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. 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. 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:
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. • 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:
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. • 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:
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. • 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:
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. 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 :
1. Amoxycillin
2. Co-amoxiclav
3. Cefuroxime
4. Cotrimoxazole
5. Ciprofloxacin
6. 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
– 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