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 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 Colorimetric Assay for Antifungal Susceptibility Testing of Aspergillus Species

2001 ◽  
Vol 39 (9) ◽  
pp. 3402-3408 ◽  
Author(s):  
J. Meletiadis ◽  
J. W. Mouton ◽  
J. F. G. M. Meis ◽  
B. A. Bouman ◽  
J. P. Donnelly ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Colorimetric Assay ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Aspergillus Species

scholarly journals Comparative Evaluation of Micronaut-AM and CLSI broth micro-dilution method for antifungal susceptibility testing of Aspergillus species against four commonly used antifungals

2019 ◽  
Author(s):  
Ali Nuh ◽  
Newara Ramadan ◽  
Silke Schelenz ◽  
Darius Armstrong-James
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Colorimetric Method ◽  
Laboratory Method ◽  
Aspergillus Species ◽  
Dilution Method ◽  
Reference Laboratory ◽  
Suitable Alternative ◽  
Broth Microdilution Method

AbstractThe aim of this study was to evaluate a colorimetric method, Micronaut-AM, for determining susceptibility testing of anidulafungin, amphotericin, voriconazole and itraconazole by comparing the Minimum Inhibitory (Effective) Concentrations (MICs/MECs) obtained by this method to those generated by the reference Clinical Laboratory Standard Investigation (CLSI) method. 78 clinical isolates of Aspergillus species, nine of them azole-resistant, were tested against above antifungals. A fumigatus ATCC 204305 was used as a reference strain and test was performed in accordance with slightly modified yeast susceptibility testing instruction of the manufacture; conidia suspension inoculum and alamarBlue concentration were optimised. These same isolates were referred to Bristol Mycology reference laboratory and tested by CLSI method. The MICs and MECs generated by the two methods were compared using concordance analysis.Micronaut-AM (MN) showed significant concordance (P< 0.0001) with CLSI method and overall agreement was high (≥ 90%). In addition, Micronaut-AM produced echinocandin MECs results within 18-24h incubation time and reliably detected azole resistant isolates. Essential agreement (within 2 log2 dilution of median) between MN and CLSI reference laboratory method was 99 % for anidulafungin, 100 % for amphotericin; 90% for voriconazole and 87 % for itraconazole. Categorical agreement for anidulafungin, amphotericin B, voriconazole and itraconazole were 100%, 96%, 97% and 99% respectively.Micronaut-AM showed very good agreement with the reference broth micro-dilution method results for all antifungal agents tested and was able to detect azole resistance. This colorimetric method is very promising and appears to be a suitable alternative susceptibility testing method to labour intensive broth microdilution method for Aspergillus species.

scholarly journals Differential Fungicidal Activities of Amphotericin B and Voriconazole against Aspergillus Species Determined by Microbroth Methodology

2007 ◽  
Vol 51 (9) ◽  
pp. 3329-3337 ◽  
Author(s):  
Joseph Meletiadis ◽  
Charalampos Antachopoulos ◽  
Theodouli Stergiopoulou ◽  
Spyros Pournaras ◽  
Emmanuel Roilides ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Fungicidal Activity ◽  
Antifungal Agents ◽  
Susceptibility Testing ◽  
Colorimetric Assay ◽  
Antifungal Susceptibility ◽  
Colorimetric Method ◽  
Reference Method ◽  
Fungicidal Action ◽  
Testing Reference

ABSTRACT Antifungal agents may differ in their fungicidal activities against Aspergillus spp. In order to compare the fungicidal activities of voriconazole and amphotericin B against 40 isolates of Aspergillus fumigatus, A. flavus, and A. terreus, we developed a new microbroth colorimetric method for assessing fungicidal activities and determining minimal fungicidal concentrations (MFCs). This methodology follows the antifungal susceptibility testing reference method M-38A for MIC determination. After drug removal and addition of fresh medium, growth of viable conidia adhering to the bottoms of the microtitration wells was assessed by a colorimetric assay of metabolic activity after 24 h of incubation. The new method was faster (six times), reproducible (92 to 97%), and in agreement with culture-based MFCs (91 to 100%). Differential fungicidal activities of voriconazole and amphotericin B were found among the three Aspergillus species, with A. fumigatus and A. flavus having the lowest (1 and 2 mg/liter, respectively) and A. terreus the highest (>16 mg/liter) median amphotericin B MFCs; A. flavus had a lower median voriconazole MFC (4 mg/liter) than the other species (>8 mg/liter; P < 0.05). Amphotericin B was fungicidal (MFC/MIC ≤ 4) against all A. fumigatus and A. flavus isolates but no A. terreus isolates, whereas voriconazole was fungicidal against 82% of A. flavus isolates and fungistatic (MFC/MIC > 4) against 94% of A. fumigatus and 84% of A. terreus isolates. The new methodology revealed a concentration-dependent sigmoid pattern of fungicidal effects, indicating that fungicidal activity is not an all-or-nothing phenomenon and that some degree of fungicidal action can be found even for agents considered fungistatic based on the MFC/MIC ratio.

scholarly journals Comparative Evaluation of MIRONAUT-AM and CLSI broth microdilution method for antifungal susceptibility testing of Aspergillus species against four commonly used antifungals

2020 ◽  
Vol 58 (8) ◽  
pp. 1085-1090
Author(s):  
Ali Nuh ◽  
Newara Ramadan ◽  
Silke Schelenz ◽  
Darius Armstrong-James
Keyword(s):  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Colorimetric Method ◽  
Reference Method ◽  
Aspergillus Species ◽  
Reference Laboratory ◽  
Broth Microdilution ◽  
Suitable Alternative ◽  
Microdilution Method ◽  
Broth Microdilution Method

Abstract The aim of this study was to evaluate a colorimetric method, MIRONAUT-AM, for determining susceptibility testing of anidulafungin, amphotericin, voriconazole, and itraconazole by comparing the minimum inhibitory (effective) concentrations (MICs/MECs) obtained by this method to those generated by the reference Clinical Laboratory Standard Institute (CLSI) broth microdilution method. In sum, 78 clinical isolates of Aspergillus species, nine of them non-wild type (non-WT) with itraconazole MIC ranging from 2 mg/l to &gt;16 mg/l, were tested against above antifungals. A. fumigatus ATCC 204305 was used as a reference strain, and test was performed in accordance with slightly modified yeast susceptibility testing instruction of the manufacture; conidia suspension inoculum and alamarBlue concentration were optimized. These same isolates were referred to Bristol Mycology reference laboratory and tested by CLSI method. The MICs and MECs generated by the two methods were compared using concordance analysis. MIRONAUT-AM showed significant concordance (P &lt; .0001) with CLSI method, and overall agreement was high (≥90%). In addition, MIRONAUT-AM produced echinocandin MECs results within 18–24 hours incubation time and correctly detected all non-WT isolates except one isolate. This colorimetric method is very promising and appears to be a suitable alternative susceptibility testing method to labor intensive broth microdilution reference method for Aspergillus species.

The Need for Using An Enzymatic Colorimetric Assay in Creatinine Determination of Peritoneal Dialysis Solutions

1990 ◽  
Vol 10 (1) ◽  
pp. 89-92 ◽  
Author(s):  
Liliane Larpent ◽  
Christian Verger
Keyword(s):  
Peritoneal Dialysis ◽  
Reliable Method ◽  
Colorimetric Assay ◽  
Colorimetric Method ◽  
Peritoneal Membrane ◽  
Creatinine Kinetics ◽  
Dialysis Solutions ◽  
Peritoneal Dialysis Solutions ◽  
Enzymatic Test

The fate of the peritoneal membrane on continuous ambulatory peritoneal dialysis (CAPD) is usually evaluated through the modification of its permeability to various solutes as glucose, creatinine, and urea. Therefore, the accuracy of the methods used for measurements of creatinine is of great importance. A particular problem does exist for creatinine determination as it may be influenced by the presence of glucose. We studied a new enzymatic colorimetric method for creatinine determination in peritoneal dialysis solutions which contain high dextrose concentrations. Creatinine was measured in plasma, urine, and dialysate from 18 patients on CAPD and in pure dextrose solutions, with an enzymatic test (Boehringer Mannheim) and with Jaffe's reaction on two different analyzers: Astra (Beckman) and Eris (Merck). Creatinine results were similar with both assays (Jaffe's reaction and enzymatic test) when measured in blood and urine. However the Jaffe's reaction gave higher creatinine results than the enzymatic test (p < 0.001), when assays were performed in peritoneal dialysis solutions and in pure glucose solutions. In addition, it appeared that other components of dialysis solutions, mainly calcium chloride, influenced unpredictably the results of creatinine with the Jaffe's reaction. We conclude that specific enzymatic test is a more accurate and reliable method to evaluate creatinine kinetics through the peritoneal membrane when determined in CAPD solutions.

Use of spectrophotometric reading for in vitro antifungal susceptibility testing ofAspergillusspp.

1999 ◽  
Vol 45 (10) ◽  
pp. 871-874 ◽  
Author(s):  
Eric Dannaoui ◽  
Florence Persat ◽  
Marie-France Monier ◽  
Elisabeth Borel ◽  
Marie-Antoinette Piens ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Antifungal Susceptibility Testing ◽  
Aspergillus Species ◽  
National Committee ◽  
Broth Microdilution Method

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.Key words: antifungal, susceptibility testing, Aspergillus, spectrophotometric reading.

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.

Quantitative Determination of Thiourea in Citrus Fruits

1977 ◽  
Vol 60 (3) ◽  
pp. 699-701
Author(s):  
Bernadette Mandrou ◽  
Suzanne Brun ◽  
Amara Kingkate
Keyword(s):  
Colorimetric Assay ◽  
Colorimetric Method ◽  
Extraction Technique ◽  
Citrus Fruits ◽  
Alumina Column ◽  
Orange Peels ◽  
Ultraviolet Reflectance ◽  
Spectrometric Measurements ◽  
Colorimetric Reaction

Abstract A quantitative method for thiourea determination in orange peels is proposed, with direct reflectance spectrometric measurements on chromatoplates. Thiourea is separated on a silica gel plate with methanol-chloroform (10+90), and the spots are characterized with 2,6-dichloroquinone chloroimide; this colorimetric reaction is unstable, so thiourea is quantitated on the chromatogram by direct ultraviolet reflectance spectrometric measurements at 240 nm. Comparison between this method and the AOAC colorimetric method shows interferences and higher results in the colorimetric assay. By slightly modifying the extraction technique, such as purifying the extract on an alumina column, it is possible to decrease these interferences and to avoid erroneous results with the AOAC colorimetric method. For both methods, colorimetry in solution and reflectometry on chromatoplates, applied after the proposed extraction technique, the sensitivity is about 1 ppm and the reliabilities are similar.

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