Brucella Agglutination test: Principle, Procedure and Result Interpretation

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Introduction and principle of Brucella agglutination test

Brucella agglutination test is a serological test for brucellosis. Other serological tests for brucellosis are the Standard Agglutination test, 2 Marceptoethanol (2ME) Agglutination test, Coombs’ test, Complement fixation, Radioimmunoassay, ELISA, and Rose bengal test. Standard agglutination test is very useful because blood culture may not demonstrate the organism for many weeks, the Brucella agglutinin determinations can support a presumptive diagnosis of acute brucellosis. Isolation of the organism, usually from blood, provides definitive proof of infection. Both IgM and IgG antibodies continue to rise during the acute stage of infection and this test turns positive 7-10 days after infection. Patients with localized brucellosis may be afebrile and may not have significant levels of Brucella agglutinin titer. The prozone phenomenon ( i.e. absence of agglutination in low dilution of serum due to the high concentration of antibodies) is common and results in a negative reaction; this can be prevented by using serial dilutions of the serum.

Requirements for Brucella agglutination test

Antigen suspensions (Brucella abortus, Brucella melitensis) in bottles fitted with droppers, Negative control serum, Positive control, serum, Applicator sticks, Glass plate, Pipettes, 50–1000 ml, Sterile saline (0.85%), Test-tubes and test-tube racks, Timer, Water-bath, temperature-controlled

Brucella agglutination test slide method

This test is preferred to the tube test as it is less complicated.
Add 0.08, 0.04, 0.02, 0.01, and 0.005 ml of serum to a row of squares on the glass plate. Place 1 drop of the appropriate well-mixed antigen suspension for the slide test on each drop of serum. Mix the serum–antigen mixture with an applicator stick, starting from the highest serum dilution. The final dilutions are correlated approximately to the macroscopic tube test dilutions and are counted as 1: 20, 1: 40, 1: 80, 1: 160, and 1: 320, respectively. Hold the glass plate with both hands and gently rotate it 15–20 times. Examine the serum–antigen mixture macroscopically for agglutination within 1 minute in a good light. The rapid slide test is a screening test designed to detect agglutinins, whereas the tube test is a confirmatory test designed to measure the agglutinin quantitatively. Any positive result obtained with the slide test should be verified with the tube test.

Brucella agglutination test tube method

Prepare serial dilutions of serum and the control sera. Place 8 test tubes in a rack for each serum to be tested. Pipette 1.9 ml of saline (0.85%) into the first tube of each row and 0.5ml into each of the remaining tubes. Add 0.1ml of the serum to tube 1 containing 1.9 ml of saline. Mix well with a pipette and transfer 0.5 ml to tube 2. Mix thoroughly. Continue serum dilution until tube 7. Mix thoroughly. Discard 0.5 ml from tube 7 after mixing thoroughly. Tube 8 is the antigen control tube. Add 0.5ml of the respective antigen to each of the 8 tubes. Shake the racks to mix the antigen and antiserum. The resultant dilutions are 1: 20 through 1: 1280, respectively. Incubate in a water bath at 37° C for 48 hours or according to the manufacturer’s instructions. Examine the tubes macroscopically for agglutination within 1 minute in a good light against a black background. Positive reactions show obvious agglutination (granulation)Negative reactions show a cloudy suspension without agglutination. The highest degree of dilution of serum in a tube showing agglutination is the titer.

Result Interpretation of Brucella agglutination test

Most patients with acute brucellosis will have an agglutinin titer of 1: 320 or greater by the end of the second week of illness. Agglutinins may be found in healthy individuals, and single sera with titers of less than 80 are of doubtful significance. Even one year after treatment, 20% of patients will continue to have a significant Brucella agglutinin titer. 1: 160 is for the non-endemic areas whereas 1: 320 endemic areas. It is generally agreed that a titer of >1:160 in the presence of a compatible illness supports the diagnosis of brucellosis. Demonstration of a fourfold or greater increase or decrease in agglutinating antibodies over 4 to 12 weeks provides even stronger evidence for the diagnosis. False-positive results may occur with sera from patients infected with Francisella tularensis or vaccinated against Vibrio cholerae.
They have also occasionally been recorded in abattoir workers. It is not possible to differentiate between B. abortus and B. melitensis infections using this test.

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