1PASTOREX STREP 6172150 TESTSLATEX TEST FOR GROUPING STREPTOCOCCI BELONGING TO GROUPS A, B, C, D, F, AND G

INTRODUCTION

The identification of streptococci rests on evaluation of the type of haemolysis surrounding colonies grown on blood agar (,) andon the detection of group-specific polyoside antigens in the cell wall.Some groups of -haemolytic streptococci are consistently associated with clinical disease (A, C, G), whereas others are pathogeniconly when found outside their natural habitat (B, D).Identification of -haemolytic antigens on the basis of group-specific polysaccharide antigens (Lancefield, 1933) can be difficult fora number of reasons:

some strains have two group antigens (C and G); some -haemolytic strains exhibit the group C antigen;

some atypical strains produce haemolytic microcolonies exhibiting A, C, or G antigens (1).However, in most instances, prompt identification of the group allows to establish the diagnosis of streptococcal infections.Streptococci are responsible for a number of infections, including pharyngitis with or without septic complications, skin infections,puerperal sepsis (2), and endocarditis. Some diseases are caused by a specific group, such as rheumatic fever and acuteglomerulonephritis (group A); urinary tract, genital, and neonatal infections (septicemia, meningitis) (group B); postsurgicalinfections (group F); and septicemia associated with intravenous substance abuse (group G) (3). Group C streptococci, which arehighly pathogenic in animals, can produce severe infections in pediatric patients (endocarditis, meningitis) (4).

PRINCIPLE OF THE TEST

PASTOREX STREP is a rapid, sensitive agglutination test for grouping -haemolytic streptococci belonging to the main Lancefieldgroups. The test involves use of latex suspensions specific for groups A, B, C, D, F, and G.Identification of -haemolytic streptococci based on group-specific polysaccharides requires previous extraction of these antigensfrom colonies obtained by primary blood agar cultures. With the PASTOREX STREP system, this requires only 15 minutes at room temperature or ten minutes at 37C. Extraction isachieved by an active enzyme that causes lysis of the cell walls, releasing the polyoside C.In the presence of the antigen, the latex particles coated with homologous antibodies agglutinate very rapidly.The speed of agglutination depends on the sensitivity of the latex particle suspensions, which is governed by the quality of theantisera raised in rabbits using Lancefield's immunisation protocol and by the amount of purified immune globulins adsorbed on thelatex particles.

PRESENTATION

1) PASTOREX STREP A, B , C, D, F, G :Kit for 50 to 60 tests, code 61721, containing:

Six vials, each of which contains 1 ml of latex suspension, specific for group A, B, C, D, F, and G streptococci, respectively. Thelatex particles are coated with group-specific rabbit immunoglobulins in suspension in a glycine buffer, pH 8.2, containing 0.01%thimerosal and 0.1% sodium azide as the preservatives.Avoid contact with eyes, skin and mucosae. Sodium azide may react with lead or copper present in plumbing conduits and thusproduce explosive metallic azides. When eliminating, rinse abundantly with water to prevent azide deposits.

Two vials of freeze-dried extraction enzyme in TRIS, containing 0.01% thimerosal. The contents of the vial should be reconstitutedwith 10 ml distilled water; the solution thus obtained can be stored for four months between +2C and +8C.

One vial containing 1.5 ml polyvalent positive control antigen, composed of a mixture of Lancefield extracts of group A, B, C, D,F, and G streptococci and 0.02% thimerosal as the preservative. (Amount for five tests using each latex suspension).

Two x 125 rods Sixty disposable agglutination cards

2) Individual latex tests (50 to 60 tests) PASTOREX STREP latex A code 61726 PASTOREX STREP latex B code 61727 PASTOREX STREP latex D code 61728

3) PASTOREX STREP : extraction enzyme code 61729

2STORAGE

+2C to +8C.The expiry date of the reagents is printed on the boxes.IN VITRO USE

NECESSARY MATERIAL NOT SUPPLIED

Pipettes for dispensing 0.3 ml Haemolysis tubes (one per strain)

PRECAUTIONS

Resuspend each regent before use. Close the vials with the appropriate closure.

TEST PROCEDURE

1) PREPARATION OF THE SPECIMENSThe PASTOREX STREP system should be used on colonies grown on blood agar and surrounded by an area of -haemolysis.

Following the demonstration of Gram-positive cocci on microscopic slides and a negative catalase test, further identification bydirect grouping can be performed if a large enough number of primary culture colonies is available.

2) PREPARATION OF THE EXTRACTS Place 0.3 ml of extraction enzyme solution in a haemolysis tube for each strain isolated. Pick off approximately five colonies and dissociate them in the enzyme solution. If the diameter of the colonies is less than 0.5 mm,

increase the size of the inoculum until cloudiness is visible with the naked eye. Incubate

- either 15 minutes at room temperature- or ten minutes at 37C.

3) GROUPING OF THE EXTRACTS Resuspend the contents of the vials containing the latex particles by shaking them vigorously for a few seconds. Transfer one drop of each latex suspension to the centre of an agglutination card. (Hold the vial upright). Use a pipette to transfer one drop of extract to the centre of each agglutination card. Use a rod to homogenise the contents of each circle. Use a different rod for each circle and discard the rods in a contaminated

waste container. Rotate the card with horizontally for a maximum of one minute. Read the result. When the reaction is positive, the latex particles agglutinate within one minute at the most. The size and speed of

development of the clumps varies with the concentration of the antigen in the extract; this concentration varies with the numberand size of the colonies picked from the agar.

4) INTERPRETATION OF RESULTS Positive reaction: red clumps on a green background.Only marked, rapid agglutination with only one of the six latex suspensions convincingly establishes the group of the strain. Negative reaction: uniform brown suspension.

5) NON-SPECIFIC REACTIONS small clumps on a brown background; multiple agglutination can be caused by the presence of other bacteria harvested from the agar with the -haemolytic colonies

(mixture of streptococci from different groups or presence of other bacteria yielding cross-reactions). When a doubtful reaction ofthis type occurs, the isolation procedure should be repeated. Biochemical tests can be used to confirm the identification in someinstances, for instance when the strain exhibits both group C and group G antigens.

QUALITY CONTROL

1) QUALITY-CONTROL OF THE LATEX SUSPENSIONSThe sensitivity of the reagents is evaluated on the basis of reactivity with the positive control, which should cause markedagglutination of the corresponding latex suspension in less than one minute.

2) QUALITY-CONTROL OF THE ENZYMEThe activity of the enzyme solution can be tested using a strain whose group is known. The antigen extracted from this strain shouldpromptly agglutinate the corresponding latex suspension.

3) VERIFICATION OF SPECIFICITYThe enzyme extract used with the heterologous latex suspensions serves as the negative control test; furthermore, the latexsuspensions should remain homogeneous after addition of a drop of extraction enzyme solution.

BIBLIOGRAPHY

1.CIMOLAI N., ELFORD R.W., BRYAN L., ANAND C. and BERGER P.Do the -haemolytic Non-Group A Streptococci cause Pharyngitis - Reviews of Infections Diseases, 1988, 10, 587-601

2.KAVIT J. and WISERGroup A-haemolytic Streptococcus causing disseminated intra vascular coagulation and maternal death, The Lancet, 1988, I,993-994

3.Mc MEEKLING A.A., HOLZMAN R.S.,Group G Streptococcal Bacteremia and Parenteral Drug Abusers - The Journal of Infections Diseases, 1988, 157, 612-613

4.ARDITI M., SHULMAN S.T., TODD DAVIS A. and YOGEV R.Group C haemolytic streptococcal infections in children. Nine pediatric cases and review. Reviews of Infection Diseases, 1989,11, 34-35

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