DRY SPOT STAPHYTECT PLUS

Intended Use
Dryspot 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 methicillinresistant 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 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 auerus 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.
Dryspot Staphytect Plus uses blue latex particles coated with both porcine fibrinogen and rabbit IgG including specific polyclonal antibodies raised against capsular polysaccharides of Staphylococcus aureus^7,8.
The reagent is dried onto the reaction card. When the reagent is mixed on the card with colonies of Staphylococcus aureus emulsified in saline 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
Dryspot Staphytect Plus Reagent Cards.
Blue latex particles coated with both porcine fibrinogen and rabbit IgG together with specific polyclonal antibodies raised against capsular polysaccharide of Staphylococcus aureus (Test Reaction Area).
Blue latex particles sensitised with non-reactive globulin (Control Reaction Area).
4 pouches each containing 10 cards and a moisture absorbent sachet. There are 3 test and 3 control reaction areas on each card. 120 tests in total.
Plastic pouch clip for storage of opened pouches.
Instruction leaflet.

Materials required but not provided:
Saline (0·85%)
Timer
Pipette or dropper (50 µl)
Sterile Microbiological Loops
A suitable laboratory disinfectant
Positive Control: S. aureus strain such as ATCC®25923
Negative Control: S. epidermidis strain such as ATCC®12228.

Precautions
This product is for in vitro diagnostic use only.
Specimen material may contain pathogenic organisms. Handle with the appropriate precautions.

Storage and Opening
This kit must be stored between 2-25°C. If stored in a cold environment, allow the pouches to reach room temperature before opening to prevent condensation of moisture on the cards. The Dryspot reagents will deteriorate and may give false results if they are allowed to absorb moisture.
Open the pouches by cutting with scissors just below the seal. Once opened remove the number of cards required for immediate testing (testing within the next 10 minutes) and reseal the pouch immediately by clamping the open end of the bag between the two halves of the plastic clip.
If fewer tests are required cut the reaction cards along the indicated lines and return the unused portions to the pouch. Do not return used portions to the pouch because they will cause contamination of remaining cards in the pouch.
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 Loop 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 Loop 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 touch the circles on the reaction cards as this may cause contamination and affect the reaction.
In a high humidity environment do not leave pouches open for more than 2 minutes. If there is evidence of moisture in the spots do not use.
Do not place the drop of saline directly onto the dry latex spots.
Pouch clips can be retained for future use to allow multiple packs to be opened. Although suitable for room temperature storage the kit or pouches must not be stored near a heat source or where exposure to sunlight may cause increased temperatures.
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. Add 1 drop (50 µl) of saline (0·85%) to the small rings (at the bottom of each oval) in both the test and control reaction areas ensuring that the liquid does not mix with the dried latex reagents.
2. Use a sterile loop to pick-up the equivalent of 5 average-sized suspect staphylococcal colonies (equivalent to 2–3 mm diameter of growth) from a culture media plate and carefully emulsify in the saline drop. Ensure that the resulting suspension is smooth.
3. Mix the suspension into the dry Control Latex spots until completely suspended and spread to cover the reaction area using the loop. 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 reaction cards safely into disinfectant.

Test Method for Oxacillin Resistance Screening Agar
1. Use a sterile loop to pick-up the equivalent of 5 average-sized suspect staphylococcal colonies (equivalent to 2–3 mm diameter of growth) from a culture media plate and smear onto the Control Reaction Area. Using the loop, spread the colony material into a thin film keeping the colony material clear of the dried latex reagents.
2. Dispense 1 drop of saline (0·85%) directly onto the thin film to form a smooth suspension and mix IMMEDIATELY.
3–6. 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 compared with the reaction of the Control Reagent.
The result is negative when there are no differences between 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 ID0580M), Staphylococcus aureus and Staphylococcus hyicus will be PYRase negative and all the other strains named above will be positive^15,16. Staphylococcus hyicus and Staphylococcus intermedius are rarely encountered in the clinical laboratory.
4. Staphylococci isolated from urine specimens which give a weak positive¹⁷ 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^18,19 non-specifically. To overcome these non-specific results a Gram-stain should be performed
so only typical staphylococci are tested.

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

Clinical Study
Oxoid Dryspot Staphytect Plus was evaluated at a large Australian teaching hospital. A total of 300 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=284). The relative sensitivity was 100% and the relative specificity was 96·3%.

Industrial Study
Oxoid Dryspot 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=603). The relative sensitivity was 98·4% and the relative specificity was 96·9%.

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. Freney, J., Brun, Y., Bes, M., Heugnier H., Grimont, F., Grimont, P.A.D., Nervie, C., & Fleurette, J. (1988). "Staphylococcus lugdunensis sp. nov. and 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". J.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 sup1:163.
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, U., 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.