Does azole prophylaxis confer resistance to amphotericin B and influence virulence?

1. Does azole prophylaxis confer resistance to amphotericin B and influence virulence? Malcolm Richardson Department of Bacteriology & Immunology Haartman Institute University of Helsinki

2. Antifungal resistance Terminology • Mycological resistance/failure • Clinical resistance/failure

3. What do we mean by resistance? Therapeutic failure + MICs rising from pretreatment levels + ?same fungal strain

4. Resistance: factors to consider • Patient status • neutrophil count • management • antifungal serum levels (compliance/absortption) • Previous and current antifungal therapy • dose • duration • route • Mycological status: rising MICs

5. Reasons for therapeutic failure • Drug resistance • Poor patient compliance • Inadequate drug absorption or distribution • Drug inactivation or interactions • Impaired host immunological responses

6. Resistance to antifungals • Low concentration at infected site • Bioavailability at infected site • Resistance in experimentally-infected animals • Prevention of access to drug target • Modification of drug target • Increased production of drug target

7. Prevention and control of antifungal resistance • Prudent use of antifungals • Appropriate treatment avoiding inappropriate doses • Therapy with combinations of existing agents • Treatment with the appropriate antifungal (in cases where the pathogen is known) • Use of surveillance studies to determine the true frequency of antifungal resistance

8. Cellular mechanisms of antifungal drug resistance • Change to a more resistant species • Change to a more resistant strain • Genetic alterations that render a strain resistant • Transient gene expression that renders a cell temporarily resistant • Alteration in cell type • Alterations in fungal population

9. Persistence of resistance • Transmission • fluconazole-resistant C. albicans in AIDS • amphotericin B-resistant C. lusitaniae • stability of resistant isolates

10. Drug resistance Acquired: C. albicans Fluconazole ?Itraconazole ?A. fumigatus ?Itraconazole ?AmB Intrinsic C. glabrata Fluconazole ?Itraconazole C. krusei Fluconazole

11. Polyene resistance Myth or reality? “Polyene resistance has not been a major clincial problem to date” White et al. Clin Microbiol Rev 1998; 11: 382-402 “Despite more than 30 years of clinical use, resistance to polyene antifungals is rare” Ghannoum & Rice Clin Microbiol Rev 1999; 12: 501-517

12. Resistance to amphotericin B: emerging clinical and microbiological patterns 1. • Rare • Candida albicans • Cryptococcus neoformans • Intrinsic resistance • Scedosporium apiospermum • Fusarium spp. • Very little acquired resistance • ?effect of azole prophylaxis Sterling & Mertz. Drug Resistance Updates 1998; 1: 161-165.

13. Resistance to amphotericin B: emerging clinical and microbiological patterns 2. • 44 published cases • AIDS • neutropenic patients • Candida spp. (6) • Trichosporon beigelli • Cryptococcus neoformans • Aspergillus fumigatus • 59% amphotericin B resistance acquired during therapy • 86% of cases did not clear infection • where infections cleared importance of host immunity Sterling & Mertz. Drug Resistance Updates 1998; 1: 161-165.

14. Mechanism of resistance to polyenes • Alteration of cell wall components: eg. Glucan in hyphae of Aspergillus species • Mutants • increasing concentrations of AmB • gradient concentration • Reduced binding of AmB to sterols in membrane • sterol substitution • masking of existing sterols

15. In vitro susceptibility of Aspergillus spp. to amphotericin B • 230 isolates • 156 A. fumigatus • 20 A. terreus • 22 A. flavus • 17 A. nidulans • 15 A. niger • Broth dilution method • No AmB MICs >2 mg/ Damaoud et al. J Antimicrob Chemother 1999; 44: 553-555.

16. Alteration of cell composition leads to amphotericin B resistance in Aspergillus flavus • AmB-resistant mutant • acquired resistance to AmB and nystatin • spheroplasts sensitive • cell wall analysis • glucans • alterations in cell wall proteins Conclusion: alterations in outer cell wall AmB resistance Seo et al.Microbiol Immunol 1999; 43: 1017-1025.

17. Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles 1. “Inadvertant clinical selection for resistance to AmB may be more likely due to prolonged azole use than to AmB therapy” • Candida species differ in their response to AmB • Preexposure to azoles decreased the susceptibilities of all Candida species that were otherwise found to be susceptible to AmB Vazquez et al. J Clin iMicrobiol 1998; 36: 2690-2695

18. Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles 2 • C. albicans was unique: preexposure to azolesallowed growth, not just survival, in the presence of AmB • fluconazole-mediated AmB tolerance established by only a few hours exposure to fluconazole • protection lasts a few days Vazquez et al. J Clin Microbiol 1998; 36: 2690-2695

19. Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles 3 “Clinicial implications of this study are apparent. If patients fail to respond to fluconazole, they are frequently switched to amphotericin B” “Our in vitro data suggess that these sequential treatments may be counterproductive” Vazquez et al. J Clin Microbiol 1998; 36: 2690-2695.

20. Isolation and characteristics of fluconazole-and amphotericin B-resistant Candida albicans from blood of two patients with leukemia • Fungaemia • Fluconazole prophylaxis 400 mg/day, 2 weeks • Empiric amphotericin B 0.5 mg/kg/day • Fluconazole MICs >64 mg/l, no history of previous exposure • Isolates resistant to amphotericin B • Successful treatment with amphotericin B 1-1.25 mg/kg/day + 5FC 150 mg/kg/day Nolte et al. Antimicrob Agents Chemother 1997; 44: 196-199

21. Isolation and characteristics of fluconazole-and amphotericin B-resistant Candida albicans from blood of two patients with leukemia Conclusion “The emergence of fluconazole- and amphotericin B-resistant strains of C. albicans is a troubling new development” Nolte et al. Antimicrob Agents Chemother 1997; 44: 196-199

22. Is there a relationship between resistance development and virulence? • Animal models: resistance appears to correlate with diminished virulence • azole-resistant C. albicans: CMC • C. albicans strains defective in sterol 5,6 desaturase • AIDS patients with OPC: sequential isoaltes: azole resistant: no decrease in virulence Conclusion: azole resistance and decrease in virulence Sanglard. Drug Resistance Updates 1998; 1: 255-265

23. Fitness of azole-resistant clinical isolates • Exposure to azole antifungals selects azole-resistant isolates • Does persistence of azole-resistant strains require continuing presence of azole antifungals? • Maintenance of azole-resistant isolates will depend on their fitness (virulence) compared to azole-susceptible isoaltes • Conclusion: azole resistance acquired by genetic alterations is not necessarily linked with a decrease in virulence

24. Alteration in cell type • Morphological forms of C. albicans differ in susceptibility to azoles • Azole drugs interfer with hyphal elongation • Do resistant yeasts form hyphae? • Are resistant strains with the ability to form hyphae, even in the presence of azoles, more pathogenic than a sensitive strain unable to form hyphae?

25. Are azole-resistant yeasts more resistant to phagocytic killing? • Candida albicans • virulent strains more resistant to phagocytosis and intracellular killing • fluconazole-resistant strains more resistant to phagocytosis and killing • ?alterations in complement/opsonin receptors • Aspergillus fumigatus ???