Brucella

And other
organisms
cause febrile
reaction

Febrile Tests
Brucella
Undulati
ng fever
Culture &
serology

Rickettsi
a
Typhus
fever
OX-19
test

Salmonella
Typhoid &
paratyphoid
fever
Widal test

Brucella
Gram negative small coccobacilli
Non-motile
Non-capsulated
Fastidious ; need special media with co and
anaerobic condition called
Castaeda medium.

Cause brucellosis disease (undulating fever).

Species of Brucella :
Brucella melitensis
Brucella abortus
Brucella suis
Brucella canis
Mode of transmission :
1. Direct contact with the body fluids of infected
animals
2. Consumption of unpasteurized milk products
(dairy products) of infected animals
3. Inhalation of aerosols from infected animals.

Diagnosis
1. Serological test
. The objective of this test is to look for antibodies against
Brucella, usually IgG is tested as IgM appear & disappear
quickly.
.
The serum agglutination test is the simplest and most
widely used testing method.
. CDC utilizes atestcalled
theBrucellamicroagglutinationtest(BMAT), a modified
version of the serum (tube) agglutinationtest(SAT), that
can detect antibodies toBrucellaspecies. This test done
after 2 weeks (10 days) of fever.

 Brucellamicroagglutination test (BMAT), a modified

version of the serum (tube) agglutination test (SAT),
that can detect antibodies toBrucellaspecies
-abortus,melitensisorsuis. There is no serological
test available to detect antibodies toB. canis.
For a diagnosis to be made using serology, two serum
samples are required. The first serum sample should be
taken when a person is acutely ill (7 days after
symptom onset); the second serum sample should be
drawn 2-4 weeks later to check for a rise in antibodies
(a fourfold or greater rise in antibodies would bean an
individual is positive for brucellosis).
Ifsubmissionof paired sera is not possible, a probable
diagnosis can be made with a single serum sample.

False Positives and Other Concerns About Reliability

There are a few reasons why diagnosing an
activeBrucellainfection can be challenging.
1. Some other types of bacteria can cause a false positive, which
means testing positive for the presence ofBrucellawhen its not
present.
2. Some immunizations can cause a test to be positive when theres
no infection.
3. A positive test doesnt always mean you have a current
infection. It could mean you were exposed toBrucellaat some
point in the past. It might also mean you have an immunity
against this type of bacteria.
4. If you were recently exposed to theBrucellaantigen, there may
be too few antibodies to be detected by the test.
5. More tests or follow-up testing may be needed to confirm or rule
out brucellosis.

The Rose Bengal test (RBT)

The Rose Bengal test (RBT) is a simple, rapid
slide-type agglutination assay performed with a
stainedB. abortussuspension at pH 3.63.7 and
plain serum.
It is often used as a screening test in human
brucellosis and would be optimal for small
laboratories with limited means.
False-negative reactions occur especially in the
early stages of acute infection.

Procedure of Rose Bengal Plate Test

Test Serum (0.03 ml) is mixed with an equal volume of
antigen on a white tile or enamel plate to produce a
zone approximately 2 cm in diameter.
The mixture is agitated gently for 4 minutes at ambient
temperature, and then observed for agglutination.
Any visible reaction is considered to be positive.
The test is very sensitive, especially in vaccinated
animals, and positive samples should be retested by a
confirmatory test such as the CF test or ELISA .
False-negative reactions may occur and can be
detected by retesting animals at intervals over a period
of at least 3months.

2.

Blood Culture :
the confirmatory test
Done in the first week of infection.
Brucella is isolated from a blood
culture on Castaeda medium.
Prolonged incubation (up to 6 week)
may be required as they are slowgrowing, but by modern automated
machines, the cultures often show +ve
results within seven days.
On gram stain they appear as dense clumps
as Gram-negative
coccobacilli and are rather difficult to see.

3. Animal inoculation test.

4- More tests or follow-up testing may be needed to

confirm or rule out brucellosis, as the following :
1. X-rays.X-rays can reveal changes in your bones and
joints.
2. Computerized tomography (CT) scan or magnetic
resonance imaging (MRI).These imaging tests help
identify inflammation or abscesses in the brain or other
tissues.
3. Cerebrospinal fluid culture.This checks a small
sample of the fluid that surrounds your brain and spinal
cord for infections such as meningitis and encephalitis.
4. Echocardiography.This test uses sound waves to
create images of your heart to check for signs of
infection or damage to your heart.

Test & Results

An agglutination test is done by mixing 50ul of sample with
1 drop of reagent.
A normal (negative) result shows no antibodies to Brucella.
Titer about 1:80 or more indicate Brucellosis (serial
dilutions).
However, during the first few days to weeks of exposure to
antigen, they may be very little antibody production & as
brucellosis progresses, more antibodies will be present.
If you suspects brucellosis, you may need to repeat the test
every 10 days or 2 weeks after the first test to notify this
rise.

Brucella antibody IgG have long

lifespan (1-2) years.
Remember

Prozone phenomena is the presence of high

antibody titer that lead to Ag block and hence,
false negative results are obtained. Dilution will
resolve this problem.
This phenomena appears obviously in Brucella
serology
test.

Weil-Felix test
OX-19 Test

Rickettsiae
Are diverse collection of obligatory intracellular
organism.
Gram negative bacteria
Found in ticks, Lice, fleas, mites, chiggers & mammals.
They include the genera Rickettsiae, Ehrlichia, Orientia
and Coxiella.
Cause zoonotic diseases that may disseminate in the
blood to many organs.
Rickettsial infection (Typhus Fever) generate
heterophilic antibodies can agglutinate some strains of
Proteus.

Laboratory diagnosis
Rickettsiosis are difficult to diagnose
both clinically and in the laboratory.
1. Culture & isolation.
Which require viable eukaryotic host
cells, such as antibiotic-free cell
cultures, embryonated eggs and
susceptible animals as guinea pigs or
.mice
2. Serologic test.
.OX-19 test ( weil-felix test )
3. Immunofluorescent antibody
technique.

Complication of Typhus infection

Lymphocytosis
Leukopenia
Thrombocytopenia
Anemia
In some cases hemoglobin may increase as a
result of hemoconcentration
Increase in kidney function tests.
In urine analysis; high RBCs cast and high protein
may present

Weil Felix test

Also named : Proteus OX-19 test, Typhus test
Is a heterophile agglutination test used to
diagnose typhus and certain other rickettsial
diseases.
Weil-Felix test is based on cross-reactions which
occur between antibodies produced in acute
rickettsial infections with antigens of OX (OX 19,
OX 2, and OXK) Strains of non motile Proteus sp.

 The basis of the test is the sharing of an Alkali

stable carbohydrate antigen of rickettsia with
certain strains of Proteus.
Serum from endemic typhus usually agglutinate
OX-19 & OX-2.
Test is negative in rickettsialpox disease.
The test is done as tube agglutination test along
with slide agglutination

Procedure
1st : Slide method

On a solid surface (glass slide, tile, card), a small

amount (50100 L) of the patients serum is
placed.
A single drop of the desired antigen is added, and
the resulting suspension is mixed and then
rotated for one minute.
Visible agglutination is indicative of a positive
result, and corresponds roughly to a titre of 1:20.
Positive results can be further titrated using the
tube method.

 2nd : Tube agglutination method

Three tubes containing two-fold dilutions of patient
serum are made .
A drop of antigen suspension (OX-19, OX-2, OX K) is
added to each tube.
The mixture is incubated at 5055C for 46 hours.
A positive tube would show visible flocculation or
granulation, which is accentuated when the tube is
gently agitated.
1:20

Start
with
dillutio

1:40

1:80

1:160

1:320

The titer corresponds to the most dilute tube in

the series that still shows positivity.
Generally, a titre of 1:80 is considered diagnostic.
A positive test may be due to Rickettsia or Proteus

Rickettsia

Proteus

Negative

positive

Deceased
(Leukopenia)

Increased
(Leukocytosis)

Lymphocytosis
Due to virus-like
behavior

Neutrophilia

Differenc
e
Culture from ear
disharge and
urine
Total WBCs
WBCs
differentiation

 The WeilFelix test suffers from poorsensitivity

and specificity.
As a result, it has largely been supplanted by
other methods of serology, including
indirectimmunofluorescenceantibody (IFA)
testing, which is the gold standard.

Widal test

Salmonella
Gram Negative bacilli
The main cause of Typhoid fever
Also implicated in food infection and food
poisoning.
There are two species of Salmonella :
1. Salmonella typhi
2. Salmonella paratyphi
S. paratyphi A
S. paratyphi B
S. paratyphi C

Antigenic structure of Salmonella

1. H( flagel) antigens
2. O (somatic) antigens
3. Vi (Virulence) capsular polysaccharide antigens

Both typhi and paratyphi have two types of antigen:

Somatic ( O ) antigen, and that is thermostable.
Flagellar( H ) antigen, and that is thermolabile.
Salmonella can be isolated from GIT, urine, blood, bile,
bone marrow, sputum, food products, and milk.
The confirmatory test for Salmonella is stool culture at the
first week of infection.

Widal test
Widal test defined as a test involving agglutination of
typhoid bacilli when they are mixed with serum
containing typhoid antibodies from a person having
typhoid fever; used to detect the presence of
Salmonella typhi and S. paratyphi .
Use to diagnose the typhoid fever that caused by
Salmonella.
In diagnosis of typhoid fever, patient serum is tested
for salmonella O and H antibodies against Ag
suspension.
The test is done after 2-3 weeks of infection or after 10
days of fever.

Principle of test :
Patients suffering from enteric fever would
possess antibodies in their sera which can react
and agglutinate serial doubling dilutions of killed
colored Salmonella antigens in a tube
agglutination test.

Widal test
Agglutinati
on

Slide
method

Qualitativ
e

Precipitati
on

Semiquantitativ
e

Tube
method

Agglutination procedures
Slide method

1st : Qualitative test :

One drop each of patients serum samples for the six

antigens are placed on the circled card and one drop
of each of the six Salmonella antigens are added
separately and gently rotated for one minute.
Positive & negative controls are also placed on their
specific circles and mixed with drops of Widal TEST
antigen suspension O.

Appearance of agglutination gives qualitative

results.
To know the titer for each of the antigens, the
test is repeated
with dilutions of serum.

2nd Semi - Quantitative test

serum
saline

Here, Titer = 160

another method for Semi Quantitative test

Reading the results of semiquantitative method

The control tubes must be examined first, where

they should give no agglutination.
The agglutination of O antigen appears as a matt
or carpet at the bottom.
Agglutination of H antigens appears loose, wooly or
cottony.
If agglutination was visible, the results were
considered positive
The highest dilution of serum that produces a
positive agglutination is taken as titer.
The titers for all the antigens are noted.

Precipitation
In this test, the result is observed as
precipitate rather than visible agglutination
Moreover, in precipitation test; S. typhi Ags are
suspended on alcohol & S. paratyphi Ags on
formalin rather than latex particles
This test is more sensitive than agglutination
test; however it is rarely performed as it is
expensive and not frequently available
The test need 18 tubes for the detection of
whole Salmonella Ags.

Precipitation procedures
Ste
p

Step

20
ul
an
seru
d
m + 1 : 10
180
8 tubes
ul
100
sali
ulne
an
samp
d
le + 1/10 * 1/10
900 = 1/100

10
ul
seru
m + 1 : 20
190
8 tubes
ul
100
sali
ul
ne
samp
le + 1/10 * 1/20
900 = 1/200

Step

Step

Repeat step 2 for the

reminder of Salmonella Ags
- Typhi H
- paratyphi A . H
paratyphi A . O
- paratyphi
. Htubes,
We
finally getB16
paratyphi Bthem
. tap
Otheat
Centrifuge
3,000
bottom
of the tube 2
- paratyphi
C . H times
rpm
for 5 min.
- paratyphi C . O
If the ppt is
resuspende
d, the the
results

If the ppt
still at the
bottom,
the results

:Other Methods
Typhiod Rapid IgM-Assay
o Detects specific IgM and IgG antibodies to S. Typhi

RT-PCR
o The PCR technology has an unparalleled sensitivity
and specificity for the diagnosis of typhoid