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Number/Ed: DVB0035E Maintained by Sales dir.: Page: 1/5 Approved by Med.dir.: Valid from: 30.04.13

A/S ROSCO

INSERT FOR Total MBL Confirm Kit

Insert for Total Metallo-β-Lactamase Confirm Kit (98016) REVISION: DATE OF ISSUE: LANGUAGE:

DBV0035E 30/04/2013 English

Total MBL Confirm kit FOR IN VITRO DIAGNOSTIC USE ONLY PRODUCT GROUP:

Kits for beta-lactamase identification

MANUFACTURER:

ROSCO Diagnostica A/S, Taastrupgaardsvej 30, DK-2630 Taastrup, Denmark.

INTENDED USE:

Tablets are used for qualitative in vitro identification of microbial resistance mechanisms by the agar tablet/disc diffusion method, in order to confirm the mechanism by which the organism has gained resistance to specific antimicrobial agents.

INTENDED USERS:

Only to be used by professionals and people trained to work with microbes and disc diffusion testing.

PRINCIPLE OF THE TEST: Five cartridges of diffusion disc/tablets containing Meropenem 10 µg, Meropenem + DPA, Imipenem 10 µg, Imipenem + DPA and Imipenem + EDTA. Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. producing Metallo-β-lactamases (MBL), hydrolyses carbapenems efficiently. MBL’s are inhibited by Dipicolinic acid (DPA). DPA has no intrinsic antimicrobial activity, as opposed to Ethylene diamine tetra-acetic acid (EDTA), and the results are easily interpreted. The use of two carbapenems in combination with DPA permits the identification of any MBL in any species. Imipenem + DPA perform best in detecting Pseudomonas and Acinetobacter spp., while Meropenem + DPA perform best in the detection of Enterobacteriaceae. IMP-8 producing Enterobacteriaceae may show false negative results with Meropenem + DPA, but not with Imipenem + DPA or Imipenem + EDTA. Imipenem + EDTA are thus included to detect these isolates. The synergistic performance of DPA is improved by the use of Mc Conkey agar, instead of Mueller – Hinton. Therefore, use Mc Conkey agar.

DETAILED INSTRUCTIONS:

CONTENT AND FORMULATION:

ROSCO’s detailed Instruction for Use for Detection of resistance mechanisms should be available in each laboratory working with ROSCO’s Diagnostic products. Latest edition of Instruction for Use can be seen in and/or printed out from ROSCO’s website www.rosco.dk. Instructions for Use and User’s Guide can be obtained free of charge from your local distributor on request, or from ROSCO Diagnostica A/S: E-mail: info@rosco.dk or Fax: +45 43 52 73 74


Number/Ed: DVB0035E Maintained by Sales dir.: Page: 2/5 Approved by Med.dir.: Valid from: 30.04.13

A/S ROSCO

INSERT FOR Total MBL Confirm Kit

1 2 3 4

5 cartridges of tablets, formulated for maximum stability, each containing approximately 50 tablets: Meropenem 10 µg, coded MRP10 Meropenem 10 µg + DPA coded MRPDP Imipenem 10 µg , coded IMI10 Imipenem 10 µg + DPA, coded IM+DP 5 Imipenem 10 µg + EDTA, coded IM10E

STORAGE/HANDLING:

Store at 2-8°C in the box provided or unopened cartridges until the expiry date shown on the product label. Allow the cartridges to acclimatize to room temperature for 30-60 minutes before the lid is removed from the cartridge. Once a cartridge has been opened and in particular when placed in a dispenser, it should be kept at room temperature for up to 2 months. If necessary, when in use for a longer period than 2 months, the cartridges can be stored at 2-8°C. Always seal the cartridges with the original green lid, and never place a dispenser in the refrigerator. When stored at 2-8°C the cartridges should be allowed to acclimatize, as described above, before use.

PRECAUTIONS: For in vitro diagnostic use only. Safety precautions should be taken and aseptic techniques used when working with potential biohazards. To be used only by adequately trained and qualified laboratory personnel. Sterilize all biohazard waste before disposal. Refer to Product Safety Data Sheet. MATERIALS REQUIRED BUT NOT PROVIDED: Standard microbial equipment such as loops, culture media, incubator etc. and biochemical reagents. PROCEDURE: 1. Using a fresh, pure culture prepare a suspension of the organism to be tested equivalent to McFarland 0.5 2. Using a sterile swap or Drigalski spatula spread the suspension uniformly over the entire area of a Mc Conkey agar plate. Iso-sensitest Agar must not be used (false negative). 3. Using a single tablet or a dispenser, place one of each tablet on the inoculated agar plate, ensuring sufficient space between individual tablets to allow for proper measurement of inhibition zones. Notice that more than one Confirm ID Kit can be tested on the same plate. 4. Incubate at 35±1°C for 18±2 hours (overnight). 5. Measure and record the diameter of the inhibition zones. No zone around a tablet corresponds to a 9 mm inhibition zone. INTERPRETATION OF RESULTS: The results are interpreted by comparing the inhibition zones of the different tablets 1. Compare the zone of inhibition around the Meropenem 10 µg tablet to the zones of inhibition of Meropenem 10 µg +DPA , and the zone of inhibition around Imipenem 10 µg to that of Imipenem + DPA and Imipenem +EDTA. If all zones are within 3 mm of each other, the organism is not expressing MBL activity. 2.

For Enterobacteriaceae: 1.

2.

Compare the zones around Meropenem and Meropenem + DPA. If a difference in zone diameter of ≥ 5 mm (MRPDP-MRP10) is observed, report the isolate as expressing MBL activity. Compare the zones around Imipenem and Imipenem + DPA. If a difference in zone diameter of ≥ 5 mm (IM+DP-IMI10) is observed, report the isolate as


Number/Ed: DVB0035E Maintained by Sales dir.: Page: 3/5 Approved by Med.dir.: Valid from: 30.04.13

A/S ROSCO

INSERT FOR Total MBL Confirm Kit

3.

expressing MBL activity. In case of suspected IMP-8 MBL: Compare the zones around Imipenem and Imipenem + EDTA. If a difference in ≥ 7 mm (IM10E-IMI10) is observed, report the isolate as expressing IMP-8 activity. MBL detection with the EDTA combination in Enterobacteriaceae should not be considered a confirmative result.

3.

For Pseudomonas aeruginosa/Acinetobacter spp.: 1. Compare the zones around Imipenem and Imipenem + DPA. If a difference in zone diameter of ≥ 5 mm (IM+DP-IMI10) is observed, report the isolate as expressing MBL activity. 2. MBL detection with the EDTA combination in Pseudomonas aeruginosa and Acinetobacter spp. should not be considered a confirmative result.

4.

For Acinetobacter: 1. 2.

5.

The isolate should be reported as MBL negative, if a difference of ≤ 3 mm for Imipenem + DPA compared to Imipenem alone (IM+DP-IMI10) is observed. The isolate could be suspected of oxacillinase activity, if the following is observed: A difference of ≤ 3 mm for Imipenem + DPA compared to Imipenem alone (IM+DP-IMI10) and a difference of ≥ 4 mm for Imipenem + EDTA compared to Imipenem alone (IM10E-IMI10). It has been shown that EDTA converts oxacillinases into the less active monomeric state, resulting in a reduction in carbapenemase activity. DPA has no activity against the oxacillinases. When using both the EDTA and the DPA combination with Imipenem, it is possible to differentiate between MBL’s and oxacillinases in Acinetobacter spp.

Use table 1, 2 and 3 to assist in the interpretation (see below)

QUALITY CONTROL: Although ROSCO Diagnostica A/S produces, by far, the most stable diffusion discs (tablets) it is necessary to perform regular quality control. This should be done with at least one organism to demonstrate a positive reaction and at least one organism to demonstrate a negative reaction. Zones of inhibition obtained using the combination tablets plus the carbapenem alone tablet against the negative control (i.e. E. coli ATCC 25922), should be within 3 mm. As positive Q. C. stains the following may be used: Klebs. pneumoniae NCTC 13438, MBL positive Klebs. pneumoniae ATCC BAA-2146, MBL positive

Table 1: Enterobacteriaceae

MBL

MBL

Meropenem 10 µg MRP10 Imipenem

Meropenem + DPA MRPDP

Imipenem + DPA IM+DP

Imipenem + EDTA IM10E

≥ 5 mm

-

-

-

≥ 5 mm

Variable


Number/Ed: DVB0035E Maintained by Sales dir.: Page: 4/5 Approved by Med.dir.: Valid from: 30.04.13

A/S ROSCO

INSERT FOR Total MBL Confirm Kit

IMP/MBL

10 µg IMI10 Imipenem 10 µg IMI10

-

≥ 5 mm

≥ 7 mm

Imipenem + DPA IM+DP

Imipenem + EDTA IM10E

-

≥ 5 mm

Variable

Meropenem + DPA MRPDP

Imipenem + DPA IM+DP

Imipenem + EDTA IM10E

-

≥ 5 mm

Variable

-

≤ 3 mm and

≥ 4 mm

Table 2: Pseudomonas aeruginosa. Mc Conkey agar Meropenem + DPA MRPDP MBL

Imipenem 10 µg IMI10

Table 3: Acinetobacter spp. Mc Conkey agar

MBL

Oxacillinases

Imipenem 10 µg IMI10 Imipenem 10 µg IMI10

Non MβL: All zones within 3 mm of each other.

REFERENCES:

www.rosco.dk

1. Dongeun Yong et al: Evaluation of double disk potentiation and disk potentiation tests using Dipicolinic acid for detection of MBL – producing Pseudomonas spp and Acinetobacter spp. J. Clin. Microbiol. 50, 3227-3232, 2012.


Number/Ed: DVB0035E Maintained by Sales dir.: Page: 5/5 Approved by Med.dir.: Valid from: 30.04.13

A/S ROSCO

INSERT FOR Total MBL Confirm Kit

LIST OF CHANGES:

Edition:

Valid from:

Changes:

A B C

10.02.2010 mpdj 23.01.2013 MLSD 25.03.2013 MLSD

D E

02.04.2013 MLSD 30-04-13 Casals

1. edition – new kit Revision of storage and handling Revision of name/title, text and interpretation of results; now comprising interpretation of Enterobacteriaceae, P.aeruginosa and Acinetobacter spp. new tables for interpretation Minor revision- marked with yellow marker.


98016%20dbv0035e%20insert%20total%20mbl%20confirm%20kit%20eng