Abstract No clinical trials for histoplasmosis have been performed with the newer azoles, leaving itraconazole as the azole of choice. In vitro studies suggest that Histoplasma capsulatum from patients that relapse on fluconazole has higher minimum inhibitory concentrations (MICs) to fluconazole and voriconazole but not itraconazole and posaconazole. The newest azole, isavuconazole, shares structural similarity to voriconazole, but to date nobody has explored emergence of resistance. In vitro susceptibilities to isavucoanzole and fluconazole were performed on previously obtained isolates from the patients who relapsed on fluconazole therapy. Susceptibilities were determined by NCCLS method M27A developed for yeasts, as modified for H. capsulatum. The change in the MIC from the primary to the relapse isolate was tested using Wilcoxon Rank-Sum for paired data. Among the primary isolates, the median MICs were 1.0 (range 0.25 to 4.0) μg/ml for fluconazole and ≤0.007 (range ≤0.007 to 0.015) μg/ml for isavuconazole. In the group of relapsed isolates, the median MICs rose to 8.0 (range 0.25 to 64.0) μg/ml for fluconazole and remained unchanged at ≤0.007 (range ≤0.007 to 0.015) μg/ml for isavuconazole (P < .001). Only one isolate exhibited a nonsignificant increase in MIC to isavuconazole. Histoplasma isolates from patients who relapsed on fluconazole did not have an elevation in MICs to isavuconazole. This differs from the results previously seen with voriconazole and suggests that despite a closer structural similarity to voriconazole than itraconazole and posaconazole, isavuconazole has a higher barrier to resistance and may be effective as therapy for histoplasmosis. Histoplasma capsulatum, treatment, azole, fluconazole, isavuconazole Introduction Histoplasmosis is the most common endemic mycosis in the United States and worldwide.1 Areas of hyperendemnicity include the Ohio-Mississippi River valley and areas of Mexico, Central and South America,2 although it is also found in Africa,3 India,4 China,5,6 and Australia.7 Itraconazole has become the most commonly used and recommended azole therapy, either as step down from amphotericin B or as initial therapy in mild to moderate cases.8 However, itraconazole has significant limitations, such as variable absorption and metabolism that make it less favorable.9,10 Fluconazole is an alternative option; however, there are concerns for development of resistance and lower efficacy.11–13 For example, one third of the isolates from patients that relapse on fluconazole have reduced susceptibilities to fluconazole,13 and 70% exhibited increased MICs when the pre- and post-treatment isolates were compared.14 The mechanism of resistance was found to be the reduction in fluconazole susceptibility of cytochrome P450-dependant enzymes 14α-demethylase (CYP51p) and 3-ketosteroid reductase, most specifically via the Y136F mutation,15 which is analogous to the Y132H mutation conferring fluconazole resistance in Candida albicans.16 In addition, patients that relapse on fluconazole have increased MICs to voriconazole, with 41% of relapsed isolates showing a fourfold or greater increase in MIC to voriconazole.14 This is not the case for itraconazole and posaconazole.14 Isavuconazole is the newest commercially available azole and is more structurally similar to fluconazole and voriconazole as compared to itraconazole and posaconazole. This raises a concern that the same mechanism of resistance may arise on treatment with isavuconazole.17 However, isavuconazole does have significant structural differences (exists as a prodrug, and contains a fourth ring that is designed to improve absorption and provide a larger barrier to resistance). Therefore, understanding how successful this development strategy has been could help clinicians decide about its use in salvage treatment of histoplasmosis. In this study, we report the in vitro susceptibility to isavuconazole of isolates from patients who relapsed on fluconazole therapy as compared to the baseline isolates, in order to ascertain for treatment emergent resistance, in order to ascertain for treatment emergent resistance. Methods Isolates Isolates were obtained from a previously described trial of fluconazole for disseminated histoplasmosis in patients with AIDS.13 Pre- and post-treatment isolates were studied in patients who relapsed while on fluconazole. The isolates were stored frozen in liquid nitrogen.12 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. IRB waiver for this analysis was obtained from the Washington University IRB. Antifungal susceptibility testing Antifungal susceptibility testing was performed using NCCLS method M27A developed for yeasts, as modified for H. capsulatum.12 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). Isavuconazole was tested at 8–0.007 μg/ml. 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. A fourfold or greater increase in MIC was considered significant. Statistical methods Due to small samples and the noncontinuous nature of the data, nonparametric tests were used throughout. The change in the MIC from the primary to the relapsed isolate was tested using Wilcoxon Rank-Sum for paired data. 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 azole were estimated along with range for these medians. Results Among the primary isolates, the median MICs were 1.0 (range 0.25 to 4.0) μg/ml for fluconazole and ≤0.007 (range ≤0.007 to 0.015) μg/ml for isavuconazole. In the group of relapsed isolates, the median MICs rose to 8.0 (range 0.25 to 64.0) μg/ml for fluconazole and remained unchanged at ≤0.007 (range ≤0.007 to 0.015) μg/ml for isavuconazole (P < .001) (Fig. 1). Figure 1. View largeDownload slide Comparison of MICs for pre-treatment versus relapse isolates. The MICs for the pre-treatment and relapse isolates are connected by a line for each patient. Of note, due to minimal changes and different baseline resistances, the scales for fluconazole and isavuconazole are different. μg, microgram; ml, milliliter. Figure 1. View largeDownload slide Comparison of MICs for pre-treatment versus relapse isolates. The MICs for the pre-treatment and relapse isolates are connected by a line for each patient. Of note, due to minimal changes and different baseline resistances, the scales for fluconazole and isavuconazole are different. μg, microgram; ml, milliliter. Of the 17 post-treatment isolates, 12 (71%) exhibited a fourfold or greater increase in MIC to fluconazole, which was not observed in isavuconazole. Only one isolate had a nonsignificant twofold MIC increase from ≤0.007 μg/ml to 0.015 μg/ml after treatment. The mean increase in MIC for fluconazole was 8.0 μg/ml (95% confidence interval [CI]: 2.0, 16.0) and could not be calculated for isavuconazole because the MICs for primary and relapse isolates were very similar. Of note, the single isolate that experienced a rise in MIC to isavuconazole of 1 dilution had a 16-fold increase in MIC to fluconazole. Discussion It was previously reported that Histoplasma capsulatum isolates that have developed fluconazole resistance through fluconazole treatment also have elevated MICs to voriconazole but not to itraconazole or posaconazole.14 The primary and relapsed isolates had a median voriconazole MIC of 0.015 μg/ml (range ≤0.007 μg/ml to 0.25 μg/ml) and 0.03 μg/ml (range ≤0.007 μg/ml to 1.0 μg/ml), with 42% of the isolates exhibiting a fourfold or higher increase in MIC.14 Since the time of that study, isavuconazole, a new azole has entered the market with activity against histoplasmosis.18,19 Even though isavuconazole is chemically more similar to fluconazole and voriconazole, we were able to demonstrate that the resistance mechanisms that lead to higher MICs in fluconazole and voriconazole do not affect in vitro susceptibility to isavuconazole. This suggests that the structural alterations, such as the addition of the fourth ring, may have led to a higher barrier to resistance in isavuconazole. The highest isavuconazole MIC observed in both the primary and the relapse isolates was 0.0015 μg/ml. This is significantly lower than the fluconazole MICs, but more importantly, in a phase 3 trial of isavuconazole, 100% of patients achieved serum levels at least 10 times higher than the highest MIC observed in our study.20 This is also lower than previously-reported MICs.21 Furthermore, unlike voriconazole,14 isavuconazole did not demonstrate any increase in MICs amongst isolates with known CYP51p Y136F mutations. Only one isolate had a rise in the MIC when comparing pre-and-post treatment, and did not meet the fourfold increase in MIC required to reach significance. This suggests that the mutations that are presumed to underlie the development of fluconazole and voriconazole MIC elevations on treatment do not have the same phenotypic effect when the organism is exposed to isavuconazole. Even though no significant isavuconazole resistance was appreciated in this study, even in isolates with elevated MICs to fluconazole and voriconazole, this should not necessarily translate to widespread clinical use of isavuconazole for treatment of histoplasmosis. To date, the only published clinical experience is the VITAL study, analyzing the effect of isavuconazole on Cryptococcus and dimorphic moulds.18 In that study, seven patients with histoplasmosis (three with primary pulmonary and four with disseminated disease, including one with central nervous system disease) were treated with isavuconazole as primary therapy, with five (71%) having a stable or resolved disease, and two (29%) with clinical progression. In trials of itraconazole response rates of 85% have been reported.22 The 15% of patients that that did not respond were lost to follow up or intolerant of itraconazole. However, these are small numbers, the patient populations are different, and the studies were performed in different underlying conditions, so no direct comparisons should be made. Further clinical studies are needed before isavuconazole is used routinely in the treatment of histoplasmosis. This study has a limitation in that in vitro fungal MICs may not predict the clinical response in patients, no animal models were performed to assess for a clinical response, and the small number of samples were available. In conclusion, Histoplasma capsulatum has low initial MICs to isavuconazole, and isolates obtained from patients who relapsed on fluconazole treatment and where resistance mutations evolved do not exhibit a significant increase in MICs to isavuconazole. These data would suggest that isavuconazole has a potential as a treatment for histoplasmosis, but clinical studies are warranted. Declaration of interest A.S. has received funding from Astellas. L.J.W. is employed by MiraVista Diagnostics. In addition, research reported in this publication was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR002345 from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. All other authors have no conflict of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Wheat LJ, Azar MM, Bahr NC et al. Histoplasmosis. Infect Dis Clin North Am . 2016; 30: 207– 227. Google Scholar CrossRef Search ADS PubMed 2. Neglected Histoplasmosis in Latin America Group. Disseminated histoplasmosis in Central and South America, the invisible elephant: the lethal blind spot of international health organizations. AIDS . 2016; 30: 167– 170. CrossRef Search ADS PubMed 3. Bahr NC, Sarosi GA, Meya DB et al. Seroprevalence of histoplasmosis in Kampala, Uganda. Med Mycol . 2016; 54: 295– 300. Google Scholar CrossRef Search ADS PubMed 4. Gopalakrishnan R, Nambi PS, Ramasubramanian V, Abdul Ghafur K, Parameswaran A. Histoplasmosis in India: truly uncommon or uncommonly recognised? J Assoc Physicians India , 2012; 60: 25– 28. Google Scholar PubMed 5. Pan B, Chen M, Pan W, Liao W. Histoplasmosis: a new endemic fungal infection in China? Review and analysis of cases. Mycoses . 2013; 56: 212– 221. Google Scholar CrossRef Search ADS PubMed 6. Wen FQ, Sun YD, Watanabe K et al. Prevalence of histoplasmin sensitivity in healthy adults and tuberculosis patients in southwest China. J Med Vet Mycol . 1996; 34: 171– 174. Google Scholar CrossRef Search ADS PubMed 7. McLeod DS, Mortimer RH, Perry-Keene DA et al. Histoplasmosis in Australia: report of 16 cases and literature review. Medicine (Baltimore) . 2011; 90: 61– 68. Google Scholar CrossRef Search ADS PubMed 8. Wheat LJ, Freifeld AG, Kleiman MB et al. Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the infectious diseases society of America. Clin. Infect. Dis . 2007; 45: 807– 825. Google Scholar CrossRef Search ADS PubMed 9. Pasqualotto AC, Denning DW. Generic substitution of itraconazole resulting in sub-therapeutic levels and resistance. Int J Antimicrob Agents . 2007; 30: 93– 94. Google Scholar CrossRef Search ADS PubMed 10. Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc . 2011; 86: 805– 817. Google Scholar CrossRef Search ADS PubMed 11. Wheat LJ, Connolly P, Haddad N et al. Antigen clearance during treatment of disseminated histoplasmosis with itraconazole versus fluconazole in patients with AIDS. Antimicrob Agents Chemother . 2002; 46: 248– 250. Google Scholar CrossRef Search ADS PubMed 12. Wheat LJ, Connolly P, Smedema M et al. Emergence of resistance to fluconazole as a cause of failure during treatment of histoplasmosis in patients with acquired immunodeficiency disease syndrome. Clin Infect Dis . 2001; 33: 1910– 1913. Google Scholar CrossRef Search ADS PubMed 13. Wheat J, MaWhinney S, Hafner R et al. 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 . 1997; 103: 223– 232. Google Scholar CrossRef Search ADS PubMed 14. Wheat LJ, Connolly P, Smedema M et al. Activity of newer triazoles against Histoplasma capsulatum from patients with AIDS who failed fluconazole. J Antimicro. Chemother . 2006; 57: 1235– 1239. Google Scholar CrossRef Search ADS 15. Wheat J, Marichal P, Vanden Bossche H, Le Monte A, Connolly P. Hypothesis on the mechanism of resistance to fluconazole in Histoplasma capsulatum. Antimicrob Agents Chemother . 1997; 41: 410– 414. Google Scholar PubMed 16. Kelly SL, Lamb DC, Kelly DE. Y132H substitution in Candida albicans sterol 14α-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett . 1999; 180: 171– 175. Google Scholar PubMed 17. Falci DR, Pasqualotto AC. Profile of isavuconazole and its potential in the treatment of severe invasive fungal infections. Infect Drug Resist . 2013; 6: 163– 174. Google Scholar PubMed 18. Thompson GR3rd, Rendon A, Ribeiro Dos Santos R et al. Isavuconazole treatment of cryptococcosis and dimorphic mycoses. Clin Infect Dis . 2016; 63: 356– 362. Google Scholar CrossRef Search ADS PubMed 19. Seyedmousavi S, Verweij PE, Mouton JW. Isavuconazole, a broad-spectrum triazole for the treatment of systemic fungal diseases. 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Medical Mycology – Oxford University Press
Published: Dec 14, 2017
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