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STREPTOCOCCAL GROUPING KIT

A latex agglutination test for the identification of streptococcal groups A, B, C, D, F, and G.
Lancefield showed that the majority of pathogenic streptococci possess specific carbohydrate antigens, which permit the classification of streptococci into groups¹. These streptococcal group antigens can be extracted from the cells and their presence demonstrated with latex particles previously coated with group-specific antibodies. These latex particles will agglutinate in the presence of homologous antigen, but will remain in smooth suspension in the absence of such
antigen. The Oxoid Streptococcal Grouping Kit is such a latex agglutination test for the identification of the streptococcal group, and reagents are provided for groups, A, B, C, D, F, and G. The use of a new enzymatic extraction procedure considerably shortens the time required for antigen extraction and much improves the antigen yield, particularly for Group D streptococci.

Reagents
DR0586 Latex Grouping Reagent A
DR0587 Latex Grouping Reagent B
DR0588 Latex Grouping Reagent C
DR0589 Latex Grouping Reagent D
DR0590 Latex Grouping Reagent F
DR0591 Latex Grouping Reagent G
DR0592 Polyvalent Positive Control
DR0593 Extraction Enzyme
DR0500 Disposable reaction cards

Precautions
These reagents are for in vitro use only. Do not freeze the latex grouping reagents.

Working Reagents
Each latex reagent is ready for use after reaching room temperature.
It is essential that the latex reagent is vigorously shaken to obtain a homogenous suspension before use.
When required for use, the enzyme reagent should be reconstituted with distilled water to the amount shown on the label. The positive control contains extracts from all six group antigens.

Preservatives
Each latex reagent contains 0.1% sodium azide.
The positive control reagent contains 0.1% sodium azide.
After reconstitution, the extraction enzyme solution contains 0.01% thiomersal.

Storage

A. Latex Reagents
The latex reagent bottles should be stored in an upright position at 2-8ºC. Under these conditions they will retain their activity until the date shown on the bottle label.

B. Extraction enzyme
The freeze-dried extraction enzyme should be stored at 2-25ºC. Under these conditions it will retain its activity until the date shown on the bottle. After reconstitution with distilled water, store the solution at 2-8ºC. Under these conditions it will retain its activity for four months.

C. Positive control
Store the polyvalent positive control at 2-8ºC. It will retain its activity until the date shown on the bottle.

Preparation of Cultures
Samples for identification should be grown on a blood agar plate overnight at 37ºC. Note the haemolytic reaction of suspect colonies. It is also advisable to carry out a Gram stainand catalase test to confirm the presence of Gram-positive, catalase-negative cocci. For further details, please consult standard texts².
For each culture to be grouped:

1. Reconstitute a bottle of Oxoid Streptococcus Extraction Enzyme (DR0593) with sterile distilled water to the amount shown on the label. Label test tubes
appropriately and dispense 0.4 ml of enzyme into each test tube.
2. Select 2-5 test colonies equivalent to 2-3 mm of growth with a bacteriological loop and emulsify in the enzyme preparation. If the culture is mixed, avoid obvious contamination.
3. Incubate for 10 minutes at 37ºC in a water bath. After 5 minutes incubation it is important to remove each tube and shake vigorously for 2-3 seconds, then
continue the incubation. Remove and allow to cool to room temperature. The extract is now ready for use.

Test Method
1. Bring the latex reagents to room temperature by warming the bottles by hand. Make sure the latex suspensions are mixed by vigorous shaking. Expel any
latex from the dropper pipette for complete mixing.
2. Dispense 1 drop from each latex reagent into the circular rings on the reaction card (DR0500).
3. Using a Pasteur pipette, add 1 drop of extract to each of the 6 rings.
4. With the mixing sticks provided, spread the mixture over the entire area of the ring using a separate stick for each ring.
5. Gently rock the card. Agglutination in 1 or more of the rings will normally take place within 30 seconds. Do not rock the card for more than 1 minute. Do not use amagnifying glass to aid reading.
6. The positive control may be used as above to check performance of latex reagents.
7. Dispose of the Reaction Card safely into a suitable disinfectant.
N.B. If fewer texts are to be performed the cards may be cut with scissors and the unused portions saved for future use.

Interpretation of Results
The test should be considered positive when agglutination occurs with one grouping reagent or when one grouping reagent gives a substantially stronger reaction than the other five. The test should be considered negative when no agglutination occurs.
Faint traces of granular material may be observed in negative reactions and should be ignored.

Limitations of the Test
False negatives can occur if an inadequate amount of culture is used for extraction.
Nearly all the beta-haemolytic streptococci isolated from the human infections possess specific carbohydrate antigens which can be recognised by serological reactions.
Attempts to extend these procedures to non beta-haemolytic streptococci have been unsuccessful except for groups B, D and N. Group N streptococci are not found in human infections⁵.
It should be noted that the Group D reagent may fail to react with some Streptococcus bovis strains and these strains would require further tests for identification.
The following flow chart describes the recommended procedure for identifying streptococci when using the Oxoid latex agglutination test.
When carrying out a serological identification of streptococci the following initial observations should be made, (i) note haemolysis^a,c, (ii) note cell morphology^b,c, (iii) assess colonial growth for purity and quantity^d.

Negative reaction on slide
Beta-haemolytic		alpha or non-haemolytic
Repeat extraction with heavier suspension		Bile-aesculin/6.5% NaCi both
Repeat test	+/+ Group D enterococcusb	+/- Group D non-enterococcus	-/- Viridans streptococci
If negative, report ‘Not group A, B, C, D, F or G’

Positive reaction on the slide
Beta-haemolytic						alpha or non-haemolytic
Reacts solely in A, B, C, F or G		Reacts in Group D		Positive in more than one group			Reacts in Group B		Reacts in Group D		Reacts in A, C, F or G
Report group		Test in 6.5% NaCI broth		Subculture and re-test			Report non-haemolytic group B		Bile-aesculin/ 6.5% NaCI broth		Biochemical identification required
+ growth Report Group D enterococcus		- growth Report Group D non- enterococcus		Biochemical identification if problem not resolvede			+/+ Group D enterococcusb		+/- Group D non-enterococcus		-/- Viridans streptococci

(a) Rule out Streptococcus pneumoniae. This streptococcus is a-haemolytic, bile soluble and optochin susceptible. Other streptococci are not bile soluble and are optochin resistant⁵.
(b) Aerococci are non ß-haemolytic, grow in 6.5% NaCI broth and give variable reactions in the bile-aesculin test. They can be differentiated from enterococci by their arrangement in tetrads or as single cells, whereas enterococci are arranged as diplococci or short chains⁵.
(c) Staphylococci and Listeria monocytogenes are ß-haemolytic and can be distinguished from streptococci by their cellular morphology and catalase reaction^6,7.
(d) Subculture, if the suspected organism is overgrown or insufficient.
(e) Strains have been found which appear to have both D and G antigens^3,4.

Performance Characteristics
The extraction enzyme reagent formulation has recently been improved. The performance of Oxoid Streptococcal Grouping Kit with the new Enzyme was
evaluated at one trial centre in South Australia The table below shows the results obtained.

Sensitivity and specificity of Streptococcal Grouping Kits

* All strains were tested with each of the six grouping reagents
1 A number of Lancefield Group D organisms yielded a D/G reaction. These results have been included in the calculations as true positive Group Ds
and false positive Group Gs. It is acknowledged in literature that Group D strains exist which also yield G antigens upon enzymatic extraction.
WARNING: This product contains sodium azide. Harmful if swallowed.

References
1. Lancefield R.C. (1938) Proc. Soc. Exp. Bio. Med. 38, 473.
2. Facklam R.R. (1980) in ‘Manual of Clinical Microbiology’, 3rd Edition. American Society for Microbiology, Washington, D.C., pp. 88-110.
3. McIllmurray M.B. (1984) Lancet, I, 1353.
4. Birch B.R., Keaney M.G.L. and Ganguli L.A. (1984) Lancet, I, 856-857.
5. Facklam R.R. and Carey R.B. (1985) in ‘ Manual of Clinical Microbiology’, 4th Edition. Eds. Lennette E.H., Balows A., Hausler W.J., Shadomy H.J., Amer. Soc. for Microbiol., Washington, D.C., pp. 154-175.
6. Kloos W.E. and Jorgensen J.H. (1985) in ‘Manual of Clinical Microbiology’, 4th Edition, pp. 143-153.
7. Bortolussi R., Schlech W.F. and Albritton W.L. (1985) in ‘Manual of Clinical Microbiology’, 4th Edition, pp. 205-208.