Typhoid serology

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1. TYPHOID SEROLOGY 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. The Genus Salmonella belongs to Enterobacteriaceae -Facultative anaerobe -Gram negative bacilli -Distinguished from other bacteria by Biochemical and antigen structure. Bacteriology – Typhoidfever

4. Antigenic structure of Salmonella H ( flagellar ) antigens O (somatic) antigens Vi (Virulence) capsular polysaccharide 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; O (somatic) antigens

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 H( flagellar ) antigens

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 Vi (virulence) antigen (Felix and Pitt)

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. ELISA 3. Detection of Antigens in urine: 1.Tubex system 2. Counter Immuno Electrophoresis, 3. Latex agglutination 4. and Co-agglu­tination

9. Georges- Fernand - Isidor Widal Widal & Sicard in 1896 described the Widal reaction. In 1896 Widal A professor of pathology and internal medicine at the University of Paris (1911–29), he developed a procedure for diagnosing typhoid fever based on the fact that antibodies in the blood of an infected individual cause the bacteria to bind together into clumps (the Widal reaction).

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 test Test can be performed by the tube dilution technique In 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 a thermostatic water bath and the highest dilution of serum showing visible agglutination is determined .

12. Agglutination how it appear after reactivity O Felix tube Round bottom O agglutination Compact granular agglutination H Dreyer’s tube Conical bottom H agglutination Loose Cotton woolly 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 37 C overnight; Observed for agglutination H : Loose , cotton woolly clumps; O : compact granular agglutination; Supernatant should be clear; Widal test

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 1 st wk ; Rise till 3 rd or 4 th 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. Widal test : result

16. The Widal test (Widal’s 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 paired sera Widal test is considered diagnostic of typhoid fever. However, paired sera are often difficult to obtain and specific chemotherapy has to be instituted on the basis of a single Widal test. Furthermore, in areas where fever due to infectious causes is a common occurrence. So false positive reactions may occur as a result of non-typhoid Limitations of Widal test

18. The Widal test is time consuming and most often it is too late to start an antibiotic regimen when diagnosis is reached . Limitation of Widal Test

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. Causes Of False-positive Widal Agglutination Tests

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. Causes of Negative Widal Agglutination Test

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. Prozone phenomenon in Agglutination tests

22. Slide Widal test is more popular as it gives rapid results. Qualitative test: 1 drop of each undiluted patient’s 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) \ Slide Widal test:

23. Quantitative test: 80µl, 40µl, 20µl, 10µl, 5µl, of patient’s 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. 80µl corresponds to 1 in 20 dilution. 40µl corresponds to 1 in 40 dilution. 20µl corresponds to 1 in 80 dilution. 10 µl corresponds to 1in 160 dilution. 5µl corresponds to 1in 320 dilution. Slide Widal test:

24. This test is performed with a loopful growth from nutrient agar emulsified in 2 drops of 0.85% normal saline. For salmonella isolated for the first time, the following sera are tested in order I to VI: I. Salmonella polyvalent ‘o’ serum(groups A-G,02-13)on one half of slide.Test suspension alone on other half as a control to exclude saline auto agglutination. II.Salmonella polyvalent H’ phases 1&2 serum and polyvalent H phase 2 are placed on separated halves of the slide. If polyvalent ‘O’ & “H reactions are negative, NO further slide tests are done. If polyvalent O& H reactions are positive for salmonella, further Slide tests are done. Slide Agglutination Test:

25. III. Test suspension is checked with individual Salmonella O group sera 02-013 separately. IV. If the culture is in phase 1that is if it reacts positively with mixed phase1 &2 serum, negatively with phase 2 H serum and if it reacts with any single O’ group serum, then it is to be tested with single H’ serum( a, b, d, i etc )--------- salmonella .Enteritidis & salmonella .Typhimurium are the common serotypes in many countries. If salmonella belongs to O-group B (02 ) then it is tested with H- i serum. If salmonella belongs to O-group D(09) then it is tested with H-d serum. Slide Agglutination Test:

26. V. If the culture is in phase2, it is passed through Craigie’s tube or Jameson strip containing phase 2 H serum and phase 2  phase 1. VI. Salmonella. Typhi fails to agglutinate with group D(09) because the bacilli are coated with vi antigen. If vi antiserum is added ,then it agglutinates. If vi positive, saline suspension of salmonella is heated for 30 min, cooled & retested with O’ antisera. VII. If the isolate is nontyphiodal salmonella, producing gas from sugars, it is tested for agglutination with O’& H’ antisera for groups A,B,C. Slide Agglutination Test:

27. Advances in the RapidDiagnosis of Typhoid fever

28. TUBEX TF (IDL Biotech): It is a 5 minute semi quantitative calorimetric test for Typhoid fever. It detects antibodies to the salmonella enterica Serotype Typhi lipopolysaccharide (LPS)o9 antigen. Tubex system is ideally suited for use in diagnosis of infections as it allows IgM antibodies to be detected early & rapidly from whole serum. The antibodies are detected by their ability to inhibit the interaction between two types of reagent particles a)colored indicator latex microspheres sensitized with an anti o9 monoclonal antibody. b)magnetic microspheres coated with S.Typhi LPS.

29. Following rapid mixing of the serum with these reagents and sedimentation of the magnetic particles by the magnetic force, the concentration of the particles left in suspension provides the measure of inhibition. Tubex test for antibody detection : Test serum (25µl) was mixed in a chamber of the reaction container provided(which contains a set of six identical ‘ V’shaped chambers) with 25µl of Brown reagent (antigen coated magnetic particles) for 2min. Then 50 µl of blue reagent (Ag coated indicator particles) is added & mixed for another 2 min. The reaction mixture was placed on the magnet stand, and the resultant color was read immediately and scored against the color chart(score 0-10). Scores of 0-2 considered negative. Scores of 3-10 considered positive .

30. Tubex test for antigen detection : 25µl of test serum is mixed with 50µl of blue reagent in the reaction well for 2 min. then 25µl of brown reagent is added& mixed for another 2 min. The results are read after sedimentation of magnetic particles. In conclusion, Tubex kit can potentially be used in several ways to diagnose typhoid fever including: 1)Antibody &Antigen detection in serum. 2)Antigen detection from urine to complement serum detection. 3)Detection or identification of whole organisms from primary culture plates or from blood culture (stool) broth.

31. Results of Tubex TF: results are scored against a color chart with a scale of 0 to 10. Score ‘0’, negative and most red. Score ‘10’, most positive and most blue. Reasonable sensitivities (75–90%) and specificities (70-97%) have been observed.

32. Dipstick assay: It consists of a strip of nitrocellulose membrane containing a 2mm wide line of immobilized antigen as a detection band & a separate line immobilized anti human IgM antibody as reagent control. It is developed in Netherlands. It is based on binding of S.Typhi specific IgM antibodies in samples to S.Typhi LPS antigen and the staining of bound antibodies by an antihuman IgM antibody conjugated to colloidal dye particles. Antigen preparation : culture of recent isolate of S.Typhi culture was grown in a Luria Bertani broth & antigen was prepared by heating a washed & 30x concentrated bacterial suspension of a 3 day old well grown culture for 30min at 95 ° c. Cell debris was removed by centrifugation.

33. The supernant containing the antigen was blotted in 2mm wide lanes on the nitrocellulose membrane by incubation for 2hrs at 40°c. At the end of incubation, the lanes of blotting apparatus were rinsed with phosphate buffered saline (PBS) to remove excess antigen. Blotted strips rinsed with PBS, blocked with 3%skimmed milk, rinsed & dried. Detection reagent: It consists of a monoclonal antihuman IgM antibody conjugated to colloidal suspension of palanyl red.

34. Procedure of Dipstick assay: It is performed by incubating a wetted dipstick in a mixture of 5µl of sample & 250µl of detection reagent for 3 hrs at room temperature. Dipsticks are thoroughly rinsed with water & dried. Staining intensity of the antigen band was then graded by comparison with a colored reference strip. When no staining is observed ----- Test is scored negative. Weak staining -----------------1+ Moderate staining ---------------- 2+,3+ Strong staining -----------------4+

35. Dipstick assay is applied to a single serum sample gives quick result (3hours). Testing of paired sera could increase the sensitivity of the assay. It is a simple test & does not require any specific equipment for its performance as the assay uses stabilized compounds. Test is sufficiently sensitive(69.8%) and specific.

36. Typhidot assay : (MalaysianBiodiagnosticsResearch,Selangor,Malaysia) : It is an Enzyme linked Immunosorbent Assay in the dot test format which detects IgM & IgG antibodies against outer membrane protein of salmonella .Typhi antigen in human whole blood, serum or plasma. It is a rapid serological procedure for the diagnosis of Typhoid fever. The advantages of Typhidot includes: • early and specific diagnosis of typhoid fever • fast, simple and reliable • simple to perform and no additional sample preparation required • no special equipment is needed • results are easy to interpret • minimal sample volume used

37. Procedure of the test : The test is based on the presence of specific IgM & Ig G antibodies to a specific 50kDa OMP antigen which is impregnated on nitrocellulose strips. Reaction tray is divided into 2 columns as G’ and M’. 250 µl of sample diluent was dispensed in each well and 2.5µl of test /control was added & then incubated for 20min. Strips were washed with wash buffer thrice. 250µl of antihuman IgG & IgM were dispensed in each well & incubated for15 min. wash& 250µl of substrate is added for color development ,incubated for 15 min. Results were interpreted.

38. Diagrammatic Representation of Assay Procedure Serum/ Plasma sample Add 30μl serum to the sample well. Make sure that there are no air bubbles. Add 1 drop of buffer after 15 seconds. Sample will start wicking up. Read result within 15 minutes. Whole Blood sample Add 40μl of whole blood into the sample well. Make sure that there are no air bubbles. Add 1 drop of buffer after 15 seconds. Sample will start wicking up. Read result within 15 minutes. [Note: If sample front stops wicking up after a while, add an additional drop of buffer]

39. Typhidot has a sensitivity of 92.3%,specificity of 98.8%,PPV-85.7%& NPV- 99.4%.The test becomes positive within 2-3 days of infection. LIMITATION OF THE TEST 1. This product is designed for use with human whole blood, serum and plasma only. 2. The test is a qualitative assay & is not for quantitative determination of antibodies concentration levels. The intensity of the band does not have linear correlation with the antibody titer of the specimen. 3. The results obtained should only be interpreted in conjunction with other diagnostic results and clinical information. 4. Due to the limitations of the test, for cases where interpretation of result seems difficult, repeating the test TYPHIDOT 1 hour or TYPHIDOT 3 hours is strongly recommended.

40. This test, derived from Salmonella Typhi O and H antigens, was performed in two ways: ( i ) as a semi quantitative slide agglutination test with visual examination as per the package insert; (ii) as a Widal test performed with a single tube, as described by Parry et al. The presence or absence of visible agglutination indicates the presence or absence of the corresponding antibody to the O and H antigens of Salmonella Typhi. They defined the positivity cut-off point for the slide and tube agglutination reactions for both O and H antigens as antibody titres  1:80. Publication: Bulletin of the World Health Organization ; June 2011, Type: Research Article ID: BLT.11.087627 . Linear Cromotest ® (Linear Chemicals, Barcelona, Spain)

41. Electro chemical Immunoassay: Recently, copper, silver, and gold-enhanced colloidal gold have been reported for immunoglobin G (IgG)determination, which is the model of electrochemical immunoassay with low detection limits ranged from 1.0ng/ml to 0.25 pg/ml . Newer Principles of Nanotechnology for Typhoid

42. Co agglutination is similar to the latex agglutination technique for detecting antigen . Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions Detection of antigen- CoAgglutination Test

43. Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria. Detection of antigen- CoAgglutination Test

44. Preparation of staphylococci: Staphylococcus aureus Cowan 1 was grown on tryptic soy agar at 37°C for about 18h. The cells were harvested, washed in phosphate buffer, exposed to 0.5% formaldehyde, and heated to 80°C and held for 5 min by the procedure of Edwards and Hildebrand. Attaching o antisera to staphylococci: One milliliter of the 10% suspension of stabilized, killed staphylococci was added to 0.2 ml of non- glycerolated , specific antiserum or to 0.4 ml of glycerolated antiserum, mixed thoroughly, and allowed to react at room temperature for 3 h, with occasional gentle shaking . The suspension was centrifuged at 800 x g for 30 min and washed twice with 0.02 M phosphate (pH 7.3)-buffered 0.85% saline (PBS). The cells were then suspended in PBS to a final volume of 5 ml, and 1 drop of 5% sodium azide was added . The antibody-conjugated staphylococci made up a COAG. The COAGs were stored at 4°C.

45. Testing Salmonella strains by co agglutination: Salmonella strains were grown overnight on blood agar base (BBL Microbiology Systems). To test for agglutination, 1 drop of COAG is placed on a slide. Growth was taken from a slant with a bacteriological loop and was mixed into the COAG drop. Strong homologous reactions usually occurr immediately. If there was no reaction, the slide was rocked gently for 1 min and then read under fluorescent light against a black background. The amount of agglutination was read as 4+, 3+, 2+, or 1+. A trace was recorded as +/-.

46. Many known carriers of typhoid bacilli possess antibody against the Vi (virulence) antigen of S. typhi . This is a surface antigen easily lost during cultivation. Vi tires seem to correlate better with the carrier state than do O or H titres. For this reason, Felix et al. suggested the use of Vi agglutination for detection of carriers. Serological detection of carriers:

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