CHAPTER 4
SEROLOGICAL
DIAGNOSIS OF ENTERIC
FEVER
By AMANUEL M.(BSc, MSc Clinical chemistry)
Learning objective
2
At the end of this chapter the students should be able to:
Describe the etiologic agent of enteric fever
Describe serological test of enteric fever
Discuss factors that causes false positive and false negative
Widal test?
Explain the source of error for Widal test.
Discuss the advantage and disadvantage of culture and Widal
test 27/06/2024
Introduction
3
Salmonella are often pathogenic for humans or animals
Transmitted from animal and animal product to humans
Three main diseases are
Enterocolitis (enteritis)
Septicemia (systemic infection)
Enteric fever (typhoid fever)
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Introduction
4
Morphology
Microscopic
• Vary in length
• Gram –Ve
• Rod shape bacilli
• Most isolates are motile
• has peritrichous flagella
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Introduction
5
Classification
More than 2500 serotypes
Four serotypes can cause enteric fever
S. paratyphi A (Serogroup A)
S. paratyphi B (serogroup B)
S. choleraesusis (serogroup C1)
S. typhi (serogroup D)
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Antigenic variation
6
Salmonella has
O-antigen ……… somatic
H-antigen……… flagellar antigen
Vi- antigen …… capsular antigen
Organism may lose H antigens and become non-motile
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Enteric fever
7
The enteric fever (typhoid and Paratyphoid)
Caused by S. typhi and S. paratyphi A, B, C
The ingested salmonella reach the small intestine
From which they enter the lymphatics Then go to
blood stream From this to many organs including
intestine The organism multiply in intestinal lymphoid
tissue And excreta stool
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Enteric fever
8
Incubation period is 10-14 days after that it causes
Fever
Malaise
Headache
Constipation
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Enteric fever
9
Fever raises to high and the spleen and liver become
enlarged
Rose spots-usually on skin of the abdomen or chest
WBC is normal or low
Some time intestinal hemorrhage and perforation at
pre-antibiotic era
Mortality rate is 10-15% 27/06/2024
Laboratory Diagnosis
10
Bacteriologic method
Blood for Culture must be taken repeatedly
Stool specimens also must be taken repeatedly
Bone marrow cultures may be useful
Urine cultures may be positive after the 2nd week
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Laboratory Diagnosis
11
1. Differential medium cultures
EMB
MacConkey agar
Deoxycholate medium
Bismuth sulfite medium
2. Selective media cultures
SS agar media
Hektoen enteric agar
XLD
Deoxycholate citrate agar
3. Enrichment cultures
Selenite F or
Tetrathionate broth
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Serological Diagnosis
12
Serological method -----Widal Test
New serological methods
Typhidot (better test)
Dipstick test
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Serological Diagnosis
13
Widal test is a serological test widely used for
diagnosis of enteric fever.
It is suspension of killed S. typhi as Ag. to detect ant-S-
typhi antibody
it has many limitations as a diagnostic tool. However, it
used to carry out sero-surveys in a community to know the
endemicity of any infection
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Serological Diagnosis
14
Widal test measures titres of serum agglutinins
against somatic (O) and flagellar (H) antigens
which usually begin to appear during the 2nd week.
In the absence of recent immunization, a high titer
of antibody to O antigen > 1:640 is suggestive but
not specific.
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Serological Diagnosis
15
While using Widal test for the diagnosis of enteric
fever, several factors need to be considered for
interpretation. For example:
Endemicity of enteric fever in an area of investigation
Administration of antibiotics
Immunization with any typhoid vaccine or a previous
infection or exposure
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Serological Diagnosis
16
The stage of the disease at the time of collection of
sample.
Early in the disease low antibody titers are found. The
antibodies start rising after 1st of illness and do so until
¾ week.
Infection with any other gram-negative bacteria may
give a false positive reaction.
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Serological Diagnosis
17
To make any logical conclusion in the diagnosis of
enteric fever on the basis of widal test, one must
submit a paired serum sample.
The 1st sample taken early in the disease
The 2nd sample at least 2 weeks later.
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Serological Diagnosis
18
Significance of Widal test
In enteric fever endemic areas Widal test are very
important to diagnose S. typhi.
When facilities for culturing are not available, the
Widal test if performed and interpreted with care can
be of value in the diagnosis of typhoid fever.
Widal test for typhoid and paratyphoid fever is an
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agglutination test.
Serological Diagnosis
19
The typhoid bacillus causes two types of
agglutinins to be produced. The agglutinins are
called:
Flagella (H) agglutinins
Somatic (O) agglutinins.
The patient serum is tested for those O and H
antibodies against the antigen suspensions.
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Serological Diagnosis
20
Salmonella antigen suspensions are commercially
available from different manufacturer.
The antigens are stable for rapid slide (screen) testing
and tube testing.
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Serological Diagnosis
21
Methods of widal tests
a. Slide method
b. Tube method
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Serological Diagnosis
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a. Slide method
Procedure
• Modification of tube test method by Welch and Mickle
at 1936
Specimen: serum/plasma
Take clean slide
Add a drop of serum, which is obtained, from non-
hemolyzed blood.
Add a drop of antigen suspension, which is non-
expired,
Mix well antigen suspension and serum.
Look for agglutination. 27/06/2024
23
Positive Result
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24
Negative result
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Serological Diagnosis
25
b. Tube method
Used to confirm slide test method
Sample: serum/plasma
Procedure
1. For each antigen arrange 10 small test tubes in a rack.
2. Place 0.9ml of saline in the 1 st tube and 0.5ml in the
remaining 9 tubes.
3. Add 0.1ml of fresh cell-free serum to the 1 st tube.
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Serological Diagnosis
26
4. Mix and transfer 0.5ml to tube 2,3,4,5,6,7,8,and
tube 9. From tube 9 discard 0.5ml.
Tube 10 will contain only saline and will serve as a
negative control (antigen control)
5. Mix antigens well and add 0.5ml to each tube. Mix
by gently shaking the tubes.
6. The final dilutions are 1:20, 1:40, 1:80, 1:160, etc.
7. Incubate the tubes at 37oC for 2-4 hrs
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Serological Diagnosis
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8. Read the negative control at the end of the
incubation period.
It should have no agglutination.
9. Read the test one row of antigen at a time. For
reading a white light shining vertically above the
tube is best and using a black background.
10. Shake the tubes gently.
The H type of agglutination is easily broken up and
may be missed. The agglutination is more granular
and not so fragile.
11. Report the highest dilution with definite clumps.
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Serological Diagnosis
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Tube dilution
0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5
0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
initial
Tube 1 2 3 4 5 6 7 8 9 10
Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120
Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120
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Serological Diagnosis
29
Reporting of widal reaction
The Widal test is reported by giving the titer for both O and
H antibodies.
The antibody titer is taken as the highest dilution of serum
in which agglutination occurs.
The type of agglutination seen with O reactions is granular
while that seen with H reactions. Both slide and tube tests
are more easily read against a dark background.
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Serological Diagnosis
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If no agglutination occurs the test should be reported as:
S. typhi O titer less than in 20 (O 1:20)
S. typhi H titer less than in 20 (H 1:20)
Interpretation of the Widal Reaction
A Negative test does not necessarily mean the patient
is not infected. Reaction occurs in infected patients
about
50% during the 1st week
80% in the 2nd week,
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90-95% in the 3rd or 4th week
Serological Diagnosis
31
Positive reactions with O antigen occur earlier in the
disease than the reaction with the H antigen. H antigen
reactions may remain sometimes for years.
A positive reaction occurs after typhoid vaccination
and lasts for 1-5 years.
In endemic regions, natural agglutinins may be
present in the serum. 27/06/2024
Serological Diagnosis
32
Some persons will not produce antibodies because
of other diseases.
E.g.
agammaglobinemia (absence of
globulin in the serum),
leukemia, and
carcinoma (malignant tumor)
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Serological Diagnosis
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Taking antibiotics may cause a decrease in the titer.
No one titer can be considered diagnostic for typhoid
fever. Arise in titer over a period of time is
significant and of diagnostic value.
Causes of false positive widal result
Causes of raised 0 and H agglutinins other than
typhoid fever include 27/06/2024
Serological Diagnosis
34
Previous Salmonella infections,
Chronic salmonellosis associated with schistosomal infection
Vaccination with TAB or typhoid vaccine.
Acute falciparum malaria (particularly in children),
Chronic liver disease associated with raised globulin levels
Disorders such as rheumatoid arthritis, myelomatosis and
nephrotic syndrome.
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Serological Diagnosis
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False negative Widal tests may be
Due to antibody responses being blocked by early
antimicrobial treatment or following a typhoid
relapse.
Severe hypoproteinaemia may also prevent a rise in
0 and H antibody titres.
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Serological Diagnosis
36
Source of error
Hemolyzed blood
Expired antigen suspension
Dirty slides or tubes
Failure in performing technique
Failure in reading of technique or interpretation
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Summary
37
Salmonella has over 2000 serovars which can cause
disease in humans.
Enteirc fever caused by S. typhi and S. paratyphi A, B, C
Widal test is a most commonly used serological diagnosis
for enteric fever.
The Widal test measures agglutinating antibody levels
against 0 (somatic) and H (flagellar) antibodies. In acute
typhoid fever, 0 agglutinins can usually be detected 6–8
days after the onset of fever and H agglutinins after 10–12
days. 27/06/2024
38
Serological test for Rickettsia
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39
Learning objective
At the end of this chapter the students should be able
to:
Discuss the etiologic agent, sign and symptom of
typhus fever
Discuss serological diagnosis of typhus fever
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Introduction
40
The genus rickettsia contains several species and sub
species.
Although classified as bacteria, rickettsia resemble
viruses in that they are mostly obligate parasites and are
unable to survive as free living organisms.
They are about the size of the largest viruses and can just
be seen with the light microscope.
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Introduction
41
Rickettsia resembles bacteria also by virtue of their
morphology and microscopic visibility.
Unlike viruses, however, rickettsia contains both RNA
and DNA, multiply by binary fission, have cell wells
that contain muramic acid, possess enzymes, and show
sensitivity to antiseptic and antibodies.
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Introduction
42
Species in the genus rickettsia have been sub
divided into three groups of antigenically related
microorganisms:
typhus group
scrub group
spotted group
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Organism Disease Host Method of transmission
R. prowazeki Louse born typhus. Man Through the feaces of louse
43
(Endemic typhus)
R. typhi Mourine typhus (Ende Rats Rates---Fleas/lice ----rat
mic typhus) --- Fleas/lice----human
Rats, field mice, vo
R. tsutsugamushi Scrubing typhus (mite Bite of infected larvae mite
les & quail, mite
typhus)
domestic & wild
R. rickettsia Spotted typhus Bite of infected tick
dogs wild rabbits,
rodents.
R. conori Fever boutonneus Dog Bite of infected tick
R. siberica Asian tick typhus Dog Bite of infected tick
R. conori pjiperi African tick typhus Dog Bite of infected tick
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Serologic diagnosis
44
The most reliable and useful serological technique
for diagnosing rickettsial infections is the indirect
fluorescent antibody (IFA) test. It is of value not
only in diagnosing acute infections but also in
serological epidemiological studies.
Another important test is CFT test, which is not
sensitive as IFA test. 27/06/2024
Serologic diagnosis
45
Weil-Felix reaction test
Weil-Felix is an agglutination test for various rickettsial
infections (as typhus and tsutsugamushi disease) using
particular strains of bacteria of the genus Proteus that
have antigens in common with the rickettsiae to be
identified Weil, Edmund (1880-1922), and Felix,
Arthur (1887-1956), Austrian bacteriologists.
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Serologic diagnosis
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During World War I Felix served as a bacteriologist
charged with the diagnosing of typhus in the
Austrian army.
As a result of his work he and Weil developed an
agglutination test for typhus in 1916.
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Serologic diagnosis
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The Weil-Felix reaction test, developed by Weil and Felix
(1916), is based on the fact that certain strains of Proteus
vulgaris and P. mirabilis share antigens with several of the
rickettsia species that produce febrile disease, such as
typhus.
Three strains of Proteus species have been found to be
useful in diagnosing rickettsial diseases; these have been
labeled OX-2, OX-19 and OX-K.
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Serologic diagnosis
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A test for diagnosis of typhus and certain other rickettsial
diseases.
The blood serum of a patient with suspected rickettsial disease
is tested against certain strains of (OX-2, OX-19, OX-K).
The agglutination reactions, based on antigens common to
both organisms, determine the presence and type of rickettsial
infection.
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Serologic diagnosis
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In 1915, Weil and Felix showed that serum of patients
infected with any member of the typhus group of
diseases contains agglutinins for one or more strains of
OX Proteus.
In cases of typhus fever the reaction usually appears
before the sixth day and reaches its height in the
second week.
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Serologic diagnosis
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Weil-Felix reaction is an agglutination test based on
the cross-reactions, which occur between
antibodies, produced in acute rickettsial infections
and the OX-19 and X-2 strains of Proteus vulgaris
and the OX-K strains of Proteus mirabilis.
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Serologic diagnosis
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In a Weil-Felix reaction a strong agglutination test
with strain OX-19 may indicate epidemic or endemic
typhus, and a weak agglutination may indicate Rocky
Mountain spotted fever, Mediterranean fever and
South African tick fever, while agglutination with
Proteus strain OX-K indicates scrub typhus.
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Organism Disease Proteus group used and
degree of reaction
52
OX-19 OX-2 OX-K
Typhus group
Endemic typhus,Brill’s disease ++++ +/- 0
R. prowazeki
Murine typhus ++++ +/- 0
R. typhi (R. mooseri)
Scrub typhus group
0 0 ++++/-
R. tsutsugamushi Scrub typhus
(R.orientalis )
Spotted fever group:
---- +/++++ 0
R. conori +/++++
R. conoripeiperi +/++++ +/++++ 0
----
R. siberica
Asian tick fever +/++++ +/++++ 0
R. rickettsia 27/06/2024
Rocky mountain spotted fever +/++++ +/++++ 0
Serologic diagnosis
53
The Weil-Felix reactions are non- specific and cannot be
fully relied on to diagnose acute rickettsial infections. False
negative Weil-Felix reactions are common especially with R.
tsutsugamushi infections.
False positive Weil-Felix reactions are may also occurs in
Proteus infections, relapsing fever, brucellosis, rat bite fever,
infectious, mononucleosis, and other acute febrile illness.
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Summary
54
Most rickettsial diseases are diagnosed serologically by
testing paired sera (acute and convalescent samples).
A range of antibody tests to investigate rickettsial
infections is available. This includes indirect
fluorescence antibody tests (IFAT) and enzyme linked
immunosorbent assays (ELISA).
Weil-Felix reaction is non-specific test based on cross-
reactions which occur between antibodies produced in
acute rickettsial infections and the OX 19, OX 2, and
OXK strains of Proteus species.
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