JAC Advance Access originally published online on April 20, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1235-1239; doi:10.1093/jac/dkl133
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Activity of newer triazoles against Histoplasma capsulatum from patients with AIDS who failed fluconazole
1 MiraVista Diagnostics/MiraBella Technologies 4444 Decatur Boulevard, Indianapolis, IN 46241, USA 2 Indiana University School of Medicine 1100 West Michigan Street, Indianapolis, IN 46202, USA 3 Schering-Plough Research Institute 2015 Galloping Hill Road, K15-4-4700, Kenilworth, NJ 07033, USA
*Corresponding author. Tel: +1-317-856-2681; Fax: +1-317-856-3685; E-mail: jwheat{at}miravistalabs.com
Received 25 November 2005; returned 24 January 2006; revised 1 February 2006; accepted 21 March 2006
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Abstract |
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Objectives: To determine the activity of newer triazoles against strains of Histoplasma capsulatum resistant to fluconazole.
Methods: Susceptibility testing was performed on 17 paired pre- and post-treatment H. capsulatum isolates from patients with AIDS who failed fluconazole.
Results: The median MICs of fluconazole, voriconazole, and posaconazole and ravuconazole for the pre-treatment isolates were 1 mg/L, 0.015 mg/L and <0.007 mg/L, respectively. A 4-fold or greater increase in the MIC of fluconazole and voriconazole was observed in 12 and 7 of the post-treatment isolates, respectively; the median fold increases in MIC were 8 and 2.1, respectively. No MIC increases were observed for posaconazole and ravuconazole. One pair of isolates exhibiting reduced susceptibility was examined in more detail. A single amino acid substitution (at tyrosine 136) was identified in the active site of the CYP51 protein from the post-treatment isolate, which is presumed to be responsible for reduced susceptibility to voriconazole and fluconazole, analogous to recent observations in Candida albicans.
Conclusions: These findings support careful monitoring for relapse in patients receiving voriconazole treatment for histoplasmosis, particularly in those who were previously treated with fluconazole.
Keywords: voriconazole , posaconazole , ravuconazole , susceptibility , resistance
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Introduction |
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Itraconazole is an effective therapeutic drug for histoplasmosis but is not well tolerated in all patients and exhibits variable drug exposure due to differences in absorption or metabolism. Fluconazole is less active than itraconazole. For example, one-third of cases relapsed in one study1 and reduced susceptibility to fluconazole occurred in the isolates from most patients who failed therapy.2 The mechanism of resistance was the reduction in susceptibility of cytochrome P450-dependent enzymes 14
-demethylase (CYP51p) and 3-ketosteroid reductase to fluconazole.3 Posaconazole, ravuconazole and voriconazole are active against Histoplasma capsulatum, and posaconazole and ravuconazole were effective in an experimental model of histoplasmosis.4,5 Voriconazole and posaconazole have not been approved for histoplasmosis but have been increasingly used in patients unable to take itraconazole (L. J. Wheat, unpublished data).
Recent studies of mechanisms of resistance to azoles in Candida albicans and Aspergillus fumigatus showed that mutations in cytochrome P450-dependent enzyme 14-demethylase (CYP51p) frequently resulted in cross-resistance to fluconazole and voriconazole but not always to itraconazole or posaconazole.6 In this article we report the in vitro susceptibility to the newer triazoles of isolates from patients who failed fluconazole therapy and the identification of a single amino acid substitution in CYP51p that may account for differences in susceptibility.
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Materials and methods |
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Isolates
The fluconazole trial was described previously.1 Pre- and post-treatment isolates were stored frozen in liquid nitrogen for 17 patients who failed fluconazole therapy.2 Informed consent was obtained from all patients in the clinical trial, in accordance with human experimentation guidelines of the US Department of Health and Human Services and the investigators' institutional policies.
Antifungal susceptibility testing
Antifungal susceptibility testing was performed using NCCLS method M27A developed for yeasts, as modified for H. capsulatum.2 The drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 times the concentration of the final drug dilution and then diluted in RPMI 1640 medium (Bio Whitakker 04-525F) containing the test strains of H. capsulatum or the control strains, which were Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258). The newer triazoles were tested at 8–0.007 mg/L. The endpoints were read visually and defined as the concentration of the drug that inhibited 80% or greater of the organism growth as compared with the no drug control. The powder formulations of the antifungal agents used for susceptibility testing were obtained from the pharmaceutical manufacturers.
DNA sequencing of CYP51 alleles
The accession number for CYP51A is AY690428 [GenBank] . The H. capsulatum genomic sequence was obtained from Washington University (http://genomeold.wustl.edu/blast/histo_client.cgi). Oligonucleotides were used to PCR-amplify the two CYP51 alleles in overlapping 600 bp segments from total genomic DNA isolated using the method of Spitzer et al.7 Both strands of each allele were sequenced in their entirety using a Beckman CEQ8000 Genetic Analyzer (Beckman Coulter, Fullerton, CA, USA), and sequence alignments and analysis were performed using Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) and Vector NTI (Informax, Carlsbad, CA, USA).
Statistical methods
The MICs for the primary isolates were compared among the four triazoles using the Kruskal–Wallis test. Sidak's adjustment for multiple comparisons was used to make pair-wise comparisons between the triazoles. The change in the MIC was defined as the relapse isolate MIC minus the primary isolate MIC. The ratio of the MICs was defined as the ratio of the relapse isolate MIC to the primary isolate MIC. The median values for the change and ratio within each triazole were estimated along with 95% confidence intervals (CI) for these medians.
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Results |
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The MICs for the 17 primary isolates obtained before starting the treatment are shown in Figure 1(a). The median MICs were 1.0 mg/L for fluconazole, 0.015 mg/L for voriconazole and <0.007 mg/L for posaconazole and ravuconazole. While the MIC of voriconazole was significantly lower than that of fluconazole, MICs of fluconazole and voriconazole were higher than those of posaconazole or ravuconazole.
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0.007 mg/L).Of the 17 post-treatment isolates, 12 and 7 isolates exhibited a 4-fold or greater decrease in susceptibility to fluconazole and voriconazole, respectively, but none showed a change in MIC of posaconazole or ravuconazole (Figure 1b). The median increase in MIC of fluconazole was 7.5 mg/L (95% CI: 1.5, 15.0), compared with 0.008 mg/L (95% CI: 0.0, 0.11 mg/L) for voriconazole. The fold increase in median MIC was 8.0 (95% CI: 2.0, 16.0) for fluconazole and 2.1 (95% CI: 1.0, 8.3) for voriconazole.
While the change in MIC of voriconazole was less than that of fluconazole, the 95% CI was above 1.0. A change in MIC could not be determined for posaconazole or ravuconazole because the MICs for primary and relapse isolates were very similar.
To determine whether the changes in susceptibility were due to alterations in the target site, a matched pair of pre-/post-treatment isolates was analysed in more detail. A CYP51 allele in H. capsulatum (designated CYP51A in this study) was previously identified.8 Interrogation of the H. capsulatum genome sequences deposited at Washington University identified a second allele, CYP51B. Although CYP51Ap and CYP51Bp are highly homologous, the start of the open reading frame of CYP51Bp was not readily apparent (Figure 2). Comparison of the CYP51Ap amino acid sequences from a fluconazole-susceptible pre-treatment isolate (MIC 1 mg/L) and a post-treatment isolate exhibiting reduced susceptibility to fluconazole (MIC 16 mg/L) identified a single substitution in the post-treatment isolate; tyrosine at position 136 was replaced by phenylalanine (Y136F). There were no substitutions in CYP51Bp in either the pre- or post-treatment isolates.
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Discussion |
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The findings in this study suggest that voriconazole may be less effective in treating histoplasmosis than itraconazole, ravuconazole or posaconazole. The median MIC of voriconazole for H. capsulatum, although lower than that of fluconazole, was higher than those of posaconazole and ravuconazole. Exposure to fluconazole has been associated with a reduction in susceptibility to voriconazole in about 40% of strains. Also important in the antifungal efficacy of voriconazole is the drug exposure. While fluconazole achieves predictable high concentrations in the serum and tissues in patients treated with the high dosages used in histoplasmosis,1 voriconazole concentrations are lower and more variable.9 Whether voriconazole will be effective in histoplasmosis, and whether it will select for resistant isolates, as seen with fluconazole, remains to be determined, since it has not been studied in a prospective clinical trial. In the absence of clinical trials establishing its efficacy, the reduction in susceptibility to voriconazole and the potential for induction of resistance support the need for careful monitoring of patients with histoplasmosis treated with voriconazole, especially if they are immunosuppressed or have been previously treated with fluconazole.
The mechanism for resistance to fluconazole in H. capsulatum has been examined.3 Cytochrome P450-dependent enzymes 14-demethylase (CYP51p) and 3-ketosteroid reductase became less sensitive to fluconazole in the relapse isolate in that study. No differences in intracellular concentrations of itraconazole or fluconazole were seen in the relapse versus the pre-treatment isolate. The recent availability of genomic sequence allowed us to determine whether the changes in susceptibility in a pair of isolates from this study were caused by amino acid substitutions in CYP51p. Sequencing identified a single substitution (Y136F) in CYP51Ap from a post-treatment isolate exhibiting reduced susceptibility to fluconazole that was absent in the matched fluconazole-susceptible baseline isolate. An alignment of CYP51Ap with CYP51p from C. albicans revealed that Y136 is analogous to Y132 in C. albicans (Figure 2). Previously, a substitution at Y132 in CYP51p from C. albicans was demonstrated to interfere with fluconazole binding to the purified protein.10 In addition to being moderately resistant to fluconazole the post-treatment isolate exhibited a 16-fold reduction in susceptibility to voriconazole but no change in susceptibility to either posaconazole or ravuconazole. We propose that the Y136F substitution in the relapse isolate is responsible for the reduction in susceptibility to fluconazole and voriconazole.
Recent studies began to elucidate the cause for different susceptibility patterns among the triazoles.6 Triazoles with a more extended structure, such as posaconazole (and presumably to a lesser degree ravuconazole), are predicted to make extensive contacts with the residues in the target protein, both in the vicinity of the active site and further afield. In contrast, the more compact triazoles, such as fluconazole and voriconazole, principally interact with residues in the region of the active site. Consequently, when mutations occur in and around the active site they impact the binding of the compact triazoles much more than they do for triazoles such as posaconazole.
In summary, fluconazole treatment of disseminated histoplasmosis in patients with AIDS was associated with induction of resistance to fluconazole and, to a lesser extent, to voriconazole. A single amino acid substitution in CYP51p at Y136 appeared to be responsible for the reduction in susceptibility seen in the relapse isolate. Additional studies are needed to determine whether voriconazole is effective for the treatment of histoplasmosis caused by strains with reduced susceptibility and whether it induces resistance. Until such studies are completed, monitoring for relapse is indicated, particularly in those previously treated with fluconazole.
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Transparency declarations |
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L. J. W. is on the speaker panel for Pfizer, Fujisawa Healthcare Inc., and Merck and is a consultant and investigator for Schering-Plough. M. G. has received honoraria and research support from Pfizer, is an investigator for Schering-Plough and a consultant for Merck. P. M., R. P. and P. M. M. are employees of Schering-Plough Research Institute.
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Acknowledgements |
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The clinical trial from which the isolates for this investigation were obtained was sponsored by The AIDS Clinical Trials Group and Mycoses Study Group of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract numbers AI 25859 and NO-1-AI-65296. Pfizer Central Research was the pharmaceutical sponsor for the clinical trial. No financial support was obtained for the susceptibility testing, and the genetic analysis was performed by the co-authors at Schering-Plough Research Institute.
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References |
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1 Wheat J, MaWhinney S, Hafner R, et al. (1997) Treatment of histoplasmosis with fluconazole in patients with acquired immunodeficiency syndrome. National Institute of Allergy and Infectious Diseases Acquired Immunodeficiency Syndrome Clinical Trials Group and Mycoses Study Group. Am J Med 103:223–32.[CrossRef][Web of Science][Medline]
2 Wheat LJ, Connolly P, Smedema M, et al. (2001) Emergence of resistance to fluconazole as a cause of failure during treatment of histoplasmosis in patients with acquired immunodeficiency disease syndrome. Clin Infect Dis 33:1910–13.[CrossRef][Web of Science][Medline]
3 Wheat J, Marichal P, Vanden Bossche H, et al. (1997) Hypothesis on the mechanism of resistance to fluconazole in Histoplasma capsulatum. Antimicrob Agents Chemother 41:410–14.[Abstract]
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Connolly P, Wheat J, Schnizlein-Bick C, et al. (1999) Comparison of a new triazole antifungal agent, Schering 56592, with itraconazole and amphotericin B for treatment of histoplasmosis in immunocompetent mice. Antimicrob Agents Chemother 43:322–8.
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Clemons KV, Martinez M, Calderon L, et al. (2002) Efficacy of ravuconazole in treatment of systemic murine histoplasmosis. Antimicrob Agents Chemother 46:922–4.
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Xiao L, Madison V, Chau AS, et al. (2004) Three-dimensional models of wild-type and mutated forms of cytochrome P450 14-sterol demethylases from Aspergillus fumigatus and Candida albicans provide insights into posaconazole binding. Antimicrob Agents Chemother 48:568–74.
7 Spitzer ED, Keath EJ, Travis SJ, et al. (1990) Temperature-sensitive variants of Histoplasma capsulatum isolated from patients with acquired immunodeficiency syndrome. J Infect Dis 162:258–61.[Web of Science][Medline]
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Revankar SG, Fu J, Rinaldi MG, et al. (2004) Cloning and characterization of the lanosterol 14-demethylase (ERG11) gene in Cryptococcus neoformans. Biochem Biophys Res Commun 324:719–28.[CrossRef][Web of Science][Medline]
9 Purkins L, Wood N, Greenhalgh K, et al. (2003) The pharmacokinetics and safety of intravenous voriconazole—a novel wide-spectrum antifungal agent. Br J Clin Pharmacol 56:2–9.
10
Kelly SL, Lamb DC, Kelly DE. (1999) Y132H substitution in Candida albicans sterol 14-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett 180:171–5.[Web of Science][Medline]
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