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STAPHYTECT PLUS

Intended Use
Staphytect Plus is a latex slide agglutination test¹ for the differentiation of Staphylococcus aureus by detection of
clumping factor, Protein A and certain polysaccharides found in methicillin resistant Staphylococcus aureus (MRSA) from those staphylococci that do not possess these properties.

Principle of the Test
Traditionally, differentiation between coagulase-positive and coagulase-negative staphylococci has been performed either with the tube coagulase test that detects extracellular staphylocoagulase or the slide coagulase test that detects the clumping factor (bound coagulase) present on the bacterial cell surface. Several other differentiation tests are also available including the passive haemagglutination test (Oxoid Staphylase – DR0595) and the DNase test.
It has been reported that approximately 97% of human strains of Staphylococcus aureus possess both bound coagulase and extracellular staphylocoagulase.
Protein A is found on the cell surface of about 95% of human strains of Staphylococcus aureus and has the ability to bind the Fc portion of immunoglobulin G (IgG)².
It has been observed that certain methicillin-resistant strains of Staphylococcus aureus (MRSA) may express undetectable levels of clumping factor and Protein A^3,4,5. It has been shown however that these strains all possess capsular polysaccharide⁶. The capsule can mask both Protein A and the clumping factor thereby preventing agglutination.
Staphytect Plus uses blue latex particles coated with porcine fibrinogen and rabbit IgG including specific polyclonal antibodies raised against capsular polysaccharides of Staphylococcus aureus^7,8.
When the reagent is mixed on a card with colonies of Staphylococcus aureus, rapid agglutination occurs through the reaction between (i) fibrinogen and clumping factor, (ii) Fc portion of IgG and Protein A, (iii) specific IgG and capsular polysaccharide. Agglutination may also occur with other species which possess clumping factor or Protein A such as Staphylococcus hyicus and Staphylococcus intermedius. If neither clumping factor, Protein A nor specific capsular polysaccharides are present, agglutination will not occur and the result will be regarded as negative. The most frequent coagulase and Protein A negative isolates of staphylococci are Staphylococcus epidermidis.

Components of the Kit (DR0850M)
DR0851M Staphytect Plus Test Reagent (5.6 ml)
Blue latex particles coated with both porcine, fibrinogen and rabbit IgG together with specific polyclonal antibodies raised against capsular polysaccharide of
S. aureus. Each bottle contains sufficient reagent for 100 tests.
DR0852M Staphytect Plus Control Reagent (5.6 ml)
Blue unsensitised latex particles. Each bottle contains sufficient reagents for 100 tests.
DR0500G Reaction Cards
There are 35 disposable reaction cards provided in the kit.
Instruction Leaflet.

Materials required but not provided
Timer
Microbiological Loop
A suitable laboratory disinfectant
Positive Control: Staphylococcus aureus strain such as ATCC® 25923
Negative Control: Staphylococcus epidermidis strain such as ATCC® 12228.

Precautions
This product is for in vitro diagnostic use only.
Do not freeze.
Reagents contain 0.095% 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 amounts of water immediately after waste disposal.
Specimen materials may contain pathogenic organisms handle with appropriate precautions.

Storage
This kit must be stored at 2–8°C away from direct sunlight or heat sources. Do not freeze.
Under these conditions the reagents will retain their activity until the expiry date shown on the kit box.

Control Procedures
On each occasion the kit is used the following control procedures must be performed:
1. Positive Control: Use a known Staphylococcus aureus strain such as ATCC® 25923 (Oxoid Culti-Loops C7010L). Follow the method given in Test Procedure. Ensure that agglutination occurs within 20 seconds.
2. Negative Control: Use a known Staphylococcus epidermidis strain such as ATCC® 12228 (Oxoid Culti-Loops C6500L). Follow the method given in Test Procedure. Ensure that the reagent remains smooth and non-agglutinated for the entire 20 seconds of the test.
Do not use the test if reactions with the control organisms are incorrect.

Important Procedure Notes
Do not allow the test latex to become contaminated by letting the dropper tip touch the specimen on the reaction card. Ensure that the caps are securely fitted
after each use to prevent contamination and drying out of the reagent.
After use return the kit to the refrigerator ensuring that the bottle is stored in an upright position.
Ensure that sufficient growth is removed from the culture plate; an insufficient quantity may give rise to false negative reactions.

Specimen Collection and Preparation
For details of specimen collection and treatment a standard text book should be consulted⁹.
Gram positive, catalase positive colonies may be tested from any of the following culture media:
Blood Agar
Nutrient Agar
Tryptone Soya Agar
Tryptone Soya Agar with 5% blood
Mannitol Salt Agar
Columbia Blood Agar
Columbia CNA Agar
Mueller-Hinton Agar with 5% blood
Baird-Parker Agar
CLED Medium
Iso.Sensitest Agar
Iso.Sensitest Agar with 5% blood
Oxacillin Resistance Screening Agar (ORSA).
The use of fresh cultures grown overnight is recommended (18-36 hours incubation). The tendency of colonies to cause autoagglutinating reactions increases with incubation beyond the recommended 36 hour period.

Standard Test Method
1. Bring the latex reagents to room temperature. Make sure that the latex reagent is mixed by vigorous shaking and expel any latex from the dropper pipette for complete mixing.
2. Dispense 1 drop of test latex onto one of the circles on the reaction card and 1 drop of control latex onto another circle.
3. Using a loop, pick up and smear the equivalent of 5 average-sized suspect Staphylococcal colonies (equivalent to 2–3 mm diameter of growth) onto a
circle from a culture media plate and mix this in the Control Latex reagent. Spread to cover the circle. Discard the loop appropriately.
4. Using a separate loop proceed in the same way with the Test Latex.
5. Pick up and rock the card for up to 20 seconds and observe for agglutination under normal lighting conditions. Do not use a magnifying glass.
6. When the test is completed dispose of the reaction cards safely into disinfectant.

Test Method for Oxacillin Resistance Screening Agar
1. As Standard Test Method.
2. Using a loop, pick up and smear the equivalent of 5 average-sized suspect Staphylococcal colonies (equivalent to 2–3 mm diameter of growth) from a
culture media plate onto a circle. Using the loop, spread the colony material into a thin film.
3. Dispense 1 drop of Control Latex directly onto the thin film and mix IMMEDIATELY.
4–6. As Standard Test Method.

Reading and Interpretation of Results
Positive Result
A result is positive if agglutination of the blue test latex particles occurs within 20 seconds. This presumptively identifies the strain as a Staphylococcus aureus.
Negative Result
A negative result is obtained if no agglutination occurs and a smooth blue suspension remains after 20 seconds in the test circle. This presumptively identifies the
strain as a non Staphylococcus aureus.
Equivocal Result
Slight graininess of the test latex accompanied by no change in the appearance of the control latex should be interpreted as an equivocal result. Strains should be re-tested following subculture onto non-selective media.
Uninterpretable Result
The test is uninterpretable if the control reagent shows agglutination. This indicates that the culture causes autoagglutination.
Granular or Stringy Reactions
Occasionally granular or stringy reactions may be seen due to the particulate nature of the test material. When such reactions are seen to occur they should be
interpreted using the following criteria:
The result is positive when using the test reagent greater clearing of the blue background is observed when compared with the reaction of the control reagent. The result is negative when there are no differences between the clearing of the blue background using the Test and Control Reagents.
Reactions occurring after 20 seconds should be ignored.

Limitations of the Procedure
1. The tendency of isolated colonies to cause autoagglutination increases with incubation times beyond the recommended 36 hour period.
2. The antibody used in Staphytect Plus has been optimised to avoid potential cross-reactions with shared antigens from coagulase negative staphylococci.
It should be noted that this has been shown to reduce sensitivity to some type 18 MRSA strains¹⁰.
3. Some species of Staphylococci other than Staphylococcus aureus notably Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus xylosus, Staphylococcus schleiferi and Staphylococcus haemolyticus^11,12,13,14 may give positive results in coagulase tests and/or rapid latex procedures. If necessary the species may be identified by biochemical test procedures e.g. using a test for PYRase activity (Oxoid O.B.I.S. PYR ID0580), Staphylococcus aureus and Staphylococcus hyicus will be PYRase negative and all the other strains named above will be positive^15,16,17. Staphylococcus hyicus and Staphylococcus intermedius are rarely encountered in the clinical laboratory.
4. Staphylococci isolated from urine specimens which give a weak positive¹⁸ result with Staphytect Plus may be Staphylococcus saprophyticus. Further identification of such isolates may be conducted using biochemical tests and novobiocin sensitivity (Staphylococcus saprophyticus is resistant to novobiocin).
5. Some streptococci and possibly other organisms possessing immunoglobulin or plasma binding factors may react in the latex test and some species such as
Escherichia coli are able to agglutinate latex particles^19,20 non-specifically. To overcome these non-specific results a Gram-stain should be performed to
ensure only typical Staphylococci are tested.

Performance Characteristics
The performance characteristics of Oxoid Staphytect Plus have been determined using data from the studies detailed below. It is important to note, however, that Staphylococcus aureus is known to show considerable antigenic variation with respect to different geographical locations.

Clinical study
Oxoid Staphytect Plus was evaluated at a large French hospital. A total of 299 isolates was tested using tube coagulase as the gold standard method. In the following data analysis, results from strains known to be cross reactors^{11, 12,13,14} and results from autoagglutinating strains have been omitted (n=283). The relative sensitivity was 96.5% and the relative specificity was 97.6%.

Industrial study
Oxoid Staphytect Plus was evaluated in food laboratories in a multi-centre study in the United Kingdom. A total of 621 samples was evaluated, these were colonies taken from Baird-Parker Agar, which had been inoculated with food or environmental material. The gold standard method was tube coagulase. In the following data analysis, results from strains known to be cross reactors^{11, 12,13,14} and results from autoagglutinating strains have been omitted (n=604). The relative sensitivity was 97.6% and the relative specificity was 97.1%.

WARNING: This product contains sodium azide. Harmful if swallowed.

References:
1. Essers, L. and Radebold, K. (1980). "Rapid and Reliable Identification of Staphylococcus aureus by a Latex Agglutination Test". J.Clin.Microbiol. 12: 641-643.
2. Taussig, M.J. (1984). Processes in Pathology and Microbiology 2nd Ed. 520-530. Blackwell,Oxford.
3. Ruane, P.J., Morgan, M.A., Citron, D.M. and Mulligan, M.E. (1986). "Failure of Rapid Agglutination Methods to Detect Oxacillin-Resistant Staphylococcus aureus". J.Clin.Microbiol. 24: 490-492.
4. Roberts, J.I.S. and Gaston, M.A. (1987). "Protein A and coagulase expression in epidemic and non-epidemic Staphylococcus aureus". J.Clin.Pathol. 40: 837-840.
5. Wanger, A.R., Morris, S.L. Ericsson, C., Singh, K.V. and LaRocco, M.T. (1992). "Latex Agglutination-Negative Methicillin-Resistant Staphylococcus aureus Recovered from Neonates: Epidemiologic Features and Comparison of Typing Methods". J.Clin. Microbiol. 30: 2583-2588.
6. Fournier, J.M., Boutonnier, A. and Bouvet, A. (1989). "Staphylococcus aureus Strains Which Are Not Identified by Rapid Agglutination Methods Are of Capsular Serotype 5". J.Clin.Microbiol. 27: 1372-1374.
7. Fournier, J.M., Bouvet, A. Boutonnier, A., Audurier, A. , Goldstein, F., Pierre, J., Bure, A., Lebrun, and L., Hochkeppel, H.K. (1987). Predominance of Capsular Polysaccharide Type 5 among Oxacillin-Resistant Staphylococcus aureus". J.Clin.Microbiol. 25: 1932-1933.
8. Karakawa, W.W., Fournier, J.M., Vann, W.F., Arbeit, R., Schneerson, R.S., and Robbins, J.B. (1985). "Method for the Serological Typing of the Capsular Polysaccharides of Staphylococcus aureus ". J.Clin.Microbiol. 22: 445-447.
9. Kloos, W.E., Jorgensen J.H.(1988). Staphylococci. pp.143-153. In Manual of Clinical Microbiology. 4th Edn. (Eds) Lennette, E.H., Balows, A.Hauser W.J., and Shadomy, H.J. Assoc.Amer.Microbiol. Washington.
10. Data on file at Oxoid Ltd.
11. Jean-Pierre, H., Darbas, H., Jean-Roussenq,. A. & Boyer, G.(1989). "Pathogenicity in Two Cases of Staphylococcus schleiferi, a Recently Described Species". J.Clin.Microbiol.27: 2110-2111.
12. Frenney, J., Brun, Y., Bes, M., Meugnier H., Grimont, F., Grimont, P.A.D.,Nervie, C., & Fleurette, J. (1988). "Staphylococcus schleiferi sp. nov. Two species from Human Clinical Specimens". Int.J.Sup Bacteriol. 38: 168-172.
13. Phillips, W.E., and Kloos, W.E. (1981). "Identification of Coagulase-Positive Staphylococcus intermedius and Staphylococcus hyicus. subsp. hyicus Isolates from Veterinary Clinical Specimens". Clin.Microbiol:14: 671-673.
14. van Griethuysen, A., Bes, M., Etienne, J., Zbinden, R. and Kluytmans, J. (2000). "An International Multicenter Evaluation of a new Latex Agglutination Test for Identification of Staphylococcus aureus". Clin Microbiol. and Infect. 6 sup 1: 163.
15. Schnitzler, N., Rainer, M., Conrads, G., Frank, D. and Haase, G. (1988). “ Staphylococcus lugdunensis: Report of a case of Peritonitis and an Easy-To-
Perform Screening Strategy”. J. Clin.Microbiol. 26: 1939–1949.
16. Ann-Herbert, A., Crowder, C. G., Hancock, G. A., Jarvis, W. R. and Thornsberry, C. (1998). “Characteristics of Coagulase-Negative-Staphylococci
That Help Differentiate These Species of the Family Micrococcaceae”. J. Clin.Microbiol. 36: 812–813.
17. Gregson, D. B., Low, D. E., Skulnick, M.and Simor, A. E. (1988). “Problems with Rapid Agglutination Methods for Identification of Staphylococcus aureus
When Staphylococcus saprophyticus Is Being Tested”. J. Clin. Microbiol. 26:1398–1399.
18. Myhre, E. B. and Kuusela, P. (1983).“ Binding of Human Fibronectin to Group A, C and G Streptococci”.Infect. Immun. 40: 29–34.
19. Runehagen, A., Schonbeck, C., Hedneru, Hessel, B. and Kronvall, G.(1981). “Binding of Fibrinogen Degradation Products to S. aureus and to ß-Hemolytic Streptococci Group A, C and G”. Acta. path. microbiol. Scand., Sect B. 89: 49–55.