Ana Espinel-Ingroff 1, Elizabeth Johnson 2, Hans Hockey 3 and Peter Troke 4 *

Journal of Antimicrobial Chemotherapy (2008) 61, 616 620 doi:10.1093/jac/dkm518 Advance Access publication 25 January 2008 Activities of voriconazole, itraconazole and amphotericin B in vitro against 590 moulds from 323 patients in the voriconazole Phase III clinical studies Ana Espinel-Ingroff 1, Elizabeth Johnson 2, Hans Hockey 3 and Peter Troke 4 * 1 Virginia Commonwealth University, Medical Centre, Richmond, VA, USA; 2 Health Protection Agency, Myrtle Road, Kingsdown, Bristol, UK; 3 Nevada Road, Hamilton 3216, New Zealand; 4 The Old Court, Kingsgate, CT10 3LW Kent, UK Received 3 October 2007; returned 9 November 2007; revised 26 November 2007; accepted 5 December 2007 Introduction: Fungal pathogens from the voriconazole trials were identified and tested for susceptibility at two reference laboratories. Methods: MICs were measured using CLSI M38-A 48 h microdilution methodology. Results: Moulds from 29 genera and 38 species were isolated from 18 countries. Aspergillus spp. predominated (69%), followed by Scedosporium spp. (11.5%). Aspergillus fumigatus (292/590, 49.5%) was the most common species, followed by Scediosporium apiospermum (9.7%) and Aspergillus terreus (7.3%). The bronchi, lungs and sinuses yielded 45% of the isolates (57% of aspergilli), with 24% from the oropharynx/oesophagus. Other sites included blood/catheter (7.3%) and CNS (5.2%). MIC 90 s of itraconazole and voriconazole for Aspergillus spp. were the same (0.5 mg/l), but 17 Aspergillus isolates were itraconazole-resistant (MICs 1 16 mg/l). Additionally, in 31 A. fumigatus and 23 A. terreus isolates, amphotericin MICs were 2.0 mg/l. Voriconazole MICs exceeded 4 mg/l in only 5.8% (34/590) of the isolates, including one A. fumigatus (8.0 mg/l), 9/11 Scedosporium prolificans, 10/13 Fusarium solani and all 9 Zygomycetes. Most were also not susceptible to itraconazole or amphotericin B. A notable increase in MIC (more than two doubling dilutions) during voriconazole therapy was seen for one A. fumigatus isolate. The response rate of voriconazole-treated patients with isolate MICs 4.0 mg/l was 38% when compared with 52% for those with MICs <4.0 mg/l. Conclusions: Voriconazole shows activity, in vitro, similar to that of itraconazole against a wide range of moulds. It is also active against some isolates not susceptible to itraconazole or amphotericin B, but not the Zygomycetes. The relationship between voriconazole MIC and clinical outcome requires further study. Keywords: Aspergillus, Scedosporium, Fusarium, Zygomycetes, MIC, clinical outcome Introduction Voriconazole is a wide-spectrum triazole antifungal agent that is approved for treating a range of fungal infections including those caused by the moulds Aspergillus, Scedosporium and Fusarium. There is an extensive literature detailing the in vitro susceptibility of fungi to voriconazole. 1 9 However, there are few data and mostly from case reports, examining the susceptibility of clinical mould isolates from patients treated with voriconazole. 10 20 The aim of the in vitro study reported here was to examine the worldwide susceptibility of moulds to voriconazole in comparison with the standard agents such as itraconazole and amphotericin B, to monitor for resistance during therapy and to examine any relationship between MIC and clinical response. Data for some of these isolates have been reported previously. 11 Methods The Voriconazole Mycology Reference Laboratories (A. E.-I., in Richmond, Virginia, USA and E. J. in Bristol, UK) confirmed isolate identity using standard methods and conducted susceptibility testing on 590 mould isolates from 323 patients included in the... *Corresponding author. Tel: þ44-1843-863900; E-mail: peter2troke@btinternet.com... 616 # The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Activities of voriconazole, itraconazole and amphotericin B Pfizer-sponsored, VCR Phase III global clinical studies and compassionate programmes as of October 2004. Both reference laboratories used the CLSI MIC method M38-A for moulds in its microdilution format. 21 Test strains were regularly exchanged between the two laboratories to ensure that their results were compatible. As no CLSI standard moulds were available at the time, the studies were conducted, and two Aspergillus fumigatus QC standards from the UK National Collection of Pathogenic Fungi (NCPF 7097 and NCPF 7100) were used on each test plate instead. Results Moulds from 29 genera and 38 species were isolated from 323 patients in 18 countries and 6 continents. However, most patients (71.8%) came from the USA, France and Germany. For those isolates where the numbers were large enough (Aspergillus spp. and Scedosporium spp.), there was no evidence for a significant impact of country of origin on MIC (data not shown). The most common predisposing underlying conditions were bone marrow or peripheral stem cell transplantation (32%), haematological malignancy (19%), solid organ transplantation (11%) and AIDS (9%). Most of the 590 isolates were from the bronchi, lungs or sinuses (45% overall, but 57% of the aspergilli) (Table 1). Other sites included the oropharynx/oesophagus (23%), blood/catheter (7.3%), brain/csf (5.2%) and bone, eye, liver, spleen, skin nodules and thyroid (9.0% in total). The majority of the isolates (57%) came from patients who had failed or been intolerant to prior antifungal therapies, whereas 43% came from primary therapy studies. Figure 1 shows the distribution of voriconazole, itraconazole and amphotericin B MIC values for all isolates tested, irrespective of genus. The MIC 90 s of both voriconazole and itraconazole were 1.0 mg/l and their MIC 50 s were 0.25 mg/l, whereas the corresponding values for amphotericin B were 4.0 and 1.0 mg/l, respectively. Aspergillus spp. accounted for 69% (409/590) of all mould isolates (A. fumigatus 49%, Aspergillus terreus 7.3% and Aspergillus flavus 6.1%). Scediosporium apiospermum (9.7%) was the most commonly isolated species, followed by Penicillium marneffei (5.8%; Table 2). Minor isolates (three or fewer) are given in Table 3. The voriconazole MICs for Aspergillus spp. ranged from 0.03 to 8.0 mg/l (MIC 90 0.5 mg/l). In only a single Aspergillus Table 1. Major body sites of the mould isolates Body site Moulds (n) Bronchi (bronchoalveolar lavage) 148 Oropharynx/oesophagus 135 Lung/sinuses (biopsy) 120 Other deep biopsies a 68 Skin, skin nodules, pus 44 Blood/catheter 43 Other sites b 33 a Brain, bone, liver, stomach wall, spleen, thyroid, lymph node and eye. b Faeces, nasal swabs, open wounds, unknown (7). Figure 1. Distribution of voriconazole MICs for all mould isolates. VCR, voriconazole; ITR, itraconazole; AMB, amphotericin B. isolate (A. fumigatus) did the MIC exceed 2.0 mg/l. This isolate also had a raised MIC of itraconazole (MIC range 0.25 4.0 mg/ L). However, 17 Aspergillus isolates were itraconazole resistant (MICs 1 16 mg/l). Additionally, 31 A. fumigatus and 23 A. terreus isolates had amphotericin MICs 2.0 mg/l. Scedosporium prolificans (MIC range 2.0 8.0 mg/l) and various Fusarium spp. (MIC range 1.0 16.0 mg/l) also yielded isolates with voriconazole MICs above the MIC 90 (Table 3). The few biphasic moulds (50 isolates) included in the database (Blastomyces dermatiditis, Histoplasma capsulatum, Paracoccidioides brasiliensis and P. marneffei) were all highly susceptible to voriconazole (Tables 2 and 3). The voriconazole, itraconazole and amphotericin B MICs for the 18 infrequently isolated genera including species of Acremonium, Alternaria, Blastomyces, Cladosporium, Curvularia, Cylindrocarpon, Exophiala, Fonsecaea, Microascus, Mycoleptodiscus, Neosartorya, Penicillium, Phialophora, Phoma, Pithomyces, Scopulariopsis, Trichoderma and Wangiella are given in Table 3. Voriconazole MICs were 2.0 mg/l for all isolates, except Scopulariopsis brevicaulis. In contrast, in 9 of these 18 isolates, itraconazole MICs were 2.0 mg/l, whereas in 12 of 18, amphotericin MICs were 2.0 mg/l. Among the 34 isolates with voriconazole MICs 4 mg/l (the resistance value established for yeasts by Pfaller et al. 22 ) were all 9 Zygomycetes (Absidia, Cunninghamella, Mucor, Rhizomucor and Rhizopus: MICs 8.0 16.0 mg/l), 10/13 Fusarium solani, 9/11 S. prolificans, 1 A. fumigatus, 1 Fusarium oxysporum, 1Fusarium proliferatum, 1Microascus cinereus, 1 S. apiospermum and 1 S. brevicaulis. However, 32 of 33 isolates also had MICs of itraconazole 1.0 mg/l and 26 of 34 had MICs of amphotericin B 2.0 mg/l. In total, 55 patients had 82 isolates from 16 genera, which were itraconazole resistant (Tables 2 and 3). There were 24 patients with high voriconazole MIC isolates (including 7 with a zygomycete) occurring at baseline or at some time during voriconazole therapy, plus an assessment of clinical efficacy. These patients showed a reduced clinical response rate (38%) to voriconazole therapy when compared with the 202 voriconazole-treated patients with clinical isolates with voriconazole MICs,4.0 mg/l (52% response). In 37 patients, multiple cultures of 38 isolates were obtained during voriconazole therapy [21 Aspergillus spp. (A. flavus, 617

Espinel-Ingroff et al. Table 2. Most commonly isolated mould spp. susceptibility of clinical isolates to voriconazole and standard agents MIC range [IC 50 ] (IC 90 ) mg/l Isolates (n) voriconazole itraconazole amphotericin B A. flavus (36) 0.12 1.0 [0.5] (0.5) 0.03 0.5 [0.0078] (0.25) 0.5 4.0 [1.0] (2.0) A. fumigatus (292) 0.06 8.0 [0.25] (0.5) 0.12 16.0 [0.25] (0.5) 0.25 4.0 [1.0] (2.0) A. nidulans (13) 0.03 2.0 [0.12] (0.12) 0.0078 0.25 [0.125] (0.25) 0.25 2.0 [1.0] (2.0) Aspergillus niger (21) 0.12 1.0 [0.31] (0.5) 0.062 0.5 [0.05] (0.5) 0.25 1.0 [0.5] (1.0) A. terreus (43) 0.03 1.0 [025] (0.5) 0.003 0.5 [0.062] (0.25) 0.5 4.0 [2.0] (2.0) Aspergillus spp. (4) a 0.12 0.5 0.12 0.5 2.0 4.0 Fusarium moniliforme (5) 1.0 2.0 2.0 16.0 1.0 4.0 F. solani (13) 1.0 16.0 [8.0] (16.0) 2.0 16.0 [16.0] (16.0) 0.5 4.0 [2.0] (4.0) Fusarium spp. (3) b 2.0 8.0 16.0 2.0 4.0 H. capsulatum (5) 0.0078 0.5 0.0078 0.5 0.0078 0.25 P. lilacinus (5) 0.06 0.125 1.0 2.0 16.0 16.0 P. brasiliensis (10) 0.003 0.06 [0.0039] (0.035) 0.003 0.03 [0.008] (0.03) 0.03 0.5 [0.0125] (0.5) P. marneffei (34) 0.003 0.003 [0.003] (0.003) 0.003 0.12 [0.03] (0.03) 0.25 1.0 [1.0] (1.0) S. apiospermum (57) 0.06 4.0 [0.25] (0.25) 0.03 2.0 [0.5] (1.0) 0.5 16 [4.0] (8.0) S. prolificans (11) 2.0 8.0 [4.0] (8.0) 4.0 16.0 [16.0] (16.0) 4.0 16.0 [16.0] (16.0) Zygomycetes (9) 8.0 16.0 0.12 16.0 0.25 2.0 a Aspergillus glaucus (2), Aspergillus sydowii (1) and Aspergillus versicolor (1). b Fusarium incarnatum (1), F. oxysporum (1), F. proliferatum (1). Zygomycetes ¼ Absidia corymbifera (2), Cunninghamella bertholetiae (2), Mucor racemosus (1), Rhizomucor pusillus (1), Rhizopus arrhizus (1) and Rhizopus microsporus (2). A. fumigatus, Aspergillus nidulans and A. terreus), 7 S. apiospermum, 3P. marneffei, 2F. solani, 2P. brasiliensis, 1 Cylindrocarpon lichenicola, 1 Fonsecaea pedrosoi and 1 Paecilomyces lilacinus]. In 20 of 37 patients, therapy exceeded 15 days (range 16 264 days, median 71 days). A notable (more than two doubling dilutions) MIC increase during therapy was detected for a single A. fumigatus isolate, obtained via sequential lung biopsies from a patient with pulmonary aspergillosis. After Table 3. Minor mould spp. susceptibility of clinical isolates to voriconazole and standard agents MIC range (mg/l) Isolates (n) voriconazole itraconazole amphotericin B Acremonium sp. (1) 0.5 16.0 2.0 Alternaria sp. (4) 0.25 1.0 0.125 0.25 0.25 2.0 B. dermatiditis (1) 0.0312 0.0078 0.5 Cladosporium cladosporiodes (1) 0.5 0.25 2.0 Curvularia sp. (1) 0.5 1.0 2.0 C. lichenicola (3) 2.0 16.0 1.0 Exophiala spinifera (1) 0.5 0.5 2.0 F. pedrosoi (4) 0.0156 0.0625 0.0312 0.0.0625 1.0 M. cinereus (1) 8.0 16.0 8.0 Mycoleptodiscus indicus (2) 0.0625 0.125 0.0625 0.0078 0.0625 Neosartorya fischeri (1) 0.25 0.125 1.0 Penicillium verrucosum (1) 2.0 1.0 0.5 Phialophora parasitica (1) 0.25 2.0 4.0 Phialophora richardsiae (1) 0.5 1.0 2.0 Phoma sp. (1) 0.5 0.5 4.0 Pithomyces sp. (2) 0.25 0.5 2.0 Scopulariopsis brevicaulis (1) 16.0 16.0 16.0 Trichoderma sp. (1) 1.0 16.0 2.0 Wangiella dermatiditis (1) 0.0625 0.0625 2.0 618

Activities of voriconazole, itraconazole and amphotericin B 148 days of therapy, the voriconazole MIC had increased 16-fold from 0.5 to 8.0 mg/l and the patient failed therapy. The isolate also became cross-resistant to itraconazole (MIC increase from 0.25 mg/l at baseline to 4.0 mg/l). Conclusions Voriconazole was highly potent in vitro against the majority of clinical isolates from the Phase III studies. These MIC results are consistent with the published data for recent isolate collections and case studies. 1 20 However, as has been established previously, the zygomycete isolates were not susceptible to voriconazole. 23 25 In general, the in vitro activity of voriconazole against the moulds in this study was comparable with that of itraconazole and somewhat better than amphotericin B. The voriconazole susceptibility ranges for the Fusarium species, especially F. solani, and S. prolificans were wide, which suggests that elevated dose levels of voriconazole or even combination therapy should be considered to treat these lesssusceptible but potentially severe infections. 26,27 However, outcome data suggest that voriconazole therapy alone may be successful in some cases. 11,28 Resistance development during long-term voriconazole therapy was detected in a single A. fumigatus isolate and this patient failed therapy. Unlike the yeasts, 22 a correlation between voriconazole mould MICs and clinical outcome remains to be established. Many of the patients in this analysis were neutropenic or had other significant immune deficits, and the overall response rate to voriconazole therapy was less than that for yeasts. 22 However, based on the current breakpoints for Candida spp. 21 and recent in vitro data for moulds, 30 plus the achievable voriconazole exposure in adults (6 mg/kg iv 12 hourly loading dose on day 1 then 4 mg/kg iv twice daily) and published efficacy against moulds, a 48 h resistance breakpoint of 4 mg/l might represent a suitable initial, experimental 11,28 30 value. Funding The data for this manuscript were generated during Pfizer-sponsored, Phase III clinical trials. Both P. T. and H. H. received funding from Pfizer in connection with the development of this manuscript. E. J. and A. E.-I. received funding from Pfizer to run the voriconazole mycological reference laboratories. Transparency declarations P. T. owns shares in, was previously an employee of, and is currently a consultant to Pfizer. He is also a consultant to and a share owner of Cytomics and has received honoraria from Rothschilds and F2G. H. H. is a statistical consultant to Pfizer. E. J. and A. E.-I. received funding from Pfizer to attend relevant conferences during the study period. E. J. has also received honoraria from Gilead, MSD, Ortho-Biotech, Pfizer, Schering-Plough and Zeneus. References 1. Espinel-Ingroff A, Boyle K, Sheehan DJ. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 2001; 150: 101 15. 2. Pfaller MA, Messer SA, Hollis RJ et al. Antifungal activities of posaconazole, ravuconazole, and voriconazole compared to those of itraconazole and amphotericin B against 239 clinical isolates of Aspergillus spp. and other filamentous fungi: report from SENTRY antimicrobial surveillance program, 2000. Antimicrob Agents Chemother 2002; 46: 1032 7. 3. Pfaller MA, Messer SA, Boyken L et al. 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