16 2 - - - -
T. tonsurans 42 3 7 18 9 4 1 -
M. canis 7 - - - - 3 2 2
E. floccosum 3 - - - - 1 2 -
All derm isolates 216 57 88 43 13 8 5 2
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Susceptibility of Clinical Trial Isolates against
Fluconazole
Organism Sample Size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample Size (n) 0.125 0.25 0.5 1 2 4 8 16 32
T. rubrum 132 - 11 36 68 11 5 - 1 -
T. mentagrophytes 32 - 1 4 8 6 6 3 1 3
T. tonsurans 42 - 1 7 14 8 8 3 1 -
M. canis 7 2 2 1 1 1 - - - -
E. floccosum 3 - - - - 1 2 - - -
All derm isolates 216 2 15 48 48 27 21 6 3 3
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Susceptibility of Clinical Trial Isolates against
Itraconazole
Organism Sample Size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample Size (n) lt0.06 0.06 0.125 0.25 0.5 1 2 4 8
T. rubrum 132 20 36 34 24 17 1 - - -
T. mentagrophytes 32 19 10 2 - 1 - - - -
T. tonsurans 42 34 8 - - - - - - -
M. canis 7 6 1 - - - - - - -
E. floccosum 3 3 - - - - - - - -
All derm isolates 216 82 55 36 24 18 1 - - -
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Susceptibility of Clinical Trial Isolates against
Griseofulvin
Organism Sample Size (n) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL)
Organism Sample Size (n) lt0.125 0.125 0.25 0.5 1 2 4 8
T. rubrum 132 1 3 21 68 20 19 - -
T. mentagrophytes 32 10 14 8 - - - - -
T. tonsurans 42 - 2 6 18 9 2 5 -
M. canis 7 - 2 1 2 2 - - -
E. floccosum 3 - - - 1 1 1 - -
All derm isolates 216 11 21 36 88 32 22 5 -
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Cumulative MIC Values of Terbinafine for ALL
Dermatophyte Isolates Tested
Organism Isolates (n) Range (?g/mL) MIC50 MIC90
T. rubrum 955 lt0.001 0.25 0.002 0.015
T. mentagrophytes 153 lt0.001 0.06 0.001 0.004
T. tonsurans 164 lt0.001 0.06 0.008 0.03
M. canis 52 0.004 0.06 0.015 0.03
E. floccosum 11 0.008 0.06 0.015 0.03
All derm isolates 1,335 lt0.001 0.25 0.002 0.015
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MIC Cumulative Data - Terbinafine
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MIC Cumulative Data (contd.)
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MIC CUMULATIVE DATA
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MIC Cumulative Data (contd.)
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Summary

• Fluconazole, griseofulvin, itraconazole, and
  terbinafine are active against the tested
  organisms in vitro
• No resistance to these drugs was detected
  (Griseo?)
• Terbinafine showed the most potent antifungal
  activity relative to the other antifungals tested
• Some clinical isolates tended to have higher MICs
  to griseofulvin compared to epidemiological
  isolates

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Terbinafine Susceptibility

• 99.4 (2,176/2,189) of dermatophyte isolates
  tested had terbinafine MIC ? 0.06 ?g/mL
• We detected a set of sequential isolates with
  elevated MICs
• These isolates were selected for further analysis

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Characterization of the Sequential T. rubrum
Isolates with Elevated MICs

• Isolates obtained from patients enrolled in a
  multi-center onychomycosis clinical trial
• 1,500 patients
• Therapy Oral terbinafine (250 mg/d) for 12 24
  weeks
• Selection criteria
• Culture positive at the initial visit
• Culture positive at one or more visits during the
  study
• Culture positive at the end of the study
• Thirty-eight patients positive for T. rubrum
  throughout the study were chosen for evaluation.
• Total of 140 sequential isolates obtained

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Patients failure could be attributed to

• a) Decrease in antifungal susceptibilities of
• the infecting organism
• b) Re-infection with a new genetically
• unrelated strain
• Host-related factors

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Is there a Decrease in Antifungal Susceptibility
of the Sequential Isolates?

• In all cases, the MICs of terbinafine from each
  patient set were either identical or within 1
  tube dilution, implying no resistance developed.
• The same results were obtained within each set
  against fluconazole, itraconazole, and
  griseofulvin, indicating there is no
  cross-resistance
• One exception was observed for the antifungal
  susceptibility to griseofulvin, with one isolate
  having a 3-fold increase in the MIC.
• Bradley M, Leidich S, Isham N, Elewski B, and
  Ghannoum M. Mycoses 1999. 42(S2)105-110

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A Representative Example of MICs of Sequential
Isolates from One Patient
MIC (?g/ml)

• Specimen Flu Itra Terb Griseo
• ____________________________________________
• 1462 .25 .125 lt.001
  .5
• 3117 .25 .125 lt.001
  .5
• 4385 .25 .125 lt.001
  .5
• 11009 .25 .125 lt.001
  .5

• Similar patterns were observed in 37 patients

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Only one patient had sequential isolates that had
elevated MICs against terbinafine
Mukherjee et al. (2003) Antimicrob. Agents
Chemother. 4782-86
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Patient Failure is Not Due to a Decrease in
Antifungal Susceptibility

• One exception where all 6 sequential T. rubrum
  isolates (including baseline isolate) were found
  to have greatly reduced susceptibility
• These sequential isolates were analyzed further

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Is There Cross-resistance to Other Classes of
Antifungals?

• No cross-resistance to azoles or griseofulvin was
  observed

Mukherjee et al. (2003) Antimicrob. Agents
Chemother. 4782-86
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Is There Cross-resistance to Other Squalene
Epoxidase Inhibitors?
Drug Isolate MIC (?g/mL)
Naftifine NFI 5147 4
NFI 5150 4
NFI 1895 0.008
Butenafine NFI 5147 64
NFI 5150 gt128
NFI 1895 0.0002
Tolnaftate NFI 5147 0.25
NFI 5150 gt128
NFI 1895 0.0002
Tolciclate NFI 5147 16
NFI 5150 gt128
NFI 1895 0.0002

• NFI 5147 visit 2
• NFI 5150 visit 7
• NFI 1895 terbinafine-susceptible reference
  strain
• Cross resistance to other squalene epoxidase
  ihibitors was observed

Mukherjee et al. (2003) Antimicrob. Agents
Chemother. 4782-86
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Are the Six Sequential Isolates Genetically
Related?

• To answer this question, we performed random
  amplified polymorphic DNA (RAPD) analysis

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DNA Extraction

• Isolates were grown in Brain Heart Infusion.
  Mycelia incubated with 1 cetyltrimethylammonium
  bromide (CTAB).
• Phenol/chloroform/iso-amyl alcohol, glass beads
  and mycelia/CTAB mixture were vortexed and
  centrifuged.
• Supernatant was precipitated with ammonium
  acetate and ethanol.
• DNA pellets were obtained by microcentrifuging.

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RAPD analysis

• PCR was performed using the primer OPK-17 and
  extracted DNA as a template.
• The PCR program cycle used 40 sec. at 94C, 45
  cycles of 20 sec. at 94C, 1 min. at 35C, 1 min.
  at 72C and an extension for 10 min. at 72C.
• The PCR products were separated by
  electrophoresis.
• Zhong et al. (1997) Jpn. J. Med. Mycol.
  38239-246

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RAPD Analysis Revealed That The Sequential
Isolates Are Genetically Identical

• All the sequential isolates exhibited similar
  banding patterns
• Therefore, the isolates obtained at sequential
  visits represent a single strain of T. rubrum.

Mukherjee et al. (2003) Antimicrob. Agents
Chemother. 4782-86
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Is Patient Failure Due to Host-Related Factors?

• Review of clinical data for the 38 patients who
  failed terbinafine therapy may be attributed to
• History of prior use of other antifungals (53.3
  of patients received prior antifungals, including
  keto, itra and griseo)
• Family history of onychomycosis (60 of patients
  had one or more member of their family with
  history of onychomycosis)
• Age (70 were over 45 years old)
• Bradley M, Leidich S, Isham N, Elewski B, and
  Ghannoum M. Mycoses 1999. 42(S2) 105-110

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Summary

• Our data indicate that failure of patients to
  clear onychomycosis is
• Not related to resistance development(with one
  exception)
• Not due to re-infection with a new T. rubrum
  strain
• May be attributed to host-related factors
  including
• family history of onychomycosis
• prior antifungal treatment
• age

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Where Are We With Dermatophyte Susceptibility?

• A method to measure antifungal susceptibility is
  now established
• Only a few studies using this method have
  addressed whether resistance exists
• Based on these studies, resistance is not a
  problem (most compelling data is from
  terbinafine)
• There is a lack of data concerning the
  susceptibility profile of agents
• For dermatophytes, unlike for yeasts, the in
  vitro-in vivo correlation is lacking
• No breakpoints established for any of the drugs
  used to treat dermatophytosis
• Information about the mechanism of resistance is
  not available

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What Needs to be Done?

• Need to establish baseline data concerning
  antifungal agents, which will allow trend
  observation
• Surveillance studies to determine the true
  frequency of antifungal resistance should be
  implemented
• Studies to establish in vitro-in vivo correlation
  should be undertaken in animal models
• Data should be collected on both clinical and MIC
  from patients treated with various agents in an
  effort to establish breakpoints for different
  antifungal agents
• MIC data, using the developed method, should be
  collected as part of drug approval process

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Contributors

• Mary Bradley, M.S.
• Nancy Isham, B.A., M(ASCP)
• Steven Leidich, Ph.D.
• Pranab Mukherjee, Ph.D.