MT 65: SEROLOGICAL DETECTION OF BACTERIAL INFECTIONS | CSU-CAHS-BSMLS-3C
GROUP A STREPTOCOCAL INFECTION (STREPTOCOCCUS PYOGENES)
INTRODUCTION
CLASSIFICATION AND STRUCTURE
❖ Gram-positive (spherical, ovoid, or lancet-shaped organisms)
❖ Catalase negative
❖ Streptococcus pyogenes is a β-hemolytic streptococci (Lancefield group A)
❖ Outermost cell wall: contains M and T protein
❖ Interior membrane: group-specific carbohydrate (basis of Lancefield groups: A-H and K-V)
VIRULENCE FACTORS
❖ M protein
major virulence factor of Group A streptococci (GAS)
inhibits phagocytosis
diminishes complement activation
❖ Most group A strains produce capsules composed of hyaluronic acid
❖ Pyrogenic Exotoxins (Erythrogenic Toxin): Streptococcal pyrogenic exotoxins A, B, and C
❖ Hemolysins (streptolysins): Streptolysin O and Streptolysin S
❖ Streptokinase (Fibrinolysin)
❖ Streptodornase (Streptococcal deoxyribonuclease): deoxyribonuclease B (DNase B)
❖ Hyaluronidase
❖ Nicotinamide adenine dinucleotidase (NADase)
CLINICAL MANIFESTATION OF GROUP A STREPTOCOCCI (GAS)
❖ Two major sites of infections:
Upper respiratory track
Skin
❖ Most common clinical manifestations:
Pharyngitis (“Strep Throat”) → fever, chills, severe sore throat, headache, tonsillar exudates,
petechiae on the soft palate, and anterior cervical lymphadenopathy
Streptococcal Pyoderma (Impetigo) → local infection of the superficial layers of skin; vesicular
lesions on the extremities
❖ Scarlet fever → associated with Streptococcal pyrogenic exotoxins A, B, and C
❖ Toxic shock syndrome → multisystem disease which may progress to shock and renal failure
❖ Necrotizing fasciitis → skin infection that invades the muscles of the extremities or trunk
❖ Group A Streptococcal Sequelae
Acute Rheumatic Fever
Poststreptococcal Glomerulonephritis
DETECTION OF GROUP A STREPTOCOCCAL ANTIGENS
❖ Useful for diagnosis of acute pharyngitis
❖ Enzyme Immunoassays (EIA) and Latex Agglutination
❖ Lateral Flow Immunochromatographic Assays (LFA)
Detects bacterial, viral, fungal, parasitic antigens
PREPARED BY: KAREN B. ROSETE, RMT 1
MT 65: SEROLOGICAL DETECTION OF BACTERIAL INFECTIONS | CSU-CAHS-BSMLS-3C
Easy to perform
More sensitive
Strep A antigen (extracted from a throat swab) + enzyme-labeled antibody → antigen-
antibody complex + conjugated antibody → pink/purple-colored line
Positive result
o T line = pink/purple color
o C line = pink/purple color
Negative result
o T line = NO pink/purple color
o C line = pink/purple color
DETECTION OF GROUP A STREPTOCOCCAL ANTIBODIES
❖ Useful for diagnosis of acute rheumatic fever and post-streptococcal glomerulonephritis
❖ Most diagnostically important streptococcal antibodies:
• Anti-streptolysin O (ASO)
• Anti-DNase B
• Anti-NADase
• Anti-hyaluronidase (AHase)
❖ Serological evidence of GAS infection is based on elevated or rising streptococcal antibody titer
❖ Four-fold rise in titer = significant; indicates acute and/or convalescent period of infection
❖ Most commonly used tests:
• ASO Testing
• Anti-DNase B Testing
❖ Recommended: use of at least two tests for antibodies to different exotoxins
ANTISTREPTOLYSIN O (ASO) TESTING: Detects antibodies to Streptolysin O enzyme
1. ASO Neutralization Test
Classic hemolytic method for determining ASO titer
Specimen: patient’s serum
Reagent: Streptolysin O
Indicator: Red blood cells (RBCs)
Based on the ability of streptococcal antibodies (in the patient’s serum) to neutralize
the hemolytic activity of streptolysin O
PREPARED BY: KAREN B. ROSETE, RMT 2
MT 65: SEROLOGICAL DETECTION OF BACTERIAL INFECTIONS | CSU-CAHS-BSMLS-3C
Procedure:
1. Perform serial dilutions on patient’s serum
2. Add streptolysin O reagent
3. Incubate
4. Add indicator system → red blood cells (RBCs)
5. Observe for hemolysis
Patient’s serum (contains Abs to Streptolysin O or ASO) + streptolysin O (SO) → ASO-SO
complex formed (during incubation) → add RBCs → no free SO to lyse RBCs → no hemolysis
Patient’s serum (no Abs to Streptolysin O or ASO) + streptolysin O (SO) → no ASO-SO
complex formed (during incubation) → add RBCs → SO is free to lyse RBCs → hemolysis
occurs
ASO titer → reciprocal of the highest dilution demonstrating no hemolysis
ASO titer can either be expressed as Todd units (when streptolysin reagent is used) or
International Units (IU) (when WHO international standard is used)
Moderately elevated ASO titers:
• Adult: 240 Todd units
• Child: 320 Todd units
PREPARED BY: KAREN B. ROSETE, RMT 3
MT 65: SEROLOGICAL DETECTION OF BACTERIAL INFECTIONS | CSU-CAHS-BSMLS-3C
2. ASO Rapid Latex Agglutination Test
Principle: Latex particles coated with Streptolysin O agglutinate when mixed with patient’s serum
containing ASO antibody
3. ASO Testing by Nephelometric Method (Automated)
Rapid, quantitative measurement of ASO titers
Uses purified recombinant streptolysin (reagent)
Antibody-positive patient serum + antigen reagent → Ag-Ab complex → ↑ light
scattering → ↑ signal strength detected
Results are reported in International Units
ANTI-DNASE B TESTING: Detects antibodies to DNase B
❖ Useful in patients suspected with glomerulonephritis preceded by streptococcal skin infections
1. Neutralization Method (Classic Test)
Specimen: patient serum
Reagent: DNase B
Indicator: DNA-methyl-green conjugate
Patient’s serum (contains Abs to DNase B) + DNase B → Anti-DNase-DNase B complex
formed (during incubation) → add DNA-methyl-green conjugate → no free DNase B to
hydrolyze DNA → color of indicator remains intact → green color (POSITIVE)
Patient’s serum (NO Abs to DNase B) + DNase B → no Anti-DNase-DNase B complex
formed (during incubation) → add DNA-methyl-green conjugate → DNase B is free to
hydrolyze DNA → color of indicator (green) becomes colorless → colorless (NEGATIVE)
Grading: ranges from 4+ (unchanged color) to 0 (total loss of color)
Result is reported as the reciprocal of the highest dilution demonstrating a color
intensity of between 2+ to 4+.
Normal titer (Children: 2-12 years old) → 240 to 640 units
PREPARED BY: KAREN B. ROSETE, RMT 4
MT 65: SEROLOGICAL DETECTION OF BACTERIAL INFECTIONS | CSU-CAHS-BSMLS-3C
2. Anti-DNase B Testing by Nephelometry (Automated)
Rapid, quantitative measurement of anti-DNase B
Antibody-positive patient serum + antigen reagent → Ag-Ab complex → ↑ light scattering → ↑
signal strength detected
Results are reported in International Units per mL
STREPTOZYME TESTING
❖ slide agglutination screening test for the detection of streptococcal antibodies
❖ It measures the following antibodies:
Anti-streptolysin (ASO)
Anti-hyaluronidase (AHase)
Anti-streptokinase (ASKase)
Anti-nicotinamide-adenine-dinucleotidase (anti-NADase)
Anti-DNase B
❖ Procedure:
Diluted serum (1:100) + sheep RBCs coated with streptolysin, streptokinase, hyaluronidase, DNase
and NADase → observe for agglutination
Positive: hemagglutination
Negative: no hemagglutination
PREPARED BY: KAREN B. ROSETE, RMT 5
