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 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 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 EUCAST rapid antimicrobial susceptibility testing (RAST) in blood cultures: validation in 55 European laboratories

2020 ◽  
Vol 75 (11) ◽  
pp. 3230-3238
Author(s):  
Anna Åkerlund ◽  
Emma Jonasson ◽  
Erika Matuschek ◽  
Lena Serrander ◽  
Martin Sundqvist ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Total Error ◽  
Bloodstream Infections ◽  
Error Rates ◽  
Blood Cultures ◽  
Antimicrobial Susceptibility Testing ◽  
Fine Tuning ◽  
Resistant Bacteria ◽  
Disc Diffusion

Abstract Objectives When bloodstream infections are caused by resistant bacteria, rapid antimicrobial susceptibility testing (RAST) is important for adjustment of therapy. The EUCAST RAST method, directly from positive blood cultures, was validated in a multi-laboratory study in Europe. Methods RAST was performed in 40 laboratories in northern Europe (NE) and 15 in southern Europe (SE) from clinical blood cultures positive for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus or Streptococcus pneumoniae. Categorical results at 4, 6 and 8 h of incubation were compared with results for EUCAST standard 16–20 h disc diffusion. The method, preliminary breakpoints and the performance of the laboratories were evaluated. Results The total number of isolates was 833/318 in NE/SE. The number of zone diameters that could be read (88%, 96% and 99%) and interpreted (70%, 81% and 85%) increased with incubation time (4, 6 and 8 h). The categorical agreement was acceptable, with total error rates in NE/SE of 2.4%/4.9% at 4 h, 1.1%/3.5% at 6 h and 1.1%/3.3% at 8 h. False susceptibility at 4, 6 and 8 h of incubation was below 0.3% and 1.1% in NE and SE, respectively, and the corresponding percentages for false resistance were below 1.9% and 2.8%. After fine-tuning breakpoints, more zones could be interpreted (73%, 89% and 93%), with only marginally affected error rates. Conclusions The EUCAST RAST method can be implemented in routine laboratories without major investments. It provides reliable antimicrobial susceptibility testing results for relevant bloodstream infection pathogens after 4–6 h of incubation.

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 Evaluation of the Accelerate Pheno System for Fast Identification and Antimicrobial Susceptibility Testing from Positive Blood Cultures in Bloodstream Infections Caused by Gram-Negative Pathogens

2017 ◽  
Vol 55 (7) ◽  
pp. 2116-2126 ◽  
Author(s):  
Matthias Marschal ◽  
Johanna Bachmaier ◽  
Ingo Autenrieth ◽  
Philipp Oberhettinger ◽  
Matthias Willmann ◽  
...  
Keyword(s):  
Blood Culture ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Bloodstream Infections ◽  
Test System ◽  
Blood Cultures ◽  
Antimicrobial Susceptibility Testing ◽  
Diagnostic Tools ◽  
Gram Negative ◽  
Content Type

ABSTRACT Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillin-tazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli ( n = 7) and multidrug-resistant Pseudomonas aeruginosa ( n = 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h ( P < 0.0001) and for AST by 40.39 h ( P < 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gram-negative BSI.

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