MICROBIOLOGY AND DERMATOLOPHYTE RESISTANCE RELATED TO THE TREATMENT OF TINEA PEDIS

1. 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

2. 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

3. 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.

4. 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

5. 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.

6. 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

7. 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

8. 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)

9. 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

10. Dermatophytes from Epidemiological Study: Terbinafine Susceptibility

11. Dermatophytes from Epidemiological Study: Fluconazole Susceptibility

12. Dermatophytes from Epidemiological Study: Itraconazole Susceptibility

13. Dermatophytes from Epidemiological Study: Griseofulvin Susceptibility

14. Susceptibility of Clinical Trial Isolates against Terbinafine

15. Susceptibility of Clinical Trial Isolates against Fluconazole

16. Susceptibility of Clinical Trial Isolates against Itraconazole

17. Susceptibility of Clinical Trial Isolates against Griseofulvin

18. Cumulative MIC Values of Terbinafine for ALL Dermatophyte Isolates Tested

19. MIC Cumulative Data - Terbinafine

20. MIC Cumulative Data (cont’d.)

21. MIC CUMULATIVE DATA

22. MIC Cumulative Data (cont’d.)

23. 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

24. 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

25. 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

26. 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 • Host-related factors

27. 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

28. A Representative Example of MICs of Sequential Isolates from One Patient MIC (g/ml) • Similar patterns were observed in 37 patients 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

29. Only one patient had sequential isolates that had elevated MICs against terbinafine Mukherjee et al. (2003) Antimicrob. Agents Chemother. 47:82-86

30. 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

31. 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

32. Is There Cross-resistance to Other Squalene Epoxidase Inhibitors? • 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. 47:82-86

33. Are the Six Sequential Isolates Genetically Related? • To answer this question, we performed random amplifiedpolymorphic DNA (RAPD) analysis

34. 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.

35. 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

36. 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

37. 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 Is Patient Failure Due to Host-Related Factors?

38. 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

39. 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

40. 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

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