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PPT DEVELOPMENT OF RESISTANCE TO LAMISIL TO WORRY OR NOT TO

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PPT DEVELOPMENT OF RESISTANCE TO LAMISIL TO WORRY OR NOT TO Powered By Docstoc
					     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
              Conflict of Interest
   Funding from various pharmaceutical and biotech
    companies in the form of grants and contracts
   Act as consultant and speaker’s bureau member
    for different companies
   Companies relevant to this presentation include:
    –   Pfizer
    –   Novartis Consumer Health and Pharma
    –   Schering-Plough
    –   Aventis
    –   Merck
       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.
Annual Number of Reports on Medline Search
  Annual number of English language articles retrieved by Medline
           for Antifungal Resistance
                 search for antifungal resistance
                             50

                             45
                                     Total AF
                             40      Azoles
                                     5FC
                             35      A mph B                                                    I TR A
        # of hits per year




                             30
                                                                M ICO
                             25                                                          FLU

                             20
                                                5 FC
                             15                                         K ETO

                             10

                              5

                              0
                              1966    1970             1974   1978         1982   1986   1990            1994
                                                                          Ye ar



      Resistance is reported two years after the introduction of a
       new antifungal agent
      Most studies are limited to systemic antifungals
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.
        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
 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 - 35°C


 Norris et al. (1999) J.Amer.Acad. Derm. 40:S9-S13
 Jessup et al. (2000) J. Clin. Microbiol. 38:341-344
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)
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
Dermatophytes from Epidemiological Study:
       Terbinafine Susceptibility

                                            MIC (μg/mL)
Organism            Sample size (n)
                                      <0.001 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
Dermatophytes from Epidemiological Study:
       Fluconazole Susceptibility

                    Sample size                        MIC (μg/mL)
Organism
                       (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
Dermatophytes from Epidemiological Study:
       Itraconazole Susceptibility

                      Sample size               MIC (μg/mL)
  Organism
                         (n)        <0.06 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
Dermatophytes from Epidemiological Study:
       Griseofulvin Susceptibility

                      Sample                  MIC (μg/mL)
  Organism
                      size (n)   <0.125 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
    Susceptibility of Clinical Trial
     Isolates against Terbinafine
Organism            Sample               MIC (μg/mL)
                     Size
                      (n)  <0.001 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
            Susceptibility of Clinical Trial
             Isolates against Fluconazole
Organism            Sample                        MIC (μg/mL)
                     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
            Susceptibility of Clinical Trial
             Isolates against Itraconazole
Organism            Sample                          MIC (μg/mL)
                     Size
                      (n)    <0.06   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   -   -   -
       Susceptibility of Clinical Trial
        Isolates against Griseofulvin
Organism`           Sample                     MIC (μg/mL)
                     Size
                      (n)  <0.125 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   -
Cumulative MIC Values of Terbinafine
for ALL Dermatophyte Isolates Tested
Organism            Isolates  Range     MIC50 MIC90
                      (n)    (g/mL)
T. rubrum             955 <0.001 – 0.25 0.002 0.015

T. mentagrophytes    153    <0.001 – 0.06 0.001 0.004

T. tonsurans         164    <0.001 – 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   <0.001 – 0.25 0.002 0.015
                  MIC Cumulative Data - Terbinafine
                              Terbinafine MIC Cumulative Data

                 180
                          T. rubrum
                 160
                          T. mentagrophytes
                 140
Total Isolates




                 120      T. tonsurans
                 100      M. canis
                  80      E. floccosum
                  60
                  40
                  20
                   0
                       0.03   0.015   0.008   0.004   0.002     0.001   <.001
                                              ug/ml
MIC Cumulative Data (cont’d.)

                             Fluconazole MIC Cumulative Data

                 200
                           T. rubrum
                 180
                 160
                           T. mentagrophytes
                 140       T. tonsurans
Total Isolates




                 120       M. canis
                 100       E. floccosum
                 80
                 60
                 40
                 20
                  0
                       1      2        3   4    5      6   7   8   9
                                               ug/ml
                 MIC CUMULATIVE DATA

                             Itraconazole MIC Cumulative Data

                 140
                       T. rubrum
                 120
                       T. mentagrophytes
                 100   T. tonsurans
Total Isolates




                  80   M. canis
                  60   E. floccosum
                  40

                  20

                   0
                       1          0.5      0.25           0.125   0.06   <.06
                                                  ug/ml
                       MIC Cumulative Data (cont’d.)

                                 Griseofulvin MIC Cumulative Data
                 200
                 180       T. rubrum
                 160
                           T. mentagrophytes
Total Isolates




                 140
                 120
                           T. tonsurans
                 100       M. canis
                 80        E. floccosum
                 60
                 40
                 20
                  0
                       4         2        1     0.5     0.25    0.125   <.125
                                               ug/ml
                    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
     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
      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
       Patient’s failure could be
             attributed to:
a) Decrease in antifungal susceptibilities of
     the infecting organism
b) Re-infection with a new genetically
   unrelated strain
c) Host-related factors
   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
A Representative Example of MICs of
 Sequential Isolates from One Patient

                       MIC (g/ml)
  Specimen      Flu     Itra     Terb Griseo
  ____________________________________________

     1462        .25     .125    <.001   .5
     3117        .25     .125    <.001   .5
     4385        .25     .125    <.001   .5
    11009        .25     .125    <.001   .5

  • Similar patterns were observed in 37 patients
 Only one patient had sequential isolates that had
       elevated MICs against terbinafine




Mukherjee et al. (2003) Antimicrob. Agents Chemother. 47:82-86
   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
     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. 47:82-86
     Is There Cross-resistance to Other
      Squalene Epoxidase Inhibitors?
                                             Drug         Isolate    MIC (g/mL)
      NFI 5147 – visit 2                    Naftifine    NFI 5147        4
      NFI 5150 – visit 7                                 NFI 5150        4
      NFI 1895 – terbinafine-                            NFI 1895      0.008
       susceptible reference strain          Butenafine NFI 5147         64
                                                          NFI 5150      >128
                                                          NFI 1895     0.0002
                                             Tolnaftate   NFI 5147      0.25
      Cross resistance to
                                                          NFI 5150      >128
       other squalene                                     NFI 1895     0.0002
       epoxidase ihibitors                   Tolciclate   NFI 5147       16
       was observed                                       NFI 5150      >128

                                                          NFI 1895     0.0002
Mukherjee et al. (2003) Antimicrob. Agents
Chemother. 47:82-86
    Are the Six Sequential Isolates
         Genetically Related?

   To answer this question, we performed
    random amplified polymorphic DNA
    (RAPD) analysis
                 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.
                 RAPD analysis:
 PCR was performed using the primer OPK-
  17* and extracted DNA as a template.
 The PCR program cycle used: 40 sec. at
  94°C, 45 cycles of 20 sec. at 94°C, 1 min. at
  35°C, 1 min. at 72°C and an extension for
  10 min. at 72°C.
 The PCR products were separated by
  electrophoresis.

   Zhong et al. (1997) Jpn. J. Med. Mycol. 38:239-246
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. 47:82-86
      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
                  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
           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
           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
     Contributors:

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

				
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Description: PPT DEVELOPMENT OF RESISTANCE TO LAMISIL TO WORRY OR NOT TO