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

2019 ◽  
Vol 74 (8) ◽  
pp. 2247-2254 ◽  
Author(s):  
Joseph Meletiadis ◽  
Maria Siopi ◽  
Lamprini Kanioura ◽  
Karin Meinike Jørgensen ◽  
David S Perlin ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Screening Method ◽  
Colony Growth ◽  
Tween 20 ◽  
Optimal Test ◽  
And Control ◽  
Drug Free ◽  
Minimal Effective Concentration

Abstract 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.

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

2020 ◽  
Vol 75 (7) ◽  
pp. 1799-1806
Author(s):  
Joseph Meletiadis ◽  
Maria Siopi ◽  
Lamprini Kanioura ◽  
Karin Meinike Jørgensen ◽  
David S Perlin ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Colorimetric Assay ◽  
Antifungal Susceptibility ◽  
Colorimetric Method ◽  
Aspergillus Species ◽  
Subjective Time ◽  
Optimal Conditions ◽  
Drug Free ◽  
Minimal Effective Concentration

Abstract 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 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.

scholarly journals Evaluation of a Modified EUCAST Fragmented-Mycelium Inoculum Method forIn VitroSusceptibility Testing of Dermatophytes and the Activity of Novel Antifungal Agents

2015 ◽  
Vol 59 (6) ◽  
pp. 3675-3682 ◽  
Author(s):  
B. Risslegger ◽  
C. Lass-Flörl ◽  
G. Blum ◽  
M. Lackner
Keyword(s):  
Antifungal Agents ◽  
Susceptibility Testing ◽  
Preparation Method ◽  
Antifungal Susceptibility ◽  
Broth Microdilution ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Inoculum Preparation ◽  
Minimal Effective Concentration

ABSTRACTFor antifungal susceptibility testing of nonsporulating or poorly sporulating dermatophytes, a fragmented-mycelium inoculum preparation method was established and compared to broth microdilution testing according to CLSI and EUCAST guidelines. Moreover, thein vitroactivity of new antifungal agents against dermatophytes was evaluated. Agreement between the mycelial inoculum method and the CLSI broth microdilution method was high (93% to 100%). Echinocandins (minimal effective concentration [MEC], ≤0.5 mg/liter) and posaconazole (MIC, ≤3.00 mg/liter) showed good activity against all tested dermatophytes.

scholarly journals Impact of Morphological Sectors on Antifungal Susceptibility Testing and Virulence Studies

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Emina Jukic ◽  
Michael Blatzer ◽  
Ulrike Binder ◽  
Lisa Mayr ◽  
Cornelia Lass-Flörl ◽  
...  
Keyword(s):  
Aspergillus Terreus ◽  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Antifungal Susceptibility ◽  
Survival Rates ◽  
Drug Efficacy ◽  
Content Type ◽  
Morphological Heterogeneity ◽  
Survival Studies ◽  
Drug Free

ABSTRACT Morphological heterogeneity of Aspergillus terreus cultures was observed during continued cultivation of amphotericin B (AMB)-resistant isolates on drug-free medium. Outgrowth leads to the emergence of multiple sectors that might result from increased growth rates at drug-free conditions. We evaluated the differences in AMB susceptibility and virulence between sector subcultures (ATSec), AMB-resistant (ATR) strains, and AMB-susceptible (ATS) strains. By comparing A. terreus AMB-resistant (ATR) strains and A. terreus sector (ATSec) cultures we observed a highly significant reduction of AMB MICs in ATSec (ATR MIC, 2 to 32 μg/ml; ATSec MIC, 0.12 to 5 μg/ml). Furthermore, Galleria mellonella survival studies revealed an enhanced virulence of ATSec, which was comparable with that of AMB-sensitive Aspergillus terreus strains (median survival rates for ATS isolates, 72 h; for ATSec isolate ATSecG1, 84 h; for ATR isolates, 144 h). Our findings clearly demonstrate that spontaneous culture degeneration occurs in A. terreus and, most importantly, crucially impacts drug efficacy and virulence.

scholarly journals Ευαισθησίες των υφομυκήτων σε νεότερα αντιμυκητικά φάρμακα

2014 ◽  
Author(s):  
Ευαγγελία Πετρίκκου
Keyword(s):  
Filamentous Fungi ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Inoculum Size ◽  
Tween 20 ◽  
Second Phase ◽  
Registry Study ◽  
Broth Microdilution Method ◽  
Invasive Mycoses ◽  
Inoculum Preparation

The continuing increase of systemic and invasive mycoses due to filamentous fungi (moulds) such as species of Aspergillus, zygomycetes of the order Mucorales (Rhizopus, Mucor, θιπ.) and rare hyalohyphomycetes such as Fusarium and Scedosporium, and their difficult management because of their resistance in most antifungals, needed the development and standardization of a method to determine susceptibility to these drugs. The present dissertation aimed to the development of an antifungal susceptibility testing method which would be reliable and reproducible, in order to be used as a standard method. This method should be applicable to all filamentous fungi,The strains used for this study were isolates from cases with systemic mycoses with known antifungal susceptibility and outcome, as well as from a prospective registry study. The experiments of the first phase of the study aimed to verify the optimal conditions for the preparation of the inoculum of conidia to be tested for antifungal susceptibility with the broth microdilution method. The main conclusions of this phase were that the haemocytometer counting of conidia was a more reliable method of inoculum preparation than the spectrophotometric adjustment, and that Tween 20, a nonionic surfactant, should be used as a dispersing agent for the optimal suspension of hydrophobic conidia. The ideal inoculum size was 1.0 × 106 -5.0 × 106 CFU/ml. In the second phase, this new method was evaluated in three independent laboratories and its reliability and reproducibility were confirmed, with interlaboratory agreement of 89.2% and a narrow 95% CI (2.20 -2.65). In the third phase of the study the method was used for the susceptibility testing of strains from a prospective registry study of rare invasive mycoses and performed very well. The results of this study have been adopted by the EUCAST and constitute the basis of the guidelines for the determination of the MICs of antifungals against conidia forming moulds (EUCAST DEFINITIVE DOCUMENT E.DEF 9.1).

scholarly journals Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes

2020 ◽  
Vol 75 (7) ◽  
pp. 1807-1819 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Karin Meinike Jørgensen ◽  
Jesus Guinea ◽  
Katrien Lagrou ◽  
Erja Chryssanthou ◽  
...  
Keyword(s):  
Target Gene ◽  
Susceptibility Testing ◽  
Trichophyton Rubrum ◽  
Antifungal Susceptibility ◽  
Error Rates ◽  
High Reproducibility ◽  
Upper Limits ◽  
Gene Mutant ◽  
Testing Error

Abstract 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.

scholarly journals Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin

2016 ◽  
Vol 60 (8) ◽  
pp. 5088-5091 ◽  
Author(s):  
M.-E. Bougnoux ◽  
E. Dannaoui ◽  
I. Accoceberry ◽  
A. Angoulvant ◽  
E. Bailly ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Antifungal Susceptibility Testing ◽  
Medical Mycology ◽  
Single Center ◽  
Content Type ◽  
Valuable Alternative

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.

scholarly journals Determination of Antimicrobial Properties of Myristica fragans (Nutmeg) on Microorganisms Isolated from Veritas University Hostels Bathroom and their Surfaces

2019 ◽  
pp. 1-7
Author(s):  
T. O. Ozoude ◽  
C. C. Mbah ◽  
E. U. Amacheree
Keyword(s):  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antimicrobial Properties ◽  
Diffusion Method ◽  
Fungal Contamination ◽  
Male And Female ◽  
Antibacterial Susceptibility ◽  
Agar Diffusion Method ◽  
Paper Method

The bathroom which is a place for people to clean up themselves is also prone to contamination. The aim of this study was to investigate the antimicrobial properties of nutmeg against isolates from the bathroom using different solvents such as acetone, ethanol and water for the extraction of the nutmeg. In this study, samples from different areas in Veritas University male and female hostel bathrooms were screened for bacterial and fungal contamination and results from the study showed that bacterial genera such as Escherichia coli, Salmonella, Staphylococcus aureus, Bacillus, Pseudomonas, and fungus Aspergillus were found in this public bathroom surfaces. The Cup-plate Agar Diffusion method was used to determine the antibacterial potency of the nutmeg while the Filter paper method was used to determine its antifungal properties. The results proved that nutmeg has great anti-microbial properties thus was capable of inhibiting the growth of these isolates. Results from this study also revealed that when acetone was used as the solvent at 75% concentration, it was the most effective for the antibacterial susceptibility test while ethanoic solvent at the same concentration was the most effective for the antifungal susceptibility testing.

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