Subject: MMIC
Topic: Methicillin Resistant Staphylococcus Aureus (MRSA) Rapid Screening

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body. It's tougher to treat than most strains of staphylococcus aureus -- or staph -- because it's immune to some commonly used antibiotics.

Recent research suggests that in the identification of MRSA, it is more accurate to either directly detect the gene encoding the methicillin resistance determinant (mecA) or its product, penicillin-binding protein 2' (2a), or PBP2' (PBP2a), which is found in the cell membrane of MRSA. However, as nucleic acid hybridization and DNA amplification techniques such as PCR for detecting the mecA gene are expensive and technically demanding, simple and more inexpensive techniques are required for routine use. MRSA-Screen was developed expressly for this purpose, providing results in 15 minutes with minimal labor and no specialized equipment.

Principle
MRSA Screen is a rapid slide latex agglutination assay which detects the penicillin binding protein 2 (PBP2’) of methicillin-resistant S.aureus. Latex particles are sensitized with a monoclonal antibody against PBP2’ and will specifically react with MRSA to cause agglutination visible to the unaided eye. It will therefore enable the rapid and appropriate antimicrobial therapy for serious MRSA infections.

Equipment & Materials
NMRSA Screen kit
Sensitised Latex 2.5 ml x 1
Control Latex 2.5 ml x 1
Extraction Reagent 1 10 ml x 2
Extraction Reagent 2 5 ml x 1
Test card 55
Mixing stick 220
BAP
Micropipette and tips (50ml)
A beaker, tripod, wire net and a burner for boiling the specimen


Procedures
A. Sample Preparation
1) Colonies believed to be S. aureus taken from BAP or other media, after incubating for 18-24 hours at 35°C are used.

B. PBP2’ Extraction
2) Add 4 drops of Extraction Reagent 1 into a test tube.
3) 4 drops of Extraction Reagent 1 are added into a test tube.
4) A loopful of cells are taken using a platinum loop or other appropriate device and suspended in the test tube.
5) The test tube is placed into a boiling water bath or heating block and heated for 3 minutes.
6) The test tube is removed and allowed to cool to room temperature.
7) A drop of Extraction Reagent 2 is added into the test tube and mixed well.
8) The test tube is centrifuged at 1500 xg for 5 minutes.
9) The supernatant is used as specimen.

C. Latex Agglutination
10) Circles on test card are labeled with Sensitised latex and Control latex for each specimen.
11) 50 ml of specimen is placed onto the test circles
12) Sensitised latex or control latex is added into each respective test circles.
13) They are mixed well using a mixing stick
14) The test card is rotated by hand for 3 minutes and agglutination patterns are observed.

Interpretation
Agglutination is seen with Sensitized but not Control Latex: PBP2’ positive (MRSA)
No agglutination is seen with either latex reagent : PBP2’ negative (MRSA)



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