Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Laboratory diagnosis of salmonella

10,548 views

Published on

Laboratory diagnosis of Salmonella

Published in: Health & Medicine
  • Be the first to comment

Laboratory diagnosis of salmonella

  1. 1. Laboratory diagnosis of Salmonella By, Dr.M.Malathi Postgraduate Department of Microbiology Chengalpattu Medical College
  2. 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. 3. Clinical Manifestations • Typhoidal salmonella – Enteric fever • Non typhoidal salmonella – Gastroenteritis • Bacteremia • Osteomyelitis • Localised infections • Carriers
  4. 4. Laboratory diagnosis of Enteric fever • Typhoid fever + Paratyphoid fever • Typhoid fever – S.Typhi • Paratyphoid fever – S.Paratyphi A, B, and C
  5. 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. 6. Specimen collection Blood Serum Urine Feces BoneMarrow Bile Pus CSF Sputum Gall bladder Liver Spleen Mesentric lymph nodes
  7. 7. Ideal specimen First week Blood (culture) Second week Serum (Antibodies) Third week Stool Fourth week Urine
  8. 8. Chance of isolation Specimens First week Third week Blood 50 to 80% 30% Feces 40 to 50% 80% Urine - 25%
  9. 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. 10. Blood culture bottles
  11. 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. 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. 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. 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. 15. 1. Salmonella polyvalent O (Groups A-G) 2. Salmonella polyvalent H phases 1 and 2 serum and polyvalent H phase 2 serum 3. Individual Salmonella O group sera O2 – O13 4. Single factor H sera Unusual Serotype ? Send to National Salmonella Reference Centre Central Research Institute, Kasauli
  16. 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. 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. 18. Interpretation PYRase negative NPA negative Salmonella PYRase positive NPA negative Citrobacter NPA positive PYRase negative Proteus, Morganella and Providencia
  19. 19. Automated systems • BACTEC • BacTalert • Vitek
  20. 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. 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. 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. 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. 24. • Procedure: 1. Serial dilutions of equal volumes of serum and antigen mixed. 2. Put controls 3. Incubated overnight at 37degC 4. Read the results 5. No agglutination in controls 6. For O antigen – disc like pattern 7. For H antigen – loose, cotton wooly clumps
  25. 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. 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. 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. 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. 29. • Test Pack: 1. Sets of V shaped tubes – six samples per set – tested simultaneously 2. Reagent A, magnetic particles coated with S.Typhi LPS 3. Reagent B, Blue coloured latex particles coated with a monoclonal antibody specific for the O9 antigen
  30. 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. 31. IDL Tubex ® test
  32. 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. 33. Typhidot ® rapid assay
  34. 34. IgM dipstick test • Detects IgM antibodies in serum and whole blood • Materials: 1. Dipstick 2. Lyophilised non enzymatic detection reagent 3. Liquid to reconsitute the detection reagent 4. Liquid to wet the test strip of dipstick
  35. 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. 36. Enterocheck - WB • Immunochromatographic test in cassette from • 30 minutes test • Sensitivity – 79.3% • Specificity – 90.2%
  37. 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. 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. 39. Feces • Collected in a container • Spoonful amount • Transport immediately • 6ml of buffered glycerol saline transport medium Alternate specimen: • Rectal swabs • Fecel swabs
  40. 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. 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. 42. Interpretation Provisional report – given on third or fourth day and inform the clinician Secondary confirmation test panel: 1. Citrate agar slope 2. Lysine decarboxylase medium with control 3. Salicin peptone water 4. ONPG 5. Mac Conkey secondary purity plate 6. Nutrient agar slope 7. Sensitivity agar plate
  43. 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. 44. Final report S.enterica subsp. enterica serovar Typhi
  45. 45. Other specimens • Vomitus • Bile • Pus • Bone marrow (Gold standard specimen)
  46. 46. Antimicrobial susceptibility testing Drugs : 1. Amoxycillin 2. Co-amoxiclav 3. Cefuroxime 4. Cotrimoxazole 5. Ciprofloxacin 6. Chloramphenicol
  47. 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. 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. 49. Molecular methods • PCR is sensitive, but not widely used
  50. 50. Miscellaneous tests • Leucopenia with relative lymphocytosis
  51. 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. 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. 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. 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. 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. 56. Pasteurised eggs
  57. 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. 58. Molecular methods: • PFGE • MLEE • IS 200 profilinng • Random amplified polymorphic DNA analysis
  59. 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. 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. 61. References • Mackie & McCartney – Practical Medical Microbiology – 14th Edition • Konemann – Colour atlas of diagnostic microbiology – 6th edition • Harrisons – principle of internal medicine – 18th edition • District laboratory practive in tropical countries – 2nd 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

×