EUCAST Determination of Olorofim (F901318) Susceptibility of Mold Species, Method Validation, and MICs

ABSTRACT

Olorofim is a novel antifungal agent with in vitro activity against Aspergillus and some other molds. Here, we addressed technical aspects for EUCAST olorofim testing and generated contemporary MIC data. EUCAST E.Def 9.3.1 testing was performed comparing two plate preparation methods (serial dilution in medium [serial plates] versus predilution in DMSO [ISO plates]), two lots of olorofim, visual (visual-MIC) versus spectrophotometer (spec-MIC) reading, and four polystyrene plates using 34 to 53 Aspergillus isolates from five genera. Subsequently, olorofim MICs were compared to itraconazole, voriconazole, posaconazole, and amphotericin B MICs for 298 clinical mold isolates (2016 to 2017). Wild-type upper limits (WT-UL) were determined following EUCAST principles for epidemiologic cutoff value (ECOFF) setting. Olorofim median MICs comparing serial plates and ISO plates were identical (25/36 [69%]) or one dilution apart (11/36 [31%]). Interperson agreement for visual-MICs was 92% to 94%/100% for ≤1/≤2 dilutions, respectively. The visual-MIC values across tested microtiter plates and olorofim lots revealed only discrete differences (≤1 dilution lower for treated plates). No single spec-MIC criterion was applicable to all species. Olorofim MICs were low against 275 Aspergillus species isolates (modal MIC, 0.06 mg/liter; MIC range, < 0.004 to 0.25 mg/liter) and three dermatophytes (MICs 0.03 to 0.06 mg/liter). MICs against Fusarium were diverse, with full inhibition of F. proliferatum (MIC, 0.016), 50% growth inhibition of Fusarium solani at 1 to 2 mg/liter, and no inhibition of F. dimerum. Olorofim displayed potent in vitro activity against most mold isolates and was associated with limited variation in EUCAST susceptibility testing.

INTRODUCTION

Olorofim (formerly F901318) is a novel antifungal agent belonging to the new orotomide class of drugs which targets an important enzyme for pyrimidine biosynthesis, dihydroorotate dehydrogenase. It has in vitro activity against Aspergillus and other difficult-to-treat molds, including Scedosporium, Lomentospora, and Fusarium species, but no activity against Candida, Cryptococcus, or Mucorales species due to differences in the drug targets (1–3). For Aspergillus species specifically, in vitro activity has been demonstrated against azole-susceptible wild-type (WT) isolates as well as azole-resistant cyp51A mutant Aspergillus fumigatus isolates and also against other Aspergillus species, including Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Aspergillus flavus complex, and the difficult-to-treat Aspergillus calidoustus and Aspergillus tubingensis species, using the standard EUCAST or CLSI reference methodologies and a limited number of isolates (1, 2). Interestingly, in vivo experimentation suggests potent efficacy against pulmonary aspergillosis in a mouse model involving WT A. fumigatus and azole-resistant TR₃₄/L98H A. fumigatus, as well as activity against A. flavus (2, 4), in a murine model of sinopulmonary aspergillosis. Olorofim has successfully progressed through 90-day toxicology studies in two species as well as phase I healthy volunteer trials. Initial patient studies are planned starting in 2018. Recently, olorofim has been granted orphan drug status for the treatment of invasive aspergillosis and rare mold infections caused by Scedosporium species by the European Medicines Agency (EMA) Committee for Orphan Medicinal Products.

Establishing a robust susceptibility testing procedure, associated endpoint reading method, and MICs for contemporary isolates are crucial parts of the clinical breakpoint setting process. CLSI and EUCAST have both provided methods for the licensed antifungal compounds (5, 6). Still, both organizations also recognize that despite standardized reference protocols, sources of variations still need to be thoroughly investigated and, if possible, controlled for and that not all compounds can be read with the same endpoint definition. For example, a morphological aberrant growth MEC (minimum effective concentration) endpoint is acknowledged for the echinocandins against Aspergillus, in contrast to a full visual inhibition MIC endpoint adopted for amphotericin B and azoles against Aspergillus and other molds. Moreover, some of the MICs obtained for some compounds have been found to vary with the type of microtiter plate used for microdilution, as exemplified by caspofungin and amphotericin B (7). This was recently also suggested for another investigational antifungal compound (CD101, rezafungin) and prohibited epidemiologic cutoff value (ECOFF) determination for the most susceptible Candida species (8). Finally, variations across different lot numbers have also been reported, particularly for caspofungin (7, 9).

Here, we investigated the influence of various technical issues related to EUCAST MIC determination for the novel compound olorofim, including inter-reader variability in visual endpoint determination, performance of spectrophotometric endpoint reading, endpoint variation across two lots of olorofim, and finally, the impact of various brands and types of polystyrene plates. Subsequently, the in vitro susceptibilities among prospectively collected contemporary clinical mold isolates to olorofim were investigated in comparison with that of the licensed antimold agents using the EUCAST E.Def 9.3.1 method.

RESULTS

Technical aspects.

Olorofim susceptibility was first evaluated by comparing MICs generated using plates prepared by serial dilution (serial plates) and the ISO method (ISO plates) and by using visual (comparing two readers) and spectrophotometric endpoint reading. Median MICs and ranges were low and similar across the species and across A. fumigatus with or without Cyp51A alterations (Table 1). Species- and Cyp51A-specific median MICs for the two plate preparation methods were either identical (25/36 [69%]) or one dilution apart (11/36 [31%]). The closest agreement was seen for the visual reading for which 11/12 median MICs were identical using serial and ISO plates and the lowest for spectrophotometric reading using 50% endpoint for which 1/6 median MICs were identical using serial or ISO plates. If adopting MIC ranges spanning ≤3 dilutions as an indicator of low technical variability, this target was met for the visual endpoint method and for both plate preparations in all cases except for A. niger and A. terreus. Similarly, this target was met for the spectrophotometer reading method except (i) when 50% endpoint was adopted, (ii) for A. niger in general, and (iii) for the 95% endpoint reading of non-WT A. fumigatus (Table 1).

TABLE 1.

Comparison of olorofim MIC determined using plates prepared using serial dilution or ISO dilution and by adopting visual full inhibition or spectrophotometer endpoint criteria

Isolate (n)	MIC (mg/liter)
	Serial dilution plates						ISO plates
	Full inhibition		Spectrophotometric inhibition (%)				Full inhibition		Spectrophotometric inhibition (%)
	Reader 1	Reader 2–4a	95	90	85	50	Reader 1	Reader 2–4a	95	90	85	50
A. flavus (10)
Median	0.03	0.03	0.03	0.016	0.016	0.001	0.03	0.03	0.016	0.016	0.016	0.002
Range	0.03	0.03	0.016–0.03	0.016–0.03	0.008–0.03	0.0005–0.008	0.3–0.125	0.016–0.06	0.016–0.03	0.008–0.016	0.004–0.016	0.00024–0.008
No. 2-fold dilutions	1	1	2	2	3	5	3	3	2	2	3	6
A. fumigatus WT (5)
Median	0.06	0.03	0.03	0.03	0.016	0.016	0.03	0.03	0.03	0.016	0.016	0.008
Range	0.06	0.016–0.06	0.03	0.016–0.03	0.016–0.03	0.004–0.016	0.03–0.06	0.016–0.06	0.016–0.03	0.016–0.03	0.008–0.016	0.001–0.008
No. 2-fold dilutions	1	3	1	2	2	3	2	3	2	2	2	4
A. fumigatus mutant (5)
Median	0.06	0.03	0.03	0.03	0.03	0.004	0.06	0.03	0.03	0.03	0.03	0.004
Range	0.06–0.125	0.03–0.125	0.03–0.125	0.016–0.03	0.016–0.03	0.004–0.03	0.03–0.06	0.03–0.06	0.03 to >1	0.016–0.06	0.016–0.06	0.004–0.03
No. 2-fold dilutions	2	3	3	2	2	4	2	2	7	3	3	4
A. nidulans (10)
Median	0.06	0.06	0.03	0.03	0.03	0.008	0.06	0.06	0.03	0.03	0.016	0.004
Range	0.03–0.125	0.03–0.125	0.03–0.06	0.016–0.03	0.016–0.03	0.004–0.016	0.03–0.125	0.3–0.125	0.016–0.06	0.016–0.03	0.016–0.03	0.002–0.016
No. 2-fold dilutions	3	3	2	2	2	3	3	3	3	2	2	4
A. niger (10)
Median	0.06	0.06	0.5	0.06	0.03	0.016	0.06	0.06	0.5	0.06	0.03	0.008
Range	0.03–0.25	0.03–0.125	0.03 to >1	0.016 to >1	0.016–0.25	0.008–0.032	0.03–0.125	0.03–0.25	0.06 to >1	0.016 to >1	0.016–0.25	0.002–0.03
No. 2-fold dilutions	4	3	7	8	5	3	3	4	6	8	5	4
A. terreus (10)
Median	0.03	0.016	0.016	0.016	0.008	0.004	0.03	0.016	0.008	0.008	0.008	0.002
Range	0.016–0.06	0.008–0.125	0.008–0.03	0.008–0.016	0.004–0.016	0.0005–0.008	0.008–0.06	0.008–0.03	0.004–0.016	0.004–0.008	0.004–0.008	0.00024–0.004
No. 2-fold dilutions	3	5	3	2	3	5	4	3	3	2	2	5

Species-specific MIC ranges broader than the optimal target (spanning ≤3 dilutions) are underlined.

^aReader 1 was constant throughout the study, whereas the second reader was any of 3 different individuals (Reader 2 to 4).

Interperson variability for visual endpoint determination was investigated by comparing individual endpoints across all species and Cyp51A types read by reader 1 with those of a second reader (reader 2, 3, or 4, with the second reader varied on different days). A 100% agreement was obtained within ±1 dilutions and ≤1 dilution apart in 92% to 94% of the cases independently if the serial dilution or the ISO method was adopted for plate production (see Table S1 in the supplemental material).

Spectrophotometric determinations of MIC endpoints (spec-MICs) using 95%, 90%, 85%, and 50% growth inhibition criteria are displayed in Table 1 and are the following compared to the visual MICs. Median spec-MICs using a 95% inhibition endpoint were ≤1 dilution apart from the visual endpoint on all occasions except for A. niger (using both serial dilution and ISO method prepared plates) and A. terreus (using the ISO method prepared plates). However, this strict endpoint resulted in a wide spec-MIC distribution for the A. fumigatus mutants (7 versus 2 dilutions for the visual MIC reading of ISO plates) and an artificially elevated median spec-MIC for A. niger (0.5 mg/liter versus 0.06 mg/liter) (Table 1). With a 90% inhibition endpoint, the overall median spec-MIC was one step below that for visual reading, but the spec-MIC ranges for all isolate groups, except A. niger, were acceptable (≤3 dilutions). For A. niger, the 85% inhibition endpoint was superior.

The impact of polystyrene plate type and variation between different lots of pure olorofim substance was next examined. Visual MICs were obtained using four different microtiter plates (treated and untreated Nunc and Greiner plates) prepared using serial as well as ISO methods, respectively, and with two different lots of olorofim pure substance. The MICs determined were overall similar across the different plate types (see Table S2). However, discrete differences of ≤1 dilution were noted, as the use of treated plates tended to lead to slightly lower median MICs, particularly when the plates were prepared using 2-fold serial dilutions. There were no differences in MICs obtained using two different batches of olorofim.

Olorofim EUCAST reference method MICs for contemporary mold isolates.

In total, 298 mold isolates were referred for susceptibility testing March 2016 to October 2017 and thus tested for olorofim susceptibility. The isolates were derived from airway samples (239 [80%]), pleura (1), sinus (2), blood, brain, or cerebral spinal fluid (CSF) (1 each), ear (20 [7%]), eye (8 [3%]), skin, hair, or nail (5), other (11 [4%]), or unknown sites (9 [3%]). The referred isolates included 275 Aspergillus species, 10 Fusarium species, 10 Mucorales isolates, and 3 dermatophytes.

Olorofim displayed potent in vitro activity across the 275 isolates belonging to eight different Aspergillus spp., with an overall modal MIC of 0.06 mg/liter, geometric MIC (GM-MIC) of 0.037 mg/liter, and MIC range of <0.004 to 0.25 mg/liter (visual endpoint as per reference standard) (Table 2). For A. fumigatus specifically, the GM-MIC was 0.037 mg/liter and the wild-type upper limit (WT-UL) was 0.25 mg/liter unless the statistical method with the 99% endpoint was used (0.5 mg/liter) (Table 2). The olorofim in vitro activity against A. fumigatus was unaffected by concomitant azole resistance. Thus, olorofim susceptibility against the wild type was a modal MIC of 0.06 mg/liter, GM-MIC of 0.037 mg/liter, and MIC range of <0.004 to 0.25 mg/liter compared to a modal MIC of 0.06 mg/liter, GM-MIC of 0.042 mg/liter, and a range of 0.004 to 0.125 mg/liter against the non-wild type (Table 2). The MICs were similar for A. flavus and A. niger (modal MIC, 0.03 and 0.03 to 0.06 mg/liter, and GM-MICs, 0.050 and 0.052 mg/liter, respectively) and slightly lower for A. terreus (range, 0.008 to 0.03 mg/liter; GM-MIC, 0.022 mg/liter). A similar potent in vitro activity was observed against the three dermatophyte isolates (MICs, 0.03 to 0.06 mg/liter).

TABLE 2.

EUCAST E.Def 9.3.1 MICs of olorofim against contemporary prospectively collected clinical mold isolates 2016 to 2017

Isolate	No.	No. of isolates at MIC (mg/liter) of:a										MIC (mg/liter)b
		<0.004	0.004	0.008	0.016	0.03	0.06	0.125	0.25	0.5	≥1	Range	Modal	GM	WT-UL visual	WT-UL 97.5%	WT-UL 99%
Aspergillus spp.
A. flavus	12	1			1	8	2					<0.004–0.06	0.03	0.050	0.125	0.06	0.06
A. fumigatus	235	4	11	12	33	48	90	32	5			<0.004–0.25	0.06	0.037	0.25	0.25	0.5
WT for azoles	213	4	9	12	30	45	81	27	5			<0.004–0.25	0.06	0.037	0.25	0.25	0.5
Non-WT for azoles	22		2		3	3	9	5				0.004–0.125	0.06	0.042	0.25	2	4
A. giganteus	1				1
A. nidulans complex	1					1
A. niger	17			1		6	6	3	1			0.008–0.25	0.03/0.06	0.052	0.25	0.25	0.25
A. persii	1	1
A. terreus	5			2	1	2						0.008–0.03		0.022
A. tubingensis	2				1		1					0.016–0.06
A. turcosus (A section fumigati)	1				1
Fusarium spp.
F. dimerum	1										1
F. solani complex	8										8	>0.5	>0.5
F. proliferatum	1						1
Dermatophytesc	3					2	1
Aspergillus, Fusarium, and dermatophytes	288	6	11	15	38	67	101	35	6	0	9	<0.004 to >0.5	0.06	0.038	0.25	0.25	0.5
Mucoralesd	10										10
In total	298	6	11	15	38	67	101	35	6	0	19	<0.004 to >0.5	0.06	0.038	0.25	0.25	0.5

^aModal MICs are highlighted in bold. MICs were determined visually as per E.Def 9.3.1 protocol.

^bWT-UL, wild type upper limit.

^cOne each of Microsporum canis, Trichophyton interdigitale, and Trichophyton rubrum.

^dOne each of Absidia corymbifera, Circinella muscae, Lichtheimia ramose, and Rhizomucor miehei, two Rhizomucor pusillus, and four Rhizopus microsporus isolates.

The in vitro activity for Fusarium spp. was more diverse and species specific. The most susceptible was F. proliferatum (olorofim MIC, 0.016 mg/liter) (Table 2). In contrast, the activity against the other Fusarium species was less potent on a mg/liter basis, with MICs of ≥1 mg/liter for all. Partial growth inhibition was observed, however, against Fusarium solani isolates. Repeated testing of F. solani and F. dimerum isolates using an extended concentration range and a 50% growth inhibition endpoint revealed activity against F. solani complex isolates (MIC₅₀ = 1 mg/liter; range, 1 to 2 mg/liter) but no inhibition against F. dimerum. Finally, no inhibition was observed against Mucorales for which the drug target is known to be different.

When the in vitro activity was compared to that of the mold-active azoles and amphotericin B (Table 3), olorofim was the most active agent on a mg/liter basis against all the mold species except the Mucorales and the group of other mold species. If applying the WT-UL/ECOFF values for A. fumigatus as an indicator of susceptibility (assuming that isolates with in vitro susceptibility comparable to that of A. fumigatus are likely susceptible), the proportion of isolates in this prospective Danish collection with MICs above the A. fumigatus WT-UL/ECOFFs was overall 7.3% for F901318 compared to 4% for amphotericin B, and 13% to 15% for itraconazole, posaconazole, and voriconazole.

TABLE 3.

In vitro susceptibility of contemporary mold isolates to olorofim and mold active azoles presented as MIC ranges, modal MICs, and the proportion of isolates with MICs above the A. fumigatus WT-UL (olorofim) and EUCAST ECOFF (licensed comparators), respectively^a

Isolate (n)	Olorofim			AMB			Itraconazole			Posaconazole			Voriconazole
	MIC range	Modal MIC	% or proportion >0.25 mg/liter	MIC range	Modal MIC	% or proportion >1 mg/liter	MIC Range	Modal MIC	% or proportion >1 mg/liter	MIC Range	Modal MIC	% or proportion >0.25 mg/liter	MIC Range	Modal MIC	% or proportion >1 mg/liter
Aspergillus spp.	<0.004–0.25	NAb	0	0.125–4	NA	2	<0.125 to >16	NA	11	<0.06 to >4	NA	8	0.25–16	NA	7
A. flavus (12)	<0.004–0.06	0.03	0	0.5–4	1	25	0.125–0.5	0.125	0	0.06–0.25	0.125	0	0.5–2	1	8
A. fumigatus (235)	<0.004–0.25	0.06	0	0.125–1	0.5	0	<0.125 to >16	0.25	8	<0.06 to >4	0.125	7	0.25–16	0.5	5
A. niger (17)	0.008–0.25	0.03/0.06	0	0.125–0.5	0.25	0	0.5 to >16	1	41	0.125–0.5	0.25	25	0.5–4	1	50
A. terreus (5)	0.008–0.03	NA	0	1–4	1	40	<0.125–0.5	0.125	0	<0.06–0.25	0.06	0	0.5–1	1	0
Other Aspergillus sp. (6)	0.004–0.6	NA	0	0.125 to >4	NA	1/6	0.125 to >16	NA	3/6	0.06–0.5	NA	2/6	0.25–2	NA	2/6
Fusarium spp. (10)	0.06 to >8	NA	90	1 to >4	NA	50	4 to >16	NA	100	>4	NA	100	8 to >16	NA	100
Dermatophytes (3)	0.03–0.06	NA	0	0.25–1	NA	0	<0.125–1	NA	0	<0.06–0.5	NA	1/3	0.125–1	NA	0
Mucoralesc (10)	>0.5	NA	100	0.016–1	NA	0	<0.125 to >16	NA	60	<0.06–4	NA	50	4 to >16	NA	100

^aMICs were determined visually as per E.Def 9.3.1 protocol.

^bNA, not applicable.

^cOne each of Absidia corymbifera, Circinella muscae, Lichtheimia ramose, and Rhizomucor miehei, two Rhizomucor pusillus, and four Rhizopus microsporus isolates.

DISCUSSION

Technical aspects.

Overall, the examined technical variables compared in this study had little impact on olorofim MICs. Interperson agreement was high for the visual MIC determinations, and MICs were comparable across the two methods for microtiter plate preparation, suggesting that the serial 2-fold dilution plates (where pipette tips were changed halfway through the dilution series) is equally appropriate for MIC determinations of olorofim against the tested Aspergillus isolates. Finally, spectrophotometric endpoint determination resulted in MIC endpoints comparable, although one dilution lower, to those obtained by visual reading if a 90% endpoint was adopted for A. flavus, A. fumigatus, A. nidulans, and A. terreus, whereas a less stringent 85% endpoint was required for A. niger. Altogether, these data suggest that the EUCAST E.Def 9.3.1 susceptibility testing is a robust method for MIC determinations of olorofim with both preparation types and reading methods.

Spectrophotometric endpoint reading was recently evaluated for A. fumigatus and mold-active azoles (10). In that study, the best correlation between visual and spec-MICs was found using a 95% growth inhibition and not 90% inhibition. Various factors may help explain the differences observed with the different endpoint methods. First, the spectrophotometric 95% endpoint is a very strict endpoint criterion. Not only is 5% growth very discrete, but the calculation of such an endpoint is normally done compared to a medium control, which does not take into account the contribution to the optical density (OD) provided by the inoculum itself. This is especially relevant for A. niger, which is colored, thus increasing the background OD further even before any growth has occurred. Second, a very strict endpoint will inevitably lead to notably elevated spec-MICs for compounds with static or “delayed-cidal” activity (the latter behavior is shown by F901318), because background levels will increase until growth arrests and thus, in such cases, may be elevated above a strict 95% endpoint calculated using medium as the background reference (11). We observed slight trailing growth and an occasional occurrence of tiny dots in the center surface of the wells in supra-MIC wells. Although this did not lead to significant variation in endpoint determination in this study, it may challenge spectrophotometer reading with a strict spectrophotometric endpoint and also accurate and reproducible visual endpoint determination between readers and laboratories, particularly when plates are read by people less familiar with antifungal susceptibility testing (AFST) in general and olorofim in particular.

Taken together, these data suggest 85% inhibition as an attractive endpoint, which could apply for hyaline as well as pigmented aspergilli. This would lead to a systematic but discrete MIC decrease of approximately 1 dilution step compared to those obtained by standard visual reading and in parallel serve to align EUCAST and CLSI olorofim MICs, as previous studies have shown that CLSI MICs may be lower than those obtained by the EUCAST method (using visual 100% growth inhibition) (12). However, a systematic MIC decrease might impact susceptibility classification unless separate EUCAST breakpoints for visual versus spectrophotometric reading were established.

A limitation associated with our evaluation of technical issues related to EUCAST testing of olorofim is that we have only evaluated the performance of visual and spectrophotometer endpoints for isolates with a WT phenotype for olorofim, because no resistant mutants have as yet been identified. Therefore, it is yet unknown how such isolates would behave in EUCAST AFST and if the individual endpoint criteria differentiate in their performance with respect to correctly identifying isolates with reduced clinical susceptibility. Moreover, a confirmation of our results in a multicenter study is warranted.

Olorofim in vitro activity against contemporary isolates.

Olorofim displayed uniform and potent in vitro activity against all A. fumigatus isolates, including those with azole resistance. This aligns with recent mouse and rabbit model studies demonstrating in vivo efficacy of olorofim against wild-type A. fumigatus isolates as well as isolates harboring TR₃₄/L98H or G138C Cyp51A alterations (13). The pharmacokinetic/pharmacodynamic (PK/PD) index that best linked drug exposure with observed effect in these studies was the ratio of the minimum total plasma concentration/MIC (C_min/MIC). On the basis of studies in murine and rabbit models of invasive pulmonary aspergillosis, an average olorofim C_min (mg/liter) and olorofim C_min/MIC of 0.1 to 0.3 and 3.3 to 9.1 were shown to have effects comparable to the effects of posaconazole and isavuconazole at efficacious human exposure levels (13).

A. fumigatus is the most prevalent mold causing invasive mold infections. This may suggest it is also the most pathogenic mold in human; thus, other molds against which olorofim MICs are equal to or lower than those against A. fumigatus are likely to be clinically susceptible as well. Such species include A. niger, which is less susceptible to azole compounds than A. fumigatus, and A. terreus and A. flavus, which are both less susceptible to amphotericin B. These findings are highly promising, with the limited armamentarium of antimold compounds in mind, as alternatives are few and currently no oral alternatives are available for isolates with decreased azole susceptibility (14).

Olorofim was active against the three dermatophyte isolates tested. In light of increasing terbinafine resistance in Trichophyton rubrum and Trichophyton interdigitale, further investigation of olorofim activity against dermatophytes is warranted (15–19). Although full growth inhibition was only obtained against the single F. proliferatum isolate among the Fusarium species included, partial growth inhibition was observed for F. solani in the 1- to 2-mg/liter concentration range. Fusarium spp. are, in general, much less susceptible than A. fumigatus to the antifungal compounds currently licensed, and infections are often difficult to treat. Voriconazole is the first-line therapy, but MICs above 16 mg/liter are not uncommon, and indeed, were also found in this study, where MICs were 8 to >16 mg/liter (20). From this perspective, partial inhibition seemed worthy of investigation as a basis for possible mono- or combination therapy scenarios. Thirteen isolates (4%) were in vitro resistant to olorofim and included 10 Mucorales isolates. This minority of isolates in our setting is thus unlikely an appropriate target for future therapy with olorofim.

In conclusion, this study suggests that in vitro testing is reproducible across various types of polystyrene microtiter plates, associated with low inter-reader variation in MIC determination using the traditional visual endpoint criteria and potentially also using spectrophotometric reading, although further likely requiring the adoption of an 85% endpoint criteria at least for A. niger. Moreover, the real-time testing in the routine setting suggested a broad activity, with more than 90% of recent mold isolates received by our reference laboratory being susceptible as wild-type A. fumigatus.

MATERIALS AND METHODS

Susceptibility testing.

Stock solutions of antifungal compounds were prepared in dimethyl sulfoxide ([DMSO] 5,000 mg/liter; Sigma-Aldrich) and microtiter plates with 2-fold dilutions prepared in double-concentrated medium according to the EUCAST methodology (5). Cell culture-treated Nunc MicroWell 96-well microplates (cat. no. 167008; Thermo Fisher Scientific) were used throughout and frozen at −80°C prior to use, as is part of routine practice in most laboratories (see below regarding additional plates used for the plate comparison study).

Susceptibility testing methodological studies. (i) Plate preparation and endpoint reading.

Plates were prepared using (i) a 2-fold serial dilution of olorofim (F2G; Manchester, UK) in microtiter plates in double-concentrated EUCAST medium supplemented with 1% DMSO (serial plates) (23) and (ii) the ISO method, performing predilution in DMSO and subsequently diluting 1:100 in double-concentrated growth medium for each drug dilution before transferring to the microtiter plates (ISO plates) (5, 6, 19). In total, 53 isolates were included: 50 clinical Aspergillus isolates, including 10 A. fumigatus, 5 of which harbored cyp51A alterations (one each of TR₃₄/L98H, TR₄₆/Y121F/T289A, G54W, M220I, and a double mutant [M220I+V101F]), 10 A. flavus, 10 A. nidulans, 10 A. niger, and 10 A. terreus, and three EUCAST QC strains, including A. fumigatus ATCC 204305, A. flavus ATCC 204304, and A. flavus CNM-CM-1813. Serial and ISO plates were tested in parallel and read visually by two independent readers (the first reader was constant throughout the study, whereas the second reader was any of 3 different individuals) after 2 days of incubation (full inhibition endpoint) as well as spectrophotometrically using a single reading point per well followed by calculation of 95%, 90%, 85%, and 50% endpoints.

(ii) Impact of various polystyrene plates and comparison of two lots of olorofim pure substance.

Four flat-bottomed polystyrene plate types produced by two different companies were compared: (i) Nunc MicroWell 96-well microplates, nontreated (cat. no. 243656; Thermo Fisher Scientific), (ii) Nunc MicroWell 96-well microplates, cell culture treated (cat. no. 167008; Thermo Fisher Scientific) (SSI-standard plates), (iii) Greiner 96-well plates, nontreated (cat. no. 655161; Sigma-Aldrich), and (iv) Greiner Cellstar 96-well plates, tissue culture treated (cat. no. 655180; Sigma-Aldrich). Two lots of olorofim (lot 5 and lot 6) were tested against 34 Aspergillus isolates, including 5 A. fumigatus, 5 A. flavus, 5 A. nidulans, 6 A. niger, 5 A. terreus, and 5 A. fumigatus harboring cyp51A alterations (one each of TR₃₄/L98H, TR₄₆/Y121F/T289A, G54W, M220I, and a double mutant [M220I+V101F]), and three EUCAST mold control strains, including Aspergillus fumigatus ATCC 204305, Aspergillus flavus ATCC 204304, and Aspergillus flavus CNM-CM-1813.

(iii) Data management.

For comparison of MICs obtained by different endpoints, plate preparation method, or drug lots, the percentage agreement between median MICs within 2-fold dilutions were calculated per species and Cyp51A genotype (WT or non-WT). The wideness of MIC ranges indicated as number of dilutions of MICs spanned for a given species and Cyp51A type were also compared using log₂ transformation of individual MICs. MIC ranges spanning a maximum of 3 dilutions were regarded as a parameter for low technical variation.

Prospective evaluation of olorofim in vitro susceptibility of cont3emporary mold. (i) Isolates.

MICs were determined prospectively in parallel with routine susceptibility testing following the E.Def 9.3.1 method (with visual endpoint reading as per protocol) for olorofim (final concentration range, 0.004 to 0.5 mg/liter), amphotericin B (0.016 to 4 mg/liter), itraconazole (0.125 to 16 mg/liter), posaconazole (0.06 to 4 mg/liter), and voriconazole (0.125 to 16 mg/liter). The exceptions were that azole EUCAST MICs were not determined for A. fumigatus isolates categorized as azole susceptible using the recently validated 4-well VIPcheck screening test (VIPcheck, Nijmegen, The Netherlands) (21). For A. fumigatus, olorofim activity was examined individually for azole wild-type and non-wild type organisms (non-wild type defined as itraconazole MIC of >1 mg/liter, posaconazole MIC of >0.25 mg/liter, voriconazole MIC of >1, or isavuconazole MIC of >2 mg/liter) (5, 22). The ECOFFfinder program (available upon request from the EUCAST secretary via http://www.eucast.org/organization/steering_committee/) was used for determining a statistical olorofim wild-type upper limit WT-UL for species represented with at ≥10 isolates. For Fusarium species isolates, the MIC₅₀ was also determined using an extended concentration range (8 to 0.03 mg/liter), a spectrophotometer reading, and a 50% growth inhibition endpoint. Finally, the proportion of isolates with MICs above the WT-UL/ECOFF for A. fumigatus was calculated.