05_Serology.pdf

Hand-out for antibiotic sensitivity test http://www.eucast.org/clinical_breakpoints/ Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] ...

Hand-out for antibiotic sensitivity test http://www.eucast.org/clinical_breakpoints/ Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] S. aureus Zone diameter E. coli Zone diameter P. aeruginosa Zone diameter breakpoints (mm) breakpoints (mm) breakpoints (mm) S≥ R< S≥ R< S≥ R< Penicillin G 26 26 Ampicillin 14 14 Piperacillin 18 18 Oxacillin - MIC>2mg/L Cefixime 17 17 Ceftazidim 16 16 Erythromycin 21 18 Cefuroxime 18 18 Imipenem 20 17 Amikacin 18 16 Imipenem 22 16 Ciprofloxacin 25 22 Norfloxacin 17 MIC Ciprofloxacin 22 19 Amikacin 18 15 Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Serological reactions  Serological tests What can we use for?: for demonstration of diseases for demonstration of previous diseases complete the biochemical identification for detection of the pathogenic members of the species for getting data about spreading of the serotypes for epidemiological research by known antigen (Ag) – detection of unknown antibodies (Ab) by known Ab – detection of unknown Ag Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Background: Antigen+Antibody binding specific Antigen: enzyme, toxin cellular components: O antigen - cell wall H antigen – flagella K antigen – capsule F antigen - fimbria Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Complement: is the part of the normal sera, is heat labile (56C, 30 minutes can inactivate) together with specific sera can cause lyses on the sensitized red blood cells can help in opsonisation is not produced by Ag stimulus we can not increase the concentration in sera Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] C reactive protein is an acute phase protein is produced by the effect of IL-1, IL-6 produced by hepatocytes after 48 hours reach the maximum o f the concentration values:10 mg/l normal 30-40 mg/l viral infection 200-250 mg/l bacterial infection 250-300 mg/l fungal infection Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Detection of Ag: direct from specimen from culture Detection of Ab: for demonstration of diseases for demonstration of the effect of vaccines Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Classification of serological tests Types: - qualitative - quantitative - visible: - agglutination - precipitation - invisible, detection by other methods - Immunofluorescence (IF) - Complement Fixation test (CFT) - Radioimmuno Assay (RIA) - Enzyme Linked Immunosorbent Assay (ELISA) Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Agglutinations: Slide agglutination (qualitative) Ag: unknown Ab: known (specific sera) results: visible Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Tube agglutination (quantitative) Ag: known Ab: unknown (dilution of patient sera) ex: Gruber-Widal test in the case of typhoid fever Wright reaction in the case of Brucella Weil-Felix reaction in the case of Rickettsia prowazekii Titer: the highest dilution fold (the lowest Ab concentration) where we can see agglutination Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Indirect agglutination – Latex agglutination Ab: binding to the surface of the carrier carrier: - red blood cells - hem agglutination - latex granule - latex agglutination - S. aureus – co-agglutination S. aureus produce the Protein-A which can bind the IgG at Fc receptors Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Precipitation Ag: is soluble (enzyme, toxin, virus)! In fluid medium: ring precipitation In agar medium: Ag and Ab diffuse into the gel, at the meeting point will form precipitation arch. ex: Elek test Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Ring precipitation quantitative Ag: soluble Ab: patient sera at Ag + Ab meeting point will form a precipitation ring Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Elek test (detection of toxin) Ag: Corynebacterium culture Ab: filter paper with Ab produced against diphtheria (antitoxin) if the strain is toxin producer will appear precipitation arch Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Quellung reaction Ag: unknown (bacteria with capsule) Ab: antibody produced against capsule if positive the capsule become opac and bigger Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] ELISA Ag: known, artificial Ab: unknown, patient sera Ab*: antihuman globulin labeld with enzyme Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] ELISA Ab-enzyme Ab from patient sera X Y Artificial Ag Ag Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Immunfluorescent method (IF) - Ag: unknown, unlabeld - Ab: labeled with fluorescent dye (rhodanin, izothiocianat) - Ag + Ab fluorescence in UV light Methods: - direct: - Ag: unknown (Chlamydia) - Ab: known, labeled - indirect: - Ag: known - Ab: unknown - Ab*: labeled antiglobulin Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Direct IF Labeled Ab Ag Cell surface Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Indirect IF Labeled antihuman Ab Unlabeled Ab Ag Cell surface Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]  Dr. Agoston Ghidan Institute of Medical Microbiology [email protected] Vaccines Dr. Agoston Ghidan Institute of Medical Microbiology [email protected]