salmonella typhi lab diagnosis and pathogenesis

1. SALMONELLA

2. MORPHOLOGY
• Gram negative rods, 1-3
µm x 0.5 µm,
• Motile with peritrichate
flagella (except for
S. gallinarum, S
pullorum) which is
always NM
• No capsule or spores
• May possess fimbriae

3. • 2463 species
• Enteric fever group : typhoid and paratyphoid
bacilli exclusively or primarily human
pathogen. S. enterica : serotypes Typhi,
Paratyphi A, B, C
• Non typhoidal : Food poisoning group
essentially animal parasites but which can also
infect human beings, producing
gastroenteritis, septicemia or localised
infections. Eg. S.enteritidis, S. typhimurium

4. Cultural Characteristics
• Aerobic and facultative anaerobic
• Media of pH 6.8
• Temp ranges from 15-40° C (opt 37°C)
• Colonies are large, 2-3mm in diameter, circular,
low convex and smooth
• More translucent than coliform colonies
• MA, DCA – NLF colourless colonies
• Wilson and Blair bismuth sulphite media : black
colonies with a metallic sheen due to production
of H2S ( S paratyphi A gives green colonies)

6. Biochemical reactions
• Ferment glucose, mannitol and maltose
(acid+gas). Exception is S Typhi which is
anaerogenic
• Lactose, sucrose and salicin : not produced
• Indole is not produced
• MR positive, VP negative
• Citrate : utilised (except Typhi and Paratyphi)
• Urea is not hydrolysed
• H2S is produced except by S Paratyphi A,
S. cholerasuis

7. RESISTANCE
• Killed at 55 C in 1hr or at 60 C in 15 mins
• Boiling or chlorination of water and
pasteurization of milk destroy the bacilli
• In polluted water and soil, they survive for
weeks and in ice for months
• If prevented from drying culture survive for yrs
• Killed within 5 mins by 5% phenol

8. ANTIGENIC STRUCTURE
• Flagellar antigen : H
• Somatic antigen : O
• Surface antigen : Vi

9. H ANTIGEN
• Present on the flagella
• Heat labile
• Destroyed by boiling or by treatment with
alcohol but not by formaldehyde
• When mixed with antisera, large loose fluffy
clumps of agglutinate
• Immunogenic, antibody in high titre following
infection or immunisation

10. O Antigen
• Somatic O antigen is a phospholipid-protein-
polysachharide complex
• Integral part of the cell wall
• Identical to endotoxin
• extracted by treatment with trichloroacetic acid
• Unaffected by boiling, alcohol or weak acids
• When mixed with antisera, suspension forms
compact chalky, granular clumps
• Less immunogenic and the titre of antibody is
generally lower
• Classified into no of groups based on the
characteristics O antigens on the bacterial surface

11. Vi antigen
• Surface Polysachharide antigen enveloping the
O ag
• Heat labile analogus to the K antigens of
coliform
• bacilli inagglutinable with the O antiserum
become agglutinable after boiling or heating
at 60 C for 1 hr
• Unaffected by alcohol or 0.2% formal

12. Vi Antigen
• Tends to be lost on serial subculture
• Acts as a virulence factor by inhibiting
phagocytosis, resisting complement activation
and bacterial lysis by the alternative pathway
and peroxidase mediated killing
• Poorly immunogenic
• Persistence of Vi antibody indicates the
development of the carrier state.

13. Pathogenicity
• S Typhi, S Paratyphi A and B : human sources
• Enteric fever
• Septicemia
• Gastroenteritis or food poisoning

14. Virulence factors
• Type III secterion system (bacterial proteins):
mediate initial invasion
• O antigen: endotoxin
• Vi antigen
• Invasins: adherence, penetration

15. ENTERIC FEVER
• Infection acquired by ingestion
• Infective dose : 103 to 106 bacilli
• Incubation period: 7-14 days (3-56)
• On reaching the gut, the bacilli attach
themselves to the lamina propia and
submucosa
• Phagocytosed by macrophages and polymorphs
• Ability to resist intracellular killing and to
multiply within these cells

16. • Enter the mesenteric LNs where they multiply
and via thoracic duct enter the bloodstream
• Transient bacteremia follows during which the
bacilli are seeded in the liver, gall bladder, spleen,
BM, LNs, lungs and kidneys where further
multiplication takes place
• Towards the end of the incubation period (10th),
there occurs massive bacteremia (secondary
bacteremia) from these sites of multiplication,
heralding onset of clinical disease

17. • Bile : multiplies abundantly in the GB and is
discharged continuously into the intestine
where it involves the Peyers patches and
lymphoid follicles of the ileum
• They become inflamed, undergo necrosis and
slough off, leaving behind the characteristic
typhoid ulcers
• Ulceration : intestinal perforation and
hemorrhage

18. E N T E R I C ( T Y P H O I D ) F E V E R
• Typhoid fever has a slow, insidious onset and
if untreated, lasts for weeks. It ends either by
a gradual resolution or in death due to
complications (eg, rupture of the intestine or
spleen).
• Inc period : 7-14 days ( 3-56days)
• Clinical course : mild undifferentiated pyrexia
(ambulant typhoid) to a rapidly fatal disease

19. • Onset is ususally gradual, with headache,
malaise, anorexia, a coated tongue and
abdominal discomfort with either constipation or
diarrhea
• Step ladder pyrexia with relative bradycardia and
toxemia
• Hepatomegaly
• Soft palpable spleen
• Rose spots (3rd -4th wk)

20. • Complications :
• Perforation, hemorrhage, circulatory collapse
• Cholecystitis, arthritis, abscesses, periosteitis,
nephritis, hemolytic anemia, venous
thromboses, peripheral neuritis
• Some develop psychoses, deafness or
meningitis
• Convalescence is slow
• In about 5-10% relapse occur

21. • S. paratyphi A and B cause paratyphoid fever
which resembles typhoid fever but is generally
milder

22. EPIDEMIOLOGY
• Typhoid fever : still an important cause of
morbidity and mortality worldwide
• Strictly a human disease
• Chronic carriers of serotype Typhi are the
primary reservoir.
• The pathogen can be transmitted in the water
supply in developing endemic areas or where
defects in any system allow sewage from
carriers to contaminate drinking water.
• Transmission is by the fecal–oral route.

23. • Pt who continue to shed typhoid bacilli in
feces for 3 wks to 3 months after clinical cure :
convalescent carriers
• Those who shed the bacilli for more than 3
mths but less than 1 yr : temporary carriers
• Over a yr : chronic carriers (2-4%)
• Some persons may become carrier following
inapparent infn : symptomless excretors

24. • Food handlers or cooks who become carriers
are particularly dangerous
• Mary Mallon (Typhoid Mary) : over a period of
15 yrs caused at least 7 outbreaks affecting
over 200 persons

26. Laboratory Diagnosis
• Isolation of the organism from the patient
– Blood culture
– Stool culture
– Urine culture
– Others- bile, duodenal juice
• Demonstration of antibodies
• Demonstration of antigen in blood or urine

27. BLOOD CULTURE
• +ve in 90% of the cases in 1st wk, 75 % in 2nd
wk, 60% in 3rd wk and 25% in 4th wk. Becomes
negative on treatment with antibiotics
• 5-10ml of blood in 50-100ml of bile broth (
BHI broth, glucose broth)
• Sodium polyanethol sulphonate counteracts
the bactericidal action of blood

28. • Incubation at 37C overnight , subcultured on
MacConkey agar
• Pale NLF colonies may appear
• Motility and biochemical test
• Identification of the isolate is confirmed by
slide agglutination

29. SLIDE AGGLUTINATION
• A loopful of the growth from an agar slope is
emulsified in 2 drops of saline on a slide
• If S typhi is suspected, a loopful of typhoid O
antiserum ( factor 9/group D) is added
• Agglutination is looked for
• If suspected of S paratyphi, then agglutination is
done with O and H antisera

30. • If not isolated from 1st subculture from bile
broth, subcultures should be repeated every
other day till growth is obtained
• Cultures should be declared negative only
after the incubation for 10 days
• Castaneda’s method : to avoid contamination
and also for economy and safety
• Clot culture yields higher rate of isolation

31. FECES CULTURE
• Org. shed throughout the disease and also
during convalescent
• Almost as valuable as blood culture
• Use of enrichment (tetrathionate or selenite
broth) and selective media
• Usually helpful in isolation of the patient with
antibiotics
• Plated to MacConkey, DCA and Wilson Blair
media

32. URINE CULTURES
• Salmonella are shed in urine infrequently and
irregularly
• Less useful
• Cultures are positive only in 2nd and 3rd wk
• Repeated sampling improves the rate of
isolation
• Clean voided urine samples are centrifuged
and the deposit inoculated into enrichement
and selective media

33. Demonstration of Antibodies
(WIDAL TEST)
• Test for the measurement of O and H
agglutinins for typhoid and paratyphoid bacilli
in the patients sera
• 2 types of tubes :
• Dreyers tube : a narrow tube with a conical
bottom for the H agglutination
• Felix tube : short round bottomed tube for O
agglutination

34. Dreyers tube

35. Widal test
• Equal volumes of serial dilution of serum and the
H and O Ag are mixed and incubated in a water
bath at 37 C overnight
• Agglutination titre of the serum are read
• H agglutination : loose, cottony woolly clumps
• O agglutination : disc like pattern at the bottom
of the tube
• Supernatant is clear in both
• Antigens used are O and H antigens of S typhi and
H antigens of S Paratyphi A and B

37. Widal test Interpretation
• Agglutination titre depend on the stage of the
disease
• Testing of 2 or more samples is more meaningful
than a single test ( rise in titre)
• Difficult to lay down the level of significance
though 1:80 for O and 1:160 for H
• Agglutinins are present on account of prior
disease, inapparent infection or immunisation

38. • Persons who have had prior infection or
immunisation may develop an anamnestic
response during an unrelated fever. This may
be differentiated by repetition of the test after
a wk. (transient rise)
• Bacterial suspensions used as antigens should
be free from fimbriae ( False positive results)
• Cases treated early with chloramphenicol may
show a poor agglutinin response

39. Demonstration of Circulating Antigen
• Sensitised Staphylococcal coagglutination test
• Staph aureus (Cowan I strain) containing protein
A is stabilised with formaldehyde and coated with
S typhi antibody
• When 1% suspension of such sensitised
staphylococcal cells are mixed with serum from pt
in the 1st wk of the disease = visible
agglutionation within 2 minutes

40. Prophylaxis and Treatment
• Improvements in sanitation
• Detection of carriers
• Provision of protected water supply
• Chloramphenicol
• Fluoroquinolones
• Third generation cephalosporins

41. Vaccines
1. Killed whole cell vaccine :
– Heat killed, phenol preserved, whole cell
containing mixture of S. Typhi and S Paratyphi A
and B (TAB)
– Injected s/c in 2 doses of 0.5ml each at an
interval of 4-6 wk followed by a booster dose
every 3 yrs
– Effication 60-70% protection for 3-7 years
2. Vaccines of purified Vi antigen :
– Contains purified Vi ag which is injected
intramuscularly in a dose of 25µg (typhim- vi)-
75%

42. 3. Live vaccine:
• live avirulent vaccine from a mutant strain of S. typhi
(Ty2 1a)
• to children
• Efficacy : 65- 96%
• 3 oral doses on alternate day one hour before food
with a glass of milk

43. Salmonella Gastroenteritis
• Food poisoning caused by any Salmonella
except S Typhi
• Source of infection : animal products
• S Typhimurium, S enteritidis, S. anatum, S
haldar, S heidelberg
• Results from contaminated food
• Eggs, egg products, meat, milk and milk
products

44. Gastroenteritis
• Disease develops after a short inc period of 24
hrs or less, with diarrhea, vomiting, abdominal
pain and fever.
• May vary in severity from the passage of one or
two loose stools to an acute cholera like disease
• Usually subsides in 2-4 days but in some cases a
more prolonged enteritis develops, with passage
of mucus and pus in feces, resembling dysentery

45. Laboratory diagnosis
• Isolating the organism from the feces
• In outbreaks, causative article of food can
often be identified by taking a proper history
• Isolation of salmonella from the article of food
confirms the diagnosis

46. Control
• Prevention of food contamination
• Proper cooking destroys salmonella
• Large problem in developed countries largely
because of their food habits and living
conditions

47. Salmonella septicemia
• S choleraesuis may cause septicemic disease
with focal suppurative lesions such as
osteomyelitis, deep abscesses, endocarditis,
pneumonia and meningitis
• Can be isolated from blood or pus
• Should be treated with chloramphenicol or
other antibiotics as determined by sensitivity
tests.

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