JAC
Journal of Antimicrobial Chemotherapy (2000) 46, 323–342
Correspondence Azole cross-resistance to ketoconazole, fluconazole, itraconazole and voriconazole in clinical Candida albicans isolates from HIV-infected children with oropharyngeal candidosis J Antimicrob Chemother 2000; 46: 338–341 Frank-Michael C. Müllera,b, Michael Weigc, Joanne Petera and Thomas J. Walsha a
Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; b Universitätskinderklinik and Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg; c Institut für Hygiene und Mikrobiologie, Universität Würzburg, Würzburg, Germany *Correspondence address. Universitätskinderklinik, Universität Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany. Tel: -931-201-5831; Fax: 49-931-201-5833; E-mail:
[email protected] Sir, The occurrence of azole cross-resistance in clinical Candida albicans isolates has been demonstrated in human immunodeficiency virus (HIV)-infected adults suffering recurrent oropharyngeal candidosis (OPC).1,2 However, little attention has been drawn so far to the clinical significance of azole cross-resistance in paediatric patients. In this study we report the development of crossresistance to various azoles in paediatric HIV patients with OPC. As part of a prospective study, conducted at the National Cancer Institute, 29 paired and serial C. albicans isolates from the oral mucosa of 10 HIV-infected children with recurrent OPC were obtained (Table). The children had acquired HIV either vertically (patients 1–3, 5, 6 and 9) or by blood transfusion (patients 4, 7, 8 and 10) and had a history of symptomatic OPC. Recurrent episodes had been managed with treatment courses of clotrimazole, ketoconazole, itraconazole, fluconazole, amphotericin B and cyclodexrine itraconazole. None of the patients had received voriconazole during the study period. All isolates were identified by the Clinical Microbiology Laboratory of the Warren Grant Magnuson Clinical Center of the National Institutes of Health. The 20C Analytic Profile Index strip
(bioMérieux, Marcy l’Etoile, France) was used to identify C. albicans. Polyethylene glycol 400 was used to solubilize ketoconazole and itraconazole (Janssen Pharmaceutica, Piscataway, NJ, USA), while DMSO (Merck, Darmstadt, Germany) was used to solubilize voriconazole (Pfizer, Sandwich, UK) and amphotericin B (Bristol-Myers Squibb, Princeton, NJ, USA). Stock solutions of amphotericin B, fluconazole (Pfizer, Groton, CT, USA), ketoconazole and itraconazole were prepared using RPMI-1640 buffered with 0.165 M MOPS t pH 7.0 (Biowhittacker, Walkerville, MD, USA), whereas voriconazole was prepared in HR antifungal compounds were 0.03–16 mg/L for amphotericin B, ketoconazole, itraconazole and voriconazole, and 0.125–64 mg/L for fluconazole. Broth microdilution testing was performed according to the NCCLS M27-A reference method. The C. albicans inoculum size ranged between 0.5 103 and 2.5 103 cfu/mL. In each case, the inoculum size was verified by colony counting. The 96-well plates were incubated at 37°C in air. The MICs were recorded at 24 and 48 h. ATCC strains 90028 (C. albicans), 6258 (Candida krusei) and 22019 (Candida parapsilosis) were included as controls. All isolates were biotyped by random amplified polymorphic DNA, inter-repeat PCR and electrophoretic karyotype for genetic alterations. In nine patients, consecutively obtained C. albicans isolates were found to be isogenic by all three methods. In patient 1, the first four isolates (mb4389, mb1307, mb8808 and mb8456) were isogenic, whereas isolates mb4544 and mb8577 showed a different karyotype. According to NCCLS breakpoints, all C. albicans isolates of three children (patients 1, 4 and 9) developed crossresistance to ketoconazole, fluconazole and itraconazole, as well as high voriconazole MICs consistent with resistance. All isolates remained susceptible to amphotericin B (Table). All isolates that were cross-resistant to ketoconazole, fluconazole (MIC 16 mg/L). However, in patient 1 a correlation between an altered karyotype of isolates mb4544 and mb8577 and greater susceptibility to all four azoles was observed. The isolates from three children (patients 2, 3 and 7) developed increased MICs or resistance to ketoconazole and fluconazole, but remained susceptible, dose dependently, to itraconazole and voriconazole, and were highly susceptible to amphotericin B. C. albicans isolates from four children (patients 5, 6, 8 and 10) were susceptible to all azoles, but the MICs of fluconazole either increased four-fold (patients 5, 6 and 8) or were already ‘susceptible dose-dependent’ (patient 10), whereas all isolates remained susceptible to ketoconazole, itraconazole, voriconazole and amphotericin B.
338 © 2000 The British Society for Antimicrobial Chemotherapy
Correspondence Table. Candida albicans isolates from 10 HIV-infected children with recurrent OPC MIC (mg/L) Patient
Isolate
Datea
1
mb4389 mb1307 mb8808 mb8456 mb4544 mb8577
06/10/93 08/05/93 10/18/93 01/12/95 02/01/96 07/31/97
2
mb2771 mb8586 mb6935 mb4657 mb2421
11/15/93 01/11/94 10/18/94 04/18/95 06/26/96
0.03 0.06 0.25 0.125 1
3
mb1873 mb5550
05/14/93 06/12/93
0.5 0.25
4
mb1672 mb8781 mb5330
11/03/93 04/20/94 06/29/94
0.5 4 4
5
mb4074 mb74080
08/31/93 10/04/93
0.125 0.03
6
mb1447 mb2771 mb7040
08/06/93 05/31/94 05/17/95
0.06 0.125 0.125
7
mb9329 mb8292
04/28/93 07/19/93
0.25 0.5
8
mb2143 mb4821
08/13/93 03/07/94
0.03 0.03
9
mb4256 mb3961
09/01/93 02/25/94
0.5 0.5
16 64
10
mb4738 mb5645
06/23/94 07/03/94
0.5 0.03
16 16
a
ketoconazole
fluconazole 64 64 64 64 32 32
8 8 8 16 1 0.25
2 2 8 8 64 8 32 2 64 64 0.5 2 2 4 8 16 64 0.5 2
itraconazole 16 16 16 16 2 1 0.06 0.06 0.26 0.125 0.25 32 32 0.125 16 16 0.125 0.125
voriconazole 16 16 16 16 1 1 0.03 0.03 0.06 0.06 0.25 0.25 0.5 16 16 16 0.03 0.03
amphotericin B 0.25 0.25 0.25 1 0.5 1 0.25 0.25 0.5 0.25 0.5 0.5 0.125 1 0.5 1 0.5 0.5
0.1125 0.25 0.125
0.06 0.125 0.125
0.5 0.25 0.5
0.5 0.5
0.5 1
2 1
0.03 0.03
0.03 0.03
0.5 0.5
0.25 16
0.5 1
1 0.25
0.125 0.125
1 0.5
0.125 0.06
Month/day/year.
In our study, high cross-resistance to all azoles tested was found in the isolates from three children. It seems likely that, although the newer azoles, fluconazole, itraconazole and voriconazole bind more selectively to fungal cell enzymes and are not associated with endocrine toxicity, the common mode of action by inhibition of the cytochrome P-450-dependent 14-sterol demethylase may be an important factor for development of cross-resistance. In a previous study using isolates from adult patients, Odds noticed that resistance of a Candida sp. to one azole derivative implies cross-resistance to the other azole antifungal agents.3 Likewise, in several other studies on adult patients with
chronic mucocutaneous candidosis refractory to ketoconazole therapy, C. albicans isolates were cross-resistant to various azoles. In this study, we observed children with isolates that were resistant to ketoconazole and fluconazole, but remained susceptible dose-dependently to itraconazole and voriconazole. Another group of our patients harboured strains that were susceptible to all azoles, but the MICs of fluconazole either increased over the time or were already ‘susceptible dose-dependent’. These data indicate that azole crossresistance develops gradually during antifungal therapy. The data of Cartledge et al.2 are in accordance with our
339
Correspondence findings. In that study, 700 clinical C. albicans isolates from HIV-positive adult patients with OPC were examined for azole cross-resistance: 431 isolates were fully susceptible to all azoles (ketoconazole, fluconazole and itraconazole), 100 were resistant to fluconazole alone, 94 were resistant to fluconazole and ketoconazole but were susceptible to itraconazole, and 50 were resistant to all three azoles tested.2 No isolate was found to be resistant to ketoconazole without being fluconazole resistant, and no itraconazole resistance was detected without ketoconazole resistance.2 A significant number of fluconazole-resistant strains of C. albicans from adult patients with AIDS remained susceptible to ketoconazole and itraconazole in studies by Barchiesi et al.4 and Johnson et al.5 Vanden Bossche et al. described crossresistance of C. albicans strains to itraconazole limited to a few fluconazole-resistant isolates.6 Other studies have demonstrated that voriconazole could be active against fluconazole-resistant C. albicans isolates. In our study, we were able to demonstrate resistance to voriconazole in isolates that were cross-resistant to all three azoles tested. The MICs of voriconazole in fluconazole-resistant C. albicans isolates were generally high (16 mg/L). The emergence of cross-resistance to antifungal azoles in paediatric patients with OPC, including azoles not previously used in children, does therefore occur. These findings underscore the importance of standardized susceptibility testing of C. albicans and for the development of alternative treatment strategies in paediatric patients.
Acknowledgement F.-M. C. Müller was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF).
References 1. Stevens, D. A. & Stevens, J. A. (1996). Cross-resistance phenotypes of fluconazole-resistant Candida species: results with 655 clinical isolates with different methods. Diagnostic Microbiology and Infectious Diseases 26, 145–8. 2. Cartledge, J. D., Midgley, J. & Gazzard, B. G. (1997). Clinically significant azole cross-resistance in Candida isolates from HIVpositive patients with oral candidosis. AIDS 11, 1839–44. 3. Odds, F. C. (1993). Resistance of yeasts to azole-derivative antifungals. Journal of Antimicrobial Chemotherapy 31, 463–71. 4. Barchiesi, F., Colombo, A. L., McGough, D. A., Fothergill, A. W. & Rinaldi, M. G. (1994). In vitro activity of itraconazole against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients infected with human immunodeficiency virus. Antimicrobial Agents and Chemotherapy 38, 1530–3. 5. Johnson, E. M., Warnock, D. W., Luker, J., Porter, S. R. & Scully, C. (1995). Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis. Journal of Antimicrobial Chemotherapy 35, 103–14. 6. Vanden Bossche, H., Dromer, F., Improvisi, I., Lozano-Chiu, M., Rex, J. H. & Sanglard, D. (1998). Antifungal drug resistance in pathogenic fungi. Medical Mycology 36, 119–28.
340
