DMLT- 70 Salmonella and Lab Diagnosis.pptx

The study of salmonella and its diagnosis

1. Salmonella and lab diagnosis

2. Introduction to salmonella Morphology of salmonella Cultural characteristics Antigenic structure and pathogenesis of salmonella Diagnosis of typhoid fever Learning objectives

3. Enteric fever Introduction to salmonella The salmonella are primarily intestinal organism, which causes Enteric fever The causative agents of enteric fever are salmonella typhi which causes typhoid fever and S. Paratyphi A, B, and C which causing paratyphoid fever.

4. Gastroenteritis It causes the inflammation of the stomach and intestines, bacterial toxins causing vomiting and diarrhoea. Septicemia It cause septicemia means invasion of the bloodstream by virulent microorganisms and especially bacteria along with their toxins. Gastroenteritis Septicemia Introduction to salmonella

5. Introduction to salmonella Species of salmonella are: Salmonella typhi Salmonella paratyphi A Salmonella paratyphi B Salmonella paratyphi C

6. Morphology of Salmonella Salmonella are gram negative bacilli measuring 1-3 µm x 0.5 µm. They are motile, non- sporing and non-capsulated. Motility is due to presence of peritrichous flagella.

7. Cultural characteristics of salmonella Salmonella grow on ordinary culture media at optimum temperature of 37 The range of the temperature is 15-41 . pH of media is 6-8. They produce colonies of 2-3 mm in diameter, circular, translucent low convex and smooth.  

8. Salmonella growth on MacConkey agar Cultural characteristics of salmonella Growth on MacConkey agar They grow as non-lactose fermenting colonies( appear  transparent  and colorless ) colonies.

9. Black colonies of salmonella on DCA Cultural characteristics of salmonella Growth on deoxycholate citrate media (DCA) Pale colonies show black head due to H S (hydrogen sulphide) production .  

10. Cultural characteristics of Salmonella Growth on bismuth sulphite medium Jet black colonies with metallic sheen due to production of H₂S. Enrichment media Enrichment media are liquid media, which suppresses the commensal bacteria, while allowing the pathogen to remain viable and grow. Selenite F and tetrathionate broth used as enrichment media.

11. Cultural characteristics of Salmonella Resistance Bacilli are killed at 55 ᵒC in one hour or at 60 ᵒ C in 15 min. Boiling or chlorination of water and pasteurization of milk destroy the bacilli. In polluted water it may survive for weeks and in ice for months.

12. Biochemical reaction Salmonella ferment glucose, mannitol and maltose forming acid and gas. Salmonella are indole and VP negative, MR and citrate positive and urease negative. Salmonella produce H 2 S except paratyphi A.

13. Antigenic structure Salmonella posses the following antigens based on which they are classified & identified – Flagellar antigens H Somatic antigens O Surface antigens vi, found in some species.

14. Antigenic structure H Antigens This antigen present on the Flagella is a heat labile protein. The H antigens is strongly immunogenic & induce antibody formation rapidly & in high titre following infection or immunization.

15. Antigenic structure O antigens The somatic O antigens is a phospholipid protein polysaccharide complex which forms an integral part of the cell wall. The O antigen is less immunogenic than the h antigen & the titre of O antibody induced after infection or immunization is generally lower than that of the h antibody. Vi antigens Surface antigen & believed to be related to virulence.

16. Pathogenesis Transmission is via fecal-oral route, i.e. ingestion of contaminated food or water. The salmonellae are primarily intestinal organism, which causes: Enteric fever Gastroenteritis Septicemia

17. Enteric fever Pathogenesis Enteric fever It is also known as typhoid fever. The term enteric fever includes typhoid fever caused by S. Typhi and paratyphoid fever caused by S. Paratyphi A, B and C.

18. Pathogenesis Enteric fever The infection is acquired by ingestion through contaminated food and water. The incubation period is usually 7-14 days. Towards the end of the incubation period, a massive bacteraemia (presence of bacteria in blood) occurs .

19. Pathogenesis Symptoms of typhoid fever Typhoid fever and paratyphoid fever have similar symptoms : A sustained fever (one that doesn’t come and go) that can be as high as 103-104°F (39-40°C). Stomach pain Headache Weakness

20. Loss of appetite Rashes on skin Pathogenesis Symptoms of typhoid fever Typhoid fever and paratyphoid fever have similar symptoms Diarrhoea or constipation Cough Loss of appetite Some people with typhoid fever or paratyphoid fever develop a rash of flat, rose-coloured spots.

21. Diagnosis of typhoid fever Blood cultures are positive during the first week . The Widal test is done for titres of antibodies against salmonella typhi and salmonella paratyphi . Stool cultures and sometimes urine cultures are positive after the second week Typhidot It is a rapid serological test done for the diagnosis of typhoid fever and only gives result as positive or negative. It is a dot Elisa kit that detects IgM and IgG antibodies against the outer membrane protein (OMP) of the salmonella typhi .

22. Widal Test Diagnosis of typhoid fever Widal test It is an agglutination test for detection of agglutinins (H and 0) in patients with enteric fever. Antibodies of salmonella appears in the serum at the end of first week and rises thereafter. It can be done by slide and test tube method. The slide agglutination test is used as a primary screening procedure. Tube test is quantitative method and is a method of choice.

23. Diagnosis of typhoid fever Principle of Widal test Serum sample of the patient is tested for ‘0’ and ‘H’ antibodies by using antigenic suspensions of salmonella typhi O and salmonella typhi H respectively. In typhoid fever, an increase in the agglutinations (1:160 and above) is observed. Antigen o is common in both typhi and paratyphi species, so species is identify through h antibodies. For typhi identification, the antigen suspension is used th. For paratyphoid species identification,, the antigen suspensions used are S.Paratyphoid AH and S.Paratyphoid BH.

24. Glass plates with ceramic rings Serum Diagnosis of typhoid fever Procedure by slide method Specimen Fresh serum Requirements Glass plates with ceramic rings Droppers Widal kit consisting of controls and antigenic reagents Applicator sticks

25. Diagnosis of typhoid fever Procedure of Widal test by slide method Before performing the test, bring all reagents to room temperature and mix well. Mark the circles of slides as PC (positive control), NC (negative control), O, H, AH, BH as per antigen solutions used for testing. Add 1 drop of positive control (20μl) into the circle marked as PC of given glass slide. Then add 1 drop of negative control (20μl) into the reaction circle marked as NC.

26. Diagnosis of typhoid fever Procedure of Widal test by slide method Add 1 drop of test sample (25μl) into each reaction circle labelled as O, H, AH, BH according to given antigen solution. Add antigen solutions of salmonella typhi ‘O', salmonella typhi ‘H', salmonella paratyphi ‘AH' and salmonella paratyphi ‘BH', respectively in test serum and PC and NC. Mix the content of each reaction circle uniformly with separate mixing stick. Rotate the glass slide gently (approximately for one minute) and observe .

27. Diagnosis of typhoid fever Observation and result of Widal test by slide method After mixing the antigen solution with test sample, positive control, negative control separately, observe for the agglutination reaction.

28. Diagnosis of typhoid fever Interpretation of Widal test by slide method When the test sample is treated with attenuated salmonella antigen suspensions, the antibodies present in the sample react with the antigen suspension to give clearly visible agglutination which can be seen through naked eye. Formation of agglutination indicates positive result, means the presence of salmonella antibodies in the test sample. No agglutination is a negative test result indicates the absence of salmonella antibodies.

29. Diagnosis of typhoid fever Procedure of Widal test by test tube method The tube agglutination test is a quantitative method which is used for the determination of titre values of antibodies present in the patient serum.

30. Diagnosis of typhoid fever Requirements Patient serum Reagent: 0.85% sterile saline Other requirements: incubator, micropipettes, tips. Widal test kit Two types of tubes Dreyer’s tube - A narrow tube with conical bottom for H agglutination. Felix tube - A short tube with rounded - bottomed for O agglutination.

31. Diagnosis of typhoid fever Procedure of Widal test tube method Before starting the experiment, bring all reagents to room temperature and mix well. Prepare 4 sets of test tubes for individual antigen. Each set contains 8 tubes. Add 1.9 ml of 0.85% sterile saline to tube no. 1 of each antigen set. To tube no. 2-8 of all sets add 1 ml of physiological saline. To tube no. 1 of all sets add 0.1 ml of test sample to be tested and mix well. Transfer 1 ml of the diluted serum sample from tube no. 1 to tube no. 2 and mix well.

32. Diagnosis of typhoid fever Procedure of Widal test tube method Transfer 1 ml of the diluted serum sample from tube no. 2 to tube no. 3 and mix well. Continue this serial dilution till tube no. 7 in each set of antigen. Discard 1.0 ml of the diluted serum from tube no.7 of each set. So the dilutions of the serum sample from tube no. 1 to 7 respectively in each antigen set are 1:20, 1:40,1:80, 1:160, 1: 320, 1:640, 1: 1280. Tube no. 8 is negative control with 0.85% sterile saline.

33. Diagnosis of typhoid fever Procedure of Widal test tube method To one set i.e. From tube no.1- 8 add 50 μ l of salmonella typhi 'O' antigen. In second set i.e. From tube no.1- 8 add 50 μ l of salmonella typhi 'H' antigen. Respectively for third and fourth sets, add salmonella paratyphi ‘AH' and salmonella paratyphi ‘BH' to all tubes from 1-8. Mix well, cover and incubate these tubes overnight at 37°C (approximately 18 hours). After incubation, observe for agglutination.

34. Note:- Add 1 drop reagent to all tubes Diagnosis of typhoid fever

35. Diagnosis of typhoid fever Observation of Widal test by test tube method Note down the titre value of the antibody in the given test sample for all the antigens.

36. Diagnosis of typhoid fever Interpretation H agglutination leads to the formation of loose, cotton-woolly clumps. O agglutination is seen a disc like pattern at the bottom of the tube. Agglutinin titre (1:160 and above) is considered as significant infection and low titres indicate absence of infection. Interpretation of widal by tube method

37. Diagnosis of typhoid fever Treatment General measures Typhoid fever can be effectively controlled by sanitary measures for disposal of sewage, clean water supply and supervision of food processing and handling. Vaccination Tab vaccine (Typhoid-Paratyphoid A and B) It is heat killed cell vaccine which contains S. Typhi 1,000 millions, S. Paratyphi A and B,750 millions each per ml and preserved in 0.5 per cent phenol.

38. Diagnosis of typhoid fever Dose schedule - The vaccine is given in two doses of 0.5 ml subcutaneously at an interval of 4-6 weeks. Booster every 3 years . “Booster” basically refers to an extra dose of a vaccine after the original (or primary) dose is administered. These boosters can be weeks, months or years later. Booster shots are administered to remind the body’s immune system about the virus it needs to defend against. This improves or gives the immune system a boost.

39. Time for the quiz - 1 Q. The colonies of salmonella on MacConkey agar are _______ Lactose fermenting Non lactose fermenting Both of them None of them

40. Time for the quiz - 2 Q. Enrichment media for salmonella are? Selenite F Tetrathionate broth DCA Both A & B

41. Time for the quiz - 3 Q. Typhoid fever is acquired by ingestion through_______ Contaminated food Water Both A & B None of the above

42. Time for the quiz - 4 Q. Which test is done for the demonstration of salmonella anti -bodies in serum___________? VDRL Oxidase Widal CRP

43. Time for the quiz - 5 Q. In Widal test by tube method, dreyer’s tube is used for_____ H agglutination O agglutination None of these Both of them

44. Answers to the quiz B D C C A

45. The salmonellae are primarily intestinal organism causes enteric fever, gastroenteritis and septicemia . The most important species of salmonella are salmonella typhi , salmonella paratyphi a, salmonella paratyphi b and salmonella paratyphi c. The term enteric fever includes typhoid fever caused by s. Typhi and paratyphoid fever caused by S. Paratyphi A,B and C. Summary

46. Typhoid fever is acquired by ingestion of contaminated food and water. Antibodies of salmonella appears in the serum at the end of first week and rise sharply during the third week of enteric fever. Widal test is an agglutination test for detection of antibodies against (H and 0) in patients with enteric fever. Summary

47. Https://www.Cdc.Gov/typhoid-fever/symptoms.Html Text book of microbiology by Ananthanarayan & Paniker 9 th edition Textbook of microbiology by Dr Baveja fifth edition Textbook of medical laboratory technology by Dr Godkar 2 nd edition Himedialabs.Com /TD/HTI018.Pdf Himedialabs.Com /TD/HTI017.Pdf Https://www.Portea.Com/labs/diagnostic-tests/widal-typhoid-test-typhidot-124/ References