LAB

DIAGNOSIS
‰ Culture isolation

Blood and bone marrow culture (in first week of illness):

•
 Conventional: BHI broth/agar
 Automated blood culture systems—BACTEC or BacT/
ALERT).

Stool culture (in 3–4 weeks of illness):

 Enrichment broth such as Selenite F broth,
tetrathionate
broth and gram-negative broth
 Low selective medium: MacConkey agar (translucent
NLF colonies)
 Highly selective media: DCA, XLD agar and Wilson
Blair’s Bismuth sulphite medium.

Urine culture (in 3–4 weeks of illness)—on MacConkey

agar.
Culture smear and motility:
.

Motile, gram-negative bacilli

‰. Biochemical identification
¾. Catalase positive and oxidase negative
¾. ICUT: Indole(–), Citrate(+/–), Urease(–)

TSI: K/A, gas(+) except in S. Typhi, H2S (S. Typhi- small speck,
S. Paratyphi A-absent, S. Paratyphi B-abundant)
.
‰. Slide agglutination test:
To confirm the serotype

‰.Serum antibody detection (Widal test): 2–3
weeks of illness
Antibodies are detected against TO, TH, AH, BH
antigens
¾. In S. Typhi infection: ↑TO and TH antibodies

¾. In S. Paratyphi A infection: ↑TO and AH antibodies

¾. In S. Paratyphi B infection: ↑TO and BH antibodies.

Result and interpretation
¾. O antibodies: Produce granular chalky clumps when react with O Ag
¾. H antibodies: Produce cottony woolly clumps when react with H Ag

.
‰. Antigen detection (serum and urine):
By ELISA

‰. Molecular methods:
PCR detecting flagellin gene, iro B and fliC gene

‰. Nonspecific findings:
For example, neutropenia

‰. Antimicrobial susceptibility testing.

LABORATORY DIAGNOSIS

 Type of specimen to be collected depends

on the duration of illness. The preferred
specimen(s) to be collected are:
 ™. First week of illness: Blood culture,
bone marrow or duodenal aspirate culture
 ™. Second/third week of illness: Serum
specimen for serology (e.g. Widal test)
 ™.Third/fourth week of illness: Urine and
stool culture
 CULTURE AND IDENTIFICATION
 Blood Culture
Blood culture is the ideal method for diagnosis in the first week
of fever, which becomes positive in about 90% of cases. There
after the positivity declines to 75% in the second week and
60% in the third week and 25% till the fever subsides.
 ™. Blood culture bottles: 8–10 mL of blood may be collected
in blood culture bottle; either conventional bottle (brain heart
infusion medium—monophasic or biphasic) or automated
bottle (e.g. BacT/ALERT)
 ™ Incubation: Blood culture bottles are incubated at 37°C.

Salmonellae are nonfastidious, growth occurs within 24

hours. From positively flagged blood culture broth, subcultures
are made on to blood agar and MacConkey agar
 ™ Colony appearance:
 „. Blood agar: Nonhemolytic moist colonies
 „. MacConkey agar: Colonies are round, translucent,pale and
non-lactose fermenting
 Stool and Urine Culture
• Urine culture seldom becomes positive as
salmonellae
are shed in urine infrequently. Urine is centrifuged
and the deposit is inoculated onto MacConkey agar.
• Stool culture is done similar to the method followed
for Shigella. Appropriate media should be used to
inhibit the commensals in the stool.

 ™ Enrichment broth such as Selenite

Fbroth ,tetrathionate broth and gram-negative
broth are used
 ™ Selective media such as:
„. Low selective media such as MacConkey
agar
 ™ Selective media such as:
• „ Low selective media such as MacConkey
agar„.

• Highly selective media: Growth of S. Typhi

occurs
DCA (deoxycholate citrate agar):
Produces non lactose fermenting pale colonies
with black center
XLD agar (xylose lysine deoxycholate):
Produces red colonies with black center
XLD DCA
Wilson Blair’s brilliant green bismuth sulfite
medium is particularly useful for the isolation
of S. Typhi from heavily contaminated
specimens. S. Typhi produces characteristic
jet black colored colonies with a
metallic sheen due to production of
H2S. S. Paratyphi A and others that do not
form H2S produce green colored colonies.
 Other Specimens
 ™. Bone marrow culture is employed during the
first week of illness (55–90% sensitive) when blood
culture is negative, especially when patient is on
antibiotics
 ™. Duodenal aspirate culture is recommended
during first week of illness if both blood and bone
marrow cultures turn negative
 ™. Combination of blood, bone marrow, and
intestinal secretions culture is the best method in
the first week which shows a sensitivity of more
than 90%
 ™. Other specimens from which salmonellae can
be isolated are rose spots, pus from suppurative
lesions, cerebrospinal fluid (CSF), sputum and
autopsy specimens such as gallbladder, liver and
 Culture Smear and Motility Testing
Gram-stained smear made from colonies reveals gram
negative,
non-sporing and non–capsulated bacilli. They are motile with
peritrichous flagella.
 Identification
Identification of Salmonella from the colonies is made either
by automated identification systems such as MALDITOFor
VITEK; or by conventional biochemical tests as
 ™. It is catalase positive and oxidase negative

 ™. Indole test (negative), citrate test (negative), ureas

test(negative)
 ™. TSI (triple sugar iron test) shows:

„. Alkaline/acid
. Gas present (except for S. Typhi, which is anaerogenic)
 „. Abundant H2S present except for:

 S. Paratyphi A: H2S not produced

 ™. MALDI-TOF can identify Salmonella up to genus level.
However they poorly differentiate between serotypes as
they share the same ribosomal proteins.

 Slide Agglutination Test

 Identification of Salmonella at genus level can be confirmed
by slide agglutination using polyvalent O antisera. Then the
serotypes can be identified by using type specific O
antisera.
 ™. S. Typhi: Agglutinates with O9 antisera
 ™. S. Paratyphi A: Agglutinates with O2 antisera
 ™. S. Paratyphi B: Agglutinates with O4 antisera.
 Flagellar antigens can also be determined by using type
specific H antisera.

 Antimicrobial Susceptibility Testing (AST)

AST is performed by disk diffusion method (on Mueller–
Hinton agar) or by automated MIC detection method by
microbroth dilution (e.g. by VITEK).
DEMONSTRATI
ON OF SERUM
ANTIBODIES
 WIDAL TEST
 ™. Principle: It is an agglutination test
where H and O antibodies against S. Typhi
and S. Paratyphi A and B are detected and
measured in the patient’s sera by using O
and H antigens
 ™. Antigens used: Four antigens are used
1. O antigens of S.Typhi (TO)
2. H antigens of S.Typhi (TH)
3. H antigens of S.Paratyphi A (AH)
4. H antigens of S.Paratyphi B (BH).

™. Procedure:
 Patient’s serum is serially diluted in normal saline in
test tubes from 1 in 10 to 1 in 640 dilutions. Four such sets
are made.
 To each set of diluted sera, respective four antigen
suspensions (TO, TH, AH, BH) are added
 „ Test tubes are incubated in water bath at 37°C overnight.

 . Results
 „. O agglutination appears as compact granular chalky
clumps (disk-like pattern), with clear supernatant fluid
 „. H agglutination appears as large loose fluffy
cottonwoolly clumps, with clear supernatant fluid
 „. If agglutination does not occur, button formation occurs
due to deposition of antigens and the supernatant fluid
remains hazy
 „. Titer: The highest dilution of sera, at which agglutination
occurs, is taken as the antibody titer.
 ™ Interpretation

„Significant titer: Any titer is not significant.

Higher titers are only significant.
Significant titer in most of the places in India is
taken as:
  H agglutinin titer more than 200 and
  O agglutinin titer more than 100.
  Low titers should be ignored and
considered as baseline titers in endemic
areas.
INTERPRETATION OF
WIDAL TEST.
Widal test result Suggestive of
Rise of TO and TH antibody Enteric fever due to S. Typhi
Rise of TO and AH antibody Enteric fever due to S.
Paratyphi A

Rise of TO and BH antibody Enteric fever due to S.

Paratyphi B

Rise of only TO antibody Recent infection: Due

to S. Typhi or S.Paratyphi A or B

Rise of only TH antibody Convalescent stage/anamnestic

response

Rise of all TH, AH, BH Post TAB vaccination

antibodies
 False-positive: Widal test may occur due to:
  Anamnestic response: It refers to a transient
rise of titer due to unrelated infections (malaria,
dengue) in persons who have had prior enteric
fever
 If bacterial antigen suspensions are not free from
fimbriae
  Persons with inapparent infection or
  Persons with prior immunization (with TAB
vaccine

False-negative: Widal test may occur in:

  Early-stage (1st week of illness)
  Late-stage (after fourth week)
  Patients on antibiotics
  Due to prozone phenomena
OTHER TESTS
 Rapid antibody dection test
 ™ Typhidot test
 ™ IDLTubex test
 ™ IgM dip stick test and ELISA
 ™ Dot blot assay
 Demonstration of Serum Antigens
ELISA
 Molecular Methods
Several polymerase chain reaction (PCR) based methods
(e.g. nested PCR)
 Other Nonspecific Methods

™WBC count: Neutropenia is seen in 15–25% of cases.
Leukocytosis is more common among children, during early phase
 Antimicrobial Susceptibility Testing
It is done by disk diffusion method on Mueller Hinton agar or
MIC based method (e.g. VITEK)
 DETECTION OF CARRIERS

™ Culture: By stool and bile culture (detects fecal
carriers) and urine culture (detects urinary carriers)
 ™ Detection of Vi antibodies: It is done by tube
agglutination test by using S. Typhi suspension
carrying Vi antigen (Bhatnagar strains). Even a titer of
1:10 is also considered as significant.
 ™Isolation of salmonellae from sewage : It is carried
out to trace the carriers in the communities. It can be
done by:
1. Sewer–swab technique: Gauze pads left in sewers
are cultured on highly selective media, such as
Wilson and Blair media
2. Filtration: Sewage can be filtered through millipore
membranes and the membranes are cultured on
highly selective media.