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are cell wall antigens of Lancefield group A Gram positive (+) cocci, catalase negative (-)

hemolytic
divided into serogroups A through O contains an important surface component called FIMBRIAE

arise near the plasma membrane and project through the cell wall and capsule contains LIPOTECHOIC ACID important for adherence to human epithelium for initiation of infection

M Antigens
major virulence factor
strongly anti-phagocytic

contains hyaluronic acid in its capsule


could aid in the classification of the specific strain causing the infection

R Antigens
no known biologic role

Upper Respiratory Infection:


Cervical adenopathy Streptococcal pharyngitis Scarlet Fever Skin Infections

depends on the AGE of the subject

Upper Respiratory Infection:


INFANTS and YOUNG CHILDREN: rhinorrhea runny nose coughing fever vomiting anorexia

Scarlet Fever

pharyngeal infection caused by erythrogenic toxin by GAS produces characteristic rash develops on the 2nd day of illness results in hyperkeratosis with subsequent peeling

Scarlet Fever characteristics: Sore throat


Fever Bright red tongue with a strawberry appearance

Scarlet Fever Rash:


most striking sign

fine, red, and rough-textured


appears 1248 hours after the fever
generally starts on the chest, armpits, and behind the ears

Skin Infection
Impetigo lesion that itches then crusts over and heals Cellulitis subcutaneous infection - characterized by warm, red, tender area that is slightly swollen

Skin Infection
Erysipelas distinct cellulitis syndrome - involves the face - associated with pharyngitis - characterized by toxicity and high fever - fatal if untreated

Acute Rheumatic Fever


caused by M serotypes infects the throat prognosis is good upon initial infection if carditis is absent develops after 2 - 3 weeks after infection common in children between the age of 6 and 15 similar to rheumatism

Glomerulonephritis
may occur after pharyngitis or skin infection inflammation of the glomeruli - small blood vessels in the kidney Primary causes are ones which are intrinsic to the kidney Secondary causes are associated with certain infections

Streptolysin O (SLO)
bacterial strains produced by

Streptococcus pyogenes

released during infection which is an indication of the production of antibody in reduced rate causes the lysis of the red and white blood cells

Streptolysin O (SLO)
oxygen labile hemolytically inactive in oxidized form

are activated by sulfhydryl (SH) compounds antigenically related

Streptolysin O (SLO)
low concentrations of cholesterol and related sterols is inhibited by their biological activity

hemolysis od erythrocyte occurs within minutes after addition of Streptolysin O

Streptolysin O (SLO)
Cardiotoxic may cause interstitial myocarditis in experimental animal - causes systolic arrest of perfused mamalian heart - caused by inducing the release from the atria of ACETYLCHOLINE, that poisons the ventricles

Streptolysin O (SLO)
f site - 2 cystine residues - responsible for the attachment of the molecule to red blood cell t site - hemolytic event

Streptolysin O (SLO)
membrane cholesterol binding site exogenous cholesterol inhibits toxic action

alfalfa saponin or filipin treatment of erythrocyte membrane - Inhibits absorption of SLO

Anti-Streptolysin O
-valuable and reliable indicator of Streptococcal infection

Streptolysin O
- antigenic - elicits formation of antibody that effectively neutralize hemolytic reaction

Streptolysin O
Oxygen labile Antigenic Synthesized by growing Streptococcus

Streptolysin S
Oxygen stable Non antigenic peptide Sythesized both by growing and resting cells

Not inhibited by cholersterol


Red cells with SLO: shows distinct lesion

Inhibited by lecithin and lypoproteins


Red cell with SLS: show no lesion

Causes necrosis of the liver and kidney tubules and massive intracellular hemolysis upon injection intravenously
Induce chronic arthritis upon injection intra- articularly

Neutralization test

detect ASO in serum ASO can be specifically fixed to SLO, in vitro hemolytic activity is neutralized by doubling dilution, estimates the amount of antibody with the presence of a constant dose of SLO, completely inhibits hemolysis

Variations: age

severity of infection previous exposure to Streptococcal infection individuals ability to respond immunologically to the toxin

ASO Titers

in children's, fluctuates from 5 125 Todd units 30% rise over the previous level is considered significant. increases due to rheumatic fever and glomerulonephritis is seen during symptomfree period preceding attack of the illness

ASO Titers

in rheumatic fever 300 to 2, 500 Todd units - maintained at high levels for 6 months increased amount is found in: -Scarlet fever - Cholera minor - Tuberculosis disease - Pneumococcal pneumonia - gonorrhea

defines a minimal hemolytic dose of SLO as

that amount of toxin will completely hemolyze 0.5 ml of a 5% suspension of rabbit blood cells, measured in Todd units

MATERIALS

Saline 0.85% Streptolysin O buffer: 7.4 gm sodium chloride 3.17 gm potassium 1, 081 gm sodium phosphate add 1, 000 ml of distilled water

final pH = 6.5 6.7 buffer is stored at 4C until 1 week

STREPTOLYSIN O

if in dehydrated form must be rehydrated before use once rehydrated the solution should not be subjected to vigorous shaking, and must be used within a period of 1 hour or discarded active reagent is subject to inactivation by oxidation

RED BLOOD CELL

5% suspension of fresh human RBC (grp O) Rabbit BBC equally sensitive to SLO cells must be washed 3 times in diluent buffy coat must be removed Final centrifugation - 1, 500 rpm for 10 minutes Packed RBC = 5% suspension Final suspension in SLO buffer

TEST TUBE
must be 12 x 100 mm round bottom

PROCEDURE:
1. Prepare dilutions of fresh or inactivated serum, using SLO buffer as diluent:

1:10 - 0.5 ml of serum + 45 ml of buffer


1:100 1.0 ml of 1:10 serum dilution + 9.0 ml of buffer 1:500 2.0 ml of 1:100 serum dilution + 8.0 ml of buffer

PROCEDURE:
1. Prepare dilutions of fresh or inactivated serum, using SLO buffer as diluent:
1:10 - 0.5 ml of serum + 45 ml of buffer

1:100 1.0 ml of 1:10 serum dilution + 9.0 ml of buffer


1:500 2.0 ml of 1:100 serum dilution + 8.0 ml of buffer

The first 2 serum dilutions are usually sufficient for preliminary titrations

PROCEDURE:
2. Set up the test according to the protocol given TABLE 9.1 PAGE 188

INTERPRETATION:
the ASO titer expressed in Todd units is the reciprocal of the serum dilution that completely neutralizes the SLO.
before reporting results, always ensure that the controls give the expected results

PRINCIPLE:if polystyrene latex particles are coated with SLO antigen, visible agglutination will be exhibited in the presence of the corresponding ASO antibody

MATERIALS ASO latex reagent coated with SLO -store at 2 to 8

0.9% NaCl solution -contains sodium azide as preservative Positive (+) control serum - contains at least 200 U/ml of ASO - should exhibit visible agglutination at the end of 3 min test period

MATERIALS
Negative (-) control serum -Prediluted serum with less than 100 U/ml of ASO
Glass slides with 6 wells

ADDITIONAL MATERIALS
1. 2. 3. 4. 5. 6. 7. Applicator sticks Timer 12 x 75 mm test tubes Pasteur pipettes and rubber bulb Serologic pipettes and safety bulb 50 l disposable pipettes and safety bulb High-intensity direct light

PROCEDURE Pipette 1 ml of saline into each test tube

Add 2 drop of patient serum Cover the tube and mix the dilution thoroughly by inverting the tube several times Label 1 division of the 6-cell slide for the positive control, negative control, and the respective patient sera Pipette 50 l of the controls and patient sera

PROCEDURE Add 1 drop of latex agent

Mix each specimen with a separate applicator stick. Spread the mixture evenly on the cell. Rotate the slide for exactly 3 minutes. Examine immediately with a bright source of direct light

INTERPRETATION

AGGLUTINATION indicates a positive result


NO AGGLUTINATION

-demonstrates 200 U/ml or more ASO indicates a negative result

INTERPRETATION Positive result should be retested


quantitatively Semiquantitative testing - the U/ml of the highest dilution of serum to produce visible agglutination is the reported value

DISCUSSION
False Positive reactions - result of bacterial contamination - if the reaction is absorbed after 3 minutes - Lipemic serum or plasma

DISCUSSION
RHEUMATIC FEVER or GLOMERULONEPHRITIS - titer with 200 U/ml or greater
elevated titer should be retested over a period of 4 to 6 weeks to plot the course of titer