universe84a

Brucella Test for Brucellosis Introduction

Brucella test: The most common test is the latex agglutination test also called the Rose Bengal card test ( using dye Rose Bengal but in this test kit other color used) for brucellosis as shown above picture. It is a slide agglutination test for the qualitative and semi-quantitative detection of antibodies anti-Brucella in human serum. The stained bacterial suspension agglutinates when mixed with samples containing specific lgG or IgM antibodies present in the patient sample. Undulant fever or Malta fever, or Gibraltar fever, or Diurnal and Mediterranean fever are the other names of  Brucellosis and it is a worldwide zoonosis caused by the bacteria,  genus Brucella.  These organisms are present in the reproductive organs of host animals, causing abortions and sterility. They are shed in large numbers in the animal’s urine, milk, and placental fluid.  Exposure to infected animals and animal products causes the disease in humans. Brucellosis was first diagnosed by Wright and Smith in 1897. Species of this genus are pathogenic to animals from which they are transmitted to humans causing brucellosis are Brucella abortus causing an abortion in the cattle, Brucella melitensis causing infection in goats and sheep,  Brucella susis causing infection in the pigs, and Brucella canis causing infection in dogs. Among them, Brucella abortus and Brucella melitensis are the most common causative agents of human brucellosis. It can be transmitted by various routes like ingestion of contaminated food such as raw milk or dairy products and cheese made from raw milk (unpasteurized), and similarly, farmers and butchers are infected by contact with diseased animals. Following are clinical features of brucellosis- high fever, malaise, anorexia, arthralgia, fatigue, headache, sweating, weight loss, and depression. Specific antibodies to the Brucella species are detectable a few weeks after exposure and are of considerable importance in the diagnosis of Brucellosis. Information regarding the titer of antibodies can be obtained.

Principle of Brucella Test

It works on the principle of agglutination. 25 IU/mL in the patient serum will react with the antigen suspension to produce an agglutination reaction. No agglutination indicates the absence of detectable levels of the smooth, colored, killed Brucella antigen suspension is mixed with the patient serum.

Test Requirements for Brucella Test

1. Test kit contains-

• Antigen suspension: This reagent contains smooth, killed buffered suspensions of Brucella abortus strain 99, colored with rose bengal, standardized against anti-Brucella abortus.
• Instruction manual

2. Additional material required

• Stopwatch
• Positive control
• Isotonic saline
• Glass slide with clear (white background, appropriate)
• Pipettes / Micropipettes
• Mixing sticks
• A High-intensity direct light source

3. Specimen: Blood-freshly collected serum is preferred, samples can be stored at 2-8 °C, for 24 hours, or frozen for 8 days should a delay in testing occur.  Do not use hemolysed serum samples.

Reagent Storage and Stability

Store the reagent at 2-8° C and do not freeze because frozen reagents could change the functionality of the test. Use it until expires.

Procedure of Brucella Test

Qualitative Method

1. Bring all reagents to room temperature.
2. The sensitivity of the test may be reduced at low temperatures.
3. Shake and mix t antigen suspension well before dispensing.
4. Place one drop of Positive control onto the reaction circle of glass slide
5. Place 50µl of saline onto the next reaction circle of the glass slide.
6.  Place 50µl of patient serum to be tested onto the next reaction circle.
7. Add one drop of well-mixed antigen suspension in each of the above circles containing positive control, isotonic saline and patient serum to be tested.
8. Mix the contents of each circle uniformly over the entire circle with separate mixing sticks.
9.  Gently rock the slide back and forth for 2 minutes and observe for agglutination macroscopically against a white background.

Semi-quantitative Method 

1. Follow steps 1-3 of the qualitative method.
2. Make serial two-fold dilutions of the sample in 0.9% normal saline solution.
3. Place one drop of antigen suspension in each circle.
4.  Mix the contents of each circle uniformly over the entire circle with separate mixing sticks.
5. Gently rock the slide back and forth for 2 minutes and observe for agglutination macroscopically against a white background.

Result Interpretation of Brucella Test

Qualitative method

Presence of Agglutination:  Test Positive( i.e.  the presence of antibodies to Brucella in concentration 25 IU/ml in the patient serum)

Absence of agglutination: Test negative ( i.e. absence of antibodies to Brucella in the patient serum)

Semi-quantitative method

Agglutination is a positive test result. The titer in the semi-quantitative method is defined as the highest dilution showing a positive result.

Calculation

The approximate antibody concentration in the patient sample is calculated as follows.
25 x anti-Brucella titer= IU/ml

For example,  suppose the positive titer is 1:8.

Then, 25× 8=200 IU/ml

Performance Characteristics of this test  kit

Analytical sensitivity: 25 (±5) IU/ml, under described assay conditions.
Specificity: 100%

Clinical Significance of Brucella test

Brucella diagnostic may be assessed either by microorganism isolation in blood or stools or by titration of specific antibodies in the patient serum. The reagent, because of its formulation in an acid buffer, is reactive with both IgM and IgG antibodies and very useful for the diagnosis of chronic individuals, which present a high level of IgG antibodies. Other diagnostics methods are the cultivation of organisms, ELISA, and PCR test. These all methods are cumbersome, time taken whereas cultivation needs higher biosafety laboratory or safety cabinet and even highly trained staff because of being high-risk group organism and theses are replaced by a simple and inexpensive latex agglutination method.

Keynotes on Brucella Test

1. Both B. abortus and B. melitensis share a common Brucella antigen. A sample giving a positive result with the Rose Bengal reagent should be tested using antigen suspensions by rapid slide test and confirmed by the tube test to determine the type of Brucella antibody detected.
2.  Agglutinins are found in a high proportion of normal individuals and concentrations less than 25 IU/ml are of doubtful significance.
3. A rising titer is more significant than a single high titer.
4. False-positive reactions may occur in sera of patients infected with Pasteurella tularensis or vaccinated with vibrio cholerae.
5.  Cross-reactions between Brucella antigens and other organisms have been reported. These include Yersinia enterolitica, Escherichia coli (0:157), and Francisella tularensis.
6. False-positive results are likely if the test is read more than four minutes after mixing on the slide test.
7. Prozoning may sometimes be encountered in serum-containing very high titers on slide tests.
8.  Serological findings are not intended as a substitute for culture. An appropriate attempt should be made to recover and identify the etiologic organisms through various culture and biochemical tests.
9. Since techniques and standardization vary from laboratory to laboratory on tube differences in titers can be expected.
10. Use a separate disposable tip for each sample to prevent cross-contamination.
11. Turbid and contaminated sera should not be used for testing.
12.  After usage, the antigen suspension should be immediately recapped and replaced at 2-8 °C.
13. Reagent vials that have leakage/ breakage problems should be discarded.
14.  Only qualified and well-trained staff should use the reagents.
15. It is recommended that the results of the tests should be correlated with clinical findings to arrive at the final diagnosis.
16. The performance of the reagents should be validated periodically using known positive control. Good physiological saline may be used as a negative control.
17. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
18. The reagent contains 0.01% thimerosal as a preservative. Avoid contact with skin and mucosa. On disposal flush with large quantities of water.
19.  The reagent can be damaged due to microbial contamination or exposure to extreme temperatures. It is recommended that the performance of the reagent be verified with the positive and negative controls.
20. Shake the reagent vials well before use to disperse the antigen suspension uniformly and improve test readability.
21.  It is necessary to use the calibrated dropper provided in the reagent vial to dispense a reagent drop.
22. Only clean and dry glass slides must be used. Clean the glass slide with distilled water and dry.
23. Brucella antigen suspensions are not from human sources hence contamination due to HBsAg and HIV is practically excluded.
24. Do not use damaged or leaking reagents.
25. There is no serological test available to detect antibodies to Brucella canis.
26.  Serological diagnosis of brucellosis needs two serum samples. The first serum sample should be taken when a person is acutely ill (≤7 days after symptom onset) while 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 mean an individual is positive for brucellosis.

References

1. https://www.cdc.gov/brucellosis/clinicians/serology.html
2. https://journals.sagepub.com/doi/full/10.1177/1535676018771983
3. http://www.tulipgroup.com/Tulip_New/product_immunology_Reagents.html
4. https://www.rapidlabs.co.uk/product/rose-bengal-agglutination-test-kit/
5. https://pubmed.ncbi.nlm.nih.gov/17258083/