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

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Karin Meinike Jørgensen ◽  
Karen M. T. Astvad ◽  
Rasmus Krøger Hare ◽  
Maiken Cavling Arendrup

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.

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.


2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Anuradha Chowdhary ◽  
Karen M. T. Astvad ◽  
Karin Meinike Jørgensen

ABSTRACT APX001A is the active moiety of the first-in-class drug candidate APX001. So far, most susceptibility testing studies have examined ≤30 isolates/species, and only one used the EUCAST method. Here, we investigated the in vitro activity of APX001A and five comparators against 540 candidemia and 122 C. auris isolates. Isolates (17 Candida and 3 yeast species) were identified using CHROMagar, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) and, when needed, internal transcribed space (ITS) sequencing. EUCAST E.Def 7.3.1 susceptibility testing included APX001A, amphotericin B, anidulafungin, micafungin, fluconazole, and voriconazole. Wild-type upper limits (WT-UL) were established following the EUCAST principles for epidemiological cutoff value setting for APX001A, allowing classification as wild type (WT) or non-WT. APX001A MIC50 values (mg/liter) were as follows: Candida albicans, Candida dubliniensis, and Candida tropicalis, 0.004 to 0.008; Candida parapsilosis and Candida auris, 0.016; Candida glabrata, 0.06; and Candida krusei, >0.5. APX001A MICs against the rare species varied from ≤0.0005 (C. pelliculosa) to >0.5 (Candida norvegensis). APX001A was equally or more active in vitro than the comparators against all species except C. krusei and C. norvegensis. Four isolates were APX001A non-WT; all were fluconazole resistant. A correlation was observed between APX001A and fluconazole MICs across all species except Candida guilliermondii and C. auris, and when comparing high and low fluconazole MIC isolates of C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, and C. auris. APX001A showed promising in vitro activity against most Candida and other yeast species, including C. auris, compared to five comparators. WT-UL were suggested for the common species, and a new and unexplained correlation to fluconazole susceptibility was observed.


2019 ◽  
Vol 63 (3) ◽  
Author(s):  
William R. Wilson ◽  
Ellen G. Kline ◽  
Chelsea E. Jones ◽  
Kristin T. Morder ◽  
Roberta T. Mettus ◽  
...  

ABSTRACT Meropenem-vaborbactam is a new agent with the potential to treat carbapenem-resistant Enterobacteriaceae (CRE) infections. We describe the in vitro activity of meropenem-vaborbactam against representative CRE genotypes and laboratory-engineered Escherichia coli isolates harboring mutant blaKPC genes associated with ceftazidime-avibactam resistance. We also compared disk diffusion and gradient strip testing methods to standard broth microdilution methods. Against 120 CRE isolates, median ceftazidime-avibactam and meropenem-vaborbactam MICs were 1 and 0.03 µg/ml, respectively. Ninety-eight percent (117/120) of isolates were susceptible to meropenem-vaborbactam (MICs ≤ 4 µg/ml). Against Klebsiella pneumoniae isolates harboring mutant blaKPC, the addition of vaborbactam lowered the meropenem MICs in 78% of isolates (14/18); 100% were susceptible to meropenem-vaborbactam. Median meropenem-vaborbactam MICs were higher against K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates with mutant ompK36 porin genes (n = 26) than against those with wild-type ompK36 porin genes (n = 54) (0.25 versus 0.03 µg/ml; P < 0.0001). Against E. coli TOP10 isolates with plasmid constructs containing wild-type blaKPC or mutant blaKPC, the addition of vaborbactam at 8 µg/ml lowered the meropenem MICs 2- to 512-fold, resulting in meropenem-vaborbactam MICs of 0.03 µg/ml. The rates of categorical agreement with broth microdilution for disk diffusion or gradient strips ranged from 90 to 95%. Essential agreement rates were higher for research-use-only (RUO) gradient strips manufactured by bioMérieux (82%) than for those manufactured by Liofilchem (48%) (P < 0.0001). Taken together, our data highlight the potent in vitro activity of meropenem-vaborbactam against CRE, including isolates resistant to ceftazidime-avibactam. Vaborbactam inhibited both wild-type and variant KPC enzymes. On the other hand, KPC-producing K. pneumoniae isolates with ompK36 mutations displayed higher meropenem-vaborbactam MICs than isolates with wild-type ompK36. The results of susceptibility testing with RUO bioMérieux gradient strips most closely aligned with those of broth microdilution methods.


2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Anuradha Chowdhary ◽  
Karin Meinike Jørgensen ◽  
Joseph Meletiadis

ABSTRACT Fosmanogepix is a novel prodrug in a new class of antifungal agents. Manogepix is the active moiety. We evaluated the CLSI and EUCAST MICs of manogepix and eight comparators against Candida auris. CLSI M27-A3 susceptibility testing of manogepix was performed for 122 C. auris isolates and compared to CLSI and EUCAST MICs for manogepix and eight comparators. Differences and agreement were calculated for each compound. Wild-type upper limits (WT-ULs; the upper MIC where the wild-type distribution ends) for manogepix and correlations with other drugs’ MICs were determined. Manogepix MICs (CLSI/EUCAST [mg/liter]) and WT-ULs were as follows: MIC50s, 0.008/0.016; MIC90s, 0.03/0.03; ranges, 0.001 to 0.25/0.001 to 0.125; 97.5% and 99% WT-ULs, 0.03/0.125 and 0.06/0.125, respectively. The manogepix CLSI/EUCAST MIC distributions spanned 9/8 dilutions, respectively. Significant correlation was found for all azoles, particularly fluconazole (r = 0.22 to 0.74, P < 0.05). Isolates with EUCAST manogepix MICs of ≤0.004 had 7.6-/10.2-fold-lower fluconazole CLSI/EUCAST MICs than the remaining isolates that had higher manogepix MICs. The highest essential agreement between CLSI and EUCAST results was observed for manogepix and fluconazole, with a median difference of −1 to 0 2-fold dilutions, 90th percentile absolute difference of 1, and 90 to 92% and 98 to 100% agreement within ±1 and ±2 dilutions. The lowest agreements within ±1 and ±2 dilutions were found for isavuconazole and anidulafungin (44 to 50% and 69 to 76%). The correlation between CLSI and EUCAST manogepix MICs against C. auris was excellent. Differential MICs were found, and these correlated with fluconazole MICs, suggesting that the C. auris population is a mix of wild-type isolates and non-wild-type isolates with low-grade manogepix MIC elevation, probably involving efflux pump expression. However, manogepix was the most potent agent against C. auris in this in vitro study.


2020 ◽  
Vol 65 (1) ◽  
pp. e01527-20
Author(s):  
Karen Marie Thyssen Astvad ◽  
Karin Meinike Jørgensen ◽  
Rasmus Krøger Hare ◽  
Raluca Datcu ◽  
Maiken Cavling Arendrup

ABSTRACTOlorofim is a novel antifungal drug in phase 2 trials. It has shown promising in vitro activity against various molds, except for Mucorales. Initially, we observed a broad range of EUCAST MICs for Aspergillus fumigatus. Here, we explored the MIC variability in more detail and prospectively investigated the susceptibility of contemporary clinical mold isolates, as population data are needed for future epidemiological cutoff (ECOFF) settings. Fifteen A. fumigatus isolates previously found with low/medium/high MICs (≤0.002 to 0.25 mg/liter) were tested repeatedly and EUCAST MICs read in a blinded fashion by three observers. pyrE, encoding the olorofim target enzyme dihydroorotate dehydrogenase (DHODH), was sequenced. A total of 1,423 mold isolates (10 Aspergillus species complexes [including 1,032 A. fumigatus isolates] and 105 other mold/dermatophyte isolates) were examined. Olorofim susceptibility (modal MIC, MIC50, MIC90, and wild-type upper limits [WT-ULs] [species complexes with ≥15 isolates]) was determined and compared to that of four comparators. MICs (mg/liter) were within two 2-fold dilutions (0.016 to 0.03) for 473/476 determinations. The MIC range spanned four dilutions (0.008 to 0.06). No significant pyrE mutations were found. Modal MIC/WT-UL97.5 (mg/liter) values were 0.03/0.06 (A. terreus and A. flavus), 0.06/0.125 (A. fumigatus and Trichophyton rubrum), and 0.06/0.25 (A. niger and A. nidulans). The MIC range for Scedosporium spp. was 0.008 to 0.25. Olorofim susceptibility was similar for azole-resistant and -susceptible isolates of A. fumigatus but reduced for A. montevidensis and A. chevalieri (MICs of >1). With experience, olorofim susceptibility testing is robust. The testing of isolates from our center showed uniform and broad-spectrum activity. Single-center WT-ULs are suggested.


2015 ◽  
Vol 60 (1) ◽  
pp. 532-536 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Rasmus Hare Jensen ◽  
Manuel Cuenca-Estrella

ABSTRACTASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity againstAspergillusandCandida glabrata. We compared itsin vitroactivity against wild-type and azole-resistantA. fumigatusandA. terreusisolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-typeA. fumigatusisolates, 24A. fumigatusisolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), andA. terreusisolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) againstA. fumigatusCYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistantA. fumigatusisolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active againstA. terreusCYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayedin vitroactivity againstA. fumigatusandA. terreusisolates which was independent of the presence or absence of azole target gene resistance mutations inA. fumigatus. The findings are promising at a time when azole-resistantA. fumigatusis emerging globally.


2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Karin Meinike Jørgensen ◽  
Rasmus Krøger Hare ◽  
Anuradha Chowdhary

ABSTRACT Ibrexafungerp (SCY-078) is a novel first-in-class antifungal agent targeting glucan synthase. Candida auris is an emerging multidrug-resistant species that has caused outbreaks on five continents. We investigated the in vitro activity of ibrexafungerp against C. auris by applying EUCAST E.Def 7.3.1 methodology. C. albicans and C. glabrata, as well as anidulafungin, micafungin, amphotericin B, fluconazole, voriconazole, and isavuconazole, were included as comparators. Three C. auris reference strains (CBS12372, CBS12373, and CBS10913) and 122 C. auris, 16 C. albicans, and 16 C. glabrata isolates were evaluated. C. albicans ATCC 64548, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 served as quality control strains. Echinocandin-resistant isolates were fks sequenced. MIC ranges and modal MIC and MIC50 values were determined. Wild-type upper limits (the upper MIC value where the wild-type distribution ends) were determined according to EUCAST principles for setting ECOFFs. Nine repetitions of three QC strains and MICs for C. albicans and C. glabrata yielded narrow MIC ranges with modal MICs in agreement with established EUCAST modal MICs, confirming a robust test performance. The ibrexafungerp MICs against C. auris isolates displayed a Gaussian distribution with a modal MIC (range) of 0.5 mg/liter (0.06 to 2 mg/liter), suggesting uniform susceptibility. Of 122 isolates, 8 were echinocandin resistant and harbored the S639F Fks1 alteration. All but one were fluconazole resistant, and the MIC distributions for voriconazole and isavuconazole were multimodal confirming variable susceptibility. Ibrexafungerp demonstrated promising activity against C. auris, including isolates resistant to echinocandins and/or other agents. The MICs were similar to those reported for the Clinical and Laboratory Standards Institute method, suggesting that a common clinical breakpoint may be appropriate.


2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Alita A. Miller ◽  
Maria M. Traczewski ◽  
Michael D. Huband ◽  
Patricia A. Bradford ◽  
John P. Mueller

ABSTRACT This report describes the results of two different, multilaboratory quality control (QC) studies that were used to establish QC ranges for the novel gyrase inhibitor zoliflodacin against the ATCC strains recommended by the Clinical and Laboratory Standards Institute (CLSI). Following the completion of an eight-laboratory, CLSI document M23-defined tier 2 study, the agar dilution MIC QC range for zoliflodacin against the Neisseria gonorrhoeae QC strain ATCC 49226 was defined as 0.06 to 0.5 μg/ml and was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. This QC range will be used for in vitro susceptibility testing of zoliflodacin during phase 3 human clinical trials and surveillance studies, and eventually it will be implemented in clinical labs. In a separate study, broth microdilution MIC quality control ranges for zoliflodacin against additional QC strains were determined to be 0.12 to 0.5 μg/ml for Staphylococcus aureus ATCC 29213, 0.25 to 2 μg/ml for Enterococcus faecalis ATCC 29212, 1 to 4 μg/ml for Escherichia coli ATCC 25922, 0.12 to 0.5 μg/ml for Streptococcus pneumoniae ATCC 49619, and 0.12 to 1 μg/ml for Haemophilus influenzae ATCC 49247. These MIC QC ranges were also approved by CLSI for use in future in vitro susceptibility testing studies against organisms other than N. gonorrhoeae.


2017 ◽  
Vol 55 (10) ◽  
pp. 3021-3027 ◽  
Author(s):  
Meredith A. Hackel ◽  
James A. Karlowsky ◽  
Dana Dressel ◽  
Daniel F. Sahm

ABSTRACTDisk diffusion and MIC quality control (QC) ranges were determined for nafithromycin, a new lactone-ketolide, following the completion of a nine-laboratory, Clinical and Laboratory Standards Institute (CLSI) document M23-defined tier 2 study. Five QC strains consistent with the spectrum of activity of nafithromycin were tested:Staphylococcus aureusATCC 25923 (disk only),S. aureusATCC 29213 (broth only),Enterococcus faecalisATCC 29212 (broth only),Streptococcus pneumoniaeATCC 49619 (disk and broth), andHaemophilus influenzaeATCC 49247 (disk and broth). Nafithromycin disk diffusion QC ranges were determined to be 25 to 31 mm forS. aureusATCC 25923, 25 to 31 mm forS. pneumoniaeATCC 49619, and 16 to 20 mm forH. influenzaeATCC 49247. Nafithromycin MIC QC ranges were determined to be 0.06 to 0.25 μg/ml forS. aureusATCC 29213, 0.016 to 0.12 μg/ml forE. faecalisATCC 29212, 0.008 to 0.03 μg/ml forS. pneumoniaeATCC 49619, and 2 to 8 μg/ml forH. influenzaeATCC 49247. All disk diffusion and MIC QC ranges established in this study were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting and were initially reported in the 2017 M100S document. The QC ranges established in this study should be used for determining thein vitroactivity of nafithromycin in phase 2 and phase 3 human clinical trials and subsequently for testing patient isolates and isolates in phase 4 surveillance studies.


2016 ◽  
Vol 60 (8) ◽  
pp. 5088-5091 ◽  
Author(s):  
M.-E. Bougnoux ◽  
E. Dannaoui ◽  
I. Accoceberry ◽  
A. Angoulvant ◽  
E. Bailly ◽  
...  

ABSTRACTIn vitrosusceptibility of 933Candidaisolates, from 16 French hospitals, to micafungin was determined using the Etest in each center. All isolates were then sent to a single center for determination of MICs by the EUCAST reference method. Overall essential agreement between the two tests was 98.5% at ±2 log2dilutions and 90.2% at ±1 log2dilutions. Categorical agreement was 98.2%. The Etest is a valuable alternative to EUCAST for the routine determination of micafungin MICs in medical mycology laboratories.


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