Brucella Serology

Brucellosis is a zoonosis caused by Brucella melitensis (goats and sheep), Brucella abortus (cattle), Brucella suis (pigs) and Brucella canis (dogs). Humans are infected by unpasteurised dairy products, by contact with infected animal tissue (farmers, veterinarians, abattoir and laboratory workers) and by inhalation.

Serology is the most used diagnostic method and includes the Rose Bengal plate test (screening), the tube agglutination test (TAT, with titre), 2-mercaptoethanol agglutination, Coombs (antihuman globulin) test, ELISA for IgM, IgG and IgA, and lateral flow assays. Blood culture and PCR are used for definitive diagnosis.

There are over 1.6 to 2.1 million new human cases each year, most in the Mediterranean, Middle East, Central Asia, sub-Saharan Africa, India and Latin America. It causes undulant fever with sweats, arthralgia, back pain, weakness, weight loss, hepatosplenomegaly and epididymo orchitis. Complications include sacroiliitis and spondylitis, endocarditis (the main cause of death), neurobrucellosis and relapse in 5 to 15% after treatment.

Indications. Fever of unknown origin, particularly with sweats, arthralgia, back pain and weight loss in a person with animal exposure or a history of unpasteurised dairy intake. Epididymo orchitis, sacroiliitis or spondylodiscitis with fever. Culture negative endocarditis. Chronic meningitis. Laboratory exposure to Brucella. Abortion in livestock handlers. Follow up of treated patients for relapse. Screening of exposed workers in endemic areas.

Specimen. Serum 1 to 2 mL, no fasting. Stable for days at 2 to 8 °C. Blood culture (prolonged incubation for 4 weeks in automated systems, with the laboratory notified of suspicion because of biosafety risk), bone marrow culture (highest yield), CSF, joint fluid and tissue for PCR and culture.

Reference values.

• Rose Bengal plate test: negative (no agglutination).
• Tube agglutination test: titre under 1 in 80 in non-endemic areas (negative), with endemic thresholds of 1 in 160 or higher.
• Coombs test: titre under 1 in 160.
• ELISA IgM and IgG: negative under the manufacturer's cut off.
• Brucella DNA: not detected.

Interpretation.

• A tube agglutination test titre of 1 in 160 or more (or 1 in 80 in non-endemic regions) with compatible illness is diagnostic of active brucellosis. A fourfold rise between acute and convalescent samples 2 to 4 weeks apart also confirms acute infection.
• Rose Bengal positive: a sensitive screening test (sensitivity over 95%) that needs confirmation by tube agglutination or ELISA.
• Raised IgM with low IgG: acute infection. Rising IgG, IgA and Coombs titres with falling IgM: chronic or relapsing disease. Persistent IgG after treatment can last for months to years.
• Coombs test and 2-mercaptoethanol positivity at high titre (over 1 in 160) indicate chronic or complicated brucellosis, and detect blocking (incomplete) antibodies that tube agglutination misses.
• Negative serology in early infection (first 1 to 2 weeks) or in the prozone phenomenon (very high antibody titres inhibiting agglutination at low dilutions): repeat with serial dilution, and culture.
• Blood culture positivity (50 to 90% in acute disease, lower in chronic) confirms the diagnosis, and bone marrow culture is more sensitive.
• PCR on blood, serum or tissue gives rapid, sensitive diagnosis (over 90%), including in culture negative disease.
• Neurobrucellosis: CSF lymphocytic pleocytosis, high protein, low glucose and antibodies in the CSF.
• Seropositivity persists in cured people living in endemic areas, so serology must be interpreted with clinical findings.

Critical values and findings needing immediate action.

• Fever, new murmur, embolic phenomena or heart failure with positive Brucella serology: brucella endocarditis, urgent echocardiography and combined medical surgical management.
• Headache, neck stiffness, cranial nerve palsy, confusion or myelopathy: neurobrucellosis, CSF analysis and prolonged combination antibiotic therapy.
• Severe back pain with neurological signs: spinal brucellosis with epidural abscess, urgent MRI.
• Laboratory exposure (open bench handling of culture): post-exposure risk assessment, serological follow up and prophylaxis (doxycycline with rifampicin) for high risk exposures.
• Pregnant woman with brucellosis: risk of miscarriage, treated with rifampicin and co-trimoxazole under specialist supervision.

Interferences and pitfalls.

• Cross-reactions occur with Yersinia enterocolitica O:9, Francisella tularensis, Vibrio cholerae, Escherichia coli O157, Salmonella and Bartonella, mainly affecting agglutination and Rose Bengal tests. ELISA has fewer cross-reactions.
• Brucella canis is not detected by the usual antigens (smooth lipopolysaccharide), and specific rough antigen is required.
• The prozone effect gives false negative agglutination at low dilutions, so serial dilutions to 1 in 1280 or more are tested.
• Titres vary by region, and endemic thresholds differ from non-endemic ones. Past infection, occupational exposure and vaccination of animals (not humans) alter baselines.
• Antibody titres fall slowly after treatment, and persistently high titres do not always mean relapse. Clinical assessment, culture and a rising titre or IgG/IgA are used.
• Culture is hazardous to laboratory staff (a class 3 pathogen), so the laboratory is warned of suspected brucellosis.
• Sensitivity of blood culture falls with prior antibiotics and chronic disease.
• Treatment requires at least two drugs for 6 weeks (doxycycline with rifampicin or streptomycin, or in complicated disease for 3 to 6 months), and monotherapy fails.

Special groups.

• Children: more often present with fever and arthritis, and doxycycline is replaced with co-trimoxazole in young children.
• Pregnancy: miscarriage, stillbirth and neonatal infection. Breastfeeding can transmit infection.
• Older adults: spondylitis and endocarditis.
• Occupational risk: farmers, abattoir workers, veterinarians and laboratory staff.
• Regions: highest burden in Mediterranean countries, the Middle East, Central Asia and East Africa. Control rests on animal vaccination and pasteurisation of milk.

Follow up tests. Blood and bone marrow cultures, Brucella PCR, Coombs and ELISA IgM, IgG and IgA, repeat serology at 4 weeks and at 3 and 6 months, full blood count, liver enzymes, ESR and C-reactive protein, echocardiography, MRI of the spine and sacroiliac joints, CSF analysis, scrotal ultrasound, abdominal ultrasound, chest radiograph, and tuberculosis tests for differential diagnosis.