Laboratory diagnosis of salmonella

Laboratory diagnosis of Salmonella

1. Laboratory diagnosis of Salmonella By, Dr.M.Malathi Postgraduate Department of Microbiology Chengalpattu Medical College

2. Introduction The genus Salmonella consists of bacilli that parasitise the intestines of a large number of vertebrate species and infect human beings, leading to enteric fever, gastroenteritis, septicemia with or without focal suppuration and the carrier state

3. Clinical Manifestations Typhoidal salmonella – Enteric fever Non typhoidal salmonella – Gastroenteritis Bacteremia Osteomyelitis Localised infections Carriers

4. Laboratory diagnosis of Enteric fever Typhoid fever + Paratyphoid fever Typhoid fever – S.Typhi Paratyphoid fever – S.Paratyphi A, B, and C

5. Confirmed case of typhoid fever is defined(WHO), as a patient with fever (> 38°C) that has lasted for at least three days, with a laboratory confirmed positive culture of S.Typhi . Probable case of typhoid fever is a patient with fever (> 38°C) that has lasted for > 3 days, with a positive serodiagnosis or antigen detection test but without S.Typhi isolation. Chronic carrier is determined as excretion of S.Typhi in stools or urine for longer than one year after the onset of acute typhoid fever.

6. Specimen collection Blood Serum Urine Feces BoneMarrow Bile Pus CSF Sputum Gall bladder Liver Spleen Mesentric lymph nodes

7. Ideal specimen First week Blood (culture) Second week Serum (Antibodies) Third week Stool Fourth week Urine

8. Chance of isolation Specimens First week Third week Blood 50 to 80% 30% Feces 40 to 50% 80% Urine - 25%

9. Blood culture Volume of blood : 10 to 15 ml from adults and adolescents , 2 to 4 ml in children Ratio of blood to bile broth: 1:10 Or add saponin to BHI broth with 0.05% SPS Inoculate the blood immediately Transport immediately, never store under 15degC Incubate as soon as possible

10. Blood culture bottles

11. When blood culture bottles are not available, direct plating of blood buffy coat from 5 to 10ml sterile heparinised blood onto columbia agar plates containing 0.05% saponin is recommended( Wain , J et al)

12. Check for turbidity and evidence of growth after 1,2,3 and 7 days Bottles showing signs of growth – Do culture on solid media Subculturing done in Mac Conkey agar and Blood agar On day 7, all the bottles subcultured before being discarded as negative

13. Casteneda`s method Blood agar – Non hemolytic, 2 to 3mm, smooth white colonies Mac Conkey agar – non lactose fermenting colonies Confirmed by biochemical reactions and slide agglutination test with high titre sera

14. Slide agglutination test - Serotyping Prepare a milky suspension of overnight slope culture with saline Place a drop in clean glass slide Check autoagglutination Add diagnostic sera in the following order for serotyping

15. Salmonella polyvalent O (Groups A-G) Salmonella polyvalent H phases 1 and 2 serum and polyvalent H phase 2 serum Individual Salmonella O group sera O2 – O13 Single factor H sera Unusual Serotype ? Send to National Salmonella Reference Centre Central Research Institute, Kasauli

16. Rapid detection tests from culture MUCAP test – 4 methylumbelliferyl caprylate test – rapid identification of Salmonella strains directly from agar plates The substrate combines with Salmonella C8 esterase – releases the umbelliferone – strongly fluoresent at 365 nm Apply a drop the reagent directly over the suspected colonies on agar plat and observe under a wood`s lamp within 5 minutes 100% sensitivity and specificity

17. OBIS Salmonella test – Oxoid Biochemical Identification System – rapid colorimetric spot test For the determination of PYRase and NPA activity Sample from the colony on an agar plate and applied to the PYR and NPA test areas on the card Drop of buffer solution added to both test areas, after 5 minutes, one drop PYR reagent added in PYR test area, NPA reagent in NPA area

18. Interpretation PYRase negative NPA negative Salmonella PYRase positive NPA negative Citrobacter NPA positive PYRase negative Proteus, Morganella and Providencia

19. Automated systems BACTEC BacTalert Vitek

20. Clot culture Allow the blood to clot and serum pipetted off and used for widal test Clot is broken up with sterile glass rod and added to bottle of bile broth Add streptokinase (alternative) Higher rate of isolation than blood cultures (bactericidal action of the serum is obviated)

21. Serum 1 to 3 ml of blood inoculated into a tube without anticoagulant Second sample should be collected during convalescent phase Used for serological assays

22. Widal test Aim: Measurement of H and O agglutinins for typhoid and paratyphoid Principle : Tube Agglutination Requirements: Serum, Tubes, Antigen, Incubator, Waterbath Tubes : Dreyer`s tube and Felix tube

23. Antigens O antigen of S.Typhi H antigen of S.Typhi H antigen of S.Paratyphi A and B Strain used to prepare : S.Typhi 901, O and H

24. Procedure: Serial dilutions of equal volumes of serum and antigen mixed. Put controls Incubated overnight at 37degC Read the results No agglutination in controls For O antigen – disc like pattern For H antigen – loose, cotton wooly clumps

25. Highest dilution – TITRE Moderate sensitivity and specificity 30% of culture proven cases found to be Widal negative ( WHO – TFguide ) Slide Widal test – undiluted patient serum and antigens

26. CAUTION Stage of disease Prior antibiotics Prior immunisation Anamnestic response Antigen preparation – free from fimbriae False positive – typhus, acute falciparum malaria, chronic liver disease, rheumatoid arthritis, nephrotic syndrome False negative – antibiotics, severe hypoproteinemia

27. Interpretation Always rising titre by testing paired sera – fourfold rise in the titre needed Single report with caution Baseline titre in endemic areas O - > 1:100 H - > 1:200

28. IDL Tubex ® test Simple, Rapid Slide latex agglutination test O 9 antigen – Highly specific for S.Typhi , used here , immunodominant epitope Only for Typhoid fever, does not give positivity for S.Paratyphi Detects IgM antibodies

29. Test Pack: Sets of V shaped tubes – six samples per set – tested simultaneously Reagent A, magnetic particles coated with S.Typhi LPS Reagent B, Blue coloured latex particles coated with a monoclonal antibody specific for the O9 antigen

30. Test serum ( one drop ) + Reagent A (one drop) – 1 minute – mix Then add two drops of Reagent B Keep the tubes in magent embedded stand, and slid it several times Read the results immediately Based on the colour of the reaction – compared with the chart – Titre value noted Stored sera has a better result in tubex than widal

31. IDL Tubex ® test

32. Typhidot ® test Simple, speed, economical Sensitivity is 85.9%, Specificity 96.7% To detect specific IgM and IgG antibodies to S.Typhi Typhidot - M ® - To detect IgM alone Replaces the widal when used in conjunction with the culture (Gold Standard) High negative predictive value – useful in high endemic areas

33. Typhidot ® rapid assay

34. IgM dipstick test Detects IgM antibodies in serum and whole blood Materials: Dipstick Lyophilised non enzymatic detection reagent Liquid to reconsitute the detection reagent Liquid to wet the test strip of dipstick

35. Wet the test strip in a mixture of serum and detection reagent ( 1:50) Incubate for 3 hours at RT Rinse the test strip with water Allow it to dry Compare the color with reference strip Grade it as 1+, 2+, 3+ and 4+ Sensitivity – 65% to 77% Specificity – 95% to 100%

36. Enterocheck - WB Immunochromatographic test in cassette from 30 minutes test Sensitivity – 79.3% Specificity – 90.2%

37. Coagglutination test Demonstration of circulating antigen Done in the blood and in urine Done in early phase of the disease S.aureus (Cowan I Strain) which contains protein A is stabilised with formaldehyde and coated with S.Typhi antibody 1% above suspension + patient serum – in a slide – visible agglutination (2 min) – positive

38. Urine Irregular and infrequent shedding of bacilli Positive only in second and third weeks 25% cases + Clean voided urine samples are inoculated into enrichment and selective media

39. Feces Collected in a container Spoonful amount Transport immediately 6ml of buffered glycerol saline transport medium Alternate specimen: Rectal swabs Fecel swabs

40. Shed throughout the course of the disease and also in convalescence Valuable in patients on antibiotics ( drug does not eliminate the bacilli from the gut) Fecal samples plated directly on MacConkey DCA / XLD Wilson Blair Media Enrichment also done in selenite or tetrathionate broth , incubated for 6 to 8 hours and subcultured .

41. Pale non lactose non sucrose fermenting colonies – DCLS Red, black centred colonies – XLD Rule out proteus by urease test Check for purity by subculturing in nutrient agar Do biochemical reactions and sugars Do serotyping by slide agglutination test

42. Interpretation Provisional report – given on third or fourth day and inform the clinician Secondary confirmation test panel: Citrate agar slope Lysine decarboxylase medium with control Salicin peptone water ONPG Mac Conkey secondary purity plate Nutrient agar slope Sensitivity agar plate

43. If the secondary tests – confirm – pure culture of Salmonella  seed it on to two Dorset egg slopes and send one to a Salmonella Reference Laboratory for final serotyping . Send a confirmation report to clinician

44. Final report S.enterica subsp. enterica serovar Typhi

45. Other specimens Vomitus Bile Pus Bone marrow (Gold standard specimen)

46. Antimicrobial susceptibility testing Drugs : Amoxycillin Co- amoxiclav Cefuroxime Cotrimoxazole Ciprofloxacin Chloramphenicol

47. Most of the strains are sensitive Resistant – depends on serotype, phage type and country of origin 1990 – 20% strains resistant to Chlorampenicol isolated in UK 90% of strains are resistant to ampicillin and trimethoprim In Multidrug resistant areas – Ciprofloxacin is the drug of choice

48. A multidrug resistant strain of S.Typhimurium definitive type 104 that is resistant to five antibiotics emerged around 1990s 50% of the S.Typhimurium isolates were resistant to one or more drugs and 28% had a five drug resistance pattern 1998 – S.Newport – emerged as a major MDR pathogen

49. Molecular methods PCR is sensitive, but not widely used

50. Miscellaneous tests Leucopenia with relative lymphocytosis

51. Diagnosis of Carriers High incidence due to carrier state Contamination of food by food handlers (Carriers) Convalescent carriers Temporary carriers Chronic carriers ( 2 to 5%) Bacilli persists in the Gallbladder and kidney Intermittent shedding

52. Repeated sampling – Bile or Faeces , urine for culture (Confirmatory) Demonstration of antibodies to Vi antigens (Screening test) IgG is the primary indicator of carriers IgA and secretory IgA are seen. In Vaccinated – No secretory IgA Hence High IgA content indicate typhoid carrier state

53. Public health ? In cities – tracing of carriers – Sewer Swab technique Sewage – Filtration through Millipore membrane – culture in Wilson and blair media Food safety – Carriers in hotels – Eg : Typhoid mary

54. Salmonella and Eggs According to the Centers for Disease Control, 1 in 10,000 eggs contain Salmonella. Experts say that chickens carry the bacteria in their own bodies, and pass Salmonella along to the yolk and white while the egg is forming in the ovaries. Chickens can also pass bacteria to the eggshell—and through the shell pores into the inner egg—when the egg is laid.

55. The eggs are then submerged in all-natural water bath, where computer-controlled temperature zones monitor & heats the eggs in their shells to the exact temperature needed to destroy all bacteria, without cooking the egg. After pasteurization, the eggs are sealed with an FDA-approved, food-grade wax coating to prevent contamination and preserve product freshness . After pasteurization, the eggs are dried, cooled, and then stamped as P , which identifies them as pasteurized .

56. Pasteurised eggs

57. Typing methods Bacteriophage typing : Depends on Vi antigen Used in epidemiological surveillance National Salmonella Phage Typing Centre – Lady Hardinge Medical College S.Typhi phage types A and E1 – common in India S.Paratyphi A , types 1 and 2

58. Molecular methods: PFGE MLEE IS 200 profilinng Random amplified polymorphic DNA analysis

59. Summary Culture – Gold standard – Late results – AST Widal – Duration – Endemicity – paired sera Slide tests – Discrepancies between labs Ideal diagnostic test rapid, specific, sensitive TYPHIDOT EIA

60. In this study , 66% blood culture + 66% Widal test + 74% Typhidot + Typhidot is found to have high sensitivity and good specificity , alternate to blood culture

61. References Mackie & McCartney – Practical Medical Microbiology – 14 th Edition Konemann – Colour atlas of diagnostic microbiology – 6 th edition Harrisons – principle of internal medicine – 18 th edition District laboratory practive in tropical countries – 2 nd edition – Monica cheesbrough Wain J et al, Specimens and culture media for the laboratory diagnosis of typhoid fever WHO – Salmonella surveillance report Nitte journal of health science Malaysian journal of medical sciences