Diagnosis of Brucella

Prepared by:

Bahaa Mostafa Kamel 415

Diagnosis of Brucella

Symptoms and signs of brucellosis are unspecific; cultures and serology are usually

necessary for diagnosis. Some general laboratory findings might suggest the diagnosis

(leukopenia, relative lymphocytosis and pancytopenia). Slight elevation in liver enzymes is a

very common finding. The criterion standard test for diagnosis of brucellosis is the isolation

of the organism from the blood or tissues (bone marrow, liver aspiration).

Laboratory Studies

Organism isolation

The Brucella abortus organism can be isolated from fetal lymph nodes, placenta, milk,

vaginal mucus, uterine exudate or semen. Also any fluid can be cultured (synovial, pleural

and cerebrospinal). Recovery of the organism requires the use of enriched culture media

and incubation in 10% CO2.

Three sulphonated broth tubes are inoculated with the patient's blood

- A blood broth tube incubated aerobically.

- A blood broth tube incubated in air + 10% CO2.

- A blood broth tube incubated anaerobically.

The sensitivity of blood cultures with improved techniques such as the Castaneda bottles is

further improved by the lysis-centrifugation technique. With these methods, the sensitivity

is approximately 60%.

Subcultures are still advised for at least 4 weeks; thus, if brucellosis is suspected, the

laboratory should be alerted to keep the cultures for 3-4 weeks, which is not done routinely

for most bacterial cultures.

Bone marrow culture is thought to be the criterion standard, since the reticuloendothelial

system holds a high concentration of brucellae.

CSF evaluation: This reveals a mild-to-modest lymphocytic pleocytosis in 88-98% of in

patients with neurobrucellosis. Protein levels are elevated in conjunction with normal

glucose levels.

Serological tests

Serological testing is the most commonly used method of brucellosis diagnosis.

These tests detect antibodies present in serum, milk, whey, vaginal mucus and seminal

plasma. The serological diagnosis is considered unreliable when performed during the

period of 2 to 3 weeks before and after abortion or calving.

-Serum agglutination is considered the standard test at this time. The serum agglutination

test will detect non-specific antibodies as well as those that are specific for Brucella abortus

infection and vaccination.

-Prozone phenomenon (which is the inhibition of agglutination with low titers and the

presence of agglutination with high titers), may occur secondarily to hyperantigenemia and

formation of blocking antibodies (IgA) interfering with IgG or IgM, possibly leading to false-

negative results, so routine dilution of the serum beyond 1:320 would help to prevent such a

problem.

-Rose Bengal test (buffered plate antigen or card test), This is a rapid and simple screening

test that is useful for detecting early infections.

-Tray agglutination (TAT) and modified TAT are also popular. Titers of more than 1:160 in

conjunction with compatible clinical presentation is considered highly suggestive of

infection. Titers of more than 1:320 are considered to be more specific, especially in

endemic areas. Seroconversion and evolution of the titers can also be used for diagnosis.

-Complement Fixation Test, This test has good specificity and is the most definitive test at

this point in time aside for bacterial isolation. It is used to confirm agglutination test and in

cases where agglutination test is negative.

-ELISA (Enzyme Linked Immunosorbent Assay)

This test has been useful during eradication programs after vaccination has ceased and is

used for screening or as a supplemental test to the complement fixation test. The ELISA test

has superior sensitivity and reliably detects true negative results.

-Milk Ring Test (antibody detection in milk), this test is a

satisfactory and inexpensive test used for surveillance of dairy

herds for brucellosis.

-Polymerase chain reaction (PCR): PCR testing for brucellae is

a recent advance with promising potential. It would allow for

rapid and accurate diagnosis of brucellosis. Two major genetic targets are the Brucella gene

BCSP31 and the 16S-23S rRNA operon. The 16S-23S rRNA operon has been shown in studies

to be more reliable in terms of sensitivity but is not yet widely used in clinical practice and

needs more standardization.

Other promising tests include nested PCR, real-time PCR and PCR-ELISA, but the clinical role

for these tests remains to be defined.

-Brucellin test: it is an allergic skin test produced by intradermal injection of:

1. A culture filtrate of Brucella

2. Brucella extract (Brucellin)

3. Purified protein

The reaction resembles the tuberculin reaction, it gives rise to an edematous indurated area

of at least 5 mm after 48 hours.

Imaging Studies

Chest radiography

Radiographic findings are typically absent in brucellosis, even in patients with prominent

respiratory symptoms.

Findings observed in patients with active pulmonary involvement include hilar and

paratracheal lymphadenopathy, pulmonary nodules, pleural thickening, and pleural effusion.

Spinal radiography

Radiographic findings in patients with osteoarticular disease occur later in the course of

illness, usually 2-3 weeks after the onset of symptoms.

In patients with sacroiliitis, the most commonly observed abnormalities include blurring of

articular margins and widening of the sacroiliac spaces.

Spondylitis-related abnormalities include anterosuperior vertebral angle epiphysitis, spinal

straightening, narrowing of the intervertebral disc spaces, end-plate sclerosis, and

osteophytes.

Radionuclide scintigraphy

This study is more sensitive for revealing skeletal abnormalities, especially early in the

disease, when standard radiographic findings are usually normal.

Radionuclide scintigraphy may be especially helpful in distinguishing hip involvement from

sacroiliitis.

To facilitate prompt diagnosis, this study also may have a role in screening for newly onset

brucellosis and musculoskeletal symptoms.

Histologic Findings

Histologic findings in brucellosis usually include mixed inflammatory infiltrates with

lymphocytic predominance and granulomas (in up to 55% of cases) with necrosis.

References

- Brucella - Molecular and Cellular Biology

- JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1997, Vol. 35-No. 9

- RESEARCH ARTICLE - INTERNATIONAL MICROBIOLOGY (2004) 7:53–58

- http://emedicine.medscape.com/article/213430-diagnosis

- Brucellosis in humans and animals - WHO/CDS/EPR/2006.7

Prepared By:

Bahaa Mostafa Kamel - No. 415