salmonella. general ch. inhabitant of human and animal intestine. gram-negative bacilli, non...
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Salmonella
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SalmonellaGeneral Ch.Inhabitant of human and animal intestine.
Gram-negative bacilli, non capsulated, motile, non spore forming.
Non lactose fermenter (NLF).
Produce H2S from ‘thiosulphate’.
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Pathogenicity1- Enteric fever (Typhoid fever, Paratyphoid fever)
• (S. typhi, S. paratyphi A,B,C).
• No animal reservoir.
2- Food poisoning (Gastroenteritis)
• (S. typhimurium, S. enteretidis).
• Zoonotic infection (animal).
• No systemic infection.
3- Septicemia
• S. cholerasuis.
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Enteric fever
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Salmonella: >1000 species acc. to diff. Ag (Somatic, flagellar, virulence)
-O (Somatic or cell wall
antigen).
-H (flagellar antigen).
-Vi (polysaccharide
virulence).
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Laboratory Diagnosis of
Enteric Fever
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I. Diagnosis of A case of Enteric Fever
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A) Isolation of the organism
1- Sample:
• Blood (1st week).
• Stool (2nd ,3rd week).
• Urine (3rd week)
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a. From blood:• Blood culture or clot culture
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b. Culture of faeces:
• In at least 50% of the cases culture of faeces is
positive in first week.
• The isolation of S. typhi greatly increases in the
second or third week.
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c. Urine culture:
• Urine culture is positive in one third of
cases, and in great majority of cases, it will
be free from organisms, before the faeces
becomes negative.
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2. Direct film stained by Gram:
• Gram-negative bacilli.
• motile.
• non capsulated.
• non sporulated.
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a) Culture characters
O2facultative anaerobe
CO2
Temp37°C
3. Culture:
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b) Media Selective
Enrichment
Indicator
nutrient agar
• Selenite broth
• tetrathionate
Mackonkey, DCA
SS, HE, XLD
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b) Culture media
• On nutrient agar: Grow as smooth
colonies, 2-4 mm in diameter.
• Maximal recovery of salmonella is obtained
by using enrichment broth medium as
selenite or tetrathionate broth.
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• Selective media: for Salmonella are SS
agar, Hekton enteric (HE) medium, and
xylose-lysine-deoxycholate (XLD) agar,
which contain selective ingredients.
• Indicator media: On MacConkey and DCA
media: they produce pale colonies (NLF).
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Salmonella- XLD media
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Salmonella- SS media
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4.Identification of growth
Morphology
BR
Slide aggl
Molecular
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4. Identification of the obtained colonies by:
1. Film stained by Gram:
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2. Biochemical reactions:
• Ferment glucose, mannitol, and maltose with
acid (S. typhi) and A+gas (S. paratyphi) .
• Lactose and sucrose is not fermented .
• H2S produced from thiosulfate.
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3. Slide agglutination: • using ‘O’ and ‘H’ antisera against salmonellae.
4. Molecular:
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B)_ Serodiagnosis:
Widal test ( tube agglutination test):
Use:
Widal test is an agglutination test for detection of antibodies
against Salmonella typhi and Salmonella paratyphi, the
common causal agents of enteric fevers.
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Principle:
• When serum sample containing antibodies against
S.typhi and S.paratyphi A, B or C are mixed with
respective antigens, agglutination will take place.
• In S.typhi and S.paratyphi two types of antigens are
recognized as diagnostically important:
• ‘O’ antigen “Somatic” antigen.
• ‘H’ antigen “Flagellar” antigen.
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• O antigens of various species have components in common
and hence only one O antigen i.e. non species specific.
• H antigens of Salmonella spp. are species specific, and
hence the H antigens of all S.typhi, S.paratyphi A and
S.paratyphi B and S.paratyphi C are employed in the test.
• Serum antibodies against H and O antigens of salmonella
usually appear by the 7th-10th day of infection and the
titer reaches maximum during the 4th week.
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Method• Five sets of agglutination tubes are prepared.
• To each set, a different H suspension of the salmonella,
i.e. H suspension of S. typhi, H suspension of S.
paratyphi A, H of S. paratyphi B, H of S. paratyphi C and
one set for O suspension of any species because it is a
common antigen.
• Serial dilutions of patient’s serum (1/10, 1/20, 1/40, 1/80
…..) are made in each set of tubes.
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Interpretation:
• The endemicity of the disease in the area. In Egypt,
titers below 1/80 are of no significance. They are
due to previous subclinical infection.
• Agglutination of O suspension indicates recent
infection.• The O suspension will not be agglutinated if vaccination
was done a long time ago.
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• Recently vaccinated individuals possess
agglutinins to S. typhi and S. paratyphi.
Agglutination will occur with more than
one suspension.
•
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False –ve:
• If the test was done during the first week, it
gives false negative results as the antibodies
start to appear during the second week.
• If the patient received antibiotic treatment
early in the disease, the antibody titer will be
suppressed.
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• False +ve: Non-enteric infections may cause a non-
specific rise on antibody titer; however, the titer is
low and falls rapidly on recovery (anamnestic
reaction).
• For proper interpretations of the Widal test, two
serum samples separated by 10 days interval should
be tested. The detection of a rising titer in the second
serum sample indicates active enteric infection.
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Percautions:
1-endemicity2-antibiotics
3-vaccinations4-anamnestic reaction
5-time
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Tube agglutination ‘Widal test’
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O Ab H Ab ‘typhi’
H Ab ‘paratyphi
A’
H Ab ‘paratyphi
B’
H Ab ‘paratyphi
C’
Result
+ + - - - S. Typhi inf.
+ - + - - S. P.typ. A inf.
+ - - + - S. P.typ. B inf.
+ - - - + S. P.typ. C inf.
+ + + + + Recent vaccine
- + + + + Old vaccine
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+ve widal S paratyphi B
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+ve widal old vaccination
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II. Diagnosis of Typhoid Carrier
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- 5% of cured- Asymptomatic transmission.- Difficult treatment.
• In order to label a person as a typhoid carrier, the
isolation of the organism should be done from
urine or faeces.
• If these are repeatedly negative, bile or duodenal
aspirate can be used.
• Vi antibodies present in a titre of more than 1:10 is
also suggestive of chronic typhoid carrier.
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Thank you