brucellosis

1. LABORATORY DIAGNOSIS OF
BRUCELLOSIS

2. Background
Brucellae are small, nonmotile, non-spore-forming, and slow-growing Gram-negative
coccobacilli belonging to the Brucellaceae family that cause brucellosis.
Brucellae can infect several animal species and currently comprise 12 well-classified species, 4
of which causing almost the totality of human infections:
B. melitensis, B. abortus, B. suis, and B. canis, with B. melitensis being the most virulent.
Serologically, there is no difference between B. abortus, B. melitensis and B. suis
Brucellosis is also known as “undulant fever’’, “Mediterranean” or “malta fever”

3. Background
The clinical stages are acute, subacute and chronic stage
It can affect any organ and body site, occurring in animals and humans, as an occasional host,
with very rare cases of human-to-human transmission.
Brucellosis represents the most common zoonotic infection
Because of their potential application in bioterrorism, Brucella spp. are considered category B
select biological agents by the CDC.
Category B agents are easy to disseminate and cause moderate morbidity but low mortality.

4. Brucellosis in Tanzania
In humans, brucellosis has been reported in different regions and zones of
Tanzania with seroprevalence estimates ranging from 0.7% to 20.5%.
Different studies have been performed utilizing various tests.
The most utilized tests are the Rose Bengal Test and the Microscopic
Agglutination Test

5. Summary of the publications where human testing for clinical brucellosis was conducted in accordance with WHO
guidelines showing test used, prevalence established, and relevance of test.
Reference Location YOS Test used Prevalence Relevance of test (WHO)
Kunda et al 2007 Arusha and Manyara 2002-2003 RBPT
cELISA
No results
6.2
+++ (S)
+++ (C)
Kunda et al 2010 Arusha and Manyara 2002-2003 RBPT
cELISA
No results
7.7
+++ (S)
+++ (C)
Bouley et al., 2012 Moshi 2007-2008 MAT 3.5 +++ (S)
Wankyo, 2013 Morogoro 2018 RBPT 23.9 +++ (S)
Chipwaza et al., 2015 Morogoro 2012 IgG ELISA
IgM ELISA
15.4
7
+++ (C)
+++ (C)
Orsel et al., 2015 Arusha/NCA 2011 SAT/FBAT
IgG ELISA
IgM ELISA
5.7
34
2.5
n/c
+++ (C)
+++ (C)
Cash-Goldwasser et al., 2018 Moshi 2012-2014 MAT 8.9 +++ (S)
Carugati et al., 2018 Moshi 2007/2008
2012/2014
MAT 2.2 (2007/08)
2.9 (2012/14)
+++ (S)
Bodenham et al., 2020 Arusha/NCA 2016-2017 SAT 6.1 +++ (S)

6. Current Challenges Associated With Brucella Diagnosis In
Our Setting
In Tanzania, a systematic review by Mengele et al revealed the following
challenges associated with the diagnosis that cause under-reporting of the disease
situation
inadequate knowledge of brucellosis among stakeholders in the livestock value chain,
limited diagnostic capacity for brucellosis,
challenges associated with diagnostic tests, and
uneven distribution of brucellosis surveillance studies in the country.

7. Laboratory Diagnosis of Brucellosis
The correct diagnosis of brucellosis in humans is crucial for early and adequate patient
management.
It also has serious public health significance, as it may reveal exposure to sick animals,
consumption of contaminated food, breach of laboratory safety practices, or the intentional
release of brucellae as a biological weapon.
The laboratory diagnosis relies on three different approaches and microbiological
procedures: direct diagnosis by culture, indirect diagnosis by serological tests, and rapid
diagnosis by molecular PCR-based methods.

8. Laboratory Diagnosis of Brucellosis
Indirect diagnosis by serological tests can be further divided into:
1. Screening methods and
2. Confirmatory methods

9. Laboratory Diagnosis of Brucellosis
CDC Laboratory criteria for Diagnosis:
Definitive
1. Culture and identification of Brucella spp. From clinical specimens
2. Evidence of a four-fold or greater rise in Brucella antibody titer between acute and convalescent phase
serum specimens obtained greater than or equal to 2 weeks apart
Presumptive
1. Brucella total antibody titer of greater than or equal to 1:1160 by standard tube agglutination test (SAT)
or Brucella microagglutination test (BMAT) in one or more serum specimens obtained after onset of
symptoms
2. Detection of Brucella DNA in a clinical specimen by PCR assay

10. Laboratory Diagnosis of Brucellosis
Direct Diagnosis by Culture:
Laboratory specimen:
Cerebrospinal fluid ,pus ,bone marrow and blood
Blood is the specimen most frequently used for bacteriological culture
Culture:
Incubation; in air supplemented with 5% of CO2, 37°C
The newer semi-automatic methods (BACTEC series) can detect the presence of Brucella by the third
day of incubation

11. Conventional Identification Of Brucella spp. from culture
Colony Morphology Gram Stain Biochemical/Test Reactions
Growth seen on Blood Agar
(BA), Chocolate Agar (CA)
Tiny, often faintly stained
gram-negative coccobacilli
Urea: Positive (within 4 hours)
Non-hemolytic, small
smooth, raised, and
translucent colonies appear;
may require 48-72 h for
discrete colonies to become
evident
May be mistaken for cocci Oxidase: Positive
CO2 enhances growth
May retain crystal violet
stain in blood culture
smears
Catalase: Positive
Slow grower; punctate
colonies in 48 h

12. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
A. Screening Methods
Rose Bengal plate test (RBT)
• It is a sensitive rapid screening test, but the results should be confirmed. The sensitivity
of RBT is over 99% but can give false positive reactions with sera from patients infected
with cross-reacting bacteria.
• The screening test prove negative in the face of a history and clinical presentation.

13. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
Rose Bengal Plate Test (RBT) Procedure
The serum samples and antigen were brought at room temperature from the freezer and
refrigeration respectively
Each sample (20µl) was placed on an enamel plate,
Add the same of the rose Bengal antigen to each serum
Mix by plastic rod for each test ,agitate gently for 4 minutes on a rocker after that the test
was read immediately .
Any visible agglutination was considered positive

14. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
Rose Bengal plate test (RBT)
A: agglutination
B: no agglutination

15. Advantages of Rose Bengal test over others
Its cheap and rapid
It can detect both non agglutinating and agglutinating antibodies
Doesn’t have drawback of prozone effect
Doesn’t require technical expertise or special laboratory equipment
Highly sensitive >99% irrespective of the stage of brucella
Major disadvantages:
It can give spurious positive reaction in patients infected with cross-reacting bacteria

16. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
Serum tube agglutination test (SAT)
• The standard agglutination test (SAT) is the most common serodiagnostic assay used for
diagnosing B. abortus, B. melitensis, and B. suis infections.
• It detects antibodies to brucellar S-LPS.
• It is performed by mixing serial 2-fold dilutions of patient’s serum with Brucella antigen
derived from heat-phenol-killed B. abortus strain 119-3 in test tubes or in the wells of a
microtiter plate.

17. Laboratory Diagnosis of Brucellosis
Serum tube agglutination test (SAT)
• If agglutination has occurred, the clumps of antigen and antibody complexes will settle, leaving
a clean supernatant.
• In case of a negative test, the suspension remains unchanged and cloudy. SAT titers of ≥:160 are
considered diagnostic when coupled with a compatible clinical presentation.

18. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
Microagglutination Test
• The traditional SAT has been miniaturized so as to
be performed in a microtiter plate format.
• This enables the use of small amounts of reagents
and low serum volumes, which allows for
simultaneous testing of multiple samples.

19. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
2-mercaptoethanol test
• The 2-ME assay is performed identically to the SAT and is run in parallel, employing as a
diluent phosphate buffer containing 2-ME or DTT (dithiothreitol) at a final concentration of
0.05 M in each test tube.
• Addition of 2-mercaptoethanol disrupts disulphide bond of IgM and only IgG is detected.

20. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
2-mercaptoethanol test
• The main use of the 2-ME is for the serological monitoring of the response to antibiotic therapy
in already-diagnosed patients.
• A 2-ME titer of ≥ :20 is considered negative,
• Titers in the range from 1:40 to 1:80 are indicative of active brucellosis in regions of low
incidence of disease, and antibody titers of ≥ :80 are interpreted as diagnostic in areas of
endemicity.

21. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
ELISA
• Involves detection of antibodies reacting
against S-LPS (indirect ELISA)
• Most sensitive test for detection of IgM,
IgA, and IgG brucella antibodies during
acute and chronic brucellosis

22. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
ELISA
Brucellacapt (Vircell, Spain) Brucella ELISA (Vircell, Spain)

23. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
Complement Fixation
• Used but not recommended in small laboratories because of its technical complexity (much
greater than that of SAT and ELISA)
• Other test for the diagnosis of Brucella is Coombs antiglobulin

24. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
Febrile Brucella Agglutination Test
• Many commercially available plate agglutination tests, also known as rapid or febrile
antigen Brucella agglutination tests are widely used in human health facilities in the East
African region.
• These rapid agglutination tests are variants of the slide Brucella agglutination test (SAT).
• Examples include:
Amitech (Amitech Diagnostics, Ontario-Canada)
Eurocell (Euromedi Equip, Middlesex-UK)

25. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
Febrile Brucella Agglutination Test
Used at KCMC Used at Bugando

26. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
Febrile Brucella Agglutination Test
• A study by Lukambagire et al in Northern Tanzania revealed markedly poor diagnostic
performance of the commercial plate agglutination tests currently available and widely used in
Tanzania.
• The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for
the Febrile Brucella Agglutination Test, and 89.4% for the cELISA.
Lukambagire, A.S., Mendes, Â.J., Bodenham, R.F. et al. Performance characteristics and costs of serological tests for brucellosis
in a pastoralist community of northern Tanzania. Sci Rep 11, 5480 (2021). https://doi.org/10.1038/s41598-021-82906-w

27. Laboratory Diagnosis of Brucellosis
Indirect Diagnosis by Serological Tests:
B. Confirmatory Methods
• The result of a combination of tests such as SAT and Coombs antiglobulin can be used to
assess the stage of evolution of the disease at the time of diagnosis .
• The RBT ,tube agglutination and ELISA procedures are most recommended

28. Laboratory Diagnosis of Brucellosis
Rapid Diagnosis by Molecular PCR-based methods:
• Molecular methods, also called NAATs, allow for the diagnosis of brucellosis in a few hours
with high sensitivity and specificity.
• NAATs remain positive for a long time in patients apparently asymptomatic and when
clinical relevance is unclear.
• However, a positive test may not necessarily indicate an active infection but could be the
result of a low bacterial inoculum in frequently exposed healthy individuals, DNA from
dead organisms, or successfully treated patients.

29. Laboratory Diagnosis of Brucellosis
Rapid Point of Care Tests:
 Lateral Flow Assay
• This is a simplified version of an ELISA contained in a suitable plastic device. The LFA was
conceived for bedside use.
• The assay consists of a nitrocellulose detection strip containing B. arbotus LPS, flanked at one
end by a reagent pad and a reagent control applied in distinct lines.
• The reagent pad contains dried colloidal gold anti-human IgM and anti-human IgG conjugate as
the means of detections.

30. Laboratory Diagnosis of Brucellosis
Rapid Point of Care Tests:
 Lateral Flow Assay
• It employs a tiny blood drop drawn by
fingerprick;
• It does not require laboratory expertise or
special equipment
• Results are easy to interpret.
• The different test reagents have been stabilized
so no need for refrigeration

31. Laboratory Diagnosis of Brucellosis
Rapid Point of Care Tests:
 Dipstick assay
• The dipstick assay is a rapid test in use for the detection of Brucella-specific IgM antibodies.
• It consists of a strip of nitrocellulose containing S-LPS derived from B. abortus as the antigen applied in a distinct
line.
• A wetted dipstick strip is incubated for 3 h in a mixture of patient’s serum and detection agent (a monoclonal anti-
human IgM antibody conjugated to colloidal dye particles of Palanyl red.
• After completing this step, the strip is rinsed with tap water and dried at room temperature, and the staining intensity
is compared to a colored reference.
• Performance of the test is simple and does not require technical expertise or special equipment.

32. WHO Key Points on the Diagnosis of Brucellosis in Humans
• In acute brucellosis, isolation of Brucella from blood or other tissues is
definitive.
• Culture is often negative, especially in long-standing disease.
• Serology is the most generally useful diagnostic procedure approach.
• The RBT, tube agglutination and ELISA procedures are recommended.
• Methods which differentiate IgM and IgG can distinguish active and past infection.
Corbel, M. J. (2006). Brucellosis in humans and animals. World Health Organization.

34. Our Recommendation
Based On WHO Recommendations And Studies
Done In Tanzania, We Recommend:
1. An initial screening of the patient’s serum by the
sensitive RBT
2. Followed by a more specific confirmatory test such as
the SAT/MAT.

35. References
1. Di Bonaventura, G.; Angeletti, S.; Ianni, A.; Petitti, T.; Gherardi, G. Microbiological Laboratory Diagnosis of Human Brucellosis: An
Overview. Pathogens 2021, 10, 1623. https://doi.org/10.3390/pathogens10121623
2. Lukambagire, A.S., Mendes, Â.J., Bodenham, R.F. et al. Performance characteristics and costs of serological tests for brucellosis in a
pastoralist community of northern Tanzania. Sci Rep 11, 5480 (2021). https://doi.org/10.1038/s41598-021-82906-w
3. Isaac Joseph Mengele, Gabriel Mkilema Shirima, Shedrack Festo Bwatota, et al, The Status and Risk Factors of Brucellosis in Smallholder
Dairy Cattle in Selected Regions of Tanzania, Veterinary Sciences, 10.3390/vetsci10020155, 10, 2, (155), (2023).
4. Yagupsky P, Morata P, Colmenero JD. 2019. Laboratory diagnosis of human brucellosis. Clin Microbiol Rev 33:e00073-19.
https://doi.org/10.1128/CMR.00073-19
5. Brucellosis 2010 CSTE Case Definition. https://wwwn.cdc.gov/nndss/conditions/brucellosis/case-definition/2010/
6. Corbel, M. J. (2006). Brucellosis in humans and animals. World Health Organization.
7. https://health.maryland.gov/laboratories/docs/Brucella.pdf
8. https://www.cdc.gov/brucellosis/pdf/brucellosi-reference-guide.pdf

36. RBT

37. Brucella Antigenic Structure