Bacteriology- Lab diagnosis- Salmonella

1. SERODIAGNOSIS OF THE
SALMONELLA INFECTIONS
MANISHA SHRESTHA
SADIKSHYA KHAREL
RANJANA POUDEL
YOJANA POKHREL
CENTRAL DEPARTMENT OF MICROBIOLOGY, TU.
(MEDICAL MICROBIOLOGY)

2. Several serologic tests have been developed for point-of-
care diagnosis of enteric fever. Serological tests currently
in use to assist in the diagnosis of enteric fever include :
A. Widal Test
B. IgM Antibody Immunoassays
• The pathogen can be recovered from the blood culture
during the first week of illness but percentage falls
gradually after the first week of disease.
• Specific antibodies usually detectable in the patient
blood after 7-10 days if enteric fever, rises sharply
during the third week of disease.

3. WIDAL TEST
• Widal test is an agglutination method.
• Performed in suspected cases of enteric fever.
• Developed by Greembaum and Widal in 1896 for the
diagnosis of typhoid fever caused by Salmonella Typhi or
Salmonella Paratyphi.
• For the diagnosis of enteric fever, two specimens of
serum at an interval of 7-10 days is tested to
demonstrate a four fold rise in antibody titre.
• Typhoid fever antibodies are detected by mixing the
patient’s serum with dead Salmonella cells and
observed for clumping.

4. Principle : The main principle of widal test is that if
homologous antibody is present in patients serum, it will
react with respective antigen in the reagent and gives
visible clumping on the test card and agglutination in the
tube.
• The patient’s serum is tested for O and H antibodies
(agglutinins) against the following antigen
suspensions (usually stained suspensions):
a) Salmonella Typhi somatic antigen O
b) Salmonella Typhi flagellar antigen H
c) Salmonella Paratyphi A flagellar antigen AH
d) Salmonella Paratyphi B flagellar antigen BH

5. Preparation of Widal Antigens :
• H suspension of bacteria is prepared by adding 0.1%
formalin to a 24 hours broth culture or saline suspension
of an agar culture.
• For preparation of O suspensions of bacteria, the
organisms is cultured on phenol agar (1:800) to inhibit
flagella.
• Standard smooth strains of the organism are used; S Typhi
901, O and H strains are employed for this purpose.
• The growth is then emulsified in small volume of saline,
mixed with 20 times its volume of alcohol, heated at 40° C
to 50° C for 30 minutes and centrifuged.

6. • The antigens are treated with chloroform and
appropriate dyes are added for easy identification of
antigens.
Different techniques can be used for Widal test:
A. Qualitative slide agglutination
B. Semiquantitative slide agglutination
C. Quantitative tube agglutitation

7. A. QUALITATIVE SLIDE TEST
• 1 drop of positive control is placed on one reaction
circles of the slide, and 1 drop of saline on the next
reaction circle i.e. negative control.
• 1 drop of the patient serum to be tested is placed onto
the remaining four reaction circles.
• 1 drop of Widal test antigen suspension ‘H’ is added to
the first two reaction circles (PC & NC).
• 1 drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the
remaining four reaction circles.
• Contents of each circles are mixed uniformly over the
entire circle with separate mixing sticks.
• The slide is gently rocked back and forth for one minute
and observe for agglutination macroscopically.

8.  Result Interpretation :
Negative Test: - No agglutination upto 1 minute.
Positive Test : -Agglutination with in 1 minute.
-Idicates the presence of corresponding abs.
Agglutination with positive control and no agglutination
with negative control validate the test result.

9. B. SEMI – QUANTITATIVE SLIDE TEST
• 1 drop of normal saline is placed on first circle and in the
remaining five circles 0.005 ml, 0.01 ml, 0.02 ml, 0.04
ml, and 0.08 ml of test serum is taken.
• A drop of the antigen is added to each of the circlea
which showed agglutination with the test sample in the
screening slide test.
• Using separate mixing sticks, the contents of each circle
are mixed uniformly over the reaction circles.
• Slide is rocked gently back and forth and observe for
agglutination macroscopically within one minute.

10.  Result Interpretation :
The lowest volume of serum which shows clear
agglutination indicates the cut off level of the positive
test and the corresponding antibody titre.
Serum
Volume
0.08 ml 0.04 ml 0.02 ml 0.01 ml 0.005 ml
Antibody
Titre
1:20 1:40 1:80 1:160 1:320

11. C. QUANTITATIVE TUBE AGGLUTINATION TEST
• The patient’s serum to be tested is serially diluted in
normal saline as 1:20, 1:40, 1:80, and so on upto 1280
or more in Kahn tubes, in numbers corresponding to the
number of the bacterial suspension used. For example,
4 sets of 8 Kahn test tubes for O, H, AH and BH antibody
detection. Tube No. 8 in all the sets, serves as a saline
control.
• To all the tubes (1 to 8) of each sets 1 drop of the
respective Widal test antigen suspension (O, H, AH and
BH) are added and mix well.
• Tubes are covered and incubate at 37° C overnight
(approximately 18 hours).
• Dislodge the sedimented button gently and observe for
agglutination.

12. Fig., Serial dilution of serum sample .

13.  Result Interpretation :
Loose and cotton wooly clumps are formed in H
agglutination .
A disc like granular deposits are formed in O
agglutination.
Control tube shows a compact deposit.

14. • Sera of patients with typhoid syndromes generally
exhibit agglutinins at about day 10. 0-agglutinin titres
increase to 1:40 and disappear with an interval of one to
three months after recovery
• H-agglutinin titres rise to 1:800 or 1:160 and remain
increased after the recovery (low titres of e.g. 1:20 can
remain for years).
• Cross-reactions often observed between the 0-antigens
of the Group B and the 0-antigens of the Group D.
• Cross-reactions may be observed also with bacteria
belonging to a genus other than Salmonella, eg., Y.
pseudotuberculosis type 1 cross-reacts with Salmonella
of Group D and type IV with Group B.

15. Due to subclinical infection of salmonellosis in endemic
areas, low titre of agglutinnins is present in the serum of
normal individuals, which may cause positive reactions.
This is known as locla titre.
 According to the study conducted on Baseline Widal
Agglutination on Healthy Nepalese Blood Donors: the
baseline titre for O and H- agglutinin was noted to be
1:40 and that for AH and BH- agglutinin was noted to be
1:80.
Therefore, the cutoff level for O and H was ≥ 1:80 and
cut off level for AH and BH was ≥ 1:160.

16. IgM ANTIBODY IMMUNOASSAYS
• Detects IgM antibodies to S. Typhi which occur early in
the acute typhoid.
• More sensitive and specific than the Widal test
• In the absence of culture facilities, IgM antibody tests
are are more useful to help diagnose typhoid in endemic
areasparticularly after 7-days following the onset of
fever .
• Commercially available IgM antibody tests that have
been most widely studied are:
– TUBEX TF
– Typhidot

17. TUBEX TF
• Assays for antibodies to S. Typhi LPS (O9) by quantifying
inhibition of binding between O9 monoclonal antibodies
and LPS-coupled magnetic particles.
• Sensitivity of 56%–95%, specificity of 72%–95%
TYPHIDOT
• Miniaturized dot-blot ELISA that detects IgM and IgG
antibodies to a 50 kD S. Typhi outer membrane protein
(OMP).
• Typhidot-M uses the same approach to detect IgM to
OMP after removal of total serum IgG, to improve
specificity for recent infection.
• Sensitivity of 56%–84%, specificity of 31–97%

18. REFERENCES
Jason R. Andrews, and Edward T. Ryan. Diagnostics for
invasive Salmonella infections: current challenges and future
directions. PMC US National Library of Medicine National
Institutes of Health. Vaccine. 2015 Jun 19; 33(0 3): C8–C15.
Pokhrel, BM, Karmacharya, R, Mishra SK, and Koirala J.
Distribution of antibody titer against Salmonella enterica
among healthy individuals in Nepal. Ann Clin Microbiol
Antimicrob. 2009; 8: 1.
Cheeesebrough, M. (2007). District laboratory practices in
topical countries, Part-2. Second edition. Cambridge University
Press, new York.
https://microbiologyinfo.com/widal-test-introduction-
principle-procedure-interpretation-and-limitation/