Lecture 3 Biochemisrty and infection ASOT Procalcitonin and

Lecture 3 Biochemisrty and infection ASOT, Procalcitonin and CRP Dr. Obeid Shanab

Beta hemolytic Spherical, ovoid or lancet shaped

Divided into Serotypes or groups Two major outer proteins M and T Interior proteins divided into 20 defined groups known as Lancefield groupings A-H and K-T. Streptococcus pyogenes belong to Lancefield group A M protein chief virulent factor

Numerous Exoantigens are produced and excreted and include: Streptolysin O Dnase Hyaluronidase Nicotinamide Adenine Dinucleotidase Streptokinase Patients react to exoantigens by producing antibodies

Streptococcus pyogenes Organism found only in man. Leading cause of oropharyngitis which may lead to serious complications (sequelae) Rheumatic fever Acute glomerulonephritis Culture and rapid screening tests detect early infection.

Characteristics Two major sites of infection Upper respiratory tract Skin

Upper Respiratory Sore Throat Tonsillar exudate Fever Chills Headache 20% school children carriers

Skin Pyoderma Lesions on extremities Commonly on face Pustular and crusty

Suppurative Complications Suppurate -To generate pus; as, a boil or abscess suppurates. Erysipelas Cellulitis Necrotizing fasciitis Scarlet fever Puerperal sepsis

Erysipelas produces a rash that is red, slightly swollen, with very defined borders (well demarcated), warm, and tender to the touch. In this photograph, the right cheek is involved. There may be symptoms that affect the entire body (systemic) including fever and chills.

Cellulitis Diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of skin. Skin on face or lower leg most common site, but can occur anywhere on body.

Necrotizing Fasciitis – rare infection of deeper layers of skin and subcutaneous tissue

Scarlet Fever Strawberry tongue Strep bacteria produces a toxin that causes a rash Appears 12 -48 hours after fever Sandpapery Peels

Laboratory Testing Culture and identification Rapid Strep Tests from throat swab Detection of Streptococcal antibodies Anti-Streptolysin O (ASO) titer DNA probes

Rapid Strep Tests from throat swab Antigen from a swab is extracted. Test extracted antigens using ELISA or latex agglutination.

Detection of Streptococcal Antibodies Most useful in Streptococcal sequelae Organisms elaborate more than 20 exotoxins that may invoke antibody response. Most useful antibodies are: Anti-Streptolysin O (ASO) Anti-DNase B Anti-NADase Anti-Hyaluronidates

Detection of Streptococcal Antibodies Serological evidence of disease is based on elevated or rising titer of Streptococcal antibodies. Four fold (2 tube dilution) rise in titer is considered clinically significant.

Anti-DNase B Testing May appear earlier than ASO. Increased sensitivity for detection of glomerulonephritis preceded by streptococcal skin infections. Macro- and micro-titer, ELISA and neutralization techniques are available. Neutralization technique has advantage of stability of reagents.

DNA Probes Sensitive and specific Takes less time, hours versus days Many methods developed but principle the same. PCR using specific primers (probes) with tag Tag gives off signal, ie, fluorescence

ASOT ( Antistreptolysin O Titer )

Anti-Streptolysin O (ASO) titer Two of the toxins produced are Streptolysin S, which is oxygen stable, non-antigenic and Streptolysin O (SLO), which is oxygen labile and antigenic. SLO is a hemolysin which is toxic to many tissues, including heart and kidneys. Evokes an antibody response (anti-SLO) which neutralizes the hemolytic action of SLO. Specific for ASO, it does not test for antibodies to any other Streptococcal exotoxins.

FAlternative Names : • ASO • ASLO • Anti-streptolysin O ( ASO or ASLO ) is an antibody produced against an antigen produced by Lancefield group A streptococci “ streptococcus pyogens”. • Both toxins are involved in producing hemolysis, in particular, Beta-hemolysis. S. Pyogens by gram stain Beta-hemolysis

Significance of ASOT • ASO testing is a procedure that demonstrate the presence of antibodies generated by the body against infections by group A streptococcus (S. pyogen) mostly for Tonsillitis and Rheumatic fever, this m/o produce the toxin streptolysin O, which cause destruction of RBCs cell walls. • This toxin is immunogenic, that is, the body produces antibodies against it.

Note that : - Antibodies ( Ig. G ) begin to appear in patient serum 7 -10 days after infection. - The ASO antibodies may be found in the blood weeks or months after the strep. infection has gone away. ▷ Incidence of detect a positive result during the : ◌ ◌ 3 rd weak of infection 2 month 6 month 12 month 90 -95 % 70 -75 % 35 % 20 % Incidence decreases as the time increseases ▷ If the titer doesn’t decrease with time ; this means that a recurrent or chronic infection may exist.

ASOT test ◌ What is it ? It is a latex agglutination test for the qualitative screening and semi -quantitative determination of anti-streptolysin O antibodies in serum. - Specimen should be Serum in plain tubes. ◌ Principle of the test : A stabilized buffered suspension of polystyrene latex particles have been coated with streptolysin O when is mixed with a serum containing antibodies to streptolysin O , agglutination occur. “ the reaction will occur within minutes “

Results Interpretation At Direct agglutination step : 1. No agglutination o o serial dilution is done to exclude probability of prozone The result is written less than 200 IU/ml, o Don’t write negative result 2. Agglutination is observed o This mean that ASO Level of 200 IU/ml or more, and so Serial dilution is done to detect the exact titer. o Titer is detected by multiplying dilution factor by 200 IU/ml

Semi-quantitative method add 50 ul serum 50 ul 1 2 3 50 ul NS 1: 2 1: 4 1: 8 And so on; Total Dilution agglutination + + 400 Back to 200 800 Back to 400 + Titer ( IU/ml ) = - 1600 - Back to 800

Comments on Results FA raised or rising levels can indicate past or present infection. Elevated levels may indicate : 1. Active streptococcal infection 2. Sore throat 3. Rheumatic fever 4. Scarlet fever 5. Post-streptococcal glomerulonephritis 6. Bacterial endocarditis False positive results(rise in titer)may occur in : 1. High fibrinogen 2. Gross hemolysis 3. High lipid in serum 4. Oxizied reagent 5. Bacterial contamination “ dirty slide “

Note : The test reaction must be read immediately within 2 minutes, as Delayed readings may result in false positive result.

Procalcitonin is a 116 amino-acid peptide - • Precursor of the hormone Calcitonin 33 33

? What is Procalcitonin Precursor of hormone calcitonin Normally undetectable in healthy individuals Synthesized by thyroid cells Also released by liver, kidney, muscle, fat cells in response to bacterial toxins After exposure to toxins, serum levels of PCT increase within 2 -4 hours, peaking ~14 hours. PCT may also be elevated in non-infectious conditions (trauma, surgical procedures, pancreatitis, renal impairment).

Role of PCT in the absence of infection Release of Calcitonin in the context of endocrine regulation: Synthesis in healthy persons in the C-Cells of the thyroid PCT is enzymatically converted to calcitonin and then stored in endocrine granules Released only under certain stress (e. g. magnesium, gastrin)