MRSA LATEX TEST FOR PBP2'
BY DENKA SEIKEN
|Cat. no. DR900A||MRSA Latex Test for PBP2'||50 tests/kit|
This test is a rapid latex agglutination assay, detecting PBP2' (also called PBP2a), in isolates of Staphylococcus, as an aid in identifying methicillin-resistant Staphylococcus aureus (MRSA).(7)
Methicillin-resistant S. aureus has become a worldwide concern owing to their increasing frequency in hospitals causing serious staphyloccal infections, including sepsis and endocarditis. Rapid and appropriate antimicrobial therapy, including the administraion of vancomycin, is critical for effective treatment. However, conventional methods for identifying MRSA, such as disc susceptibility testing, are not always reliable since phenotypic expression of methicillin resistance is known to be heterogeneous, depending on such factors as incubation time, temperature, NaCl concentration, etc. Difficulties in the differentiaion of MRSA from borderline oxacillin-resistant S. aureus (BORSA), for example, may also occur. 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 hybridizaion 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.
MRSA-Screen consists of a latex reagent sensitized with monoclonal antibody against PBP2' together with reagents to rapidly extract PBP2' from the bacterial membranes of MRSA. Extracts are prepared by boiling a suspension of S. aureus cells under alkaline conditions, followed by a neutralization and a centrifugation step. The supernatant is then mixed with the latex reagent on a test card and visible clumping or agglutination within three minutes indicates the presumptive presence of PBP2'.
- Sensitized Test Latex - (dropper bottle with pink cap) Latex particles are sensitized with a monoclonal antibody against PBP2'.
- Control Latex - (dropper bottle with a blue cap) Latex particles are sensitized with a monoclonal antibody of the same IgG subclass but against a human protein showing no reactivity with proteins of Staphylococcus aureus.
- Extraction Reagent 1 - (green cap) 0.1 mol/L NaOH
- Extraction Reagent 2 - (yellow cap) 0.5 mol/L KH2 PO4
- Test Cards
- Mixing Sticks
- Package Insert
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological supplies and equipment such as 50ul micropipette and tips, 5ul microbiological loops, boiling waterbath, heating block, centrifuge, safe-lock or hinged lid microcentrifuge tubes, suitable laboratory disinfectant, loops, other culture media, swabs, applicator sticks, incinerators, etc., as well as additional serological and biochemical reagents, are not provided.
STORAGE AND SHELF LIFE
Storage: This kit must be stored at 2-10ºC. Under these conditions the reagents will retain their reactivity until the expiration date shown on the box. Ensure that the caps are tightened after use to prevent contamination and drying out of the reagent. After use, return the kit promptly to the refrigerator.
As reagents in the kit (Sensitized Latex and Control Latex) are prepared from biological materials, and due to the potential infectious nature of the isolates being tested, proper handling and disposal methods should be established and only trained personnel should be permitted to perform the procedures.
Note: The extraction procedure may not kill the bacteria; therefore the extract must be handled with caution.
1. The MRSA Latex test is intended for in vitro diagnostic use.
2. Read all instructions completely and carefully before performing the test.
3. Do not freeze reagents or use past the expiration date. Do not interchange reagents between different lot numbers or exchange caps between reagent vials.
4. Bring the kit to room temperature before use. Ensure that latex reagents are homogeneous before use by gently inverting the vials. Avoid extreme or excessive shaking, vortexing etc., as this may impair reagent performance.
5. Observe approved biohazard precautions and aseptic techniques. To be used only by adequately trained and qualified laboratory personnel. Never mouth pipette.
6. The heating time should not exceed three minutes. Heating for more than five minutes may lead to a decrease in sensitivity. Heating for only one minute or less may lead to non-specific agglutination.
7. When removing the supernatant for use in the test following centrifugation remove the pipette carefully to avoid solid material at the bottom of the tube. Carry over of this may cause non-specific agglutination.
8. Reagents contain 0.08% w/v sodium azide as a preservative. Sodium azide is toxic and may react with lead or copper plumbing to produce metal azides which are explosive by contact detonation. To prevent azide accumulation in plumbing, flush with copious quantities of water immediately after waste disposal.
9. As specimen materials may contain pathogenic organisms, handle with appropriate precautions. The extraction procedure may not kill bacteria; therefore the extract must be handled with the same precautions. Dispose of all specimens, inoculated products and equipment used in this test as biohazardous material. Disinfect any spills according to biohazard protocols.
10. Extraction Reagents 1 and 2 contain a slightly basic and a weak acid solution. 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. Seek medical attention if serious reactions develop.
Refer to the keyword "MSDS", in the Hardy Diagnostics software program HUGO™, for more information on handling potentially hazardous material.
SPECIMEN COLLECTION AND PREPARATION
For details of specimen collection a standard text book should be consulted. See listed references.(12-16)
Colonies may be tested from any of the following culture media: Tryptone Soya Agar (Tryptic Soy Agar or TSA) with 5% sheep blood (Blood Agar, Cat. no. A10), Columbia Agar with 5% sheep blood (Cat. no. A16), and Mueller Hinton Agar (Cat. no. G45). The use of fresh (18-24 hours) cultures (grown at 35ºC) is recommended.
After extraction and preparation of the test specimen, perform the test the same day. The test specimen may be stored refrigerated at 2-10ºC for batch testing later in the day or at -80ºC for long-term storage.
For Staphylococcus aureus:
The test can be performed from well-isolated colonies on the primary isolation plate, if there is sufficient growth, or from a subculture of the isolate. Other organisms that are present on the plate have not been shown to interfere with the assay. It is preferable to use hinged lid microcentrifuge tubes for the extraction/centrifugation procedures.
PBP2' EXTRACTION PROCEDURE
1. Add four drops of Extraction Reagent 1 into a microcentrifuge tube or equivalent.
2. Approximately 1.5x109 (3-5ul) cells should be tested. This may be achieved by using a sterile 5ul loop to remove sufficient growth to fill the internal diameter of the loop. Alternatively a sterile 1ul loop may be used to remove 3 portions (each to fill the internal diameter of the loop). Suspend the test culture in the microcentrifuge tube.
3. Place the tube into boiling water or heating block and heat for three minutes.
4. Remove the microcentrifuge tube and allow to cool to room temperature.
5. Add one drop of Extraction Reagent 2 into the tube and mix well.
6. Centrifuge at 1500xg for five minutes (i.e., 3000rpm at 15cm rotation radius or 4500rpm at 4.5cm rotation radius). Use the supernatant for the test.
LATEX AGGLUTINATION PROCEDURE
1. For each supernatant to be tested, label one circle on the card "Test" for testing with Test Latex and a second labeled "Control" for testing with Control Latex.
2. Place 50ul supernatant on the circle labeled "Test" and add one drop of Test Latex. Mix together well with the mixing stick provided.
3. Similarly, place 50ul supernatant on the circle labeled "Control" and add one drop of Control Latex. Mix together well with the mixing stick.
Note: Do not allow the reagents to become contaminated by allowing the dropper tip to touch the specimen on the reaction card. Ensure the caps are securely fitted after use to prevent contamination and drying out of the reagents. After use return the kit to the refrigerator ensuring that the bottles are stored in an upright position.
4. Pick up and rock the card for up to three minutes and observe for agglutination under normal lighting conditions.
5. Dispose of the reaction card(s) safely into a disinfectant solution or infectious waste receptacle.
INTERPRETATION OF RESULTS
PBP2' Positive (MRSA): Agglutination is observed with Test Latex but not observed with the Control Latex within 3 minutes.
PBP2' Negative (MSSA): No agglutination is seen with either the Test Latex or the Control Latex within 3 minutes.
Indeterminate Result : Agglutination is observed only with the Control Latex within 3 minutes.
STRENGTH OF AGGLUTINATION REACTION
Negative (-) = a homogeneous suspension of particles with no visible clumping
Weak positive (+) = small but definite clumps against a clouded background
Strong positive (+) = large and small clumps against a slightly clouded background or large clumps against a very clear background
Occasionally, negative reactions may have a finely granular appearance or reactions can be stringy. In such cases, the degree of background clearing should be used to interpret the result. An opaque background indicates a negative result and a clear background should be interpreted as a positive result.
The PBP2' test should be performed only on Staphylococcus species (gram-positive cocci). A coagulase or equivalent test must be performed in order to determine if the isolate is Staphylococcus aureus or another species of Staphylococcus (CoNS).
Indeterminate results should be re-tested with a fresh extract. If, upon retesting, the result is again indeterminate, the methicillin resistance must be determined by other methods.
False-negative results can occur if insufficient culture is used for testing. In such cases, the test should be repeated with sufficient culture.
True positive results generally have strong reactions. False-positive reactions have been known to occur rarely, but are generally limited to weak reactions. Such results can be verified by re-testing with a fresh culture.
Modified S. aureus (MODSA), and borderline resistant strains of S. aureus (BORSA) do not possess PBP2' and are not expected to react in this assay.
Some organism strains may have a low level methicillin-resistance or, in rare cases, produce PBP2' in low amounts, and give a false-negative result.
Because of limitations in sensitivity and specificity of the CLSI susceptibility test methods for coagulase-negative staphylococci, particularly for strains other than Staphylococcus epidermidis, the results with the PBP2' assay may not agree with the results of standard susceptibility testing.(8) Strains with MICs of 4ug/ml should be considered methicillin-resistant, regardless of the PBP2' assay results.
Check for signs of contamination and deterioration. Do not use kits beyond their expiration date. For each new lot of the kit and weekly thereafter, the following control procedures must be performed. Do not use the test if the reactions with the control organisms are incorrect.
Positive Control: Use a known MRSA strain, such as Staphylococcus aureus ATCC ® 43300, and follow the method given in the test procedure. Ensure that the agglutination occurs within 3 minutes.
Negative Control: Use a known MSSA strain, such as Staphylococcus aureus ATCC ® 25923, and follow the method given in the test procedure. Ensure that the agglutination occurs within 3 minutes.
ATCC ® 43300
|**||3min||Aerobic||Agglutination is observed with Test Latex but not observed with the Control Latex|
ATCC ® 25923
|**||3min||Aerobic||No agglutination is seen with either the Test Latex or the Control Latex|
** Refer to the Latex Agglutination Procedure listed above.
1. The Denka Seiken Penicillin Binding Protein Latex Agglutination Test has been evaluated in four geographically diverse laboratories with fresh clinical isolates of Staphylococcus aureus.
Two hundred and one isolates were tested with CLSI methods and with the Denka Seiken PBP2' test from each of three media. One weak false-positive Denka Seiken Latex reaction (negative on repeat) was found from TSA with blood. All positive reactions were strong, except 3 weak (but positive) reactions from Mueller Hinton Agar.
The sensitivity of the Latex test in detecting MRSA on each medium was 100%; the specificity was 99% for TSA with blood and 100% for tests from all other media.
|Results of testing of 201 fresh clinical isolates of S. aureus from 4 laboratories:|
||Number Tested||Growth on Oxacillin Salt Agar (NCCLS)||MIC > 4 ug/ml (NCCLS)||Positive Latex from TSA Blood||Positive Latex from Columbia Blood||Positive Latex from Mueller Hinton|
A MICs were 2ug/ml and mecA was negative.
B Number of weak reactions shown in parentheses.
2. Testing of challenge strains of S. aureus in three geographically distinct areas:
Three laboratories each examined a set of previously collected challenge strains of S. aureus and compared the PBP2' results to the CLSI methods of determination of MRSA. A total of 724 strains was tested from TSA with blood. The sensitivity of the Denka Seiken PBP2' test was 98.5% and the specificity was 100%. Sensitivities of the agar screen and MIC methods were 98.7% and 99.2% and specificities were 90.0% and 88.7%.
Two strains of MODSA were also tested separately; both strains gave a negative result with the Latex test.
3. Testing coagulase-negative staphylococci:
Two laboratories tested coagulase-negative staphylococci for PBP2' after induction with oxacillin discs. One laboratory tested 115 methicillin-resistant strains and 45 methicillin-susceptible strains, including 58 fresh clinical isolates. The sensitivity was 96.5% for testing from TSA blood and 95.6% for Mueller Hinton Agar. The specificity was 100% from TSA blood and 98% from Mueller Hinton Agar. Obtaining a good inoculum was more difficult with Mueller Hinton Agar. The second laboratory tested 212 methicillin-resistant strains and 203 methicillin-susceptible strains with a sensitivity of 99.5% and specificity of 99.5%, using growth on Columbia Agar for the inoculum.
Ten different well-characterized S. aureus strains (3 MRSA, 3 MSSA, 3 BORSA and 1 MODSA) were sent to three geographically diverse laboratories with each strain submitted 5 times in a coded and blinded fashion. All 150 test results agreed with the expected results for 100% reproducibility. The three mecA positive strains were positive each time they were tested (45/45 tests). The three MSSA and the three BORSA gave negative results each time the test was performed (90/90 tests). The MODSA had an MIC of 16ug/ml to oxacillin and gave a negative result in the Denka Seiken Latex Test, as expected, each time the test was performed (15/15 tests).
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This document is provided for general product information only. It does not replace the manufacturer's product insert. Always refer to the actual product insert for procedural use and for most recent product information.
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