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OXOID PENICILLIN BINDING PROTEIN (PBP2′) LATEX AGGLUTINATION TEST

Code DR0900

This test is a rapid latex agglutination assay, detecting PBP2′ (also called PBP2a) 7,in isolates of Staphylococcus, as an aid in identifying methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci.

Introduction
Staphylococci are a leading cause of nosocomial and community-acquired infections worldwide 2. In many institutions, approximately 25% to 50% of S. aureus strains and 75% of coagulase-negative staphylococci (CoNS) are resistant to methicillin 12. MRSA are of particular concern because of the ease with which certain epidemic strains spread and colonise debilitated patients. Treatment of sensitive strains with penicillinase-resistant penicillins (PRP), is preferred as beta-lactam drugs are more easily absorbed into body fluids and tissues, cause fewer complications from treatment, and do not select for vancomycin-resistant organisms. Reliable identification of methicillin-resistance is therefore important.

Strains of S. aureus with reduced susceptibility to PRP are categorized as follows:

(i) methicillin-resistant S. aureus (MRSA), which produce the low-affinity penicillin binding protein PBP2′, encoded by the mecA gene 3,6,
(ii) borderline methicillin-resistant S. aureus (BORSA), generally considered to be due to hyperproduction of type A - beta lactamase 10; and
(iii) strains with modified PBPs due to altered penicillin binding capacity or hyperproduction of PBPs (MODSA) 1,2 . MODSA have only rarely been isolated and their clinical response to beta-lactam therapy has not been well studied. Thus for clinical purposes, with rare exceptions, the presence of PBP2′ is responsible for methicillin-resistance in the treatment of infections with S. aureus and CoNS 2,6.

The methicillin-resistant phenotype can be highly heterogeneous, making it difficult to detect by conventional antimicrobial susceptibility test methods, such as Minimum Inhibitory Concentration (MIC), disc and agar screen. The accuracy of these methods is affected by inoculum size, incubation time and temperature, medium, pH, salt concentration and other factors 8, 9. In addition, these culture methods require 24h incubation for accurate results. CoNS often produce lower amounts of PBP2′ and require induction by exposure to one of the PRPs to produce sufficient product to be detected 2,3,12 .

Detection of the mecA gene has been considered the gold standard in the determination of methicillin-resistance because of its accuracy, but this method is labour-intensive and expensive to perform 1, 6. The Oxoid PBP2′ Latex test has the advantage of direct detection of the PBP2′ protein performed in a rapid timeframe with minimal labour. It has the potential for being even more accurate than the detection of the mecA gene, as false-positive results will not occur with strains that possess mecA but are unable to produce the protein product of the gene. In addition, the assay does not detect strains that are hyperproducers of either beta-lactamase or PBPs.

The Oxoid PBP2′ test has previously been evaluated world-wide, demonstrating its high sensitivity and specificity 4, 5, 11. Latex particles sensitized with a monoclonal antibody against PBP2/ will specifically react with methicillin-resistant staphylococci to cause agglutination visible to the unaided eye.

Components of the Kit

DR0901: Test Latex sensitised with a monoclonal antibody against PBP2/
DR0902: Control Latex sensitised with a monoclonal antibody of the same IgG subclass showing no reactivity with proteins of S. aureus.
DR0903: Extraction Reagent 1
DR0904: Extraction Reagent 2
Test Cards
Mixing Sticks
Instruction Leaflet

Materials required but not provided
Micropipette and tips (50ml)
Microbiological loops (5m l/1ml)
Boiling water bath or heating block
Centrifuge (1500 x g)
Microcentifuge tubes (safe lock)
Suitable laboratory disinfectant
For full proceedure see product insert.

Storage
Store the kit at 2-8° C. Do not freeze.the kit should not be used after the expiry date printed on the outside of the carton.

Precautions
The extraction procedure may not kill bacteria; therefore the extract must be handled with the same precautions.
Extraction Reagents 1 and 2 contain a mild irritant and a weak acid. Avoid direct contact by wearing suitable protective equipment. If the material comes into contact with the skin, mucous membranes or eyes immediately wash the area by rinsing with plenty of water.

Control procedures
For each new lot of the kit and weekly thereafter, the following control procedures must be performed.

Positive Control - Use a known MRSA strain such as ATCC® 43300 Oxoid Culti-Loops® C9022L. Follow the method given in the test procedure. Ensure that agglutination occurs within 3 minutes.
Negative Control - Use a known Methicillin-Sensitive Staphylococcus aureus (MSSA) strain such as ATCC® 25923 or ATCC® 29213, Oxoid Culti-Loops® C7010L or C7011L. Follow the method given in the test procedure. Ensure that no agglutination occurs within 3 minutes. Do not use the test if reactions with the control organisms are incorrect.
Do not use kits beyond their expiry date.

References
1. Bignardi, G. E., N. Woodford, A. Chapman, et al. (1996) J. Antimicrobiol. Chemother. 37:53-63
2. Chambers, H. F. (1997) Clin. Microbiol. Rev. 10:781–791
3. Gerberding, J. L., C. Miick, H. H. Liu, et al. (1991) Antimicrob. Agents Chemother. 35:2574–2579.
4. Hussain, Z., L. Stoakes, S. Garrow, et al. (2000) J. Clin. Microbiol. 38:2051-2054.
5. Louie, L., S. O. Matsumura, E. Choi, et al. (2000) J. Clin. Microbiol. 38:2170–2173.
6. Murakami, K., W. Minamide, K. Wada, et al. (1991) J. Clin Microbiol. 29:2240-2244.
7. Nakatomi, Y., and J. Sugiyama. (1998) Microbiol. Immunol. 42:739-743
8. National Committee for Clinical Laboratory Standards. (2000 Approved Standard Fifth Edition (M7-A5). National Committee for Clinical Laboratory Standards, Wayne, Pa.
9. Tenover, F.C., R. N. Jones, J. M. Swenson, et al. (1999) J. Clin Microbiol. 37:4051-4058.
10. Thornsbury, C. and McDougal, L. (1983) J. Clin. Microbiol. 18: 1084 - 1091.
11. Yamazumi, T., S. A. Marshall, W. W. Wilke, et al. (2001) J. Clin. Microbiol. 39:53-56.
12. York, M. K., L. Gibbs, F. Chehab, et al. (1996) J. Clin. Microbiol. 34:249–253.

 
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