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34 Recent Patents on Anti-Infective Drug Discovery, 2013, 8, 34-41

Clinical Presentations and Diagnosis of Brucellosis

Aysegul Ulu Kilic*, Gökhan Metan and Emine Alp

Department of Infectious Disease and Microbiology, Erciyes University Faculty of Medicine, 38039-Kayseri/Turkey

Received: May 25, 2012; Revised: July 20, 2012; Accepted: July 23, 2012
Abstract: Brucellosis is a worldwide zoonosis caused by Brucella species. The disease remains a significant economic
and public health problem particularly in the Mediterranean countries. Clinical manifestations of brucellosis are variable
and often nonspecific, simulating infectious and noninfectious diseases. Osteoarticular involvement is the most common
focal complication of brucellosis and morbidity. Mortality rate due to brucellosis is low, mostly secondary to endocarditis
and central nerve involvement of disease.
The diagnosis of brucellosis depends on the clinical presentations and laboratory tests. Detection of Brucella species by
culture method is sometimes unsuccessful; therefore, serological tests are preferred. These tests are easy to perform, and
results can be obtained within a short span of time. Several serologic tests have been developed for the diagnosis of hu-
man brucellosis, including the standard agglutination tube (SAT) test, anti-human globulin (Coombs) test, indirect fluo-
rescence antibody (IFA) test, and enzyme-linked immunosorbent assay (ELISA). SAT is the primary test used in many
clinical laboratories. IFA and ELISA are simple and reliable for the detection of immunoglobulin classes especially in
complicated cases. Polymerase chain reaction (PCR) technique is highly sensitive and specific for the determination of
Brucella spp. from peripheral blood and other tissues.
Recent patents are especially based on molecular assays in the diagnosis of brucellosis. However, PCR is still expensive
and may not be appropriate for daily practice.
Keywords: Brucellosis, clinical manifestations, diagnosis, patents.

CLINICAL PRESENTATIONS OF BRUCELLOSIS
Brucellosis is a systemic disease affecting various organs
or body systems and simulating other diseases. Symptoms
are nonspecific including fever, sweating, malaise, headache,
back pain, loss of appetite and they can develop suddenly,
evenly or over a week period. Involvement of any organ is
often referred as localized disease and can be assessed as a
complication of acute brucellosis or manifestation of chronic
brucellosis [1, 2].
The incubation period may differ according to the viru-
lence of organism, route of entry and infectious dose. Ac-
cording to the duration of symptoms, cases of brucellosis are
classified arbitrarily as "acute" (less than eight weeks), "sub
acute" (from eight to 52 weeks), and chronic (more than 52
weeks). The disease is acute in about half of the cases, with
an incubation period of two to three weeks. In the other half,
the onset is insidious, with signs and symptoms developing
over a period of weeks to months from the infection [1, 3].
Clinical spectrum of brucellosis ranges from very mild,
febrile illness to severe multisystem involvement. Fever is
the most common clinical feature; in fifty percent of indi-
viduals, sudden onset of fever occurs during evening. Pa-
tients untreated for a long time present undulant fever pat-
terns with mounting temperatures and chills.
*Address correspondence to this author at the Department of Infectious
Disease and Microbiology, Erciyes University, Faculty of Medicine, 38039
Kayseri/Turkey; Tel: (+90) 352 3474937–22057; Fax: (+90) 352 437 52 73;
E-mail: draysegululu@yahoo.co.uk
Any organ or tissue can be involved causing morbidity,
but mortality is rare and usually results from infection of the
heart or brain [1].
Acute brucellosis is especially marked by an intermittent
or remittent fever. Malaise, headache, weight loss, arthralgia,
myalgia, constipation, anorexia and backache are other
symptoms of acute brucellosis. Acute presentation is more
commonly seen with Brucella melitensis than with other
species. Brucella spp. is more frequently isolated during this
period and diagnosed by serological methods.
Subacute brucellosis is typical and classical form with
undulant fever described in endemic areas. Symptoms are
mild with fatigue, headache and myalgia. Furthermore, local-
ized infections such as epididymitis, orchitis and osteoarticu-
lar complications are more commonly seen. Patients with
incomplete treatment are also included in subacute form.
These patients are generally investigated as a case of fever of
unknown origin. Brucella spp. is isolated in 40-70% of serial
blood cultures.
Chronic brucellosis is usually due to the persistence of a
deep-seated focus of infection in a bone, joint, kidney, liver
or spleen. The common symptoms observed in chronic bru-
cellosis are weakness, fatigue, emotional lability, depression,
headache and insomnia. Chronic brucellosis has similar fea-
tures to the chronic fatigue syndrome, seen especially in eld-
erly. The rate of isolation of Brucella spp. from patients with
chronic form of the disease remains low; blood cultures of-
ten remain 10%-20% positive.

2212-4071/13 $100.00+.00 © 2013 Bentham Science Publishers

Brucellosis; Clinic and Diagnosis
Subclinical or asymptomatic infection has been docu-
mented more frequently in farmers, abattoir workers, and
veterinarians. It is necessary to isolate the causative agent or
demonstrate some type of specific serological response in
order to mention these terms.
A relapse is considered to be either a positive blood cul-
ture after completing the treatment or reappearance of com-
patible symptoms, not otherwise explained together with an
increase in the previous serological titers. Bacteriologic re-
lapse generally occurs within 3 to 6 months after discontinu-
ing therapy and usually not caused by antibiotic resistance
[2].
The case definition and diagnosis defined by World
Health Organization (WHO) depend on the combination of
presence of clinical and laboratory features [3]. The disease
can be diagnosed easily in brucella-endemic regions, how-
ever, in developed countries (brucellosis controlled areas)
diagnosis may be difficult, and patients are generally inves-
tigated as a case of fever of unknown origin. Exposure his-
tory to a known or probable source of Brucella spp., occupa-
tional history or visiting endemic countries become more
important in diagnosis of brucellosis.
MULTIORGAN SYSTEM INVOLVEMENT
1. Osteoarticular System
Osteoarticular involvement is the most common focal
complication of brucellosis. The incidence of osteoarticular
involvement is 10-85% in most series [1, 2, 4-9]. Sacroiliac
joints are the most common site involved in younger pa-
tients, whereas spondylitis and peripheral arthritis usually
occur in older patients [1, 7-10]. In a previous study, the in-
cidence of osteoarticular involvement was 19% and most
common complications were sacroiliitis (54%), peripheral
arthritis (33%) and spondylitis (10%) [6]. Arthritis and sac-
roiliitis are usually presentations of an acute form and fre-
quently respond to standard therapeutic regimens. On the
other hand, spinal column is generally affected in subacute
and chronic form of brucellosis, and treatment is more diffi-
cult [11]. The incidence of spinal brucellosis is highly vari-
able (2-54%), depending on the study population, the species
of Brucella involved and the methods used for diagnosis [6-
13]. Spinal column can be affected at any level, whereas
lumbar spine is the most common site involved, particularly
L4-L5 and L5-S1 [1, 6, 9, 12-14]. Moreover, multiple site
involvement can be observed with 9-30% incidence [1, 12,
15, 16]. Paravertebral and/or epidural abscess formation has
been rarely reported in the past [11], but in recent years, with
highly sensitive diagnostic techniques (computed tomogra-
phy or magnetic resonance imaging-MRI), it has become
more evident (21-42%) [9, 17]. In Brucellar arthritis, any
joint can be involved however, large weight-bearing joints
(hip and knees) are most commonly affected peripheral
joints. Prosthetic joint can be involved during Brucella bac-
teremia and excisional arthroplasty may be necessary for
cure. Tenosynovitis and bursitis have also been reported in
the literature [2].
Complaints of patients in osteoarticular brucellosis may
be insidious and nonspecific. The most common are arthral-
gia, joint swelling, redness around the skin of a joint, fever,
Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1 35
sweating, malaise, back pain and anorexia. Paraparesis,
paresthesia or reflex changes can occur due to cord and neu-
ral compression in spinal brucellosis. Furthermore, neuro-
brucellosis may develop as a complication. On physical ex-
amination, fever, reduced joint mobility, hepatomegaly,
splenomegaly, and positive Laseque test are the findings [1,
2, 6, 8, 18].
Patients are usually diagnosed by increased serum anti-
body levels and radiological signs. Early radiological signs
of osteoarticular brucellosis which are nonspecific may ap-
pear after the onset of symptoms [19]. The appearance of
destructive changes on direct radiographs will be about in
three months, and computed tomography may not be always
helpful in the differential diagnosis from other degenerative
diseases and disc herniation [12, 18]. MRI is the most useful
method for diagnosis, assessment and management of os-
teoarticular brucellosis. Furthermore, it has high sensitivity
for differential diagnosis and identifies the extent of brucel-
lar spondylitis [19]. The characteristic Pons sign (a step like
erosion of the anterosuperior vertebral margin) can be seen
in imaging techniques. The earliest features are characterized
by osteoporosis of affected vertebral body and erosion of the
anterior-superior endplate. This early form of brucellosis is
followed by narrowing of the joint spaces. Posterior ele-
ments are rarely involved. Epidural involvement is also fre-
quent in spinal brucellosis. Synovial fluid contains a prepon-
derance of lymphocytes. For the differential diagnosis, tu-
berculosis, salmonellosis, other pyogenic infections, disc
herniation, metastatic lesions should be kept in mind [1, 19].
2. Nervous System
Neurobrucellosis is a rare but serious complication of
brucellosis. Nervous system involvement has been detected
in less than 5% of the cases with brucellosis [2]. However,
the incidence of neurobrucellosis can be influenced by dif-
ferent sample size of study populations and, epidemiological
features of the centers and country. A high rate of 17.8% was
reported from a research hospital located at the capital city of
Turkey, while it was 3.9% in a retrospective analysis of 917
patients with brucellosis from another hospital in Central
Turkey [20, 21]. The clinical presentation of neurobrucello-
sis is not specific. Nuchal rigidity occurs in less than 50% of
cases [11, 21]. In a pooled analysis of 187 cases with neuro-
brusellosis; headache, fever, sweating, weight loss, and back
pain were the predominant symptoms [22]. Several psychiat-
ric symptoms and particularly depression were reported in
patients with neurobrucellosis which improved by antibacte-
rial therapy without any anti-depressive or antipsychotic
therapy [23]. A single center study including 18 patients re-
ported unusual initial clinical manifestations such as pseudo-
tumor cerebri, white matter lesions and demyelinating syn-
drome, intracranial granuloma, transverse myelitis, sagittal
sinus thrombosis, spinal arachnoiditis, left mild sequelae
including aphasia, hearing loss, and hemiparesis [24].
The diagnosis of neurobrucellois is based on symptoms
or clinical findings of neurologic syndrome not explained by
any other neurological disease, isolation of Brucella spp.
from the cerebrospinal fluid (CSF) or/and demonstration of
antibodies to Brucella in CSF (at any titer), the presence of
any abnormality in CSF such as pleocytosis, increased pro-
36 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1
tein levels and decreased glucose levels with positive serol-
ogy for brucellosis [22, 25]. Isolation of Brucella spp. from
the CSF culture is the golden standard for the diagnosis.
However, CSF cultures were positive in 15-30% of the pa-
tients in different case series [21, 22]. CSF tube agglutina-
tion was positive in 133 out of 187 (71%) patients with neu-
robrucellosis while Rose Bengal test was positive in only
21% [22]. In a small study which compared the performance
of agglutination tests with PCR, all six patients with neuro-
brucellosis (three meningitis and three meningoencephalitis)
had a positive real time PCR assay, whereas CSF cultures
and Wright seroagglutination tests were positive in only two
and four cases, respectively [26]. ELISA has been also rec-
ommended for the diagnosing neurobrucellosis [27]. How-
ever, the ELISA is not as widely available throughout the
world as agglutination tests; it can be a useful adjunct when
agglutination results are equivocal or negative [28].
Brucellosis can cause meningitis, meningoencephalitis,
neuritis, brain abscess, transient ischemic attacks, vascular
occlusive disease, and patients can admit with several non-
specific symptoms such as sensorineural hearing loss, and
vertigo. Acute or chronic meningitis is the most frequent
nervous system complication [22]. Although rare, hydro-
cephalus and intracranial hypertension are concerning com-
plication of neurobrucellosis [29-31]. Cranial nerve in-
volvement has been described during the treatment of neuro-
brucellosis, and also, it has been reported as the initial symp-
tom [32, 33]. Vestibulocochlear nerve and Nervous abducens
are the most commonly involved cranial nerves [11, 22, 24].
The role of neuroimaging in patients with uncomplicated
neurobrucellosis is limited. The imaging findings are not
specific for neurobrucellosis and can mimic other infectious
or inflammatory disorders. Granulamatous lesions, abnormal
enhancement of the meninges, white matter changes, and
vascular changes were reported in neurobrucellosis. Neuroi-
maging is indicated in the case of unclear clinical diagnosis,
neurologic deterioration, abnormal neurological findings at
physical examination and slow clinical improvement [25].
Contrast Magnetic resonance imaging (MRI) is superior to
computerized tomography. The complications of neurobru-
cellosis such as abscess, ischemia, subdural hematoma, suba-
rachnoid hemorrhage, and hydrocephalus can be detected by
MRI. White matter involvement of neurobrucellosis can
mimic other infectious diseases and inflammatory disorders
such as Lyme diseases, acute disseminated encephalomyeli-
tis, or multiple sclerosis [25, 34, 35]. The meningeal en-
hancement usually disappears, but the white matter and
ischemic changes can persist despite almost complete clini-
cal recovery [36, 37].
Relapse is a rare condition after successful treatment of
neurobrucellosis. Relapsing neurobrucellosis was reported in
a patient after implantation of a ventriculoperitoneal shunt
for hydrocephalus [21]. The rate of relapse was 1.8% in 215
patients with neurobrucellosis at a multi-center study includ-
ing 28 hospitals from four different countries [38]. Mortality
of neurobrucellosis was reported as low as 0.5% in the
pooled analysis of 187 patients [22]. In the case series of
neurobrucellosis released from another center, the mortality
rate was 8.3%. Three out of 36 patients died due to late term
problems other than brucellosis [21]. However, morbidity
due to complications and sequelae is of concern.
Ulu Kilic et al.
Tuberculosis meningitis should be considered in the dif-
ferential diagnosis of neurobrucellosis, because of similar
endemicity. All attempts such as Ehrlich-Ziehl-Neelsen
(EZN) stain, mycobacterium culture in specific medium, and
PCR should be performed to rule out tuberculosis in case of
limited laboratory evidence of neurobrucellosis [39].
3. Cardiovascular System
Cardiovascular complications of brucellosis (endocardi-
tis, myocarditis and pericarditis) are rare (1% of cases) [2].
Endocarditis is the most common cardiovascular in-
volvement of the disease and also cause of brucellosis-
related deaths [2, 40, 41]. The rates of endocarditis after
Brucella infection vary (0.4-11%) [42]. Clinical symptoms
observed commonly are fever, heart murmur, liver and
spleen enlargement. B. melitensis and B. abortus are most
frequently isolated species. Infection may occur in both natu-
ral and prosthetic valves. Disease usually affects the left side
of the heart and predominantly involved valve is aortic one.
In patients with mitral involvement, several rhemautical dis-
orders were described as a coexisting problem. Brucellar
endocarditis leads to degeneration of the native valve and
causes heart failure within 3 to 11 months after the onset of
symptoms [40-42]. Cardiac abscesses and aneurysm were
reported in autopsy findings. When compared with other
bacterial pathogens Brucellar endocarditis is characterized
with a greater tendency toward fibrosis, hyalinization, and
calcification involving the cardiac valves. Therefore, due to
its rapid and wide tissue destruction, higher mortality rates
were observed during the course of this disease [43, 44].
Surgery is generally indicated in the treatment of brucellar
endocarditis because of high rate of medical treatment fail-
ure. Early diagnosis of endocarditis with new blood culture
techniques and echocardiography has decreased need for
surgical intervention vice versa high mortality rate might be
due to late diagnosis of the infection. Congestive heart fail-
ure is the most frequent complication. Valve replacement is
indicated when signs of heart failure appear, despite appro-
priate antibiotic treatment. Septic and embolic complications
are rare [43-47]. Myocarditis and pericarditis are usually
accompanied by endocarditis. These complications are also
rare and life threatening. Aortic and mycotic aneurysms of
other vessels and deep venous thrombosis can also be seen
during the course of disease [2].
4. Gastrointestinal System
In humans, the most common route of entry of brucella
bacteria is through gastrointestinal tract. After entry into the
body, bacteria localize in the tissues of the reticuloendothe-
lial system. The liver is frequently affected as being the larg-
est organ of the reticuloendothelial system [41, 48]. Hepatic
enlargement is the most frequent gastrointestinal manifesta-
tion of brucellosis, has been reported 32- 63% of cases.
Hepatitis, granuloma and/or abscess formation due to brucel-
losis are rare conditions. Hepatobiliary involvement of bru-
cellosis ranges from mild elevation of transaminases, alka-
line phosphatase, total serum bilurubine and jaundice to cir-
rhosis. Isolation of B. canis has been reported especially
from hepatic abscess and suppurative lesions [1, 2, 41, 48].
Acute and chronic cholecystitis due to brucellosis is ex-
tremely rare [49].
Brucellosis; Clinic and Diagnosis
The incidence of splenomegaly is reported between 29
and 56.6% of the cases. The disease can cause granulomas
and/or abscess formation in the spleen. Surgical intervention
(splenectomy or surgical drainage) is required, especially for
patients with therapeutic failure, despite appropriate antimi-
crobial treatment. Spontaneous splenic rupture due to brucel-
losis was also reported [50]. Anorexia, vomiting, diarrhea,
constipation are other nonspecific gastrointestinal manifesta-
tions of brucellosis. On the other hand, mesenteric lympha-
denitis, pancreatitis, peritonitis and intestinal obstruction are
rare but serious complications [1, 2, 6, 51].
5. Genitourinary System
Epididymoorchitis, prostatitis and seminal vesiculitis are
genitourinary infections related with brucellosis. Brucellar
epididymoorchitis (BEO) is the most clinical condition with
serious complications such as testicular abscess, atrophy and
infertility. Incidence of disease is reported from different
countries 1.6% to 20%. BEO is dominant in younger patients
with scrotal swelling and pain. Bilateral epididymoorchitis is
observed in 59% of patients. Disease generally emerges with
acute onset but especially in endemic areas may present
without systemic symptoms [52-55]. Laboratory findings are
generally mild and nonspecific. Incidence of urinary symp-
toms varies in different studies from 22% to 69%. In most of
the case, series of urine analyses were normal and culture did
not yield Brucella spp. [52-56]. However, bacteria can be
recovered from urine with appropriate techniques [57]. Ne-
phritis or glomerulonephritis is usually observed as a com-
plication of endocarditis [1, 2, 58]. Ultrasonography is rec-
ommended for the diagnosis of BEO.
Epididymoorchitis due to brucellosis should be distin-
guished from mumps and surgical problems, such as torsion
and tumors. Orchiectomy is performed to the patients with
therapeutic failure, despite appropriate antimicrobial treat-
ment. Surgical intervention is required in 0-5% of cases es-
pecially with testiscular abscess. The rate of therapeutic fail-
ure and relapse was reported between %0 and 40% [52].
Patients with prostatitis and seminal vesiculitis, usually
present long standing systemic features such as dysuria,
chills, fever, perianal and pelvic discomfort. Brucella spp.
can be recovered from culture of prostatic secretion [59, 60].
Granuloma and abscess formation are determined in renal
biopsy of these patients. Hematuria, pyuria and proteinuria
are observed due to involvement of kidneys. However, the
disease generally does not compromise renal functions. In-
terstitial nephritis and acute tubular necrosis are attributed to
immunological mechanisms described in patients with bru-
cellosis [41].
6. Respiratory System
Involvement of the respiratory system in brucellosis is
rare (< 5%), however, a variety of pulmonary manifestations
were reported, including acute bronchitis, bronchopneu-
monia, pleural effusion, empyema and lung abscess. Most
frequently reported symptoms are dry or productive cough,
dyspne, chest pain and flu like symptoms with an incidence
of 15-30% [1, 2, 41]. Respiratory transmission of organism
remains unclear. Pathophysiology is thought to be inhalation
Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1 37
of contaminated aerosols especially by laboratory staff and
most probably distribution through bloodstream infection
[41].
Brucella spp. can be rarely observed in gram staining or
isolated from sputum. Isolation of B. melitensis from pleural
fluid has also been reported. Characteristics of pleural effu-
sion due to pulmonary brucellosis are lymphocytic pleocyto-
sis, high protein concentration, increased adenine deaminase
levels [41, 61-63]. Positive blood culture was reported in 15-
80% of patients with pleural involvement. Radiological ex-
amination generally does not reveal any specific finding.
Pneumonic patches, consolidation, hilar lymphadenopathy,
pleural effusion, pneumothorax with radiography and reticu-
lonodular infiltration, diffuse glass round opacity, abscess
with computerized tomography imaging of lung have been
documented [61-65].
Tuberculosis and sarcoidosis should be considered in the
differential diagnosis of these patients [63, 64].
7. Hematologic System
Majority of patients with brucellosis have normal hema-
tological conditions. Abnormalities are usually mild and as
coincidental laboratory findings [1, 4, 6]. The incidence of
anemia has been reported as 44 to 74% in adult series in bru-
cellosis. Etiologies of anemia vary; such as infection, hyper-
splenism, autoimmune haemolysis and haemophagocytosis
[41]. Clinical picture of anemia due to chronic infection is
usually mild and pallor may be noted. The peripheral blood
morphology is hypochromia, anisocytosis and poikilocytosis.
The serum iron and iron binding capacity are usually low.
Hypersplenism can cause pancytopenia, and rarely result
with gastrointestinal bleeding. Brucellosis can induce an
autoimmune process and cause hemolysis by various mecha-
nisms, can be treated with steroids or require splenectomy.
Rituximab is suggested to be an alternative, in cases refrac-
tory to these therapies [66].
Leucocyte count is usually normal but sometimes leuco-
penia or even leukocytosis may occur. Incidence of leuco-
penia varies from 7.7% to 68% of cases [6, 67], however,
abnormalities usually revert to normal after treatment.
Incidence of thrombocytopenia varies from 5% to 13.7%
in case series [6, 67]. Severe thrombocytopenia provoking
purpuric skin rash and bleeding may occur rarely [68].
Incidence of pancytopenia varies from 3% to 21% and
usually associated with several mechanisms such as hyper-
splenism, haemophagocytosis and suppression of bone mar-
row. Cutaneous eruptions are rarely reported and should be
distinguished from hematological malignancies [6, 41, 67].
Apart from bone marrow involvement, brucellosis was
reported in patients with hematological malignancy particu-
larly from Turkey and Saudi Arabia [69-76]. The clinical
picture can be easily confused with the findings of primary
hematological disease. However, brucellosis has to be part of
the differential diagnosis of fever of unknown origin in en-
demic areas even in hematological malignancy patients.
8. Cutaneous Complications
Cutaneous complications of brucellosis are usually re-
ported in patients with occupational exposure. Brucella bac-
38 Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1
teria enter the body through an abrasion or cut in the skin. In
addition, etiology varies accordingly with hypersensitivity
phenomena, deposition of immune complex and rarely he-
matogenous spread [41]. Cutaneous manifestations of bru-
cellosis are nonspecific and rarely occurring (< 5%). Macu-
lopapular rashes, purpura and petechiaes, erythema nodo-
sum, ulcers and abscess are reported clinical features of cu-
taneous brucellosis. Abscess and ulcerative manifestations
are reported more common with B. suis [77]. Lesions occur
more frequently in acute cases, however, can appear in every
stage of disease. Brucella spp. can be isolated from culture
specimens of skin lesions. Histological findings are generally
granulomatosis formation and infiltration of nonspecific in-
flammatory cells [41, 77, 78]. A case of leukocytoclastic
vasculitis resolving after initiating therapy was also reported
[79].
9. Ocular Complications
Ocular involvement in brucellosis reported with inci-
dence of 4- 26%. Uveitis, conjunctivitis, choroiditis, kerati-
tis, papilloedema, retinal hematogenous appear either in
acute or chronic stage of disease. Direct invasion, septic
uveal emboli from endocarditis and formation of immun-
complexes are associated mechanisms. Uveitis (anterior,
posterior or panuveitis) is the most frequent form of ocular
brucellosis. Ocular damage substantially occurs in chronic
stages and consequently results in loss of vision [1, 6, 41,
80].
LABORATORY DIAGNOSIS OF BRUCELLOSIS
CURRENT & FUTURE DEVELOPMENTS
Medical history, physical examination, appropriate labo-
ratory tests and their interpretation are essential for diagnosis
of brucellosis. Cultures of blood and other tissues (bone mar-
row vs.) are the gold standards for diagnosis; however, it is
not always possible to isolate bacteria [81]. The isolation of
the organism is often unsuccessful, especially in chronic
cases or takes a long time. Thus, serological tests are used
more widely (The microbiological features of Brucella spp.
and culture methods are described in detail at the current
issue by D. Percin).
A variety of serological tests including Rose Bengal test,
the serum agglutination test (SAT), antiglobulin or Coombs’
test, microagglutination test (MAT), Brucellacapt, enzyme-
linked immunosorbent assay (ELISA) and Indirect fluores-
cent antibody test (IFA) are developed for diagnosis of bru-
cellosis.
Rose Bengale Ag is suspension of B. abortus colored
with Rose Bengale stain. Rose Bengale test (RBT) is a rapid
slide agglutination test which depends on the reaction of
serum and suspension of B. abortus. RBT is widely used as a
screening test with a high sensitivity. However, false nega-
tive results have been reported with RBT, especially in
chronic cases [81, 82]. WHO also recommends confirming
RBT positive results by the SAT, due to reduced specificity
in endemic regions [3].
SAT detects antibodies against smooth lipopolysaccha-
ride (s-LPS), the major antigen of the bacteria. These anti-
bodies persist after recovery, affecting diagnostic value of
Ulu Kilic et al.
the test. Furthermore, LPS O-specific side chains between
smooth species of other bacteria (Yersinia enterocolitica 0:9,
Vibrio cholerae, Escherichia coli O:157 and Francisella
tularensis) cause cross reactions. SAT measures the total
quantity of agglutinating antibodies (IgG, IgM and IgA).
IgM type antibodies develop stronger agglutination. There-
fore, STA test is more sensitive in the diagnosis of acute
brucellosis than chronic cases [81, 82]. Brucella abortus
antigen prepared with best agglutinated smooth colonies of
S99 strain. A new method is issued patent for production of
Brucella serum including B. abortus and B. melitensis [83].
Agglutination titers of 1:160 or greater in one or more serum
specimens obtained after the onset of clinical symptoms,
have a diagnostic value. However, titers of 1:320 and greater
are suggested more in endemic regions. Although negative
agglutination titers can be observed during course of the dis-
ease, it does not exclude brucella infection. SAT is negative
during early phase of the disease and in the presence of
blocking antibodies or "prozone" phenomenon (agglutination
can be masked in low dilutions especially with the high titers
of serum antibody). The anti-human globulin test (Coombs)
is essential to eliminate the effect of blocking or incomplete
antibodies. This test is especially good for complicated and
chronic cases [81, 82]. Brucellacapt is an immuncapture ag-
glutination method and presents an alternative to the Coombs
test with similar sensitivity and specificity. The test rapidly
detects both agglutinating and non-agglutinating IgG and
IgA antibodies in a single step and also a handy indicator for
activity of disease in follow up [84].
Antigens other than B. abortus suspension are also com-
mercially available. A recent patent is desired to comprise
immunodominant antigens from selected human pathogens
including B. melitensis and can be used as a diagnostic
marker. In particularly preferred aspects, the antigens have
quantified and known relative reactivities with respect to
sera of the infected population, and have a known associa-
tion with a disease parameter [85]. A recent innovation also
deals with obtaining diagnostic preparations and can be ap-
plied for obtaining a diagnostic kit to detect antibodies to
brucellosis antigen in tube agglutination reaction and in
lamellar agglutination upon a slide. This kit desired to obtain
higher specificity, wider sphere of application, more pro-
longed validity terms and higher convenience in usage [86].
Another patent describes a detection method which uses
Rhizopine-binding protein precursor homolog protein of the
genus Brucella bacteria; provides accurate and rapid diagno-
sis [87]. Furthermore, a strip test for rapid detection of a
Brucella specific antibody is developed recently. It is simple
to operate, convenient, rapid, and has the advantages of re-
quiring no special instruments and training. The test strip is
suitable for base course, large scale site detection of an acci-
dent and epidemiological investigation, and has auxiliary
effect on the diagnosis of Brucella infection. The test com-
prises a reaction film and a conjugate release pad. The reac-
tion film has a detection band simultaneously coated with
Brucella specific antigen bp26, and a quality control band of
polyclonal antibody coated with Brucella outer-membrane
protein bp26. The conjugate release pad is coated with col-
loidal gold labelled Brucella outer-membrane protein bp26.
A membrane chromatography double antigen sandwich
method is adopted to detect the Brucella specific antibody in
a specimen [88].
Brucellosis; Clinic and Diagnosis
ELISA is also a well standardized, rapid (4-6h) and sen-
sitive method based on detecting antibodies against s-LPS.
The test measures the total and individual quantity of agglu-
tinating antibodies (IgG, IgM and IgA). The test also detects
IgG subclasses and IgE. ELISA is particularly recommended
when other tests are negative. Adjustment of cut-off value is
required for specificity in endemic regions and also useful
for mass screening. A recent patent is issued for brucellosis
indirect ELISA antibody detection kit with moderate cost
and short reaction time. This ELISA kit is assembled by us-
ing LPS mixture of smooth and rough type. This method also
combines the requirements of actual detection and general
survey operations and suitable for both large-scale screening
tests [89]. Two recent patents were issued to new-generation
brucellosis antibody competition enzyme- linked immu-
nosorbent adsorption test kits in which s-LPS is used as an
antigen-coated enzyme-labelled plate. One of them is a com-
petition ELISA kit which is sensitive and specific, and can
accurately diagnose brucellosis [90, 91].
A test paper strip recently was issued patent which de-
tects brucellosis antibody suitable for base course, site detec-
tion and has auxiliary effect on the diagnosis of Brucella
infection [92].
IFA is also rapid (2-3h) and shows similar efficacy as
ELISA detecting IgG, IgM and IgA using fluorescent la-
belled antihuman antibodies. Positive reactions were de-
tected with the aid of a fluorescence microscope; however
interpretation is subjective and requires skilled observation
[81, 82].
Molecular Assays
Polymerase chain reaction (PCR) was developed initially
in 1990, allowing the amplification of a 635 bp fragment of a
43 kDa outer membrane protein gene from B. abortus strain
19. Many advances in molecular technology have been de-
scribed for diagnosis of brucellosis. PCR technique is a
highly sensitive and specific for the determination of Bru-
cella spp., from peripheral blood and other tissues. PCR de-
tects Brucella DNA and is utilized in the diagnosis and fol-
low-up of patients with brucellosis. However, false negative
and positive results were reported and the lack of standardi-
zation and interpretation of test results have limited its ac-
ceptance as a diagnostic tool. Further studies are expected to
simplify the procedure for standard laboratories for the de-
tection of Brucella at the species level and quantification of
bacteria [81, 82].
There are several patents recently disclosed that concern-
ing molecular techniques. A recent patent was issued to a
method, which is convenient in operation, efficient, specific
and rapid; quantitative detection within two hours. This
method could be significant for early diagnosis of brucella
disease [93].
Also a method for detecting Brucella abortus using real
time PCR is provided to reduce the detection time and rap-
idly and accurately determine the onset of brucellosis [94].
Another recent patent was issued to PCR based methods
with a set of primers, probes and a kit for molecular diagno-
sis that can be used to differentiate between Brucella spp.
and Mycobacterium tuberculosis complex [95].
Recent Patents on Anti-Infective Drug Discovery, 2013, Vol. 8, No. 1 39
Recently, two other diagnostic methods received patents
for the detection of Brucella [96, 97].
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENTS
Declared none.
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