Antistreptolysin O: Quantification, Rationale, Technique, Pathologies

The O ASO is antibody generated in response to an infection caused by the bacteria Streptococcus pyogenes , also known as Streptococcus beta-hemolytic A. This group produces two exotoxins with hemolytic activity calls streptolysins “S” and “O” .

Streptolysin S is responsible for the beta hemolysis that occurs in blood agar and, although it is toxic to certain cells of the immune system, it is not antigenic. While streptolysin O, being labile against oxygen, is responsible for the hemolysis that occurs below the blood agar and this is antigenic.

Streptococcus pyogenes / Diagram of what happens in the ASTO test (agglutination reaction). (Antibodies bound to latex particles that contain the specific antigen). Source: User: Graham Beards [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)] ]/Alejandro Porto [CC BY-SA 3.0 (https://creativecommons.org/licenses/ by-sa / 3.0)]

Therefore, when the cells of the immune system interact with streptolysin O, a specific immune response is produced, which generates the activation of B lymphocytes. These cells produce antibodies directed against streptolysin O. Therefore, the antibodies are called antistreptolysin O.

The Streptococcus pyogenes  produces various pathologies, among which are: tonsillitis, erysipelas, impetigo, puerperal fever, scarlet fever , and sepsis. Anti-streptolysin O antibodies appear 8 to 30 days after the onset of infection.

Most of these infections are very common in the population, so it is common for people to have antistreptolysin O antibodies in their blood. Low titers indicate a past infection with this bacterium, but a high or rising titre indicates a recent or ongoing infection.

Article index

  • one

    Quantification of antistreptolysin O antibodies

  • two

    ASTO Technique Basis

  • 3

    Techniques

    • 3.1

      – Semi-quantitative technique

    • 3.2

      – Quantitative technique

  • 4

    Pathologies with high titers of antistreptolysin O

    • 4.1

      Post streptococcal autoimmune diseases

  • 5

    References

Quantification of antistreptolysin O antibodies

In the laboratory, the antistreptolysin “O” antibody titer can be measured through a serological test. The antistreptolysin O (ASTO) test is based on an agglutination reaction with latex.

It can be done semi-quantitatively, reporting in crosses or the titer can also be quantified. It is normal and not significant to find values ​​up to 200 IU / ml or Todd units / ml. 
Above this value it is considered positive and is clinically significant.

This test does not require the patient to be fasting. Serum is used as a sample, that is, the patient’s blood is drawn and placed in a tube without anticoagulant, then it is centrifuged to obtain the serum.

ASTO Technique Basis

The technique uses latex particles as a support to fix the streptolysin O antigen. 
The absorbed antigen particles are reacted with the patient’s serum. If the patient has antistreptolysin O antibody, these will bind to the antigen attached to the latex particle.

This binding causes agglutination that is macroscopically visible. The intensity of the reaction is directly proportional to the concentration of antibodies present.

Techniques

– Semi-quantitative technique

The intensity of the reaction can be semi-quantified in crosses. 
To do this, a serological reaction plate is taken and placed:

50 µl of serum and 50 µl of ASTO reagent. Mix well with a wooden toothpick and place in an automatic mixer for 2 minutes. Observe. 
If an automatic rotator is not available, it must be done manually.

Interpretation

Lump-Free Suspension (Uniform): Negative

1. + = weak reaction

2. ++ = slight reaction

3. +++ = moderate reaction

4. ++++ = strong reaction

Sera that are positive with 3 and 4 crosses can be quantified.

– Quantitative technique

For the quantification of the titer, serial dilutions 1: 2, 1: 4: 1: 8, 1:16 are made.

To do this, proceed as follows: 4 test tubes or Kahn tubes are taken and 0.5 ml of physiological saline is placed on all of them. Then 0.5 ml of patient serum is added to the first tube. It mixes well. That tube corresponds to the 1: 2 dilution.

Subsequently 0.5 ml are transferred to tube 2 and mixed well. This tube corresponds to the 1: 4 dilution and so on, until the desired dilution is reached.

Take 50 µl of each dilution and react with 50 µl of ASTO reagent on an agglutination plate, as explained in the semi-quantitative technique.

Interpretation

The highest dilution in which visible agglutination is observed is taken into consideration. The calculations are carried out as follows:

ASTO = Inverse of the highest positive dilution x the sensitivity of the technique (constant).

Example: Patient with positive reaction up to 1: 8

ASTO = 8 x 200 IU / ml = 1600 IU / ml or Todd Units / ml.

Reference value

Normal adult: up to 200 IU / ml

Normal children: up to 400 IU / ml

recommendations

It is recommended that a positive and a negative control be mounted with the patients to ensure that the reagent is in optimal condition. If the positive control does not agglutinate or the negative control agglutinates, the reagent cannot be used.

The reaction must be interpreted after 2 minutes, after this time it is not valid if there is agglutination. These are false positives.

Hyperlipemic sera interfere with the reaction. They can give false positives.

An isolated ASTO value is not very helpful. It must be accompanied by the symptoms.

In addition, it is advisable to perform at least 2 ASTO measurements when rheumatic fever or post-streptococcal glomerulonephritis is suspected, this in order to demonstrate the increase in antistreptolysin O levels and thus confirm the diagnosis.

Pathologies with high titers of antistreptolysin O

Antistreptolysin antibodies are increased after suffering a Streptococcus pyogenes or group A ß-hemolytic Streptococcus infection .

These include: acute pharyngitis, scarlet fever, impetigo, erysipelas, puerperal fever, and septicemia.

Some patients who have suffered from recent or recurrent streptococcal infections may develop autoimmune diseases as a sequela or complication of post-streptococcal infection, such as acute glomerulonephritis and rheumatic fever.

Post streptococcal autoimmune diseases

Rheumatic fever

It is an inflammatory complication or sequela that can appear 1 to 5 weeks after having a streptococcal infection. Antistreptolysin O titers increase significantly 4 to 5 weeks after disease onset.

A high ASTO titer guides the diagnosis, but is not related to the severity of the disease, and its decrease does not imply improvement.

Antistreptolysin O antibodies cross-react against collagen and muscle fibers, affecting certain organs (heart, skin, joints and nervous system, among others).

This complication or sequela occurs with cardiac involvement, fever, malaise, non-suppurative migratory polyarthritis, chorea, etc.

Acute glomerulonephritis

Acute glomerulonephritis is a non-suppurative sequela that occurs due to the deposition of antigen-antibody complexes on the glomerular basement membrane.

The formation and circulation of antigen-antibody complexes (ag-ac) generated by streptococcal infections can lead to exudative changes and inflammation of the glomeruli, with proteinuria and hematuria.

These ag-ac complexes are deposited in the glomerulus and activate the complement cascade, resulting in glomerular endothelial damage. For this reason, it is considered an autoimmune disease, since the individual’s immune system damages its own tissues.

Antistreptolysin O antibodies are very high and complement levels are low.

References

  1. Wiener Laboratories. ASO latex. 2000. Available at: wiener-lab.com.ar
  2. Wikipedia contributors. “Anti-streptolysin O.”  Wikipedia, The Free Encyclopedia . Wikipedia, The Free Encyclopedia, 23 Jan. 2019. Web. 19 Jul. 2019.
  3. Kotby A, Habeeb N, Ezz S. Antistreptolysin O titer in health and disease: levels and significance.  Pediatr Rep . 2012; 4 (1): e8. Available in: ncbi.nlm.nih
  4.  Sen E, Ramanan A. How to use antistreptolysin O titre. Arch Dis Child Educ Pract Ed.  2014; 99 (6): 231-8. Available in: ncbi.nlm.nih
  5. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. (5th ed.). Argentina, Editorial Panamericana SA
  6. González M, González N. 2011. Manual of Medical Microbiology. 2nd edition, Venezuela: Directorate of media and publications of the University of Carabobo.

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