Immunologic Study Of

Streptococcus

Laboratory test used : Anti-streptolysin

(ASO) test. [Latex Agglutination]

Background :
Streptolysins are hemolytic exotoxins
produced by Streptococci strains of
bacteria
Types 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
volunteer
Blood 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 C
Hemolysed , 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.

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

 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 acidcovered 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.