The document discusses typhoid fever and methods for diagnosing the disease. It begins by describing the causative bacteria, Salmonella Typhi, and how it is transmitted. It then discusses several diagnostic tests including the Widal test, which detects antibodies in serum and was the first test developed for typhoid. The document provides details on performing and interpreting the Widal test as well as its limitations. Finally, it summarizes several other diagnostic tests for typhoid fever such as the Tubex test, dipstick assay, and Typhidot assay, which aim to improve on the Widal test by providing faster, easier results.

1. Dr.R.Jayaprada

2.  Typhoid fever, also known as typhoid, is a common worldwide
illness, transmitted by the ingestion of food or water contaminated
with the feces of an infected person, which contain the bacterium
Salmonella enterica enterica serovar Typhi.
 The bacteria then perforate through the intestinal wall and are
phagocytized by macrophages.
 The organism is a Gram negative short bacillus that is motile due
to its peritrichous flagella.
 The term "enteric fever" is a collective term that refers to typhoid
and paratyphoid

3. Bacteriology –Typhoidfever
The Genus Salmonella
belongs to Enterobacteriaceae
-Facultative anaerobe
-Gram negative bacilli
-Distinguished from other bacteria by
Biochemical and antigen structure.

4.  Antigenic structure of Salmonella
H( flagellar ) antigens
O (somatic) antigens
Vi (Virulence) capsular
polysaccharide antigens

5.  LPS in the cell wall;
 Extracted from the cell wall by treatment with
tetrachloroacetic acid
◦ (Boivin : Boivin antigen);
 Less immunogenic than H antigen;
 Agglutination with antisera:
Compact, chalky granular clumps
 Serogrouping of Salmonellae is based on characteristic
O antigen;

6.  Present in flagella;
 Heat labile;
 2 phases : phase 1 and phase 2;
 Strongly immunogenic;
 Induce rapid antibody formation in high titres;
 Agglutination with antisera:
Large, loose, fluffy clumps

7.  Surface polysaccharide expressed on certain serotypes;
Heat labile;
 Interferes with agglutination by O antisera;
 Lost on serial subcultures;
 Poorly immunogenic, BUT antibodies are protective:
◦ Detection of Vi antibody not helpful in diagnosis
but their absence in a case of typhoid  poor
prognosis;
◦ Persistance of Vi antibody : carrier state

8.  Serodiagnosis of Typhoid :
1.Detection of Antibodies in serum---1.Widal test ,2.Typhidot assay
3.Tubex system,4. Dipstick assay.
2. Detection of Antigens in serum.---
a.Tubexsystem
b.CountercurrentImmunoelectrophoresis.
c. Co-agglutination test.
d. ELISA
3. Detection of Antigens in urine: 1.Tubex system 2. Counter
Immuno Electrophoresis, 3. Latex agglutination 4. and Co-
agglu-tination

9. Georges-Fernand-Isidor Widal
Widal & Sicard in 1896
described the Widal reaction.
In 1896 Widal A professor of
pathology and internal
medicine at the University of
Paris (1911–29), he
developed a procedure for
diagnosing typhoid fever
based on the fact that
antibodies in the blood of
an infected individual cause
the bacteria to bind together
into clumps (the Widal
reaction).

10. Widal Test
 Serum agglutinins raise abruptly during the 2ndor 3rd week.
 The Widal test detects antibodies against O and H antigens.
 Two serum specimens obtained at intervals of 7 –10 days to read
the raise of antibodies.
 Serial dilutions on unknown sera are tested against the antigens for
respective Salmonella.
 False positives and False negative limits the utility of the test.
 The interpretative criteria when single serum specimens are tested
vary.
 Cross reactions limits the specificity.

11.  Widal test – A standard tube agglutination test
Test can be performed by the tube dilution technique
In this, a constant amount of the antigen is added to a series of tubes containing
serum dilutions.
After mixing, the tubes are incubated at a temperature of 37 c in a
thermostatic water bath and the highest dilution of serum showing
visible agglutination is determined.

12. Felix tube Dreyer’s tube
Conical bottom
 O Round bottom H H agglutination
O agglutination
Compact Loose
granular Cotton woolly
agglutination clumps

14.  Equal volumes of serial dilutions of serum (1/10 to 1/640)
mixed with H and O antigens in respective tubes;
 Incubated in water bath at 370C overnight;
 Observed for agglutination
◦ H : Loose , cotton woolly clumps;
◦ O : compact granular agglutination;
◦ Supernatant should be clear;

15. 0.9ml of normalsaline
+ 0.1 ml of serum
0.5 ml of S.Typhi O’ antigen control
0.5 ml NS.
0.5ml 0.5ml discarded.
0.5 ml of S.Typhi H’ antigen
0.5ml 0.5mldiscarded.

16.  Result reported as ‘titres’ : Highest dilution where
agglutination is seen;
 Titres will depend on the stage of disease:
◦ Agglutinins will appear by the end of 1st wk;
◦ Rise till 3rd or 4th wk, later decline;
◦ Demonstration in the rise of titre is significant;
 Following Titers of antibodies against the antigens are
significant when single sample is tested.
 Significant titre: O > 1 in 100
H > 1 in 200
 Testing a paired sample for raise of antibodies carries a
greater significance.

17.  The Widal test (Widal’s agglutination reaction) is routinely practiced
for the Serodiagnosis of typhoid fever by most of the laboratories.
 Several workers have expressed doubt regarding the reliability of
the test.
 Several factors have contributed to this uncertainty. These include
1.Poorly standardized antigens,
2.Sharingofantigenicdeterminants with other Salmonellae
3.Effects of immunization with TAB vaccine.
Another major problem relates to
the difficulty of interpreting Widal test results in areas where
Salmonella.Typhi is endemic and where the antibody titres of the
normal population are often not known.

18.  Classically, a four-fold rise of antibody in paired
sera Widal test is considered diagnostic of typhoid
fever.
However, paired sera are often difficult to obtain
and specific chemotherapy has to be instituted on the basis
of a single Widal test.
Furthermore, in areas where fever due to infectious
causes is a common occurrence. So false positive reactions
may occur as a result of non-typhoid

19.  The Widal test is time consuming and most often it is too
late to start an antibiotic regimen when diagnosis is
reached.

20.  Previous immunization with Salmonella antigen.
 Cross-reaction with non- typhoidal Salmonella.
 Variability and poorly standardized commercial antigen
preparation.
 Infection with malaria
 Brucellosis
 other Enterobacteriaceae sharing the same s-LPS .
 dysgammaglobulinaemia of chronic active hepatitis.
 Autoimmune diseases.

21.  The carrier state
 An inadequate inoculum of bacterial antigen in the host to
induce antibody production
 Technical difficulty or errors in the performance of the
test.
 Previous antibiotic treatment
 Variability in the preparation of commercial antigens.
 with "hidden organisms" in bone and joints.

22.  Prozone effect - Occasionally, it is observed that
when the concentration of antibody is high (i.e. lower
dilutions), there is no agglutination and then, as the
sample is diluted, agglutination occurs.

23. Slide Widal test is more popular as it gives rapid results.
Qualitative test: 1 drop of each undiluted patient’s serum sample for
the 2 antigens is placed on the circled card.
1 drop of each of 2 salmonella antigens are added
separately
 rotated gently for 1 min.
Appearance of agglutination gives qualitative results.
(test is repeated is repeated with dilutions of serum)

24.  Quantitative test:
 80µl, 40µl, 20µl, 10µl, 5µl, of patient’s serum each for 2 salmonella
antigens are placed on the circled card.
one drop of specific antigen is added to each series of serum.
Agglutination of each of these is noted.
80µl corresponds to 1 in 20 dilution.
40µl corresponds to 1 in 40 dilution.
20µl corresponds to 1 in 80 dilution.
10 µl corresponds to 1in 160 dilution.
5µl corresponds to 1in 320 dilution.

25.  This test is performed with a loopful growth from nutrient agar
emulsified in 2 drops of 0.85% normal saline.
For salmonella isolated for the first time, the following sera are tested
in order I to VI:
I. Salmonella polyvalent ‘o’ serum(groups A-G,02-13)on one half of
slide.Test suspension alone on other half as a control to exclude
saline auto agglutination.
II.Salmonella polyvalent H’ phases 1&2 serum and polyvalent H
phase 2 are placed on separated halves of the slide.
If polyvalent ‘O’ & “H reactions are negative, NO further slide tests
are done.
If polyvalent O& H reactions are positive for salmonella, further
Slide tests are done.

26. III. Test suspension is checked with individual Salmonella O group
sera 02-013 separately.
IV. If the culture is in phase 1that is if it reacts positively with mixed
phase1 &2 serum, negatively with phase 2 H serum and if it reacts
with any single O’ group serum, then it is to be tested with single
H’ serum( a, b, d, i etc )---------salmonella.Enteritidis
&salmonella.Typhimurium are the common serotypes in many
countries.
If salmonella belongs to O-group B (02) then it is tested with H-i
serum.
If salmonella belongs to O-group D(09) then it is tested with H-d
serum.

27.  V. If the culture is in phase2, it is passed through Craigie’s tube or
Jameson strip containing phase 2 H serum and phase 2 phase 1.
VI. Salmonella. Typhi fails to agglutinate with group D(09) because
the bacilli are coated with vi antigen. If vi antiserum is added ,then it
agglutinates.
If vi positive, saline suspension of salmonella is heated for 30 min,
cooled & retested with O’ antisera.
VII. If the isolate is nontyphiodal salmonella, producing gas from
sugars, it is tested for agglutination with O’& H’ antisera for groups
A,B,C.

29.  TUBEX TF (IDL Biotech):
 It is a 5 minute semi quantitative calorimetric test for Typhoid
fever.
 It detects antibodies to the salmonella enterica Serotype Typhi
lipopolysaccharide (LPS)o9 antigen.
 Tubex system is ideally suited for use in diagnosis of infections as it
allows IgM antibodies to be detected early & rapidly from whole
serum.
The antibodies are detected by their ability to inhibit the interaction
between two types of reagent particles
a)colored indicator latex microspheres sensitized with an anti o9
monoclonal antibody.
b)magnetic microspheres coated with S.Typhi LPS.

30. Following rapid mixing of the serum with these reagents and
sedimentation of the magnetic particles by the magnetic force, the
concentration of the particles left in suspension provides the measure
of inhibition.
Tubex test for antibody detection:
Test serum (25µl) was mixed in a chamber of the reaction container
provided(which contains a set of six identical ‘V’shaped chambers)
with 25µl of Brown reagent (antigen coated magnetic particles) for
2min.
Then 50 µl of blue reagent (Ag coated indicator particles) is added &
mixed for another 2 min.
The reaction mixture was placed on the magnet stand, and the resultant
color was read immediately and scored against the color chart(score 0-10).
Scores of 0-2 considered negative.
Scores of 3-10 considered positive.

32. Tubex test for antigen detection:
 25µl of test serum is mixed with 50µl of blue reagent in the reaction
well for 2 min.
then 25µl of brown reagent is added& mixed for another 2 min.
The results are read after sedimentation of magnetic particles.
In conclusion,
Tubex kit can potentially be used in several ways to diagnose typhoid
fever including:
1)Antibody &Antigen detection in serum.
2)Antigen detection from urine to complement serum detection.
3)Detection or identification of whole organisms from primary culture
plates or from blood culture (stool) broth.

33.  Results of Tubex TF:
 results are scored against a color chart with a scale of 0 to
10.
 Score ‘0’, negative and most red.
 Score ‘10’, most positive and most blue.
 Reasonable sensitivities (75–90%) and
 specificities (70-97%) have been observed.

34. Dipstick assay:
It consists of a strip of nitrocellulose membrane containing a 2mm
wide line of immobilized antigen as a detection band & a separate
line immobilized anti human IgM antibody as reagent control.
It is developed in Netherlands.
It is based on binding of S.Typhi specific IgM antibodies in samples
to S.Typhi LPS antigen and the staining of bound antibodies by an
antihuman IgM antibody conjugated to colloidal dye particles.
Antigen preparation:
culture of recent isolate of S.Typhi culture was grown in a Luria
Bertani broth & antigen was prepared by heating a washed & 30x
concentrated bacterial suspension of a 3 day old well grown culture
for 30min at 95 c.
Cell debris was removed by centrifugation.

35. The supernant containing the antigen was blotted in 2mm wide lanes
on the nitrocellulose membrane by incubation for 2hrs at 40 c.
At the end of incubation, the lanes of blotting apparatus were rinsed
with phosphate buffered saline (PBS) to remove excess antigen.
Blotted strips rinsed with PBS, blocked with 3%skimmed milk,
rinsed & dried.
Detection reagent: It consists of a monoclonal antihuman IgM
antibody conjugated to colloidal suspension of palanyl red.

36. Procedure of Dipstick assay:
It is performed by incubating a wetted dipstick in a mixture of 5µl of
sample & 250µl of detection reagent for 3 hrs at room temperature.
Dipsticks are thoroughly rinsed with water & dried.
Staining intensity of the antigen band was then graded by comparison
with a colored reference strip.
When no staining is observed ----- Test is scored negative.
Weak staining -----------------1+
Moderate staining ---------------- 2+,3+
Strong staining -----------------4+

37.  Dipstick assay is applied to a single serum sample gives quick
result (3hours).
Testing of paired sera could increase the sensitivity of the assay.
It is a simple test & does not require any specific equipment for its
performance as the assay uses stabilized compounds.
Test is sufficiently sensitive(69.8%) and specific.

38.  Typhidot assay :
(MalaysianBiodiagnosticsResearch,Selangor,Malaysia) :
 It is an Enzyme linked Immunosorbent Assay in the dot test format
which detects IgM & IgG antibodies against outer membrane
protein of salmonella.Typhi antigen in human whole blood, serum or
plasma.
 It is a rapid serological procedure for the diagnosis of Typhoid fever.
 The advantages of Typhidot includes:
• early and specific diagnosis of typhoid fever
• fast, simple and reliable
• simple to perform and no additional sample preparation required
• no special equipment is needed
• results are easy to interpret
• minimal sample volume used

39.  Procedure of the test:
 The test is based on the presence of specific IgM & Ig G antibodies
to a specific 50kDa OMP antigen which is impregnated on
nitrocellulose strips.
Reaction tray is divided into 2 columns as G’ and M’.
250 µl of sample diluent was dispensed in each well and 2.5µl of test
/control was added & then incubated for 20min.
Strips were washed with wash buffer thrice.
250µl of antihuman IgG & IgM were dispensed in each well &
incubated for15 min.
wash& 250µl of substrate is added for color development
,incubated for 15 min. Results were interpreted.

40. Diagrammatic Representation of Assay Procedure
Serum/ Plasma sample
Add 30μl serum to the sample well. Make
sure that there are no air bubbles. Add 1
drop of buffer after 15 seconds. Sample will
start wicking up. Read result within 15
minutes.
Whole Blood sample
Add 40μl of whole blood into the sample
well. Make sure that there are no air
bubbles. Add 1 drop of buffer after 15
seconds. Sample will start wicking up.
Read result within 15 minutes.
[Note: If sample front stops wicking up after
a while, add an additional drop of buffer]

41.  Typhidot has a sensitivity of 92.3%,specificity of 98.8%,PPV-85.7%&
NPV- 99.4%.The test becomes positive within 2-3 days of infection.
 LIMITATION OF THE TEST
 1. This product is designed for use with human whole blood, serum
and plasma only.
 2. The test is a qualitative assay & is not for quantitative
determination of antibodies concentration levels. The intensity of
the band does not have linear correlation with the antibody titer of
the specimen.
 3. The results obtained should only be interpreted in conjunction
with other diagnostic results and clinical information.
 4. Due to the limitations of the test, for cases where interpretation
of result seems difficult, repeating the test
 TYPHIDOT 1 hour or TYPHIDOT 3 hours is strongly
recommended.

42.  This test, derived from Salmonella Typhi O and H antigens, was performed
in two ways:
(i) as a semi quantitative slide agglutination test with visual
examination as per the package insert;
(ii) as a Widal test performed with a single tube, as described by Parry
et al.
The presence or absence of visible agglutination indicates the
presence or absence of the corresponding antibody to the O and H
antigens of Salmonella Typhi.
They defined the positivity cut-off point for the slide and tube
agglutination reactions for both O and H antigens as antibody titres
1:80.
 Publication: Bulletin of the World Health Organization; June 2011,
Type: Research Article ID: BLT.11.087627 .

43.  Electro chemical Immunoassay:
 Recently, copper, silver, and gold-enhanced colloidal gold have
been reported for immunoglobin G (IgG)determination, which
is the model of electrochemical immunoassay with low detection
limits ranged from 1.0ng/ml to 0.25 pg/ml .

44.  Co agglutination is similar to the latex agglutination technique for
detecting antigen .
 Protein A, a uniformly distributed cell wall component of
Staphylococcus aureus, is able to bind to the Fc region of most IgG
isotype antibodies leaving the Fab region free to interact with
antigens present in the applied specimens.
 The visible agglutination of the S. Aureus particles indicates the
antigen-antibody reactions

45. Agglutination test in which inert particles
(latex beads or heat-killed
S aureus Cowan 1 strain with protein A)
are coated with antibody to any of a
variety of antigens
and then used to detect the antigen
in specimens or in isolated bacteria.

46.  Preparation of staphylococci: Staphylococcus aureus Cowan 1
was grown on tryptic soy agar at 37°C for about 18h.
 The cells were harvested, washed in phosphate buffer, exposed to
0.5% formaldehyde, and heated to 80°C and held for 5 min by the
procedure of Edwards and Hildebrand.
 Attaching o antisera to staphylococci: One milliliter of the 10%
suspension of stabilized, killed staphylococci was added to 0.2 ml of
non- glycerolated, specific antiserum or to 0.4 ml of glycerolated
antiserum, mixed thoroughly, and allowed to react at room
temperature for 3 h, with occasional gentle shaking .
 The suspension was centrifuged at 800 x g for 30 min and washed
twice with 0.02 M phosphate (pH 7.3)-buffered 0.85% saline (PBS).
The cells were then suspended in PBS to a final volume of 5 ml, and
1 drop of 5% sodium azide was added . The antibody-conjugated
staphylococci made up a COAG. The COAGs were stored at 4°C.

47.  Testing Salmonella strains by co agglutination: Salmonella strains
were grown overnight on blood agar base (BBL Microbiology
Systems).
 To test for agglutination,
 1 drop of COAG is placed on a slide. Growth was taken from a slant
with a bacteriological loop and was mixed into the COAG drop.
Strong homologous reactions usually occurr immediately. If there was
no reaction, the slide was rocked gently for 1 min and then read
under fluorescent light against a black background.
The amount of agglutination was read as 4+, 3+, 2+, or 1+. A trace
was recorded as +/-.

48.  Many known carriers of typhoid bacilli possess antibody
against the Vi (virulence) antigen of S. typhi.
 This is a surface antigen easily lost during cultivation.
 Vi tires seem to correlate better with the carrier state than do O
or H titres. For this reason, Felix et al. suggested the use of Vi
agglutination for detection of carriers.