aks ppt

Immunologic Study Of Streptococcus

Laboratory test used : Anti-streptolysin (ASO) test. [Latex Agglutination]

Background :

Streptolysins are hemolytic exotoxins produced by Streptococci strains of bacteriaTypes include Streptolysin S which is Oxygen stable and Streptolysin O which is Oxygen labile.Streptolysin O is produced by many strains of bacteria particularly Streptococcus Pyogenes.

Main function : Hemolysis.Biochemically , it is highly antigenic and immunogenic thereby resulting in the formation of specific antibodies i.e. Antistreptolysin O by the affected individual in an effort to prevent the hemolysis.

Principle :The latex(polystyrene) particles used in the test are coated with purified and stabilized Streptolysin O.When the above latex suspension is mixed with serum containing elevated levels of ASO antibodies , clear agglutination is seen within 2 minutes.

Resources required :ASO test kit (AVITEX ASO Test kit used here)Serum of patient or student volunteerBlood extraction kit (syringe, needle, spirit, cotton , tourniquet)Test tubes

The Avitex ASO test kit

Procedure :Specimen Preparation and collection:Obtain a venous blood sample from the patient (here , student volunteer) and allow a clot to form.Centrifuge the clotted blood sample and collect clear serum.Serum can be stored at 2-8 degree C for upto 48 hours prior to testing.

Longer storage for 6 weeks maximum is possible at 20 degree CHemolysed , contaminated or lipaemic serum samples should be avoided as it may affect the results.

Latex Reagent

ASO latex reagent is a stabilized buffered suspension of polystyrene latex particles that have been coated with Streptolysin O.The latex when mixed with serum of a patient containing elevated ASO antibodies causes agglutination which helps in identyfying the presence of bacteria.

Reagent Preparation :Reagents should be brought to room temperature and mixed gently to resuspend latex prior to use.Foaming should be avoided.The test slide must be thoroughly cleaned as traces of detergent or previous specimen may affect the results.The reaction circles must be physically rubbed to be cleaned.

Assay Procedure :Allow test reagents and serum to come to room temperature.Transfer one drop (50 microlitre) of the collected serum to the test circle on the slide.

Mix the drops using the provided stirrer ensuring coverage of test circle with the mixture.

Shake the latex reagent and add the suspension to the slide using the dropper provided.

Rock and rotate the test slide gently and evenly tracing figure 8 motion for 2 minutes whilst examining for any signs of agglutination.

When the test slide is examined after 2 minutes The kit negative control should give a negative result after two minutes.

Result :

The kit positive control should give a positive result at a titre of 1/4th +- double dilution after two minutes.

In our case , the result came out to be positive. (Test circle 1)

Inference :A positive result is indicated by obvious agglutination pattern of the latex , in a clear solution.

Agglutination

A negative result is indicated by no change in the latex suspension on the test slide.

A positive result will be obtained at a ASO concentration of 200 IU/ml or more in the patient's serum.A negative result will be obtained at a ASO concentration of less than 200 IU/ml in the patient's serum.

Questions :Explain the occurence of agglutination.

Streptolysin O is a exotoxin produced by Streptococcus Pyogenes. It being highly antigentic and immunogenic makes the host produce antibodies namely Antistreptolysin O against the Streptolysin in an effort to prevent hemolysis caused by Streptolysin.

The latex particles are coated with Streptolysin O and if the collected serum contains ASO antibodies , agglutination takes place.

At which titre is considered abnormally high? Two reasons for an abnormally high titre.

An ASO serum titer above 160-200IU/ml is considered abnormally high.The reasons for an abnormally high titer can be A recent Streptococci infection.Persistently high antibody levels caused by an exaggerated immune response to an earlier exposure in a hypersensitive person.

Enumerate 3 clinical conditions associated with high ASO titer and give short descriptions.

Streptococcal cellulitis : Acute , rapidly spreading infection of the skin and subcutaneous tissues . Follows infections associated with burns, wounds , surgical incisions etc. Pain ,tenderness, swelling and erythema occur.

2. Streptococcal Pharyngitis:S. pyogenes adheres to the pharyngeal epithelium by means of lipoteichoic acid-covered surface pili or by hyaluronic acid in encapsulated strains. In infants - subacute nasopharyngitis with a thin serous discharge and fever.In adults - more acute and characterized by intense nasopharyngitis ,tonsillitis , intense redness and edema of mucous membranes.

3. Rheumatic fever : Most serious sequela of S pyogenes because it results in damage to heart muscle and valves.Symptoms include fever, malaise , a migratory nonsupparative polyarthritis and evidence in inflammation in all parts of the heart.