Antistreptolysin O Testing (October 2024) PDF

MICROBIOLOGY AND PARASITOLOGY ANTISTREPTOLYSIN O TESTING Dr. Hannah Leian H. Tan, RMT, MD | October 01, 2024 Interpretation: OUTLINE...

MICROBIOLOGY AND PARASITOLOGY ANTISTREPTOLYSIN O TESTING Dr. Hannah Leian H. Tan, RMT, MD | October 01, 2024 Interpretation: OUTLINE ○ Bacitracin Susceptible = No growth I. OVERVIEW IV. ASO TESTING around the antibiotic disk A. Introduction A. ASO Slide Testing ○ Isolate is likely Streptococcus pyogenes (a B. Hemolytic (Qualitative, Semi-Quantitative) beta hemolytic). Patterns ○ Bacitracin Resistant: Isolate is likely B. ASO Tube Testing II. LANCEFIELD’S Streptococcus agalactiae (Semi-Qua GROUPING ntitative, A. Sample Case Neutralizati 2. α-hemolytic III. Streptococcus on Test) ○ Partial hemolysis of RBCs pyrogenes ○ Reduction of hemoglobin to A. Sample Case methemoglobin (cont.) ○ Oxidation of iron in RBCs produces greenish or brownish color around the I. OVERVIEW colonies in the medium ○ h Procedure: INTRODUCTION ➔ Perform testing with Optochin REVIEW: Interpretation: ○ Staphylococci are catalase positive ○ Optochin Susceptible: Isolate is likely ○ Streptococci are catalase negative Streptococcus pneumoniae. After identifying our cocci as catalase negative, ○ Optochin Resistant: Isolate is likely viridans streptococci. the next thing to do would be to analyze the ○ h hemolytic patterns that it produces on blood 3. γ (non) hemolytic agar plate. ○ no lysis of RBCs ○ no change in the surrounding medium Procedure: ➔ Culture isolate in 6.5% sodium chloride solution Interpretation: ○ Growth Present: Isolate is likely enterococci. ○ No Growth: Isolate is likely Streptococcus bovis (non-hemolytic; not able to grow in that highly salinic solution) HEMOLYTIC PATTERNS 1. β-hemolytic ○ complete or true lysis of RBCs ○ seen as a clear zone around the colonies Procedure: ➔ Perform testing with Bacitracin Transcribed by: NMD 2027  ANTISTREPTOLYSIN O TESTING cleaving neutrophil extracellular traps II. LANCEFIELD’S GROUPING (NETs) Agar may have methyl green as LANCEFIELD’S GROUPING – a serological method for indicator classifying streptococci into groups designated by letter Dnas also allows S bio to use based on the presence of polysaccharides or sugars, and nucleic acids as a source of teichoic acid antigens in the bacterial cell wall. nutrition. TYPICAL GROUP SPECIES HEMOLYSIS INFECTIONS Pharyngitis, A impetigo, S. pyogenes β cellulitis, scarlet fever Neonatal sepsis and meningitis, B S. agalactiae β puerperal infection, UTI, endocarditis Enterococci: UTI, E. faecalis nosocomial D E. faecium Usually γ bacteremia, endocarditis S. bovis Viridans: Endocarditis, Variable or S. mitis dental non S. sanguis α abscess, brain Plate 1 (Top): groupable abscess S.pneumoniae RED ARROW: Original DNA Agar – DNA is directly incorporated into Trypticase soy agar. This SAMPLE CASE medium required the addition of hydrochloric acid to A 28/M presents to the Emergency Department (ED) with a detect depolymerized DNA. one-day history of sore throat and fever. Upon examination, RED CIRCLE: Hydrolysis of DNA – DNA was observed by a clearing of the Agar as can be seen tonsillar swelling is noted with pus. A throat swab is after addition of HCL. collected for culture. YELLOW CIRCLE: Area of polymerized DNA – produced a cloudy precipitate even after addition of Laboratory results: HCL. β-hemolytic colonies on the blood agar plate after 24 hours incubation Plate 2 (Bottom): A Gram stain of a colony reveals Gram-positive cocci growing in long chains GREEN ARROW: Modified DNAse Agar – Uses The organism is catalase negative and methyl green bound to DNA as an indicator. susceptible to the antibiotic/biochemical GREEN CIRCLE: Upon depolymerization of DNA, bacitracin methyl green is unconjugated and this leaves a zone of clearing around the colonies. III. Streptococcus pyrogenes ○ Streptokinase – plasminogen activator; converts plasminogen to plasmin thus allowing lysis of fibrin Associated with a variety of suppurative infections and metastasis of bacteria to other sites Virulence factors: ○ Hyaluronidase – spreading factor; allows ○ Protein M – main virulence factor; limits dissemination of bacteria through tissue phagocytosis, prevents complement ○ Pyrogenic exotoxin – superantigen; produces fixation, mediates adhesion to host cells. Streptococcal Toxic Shock Syndrome ➔ You could also think of M as ○ Streptolysin – responsible for beta-hemolysis, standing for mimicry because causes tissue death by inducing release of mimicry between streptococcal M lysosomal contents after ingestion by phagocytes protein and cardiac myosin is Streptolysin O: immunogenic; important in the pathogenesis of oxygen labile rheumatic heart disease. Streptolysin S: ➔ In here T cells and antibodies can non-immunogenic; oxygen stable be cross reactive to M protein and proteins in the myocardium leading to immune mediated SAMPLE CASE (CONT.) attack on the heart. The patient was given antibiotics. 3 weeks after being ○ DNAse – reduces host immune response, diagnosed with strep throat, he noticed his urine was determines ability of bacteria to use unusually dark reddish-brown. His face had also started nucleic acids as a nutrient source by swelling, especially around the eyes (periorbital edema). He 2 of 4  ANTISTREPTOLYSIN O TESTING sought consultation, and urinalysis revealed proteinuria. He 5. A positive reaction appears as a fine flocculation also had elevated blood pressure. (agglutination) within 2-3 minutes 6. A negative reaction shows no agglutination ASO Tube Testing (Semi-Quantitative, Neutralization Test) Procedure: IV. ASO TESTING 1. Prepare serial dilutions of patient serum, in saline, in test tubes as follows: Antistreptolysin O is a specific neutralizing antibody produced against streptolysin O of S. pyogenes o Levels rise 1-3 weeks after an infection (no value for acute streptococcal infection) Why the test is performed: o For diagnosis of post-streptococcal conditions when it’s no longer possible to culture S. pyogenes o Glomerulonephritis, Rheumatic fever, rheumatic heart disease. Bacterial endocarditis To arrive at an ASO titer using the ASO tube test, serial Based on the case: The patient, even though he was dilutions of the patient's serum are made: given treatment for his strep throat, what happened was certain streptococcal antigens persisted in the circulation and 1. Tube 1: One drop of the patient's serum (containing deposited themselves in the glomeruli. Once bound to the streptolysin O) is mixed with 9 drops of normal glomerulus, they activate the alternative complement saline solution (NSS), resulting in a total volume of pathway directly or there is binding of anti streptococcal 10 drops and a dilution of 1:10. antibodies and subsequent activation of the classical 2. Tube 2: One drop from Tube 1 is mixed with 1 drop complement pathway. So this patient was diagnosed with of NSS, making the total volume 2 drops. The post streptococcal glomerulonephritis or PSGN. dilution is calculated by multiplying the dilution of Tube 1 (1:10) by the volume of Tube 2 (2 drops), ASO Slide Testing (Qualitative, Semi-Quantitative) resulting in a final dilution of 1:20. 3. Tube 3: One drop from Tube 2 is mixed with 1 drop Principle: Immunologic reaction between of NSS. Tube 2 has a dilution of 1:20, and streptococcal exotoxins (streptolysin O) bound to multiplying it by the new volume gives a dilution of biologically inert latex particles and streptococcal 1:40 in Tube 3. antibodies in the test sample Positive: visible agglutination (indicates Ab level is at least 200 IU/mL) This process is repeated in subsequent tubes, progressively doubling the dilution to create higher dilutions of the patient's serum. 2. Add a fixed amount of streptolysin O, then add reagent sheep RBCs. Procedure: 1. Place 1 drop of the serum in a well on the disposable card. 2. Use a new dropper to add 1 drop of sensitized latex reagent. In an ASO tube test (also known as a neutralization test), an 3. Use an applicator stick to mix the two drops and antibody-antigen reaction neutralizes streptolysin O if the spread them over the entire well. patient's sample contains anti-streptolysin O antibodies. This 4. Examine for agglutination within 2-3 minutes. 3 of 4  ANTISTREPTOLYSIN O TESTING neutralization prevents the lysis of reagent sheep red blood cells (RBCs). As dilutions increase, the concentration of anti-streptolysin O antibodies decreases, leading to a reduced ability to neutralize the streptolysin O in the tube. The last tube in which RBCs remain unlysed (indicating neutralization) provides the ASO titer. For example, if the last tube without hemolysis has a dilution of 1:320, the ASO titer is reported as 320 international units per milliliter (IU/mL). The endpoint of the test is hemolysis. To arrive at a titer, positive serum samples are serially diluted and subjected to the same test to find the highest concentration of serum that fails to lyse red blood cells The last tube without lysed red blood cells after incubation is 1:320, so the ASO titer would be reported as 320 IU/mL ○ End point: hemolysis Normal values: ○ Adult /elderly = ≤160 Todd units/mL or 500 Todd units/mL indicates acute rheumatic fever or post-streptococcal glomerulonephritis FREEDOM WALL 4 of 4