In recent years, cases involving terbinafine-resistant Trichophyton isolates have been reported increasingly, particularly in India. We present 14 cases of terbinafine treatment failure in Trichophyton-infected Danish patients due to acquired resistance. Patients infected with Trichophyton rubrum (n = 12) or Trichophyton interdigitale (n = 2) with elevated terbinafine MICs during 2013–2018 were included. Antifungal susceptibility testing (AFST) was performed following a modified EUCAST E.Def 9.3.1 method (5 days of incubation) with or without cycloheximide and chloramphenicol (CC) supplementation of the growth medium. The squalene epoxidase (SE) target gene was sequenced, and 3-dimensional enzyme homology modeling was performed. Most patients (12/14 [86%]) were male. The mean age was 53.5 years (range, 11 to 77 years). The mean duration of infections was 4.8 years at the time of resistance detection. Prior systemic terbinafine treatment was documented for all patients, and topical therapy for 62% (information was missing in one case). Overall, nine isolates (64%) displayed high terbinafine resistance (MICs, 4 to >8 mg/liter), while two (14%) displayed moderate (MICs, 1 to 2 mg/liter) and three (21%) displayed low (MICs, 0.125 to 0.25 mg/liter) terbinafine resistance compared with control isolates. MICs generated with or without CC supplementation were similar, but CC prevented contamination. Known and novel SE amino acid substitutions (F397L, L393F, L393S, F415S, H440Y F484Y, and I121M V237I) were detected in resistant but not control isolates. Three-dimensional homology modeling suggested a role of the novel I121M and V237I alterations. Terbinafine resistance has been detected in Denmark using a modified EUCAST method, which facilitated susceptibility testing of dermatophytes. Action is needed for this emerging public health problem.

The dynamics of occurrence and the genetic basis of ciprofloxacin resistance were studied in a long-term evolution experiment (940 generations) in wild-type, reference strain (PAO1) and hypermutable (PAO⌬mutS and PAOMY-Mgm) P. aeruginosa populations continuously exposed to sub-MICs (1/4) of ciprofloxacin. A rapid occurrence of ciprofloxacin-resistant mutants (MIC of >12 g/ml, representing 100 times the MIC of the original population) were observed in all ciprofloxacin-exposed lineages of PAO⌬mutS and PAOMY-Mgm populations after 100 and 170 generations, respectively, and in one of the PAO1 lineages after 240 generations. The genetic basis of resistance was mutations in gyrA (C248T and G259T) and gyrB (C1397A). Cross-resistance to beta-lactam antibiotics was observed in the bacterial populations that evolved during exposure to sublethal concentrations of ciprofloxacin. Our study shows that mutants with high-level ciprofloxacin resistance are selected in P. aeruginosa bacterial populations exposed to sub-MICs of ciprofloxacin. This can have implications for the long-term persistence of resistant bacteria and spread of antibiotic resistance by exposure of commensal bacterial flora to low antibiotic concentrations.

Objectives Terbinafine resistance is increasingly reported in Trichophyton, rendering susceptibility testing particularly important in non-responding cases. We performed a multicentre evaluation of six EUCAST-based methods. Methods Ten laboratories susceptibility tested terbinafine, itraconazole, voriconazole and amorolfine against a blinded panel of 38 terbinafine WT and target gene mutant isolates. E.Def 9.3.1 modifications included: medium with/without addition of chloramphenicol and cycloheximide (CC), incubation at 25°C to 28°C for 5–7 days and three MIC endpoints [visually and spectrophotometrically (90%/50% inhibition)], generating 7829 MICs. Quality control (QC) strains were Aspergillus flavus ATCC 204304 and CNM-CM1813. Eyeball, ECOFFinder (where ECOFF stands for epidemiological cut-off) and derivatization WT upper limits (WT-ULs), very major errors (VMEs; mutants with MICs ≤WT-ULs) and major errors (MEs; WT isolates with MICs >WT-ULs) were determined. Results MICs fell within the QC ranges for ATCC 204304/CNM-CM1813 for 100%/96% (voriconazole) and 84%/84% (itraconazole), respectively. Terbinafine MICs fell within 0.25–1 mg/L for 96%/92%, suggesting high reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 to 4 (2.6%–5.2%) for Trichophyton rubrum and from 0 to 2 (0%–2.0%) for Trichophyton interdigitale. Modes for WT and mutant populations were at least seven 2-fold dilutions apart in all cases. Excluding one I121M/V237I T. rubrum mutant and two mixed WT/mutant T. interdigitale specimens, the numbers of VMEs were as follows: T. rubrum: CC visual, 1/67 (1.5%); CC spectrophotometric 90% inhibition, 3/59 (5.1%); and CC spectrophotometric 50% inhibition, 1/67 (1.5%); and T. interdigitale: none. Voriconazole and amorolfine MICs were quite uniform, but trailing growth complicated determination of itraconazole visual and spectrophotometric 90% inhibition MIC. Conclusions Although none of the laboratories was experienced in dermatophyte testing, error rates were low. We recommend the CC spectrophotometric 50% inhibition method and provide QC ranges and WT-ULs for WT/non-WT classification.

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Olorofim is a novel antifungal agent with activity against 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 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 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 were diverse, with full inhibition of (MIC, 0.016), 50% growth inhibition of at 1 to 2 mg/liter, and no inhibition of Olorofim displayed potent activity against most mold isolates and was associated with limited variation in EUCAST susceptibility testing.