ANTIBIOTIC DISCS FOR THE DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE (ESBL) CARTRIDGE OF 50 DISCS CONFIRMATION TEST FOR THE DETECTION OF ESBL

2019/07

1- CLINICAL VALUE The detection of extended spectrum Beta-lactamases (ESBL) is complicated by the wide diversity of plasmid enzymes that hydrolyze and inactivate numerous Beta-lactams. These ESBL are becoming more and more widespread in numerous species of bacteria. The utility of detecting this phenotype is two-fold: to enable appropriate treatment and to control the diffusion of this mechanism of resistance (1, 2).

2- PRINCIPLE The technique involves comparing the in vitro activity of discs of third generation cephalosporins (3GC) and of these same 3GC in association with clavulanic acid (3GC + CA). Interpretation will be based on the recommendations of the CLSI, EUCAST and CASFM-EUCAST regarding ESBL detection in Enterobacteriaceae, and notably in strains that produce large quantities of cephalosporinase.

3- PRESENTATION Bio-Rad disks are 6.5 mm disks made from superior quality absorbent paper and impregnated with precise concentrations of antimicrobial agents and inhibitor. The disks are clearly identified by a code, comprising 3 letters, printed on each side of the disk.

DISK CONTENT SYMBOL PACK SIZE ORDER N°

Ceftazidime + Clavulanic Acid 30 / 10 µg CCA 1x50 disks 68446

Cefotaxime + Clavulanic Acid 30 / 10 µg CCT 1x50 disks 68456

Cefepime + Clavulanic Acid 30 / 10 µg CFE 1x50 disks 68466

Ceftazidime + Clavulanic Acid 10 / 10 µg CCZ 4x50 disks 12010680

Cefotaxime + Clavulanic Acid 5 / 10 µg CCO 4x50 disks 12010679

4- STORAGE The expiry date applies exclusively to disks contained in intact cartridges stored according to the manufacturer's instructions. The expiry date and batch number are indicated on each packaging (cartridge and container). • Cartridges of disks must be stored in their containers at a temperature between +2°C and +8°C in a dry place. • Containers must be allowed to adjust to room temperature (18-30°C) before opening. After applying the disks, return unused cartridges to a temperature between +2°C and +8°C. • Do not use disks after the expiry date. • Do not use any cartridge of disks left at room temperature (18-30°C) for more than 8 hours without verifying an acceptable level of performance before continuing to use this cartridge (6). • If the cartridge remain in the distributor after dispatch, it is necessary to preserve it at +2-8°C in a dry place with desiccants inside.

The stability of the disks, of open cartridges placed in distributors (preserved according to the recommendations with desiccants) was validated in routine conditions. The stability in weeks is shown inside this symbol:

5- MATERIAL REQUIRED BUT NOT SUPPLIED • disk dispenser: : 7 disks ref # 50294 12-16 disks ref # 50295 • culture media (Mueller-Hinton) • reagents • bacterial strains for quality control • opacity control equivalent to the Mac Farland 0.5 standard • laboratory equipment necessary for antibiotic susceptibility testing by the agar diffusion method.

6- PRECAUTIONS Follow the instructions of the current guidelines (CLSI, EUCAST, CA-SFM/EUCAST). Always observe the current techniques and precautions concerning protection against microbiological hazards. After use, sterilize the cultures and all contaminated material.

7- PROCEDURE Samples: disks must not be used for tests performed directly on biological samples. Refer to the current guidelines (CLSI, EUCAST, CA-SFM/EUCAST) defining preparation of the inoculum (pure, fresh culture), inoculation of Petri dishes, the incubation temperature and incubation time. Follow the instructions provided by the CLSI, EUCAST and the CASFM-EUCAST to perform the antibiogram. Place the discs that are impregnated with a predetermined concentration of 3GC+CA (CCA, 30µg/10µg, CCT 30µg/10µg, CFE 30µg/10µg, CCZ 10µg/10µg, CCO 5µg/10µg), as well as those with a 3GC alone (CAZ 30µg, CTX 30µg, FEP 30µg, CZA 10µg, COX 5µg) on the surface of a suitable, inoculated medium. Good laboratory practice should also be applied at all times.

8- INTERPRETATION OF THE RESULTS • Precisely measure the diameters of the zones of inhibition observed - Ceftazidime+ Clavulanic acid / Ceftazidime - Cefotaxime + Clavulanic acid / Cefotaxime - Cefepime + Clavulanic acid / Cefepime • The bacterial strain is interpreted as having an ESBL resistance phenotype if 3GC activity is seen to be restored in the presence of CA. • A strain is ESBL positive if at least one of the differences in diameter between the (3GC+CA) and (3GC) is greater than or equal to 5mm (3, 4, 5).

9- PERFORMANCE/QUALITY CONTROL The performances of antibiotic disks are systematically controlled by using the following strains:

Klebsiella pneumoniae ATCC® Escherichia coli ATCC® 25922™ 700603™ Cefotaxime + Clavulanic Acid 30/10µg – CCT-CTX ≥ 3 mm4, 5, 6 CCT-CTX ≤ 2 mm4, 5, 6 Cefotaxime 10µg Cefotaxime + Clavulanic Acid 5/10µg – CCO-COX ≥ 3 mm4, 5, 6 Cefotaxime 10µg Ceftazidime + Clavulanic Acid 30/10µg – CCA-CAZ ≥ 5 mm4, 5, 6 CCA-CAZ ≤ 2 mm4, 5, 6 Ceftazidime 30µg Ceftazidime + Clavulanic Acid 10/10µg – CCZ-CZA ≥ 5 mm4, 5, 6 Ceftazidime 10µg

* * Cefepime + Clavulanic Acid 30/10µg – CFE-FEP ≥ 2 mm CFE-FEP ≤ 2 mm Cefepime 30µg

*: Standard Internal

10-QUALITY CONTROL OF THE MANUFACTURER All manufactured and commercialized reagents are under complete quality system starting from reception of raw material to the final commercialization of the product. Each lot is submitted to a quality control and only is released on the market when conforming to the acceptance criteria. The records relating to production and control of each single lot are kept within our company

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11- LIMITATIONS OF USE • A resistant phenotype such as an ESBL is detected by measuring the diameters in the inhibitory zones: to the nearest millimeter, the difference between the diameters of the two discs tested determines whether or not an ESBL is present. Measurements taken to the nearest millimeter give an acceptable margin of error under laboratory conditions. • The final interpretation, as for all laboratory interpretations, cannot be based on the results of one single test but on an overview of the clinical data and other antibiotic test results. • The performances of the test depend not only on the activity of the disks, but also on factors such as the use of an appropriate inoculum and control strains, appropriate and previously tested culture media and adequate storage.

12-REFERENCES 1) Bradford, P. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51. 2) Livermore D. Beta-lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557-84. 3) CLSI: Clinical and Laboratory Standards Institute. 2019. CLSI supplement M100, 28th Edition. Performance standards for antimicrobial susceptibility testing, Wayne, Pa. 4) CA-SFM/EUCAST: Comité de l’antibiogramme. French Society of Microbiology/ European Committee on Antimicrobial Susceptibility Testing. 2019 5) EUCAST: EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. Version 2.01 July 2017. 6) V. Loncle-Provot, E. Keller, M.O. Gourdin, M.L. Garrigues. Etude de la stabilité des disques antibiotiques dans les conditions d'utilisation en routine, 18th RICAI interdisciplinary meeting on anti-infectious chemotherapy, Paris, Dec.3/4 1998.

Bio-Rad 3, boulevard Raymond Poincaré 92430 Marnes-la-Coquette - France Tel. : +33 (0) 1 47 95 60 00 Fax : +33 (0) 1 47 41 91 33 www.bio-rad.com 2019/07

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