Anti-streptolysin O Titer (ASOT): Principle, Procedure, and Results

Most people infected with group A streptococci produce anti-streptolysin O (ASO) antibodies. In infections caused by β-hemolytic streptococci,  streptolysin-O is one of the two hemolytic exotoxins liberated from the bacteria that stimulate the production of  ASO antibodies in the human serum.  The presence and the level of these antibodies in the serum may reflect the nature and severity of the infection. Rheumatic fever and poststreptococcal glomerulonephritis tend to occur 2 to 3 weeks after the initial streptococcal infection.

Streptolysin O is an enzyme that lyses RBC membranes and is responsible for the zone of beta-hemolysis around colonies of group A streptococci seen on agar plates. Classically, ASO antibodies were detected by demonstrating serum’s capacity to inhibit or neutralize the lysis of RBC membranes by streptolysin O. The test principle, procedure, and interpretation presented here is based on the latex anti-streptolysin O (ASO) test.  

Assays for anti-DNase B (ADB) antibodies are also available to diagnose group A streptococcal infection. ADB test detects antibodies against the B isoenzyme of streptococcal DNases (also called streptodornase), a group of bacterial enzymes that depolymerize DNA.

Principle

The ASO titer (ASOT) tends to rise a week following infection, peaks at 3 to 5 weeks, begins to fall at 8 weeks, and returns to preinfection levels at around 8 months. Stronger ASO responses tend to occur with throat infection than skin infection, possibly because free cholesterol present in skin binds to streptolysin O, thereby decreasing its immunogenicity. Qualitative and semi-quantitative determination of anti-streptolysin-O antibodies (ASO) in serum can be performed using a rapid latex agglutination test.

ASO latex reagent is a stabilized buffered suspension of polystyrene latex particles coated with Streptolysin O. When the latex reagent is mixed with a serum containing ASO, agglutination occurs.

Ideally, titers should be measured in both the acute and convalescent phases (2 to 4 weeks apart) to observe a rise in titer. However, single titers are frequently used in low-resource settings.

The sensitivity of the latex reagent has been adjusted to yield agglutination when the level of ASO is greater than 200 IU/ml, a level determined to be indicative of disease by epidemiological and clinical studies. Sera with titers between 200 IU/ml and 3500 IU/ml are reactive.

Sample Collection and Handling

Only fresh serum specimens should be used. Plasma must not be used since fibrinogen may cause non-specific agglutination of the latex. It is preferable to test samples on the same day as collected. Serum samples may be stored at 2-8°C for up to 48 hours prior to testing. If longer storage is necessary, sera should be stored frozen at -20°C.

Materials used in the ASO Test

1. ASO antigen: A stabilized buffered suspension of polystyrene latex particles coated with Streptolysin O and 0.1% sodium azide as a preservative. Shake well prior to use.
2. ASO positive control: Human serum-containing more than 200 IU/ml ASO and 0.1% sodium azide as a preservative.
3. ASO negative control: Human serum containing 0.1% sodium azide as a preservative.
4. Sufficient disposable pipettes.
5. Glass test slide.

Procedure for Antistreptolysin O Test

1. Bring all test reagents and samples to room temperature.
2. Use a disposable pipette to draw up and place one free-falling drop of each undiluted sample into its identified circle of the slide. Retain each pipette for mixing in step 5.
3. Deliver one free-falling drop of positive and negative control into its identified circle.
4. Mix the ASO latex reagent by gently shaking. Add one free-falling drop of a reagent to each control and sample.
5. Using the flattened end of the appropriate plastic pipette as a stirrer (step 2), thoroughly mix each sample with reagent within the full area of the circle.
6. Discard the disposable pipette.
7. Slowly rock the slide for exactly two (2) minutes and observe for agglutination under a high-intensity light.
8. Record results.
9. Re-wash glass slide for future use

Test Result

A test sample is considered to contain ASO antibodies in excess of 200 IU/ml when agglutination (clumping) is observed when compared to the result of the negative control.

Interpretation

ASO titer may be interpreted either by comparison of acute and convalescent-phase titers or against reference upper limit of normal values. Rise in titer (≥ twofold) from the acute phase to the convalescent phase is the best evidence of antecedent infection with group A streptococci. In the area, where rheumatic fever or poststreptococcal glomerulonephritis is endemic, a single initial titer more than (>) upper limit of normal value is diagnostic.  

As group A streptococcal infection is more common in children than adults, age-stratified upper limit of normal reference values should be used while interpreting the serological results. ASO titer should be interpreted with considerable caution, especially in the absence of a convincing history of rheumatic fever or glomerulonephritis.

Limitations

• False-negative ASO results can occur for patients with hyperlipidemia.
• False-positive ASO results may occur due to cross-reactivity in patients with myeloma, hypergammaglobulinemia, liver disease, and autoimmune disease with elevated rheumatoid factor.