Clinical diagnosis of brucellosis is very difficult due to protean manifestation of the disease. Hence, laboratory diagnosis plays an important role in confirming the diagnosis of brucellosis.
Blood is the specimen of choice and is collected for culture and for serological test. Bone marrow and sometimes synovial fluid and pleural fluid are also collected for culture. Specimens such as liver and lymph nodes can also be cultured for isolation of Brucellaorganisms. Rarely, the bacteria can be isolated fromcerebrospinal fluid (CSF ), urine, sputum, breast milk, vaginal discharge, and seminal fluid.
Gram staining is not useful for demonstration of Brucella organisms in clinical specimens due to their small size and intracellular location.
Isolation of Brucella from blood and other clinical speci-mens is the definitive diagnostic procedure in brucellosis. Approximately, 5–10 mL of blood is collected in the 200 mL serum dextrose broth or trypticase soy broth and incubated at 378C under 5–10% of CO2. After fourth day of incubation, subcultures are made on solid media, every 3–5 days for 8 weeks before declaring the culture as negative.
Bone marrow cultures are more sensitive than blood culture. They typically are positive in the negative blood culture and serological results. Synovial fluid culture is positive in 50% of patients.
Identification of Brucella colonies is made by microscopy of Gram-stained smears of the colonies, colony morphology, biochemical tests, and reactivity with specific antibrucella sera.
Serological tests are useful for diagnosis of subclinical bru-cellosis and for cases of acute and chronic brucellosis by demonstration of specific antibodies in patient’s serum. Specific brucella antibodies, both IgG and IgM antibodies, appear in the serum 7–10 days after infection. IgM antibodies persist for up to 3 months after which these antibodies decline. Then IgG and IgA antibodies appear after 3 weeks of infection and persist for longer time. Hence, in acute stage or subclinical brucellosis, both IgG and IgM can be demonstrated; in chronic brucellosis, only IgG can be demonstrated, as IgM are absent.
As IgG antibodies persist for many months or years, demon-stration of significant rise in the antibody titer is the definitive serological evidence of brucellosis. A fourfold increase in the titer or a single high antibody titer of 1:160 is the presumptive evidence of Brucellainfection.
B. abortus is used as an antigen in brucella agglutination testsbecause the antibodies against B. melitensis or B. suis can cross-react with B. abortus antigen. B. abortus antigen not only detects specific antibodies against its own antigens but also detects antibodies produced against B. melitensis and B. suis. However, antibodies against B. abortus do not react with B. canis. Hence, specific B. canisantigen is used to detect antibodies in the serum of patients infected with this organism.
A number of serological tests have been developed and tested in serodiagnosis of brucellosis. Common methods are (a) stan-dard tube agglutination tests, (b) indirect immunofluorescent tests, and (c) enzyme-linked immunosorbent assay (ELISA).
Standard tube agglutination test: This test remains the stan-dard method and is the most commonly used serological test for diagnosis of brucellosis. It detects the presence of antibodies against LPS component of Brucella. The test uses killed strains of B. abortus as antigen and is useful for diagnosis of brucellosis caused by B. abortus, B. melitensis, and B. suis. This test is not use-ful for the infection caused by B. canis because this bacterium does not have O polysaccharide on its surface; hence it does not produce antibodies against LPS.
The tube agglutination test is considered positive when anti-body titers are greater than or equal to 1 in 160 or when a four-fold rise titer is demonstrated in convalescent sera. Rise in titer is especially important in patients coming from endemic area in which titers of 1 in 160 are not unusual in persons who are asymptomatic.
Modified tube agglutination test:In this test, 2-mercaptoetha-nol is added to the patient’s sera before testing it. Addition of mercaptoethanol causes disruption of disulfide bond of IgM; hence only IgG is detected. This modified tube agglutination test is useful for specific detection of IgG antibodies, and titers higher than 1:80 are suggestive of active infection. A high IgG antibody titer or a titer that is higher after treatment suggests relapse or persistent infection. This test is useful for diagnosis of brucellosis during convalescence.
Indirect immunofluorescent assay: It is a sensitive methodfor detection of brucella antibodies in the serum, which may be positive even in agglutination-test-negative cases.
Enzyme-linked immunosorbent assay: ELISA is the mostsensitive test for detection of IgM, IgA, and IgG brucella antibodies during acute and chronic brucellosis. In this test, LPS-depleted cytoplasmic protein antigen of Brucella is used as antigen. Use of this antigen allows accurate evaluation of relapse of brucellosis in a patient because LPS antigenemia persists for a longer duration than does protein antigen.
ELISA is extremely useful for diagnosis of neurobrucellosis by demonstration of brucella antibodies in the CSF.
Nevertheless, these serological tests may show erroneous results due to the following factors:
1. Blocking antibodies: Blocking or nonagglutinating anti-bodies in the serum may contribute to a false positive result. This can be avoided by heating the serum at 558C for 30 minutes or by using 4% saline or diluent in the test. These blocking antibodies can be diluted by adding antihu-man globulin to the antigen serum mixture (Coombs’ test).
2. Prozone phenomenon: It is a frequent problem in diagnosis of brucellosis. This occurs due to high level of brucella anti-bodies in the serum, caused by hyperantigenemia. Prozone phenomenon gives rise to a false negative test. This can be avoided by routine dilution of serum to at least 1:320 because inhibition of agglutination may occur at low dilution.
3. Cross-reactivity with V. cholerae, Y. enterocolitica serotype O9, F. tularensis, and Salmonella species can cause false positiveresults due to the presence of cross-reacting LPS in these organisms. Cholera-induced antibodies may be removed by 2-mercaptoethanol absorption.
4. A decrease in titer is observed with recovery of patient.
Brucella skin test: Brucella skin test is a delayed type of hyper-sensitivity reaction to brucella antigen. In this test, brucellin, a protein extract of the bacteria, is used as an antigen and is administered intradermally. The presence of erythema and indu-ration of 6 mm or more within 24 hours is suggestive of posi-tive reaction. This test is positive only in chronic brucellosis but negative in acute brucellosis. Repeated negative skin test excludes brucellosis.
Bone marrow examination shows erythrophagocytosis, and CSF cultures for neurobrucellosis show pleocytosis, increased protein level, and hypoglycorrhea. Anemia in 75% of patients, thrombocytopenia in 40%, and pancytopenia in 6% of patients are other features.
The diagnosis of brucellosis in animals is based essentially on the same method as that employed for the diagnosis of human infections. In addition, culture of milk and urine from infected animals may give positive results. Rapid latex agglutination test and Rose Bengal card test are the rapid diagnostic methods, and are also used for diagnosis of brucellosis in cattle population.
Milk ring test: This is a frequently used serological test fordemonstration of antibodies in the milk of an animal. This is a screening test used to detect the presence of Brucella infec-tion in infected cattle. In this test, a concentrated suspension of killed B. abortus orB. melitensis stained with hematoxylin is used as antigen. This test is performed by adding a drop of colored brucella antigen to a sample of whole milk in a test tube. Then it is mixed, and mixed suspension is incubated in water bath at 708C for 40–50 minutes. In a positive test, if antibodies are present in the milk, the bacilli are agglutinated and raised with the cream to form a blue ring at the top, leaving the milk unstained. In a negative test, the milk remains uniformly blue without formation of any colored ring.