MRSA WOUND CULTURE

Effective Date: 
Fri, 03/24/2017
Revised: 
Wed, 03/29/2017
Policy: 

Purpose and Scope:

The Alere BPB2a SA Culture Colony Test is a qualitative, immunochromatographic assay for the rapid detection of penicillin-binding protein 2a (PBP2a) in isolates identified as Staphylococcus aureus as an aid in identifying methicillin-resistant Staphylococcus aureus (MRSA).  It can identify strains that not only harbor the mecA gene but also produce the protein that confers resistance to methicillin.

Procedure: 

 

Purpose and Scope:

The Alere BPB2a SA Culture Colony Test is a qualitative, immunochromatographic assay for the rapid detection of penicillin-binding protein 2a (PBP2a) in isolates identified as Staphylococcus aureus as an aid in identifying methicillin-resistant Staphylococcus aureus (MRSA).  It can identify strains that not only harbor the mecA gene but also produce the protein that confers resistance to methicillin.

Principle of the Method

The Alere PBP2a Culture Colony Test is a rapid immunochromatographic membrane assay that uses highly sensitive recombinant monoclonal antibody fragments (rFabs) to detect the BP2a protein directly form bacterial isolates.  The rFab and a control antibody are immobilized onto a nitrocellulose membrane as two distinct lines and combined with a sample pad, a pink/purple conjugate pad, and an absorption pad to form a test strip.

Isolates are sampled directly from the culture plate and eluted into an assay tube containing Reagent 1.  Reagent 2 is then added and the test strip is placed in the assay tube.  Results are read visually at 5 minutes.

 

Reagents and Supplies

Materials Provided in the Alere PBP2a SA Culture Colony Test Kit

Test Strips
Reagent 1:  A clear, blue alkaline solution
Reagent 2:  A clear, slightly acidic solution containing sodium azide buffer and surfactants
Assay Tubes
Test Racks

 

Storage and Stability:

Store kit components at room temperature or refrigerated (2-30 deg C). The kit and reagents are stable until the expiration dates marked on their outer packaging.

 

Specimen Required

Specimens are bacterial isolates of Staphylococcus aureus.  The use of fresh (<24 hours) cultures is recommended.  The performance of the test has not been established for use with refrigerated specimens.

Culture Media

S. aureus colonies may be tested from any of the following culture media:

BAP
Columbia Agar
Mueller Hinton Agar

 

Quality Control

Daily Quality Control

PBP2a SA Culture Colony Test has built-in positive and negative procedural controls which are recorded by laboratory personnel for each test run.

 The appearance of a pink/purple line at the control line position is considered an internal positive procedural control.  If capillary flow has occurred, this line will always appear.
In comparison to the color of the control line, the background color on the test strip should be white within 5 minutes.

External Positive and Negative Controls:

External positive and negative controls must be tested and recorded for each new lot and each day of testing.

Control Procedures:

 Subculture the control stains onto a culture plate.:

                      Positive Control:  Staphylococcus aureus :   ATCC #4330

                       Negative Control:  Staphylococcus aureus:  ATCC #25923

 Incubate the plates overnight at 33-35 deg. C for 18-24 hours.
Follow the Procedure below.

 

Procedure

If refrigerated, allow reagents and test strips to equilibrate to room temperature (15-30 deg C) before testing.

The test can be performed from well-isolated colonies on the primary plate if there is sufficient growth, or from a subculture of the isolate.

 Holding the dropper bottle vertically, add two drops of Reagent 1 to an assay tube.
Take one heaped 1 ul bacteriological loop (a heavy inoculum) of sample from well-isolated colonies on the culture plate, place into the tube and thoroughly mix.
Holding the dropper bottle vertically, add two drops of Reagent 2 to the tube.
Vortex briefly.  The blue solution must turn a clear color (if the color does not change, add one more drop of Reagent 2 and mix until the sample turns clear.)
Insert the test strip into the assay tube with arrows pointed downward.
At five (5) minutes, withdraw the test strip from the tube and read the assay result.

For a Negative Sample, a PINK/PURPLE Control Line appears in the top half of the test strip.  No other line appears.

For a Positive Sample, the PINK/PURPLE Contols Line appears AND a second PINK/PURPLE line appears below it in the bottom half of the test strip.  Any Sample Line, even when very faint, is positive.

A test is invalid if the PINK/PURPLE Control line does not appear, whether a Sample Line is present or not.  Repeat invalid tests with a new test strip.  Call Alere Technical Support if the problem persists.

 

References:

 Alere PBP2a Culture Colony Test package insert. Rev. 2 2015/07
Lodise TP, McKinnon PS. Clinical and economic impact of methicillin resistance in patients with Staphylococcus aureus bacteremia. Diagn Microbiol Infect. Dis. 2005 Jun; 52(2):113-22