scholarly journals Development of a Broth Microdilution Method for Exebacase Susceptibility Testing

2021 ◽  
Author(s):  
Jun T. Oh ◽  
Jane E. Ambler ◽  
Cara Cassino ◽  
Raymond Schuch
Keyword(s):  
Test Performance ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Horse Serum ◽  
Antibacterial Drug ◽  
Broth Microdilution ◽  
Strain Collection ◽  
Antistaphylococcal Activity ◽  
Broth Microdilution Method ◽  
The Impact

Exebacase (CF-301) belongs to a new class of protein-based antibacterial agents, known as lysins (peptidoglycan hydrolases). Exebacase, a novel lysin with antistaphylococcal activity, is in Phase 3 of clinical development. To advance into the clinic, it was necessary to develop an accurate and reproducible method for exebacase MIC determination. The Clinical Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method using cation-adjusted Mueller-Hinton broth (CAMHB) produced trailing MIC end points and exebacase activity was diminished when frozen BMD panels were used. A modified BMD method was developed using CAMHB supplemented with 25% horse serum and 0.5 mM DL-dithiothreitol (CAMHB-HSD). Preliminary quality control (QC) ranges for Staphylococcus aureus ATCC 29213 of 0.25-1 μg/ml and Enterococcus faecalis ATCC 29212 of 16-64 μg/ml were determined based on the results of a CLSI M23-defined MIC QC tier 1 study. These preliminary QC ranges validated the MIC data generated from a systematic study testing a discrete S. aureus strain collection using CAMHB-HSD to investigate the impact of parameters known to influence susceptibility test results and to evaluate the exebacase MIC distribution against clinical S. aureus isolates. Presentation of these data led to the CLSI Subcommittee on Antimicrobial Susceptibility Testing (AST) approval of the use of CAMHB-HSD to determine exebacase susceptibility and commencement of a multi-laboratory (tier 2) QC study. Use of a standard BMD method and concomitant QC testing provides confidence in the assessment of test performance to generate accurate and reproducible susceptibility data during antibacterial drug development.

scholarly journals Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria

2004 ◽  
Vol 70 (4) ◽  
pp. 2398-2403 ◽  
Author(s):  
Mokhlasur Rahman ◽  
Inger Kühn ◽  
Motiur Rahman ◽  
Barbro Olsson-Liljequist ◽  
Roland Möllby
Keyword(s):  
Bacterial Growth ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
National Committee ◽  
Broth Microdilution ◽  
Gram Negative ◽  
Microdilution Method ◽  
Epidemiological Surveys ◽  
Fermentative Bacteria ◽  
Broth Microdilution Method

ABSTRACT We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log2 dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.

scholarly journals Evaluation of the Dade MicroScan MICroSTREP Antimicrobial Susceptibility Testing Panel with Selected Streptococcus pneumoniae Challenge Strains and Recent Clinical Isolates

1998 ◽  
Vol 36 (3) ◽  
pp. 788-791 ◽  
Author(s):  
J. H. Jorgensen ◽  
M. L. McElmeel ◽  
S. A. Crawford
Keyword(s):  
Streptococcus Pneumoniae ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Reference Panel ◽  
Clinical Isolates ◽  
National Committee ◽  
Broth Microdilution ◽  
Drug Concentrations ◽  
Broth Microdilution Method

The MicroScan MICroSTREP panel is a recently marketed frozen broth microdilution panel for susceptibility testing of various streptococci, including Streptococcus pneumoniae. The panel contains 10 antimicrobial agents in cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood, similar in concept to the National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution method for testing streptococci. A group of 210 isolates of S. pneumoniae were selected to include isolates with previously documented resistance to agents incorporated in the MICroSTREP panel, as well as recent invasive clinical isolates. All isolates were tested simultaneously with the MICroSTREP panel and an NCCLS reference panel whose drug concentrations were prepared to coincide with those of the MICroSTREP panel. Of the 210 isolates, 5 failed to grow in the MICroSTREP panel; 3 of those also did not grow in the reference panel. Essential agreement of MICs determined by the two methods (test MIC ± one dilution of the reference MIC) was 99.6% overall and ranged from 98.0% with chloramphenicol to 100% with penicillin, ceftriaxone, erythromycin, tetracycline, and vancomycin. There were no very major or major interpretive category errors resulting from the MICroSTREP panel tests. Minor interpretive category errors ranged from 12.2% with cefotaxime and 9.8% with ceftriaxone (due mainly to clustering of MICs for the selected strains near the breakpoints) to 0% with chloramphenicol and vancomycin. These results indicate that the MicroScan MICroSTREP frozen panels provide susceptibility results with pneumococci that are essentially equivalent to results derived by the NCCLS reference broth microdilution procedure.

scholarly journals Determination of MIC Quality Control Parameters for Exebacase, a Novel Lysin with Anti-staphylococcal Activity

2021 ◽  
Author(s):  
Maria M. Traczewski ◽  
Jane E. Ambler ◽  
Raymond Schuch
Keyword(s):  
Staphylococcus Aureus ◽  
Quality Control ◽  
Enterococcus Faecalis ◽  
Test Performance ◽  
Susceptibility Testing ◽  
Clinical Development ◽  
Broth Microdilution ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Accuracy Of Results

Exebacase (CF-301), a novel, anti-staphylococcal lysin (cell-wall hydrolase) is the first agent of this class to enter late-stage clinical development (Phase 3, NCT04160468) for the treatment of Staphylococcus aureus bacteremia including right-sided endocarditis. A multi-laboratory Clinical and Laboratory Standards Institute (CLSI) M23-defined tier 2 quality control (QC) study was conducted to establish exebacase QC ranges for a new reference broth microdilution method. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were selected as reference QC strains. Broth microdilution MIC QC ranges for exebacase spanned 4 log2 dilutions and contained 99.2% of the MIC results generated for the two reference strains. The QC ranges for exebacase were defined as 0.25–2 μg/ml and 8–64 μg/ml against S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, respectively and were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. These QC ranges established for use with the reference broth microdilution method developed for exebacase susceptibility testing will ensure the test performance and accuracy of results generated during clinical development.

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.

Susceptibility of Filamentous Fungi to Voriconazole in Malaysia Tested by Sensititre YeastOne and CLSI Microdilution Methods

2021 ◽  
Author(s):  
Xue Ting Tan ◽  
Stephanie Jane Ginsapu ◽  
Fairuz binti Amran ◽  
Salina binti Mohamed Sukur ◽  
Surianti binti Shukor
Keyword(s):  
Filamentous Fungi ◽  
Susceptibility Testing ◽  
Rhizopus Oryzae ◽  
Antifungal Susceptibility ◽  
Sporothrix Schenckii ◽  
Rank Test ◽  
Broth Microdilution ◽  
Syncephalastrum Racemosum ◽  
Microdilution Method ◽  
Broth Microdilution Method

Abstract Background: Voriconazole is a trizaole antifungal to treat fungal infection. In this study, the susceptibility pattern of voriconazole against filamentous fungi was studied using Sensititre® YeastOne and Clinical & Laboratory Standards Institute (CLSI) M38 broth microdilution method. Methods: The suspected cultures of Aspergillus niger, A. flavus, A. fumigatus, A. versicolor, A. sydowii, A. calidoutus, A. creber, A. ochraceopetaliformis, A. tamarii, Fusarium solani, F. longipes, F. falciferus, F. keratoplasticum, Rhizopus oryzae, R. delemar, R. arrhizus, Mucor sp., Poitrasia circinans, Syncephalastrum racemosum and Sporothrix schenckii were received from hospitals. Their identification had been confirmed in our lab and susceptibility tests were performed using Sensititre® YeastOne and CLSI M38 broth microdilution method. The significant differences between two methods were calculated using Wilcoxon Sign Rank test.Results: Mean of the minimum inhibitory concentrations (MIC) for Aspergillus spp. and Fusarium were within 0.25 μg/mL-2.00 μg/mL by two methods except A. calidoutus, F. solani and F. keratoplasticum. Moreover, mean of MIC for S. schenkii were around 3.00 μg/mL by two methods. In contrast, mean of MIC for Rhizopus spp., Mucor sp., P. circinans and S. racemosum were ≥6.00 μg/mL by two methods. Generally, the MIC obtained by Sensititre YeastOne was one two-fold increase or decrease compared with the results obtained by CLSI method. The overall agreement between Sensititre YeastOne and CLSI methods to test susceptibility testing of voricaonazole was more than 70% except A. sydowii. The significant differences between two methods were significant when tested on A. niger, A. flavus, A. fumigatus, A. versicolor, A. sydowii, F. solani and S. schenkii. Conclusions: In conclusion, Sensititre YeastOne method appears to be an alternative procedure for antifungal susceptibility testing for some Malaysian moulds.

scholarly journals Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

2000 ◽  
Vol 44 (10) ◽  
pp. 2752-2758 ◽  
Author(s):  
Rama Ramani ◽  
Vishnu Chaturvedi
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Broth Microdilution ◽  
Nitrogen Base ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

scholarly journals In Vitro Activities of the New Antifungal Triazole SCH 56592 against Common and Emerging Yeast Pathogens

2000 ◽  
Vol 44 (1) ◽  
pp. 226-229 ◽  
Author(s):  
Francesco Barchiesi ◽  
Daniela Arzeni ◽  
Annette W. Fothergill ◽  
Luigi Falconi Di Francesco ◽  
Francesca Caselli ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
Clinical Development ◽  
National Committee ◽  
In Vitro Activity ◽  
Broth Microdilution ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Vitro Data ◽  
In Vitro Data

ABSTRACT A broth microdilution method performed in accordance with the National Committee for Clinical Laboratory Standards guidelines was used to compare the in vitro activity of the new antifungal triazole SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and ketoconazole (KETO) against 257 clinical yeast isolates. They included 220 isolates belonging to 12 different species of Candida, 15 isolates each of Cryptococcus neoformans andSaccharomyces cerevisiae, and seven isolates ofRhodotorula rubra. The MICs of SCH at which 50% (MIC50) and 90% (MIC90) of the isolates were inhibited were 0.06 and 2.0 μg/ml, respectively. In general, SCH was considerably more active than FLC (MIC50 and MIC90 of 1.0 and 64 μg/ml, respectively) and slightly more active than either ITC (MIC50 and MIC90 of 0.25 and 2.0 μg/ml, respectively) and KETO (MIC50 and MIC90 of 0.125 and 4.0 μg/ml, respectively). Our in vitro data suggest that SCH has significant potential for clinical development.

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.

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