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

Exploring colistin pharmacodynamics against Klebsiella pneumoniae: A need to revise current susceptibility breakpoints

Objectives: Because the pharmacokinetic/pharmacodynamic (PK/PD) characteristics of colistin against Enterobacteriaceae are not well explored, we studied the activity of colistin against K. pneumoniae in an in vitro PK/PD model simulating different dosing regimens. Methods: Three clinical isolates of K. pneumoniae with MICs of 0.5, 1 and 4mg/L were tested in an in vitro PK/PD model following a dose-fractionation design over a period of 24h. A high and low inoculumof 107 and 104 cfu/mL with and without a heteroresistant subpopulation, respectively, were used. PK/PD indices associated with colistin activity were explored and Monte Carlo analysis was performed in order to determine the PTA for achieving a bactericidal effect (2 log kill). Results: The fAUC/MIC (R2"0.64-0.68) followed by fCmax/MIC (R2=0.55-0.63) best described colistin's 24 h log10 cfu/mL reduction for both low and high inocula. Dosing regimens with fCmax/MIC≥6 were always associated with a bactericidal effect (P=0.0025). However, at clinically achievable concentrations, usually below fCmax/MIC=6, an fAUC/MIC ≤25 was more predictive of a bactericidal effect. Using a dosing regimen of 9 MU/ day, the PTA for this pharmacodynamic target was 100%, 5%-70%and 0%, for isolates with MICs of ≤0.5, 1 and ≥2 mg/L, respectively. Dosing regimens that aim for a trough level of 1 mg/L achieve coverage of strains up to 0.5 mg/L (target trough/MIC=2 mg/L). Conclusions: Characterization of the pharmacodynamics of colistin against Enterobacteriaceae in an in vitro model of infection indicates that a revision of current susceptibility breakpoints is needed. Therapeutic drug monitoring of colistin to achieve pharmacodynamic targets in individual patients is highly recommended

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

How to: interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European committee on antimicrobial susceptibility testing (EUCAST)

BACKGROUND: EUCAST has revised the definition of the susceptibility category "I" from "Intermediate" to "Susceptible, Increased exposure". This implies that "I" can be used where the drug-concentration at the site of infection is high, either because of dose escalation or through other means to ensure efficacy. Consequently, "I" is no longer used as a buffer-zone to prevent technical fact

Rapid Susceptibility Testing and Microcolony Analysis of Candida spp. Cultured and Imaged on Porous Aluminum Oxide

Contains fulltext : 124300.pdf (publisher's version ) (Open Access)BACKGROUND: Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results. METHODS: Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture). SIGNIFICANCE: Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications

Aspergillus fumigatus and aspergillosis: From basics to clinics

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.AA, RGR, and DSP were supported by NIH AI 109025. MH was supported by NIH UL1TR001442. AC was supported by the Fundação para a Ciência e a Tecnologia (FCT) (CEECIND/03628/2017 and PTDC/MED GEN/28778/2017). Additional support was provided by FCT (UIDB/50026/2020 and UIDP/50026/2020), the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023), the European Union's Horizon 2020 Research and Innovation programme under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HP17/52190003. DJA was supported by CF Trust Strategic Research Centre TrIFIC (SRC015), Wellcome Trust Collaborative Award 219551/Z/19/Z and the NIHR Centre for Antimicrobial Optimisation.S

On the isoperimetric problem for the Laplacian with Robin and Wentzell boundary conditions

Doctor of PhilosophyWe consider the problem of minimising the eigenvalues of the Laplacian with Robin boundary conditions $\frac{\partial u}{\partial \nu} + \alpha u = 0$ and generalised Wentzell boundary conditions $\Delta u + \beta \frac{\partial u}{\partial \nu} + \gamma u = 0$ with respect to the domain $\Omega \subset \mathbb R^N$ on which the problem is defined. For the Robin problem, when $\alpha > 0$ we extend the Faber-Krahn inequality of Daners [Math. Ann. 335 (2006), 767--785], which states that the ball minimises the first eigenvalue, to prove that the minimiser is unique amongst domains of class $C^2$. The method of proof uses a functional of the level sets to estimate the first eigenvalue from below, together with a rearrangement of the ball's eigenfunction onto the domain $\Omega$ and the usual isoperimetric inequality. We then prove that the second eigenvalue attains its minimum only on the disjoint union of two equal balls, and set the proof up so it works for the Robin $p$-Laplacian. For the higher eigenvalues, we show that it is in general impossible for a minimiser to exist independently of $\alpha > 0$. When $\alpha < 0$, we prove that every eigenvalue behaves like $-\alpha^2$ as $\alpha \to -\infty$, provided only that $\Omega$ is bounded with $C^1$ boundary. This generalises a result of Lou and Zhu [Pacific J. Math. 214 (2004), 323--334] for the first eigenvalue. For the Wentzell problem, we (re-)prove general operator properties, including for the less-studied case $\beta 0$ establish a type of equivalence property between the Wentzell and Robin minimisers for all eigenvalues. This yields a minimiser of the second Wentzell eigenvalue. We also prove a Cheeger-type inequality for the first eigenvalue in this case

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values

ABSTRACT Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 μg/ml for the CLSI method and 0.25 μg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 μg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 μg/ml. The tentative ECOFFinder ECV with SYO was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants. KEYWORDS Aspergillus fumigatus, CLSI ECVs, ECVs, EUCAST ECVs, Etest, SYO, cyp51A mutants, posaconazole, triazole resistance, wild typ

Method-dependent epidemiological cutoff values (ECVs) for detection of triazole resistance in Candida and Aspergillus species for the SYO colorimetric broth and Etest agar diffusion methods

Although the Sensitrite Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method-agent-dependent): 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex (SC), 157 C. (Meyerozyma) guilliermondii, 676 C. krusei (Pichia kudriavzevii), 298 C (Clavispora) lusitaniae, 911 and 3,691 C. parapsilosissensu stricto (SS) and C. parapsilosisSC, respectively, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. niger, and 302 A. terreus complexes. SYO/Etest MICs for 282 confirmed non-WT isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2, CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast spp., and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for ECV definition were pooled and we proposed SYO ECVs for S. cerevisiae, 9 yeast and 3 Aspergillus species, and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 \ub5g/ml for C. albicans and the Etest itraconazole ECV of 2 \ub5g/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as overall, the SYO appears to perform better for susceptibility testing of yeast spp. and the Etest for Aspergillus spp. Further evaluations should be conducted with more Candida mutants

The Mechanism of Antifungal Action of Essential Oil from Dill (Anethum graveolens L.) on Aspergillus flavus

The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus