Immunology and Serology Lab - 05 Serologic Tests for Febrile Diseases

IMMUNOLOGY AND SEROLOGY LABORATORY • SEROLOGIC TESTS FOR FEBRILE DISEASES OUTLINE • • Febrile Diseases o Typhoid Fever o Rickettsia Widal and Weil-Felix Tests o Discussion o Procedures FEBRILE DISEASES • Febrile diseases are microbial infections characterized by fever and produce febrile agglutinins. Can be detected using Weil-Felix test (Agglutination) o Because Rickettsia are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification. o Weil-Felix test is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris, and OX-K strain of Proteus Mirabilis o Sources: ▪ OX-2 - Proteus vulgaris ▪ OX-19 - Proteus vulgaris ▪ OX-K - Proteus mirabilis o Appears of 7-10 days after infection and peaks at the 14th day o 1:80 – doubtful, 1:160- indicative o Procedure: Patient’s serum is mixed with strains of P. Vulgaris and mirabilis and tested for agglutination. o Interpretation: TYPHOID FEVER • • • • • • • • • Salmonella typhi Acquired by ingestion Antibody forms after 7-10 days against Somatic or Flagellar antigen (O or H) O antigen - hermostable (somatic), polysaccharide, endotoxin o Prepared by phenol preparation H antigen - Flagellar, protein o Prepared by formalin, withstand formalin but not phenol Vi antigen - Capsular, indicator of carrier state, virulent factor Laboratory Test o Bacterial Culture ( Gold Standard, Reference Test) o Widal or Typhidot (serologic) Widal Test ( Febrile agglutination Test) Based on direct agglutination o Blue stained antigens are specific to somatic antibodies while red stained antigens are specific to flagellar antibody o Salmonella Paratyphi A and S. Paratyphi B also possess O antigen on their cell wall and but have AH and BH antigen on their flagella respectively. o Antibody titer greater than 1: 80 is considered significant Typhidot – Serologic test based on ELISA o IgM: acute while IgG: reinfection or carrier or successful treatment RICKETTSIA • • • Gram-negative, obligate intracellular coccobacilli bacteria that infect mammals and arthropods Rickettsia are transmitted in the arthropods, which serves as both vector and reservoir. Caused by different species under Genus Rickettsia Disease Causative OX-2 agent Epidemic R. prowazekii + typhus Murine R.typhi + typhus Scrub R. typhus tsutsugamushi Rocky R. ricketsii Variable Mountain Spotted Fever o o OX-19 OX-K ++++ - ++++ - - ++ Variable Variable IFA and MicroIF are currently considered as gold standard for rickettsial antibodies Immunochromatography: Sample to be measured is applied at one end of the strip and migrates toward the distal end where absorbent pad is present to maintain capillary flow rate. An antigen or antibody is immobilized in the detection zone with known antigen or antibody and been captured to form a colored line that will indicate positive test WIDAL AND WEIL-FELIX TESTS: TESTS FOR FEBRILE DISEASES DISCUSSION • • • Febrile Diseases are bacterial infections that are usually associated with fever or pyrexia. Febrile antigens – are killed bacteria suspension which are used to detect the presence of bacterial agglutinins of certain bacterial infections associated with persistent pyrexia (fever). There are a number of such organisms but the most prominent includes the genera Salmonella, Brucella, Francisella (Pasteurella) and some members of the Rickettsia family. Cross-reaction may be observed in some cases. Salmonella is a member of the gram-negative bacilli. Salmonella species are etiologic agents of majority of food borne enteritis. Infection is spread by poor hygiene and inadequate sanitary conditions. Fecal contamination of food or water with resultant infection is most serious. Human serum containing Antibodies (Abs) produced to viable bacteria (febrile agglutinin) will visibly agglutinate a suspension (febrile antigens) of that organism. WIDAL AND WEIL-FELIX TEST PROCEDURES • • • • Materials o Febrile antigens (Salmonella, Proteus, Brucella, Francisella, etc.) o Serological pipette/Aspirator bulb o Transparent plain-glass ring slide o Applicator sticks o Test tube o Clinical rotator (optional) Procedure 1. Bring antigens to room temperature. 2. Using suitable pipette, transfer the following amounts of serum to be tested from left to right into separate wells: .08 ml., .04 ml., .02 ml., .01 ml., .005 ml. Repeat this procedure with positive and negative control area. 3. To each serum drop, add one drop of the appropriate test antigen (shake the antigen gently to ensure a uniform suspension). 4. Mix the serum and antigen using a piece of applicator stick. Use separate applicator sticks for each serum. 5. Tilt slide from side to side for no longer than 3 minutes. 6. Observe for agglutination using any good indirect light. Interpretation o 100% agglutination - ++++(4) o 75% agglutination +++(3) o 50% agglutination ++(2) o 25% agglutination +(1) o <25% agglutination + Trace o 0% agglutination - Non-reactive