LABORATORY DIAGNOSIS OF ENTERIC FEVER

A. Patient presenting in the 1st wk of fever

I

BLOOD CULTURE

a. Indications:
For the detection of septicemic conditions. Apart from the clinical setting of
Enteric fever in the 1st wk, such conditions are also encountered in:
1. Brucellosis
2. Infective endocarditis
3. Puerperal sepsis
4. Sepsis in a surgical wound
5. Meningitis
6. Osteomyelitis
7. Pneumonia
NOTE: In conditions 2 to 7, bacteria spills over into the bloodstream from the
primary site of infection.
As part of diagnostic work up in a case of pyrexia of unknown origin (PUO)
Septicemia- The presence of bacteria in the blood of a patient with clinical signs &
symptoms of infection. Signs & symptoms of septicemia may include fever or
hypothermia, chills, hyperventilation & subsequent respiratory alkalosis,
hypotension or shock, skin lesions, change in mental status & diarrhea.
PUO- Any febrile illness lasting more than a few days, the cause of which is
obscure due to the absence of obvious specific or localizing signs & symptoms.
b. Principle of Blood Culture Examination
In this technique, detection of bacteria in the bloodstream is undertaken by
inoculating a sample of venous blood into an appropriate broth. To neutralize the
antibacterial substances present in the blood ratio of 1:10 is maintained between
volume of sample & medium. This broth is incubated at 37C & monitored at
specific time intervals for the detection of turbidity. On the appearance of turbidity,
subculture is done on an appropriate solid medium for the identification & further
processing of the organism growing in the broth. In the absence of turbidity, the
broth is incubated for 7-10 days before declaring culture results as negative.
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c. Procedure
1) Care should be taken while sample collection, it should be taken before
antibiotic therapy.
2) Skin preparation Use 70% ethanol/ betadine soaked cotton pad to scrub skin
vigorously for 60 secs.( Dont touch the sterilized part again, or contamination
may give false positivity). Allow to dry.
3) Preparation of Blood culture vials- Wipe tops of vials with single alcohol swab
& allow to dry (Dont touch the sterilized part again, or contamination may give
false positivity).
4) Phlebotomy- Insert the needle into the prepared vein & collect 5-10ml blood (
in adults) & 2-5 ml blood in children. Add blood into the blood culture bottle
through the needle.
5) Transport to laboratory at room temperature with minimum delay. NEVER
REFRIGERATE.
NOTE: The addition of SPS (Sodium Polyanethol Sulphonate) in culture media is
currently being done by some manufacturers to counteract the
bactericidal action of blood.
To reduce the risk of contamination during repeated subcultures, Castanedas
method of culture or biphasic medium is used.
In this biphasic medium, broth as well as agar slant is there.
After inoculation of blood, the bottle is incubated in the upright position. For
subculture, the bottle is tilted so that the broth runs over the surface of the
agar.
It is reincubated in the upright position.
If organisms are present, colonies will appear on the slant.
Media & Inoculation
Medium

Amount of blood to be
inoculated
Adults- 10ml
Children- 5 ml
Neonates- 2ml

Aerobic bacteria
Brain Heart Infusion broth
Bile broth
Glucose broth
Anaerobic bacteria
Thioglycollate both
Robertson Cooked Meat Broth (RCM)
d. Observation
Time of

Broth observed

If positive
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If negative

Reading
After
Overnight
incubation
On 3rd day

for
Turbidity

On 5th day

Turbidity

On 7th day

Turbidity

Turbidity

Subculture on
Blood agar &
MacConkey agar
Subculture on
Blood agar &
MacConkey agar
Subculture on
Blood agar &
MacConkey agar
Subculture on
Blood agar &
MacConkey agar

Reincubate
till 3rd day
Reincubate
till 5th day
Reincubate
till 7th day
Declare as
Culture
negative
sample

Colony morphology
Culture media
1. Blood agar
2. MacConkey agar

Colony
characterstics
after
Subculture at 37 C
Grayish color & Non-Haemolytic
Pale color

e. Further tests for speciation

1. Biochemical tests
Tests

Result

1. Oxidase

Negative

2. Nitrate reduction
test
3. Catalase

Positive

4. Sugar
fermentation test

Glucose fermented : S.Typhi (with acid no gas),

S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
& gas).
Lactose not fermented
Mannitol fermented : S.Typhi (with acid no gas),
S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
& gas).
Maltose fermented : S.Typhi (with acid no gas),
S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
& gas).
Sucrose not fermented
Xylose fermented S.Typhi (with acid no gas),
S.Paratyphi A (not fermented), S.Paratyphi B (with acid &
gas).

Positive

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5. Motility
6. Indole
production
7. Methyl red
8. Voges-Proskauer
9. Citrate
Utilization test
10. Urease
11. Triple Sugar
Iron agar

12. Phenylalanine
deaminase

Motile
Indole not produced
Positive
Negative
Not utilized: S.Typhi & S.Paratyphi A
Utilized: S.Paratyphi B
Negative
Alkaline slant / Acidic butt ( K/A, no gas, H2S ): S.Typhi
Alkaline slant / Acidic butt ( K/A, gas, no H2S ):
S.Paratyphi A
Alkaline slant / Acidic butt ( K/A, gas, H2S ): S.Paratyphi
B
Not produced

2. Slide agglutination test

Choice of antisera to be guided by biochemical reactions given by the
recovered isolate.
Serotyping

O antisera

H antisera

For S.Typhi
For S.Paratyphi A
For S.Paratyphi B

D9
A2
B4

D
A
B

B. Patient presenting in the second or subsequent week of Enteric Fever:

Note
1. Following the first week of enteric fever, the sensitivity of blood culture gradually
decreases to about 75%of cases in second week of fever, 60% in the third week and
25% thereafter till the subsidence of pyrexia.
2. From the second week of enteric fever, the sensitivity of Widal test starts rising
owing to the appearance of specific antibodies in the peripheral circulation.
3. Since Salmonella is excreted in the faeces throughout the course of the disease,
faecal culture can also be done for diagnosis of enteric fever. Importance of faecal
culture rises in the later stages of the disease and convalescence,since the bacteria
is no larger present in the blood stream during that period. Faecal culture is
valuable in patients on antibiotics as drug does not eliminate the bacilli from the

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gut as rapidly as it does from the blood. However faecal culture can also be positive
in carriers.
4. The role of urine culture in enteric fever is relatively less owing to irregular and
infrequent excretion of bacteria in urine.
WIDAL TEST
Indication: For the diagnosis of enteric fever, beyond the first week of disease,
through the detection of specific antibodies to enteric fever pathogens.
Principle: It is a tube agglutination test employed for the serological diagnosis of
enteric fever. Patient serum, in doubling dilutions, is reacted with 4 antigens of
Salmonella taken in separate tubes, viz. (i) common O antigen shared between S.
typhi/ S.paratyphi A/ S. Paratyphi B (ii) Salmonella Typhi H(TH) (iii) Salmonella
Paratyphi AH (AH), (iv) Salmonella Paratyphi B H (BH)antigens. Presence of specific
antibodies is demonstrated by the formation of antigen- antibody complexes, in the
form of clumps, at the bottom of the tubes.
Procedure:
1. Test tubes are arranged in four rows, with 6 tubes in each row.
2. Patient serum is diluted (in doubling dilution) with normal saline through a series
of tubes (Felixs Tube, a short rounded bottom tube for O antigen and Dreyers
Tube, a narrow tube with a conical bottom for H antigen) from left to right,
ranging from 1:10 to 1:160.
3. In each row, a control tube is set up that contains normal saline & antigen.
4. Final dilution is done by adding a fixed amount of standard suspension of O
antigen and H antigen in each tube.
5. Tubes are incubated overnight at 37 C.
6. Tubes are examined for agglutination comparing with the respective antigen
control tubes.
Serum dilutions:
Felixs Tube for S.Typhi Oantigen

1:10

1:20

1:40

Control Tubes

1:80
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1:160

Normal Saline

Dreyers Tube for S.TyphiH antigen

1:10

1:20

1:40

1:80

1:160

Normal Saline

1:160

Normal Saline

1:160

Normal Saline

Dreyers Tube for S.Paratyphi A AH antigen

1:10

1:20

1:40

1:80

Dreyers Tube for S.Paratyphi B BH antigen

1:10

1:20

1:40

1:80

Final Dilution (After adding equal volume of respective antigen)

1:10

1:20

1:40

1:80

1:160 Normal Saline + Antigen

Observation:
The control tubes are examined first, for the absence of agglutination.
The agglutination of O antigen appears as a matt or carpet at the bottom.
Agglutination of H antigens appears as large, loose, fluffy clumps.
The highest dilution of serum that produces a visible agglutination is taken as endtitre. The titres against all four antigens are recorded.
Interpretation:
Generally antibody titers above 1:80 for O & 1: 160 for H antigens are considered
clinically and significant. However these cut- off titres show geographical variation,
depending on the endemicity of Enteric fever.
Positive Widal test (antibody titers above cut-off level) is indicative of the presence
of antibodies to Enteric fever pathogens.
False negative Widal test is observed in early stage (<1 week) of Enteric fever or
with the early administration of antibiotics.

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 False positive Widal test may be observed as an anamnestic response in several

febrile illnesses.
Repeat performance of Widal test after a gap of 7- 10 days demonstrates a drop in
antibody titer, in case of an anamnestic response. In case of Enteric fever, a repeat
test shows rise in antibody titer.
TAB vaccinated patients may show a high titre of antibodies to each of the

antigens.

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