In vitro activity of manogepix and comparators against infrequently encountered yeast and mold isolates from the SENTRY Surveillance Program (2017–2022)

ABSTRACT Manogepix is a potent new antifungal agent targeting the fungal Gwt1 enzyme. Manogepix has previously demonstrated potent in vitro activity against clinical isolates of both Candida (except Candida krusei) and Aspergillus species. This study determined the in vitro activity of manogepix and comparators against a large collection of infrequently encountered yeast and molds. Manogepix demonstrated potent in vitro activity against infrequently encountered yeasts exhibiting elevated MIC values to other drug classes, including Candida spp. (MIC50/90, 0.008/0.12 mg/L), Saprochaete clavata (Magnusiomyces clavatus) (MIC50/90, 0.03/0.06 mg/L), Magnusiomyces capitatus (MICrange, 0.016-0.06 mg/L), Rhodotorula minuta (MIC, 0.016 mg/L), and Rhodotorula mucilaginosa (MIC50/90, 0.03/0.12 mg/L). Similarly, manogepix was active against infrequently encountered mold isolates and strains exhibiting elevated MIC/MEC values to echinocandins, azoles, and amphotericin B, including Coprinopsis cinerea (MEC, 0.004 mg/L), Fusarium spp. (MEC50/90, 0.016/0.06 mg/L), Fusarium (Gibberella) fujikuroi species complex (MEC50/90, 0.016/0.03 mg/L), Lomentospora prolificans (MEC50/90, 0.03/0.06 mg/L), Microascus cirrosus (MEC, 0.008 mg/L), Paecilomyces spp. (MEC50/90, ≤0.008/0.016 mg/L), Pleurostomophora richardsiae (MEC, 0.06 mg/L), Sarocladium kiliense (MEC range, 0.016–0.12 mg/L), and Scedosporium spp. (MEC50/90, 0.03/0.06 mg/L). Manogepix demonstrated potent activity against a majority of the infrequently encountered yeast and mold isolates tested including strains with elevated MIC/MEC values to other drug classes. Additional clinical development of manogepix (fosmanogepix) in difficult-to-treat, resistant fungal infections is warranted.

on the order of that typically encountered by fluconazole-resistant strains of Candida glabrata and C. krusei (13).Cross-resistance between fluconazole and voriconazole may be more prominent for non-Candida yeast species than for Candida spp.(13).Decreased susceptibility of most non-candidal yeasts to azoles is further complicated by the fact that some species, such as Saprochaete capitata (M.capitatus), S. clavata (M.clavatus), Trichosporon asahii, and Rhodotorula spp., are intrinsically resistant to the echinocandins (8,12,14).
Rare species of fungi are unlikely to be familiar to clinicians and microbiologists alike, and there are few or no data concerning prognosis or optimal treatment strat egies.In many instances, these organisms may present as breakthrough infections in immunocompromised hosts who have already been receiving an azole or echinocandin empirically (8,18).
Besides Candida and A. fumigatus, few laboratories identify fungi to the species level, and a minority perform antifungal susceptibility testing locally for systemically active antifungal agents (19)(20)(21).The identification of emerging, uncommon, yeasts and molds is complicated by the constant change in nomenclature, which may complicate treatment (7,14).As such, there is a critical role for antifungal surveillance programs that also include the less common fungi.Differences in the regional prevalence and resistance of these fungi render local epidemiological knowledge essential for the care of patients with a suspected IFI (3,8,15).
Although Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility testing has been standardized for testing of Candida spp., Cryptococcus spp., and Aspergillus spp.(22)(23)(24)(25)(26)(27) and may be applied to rare yeasts and molds, clinical breakpoints and epidemiological cutoff values (ECVs) have not been defined for the rare organisms and any antifungal agent.Regardless, the use of these methods is recom mended for the generation of epidemiological data (7,14).Accurate species identification combined with MIC data may be of use in guiding treatment.Unfortunately, the uncommon nature of these IFIs means that only a single encounter or episode of infection may occur in any given medical center, thus limiting the ability to generate local epidemiological data in a reasonable, clinically relevant time frame.As such, the data generated by comprehensive surveillance programs, such as SENTRY (6,28), should prove to be a useful reference.
In addressing the antifungal coverage of the rare fungi, it is helpful to understand the activity of conventional antifungal agents as well as that of investigational agents that promise an enhanced spectrum and potency directed at both common and uncommon fungal pathogens (29)(30)(31).
In this study, we utilized SENTRY Antimicrobial Surveillance Program data from 2017 to 2022 to examine the in vitro activity of manogepix and comparators against 1,937 contemporary clinical fungal isolates of rare yeasts and molds from a variety of infections.The isolates were collected from 93 medical centers located in North America (NA), Europe (EU), the Asia-Pacific region (APAC), and Latin America (LA).These data expand on our previous reports from 2017 (36), providing a sizable MIC database for eventual determination of ECVs and clinical breakpoints for manogepix and other antifungal agents and a variety of infrequently encountered fungal species.

Organisms
A total of 1,937 infrequently encountered nonduplicate fungal clinical isolates were selected among 8,512 isolates included in the SENTRY Surveillance Program during 2017-2022 from 93 medical centers located in North America (34 medical centers, 806 isolates), Europe (37 medical centers, 629 isolates), the Asia-Pacific region (15 medical centers, 344 isolates), and Latin America (7 medical centers, 158 isolates).For the purposes of this study, all species of Candida less common than C. krusei, all Aspergil lus species less common than A. fumigatus, all Cryptococcus species, all non-Candida, non-Cryptococcus yeasts, and all non-Aspergillus molds were included.Fungal isolates were recovered from patients with bloodstream infections (630 isolates), respiratory tract infections (543 isolates), skin and skin structure infections (171 isolates), urinary tract infections (35 isolates), intra-abdominal infections (25 isolates), and other infection sites (533 isolates) as determined by local criteria.

Fungal identification methods
Yeast isolates were subcultured on HardyCHROM agar medium (Hardy Diagnostics, Santa Maria, CA, USA) upon arrival to confirm culture purity for Candida spp.isolates and submitted to matrix-assisted laser desorption ionization-time of flight mass spectrome try (MALDI-TOF MS) using the MALDI Biotyper (Bruker Daltonics, Billerica, MA, USA).Any yeast isolates not identified by this process were identified using sequencingbased methods for the internal transcribed spacer (ITS) region, 28S ribosomal DNA, or intergenic spacer 1 for Trichosporon spp.(39)(40)(41)(42).Mold isolates were identified by DNA sequencing when an acceptable identification was not achieved by MALDI-TOF MS.For all isolates, 28S was sequenced, and one of the following genes was analyzed: β-tubulin for Aspergillus spp., translation elongation factor (TEF) for Fusarium spp., or ITSs for all other species of filamentous fungi (39)(40)(41)(42).Nucleotide sequences were analyzed using Lasergene software (DNAStar, Madison, WI, USA) and compared to available sequences using BLAST.TEF sequences were analyzed using the Fusarium multilocus sequence-typ ing database (https://fusarium.mycobank.org/).
The identification of fungal species (common and uncommon) is complicated by frequent changes in the nomenclature, largely driven by the use of nucleic acid sequence-based identification (43,44).The adoption of the "one fungus, one name" approach aims to simplify the naming of fungi.In the interest of clinical utility, sup porters of nomenclatural stability of medically important fungi have urged caution in adopting new names for fungi (15,45).In the present survey, we have adopted the approach suggested by previous authors (43,44) to employ the more familiar name used clinically followed by the newly proposed name in parentheses.This is done the first time it is encountered with the more familiar name used subsequently throughout the manuscript.
Clinical breakpoints and ECV values have not yet been determined for manogepix against any fungal species.For comparison, manogepix MIC distributions from the SENTRY Surveillance Program performed in three prior surveys were utilized to generate tentative wild-type upper limits (WT-UL; two twofold dilutions higher than the modal MIC value of each MIC distribution) for several species of Candida and Aspergillus (28,36,37).These WT-UL values were used in the present survey as the cutoff value to define wild-type (MIC ≤ WT Ul) and non-WT (MIC > WT Ul) populations for manogepix and each species for which there were at least 10 isolates (28,33,34,36,37,47).The previously published tentative WT-UL values employed in this evaluation were 0.016 mg/L for Candida dubliniensis, 0.03 mg/L for Candida orthopsilosis, Aspergillus niger species complex, and Aspergillus terreus species complex, 0.06 mg/L for Aspergillus flavus species complex, 0.12 mg/L for Candida lusitaniae and Scedosporium spp., and 1 mg/L for Candida kefyr (28).A tentative 97.5% WT-UL cutoff for manogepix and C. auris of 0.03 mg/L have been proposed for the CLSI method by Arendrup et al (34).
Since neither clinical breakpoints nor ECVs are available for most, if not all, of these uncommon organism-antifungal agent combinations, we used the C. albicans-resistant clinical breakpoints or ECVs for fluconazole (clinical breakpoint, ≥8 mg/L), voriconazole (clinical breakpoint, ≥1 mg/L), anidulafungin (clinical breakpoint, ≥1 mg/L), micafungin (clinical breakpoint, ≥1 mg/L) and amphotericin B (ECV, ≤1 mg/L) for the rare species of Candida and other yeasts and the A. fumigatus ECVs for amphotericin B (≤2 mg/L), itraconazole (≤1 mg/L), and voriconazole (≤1 mg/L) for the rare non-Aspergillus molds and the non-fumigatus species of Aspergillus that lack ECVs as a means of identifying species or individual isolates with elevated MIC/MEC ranges (14).

Organisms
The frequency distributions and cumulative percent inhibition data for manogepix against the species and organism groups of yeast tested are listed in Tables 1 and 2 and in Tables 3 and 4 for molds.All fungal species containing ≥10 isolates were analyzed separately (Tables 2 and 4).Manogepix and comparator agent susceptibility results for fungal species with fewer than 10 isolates are listed in Tables S1 and S2.
When compared to the Candida species, the potency of manogepix was reduced against isolates of Cryptococcus spp.(Table 1).Whereas 100.0% of isolates of Cryptococcus deneoformans (formerly C. neoformans var.neoformans) were inhibited by ≤1 mg/L of manogepix, 92.1% of C. neoformans (formerly C. neoformans var.grubii) and 83.3% of Cryptococcus gattii species complex were inhibited at this concentration.A single C. gatti species complex isolate had a manogepix MIC value of 2 mg/L (Table 1).
The 776 isolates of infrequently encountered Candida spp.included 24 isolates (3.1%) of 4 different species [C.kefyr (10 isolates), C. lusitaniae (2 isolates), C. norvegensis (6 isolates), and C. inconspicua (6 isolates)] for which the manogepix MIC values were elevated at 0.5 mg/L or greater (range, 0.5-2 mg/L) (Table 1).Two of these species, C. norvegensis and C. inconspicua, are closely related to C. krusei, a species known for its intrinsic resistance not only to fluconazole but also to manogepix.Whereas none of the isolates of C. kefyr were resistant to fluconazole using the CLSI C. albicans clinical breakpoints, two C. lusitaniae, six C. norvegensis, and six C. inconspicua isolates for which the manogepix MIC values were ≥0.5 mg/L were also resistant to fluconazole (MIC range, 8-32 mg/L).
All (100.0%) of the 77 C. auris isolates were inhibited by manogepix at ≤0.06 mg/L, and 90.9% were inhibited at the WT-UL MIC cutoff value of ≤0.03 mg/L (Tables 1 and  2).Using the CDC tentative clinical breakpoints, 85.7% of the isolates were resistant to fluconazole, 14.3% were resistant to amphotericin B, and 1.3% were resistant to anidulafungin and micafungin (Table 2).Application of the CLSI-resistant breakpoints for C. albicans to this collection showed 89.6% were resistant to fluconazole, 51.9% were resistant to voriconazole, 14.3% were resistant to amphotericin B, 5.2% were resistant to anidulafungin, and 1.3% were resistant to micafungin.Notably, 58.0% of fluconazoleresistant isolates were also resistant to voriconazole.Of the 77 isolates of C. auris, 22 (28.6%) were from North America (5 states), 32 (41.6%) were from Europe (4 countries, 5 sites), 22 (28.6%) were from Latin American [1 country (Panama), 1 site], and 1 (1.3%) was from the Asia-Pacific region [1 country (Japan), 1 site].The antifungal resistance/non-wild type (NWT) varied considerably by geographic region.Whereas none of the isolates from Latin America (Panama) or the single isolate from Japan expressed an NWT manogepix phenotype [MIC >0.03 mg/L (> WT Ul)], 6.2% of the isolates from Europe and 22.7% of the isolates from North America were NWT to manogepix.Resistance to fluconazole and amphotericin B was detected in isolates from North America (90.9% fluconazole resistant and 36.4% amphotericin B resistant), Europe (100.0%fluconazole resistant and 6.2% amphotericin B resistant), and Latin America (59.1% fluconazole resistant and 4.5% amphotericin B resistant) (data not shown).Aside from the Latin American isolates (4.5% resistant to anidulafungin and micafungin), none of the remaining isolates of C. auris in this survey were resistant to the echinocandins.The single C. auris isolate from the Asia-Pacific region was WT to manogepix, resistant to fluconazole, and susceptible to the echinocandins and amphotericin B.
Aside from C. fabianii and C. kefyr, the remaining species of rare Candida shown in  1 and 2).The cryptococci are intrinsically resistant to the echino candins and are generally susceptible to the azoles and amphotericin B. Overall, 98.5% of the isolates tested were inhibited by ≤2 mg/L of manogepix.The three isolates for which the manogepix MIC value was 4 mg/L were all C. neoformans and were WT to fluconazole.The two most common species groups of Cryptococcus in this survey were C. neoformans (n = 178) and C. deneoformans (n = 13).Both species showed a predomi nantly WT profile to fluconazole [98.9% and 100.0%WT (MIC ≤8 mg/L), respectively], voriconazole [100.0%WT (MIC ≤0.25 mg/L) for both], and amphotericin B (all isolates MIC ≤1 mg/L).The six isolates of C. gattii species complex were all inhibited by ≤2 mg/L of manogepix and were WT to fluconazole, voriconazole, and amphotericin B (Table S1).

In vitro activity of manogepix against infrequently encountered mold isolates
The rare molds included in this survey were 24 species or species complexes of Aspergillus [non-A.fumigatus (n = 548 isolates)] and 41 species/species complexes of non-Aspergillus molds (n = 304 isolates) (Tables 3 and 4).
The most common non-fumigatus species of Aspergillus in this 2017-2022 survey that were tested against manogepix included the following four Aspergillus species complex in the order of frequency: A. niger species complex (n = 185), A. flavus species complex (n = 173), A. terreus species complex (n = 71), and Aspergillus nidulans species complex (n = 42) (Tables 3 and 4).Manogepix exhibited potent in vitro activity against all four Aspergillus species complex shown in Tables 3 and 4, with MEC 90 values of 0.016-0.06mg/L.The WT-UL determined previously for three of these groups was ≤0.03 mg/L for A. niger species complex (95.7%WT) and A. terreus species complex (100.0%WT) and ≤0.06 mg/L for A. flavus species complex (98.8%WT) (Tables 3 and 4).
treatment for immunosuppression as well as those individuals suffering from severe respiratory viral infections (e.g., influenza and/or SARS-CoV-2) and the elderly (2)(3)(4)(5)(48)(49)(50).The application of molecular and proteomic methods for fungal identification and resistance testing has allowed clinical microbiology laboratories to characterize even rare fungi to a degree not possible 20 years ago (21).
Current antifungal agents cover most opportunistic fungal pathogens, including but not limited to C. albicans, A. fumigatus, and C. neoformans (51); however, breakthrough infections do happen and increasingly include relatively uncommon yeasts and molds (8,15).The use of mold-active antifungal agents for fungal prophylaxis in individuals at high risk for IFI has reduced the frequency of candidemia and invasive aspergillosis (IA) (4,8,15); however, this selective pressure has resulted in an increase in breakthrough infections caused by infrequently encountered species of Candida (C.auris, C. guilliermon dii, and C. rugosa), and Aspergillus (A. lentulus, A. udagawae, and A. ustus species complex), non-Candida yeasts (Saprochaete, Rhodotorula, Trichosporon, and Saccharomyces spp.), and non-Aspergillus molds (Mucorales, L. prolificans, Fusarium, and Scedosporium spp.) (18).Optimal treatment regimens against unusual fungal pathogens have not been established, and anecdotal reports of successes have been published with various agents alone or in combination (8,15).Some of these fungi (e.g., Fusarium, Scedosporium, Lomentospora, Trichosporon, and C. auris) are among the most drug-resistant fungal organisms encountered in clinical practice (8,15).It is worth noting that, in very high-risk patients (e.g., HSCT), the organisms with the greatest intrinsic resistance to the antifungal agent administered will ultimately emerge as a cause of infection (52).
The results of the present survey confirm and extend those previously reported in 2017 (28,36,37), regarding the in vitro activities of manogepix and comparator antifungals against contemporary yeast and mold pathogens.In this survey, we have focused on the less frequently encountered yeasts and molds, demonstrating decreased susceptibility of the rare yeasts to azoles and echinocandins, and of the rare molds to the azoles.Resistance to amphotericin B is difficult to characterize but appears to be intrinsic to several species such as A. lentulus, A. terreus species complex, Scedosporium spp., L. prolificans, among others (Tables 2 and 4).
Whereas most of the non-A.fumigatus species of Aspergillus expressed a WT phenotype for the echinocandins, azoles, and amphotericin B, both A. ustus species complex and A. tubingensis show decreased susceptibility to itraconazole (MEC 90 , >8 mg/L for both) and voriconazole (MEC 90 , 8 and 4 mg/L, respectively) (Table 4).The non-Aspergillus molds generally show resistance or decreased susceptibility to one or more classes of antifungal agents (Table 4; Table S2).The Mucorales show resistance to echinocandins, itraconazole, and voriconazole but not amphotericin B. Fusarium spp.and L. prolificans are intrinsically resistant to echinocandins, azoles, and amphotericin B, although voriconazole with or without a second agent in combination is often used in treatment of infections due to these organisms (15).Scedosporium spp.are resistant to amphotericin B, and isolates of P. variotii, Purpureocillium lilacinum, and Rasamsonia argillacea species complex generally show low MEC values for the echinocandins but are variable in their susceptibility to itraconazole, voriconazole, and amphotericin B (Table 4).
The in vitro susceptibility of the infrequently encountered yeasts and molds to manogepix in the present survey expands upon the data presented previously and documents the WT manogepix MIC distribution for several uncommon, and drug-resist ant, fungi (28,36,37).In previous surveys of the in vitro activity of manogepix against yeasts and molds, small numbers of the less common species were included but not displayed in detail.We have expanded our analysis in the present survey to include 1,937 isolates of the less commonly encountered fungi and confirm the excellent potency and spectrum of manogepix against azole-and echinocandin-resistant yeasts and azole-resistant molds.Notably, we show that manogepix is active (MIC/MEC, ≤0.12 mg/L) against several MDR fungi, including C. auris, Saprochaete spp., Rhodotorula spp., A. terreus species complex, A. ustus species complex, Fusarium spp., Scedosporium spp., and L. prolificans.C. krusei is known to be intrinsically resistant to manogepix, and it appears from this survey that the closely related species C. inconspicua and C. norvegensis also may be as well.The only other rare yeast that may not be a target for manogepix is Trichosporon spp.(MIC 50/90 , >2/>2 mg/L).Manogepix shows potent activity against the uncommon species of Aspergillus, including antifungal-resistant species such as A. lentulus and A. ustus species complex.The Mucorales (MEC 90 , >4 mg/L) are considerably less susceptible to manogepix when compared to the rare species of Aspergillus (MEC 90 , 0.03 mg/L) (Table 4).
The broad spectrum of manogepix is notable for its activity against many less common and often antifungal-resistant yeast and mold strains.Clinical development of the prodrug fosmanogepix has focused on the treatment of infections due to Candida, Aspergillus, and the rare molds.Phase 1 studies in healthy volunteers have demonstrated high (>90%) oral bioavailability of manogepix and maintenance of manogepix plasma drug exposures above estimated antifungal target levels for 7-42 days, even when switching from intravenous (IV) to oral dosing (32).Following IV administration of 600 mg fosmanogepix over a 3-hour infusion period, the observed geometric manogepix C max on day 7 was 7.9 mg/L and on day 14 was 6.3 mg/L (53).These exposures were similar with either IV or oral administration in both healthy volunteers and patients with neutropenia (53).The US Food and Drug Administration has granted Fast Track designation for intravenous and oral formulations of fosmanogepix for seven different indications, including treatment of invasive candidiasis, invasive aspergillosis, scedospor iosis, fusariosis, mucormycosis, cryptococcosis, and coccidioidomycosis (32).Three phase 2 clinical trials have been completed.The first (NCT03604705; clinicaltrials.gov)was an open-label, non-comparative study assessing the safety and efficacy of fosmanogepix in the treatment of non-neutropenic patients with candidemia which met its primary efficacy endpoint with a treatment success rate of 80% with an acceptable safety profile (54).The second study (NCT04148287; clinicaltrials.gov)tested the safety and efficacy of fosmanogepix treatment in patients with candidemia caused by C. auris (55).Treatment success at the end of study treatment and day 30 survival were 89% with no treatment-related adverse events or study drug discontinuations reported.The third study (NCT04240886) examined the use of fosmanogepix for the treatment of patients with invasive mold infections caused by Aspergillus species or rare molds.Results of this study are awaiting publication.Continued development of fosmanogepix for the treatment of IFI, including MDR strains, is warranted.

8 TABLE 2 9 TABLE 2
In vitro activity of manogepix and comparators against infrequently encountered yeast isolates from the SENTRY Surveillance Program (2017-2022) (Continued) In vitro activity of manogepix and comparators against infrequently encountered yeast isolates from the SENTRY Surveillance Program (2017-2022) (Continued)

CTABLE 2
MIC (mg/L)No.and cumulative percent of isolates inhibited at MIC (mg/L) of: .deneoformans(13) (C.neoformans var.neoformans) on next page) Full-Length Text Antimicrobial Agents and Chemotherapy February 2024 Volume 68 Issue 2 10.1128/aac.01132-2311In vitro activity of manogepix and comparators against infrequently encountered yeast isolates from the SENTRY Surveillance Program (2017-2022) (Continued) MIC (mg/L) No. and cumulative percent of isolates inhibited at MIC (mg/L) of: on next page) Full-Length Text Antimicrobial Agents and Chemotherapy February 2024 Volume 68 Issue 2 10.1128/aac.01132-2312

TABLE 1
Antifungal activity and cumulative percent inhibition data for manogepix against yeast isolates a

TABLE 1
Antifungal activity and cumulative percent inhibition data for manogepix against yeast isolates a

TABLE 2
In vitro activity of manogepix and comparators against infrequently encountered yeast isolates from the SENTRY SurveillanceProgram (2017Program ( -2022)   )

TABLE 2
In vitro activity of manogepix and comparators against infrequently encountered yeast isolates from the SENTRY Surveillance Program (2017-2022) (Continued)

TABLE 3
Antifungal activity and cumulative percent inhibition data for manogepix against mold isolates a

TABLE 3
Antifungal activity and cumulative percent inhibition data for manogepix against mold isolates a

TABLE 4
In vitro activity of manogepix and comparators against infrequently encountered mold isolates from the SENTRY SurveillanceProgram (2017Program ( -2022) ) m MIC (mg/L) No.

TABLE 4
In vitro activity of manogepix and comparators against infrequently encountered mold isolates from the SENTRY Surveillance Program (2017-2022) m

TABLE 4
In vitro activity of manogepix and comparators against infrequently encountered mold isolates from the SENTRY Surveillance Program (2017-2022) m