ASO

ASO Test (Anti-Streptolysin O)




Dr. M. MohammadiTeheran University of Medical SciencesSchool of MedicineDepartment of Immunology

Outline

  • Introduction to streptococcal infection

  • Properties of streptolysin (SLO)

  • Detection of anti-streptolysin O

Serology


Definition: Serological reactions are interactions between antigen and antibody under in vitro conditions.Methods based on specific binding of antibodies:

  • Detection of suspected antigen with known antibody

  • Detection of suspected antibody with known antigen

Types of Streptococci

  1. Hemolytic Streptococci (Groups A, B, C, D, F, etc.)

  2. Alpha Hemolytic Streptococci

  3. Gamma Hemolytic Streptococci

  4. Pepto Streptococci

Group A Streptococcus (Streptococcus pyogenes)

Associated conditions:

  • Pharyngitis & Tonsillitis

  • Acute Glomerulonephritis

  • Acute Rheumatic Fever

  • Pyoderma & Impetigo

Overview of ASO Test

The ASO test is a serologic method used for diagnosing:

  • Acute Rheumatic Fever (ARF)


  • Acute Glomerulonephritis (AGN)The test follows an infection with group A Streptococcus or Streptococcus pyogenes. This bacteria can lead to serious post-infectious sequelae, thus the importance of diagnosing the infection through the ASO test.

Characteristics of Streptococcus pyogenes

  • Exclusively found in humans.

  • Primary infection sites: Upper respiratory tract and skin.

  • Induces hypersensitivity leading to autoimmune responses.

  • High virulence factors assist in the dissemination and colonization of the pathogen, enhancing its pathogenesis.

Toxins and Enzymes

Key toxins and enzymes produced by Streptococcus include:

  • Hemolysin: (SLO, SLS) - responsible for the lysis of red blood cells.

  • Erythrogenic toxin: - associated with scarlet fever, inducing a rash and systemic inflammation.

  • Streptokinase: - promotes dissolution of blood clots, aiding bacterial spread.

  • Nuclease (DNase B): - contributes to DNA degradation, facilitating immune evasion.

  • Hyaluronidase: - breaks down hyaluronic acid in connective tissues, enhancing tissue penetration.

  • Antibody response is activated by exoantigens, leading to specific immune reactions against these components.

Streptococcal Antibodies

Streptococcus produces over 20 exotoxins leading to antibody production. Key antibodies include:

  • Anti-streptolysin O (ASO)

  • Anti-DNase B


  • Anti-HyaluronidaseThese antibodies are critical for diagnosing streptococcal infections post-infection, aiding in the identification of prior infections that can lead to diseases such as ARF and AGN.

Laboratory Tests for Streptococcal Infection

Main laboratory tests include:

  • Blood Agar - culture medium that supports the growth of streptococci.

  • Anti-DNase B Antibodies

  • Anti-Streptolysin O Antibodies (ASO)

  • Anti-Hyaluronidase Antibodies

ASO Antibody Testing Methods

Tests for ASO antibodies include:

  1. Latex agglutination: - a rapid test detecting ASO by visual agglutination.

  2. Nephelometry: - measures light scattered by ASO-antigen complexes in a solution, quantifying antibody concentration.

  3. Turbidimetry: - assesses the turbidity in a sample to estimate antibody levels.

  4. Neutralization: - evaluates the ability of antibodies to inhibit the activity of SLO.

Effector Functions of Antibodies

  1. Neutralization: - blocking of microbes and toxins to prevent infection.

  2. Opsonization: - enhances phagocytosis of microbes by marking them for destruction by immune cells.

  3. Antibody-dependent cellular cytotoxicity (ADCC): - engages natural killer (NK) cells for the lysis of microbes.

  4. Complement Activation: - triggers inflammation and assists in opsonization of pathogens.

Principle of the ASO Test

Key components of the ASO test:

  • Antigenic red blood cells

  • Complex formation of Ag+Ab

  • Induction of hemolysis by streptolysin-O (SO).

ASO Test Procedure

  • Positive control: - uses RBC + SLO to ensure the assay can detect hemolysis.

  • Negative control: - uses RBC + buffer (should display no lysis) to confirm specificity.

Titer Measurement (Todd Units)


Titration measures the final concentration preventing red blood cell lysis, recorded as Todd units.Components of the titration:

  • Stock

  • Dilution

  • Total Volume

  • Volume of Serum Added

ASO Test Controls

  • Negative Control: - RBC + buffer (no lysis).

  • Positive Control: - RBC + SLO (results in lysis).

ASO Test Procedure Breakdown

Tube setup with varying concentrations and controls:

  • Tube NO. 1: (8/1000)

  • Tube NO. 2: (6/1000)

  • Tube NO. 3: (4/1000)

  • Tube NO. 4: (3/1000)

  • RBC Control


  • SLO ControlKey variables: include serum dilutions, ASO buffer, incubation times, etc.

ASO Test Repetition

The experiment is repeated with the same conditions from previous tube setup to verify results and ensure accuracy.

Lysis Observation

Responses observed in tube setups regarding lysis or no lysis are critical for determining the presence of ASO antibodies in the patient's serum.

Interpretation of ASO Test Results


Patient Titer: - highest dilution inhibiting hemolysis.Todd Unit: - minimum hemolytic dose of SLO leading to lysis of rabbit RBCs. Interpretation helps confirm or rule out recent streptococcal infection.

Significance of ASO Test

SLO is produced by most strains of group A Streptococci. The commercial availability of SLO antigen provides a reliable test standard, enhancing diagnostic accuracy for streptococcal infections.

Precautions in ASO Testing

Antibiotic or immunosuppressive therapy may affect ASO titers. Early penicillin injection prevents titer rise. Certain conditions can lead to false titer results. Precautions in clinical settings include:

  • Nephrotic syndrome and bacterial contamination can lead to misleading ASO titers.

Acute Rheumatic Fever (ARF)

Occurs in 2-3% of patients following pharyngeal infection. Symptoms typically arise 20 days post sore throat and may include fever, joint pain, and carditis. Potentially causes significant cardiac damage, emphasizing the importance of timely diagnosis and treatment.

Acute Glomerulonephritis (AGN)


Latency of 10 days following group A strep pharyngitis, and up to 3 weeks post group A strep skin infections. Characterized by glomeruli damage due to inflammatory responses.Diagnosis and Testing: includes isolation of group A strep from throat or skin lesions, and elevated streptococcal antibodies observed in patient tests.

Properties of SLO and SLS

  • SLO: Oxygen-labile, molecular weight 70,000, causes lysis in erythrocytes and cardiotoxicity.

  • SLS: Oxygen-stable, molecular weight 2,800, synthesized by both growing and resting streptococci. Understanding these properties assists in the clinical implications of streptococcal infections and their management.