Diagnosis of Brucella Infection

What sample is needed for brucellosis?

1. It is performed on the patient’s serum.
2. The serum is taken in the first week of illness and later in the 3 to 4 weeks.
3. Brucella can be cultured from the blood, sputum, bone marrow, CSF, tissue, lymph node, and urine.

What are the indications for brucellosis?

1. The patient has a fever of unknown origin.
2. The patient has a suspected history of contact with cattle.
3. The patient exhibits signs and symptoms consistent with a Brucella infection.

How will you define Brucellosis?

1. It is a chronic granulomatous intracellular infection caused by small, gram-negative, aerobic coccobacilli.
2. Three members of the genus Brucella produce an uncommon febrile disease known as Brucellosis (Undulant fever).
3. It is a chronic granulomatous intracellular infection due to small gram-negative aerobic coccobacilli seen in:
   1. Brucella melitensis  = By goats and sheep.
   2. Brucella suis  = By swine.
   3. Brucella abortus = By cattle.
   4. Brucella canis = By dogs.
4. It spreads through infected animals or by eating contaminated animal products.
5. Brucellosis (Undulant fever) is found as follows:
   1. Brucella suis is pig-associated.
   2. Brucella melitensis is goat-associated.
   3. Brucella Canis is dog-associated.
   4. Brucella abortus is cattle-associated.

How will you discuss the microbiology of Brucellosis?

1. The Brucella abortus, B. Suis, B. Melitensis, or B. Canis causes brucellosis.
2. Brucella is a slow-growing aerobic gram-negative coccobacillus.
   1. Short, slender, pleomorphic, gram-negative bacilli (coccobacillus).
   2. Brucella is an oxidase-positive urease variable.
   3. Brucella is nonmotile.
   4. These are non-sporing.
3. Culture media:
   1. Enriched medium such as glucose serum, liver infusion broth, or agar.
   2. Small transparent colonies develop after several days of incubation at 37 °C  in aerobic conditions.
   3. CO2 is needed for the growth of B. abortus.
4. There is growth in selective buffered charcoal yeast extract and Thayer-Martin medium.
5. Brucella canis is very rare and is typically contracted through exposure to dogs.

What are the characteristic features of the Brucella organism?

What is the mode of spread of Brucellosis?

1. It is by ingesting contaminated milk and milk products, especially goat milk.
2. Their milk is also contaminated when sheep, goats, cows, or camels are infected with these bacteria.
3. These names are based on infected animals.
4. Brucella is primarily transmitted through unpasteurized milk or raw dairy products.
5. Direct puncture of the skin of butchers and farmers. These bacteria can enter through the skin and mucous membranes.
6. Brucella can penetrate the skin, conjunctiva, lungs, and GI Tract.
7. Brucella can spread through inhalation.
8. Brucella also spreads among meat industry workers, especially with swine (pigs).
9. Veterinarians and dairy farmers are also at risk.
10. Penetration is followed by the lymphatics spread.
    1. There is facultative intracellular growth in the macrophages.
    2. It also involves the organs.

What are the types of Brucella?

1. There are the following types of Brucella:
2. Brucella abortus is sourced from cattle and exhibits moderate pathogenicity.
3. Brucella melitensis is sourced from sheep, and these are highly pathogenic.
4. Brucella Canis is sourced from dogs and has moderate pathogenicity.
5. Brucella suis is a source in pigs, and these are highly pathogenic.

What is the antigenic structure of Brucella?

1. These Brucella antigens are classified into :
   1. Surface antigens.
   2. Cytoplasmic antigens.
   3. Virulence-associated structures.
2. Three species share two antigens, A and M, which are O-polysaccharide antigens present on the smooth polysaccharides (L-LPS) of Brucella.
   1. B. melitensis contains an excess of M antigen.
   2. B. abortus and suis contain an excess of A  antigen.
3. Monospecific antisera can be prepared and used for identification.

What are the clinical Signs and Symptoms of Brucellosis?

1. This may be the acute or insidious onset of the symptoms.
2. The acute or insidious onset of signs and symptoms characterizes this multisystem disease.
3. There may be fever, chills, and night sweats.
   1. The fever peaks in the evening and slowly returns to normal by morning.
4. Undulant fever (wavy pattern of the fever) is characterized by a slow rise in temperature during the day, declining at night.
5. There is undue fatigue.
6. There is anorexia and weight loss.
7. There may be a headache and arthralgia.
   1. 25% of the patients develop single-joint arthralgia.
   2. There may be pain in muscles, joints, or the back. Myalgia may be the first symptom.
8. Sometimes there is lymphadenopathy. But most lymph nodes are not enlarged.
9. Approximately 20% of patients may exhibit splenomegaly.
10. Some patients may develop pneumonia.
11. If not treated, then these patients may have:
    1. Arthritis.
    2. Recurrent fever.
    3. Swelling of the scrotal area and testes.
    4. Depression.
    5. Chronic fatigue.
    6. May develop endocarditis.
    7. Splenomegaly and hepatomegaly are common findings.
    8. Spondylitis is also common.
12. These symptoms may last months to years, but the outcome is not fatal.
13. Laboratory findings due to involvement of various organs:
    1. Liver >50% have increased LDH, SGOT, and SGPT>
    2. CNS has been involved <7% of the cases.
    3. The principal cause of death is due to endocarditis and involvement of the aortic valve.

What are the complications of brucellosis?

1. The patient may develop endocarditis.
2. There may be arthritis and osteomyelitis.
3. The infection of the testes gives rise to epididymal orchitis.
4. CNS involvement may lead to meningitis or encephalitis.
5. Inflammation of the liver and spleen leads to splenomegaly and hepatomegaly.
6. Relapses occur within the first year in about 10% of the cases.

How will you prevent Brucellosis?

1. Please avoid the following:
   1. Use of unpasteurized milk and dairy products (cheese, ice cream).
   2. When handling animals, use preventive measures such as gloves.

How will you diagnose Brucella Infection?

1. WBC count is usually normal or decreased. There may be decreased WBC with relative lymphocytosis.
   1. There is mild anemia. There may be anemia in <75% of the cases with the localized type of disease.
   2. There is relative lymphocytosis.
   3. ESR may be increased in <25% of the cases.
   4. There is thrombocytopenia in <20% of the cases.
2. Liver function tests are abnormal.
3. B. melitensis infection is more severe, giving higher AST, ALT, and LDH, and may give rise to thrombocytopenia.
4. By serological test detecting the Brucella antibodies.
5. A skin test with brucellergin is available, but a positive result only indicates exposure to the organism and does not prove active disease.
6. Culture for Brucellosis:
   1. Multiple blood cultures are necessary when a high agglutination titer is present.
   2. Blood culture. This is positive in 30% to 40% of the cases.
   3. Blood culture for B. abortus needs 10% CO2.
   4. Blood culture must be kept for at least four weeks.
   5. About 70% are positive.
7. Bone marrow:
   1. Bone marrow culture may be positive when blood culture and serologic tests are negative.
   2. Bone marrow culture has more positivity.
8. Cerebrospinal fluid (CSF) examination.
9. Sputum.
10. Food specimen.
11. Biopsy and culture of affected tissue and organs.
    1. On biopsy, one may see a nonspecific granuloma, suggesting the diagnosis of Brucellosis.
12. By immunofluorescent demonstration of the organism in the clinical specimens.
13. Serology values:
    1. Test results greater than 1:80 are suspicious for brucellosis. The serologic tests become positive during the second to the third week of illness.
    2.  90% of the patients have a titer of ≥1:160.
    3. The rising titer is diagnostic.
    4. False-negative results are rare.
14. In chronic localized brucellosis, the titer may be negative or ≤1:200.
    1. These tests remain positive for a long time after the infection is cured.
15. EIA is the method of choice to detect specific IgM and IgG antibodies.
16. A failure to decline the titer indicates an incomplete response to treatment.
17. With a high titer, multiple blood cultures are positive.

What are the normal serological values for Brucellosis?

1. Source 1
   1. This test detects the presence of antibodies against the Brucella antigen.
   2. Borderline cases  1: 160. Ninety percent of patients have this titer. The agglutination reaction becomes positive during the second to third weeks of illness.
   3. Significant level = >1:160
   4. Highly suggestive of active infection = > 1: 320.
   5. The rising titer is of diagnostic significance.
   6. In chronic localized brucellosis, the titer may be negative or <1:200.
2. False-negative results are rare.
3. False-positive serological tests are seen with tularemia, cholera vaccination, or after the Brucellin skin test.
4. Source 2
   1. A rising titer ≥ 1:160 suggests infection, either past or present.
   2. A single titer of 1:160 or 1:320 may be suggestive of Brucellosis, if there are clinical S/S of brucellosis.
   3. It can prevent the prozone phenomenon by diluting the serum  ≥1: 1280.
5. Antibody levels decrease within 3 months or with the use of antibiotics, but persist at low levels for years, especially in chronic infections.
   1. The antibody titer may persist for 1 to 2 years after recovery.

How will you perform a Skin test for the diagnosis of Brucellosis?

1. Brucellergen (these are killed bacteria) is injected into the skin.
2. After 24 to 48 hours, it shows a reaction greater than 5 mm.
3. A definitive diagnosis requires the isolation of the organism through blood culture or a tissue sample.
4. Real-time PCR is more specific.

How will you treat brucellosis?

• These bacteria are sensitive to a combination of tetracycline and streptomycin. This combination is the choice of treatment.

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