In Vitro Activity of the Novel Antifungal Olorofim against Scedosporium and Lomentospora prolificans

ABSTRACT Scedosporium spp. and Lomentospora prolificans are an emerging group of fungi refractory to current antifungal treatments. These species largely affect immunocompromised individuals but can also be lung colonizers in cystic fibrosis patients. Although Scedosporium apiospermum is thought to be the predominant species, the group has been expanded to a species complex. The distribution of species within the S. apiospermum species complex and other closely related species in the United States is largely unknown. Here, we used β-tubulin and ITS sequences to identify 37 Scedosporium isolates to the species level. These Scedosporium isolates as well as 13 L. prolificans isolates were tested against a panel of nine antifungal drugs, including the first in novel class orotimide, olorofim. IMPORTANCE Scedosporium and Lomentospora infections are notoriously hard to treat as these organisms can be resistant to numerous antifungals. The manuscript contributes to our knowledge of the activity of the new antifungal agent olorofim and comparator agents against Lomentospora and against Scedosporium isolates that have been molecularly identified to the species level. The efficacy of olorofim against all species of Scedosporium and Lomentospora was confirmed.

IMPORTANCE Scedosporium and Lomentospora infections are notoriously hard to treat as these organisms can be resistant to numerous antifungals. The manuscript contributes to our knowledge of the activity of the new antifungal agent olorofim and comparator agents against Lomentospora and against Scedosporium isolates that have been molecularly identified to the species level. The efficacy of olorofim against all species of Scedosporium and Lomentospora was confirmed.
KEYWORDS Lomentospora prolificans, Scedosporium, amphotericin B, olorofim, triazole S cedosporium species and Lomentospora prolificans are globally emerging, rare opportunistic fungal pathogens that can cause fungal infections that range from superficial colonization to invasive and disseminated infection (1). The majority of infections occur in patients with immunocompromising conditions, but trauma-associated cases, mycetoma, and lung colonization have also been identified in nonimmunocompromised individuals (2). Scedosporium species are also a frequent colonizer of the lungs of cystic fibrosis patients (3,4). The genus Scedosporium includes the species S. aurantiacum, S. minutisporum, S. desertorum, S. cereisporum, S. dehoogii, and the S. apiospermum species complex, including Pseudallescheria angusta, S. apiospermum, S. boydii, P. ellipsoidea and S. fusoidium (1,5,6). The genus Lomentospora contains the single clinically relevant species L. prolificans (formerly Scedosporium prolificans). Effective treatment options are limited for both Scedosporium and Lomentospora infections, and despite surgical intervention the mortality rate is high (7). Scedosporium is refractory to current antifungal treatment options, while L. prolificans is considered pan-antifungal resistant (8)(9)(10). Limited availability of viable antifungals for treatment of these infections highlights the need for development of novel antifungal agents.
Olorofim is a newly developed antifungal of the novel orotimide drug class. Olorofim (formerly F901318) was developed by F2G Limited (Manchester, England) and has been granted orphan drug approval by FDA for treatment of invasive scedosporiosis and invasive lomentosporiosis and breakthrough therapy designation by the FDA (11). Olorofim inhibits fungal dihydroorotate dehydrogenase (DHODH), a unique drug target that makes it less likely to be affected by acquired mutations which confer resistance to other antifungals and the fungal DHODH differs significantly from human DHODH minimizing target-based toxicity (12). Published data show olorofim has in vitro activity against Penicillium, Fusarium, Aspergillus, Scedosporium, Lomentospora, and other rare molds (13)(14)(15)(16)(17)(18)(19)(20).
Recent studies have shown the efficacy of olorofim against difficult to treat Scedosporium and Lomentospora infections (21)(22)(23). Our study expands upon recent findings on the efficacy of olorofim with the addition of new species of Scedosporium and additional isolates from the United States of both Scedosporium and Lomentospora. These fungi are often identified only to the genus or species complex level, but to establish proper breakpoints and epidemiological cutoff values, identification to species is essential. Here, we provide a careful species analysis of Scedosporium spp. and describe the efficacy of olorofim and five comparator antifungals against these isolates and isolates of L. prolificans.

RESULTS
Isolates of Scedosporium and Lomentospora prolificans were identified using ITS and b-tubulin gene sequencing. A phylogenetic tree using concatenated beta tubulin gene and ITS locus was constructed for definitive identification of isolates through phylogenetic clustering (Fig. 1). The phylogenetic tree showed that the isolates fell into 4 distinct groups; Lomentospora prolificans (n = 13), S. dehoogii (n = 2), S. aurantiacum (n = 1), and S. apiospermum species complex. The S. apiospermum species complex additionally had four clusters, which included S. apiospermum (n = 16), P. angusta (n = 4), P. ellipsoidea (n = 3), and S. boydii (n = 11) ( Table 1). The predominant species in the collection were S. apiospermum and L. prolificans, comprising 31% and 25% of all isolates, respectively. The S. apiospermum species complex as a whole comprised 92% of all isolates of Scedosporium.
Antifungal susceptibility testing for olorofim. Distribution of olorofim MIC results for each species are listed in Table 2. Overall, olorofim activity ranged from 0.008 to >2 mg/mL. Across all species tested, the modal MIC value was 0.06 mg/mL. There was no discernible difference in MIC ranges across species. Only two isolates, one S. apiospermum and one L. prolificans, had MIC values over 0.25 mg/mL, and both of those were >2 mg/mL. There was no species-specific pattern to the susceptibilities for olorofim. For all species with greater than 10 isolates (L. prolificans, S. apiospermum and S. boydii), the modal MIC value was 0.06 mg/mL.
AFST for azoles and amphotericin B. Table 2 summarizes the AFST results for the mold-active azoles and amphotericin B. In general, L. prolificans responded poorly to both the azoles and amphotericin B. A single isolate, B4654, had MIC values that were

DISCUSSION
This report provides MIC data for both currently available antifungal agents and the new antifungal olorofim against Scedosporium spp. and L. prolificans. The emergence of Scedosporium spp. and L. prolificans poses a clinical challenge as most isolates are multidrug resistant with particular resistance to amphotericin B, and the triazoles itraconazole and isavuconazole and response to antifungal treatment is generally poor (2,8,24). Lomentospora prolificans infections remain rare, but because of limited treatment options the mortality rate is greater than 90% (24). ESCMID and ECMM have issued joint guidelines on the use of voriconazole as first-line treatment but give no solid recommendation as outcomes remain poor (25). Recent studies have confirmed the relative effectiveness of voriconazole compared to the echinocandins and amphotericin B (15,26). Combination therapy, such as voriconazole with amphotericin B or terbinafine, is often used for L. prolificans and Scedosporium cases. There are no studies beyond case registries which might indicate a positive shift in outcome due to combination therapy, although at least one registry noted a positive trend in survival for patients treated with voriconazole and terbinafine for L. prolificans (7,8). Many other combination therapies have been tried but most of the data are single case descriptions (1). A large study of 264 Scedosporium isolates found that the MIC values for olorofim were lower than either voriconazole or posaconazole (7). Our data for a smaller number of Scedosporium isolates are similar. Our isolates collected from across the United States demonstrated a similar AFST resistance profile with olorofim being the most active antifungal across all Scedosporium species and L. prolificans.
Preliminary in vitro data on the antifungal activity of olorofim against mold species has been encouraging. Recent studies have demonstrated the in vitro efficacy of olorofim to Scedosporium spp. and L. prolificans (10,13,15,20,26 a The mode is provided only for species with more than 10 isolates. b Vori, voriconazole; Itra, itraconazole; Isav, isavuconazole; Posa, posaconazole; Amp B, Amphotericin B. and L. prolificans (15). Limited data are available regarding antifungal activity against P. angusta, and no study has reported the activity of olorofim. Our study shows that the activity of olorofim to P. angusta is similar to that of other Scedosporium spp. This report supports previous data, which suggest there are few antifungal options available for the treatment of Scedosporium species and L. prolificans, but also showcases the potential impact of the new antifungal olorofim. During a recent phase 2 clinical trial, olorofim was used with positive clinical outcomes for two cases of invasive lomentosporiosis (22,23). In order to establish both species specific breakpoints and epidemiological cutoff values for olorofim, a tremendous amount of MIC data from diverse laboratories is essential. These data set, with well-defined species, contributes to that essential data and shows that olorofim has good activity across Scedosporium species and L. prolificans.

MATERIALS AND METHODS
Mold isolates. A total of 50 historical isolates in the CDC collection, received predominantly from U.S. states, were included in this study: Lomentospora prolificans (n = 13), Pseudallescheria angusta (n = 4), Scedosporium apiospermum (n = 16), Scedosporium aurantiacum (n = 1), Scedosporium boydii (n = 11), Scedosporium dehoogii (n = 2), and Pseudallescheria ellipsoidea (n = 3). Isolates were received between 1951 and 2019 and were collected from clinical and environmental sources (Table 1). Isolates were collected during routine identification in the fungal reference laboratory at the Centers for Disease Control and Prevention and stored at 280°C. Isolates were grown on Sabouraud dextrose agar and DNA was extracted using the DNeasy kit (Qiagen; Valencia, CA, USA) as described (27).
Phylogenetic analysis. The b-tubulin (BT2) (GenBank submission numbers OP649685 to OP649734) and internal transcribed spacer (ITS) genes (GenBank submission numbers OP480737 to OP480790) were sequenced for each isolate (27,28). Fragments were amplified using PCR and sequenced with BigDye Terminator as suggested by the manufacturer (Life Technologies; Grand Island, NY, USA). The PCR cycle was as follows: 95°C for 5 min, 30 cycles of 95°C for 30 s, 52 to 58°C for 30 s, and 72°C for 7 min. Geneious (Biomatters Limited; San Francisco, CA, USA) was used to edit, analyze, and concatenate the DNA sequences. Basic Local Alignment Search tool (BLAST) analysis in the National Center for Biotechnology Information database was used for initial determination of species using only the tubulin sequence. The sequences were then aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) alignment and a tree of concatenated sequences was generated using Geneious 11.1.2.
Olorofim. Susceptibility testing was performed following the Clinical and Laboratory Standards Institute (CLSI) reference standard M38-A2 (29). Olorofim powder was provided by F2G, Ltd., and dilutions of the drug were prepared using DMSO. The concentration range for testing was 0.001 to 2 mg/mL in 96-well microtiter plates with a u-shaped bottom. An HP D300e digital dispenser, HP dispensing software, and HP T81 and HP D41 dispensing cassettes were used to prepare the drug plates which were stored at 280°C until used (30). On the day of testing, the plates were thawed in a 37°C incubator before use. Isolates were tested against a set of five additional antifungals using commercially produced frozen panels as previously described for isavuconazole, itraconazole, posaconazole, and voriconazole (18). Amphotericin B MICs were determined using Etest (bioMérieux, France). Plates were incubated and results were read at 72 h.

ACKNOWLEDGMENTS
We have the following conflicts of interest to declare: Derek Law and Mike Birch are employees of F2G, Ltd.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.