Typhoid serology

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Transcript of Typhoid serology

  • 1. Dr.R.Jayaprada
  • 2. Typhoid fever, also known as typhoid, is a common worldwide illness, transmitted by the ingestion of food or water contaminated with the feces of an infected person, which contain the bacterium Salmonella enterica enterica serovar Typhi. The bacteria then perforate through the intestinal wall and are phagocytized by macrophages. The organism is a Gram negative short bacillus that is motile due to its peritrichous flagella. The term "enteric fever" is a collective term that refers to typhoid and paratyphoid
  • 3. Bacteriology TyphoidfeverThe Genus Salmonellabelongs to Enterobacteriaceae-Facultative anaerobe-Gram negative bacilli-Distinguished from other bacteria byBiochemical and antigen structure.
  • 4. Antigenic structure of SalmonellaH( flagellar ) antigensO (somatic) antigensVi (Virulence) capsularpolysaccharide antigens
  • 5. LPS in the cell wall; Extracted from the cell wall by treatment with tetrachloroacetic acid (Boivin : Boivin antigen); Less immunogenic than H antigen; Agglutination with antisera: Compact, chalky granular clumps Serogrouping of Salmonellae is based on characteristic O antigen;
  • 6. Present in flagella; Heat labile; 2 phases : phase 1 and phase 2; Strongly immunogenic; Induce rapid antibody formation in high titres; Agglutination with antisera: Large, loose, fluffy clumps
  • 7. Surface polysaccharide expressed on certain serotypes; Heat labile; Interferes with agglutination by O antisera; Lost on serial subcultures; Poorly immunogenic, BUT antibodies are protective: Detection of Vi antibody not helpful in diagnosis but their absence in a case of typhoid poor prognosis; Persistance of Vi antibody : carrier state
  • 8. Serodiagnosis of Typhoid :1.Detection of Antibodies in serum---1.Widal test ,2.Typhidot assay 3.Tubex system,4. Dipstick assay.2. Detection of Antigens in serum.--- a.Tubexsystem b.CountercurrentImmunoelectrophoresis. c. Co-agglutination test. d. ELISA3. Detection of Antigens in urine: 1.Tubex system 2. Counter Immuno Electrophoresis, 3. Latex agglutination 4. and Co- agglu-tination
  • 9. Georges-Fernand-Isidor WidalWidal & Sicard in 1896described the Widal reaction.In 1896 Widal A professor ofpathology and internalmedicine at the University ofParis (191129), hedeveloped a procedure fordiagnosing typhoid feverbased on the fact thatantibodies in the blood ofan infected individual causethe bacteria to bind togetherinto clumps (the Widalreaction).
  • 10. Widal Test Serum agglutinins raise abruptly during the 2ndor 3rd week. The Widal test detects antibodies against O and H antigens. Two serum specimens obtained at intervals of 7 10 days to read the raise of antibodies. Serial dilutions on unknown sera are tested against the antigens for respective Salmonella. False positives and False negative limits the utility of the test. The interpretative criteria when single serum specimens are tested vary. Cross reactions limits the specificity.
  • 11. Widal test A standard tube agglutination testTest can be performed by the tube dilution techniqueIn this, a constant amount of the antigen is added to a series of tubes containing serum dilutions. After mixing, the tubes are incubated at a temperature of 37 c in athermostatic water bath and the highest dilution of serum showing visible agglutination is determined.
  • 12. Felix tube Dreyers tube Conical bottom O Round bottom H H agglutination O agglutination Compact Loose granular Cotton woollyagglutination clumps
  • 13. Equal volumes of serial dilutions of serum (1/10 to 1/640) mixed with H and O antigens in respective tubes; Incubated in water bath at 370C overnight; Observed for agglutination H : Loose , cotton woolly clumps; O : compact granular agglutination; Supernatant should be clear;
  • 14. 0.9ml of normalsaline + 0.1 ml of serum 0.5 ml of S.Typhi O antigen control 0.5 ml NS. 0.5ml 0.5ml discarded. 0.5 ml of S.Typhi H antigen 0.5ml 0.5mldiscarded.
  • 15. Result reported as titres : Highest dilution where agglutination is seen; Titres will depend on the stage of disease: Agglutinins will appear by the end of 1st wk; Rise till 3rd or 4th wk, later decline; Demonstration in the rise of titre is significant; Following Titers of antibodies against the antigens are significant when single sample is tested. Significant titre: O > 1 in 100 H > 1 in 200 Testing a paired sample for raise of antibodies carries a greater significance.
  • 16. The Widal test (Widals agglutination reaction) is routinely practiced for the Serodiagnosis of typhoid fever by most of the laboratories. Several workers have expressed doubt regarding the reliability of the test. Several factors have contributed to this uncertainty. These include 1.Poorly standardized antigens, 2.Sharingofantigenicdeterminants with other Salmonellae 3.Effects of immunization with TAB vaccine.Another major problem relates to the difficulty of interpreting Widal test results in areas where Salmonella.Typhi is endemic and where the antibody titres of the normal population are often not known.
  • 17. Classically, a four-fold rise of antibody in pairedsera Widal test is considered diagnostic of typhoidfever.However, paired sera are often difficult to obtainand specific chemotherapy has to be instituted on the basis of a single Widal test.Furthermore, in areas where fever due to infectiouscauses is a common occurrence. So false positive reactions may occur as a result of non-typhoid
  • 18. The Widal test is time consuming and most often it is too late to start an antibiotic regimen when diagnosis is reached.
  • 19. Previous immunization with Salmonella antigen. Cross-reaction with non- typhoidal Salmonella. Variability and poorly standardized commercial antigen preparation. Infection with malaria Brucellosis other Enterobacteriaceae sharing the same s-LPS . dysgammaglobulinaemia of chronic active hepatitis. Autoimmune diseases.
  • 20. The carrier state An inadequate inoculum of bacterial antigen in the host to induce antibody production Technical difficulty or errors in the performance of the test. Previous antibiotic treatment Variability in the preparation of commercial antigens. with "hidden organisms" in bone and joints.
  • 21. Prozone effect - Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs.
  • 22. Slide Widal test is more popular as it gives rapid results.Qualitative test: 1 drop of each undiluted patients serum sample for the 2 antigens is placed on the circled card. 1 drop of each of 2 salmonella antigens are added separately rotated gently for 1 min. Appearance of agglutination gives qualitative results. (test is repeated is repeated with dilutions of serum)
  • 23. Quantitative test: 80l, 40l, 20l, 10l, 5l, of patients serum each for 2 salmonella antigens are placed on the circled card. one drop of specific antigen is added to each series of serum. Agglutination of each of these is noted.80l corresponds to 1 in 20 dilution.40l corresponds to 1 in 40 dilution.20l corresponds to 1 in 80 dilution.10 l corresponds to 1in 160 dilution. 5l corresponds to 1in 320 dilution.