scholarly journals Validation of a Gradient Diffusion Method (Etest®) for Antimicrobial Susceptibility Testing of Aerococcus urinae to Fluoroquinolones

2021 ◽  
Author(s):  
France Emilie Roy ◽  
Tammy Berteau ◽  
Julie Bestman-Smith ◽  
Simon Grandjean Lapierre ◽  
Simon Frédéric Dufresne ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Error Rates ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
Sheep Blood ◽  
Gradient Diffusion ◽  
Reference Methods ◽  
Aerococcus Urinae

Aerococcus urinae is a urinary pathogen with well-described resistance to fluoroquinolones. This study aimed to validate the gradient diffusion (GD) method (Etest®) on cation-adjusted Mueller-Hinton agar with 5% sheep blood for Aerococcus urinae antimicrobial susceptibility testing (AST) to ciprofloxacin and levofloxacin and compare it to the broth microdilution (BMD) method from CLSI M45-A3. Agar dilution (AD), as recommended by EUCAST, was used as an alternate reference method to arbitrate discrepancies or address technical issues. Aerococcus urinae isolates from urinary specimens were prospectively collected between June 2016 and December 2017 from six Quebec hospitals (Canada) and identifications were confirmed using Vitek MS® with IVD 3.0 database. Of the 207 isolates tested using BMD, 37 (17.9%) showed trailing and 19 (9.2%) showed insufficient growth and were tested using AD. Also, 38 isolates (18.4%) for ciprofloxacin and 13 isolates (6.3%) for levofloxacin showed a lack of essential or categorical agreement between Etest® and BMD and were also tested by AD. Using a combined reference method (BMD or AD), susceptibility rate of Aerococcus urinae was 82.6% and 81.6% for ciprofloxacin and levofloxacin, respectively. Categorial agreement between GD and the combined reference methods was 95.2% for ciprofloxacin and 97.1% for levofloxacin, with no very major error identified. Major and minor error rates were 0.6% and 4.3% for ciprofloxacin, and 1.2% and 1.9% for levofloxacin, respectively. Overall, AST using Etest® on sheep blood agar showed a good agreement with reference methods and can be considered by clinical laboratories wishing to perform AST on Aerococcus urinae isolates.

scholarly journals The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles

2020 ◽  
Vol 75 (4) ◽  
pp. 968-978 ◽  
Author(s):  
Emma Jonasson ◽  
Erika Matuschek ◽  
Gunnar Kahlmeter
Keyword(s):  
Blood Culture ◽  
Positive Blood Culture ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Bloodstream Infections ◽  
Error Rates ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
Disc Diffusion

Abstract Objectives With increasing antimicrobial resistance, rapid antimicrobial susceptibility testing (RAST) becomes important, especially in patients with bloodstream infections. EUCAST decided to develop a standardized rapid method, based on EUCAST disc diffusion, to offer susceptibility reports within 4–8 h of a positive blood culture (BC). Methods BC bottles were spiked with clinical isolates (n = 332) of the seven most relevant sepsis pathogens with a variety of resistance mechanisms. RAST was performed directly from the bottle and zones read after 4, 6 and 8 h. Several variables were investigated, including the effect of using different BC bottles and of a 0–18 h delay between a positive signal and the performance of RAST. Results For five species, most inhibition zones could be read after 4 h. The proportion of results that could be interpreted increased from 75% at 4 h to 84% after 8 h. Categorical agreement against the reference method was good, with error rates of false susceptibility of 0.2%, 0.2% and 0.2% at 4, 6 and 8 h and false resistance of 1.2%, 0.2% and 0.1% at 4, 6 and 8 h, respectively. Conclusions With the EUCAST RAST method, reliable AST results can be delivered within 4–8 h of positivity of BC bottles for seven important bloodstream infection pathogens. To reduce the occurrence of errors and to absorb the variability caused by using a non-standardized inoculum, material from different manufacturers and workflow-related delays, we have introduced an area in which interpretation is not permitted, the Area of Technical Uncertainty.

scholarly journals 2149. Performance of a Gradient Diffusion Method (Etest®) on Mueller–Hinton Agar with Sheep Blood for Aerococcus urinae Antimicrobial Susceptibility Testing

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S728-S729
Author(s):  
Tammy Berteau ◽  
France Emilie Roy ◽  
Julie Bestman-Smith ◽  
Simon Grandjean-Lapierre ◽  
Jean Longtin ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Diffusion Method ◽  
Quebec City ◽  
Sheep Blood ◽  
Maldi Tof ◽  
Gradient Diffusion ◽  
Aerococcus Urinae ◽  
Agar Dilution

Abstract Background Aerococcus urinae is frequently identified by MALDI-TOF in urinary specimens. It is generally susceptible to β-lactams, but its susceptibility pattern to fluoroquinolones (FQ) remains unpredictable. The goal of this study was to evaluate the performance of the gradient diffusion method (Etest®) to determine FQ susceptibility compared with broth microdilution (BMD) and agar dilution (AD). Methods Prospectively collected isolates of A. urinae from urinary tract specimens originating from 5 hospitals in Quebec city and Montreal were identified by MALDI-TOF (Vitek-MS). All isolates were tested using BMD according to CLSI guidelines, and also with Etest® strips on MH agar w/ 5% sheep blood. Isolates showing trailing, insufficient growth or discordance between both methods were further tested by agar dilution (MH agar w/5% horse blood + β-NAD) according to EUCAST guidelines. Breakpoints were interpreted using CLSI M45-A3. Combined results of BMD and AD were then compared with Etest. Results Of the 207 isolates of A. urinae tested, 37 showed trailing (17,8%) and 19 (9,2%) insufficient growth with the BMD method and were retested using AD. Moreover, 38 isolates (ciprofloxacin) and 13 isolates (levofloxacin) showed either lack of categorical or essential agreement between Etest and BMD and were also retested using AD to arbitrate discrepancies. Susceptibility profiles combining BMD and AD are presented in Table 1. As suggested in EUCAST guidelines, readings were much clearer and growth was better with AD compared with BMD. The categorical agreement of the Etest® with BMD+AD was 95% for ciprofloxacin and 97% for levofloxacin. Essential agreement was 95% for ciprofloxacin and 97% for levofloxacin. No very major errors were identified. Two major errors were identified for levofloxacin (1,2%) and one for ciprofloxacin (0.6%). Conclusion Gradient diffusion method using Etest® strips on MH agar w/ sheep blood is a valid method to determine susceptibility to FQ for urinary tract isolates. As a reference method, AD provides clearer endpoints and better growth than BMD for FQ susceptibility testing. Disclosures All authors: No reported disclosures.

scholarly journals Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories

2021 ◽  
Vol 12 ◽  
Author(s):  
Min Cao ◽  
Lin Huang ◽  
Yanyan Hu ◽  
Yinfei Fang ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Blood Culture ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Total Error ◽  
Agar Plate ◽  
Error Rates ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
High Morbidity ◽  
Clinical Strains

Bloodstream infections (BSI) are associated with high morbidity and mortality and remain a leading cause of death. Blood culture (BC) including the identification and the antimicrobial susceptibility testing of the causative microorganisms should be performed as soon as possible. In this study, we developed an in-house rapid antimicrobial susceptibility testing (rAST) protocol for positive BC. First, the rAST was performed in the simulated positive BC of standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853) at three different times to assess the reproducibility and operability by dispensing four drops of BC broth onto a Mueller–Hinton agar plate after a positive signal. Furthermore, the rAST was performed in clinical positive BCs. The results of rAST at 4, 6, 8, and 18 h of incubation were compared with results of the standard 16- to 20-h disk diffusion method, and the preliminary breakpoints of the rAST method were established according to the inhibition diameter of sensitive strains and resistant strains. Finally, the rAST was performed in the simulated positive BC of clinical strains to evaluate the availability of the preliminary breakpoints. The rAST results of standard strains were distributed evenly at three different times. Among the 202 clinical strains used to establish the preliminary breakpoints, the number of zone diameters that could be read and interpreted (60, 87, 98, and 100%) increased with incubation time (4, 6, 8, and 18 h), and the categorical agreement was acceptable, with total error rates of 3.0, 2.3, 2.1, and 1.3% at 4, 6, 8, and 18 h of incubation, respectively. In conclusion, the in-house rAST protocol for positive BC can be implemented in routine laboratories. It provides reliable antimicrobial susceptibility testing results for BSI pathogens after 4–6 h of incubation.

scholarly journals Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 975
Author(s):  
Florian Baquer ◽  
Asma Ali Sawan ◽  
Michel Auzou ◽  
Antoine Grillon ◽  
Benoît Jaulhac ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Antimicrobial Susceptibility Testing ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Broth Microdilution ◽  
Multicenter Studies ◽  
Gradient Diffusion ◽  
Agar Dilution

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

scholarly journals Implementing EUCAST rapid antimicrobial susceptibility testing method for sepsis: lessons learned in a tertiary care center

2021 ◽  
Vol 15 (06) ◽  
pp. 833-839
Author(s):  
Mohammad Aadam Bin Najeeb ◽  
Ayush Gupta ◽  
Shashank Purwar ◽  
Vishnu Teja Nallapati ◽  
Jogender Yadav ◽  
...  
Keyword(s):  
Blood Culture ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Error Rates ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
Major Error ◽  
Disk Diffusion Method ◽  
Testing Method ◽  
Micro Organisms

Introduction: We prospectively evaluated EUCAST rapid antimicrobial susceptibility testing methodology for susceptibility testing directly from blood culture bottles in comparison to CLSI disk-diffusion method. Methodology: During May-November 2019, positively flagged blood culture bottles showing Gram-negative micro-organisms were simultaneously processed by rapid antimicrobial susceptibility testing and CLSI methodology. Antibiotics tested were cefotaxime, ceftazidime, piperacillin-tazobactam, imipenem, meropenem, gentamicin, tobramycin and trimethoprim-sulphamethoxazole. Results: Overall, 80 isolates identified as Escherichia coli (n = 24, 30%), Klebsiella pneumoniae (n = 15, 18.7%), Pseudomonas aeruginosa (n = 16, 20%) and Acinetobacter baumannii (n = 25, 31.2%) were included. Categorical agreements  of rapid antimicrobial susceptibility testing at 4-, 6- and 8-hour reading times were 88.1% (304/345), 90.8% (425/468) and 92.3% (467/506), respectively. Major Error rates were 14% (21/150), 4.9% (10/206) and 4/236 (1.7%), whereas Very Major Error rates were 1.1% (2/177), 1.3% (3/232) and 3.3% (8/243), respectively. Results categorized as “Area of Technical Uncertainty” were significantly lower at 8-hour {10.2% (39/384) vs 5.2% (28/534), 4- vs 8-hour, p = 0.003, Fischer’s exact test}. Conclusions: Except for a slightly higher Very major error rate, rapid antimicrobial susceptibility testing at 8-hour is equivalent to Disk-diffusion method (CLSI-M100) using CLSI-M52 criteria for equivalence: (Categorical agreement ≥ 90%, Very major error ≤ 1.5% and Major error ≤ 3%). Poor Categorical agreements at all reading times were noted for piperacillin-tazobactam, ciprofloxacin and E. coli. Performance of rapid antimicrobial susceptibility testing methodology in resource limited settings brings unique challenge of identifying micro-organisms within 8 hours. We suggest reading and reporting of results at a single time point using rapid antimicrobial susceptibility testing method i.e. at 8-hour.

scholarly journals Multicenter Evaluation of the New Etest Gradient Diffusion Method for Piperacillin-Tazobactam Susceptibility Testing of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Sergio García-Fernández ◽  
Yohann Bala ◽  
Tom Armstrong ◽  
María García-Castillo ◽  
Carey-Ann D. Burnham ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Hospital Setting ◽  
Antimicrobial Susceptibility Testing ◽  
International Standards ◽  
Diffusion Method ◽  
Content Type ◽  
Gradient Diffusion

ABSTRACT Piperacillin-tazobactam (P/T) is a β-lactam–β-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales, P. aeruginosa, and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa, respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales, but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales, P. aeruginosa, and A. baumannii complex isolates with limited category errors.

scholarly journals Antibiotic Resistance among Escherichia coli Isolates from Hospital Wastewater Compared to Community Wastewater

Water ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 3449
Author(s):  
Cristina-Mirabela Gaşpar ◽  
Ludovic Toma Cziszter ◽  
Cristian Florin Lăzărescu ◽  
Ioan Ţibru ◽  
Marius Pentea ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
Resistant Bacteria ◽  
Hospital Wastewater ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli

This study aimed to compare the antibiotic resistance levels of the indicator bacteria Escherichia coli in wastewater samples collected from two hospitals and two urban communities. Antimicrobial susceptibility testing was performed on 81 E. coli isolates (47 from hospitals and 34 from communities) using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. Ten antibiotics from nine different classes were chosen. The strains isolated from the community wastewater, compared to those from the hospital wastewater, were not resistant to gentamicin (p = 0.03), but they showed a significantly higher susceptibility—increased exposure to ceftazidime (p = 0.001). Multidrug resistance was observed in 85.11% of the hospital wastewater isolates and 73.53% of the community isolates (p > 0.05). The frequency of the presumed carbapenemase-producing E. coli was higher among the community isolates (76.47% compared to 68.09%) (p > 0.05), whereas the frequency of the presumed extended-spectrum beta-lactamase (ESBL)-producing E. coli was higher among the hospital isolates (21.28% compared to 5.88%) (p > 0.05). The antibiotic resistance rates were high in both the hospital and community wastewaters, with very few significant differences between them, so the community outlet might be a source of resistant bacteria that is at least as important as the well-recognised hospitals.

scholarly journals Methicillin-Resistant Staphylococcus Aureus (mrsa) as a Cause of Nosocomial Wound Infections

2010 ◽  
Vol 10 (1) ◽  
pp. 32-37 ◽  
Author(s):  
Maida Šiširak ◽  
Amra Zvizdić ◽  
Mirsada Hukić
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Susceptibility ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Susceptibility Testing ◽  
Antimicrobial Susceptibility Testing ◽  
Diffusion Method ◽  
Wound Infections ◽  
Common Cause ◽  
Methicillin Resistant ◽  
Mrsa Infection

Postoperative wound infections represent about 16% of hospital-acquired infections. Staphylococcus aureus is the most common cause of nosocomial wound infections. Increased frequency of Methicillin-re- sistant Staphylococcus aureus (MRSA) in hospitalized patients and possibility of vancomycin resistance requires permanent control of MRSA spread in the hospital.The purpose of this study was to analyse the frequency of Methicillin-resistant Staphylococcus aureus (MRSA) in the swabs taken from the surgical wounds, the presence of MRSA infection in surgical departments and to examine antimicrobial susceptibility of MRSA isolates.Wound swabs were examined from January 2006 to December 2008. The isolates were identified by conventional methods. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc-diffusion method as per NCCLS guidelines.A total of 5755 wound swabs were examined: 938 (16,3%) swabs were sterile and 4817 (83,7%) were positive. Staphylococcus aureus was isolated in 1050 (22,0%) swabs and it was the most common cause of wound infections. MRSA was isolated from 12,4% samples in 2006, from 6,7% samples in 2007 and from 3,7% samples during 2008. Wound infections caused by MRSA dominated in the department of plastic surgery (24,4%) and in the department of orthopaedic surgery (24,1%). Antimicrobial susceptibility testing showed that 73% of MRSA isolates were with the same antibiotic sensitivity pattern (antibiotyp)- sensitive only to vancomycin, tetracycline, fucid acid and trimethoprim/sulfamethoxasole.Our results show decreasing of MRSA infection in the surgical wards. These results appear to be maintained with strategies for preventing nosocomial infection: permanent education, strong application of protocols and urging the implementation of strict infection control policy.

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