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GHOR ALSAFI LAB.

Brucella Testing

A.KAREEM AL-AJOURI 1


Brucella Testing
Screen, Tube and Microtiter
Methods

1. Introduction:

Brucella species cause disease in animals and human disease results from contact
with animals.
There are four species responsible for the majority of human disease: B. abortus,
B. melitensis, B. suis, and B. canis.
The disease caused is brucellosis and is characterized by fever, chills, fatigue,
weakness, and internal organ lesions.
Brucellosis can be chronic.
The organism is slow-growing, and blood cultures may need to be incubated for
4-6 weeks before they are considered negative.
Febrile antigens tests (serological tests) are valuable in identifying the causative
agent; such tests detect a group of antibodies developed during some febrile
infections caused by brucella, salmonella and rickettsia.

2. Clinical Significance:

The clinical significance of serological Brucella testing is:
Detection and quantitation of specific antibodies to Brucella
abortus and Brucella melitensis.
Investigation of febrile diseases in parallel with culture.

3. Health and Safety Precautions:

Brucella specimens should be treated as potentially infectious and handled with
all necessary precautions.
While handling specimens and performing Brucella testing, do not pipette by
mouth, wear disposable gloves, wear eye protection, and wash hands when
finished.
Disposables (pipette tips, droppers, plastic tubes, etc.) used to process the test
should be autoclaved or incinerated before disposal. Non-disposables should be
autoclaved for at least 15 minutes at 121
0
C.
Spillage of potentially infectious material should be immediately wiped with
tissue paper and the contaminated areas swabbed with a standard bacterial
disinfectant or 70% alcohol. Materials (gloves, tissue paper, swabs, etc.) used to
clean spills should be disposed of as biohazardous waste.





GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 2
4. Preanalytical Conditions:

4.1. Collection of Specimen:

Serum is the recommended sample.
Care should be taken to ensure that the blood samples are fully clotted before
centrifugation.
Hemolyzed, lipemic or contaminated samples are not suitable for testing.

4.2. Storage Considerations:

If the test cannot be carried out on the same day, the serum may be stored
between 2-8
0
C for no longer than 72 hours after collection.
For longer periods store serum frozen at 15
0
C to 25
0
C.

5. Rose Bengal Rapid Slide Screening Test:

5.1. Principle (Rose Bengal Test):

The Rose Bengal Test is a slide agglutination test for the direct detection of
Brucella abortus antibodies in serum.

5.2. Reagents (Rose Bengal Test):

Commercially available kits usually include:
Rose Bengal Reagent: a bacterial suspension (antigen) of Brucella
abortus stained with Rose Bengal and buffered at pH 3.6 with
preservative (sodium azide). The minimum detectable unit is
equivalent to 25 IU/mL.
Positive Control Serum: prepared from human serum with a high
concentration of anti-Brucella antibodies. Sodium azide is added as
a preservative.
Negative Control Serum: prepared from human serum without
anti-Brucella antibodies. Sodium azide is added as a preservative.
Because reagents contain sodium azide, skin and mucous contact with the
reagents should be avoided.

5.3. Storage of Reagents (Rose Bengal Test):

Reagents should be stored at 2-8
0
C.
Freezing and thawing of the Rose Bengal reagent lead to false-positive
agglutination.







GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 3
5.4. Procedure (Rose Bengal Test):

Table 5.4. Procedure for Rose Bengal Screening Test
5.4.1. Bring test reagents and specimens to room temperature.
5.4.2. Label 3 sections on the reaction slide with patient identification, positive
control, and negative control, respectively.
5.4.3. Resuspend the Rose Bengal reagent gently.
5.4.4. Place one drop (50 L) of the reagent on each of the three sections.
5.4.5. Add one drop (50 L) of each patient serum, positive control, and negative
control into the appropriate labeled section.
5.4.6. Mix the contents of each circle with a disposable stirrer spreading over the
entire area of each section.
5.4.7. Rotate the reaction slide slowly either by hand or by means of a mechanical
rotator for 4 minutes.
5.4.8. Read by comparing patient serum with controls.
5.4.9. A negative reaction is indicated when a smooth suspension with no visible
agglutination is observed and is comparable with the negative control.
5.4.10. A positive reaction is indicated when any degree of agglutination is visible
macroscopically and is comparable with the positive control.
5.4.11. Any positive reaction should be confirmed by titration with the stained
Brucella suspensions.

5.5. Limitations of the Rose Bengal Test:

False-negative reactions can occur in:
Early infections.
Late stages of the disease.
False-positive reactions occur with patients vaccinated against Brucella.

6. Stained Brucella Suspensions Semi-Quantitative Tests:

Two methods are used; the tube test and the microtiter plate test.
The microtiter plate is more reliable because it demonstrates clear cut patterns of
agglutinations.

6.1. Principle (Stained Brucella Suspensions):

The stained antigens are standardized suspensions of killed bacteria prepared for
the detection and semi-quantitation of Brucella antibodies in serum.
Diluted patient serum is mixed with the bacterial suspension in tubes or in
microtiter plates. If homologous antibodies (produced in response to exposure to
bacterial antigens) are present, they will react with the bacterial suspensions
(which carry the antigens) and cause visible agglutination.





GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 4





6.2. Reagents (Stained Brucella Suspensions):

The Brucella suspensions are standardized and prepared from smooth suspensions
of killed bacteria.
The suspensions have been stained to facilitate reading of agglutination tests.
Two suspensions are provided:
Brucella abortus: a stained suspension with added preservative and
supplied ready for use. It should be standardized to contain
approximately 10
10
bacteria/mL.
Brucella melitensis: a stained suspension with added preservative
and supplied ready for use. It should be standardized to contain
approximately 10
10
bacteria/mL.
Negative Control.
Positive Control.

6.2.1. Technical Notes on Reagents (Stained Brucella Suspensions):

Reagents should be stored at 2-8
0
C in the dark.
Immediately after use, the reagents should be stored at 2-8
0
C in the dark.
Do not expose reagents to strong light during storage or incubation.
Reagents must not be used beyond the expiry date.
Contaminated reagents must not be used.
Care should be taken to avoid cross-contamination of the reagents.
Reagents contain preservatives (e.g. thiomersal and formalin); such preservatives
are toxic by ingestion and skin contact. If any come in contact with skin or eyes,
the affected area should be washed with plenty of water.

















GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 5
6.3. Procedure for Brucella Stained Suspensions -Tube Test:

Table 6.3. Procedure for Brucella Stained Suspensions -Tube Test:
6.3.1. Allow all reagents and samples to reach room temperature before use.
6.3.2. Arrange 2 rows of tubes each consisting of 8 tubes, the first row for B.abortus and the
second row for B. melitensis. Label each tube with the patients name, and titer. Label
tube 8 of each row Suspension Control.
6.3.3. Using normal saline (0.85%) as a diluent, pipette 1.9 mL to the first tube (1:20) of each
row and 1.0 mL in each of the rest of tubes.
6.3.4. Pipette 0.1 mL of patients serum to the first tube (1:20) of each row.
6.3.5. Mix the contents of tube 1 and transfer 1.0 mL to tube 2. Repeat for each tube, up to tube
7 (discard 1.0 mL from tube 7) as shown in the following scheme:
Tube 1 2 3 4 5 6 7 8
Saline 1.9
mL
1.0
mL
1.0
mL
1.0
mL
1.0
mL
1.0
mL
1.0
mL
1.0
mL
Serum 0.1 mL _ _ _ _ _ _ _

1.0 mL Serial Dilutions
_


Mix &
transfer
1.0 mL to
2

Mix &
transfer
1.0 mL to
3

Mix &
transfer
1.0 mL to
4

Mix &
transfer
1.0 mL to
5

Mix &
transfer
1.0 mL to
6

Mix &
transfer
1.0 mL to
7

Mix &
discard
1.0 mL
from 7

_
Final
Titer
1:20 1:40 1:80 1:160 1:320 1:640 1:1280 Suspension
Control
6.3.6. Mix the B. abortus suspension and add one drop to each of the 8 tubes in the first row,
including the suspension control tube.
6.3.7. Mix the B. melitensis suspension and add one drop to each of the 8 tubes in the second
row, including the suspension control tube.
6.3.8. Mix all tubes thoroughly and cover tightly with parafilm. Incubate at 37
0
C, in a waterbath,
for 24 hours.
6.3.9. Examine each tube for agglutination under bright light against the suspension control tube.
6.3.10. As a positive control for each suspension, a dilution series of B.abortus and B. melitensis
antiserum may be included.
6.3.11. Hold each tube under bright light and flick it without shaking.
6.3.12. In a positive reaction, there is an obvious granular agglutination.
6.3.13. In a negative reaction, the appearance of the suspension should be unchanged and show a
typical swirl when the tube is flicked without visible agglutination. The tube should not be
shaken.
6.3.14. In the suspension control tube, the appearance of the suspension should be unchanged and
show a typical swirl when the tube is flicked without visible agglutination. The tube
should not be shaken.
6.3.15. The titer in each positive case is the dilution of the serum in the last tube showing
agglutination.
Example: B. abortus 1:1280 and B. melitensis 1:1280.
6.3.16. A high proportion of normal individuals can show positive reactions, therefore titers of
less than 1:80 are of doubtful significance.
6.3.17. If test is positive at a dilution of 1:1280, repeat the test on further dilutions of the serum:
1:2560, 1:5120, etc.



GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 6
6.4. Procedure for Stained Suspensions Brucella Microtiter Test:

6.4. Procedure for Stained Suspensions Brucella Microtiter Test:
6.4.1. Bring all reagents and specimens to room temperature.
6.4.2. Label 2 rows, row A for Brucella abortus and row B for Brucella melitensis. Follow the
scheme below for each row separately.
6.4.3. Dilute serum 1:80 with normal saline (in a 5-mL plastic tube, pipette 2 mL normal saline,
remove 25 L of those, then add 25 L patient serum and mix well on a vortex mixer, i.e.,
1975 L normal saline + 25 L serum).
6.4.4. In a microtiter plate with U-shaped wells bottom, dilute each serum specimen with normal
saline following the dilution scheme. It is recommended to carry out this step with the
same pipette, to mix carefully the diluted serum in each well and to transfer 100 L of
each dilution to the following well as demonstrated in the following scheme:
Well
No.
1 2 3 4 5 6 7 8 9 10 11 12
Final
Titer
1: 80 1:160 1:320 1:640 1:1280 1:2560 1:5120 SC NC PC
Serum
1:80
200
L
_ _ _ _ _ _ _ _ _
Saline
_
100
L
100
L
100
L
100
L
100
L
100
L
100
L
_ _

100 L Serial Dilutions




Mix &
transfer
100 L
to 2

Mix &
transfer
100 L
to 3

Mix &
transfer
100 L
to 4

Mix &
transfer
100 L
to 5

Mix &
transfer
100 L
to 6

Mix &
transfer
100 L
to 7

Mix &
discard
100 L
from 7


_


_



_

NC 100
L

PC 100
L

BS 50
L
50
L
50
L
50
L
50
L
50
L
50
L
50
L
50
L
50
L

SC: Suspension Control, NC: Negative Control, PC: Positive Control, BS: Bacterial Suspension
6.4.5. Mix the microtiter plate on a shaker for about 20-30 seconds.
6.4.6. Incubate for 16-18 hours at 37
0
C.
6.4.7. For better visual reading of the precipitate, it is recommended to place the microtiter plate
in refrigerator, after the incubation step, for 2 hours.
6.4.8. Place the microtiter plate on a flat surface avoiding shaking and read results comparing
patterns to the controls.
6.4.9. Negative reaction: a clear precipitate in the center of the well bottom.
6.4.10 Positive reaction: an agglutination spread on the well bottom.
6.4.11. Weak positive reaction: an agglutination is not uniform with precipitate in the center of
the well bottom.
6.4.12. The titer is reported as the highest dilution that shows agglutination. The next higher
dilution should be negative.
6.4.13. Titers up to 1:40 must be considered negative.
Titers from 1:80 to 1:160 are doubtful and should be repeated on a second sample taken
few days later.
Titers from 1:320 and above are positive.

GHOR ALSAFI LAB. Brucella Testing

A.KAREEM AL-AJOURI 7
6.5. Agglutination Pattern of Brucella Microtiter Reactions:

Figure 6.5. Agglutination Pattern of Brucella Microtiter Reactions

Negative Result Positive Result Weak Positive Result
Clear precipitate Agglutination Agglutination not uniform
in the center of Spread on the with precipitate in the
the well bottom well bottom. center of the well bottom.

7. Interpretation of Brucella Test Results:

The strength (titer) of the reaction is indicative of the concentration of antibodies
in the patients serum.
A rising or falling titer detected after testing two samples for the same patient on
different timing (e.g. 2 weeks apart) is more significant than a single high titer.
It is difficult to differentiate between a Brucella abortus and a Brucella melitensis
infection by serological tests, but from the point of view of treatment, this
distinction is not necessary.

8. Limitations of the Procedure:
Modification of the test procedure, incubation time or temperature would hinder
the validity and accuracy of the test.
Inactivated serum should not be used.
False positive results may occur with sera from patients infected with Francisella
tularensis or vaccinated with Vibrio cholerae.
A prozone phenomenon, which can be observed with absence of positivity at low
dilution and presence of positivity at high dilution level, may be encountered with
sera containing high antibody levels.

9. Quality Control:
It is recommended that a suspension control should be used with each run of test
samples.
It is recommended to test each suspension with a known positive serum (positive
control), that is commercially available like for example Brucella abortus antisera
and Brucella melitensis antisera, with each run.
It is recommended to test each suspension with a commercially available
negative control sera that should be used with each run.
If a suspension agglutinates with a known negative serum or fails to agglutinate
with a known positive serum it should be discarded.
The control sera are not standard sera and although titers should approach those
given on the bottle labels, the exact titers may not always be obtained but at least
titers close ( 1 titer or 2 titers) to what is stated on the bottle labels should be
obtained. One advantage of the control sera is to show the type of agglutination
that can serve as a pattern to that seen in a positive patient test.
A prozone phenomenon may sometimes be encountered with the positive control
sera as well.