Voriconazole efficacy against Candida glabrata and Candida krusei: preclinical data using a validated in vitro pharmacokinetic/pharmacodynamic model

Background: Voriconazole exhibits in vitro activity against Candida glabrata and Candida krusei (EUCAST/CLSI epidemiological cut-off values 1/0.25 and 1/0.5 mg/L, respectively). Yet, EUCAST found insufficient evidence to set breakpoints for these species. We explored voriconazole pharmacodynamics (PD) in an in vitro dynamic model simulating human pharmacokinetics (PK). Methods: Four C. glabrata and three C. krusei isolates (voriconazole EUCAST and CLSI MICs of 0.03–2 mg/L) were tested in the PK/PD model simulating voriconazole exposures (t1=2 6 h q12h dosing for 3 days). PK/PD breakpoints were determined calculating the PTA for exposure indices fAUC0–24/MIC associated with half-maximal activity (EI50) using Monte Carlo simulation analysis. Results: Fungal load increased from 3.60±0.35 to 8.41±0.24 log10 cfu/mL in the drug-free control, with a maximum effect of 1 log10 kill of C. glabrata and C. krusei isolates with MICs of 0.06 and 0.25 mg/L, respectively, at high drug exposures. The 72 h log10 cfu/mL change versus fAUC0–24/MIC relationship followed a sigmoid curve for C. glabrata (R2 =0.85–0.87) and C. krusei (R2 =0.56–0.76) with EI50 of 49 (32–76) and 52 (33–78) fAUC/MIC for EUCAST and 55 (31–96) and 80 (42–152) fAUC/MIC for CLSI, respectively. The PTAs for C. glabrata and C. kr

A multicentre study to optimize echinocandin susceptibility testing of Aspergillus species with the EUCAST methodology and a broth microdilution colorimetric method

BACKGROUND: The determination of the minimal effective concentration (MEC) of echinocandins against Aspergillus species is subjective, time consuming and has been associated with very major errors. METHODS: The MECs/MICs of 40 WT [10 each of Aspergillus fumigatus species complex (SC), Aspergillus flavus SC, Aspergillus terreus SC and Aspergillus niger SC] and 4 non-WT A. fumigatus isolates were determined with EUCAST E.Def 9.3.1 read microscopically, macroscopically, spectrophotometrically and colorimetrically in three centres. The optimal conditions for spectrophotometric (single- versus multi-point readings) and colorimetric (XTT/menadione concentration and stability, incubation time) methods were evaluated in preliminary studies using different cut-offs for the determination of macroscopic, spectrophotometric and colorimetric MIC endpoints compared with the microscopically determined MEC. Inter-centre and inter-method essential (within one 2-fold dilution) agreement (EA) and categorical agreement (CA) were determined. RESULTS: Both macroscopic and spectr

Development and multicentre validation of an agar-based screening method for echinocandin susceptibility testing of Aspergillus species

BACKGROUND: Reference antifungal susceptibility testing of echinocandins against Aspergillus spp. relies on the determination of the minimal effective concentration, which is difficult to perform, time-consuming and subjective. We developed and evaluated in a multicentre study an agar-based screening method for echinocandin susceptibility testing of Aspergillus spp. METHODS: Forty WT isolates [10 Aspergillus fumigatus species complex (SC), 10 Aspergillus flavus SC, 10 Aspergillus terreus SC and 10 Aspergillus niger SC] and 4 non-WT A. fumigatus isolates with or without known fks alterations were used. The optimal test conditions and stability over time were evaluated in preliminary studies monitoring colony growth. Twenty-microlitre aliquots of 1-2 McFarland inocula in 0.1% Tween 20 aqueous solution were added to each well and plates were incubated for 24/48 h at 35 ± 2°C. Subsequently, all isolates were tested blindly at three centres using four-well screening plates, containing anidulafungin, caspofungin, micafungin or no antifungal in each of the four wells, respectively. RESULTS: WT isolates produced fluffy colonies on drug-free agar wells only. The non-WT isolates produced fluffy colonies on echinocandin-containing and control agar wells. Using the echinocandin concentrations of 0.25 mg/L anidulafungin, 1 mg/L caspofungin and 0.125 mg/L micafungin, and the compact (non-fluffy) versus fluffy colony morphology endpoint, all centres successfully discriminated non-WT and WT strains even after 24 h. Among the three echinocandins, anidulafungin produced the clearest endpoints. CONCLUSIONS: The four-well plate agar method is suitable for echinocandin susceptibility screening of Aspergillus spp. and can be used to detect echinocandin non-WT isolates. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: [email protected]

Association Between Candiduria and Candidemia: A Clinical and Molecular Analysis of Cases

The risk of developing candidemia after candiduria is reportedly very low, but it has not been adequately investigated. The aim of this study was to examine the molecular relatedness between Candida strains isolated from adult patients with candidemia and concomitant candiduria in association with the clinical characteristics of the cases. All episodes of candidemia occurring in a tertiary care academic hospital during a 5-year period were recorded prospectively. Patients with episodes of candiduria occurring two weeks preceding to or one week following a positive for Candida blood culture were included in the study. The genotypic relatedness of Candida strains isolated from blood and urine was investigated by pulsed-field gel electrophoresis after digestion with the BssHII restriction endonuclease. We recorded 141 candidemia episodes, occurring in 134 patients. Twelve episodes of candidemia with concomitant candiduria occurred in 11 patients (8% of all candidemias). In six of these episodes, the strains in the blood–urine pairs belonged to different species. In two episodes, the isolates belonged to the same species but were not genetically related, and only in four (2.8% of all candidemias), the strains were related. All four patients were severely ill and had multiple risk factors for candidemia. These findings indicate that in hospitalized patients with candidemia, concomitant candiduria is rare and usually an independent event, confirming previous reports. In the critically ill, however, the existence of genetically related strains in blood and urine appears to be more frequent, with more probable the hematogenous dissemination. © 2017, Springer Science+Business Media B.V

Fosfomycin efficacy and emergence of resistance among Enterobacteriaceae in an in vitro dynamic bladder infection model

Background: Urinary tract infections (UTIs) are among the most common bacterial infections and a frequent indication for antibiotic use. Fosfomycin, an important oral antibiotic for outpatient UTIs, remains a viable option for MDR uropathogens. We aimed to perform pharmacodynamic profiling simulating urinary concentrations to assess the adequacy of the current dosing regimen. Methods: A dynamic in vitro bladder infection model was developed, replicating urinary fosfomycin concentrations after gastrointestinal absorption, systemic distribution and urinary elimination. Concentrations weremeasured by LC-MS/MS. Twenty-four Enterobacteriaceae strains (Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae; MIC range 0.25-64 mg/L) were examined. Pathogen kill and emergence of resistance was assessed over 72 h. Results: Observed in vitro fosfomycin concentrations accurately simulated urinary fosfomycin exposures (T max 3.8±0.5 h;C max 2630.1±245.7 mg/L;AUC 0-24 33932.5±1964.2mg.h/L). Fifteen of 24 isolates regrew, with significant rises in fosfomycin MIC (total population MIC 50 4 to 64 mg/L, MIC 90 64 to>1024 mg/L, P=0.0039; resistant subpopulation MIC 50 128 to>1024 mg/L, MIC 90 >1024 mg/L, P=0.0020). E. coli and E. cloacae isolates were killed with pharmacokinetic/pharmacodynamic EI50 of fAUC 0-24 /MIC=1922, fC max /MIC=149 and fTime>4×MIC=44 h. In contrast, K. pneumoniae isolates were not reliably killed. Conclusions: Using dynamic in vitro simulations of urinary fosfomycin exposures, E. coli and E. cloacae isolates with MIC>16 mg/L, and all K. pneumoniae isolates, were not reliably killed. Emergence of resistance was significant. This challenges fosfomycin dosing and clinical breakpoints, and questions the utility of fosfomycin against K. pneumoniae. Further work on in vitro dose optimization is required. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

A multicentre study to optimize echinocandin susceptibility testing of Aspergillus species with the EUCAST methodology and a broth microdilution colorimetric method

The determination of the minimal effective concentration (MEC) of echinocandins against Aspergillus species is subjective, time consuming and has been associated with very major errors. Methods: The MECs/MICs of 40 WT [10 each of Aspergillus fumigatus species complex (SC), Aspergillus flavus SC, Aspergillus terreus SC and Aspergillus niger SC] and 4 non-WT A. fumigatus isolates were determined with EUCAST E.Def 9.3.1 read microscopically, macroscopically, spectrophotometrically and colorimetrically in three centres. The optimal conditions for spectrophotometric (single- versus multi-point readings) and colorimetric (XTT/menadione concentration and stability, incubation time) methods were evaluated in preliminary studies using different cut-offs for the determination of macroscopic, spectrophotometric and colorimetric MIC endpoints compared with the microscopically determined MEC. Inter-centre and inter-method essential (within one 2-fold dilution) agreement (EA) and categorical agreement (CA) were determined. Results: Both macroscopic and spectrophotometric endpoint readings showed poor inter-centre EA (53%-66%) and low CA (41%-88%) in distinguishing WT from non-WT A. fumigatus SC isolates, while significant differences compared with the microscopic MECs were observed for all echinocandins (EA 6%-54%). For the colorimetric method, the optimal conditions were 400 mg/L XTT/6.25 μΜ menadione, incubation for 1-2 h until the drug-free control reached an absorbance at 450/630 nm of >0.8 and use of 50% inhibition of XTT conversion as a cut-off for all species and echinocandins. All non-WT isolates had high XTT MICs >1 mg/L, whereas the overall inter-centre EA and CA were 72%-89% and 100%, respectively. Conclusions: The XTT colorimetric assay improved the antifungal susceptibility testing of echinocandins against Aspergillus spp., reliably detecting non-WT isolates. © 2020 The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

Voriconazole efficacy against Candida glabrata and Candida krusei: Preclinical data using a validated in vitro pharmacokinetic/pharmacodynamic model

Background: Voriconazole exhibits in vitro activity against Candida glabrata and Candida krusei (EUCAST/CLSI epidemiological cut-off values 1/0.25 and 1/0.5 mg/L, respectively). Yet, EUCAST found insufficient evidence to set breakpoints for these species. We explored voriconazole pharmacodynamics (PD) in an in vitro dynamic model simulating human pharmacokinetics (PK). Methods: Four C. glabrata and three C. krusei isolates (voriconazole EUCAST and CLSI MICs of 0.03-2 mg/L) were tested in the PK/PD model simulating voriconazole exposures (t½ ∼6 h q12h dosing for 3 days). PK/PD breakpoints were determined calculating the PTA for exposure indices fAUC0-24/MIC associated with half-maximal activity (EI50) using Monte Carlo simulation analysis. Results: Fungal load increased from 3.60±0.35 to 8.41±0.24 log10 cfu/mL in the drug-free control, with a maximum effect of ∼1 log10 kill of C. glabrata and C. krusei isolates with MICs of 0.06 and 0.25 mg/L, respectively, at high drug exposures. The 72 h log10 cfu/mL change versus fAUC0-24/MIC relationship followed a sigmoid curve for C. glabrata (R2=0.85-0.87) and C. krusei (R2=0.56-0.76) with EI50 of 49 (32-76) and 52 (33-78) fAUC/MIC for EUCAST and 55 (31-96) and 80 (42-152) fAUC/MIC for CLSI, respectively. The PTAs for C. glabrata and C. krusei isolates with EUCAST/CLSI MICs ≤0.125/≤0.06 mg/L were >95%. Isolates with EUCAST/CLSI MICs of 0.25-1/0.125-0.5 would require trough levels 1-4 mg/L; isolates with higher MICs would not attain the corresponding PK/PD targets without reaching toxicity. Conclusions: The in vitro PK/PD breakpoints for C. glabrata and C. krusei for EUCAST (0.125 mg/L) and CLSI (0.06 mg/L) bisected the WT populations. Trough levels of >4 mg/L, which are not clinically feasible, are necessary for efficacy against WT isolates. © 2019 The Author(s). Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

Multicentre determination of rezafungin (CD101) susceptibility of Candida species by the EUCAST method

Objectives: Rezafungin (CD101) is a new long-acting echinocandin allowing weekly dosing, currently undergoing phase-II clinical trials for invasive candidiasis. The aim of this study was to assess rezafungin's in vitro activity against the most frequent Candida species following the EUCAST methodology. Methods: The susceptibility of 2018 clinical Candida isolates was determined at four European laboratories. In parallel, six control strains were repeatedly tested. Wild-type upper limits (WT-ULs), defined as the MIC value where the wild-type distribution ends, were determined following the principles for EUCAST ECOFF-setting. Results: The lowest rezafungin MICs (geometric MIC (GM-MIC), MIC range (mg/L)) were observed for C. albicans (0.016, 0.002–0.125) and the highest for C. parapsilosis (1.657, 0.063->4). MICs for the remaining species were in between these values (GM-MICs 0.048–0.055). Visual and statistical WT-ULs were identical for C. glabrata (0.125), C. krusei (0.125), C. parapsilosis (4), and C. tropicalis (0.25). If adopting these WT-ULs for classification into WT and non-WT populations, 1/413 C. glabrata, 1/402 C. krusei, 1/398 C. parapsilosis, and 1/402 C. tropicalis isolates were categorized as non-WT, all of which derived from Laboratory 1. For C. albicans unexplained laboratory variation was observed (WT-UL: 0.063–0.125 in Laboratories 1 and 2 versus 0.016 in Laboratories 3 and 4). A similar systematic difference was observed comparing the MICs for the three C. albicans QC strains, specifically, obtained in Laboratories 1and 2 with those in Laboratories 3 and 4. Discussion: Rezafungin displayed species-specific activity similar to other echinocandins. Interlaboratory variation was observed for the most susceptible species C. albicans clinical and QC strains, an observation that warrants further investigation. © 2018 European Society of Clinical Microbiology and Infectious Disease

In vitro antifungal susceptibility of filamentous fungi causing rare infections: Synergy testing of amphotericin B, posaconazole and anidulafungin in pairs

Objectives: Mucormycetes (formerly known as zygomycetes of the order Mucorales) and hyaline moulds such as those of the genus Fusarium or Paecilomyces are emerging as significant human pathogens. The aim of the study was to determine the in vitro antifungal susceptibility of these fungi to older and newer antifungals and to investigate the antifungal activity of amphotericin B, posaconazole and anidulafungin in dual combinations. Methods: Twenty-one clinical isolates of mucormycetes and 16 of rare hyaline moulds were tested. MICs were determined by EUCAST methodology for conidia-forming moulds and Etesting. For antifungal combinations a chequerboard method based on EUCAST methodology was used. Results: Against mucormycetes, amphotericin B exhibited the lowest MICs, followed by posaconazole. Ravuconazole was active against eight of the Rhizopus isolates (MIC 1 mg/L). Resistance to amphotericin B (MIC ≥2 mg/L) and posaconazole (MICs ≥4 mg/L) was observed in five and three Rhizopus isolates, respectively. Among Fusarium species variable susceptibility patterns were detected. Amphotericin B exhibited the lowest MICs, followed by voriconazole. Etesting for amphotericin B and posaconazole had excellent agreement with EUCAST methodology (78.6%-100%). Synergy between amphotericin B and anidulafungin was observed against two isolates (one Mucor circinelloides and one Fusarium proliferatum). Synergy or antagonism was not detected in any other combination. Conclusions: The study showed that mucormycetes and other rare hyaline moulds exhibit variable susceptibilities to antifungals, and hence antifungal testing is valuable. The fact that the combination of amphotericin B with anidulafungin was found synergistic in some cases merits further investigation. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved