Single dose pharmacodynamics of amphotericin B against Aspergillus species in an in vitro pharmacokinetic/pharmacodynamic model

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MIC with a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of A. fumigatus, A. terreus and A flavus with the same CLSI modal MICs of 1 mg/l were exposed to amphotericin B concentrations following the plasma concentration-time profile after single bolus administration with Cmax 0.6, 1.2, 2.4 and 4.8 mg/L. Fungal growth was monitored up to 72h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B Cmax ≥2.4 mg/L. At lower Cmaxs 0.6 and 1.2 mg/L, a significant growth delay of 34h and 52h was observed, respectively (pA flavus>A. terreus in the in vitro PK/PD model possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerting against these species

In Vitro–In Vivo Correlation of Posaconazole–Amphotericin B Combination against Candida albicans:In Vitro Interacting Concentrations Are Associated with In Vivo Free Drug Levels

The in vitro/in vivo correlation of antifungal combination testing is necessary in order to assess the efficacy of combination regimens. We, therefore, attempted to correlate in vitro chequerboard testing of posaconazole (POS) and amphotericin B (AMB) with the in vivo outcome of combination therapy against experimental candidiasis in a neutropenic murine model. The AMB + POS combination was tested against a Candida albicans isolate. In vitro, a broth microdilution 8 × 12 chequerboard method with serial two-fold drug dilutions was used. In vivo, CD1 female neutropenic mice with experimental disseminated candidiasis were treated with i.p. AMB and p.o. POS alone and in combination at three effective doses (ED20, ED50 and ED80 corresponding to 20%, 50% and 80% of maximal effect, respectively). CFU/kidneys after 2 days were determined. The pharmacodynamic interactions were assessed based on Bliss independence interaction analysis. In vitro, a Bliss antagonism of −23% (−23% to −22%) was observed at 0.03–0.125 mg/L of AMB with 0.004–0.015 mg/L of POS, while a Bliss synergy of 27% (14%–58%) was observed at 0.008–0.03 mg/L of AMB with 0.000015–0.001 mg/L of POS. In vivo, Bliss synergy (13 ± 4%) was found when an AMB ED20 of 1 mg/kg was combined with all POS ED 0.2–0.9 mg/kg, while Bliss antagonism (35–83%) was found for the combinations of AMB ED50 2 mg/kg and ED80 3.2 mg/kg with POS ED80 of 0.9 mg/kg. Free drug serum levels of POS and AMB in in vivo synergistic and antagonistic combinations were correlated with the in vitro synergistic and antagonistic concentrations, respectively. Both synergistic and antagonistic interactions were found for the AMB + POS combination. POS compromised the efficacy of high effective AMB doses and enhanced low ineffective AMB doses. In vitro concentration-dependent interactions were correlated with in vivo dose-dependent interactions of the AMB + POS combination. In vivo interactions occurred at free drug serum levels close to in vitro interacting concentrations.</p

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

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 factors from causing misclassifications and discrepancies in interpretations. Instead, an Area of Technical Uncertainty (ATU) has been introduced for MICs that cannot be categorized without additional information as a warning to the laboratory that decision on how to act has to be made. To implement these changes, the EUCAST-AFST (Subcommittee on Antifungal Susceptibility Testing) reviewed all, and revised some, clinical antifungal breakpoints. Objectives The aim was to present an overview of the current antifungal breakpoints and supporting evidence behind the changes. Sources This document is based on the ten recently updated EUCAST rationale documents, clinical breakpoint and breakpoint ECOFF documents. Content The following breakpoints (in mg/L) have been revised or established for Candida species: micafungin against C. albicans (ATU = 0.03); amphotericin B (S ≤/> R = 1/1), fluconazole (S ≤/> R = 2/4), itraconazole (S ≤/> R = 0.06/0.06), posaconazole (S ≤/> R = 0.06/0.06) and voriconazole (S ≤/> R = 0.06/0.25) against C. dubliniensis; fluconazole against C. glabrata (S ≤/> R = 0.001/16); and anidulafungin (S ≤/> R = 4/4) and micafungin (S ≤/> R = 2/2) against C. parapsilosis. For Aspergillus, new or revised breakpoints include itraconazole (ATU = 2) and isavuconazole against A. flavus (S ≤/> R = 1/2, ATU = 2); amphotericin B (S ≤/> R = 1/1), isavuconazole (S ≤ /> R = 1/2, ATU = 2), itraconazole (S ≤/> R = 1/1, ATU = 2), posaconazole (ATU = 0.25) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. fumigatus; itraconazole (S ≤/> R = 1/1, ATU = 2) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. nidulans; amphotericin B against A. niger (S ≤/> R = 1/1); and itraconazole (S ≤/> R = 1/1, ATU = 2) and posaconazole (ATU = 0.25) against A. terreus. Implications EUCAST-AFST has released ten new documents summarizing existing and new breakpoints and MIC ranges for control strains. A failure to adopt the breakpoint changes may lead to misclassifications and suboptimal or inappropriate therapy of patients with fungal infections

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

Multicenter Collaborative Study of the Interaction of Antifungal Combinations against BrowZine Journal Cover Candida Spp. by Loewe Additivity and Bliss Independence-Based Response Surface Analysis

Combination antifungal therapy is widely used but not well understood. We analyzed the spectrophotometric readings from a multicenter study conducted by the New York State Department of Health to further characterize the in vitro interactions of the major classes of antifungal agents against Candida spp. Loewe additivity-based fractional inhibitory concentration index (FICi) analysis and Bliss independence-based response surface (BIRS) analysis were used to analyze two-drug inter- and intraclass combinations of triazoles (AZO) (voriconazole, posaconazole), echinocandins (ECH) (caspofungin, micafungin, anidulafungin), and a polyene (amphotericin B) against Candida albicans, C. parapsilosis, and C. glabrata. Although mean FIC indices did not differ statistically significantly from the additivity range of 0.5−4, indicating no significant pharmacodynamic interactions for all of the strain−combinations tested, BIRS analysis showed that significant pharmacodynamic interactions with the sum of percentages of interactions determined with this analysis were strongly associated with the FIC indices (Χ2 646, p \u3c 0.0001). Using a narrower additivity range of 1−2 FIC index analysis, statistically significant pharmacodynamic interactions were also found with FICi and were in agreement with those found with BIRS analysis. All ECH+AB combinations were found to be synergistic against all Candida strains except C. glabrata. For the AZO+AB combinations, synergy was found mostly with the POS+AB combination. All AZO+ECH combinations except POS+CAS were synergistic against all Candida strains although with variable magnitude; significant antagonism was found for the POS+MIF combination against C. albicans. The AZO+AZO combination was additive for all strains except for a C. parapsilosis strain for which antagonism was also observed. The ECH+ECH combinations were synergistic for all Candida strains except C. glabrata for which they were additive; no antagonism was found

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

Evaluation multicentrique d'une méthode EUCAST pour tester la sensibilité antifongique des dermatophytes produisant des spores

Background: Terbinafine resistance is increasingly reported in Trichophyton rubrum and Trichophyton interdigitale rendering susceptibility testing important particularly in non-responding cases. We performed a multicentre evaluation of a recently proposed modified EUCAST method implementing medium supplemented with chloramphenicol and cycloheximide (CC) to avoid contamination. Materials/methods: A blinded panel of wild-type and squalene epoxidase (SQLE) target gene mutant T. rubrum and T. interdigitale strains were distributed to 10 European laboratories. Susceptibility to terbinafine, itraconazole, voriconazole and amorolfine) were performed according to the E.Def 9.3.1 method with and without addition of chloramphenicol and cycloheximide (final concentrations 50 mg/L and 300 mg/L, respectively). Plates were incubated at 25 °C (one laboratory used 30 °C) for 5-7 days until sufficient growth. MICs were determined visually (ignoring trailing growth for itraconazole) and spectrophotometrically with 90% and 50% endpoints yielding a total of 7,829 MICs. A. flavus ATCC 204304 and A. flavus CNM-CM1813 were included as controls. Results: 100%/96% (voriconazole) and 84%/84% (itraconazole) MIC determinations fell within the QC ranges for the two QC strains, respectively, and 96%/92% terbinafine MICs fell in a 0.25-1 mg/L 3 two-fold-dilution range suggesting a high interlaboratory reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 (2.6%) to 5 (6.6%) for T. rubrum and between 0 and 2 (2.0%) for T. interdigitale (lowest for the CC-method (2.6%-4.4%/ 0-1% for T. rubrum/T. interdigitale). The difference between the modes for the wt and mutant population were ≥7 two-fold-dilutions in all cases (Table). If excluding a I121M/V237I T. rubrum mutant, and two mixed T. interdigitale strains, the number of VMEs were CC visual: T. rubrum: 1/77 (1.3%), CC spec-90%: 3/68 (4.4%) and CC spec-50%: 1/76 (1.3%), and none for T. interdigitale. The activity of voriconazole, itraconazole and amorolfine were quite uniform against T. rubrum and T. interdigitale, but unacceptably wide MIC ranges were found for the visual and spec-90% inhibition methods for itraconazole (data not shown). Conclusions: Although none of the laboratories perform dermatophyte testing at a regular basis an acceptable interlaboratory agreement and good separation between SQLE wt and mutants were found, suggesting a robust performance of the proposed method

MixInYeast: A Multicenter Study on Mixed Yeast Infections

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions