Brucelosis Revision

7/26/2019 Brucelosis Revision

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Review

Human brucellosis

Mara Pa Franco, Maximilian Mulder, Robert H Gilman, Henk L Smits

Human brucellosis still presents scientists and clinicians with several challenges, such as the understanding ofpathogenic mechanisms of Brucellaspp, the identification of markers for disease severity, progression, and treatmentresponse, and the development of improved treatment regimens. Molecular studies have shed new light on thepathogenesis of Brucellaspp, andnewtechnologies have permitted the development of diagnostic tools that will beuseful in developing countries, where brucellosis is still a very common but often neglected disease. However, furtherstudies are needed to establish optimum treatment regimens and local and international control programmes. ThisReview summarises current knowledge of the pathogenic mechanisms, new diagnostic advances, therapeutic options,and the situation of developing countries in regard to human brucellosis.

IntroductionA renewed scientific interest in human brucellosis has

been fuelled by its recent re-emergence and enhancedsurveillance in many areas of the world,1 and from itsstatus as a class B bioterrorist agent.2The disease remainsthe worlds most common bacterial zoonosis, with overhalf a million new cases annually and prevalence rates insome countries exceeding ten cases per 100 000population.1Despite being endemic in many developingcountries,1,3 brucellosis remains underdiagnosed andunder-reported.4 Furthermore, since brucellosis is animportant cause of veterinary morbidity and mortality,the disease can also cause important economic losses indeveloping countries.5

Although brucellosis in human beings is rarely fatal, it

can be severely debilitating and disabling. Brucellosis is afebrile disease capable of masquerading as a myriad ofentities, both infectious and non-infectious. The diseasehas a tendency towards chronicity and persistence,becoming a granulomatous disease capable of affectingany organ system.6,7The timely and accurate diagnosis ofhuman brucellosis continues to challenge cliniciansbecause of its non-specific clinical features, slow growthrate in blood cultures, and the complexity of itsserodiagnosis.810

The clinical management of brucellosis is of particularconcern because of high initial treatment failure andrelapse rates. The availability of the complete genomesequences of the three main human brucellosis

pathogens, Brucella melitensis, Brucella suis, andBrucellaabortus, and the advancement of genomics andproteomics will enable scientists to better understand thediseases pathogenic mechanisms. Developments inculture and serological methods, and the availability ofadvanced molecular detection and typing methods havecontributed to improved laboratory diagnosis. Thesemolecular methods could serve as important alternativesto culture methods for the confirmation of the diseaseand may also provide valuable epidemiological tools totrace sources of infection. Some of the newer diagnosticmethods are simple, robust, and affordable, and mayprove to be essential in endemic areas with limitedfinancial resources and a limited number of laboratoryworkers.

Pathogenicity and biology of brucellosisBrucellaspecies are facultative intracellular bacteria that

can multiply within phagocytic cells with human beingsas end hosts. Brucella may enter the host via ingestion orinhalation, or through conjunctiva or skin abrasions.After infecting the host, the pathogen becomessequestered within cells of the reticuloendothelial sys-tem. The mechanisms by which brucella enters cells andevades intracellular killing and the host immune systemare the subject of much research and debate. In depthanalysis of the complete Brucella sppgenomes has failedto identify any of the classic virulence factors such astoxins, fimbriae, and capsules, which raises the possibilitythat these organisms use unique and subtle mechanismsto evade host defences, penetrate host cells, alter

intracellular traffi cking to avoid degradation and killingin lysosomes, and modulate the intracellular environmentto allow long-term intracellular survival and replication.11The smooth lipopolysaccharides that cover the bacteriumand proteins involved in signalling, gene regulation, andtransmembrane transportation are among the factorssuspected to be involved in the virulence of brucella.12

Research suggests that the smooth, non-endotoxiclipopolysaccharides help block the development of innateand specific immunity during the early stage of infection,and protect the pathogen from the microbicidal activitiesof the immune system. Rough (vaccine) strains (ie, strainswith lipopolysaccharide lacking the O-side chain) are lessvirulent because of their inability to overcome the host

defence system.13 Smooth lipopolysaccharide preventsthe synthesis of immune mediators and is much lesspotent than Escherichia colilipopolysaccharide in inducinghost release of inflammatory cytokines.14 Smoothlipopolysaccharide also has a role in cell entry andimmune evasion of the infected cell. Thelipopolysaccharide is thought to alter the capacity of theinfected cell to present foreign antigens to the MHCclass II antigen presentation system, hence preventingattack and killing of the infected cell by the immunesystem.12,15 Additionally, smooth lipopolysaccharide inbrucella may be involved in the inhibition of apoptosis(ie, programmed cell death) of infected cells,16 sinceresistance to apoptosis of infected cells has been observedin patients with acute and chronic disease.17By contrast,

Lancet Infect Dis2007; 7: 77586

Department of Neurology,

University of Minnesota,

Minneapolis, MN, USA

(M P Franco MD); Department

of Internal Medicine, Hennepin

County Medical Center,

Minneapolis, MN, USA

(M Mulder MD);Asociacin

Benfica Proyectos en

Informtica, Salud, Medicina y

Agricultura, Lima, Peru

(Prof R H Gilman MD);

Department of International

Health, Johns Hopkins

Bloomberg School of Public

Health, Baltimore, MD, USA

(R H Gilman); Wellcome Trust

Centre for Clinical Tropical

Medicine, Imperial College,

London, UK(R H Gilman); and

KIT Biomedical Research, Royal

Tropical Institute/Koninklijk

Instituut voor de Tropen (KIT),

Amsterdam, Netherlands

(H L Smits PhD)

Correspondence to:

Dr Henk L Smits, KIT Biomedical

Research, Royal Tropical

Institute/Koninklijk Instituut

voor de Tropen (KIT),Meibergdreef 39, 1105 AZ

Amsterdam, Netherlands.

Tel +31 20 5665470;

fax +31 20 6971841;

[email protected]


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rough strains do not confer host cells resistance to

apoptosis. Smooth brucella strains with mutations in thephosphoglucomutase gene involved in lipopolysaccharideO-chain synthesis show a profound attenuation ofvirulence.12,18

The two-component BvrR/BvrS gene sensing systemthat acts through a cascade of protein phosphorylation tomodulate bacterial gene expression is thought to be oneof the key factors involved in the modulation of cellbinding and penetration. The BvrR/BvrS system ofbrucella has a profound effect on the expression ofvarious cell-surface proteins including Omp25 (alsoknown as Omp3a) and Omp22 (Omp3b).19,20It is thoughtthat the altered expression of the surface proteins allowsbrucella to bind to and penetrate host cells while escaping

from the lysosomal pathway, since mutants that aredefective in this system show impaired cell penetrationand increased destruction by phagolysosomes.

In other pathogenic bacteria including Bordetellapertussis, Bartonella henselae, Helicobacter pylori, andLegionella pneumophila, the type IV secretion system(VirB)a pumping system that selectively transportsproteins or other macromolecules through membranesis essential for pathogenicity. The transported moleculein these pathogens is a classic virulence factor, such as atoxin, which is secreted into the bloodstream or injectedinto host cells.21,22 In brucella, VirB is thought to beessential for intracellular survival; however, thetransported effector substrate in brucellahas not yet beenidentified and it is very unlikely that the transportedmolecule is a classic virulence factor. The VirB pumpingsystem is built from a series of proteins encoded by theVirB operon. Many attenuated brucella strains showmutations within the VirB operon, indicating that an

intact VirB is essential for virulence.23VirB seems to have

a role in adherence of the bacterium to the host cell, cellentry, and it modulates the intracellular traffi cking andreplication of the bacterium.24,25 After binding tomacrophages, brucella is taken up by internalisationvesicles that would normally fuse with endosomes. Afteracidification, these endosomes lyse, destroying theircontents. Acidification is thought to induce VirBexpression.2527The VirB system is suspected to interactwith components of the endoplasmic reticulum,neutralising the pH and allowing the brucellae to undergoregulated cell division within the endoplasmic reticulumssafe environment.22

Heat shock protein 60 (Hsp60), a member of the GroElfamily of chaperonins, is expressed on the cell surface of

wild-type Brucella spp,but not on VirB mutants. Hsp60seems to play a part in cell adherence by binding to acellular prion molecule called PrPr. Since the exportationof Hsp60 is VirB-dependent, it has been postulated thatHsp60 could in fact be a virulence factor.28

Clinical diagnosisHuman brucellosis has a wide spectrum of clinicalmanifestations, earning it a place alongside syphilis andtuberculosis as one of the great imitators.2933 Theclinical features of brucellosis depend on the stage of thedisease, and the organs and systems involved. Brucellahas been reported to compromise the central andperipheral nervous system, and the gastrointestinal,hepatobiliary, genitourinary, musculoskeletal, cardio-vascular, and integumentary systems. Historically, onlyB melitensis, B abortus, and B suis were considered humanpathogens, but recent reports have shown that newlyrecognised marine mammal species have zoonotic

Total number

of patients

Fever or chil ls Arthralgia or

arthritis

Sweating Constitutional

symptoms*

Hepatomegaly Splenomegaly

Memish et al (2000)10 160 146 (913%) 105 (656%) 30 (188%) 70 (438%) 9 (56%) 11 (69%)

Kokoglu et al (2006)9 138 108 (783%) 107 (775%) 100 (725%) 98 (710%) 37 (268%) 50 (362%)

Mantur et al (2006)29 495 417 (842%) 117 (236%) 19 (38%) 6 (12%) 56 (113%) 95 (192%)

Ruiz-Mesa et al (2005)38 711 702 (987%) 353 (496%) 597 (840%) 533 (750%) 250 (352%) 148 (208%)

Barroso Garcia et al

(2002)30565 441 (781%) 248 (439%) 483 (855%) 472 (835%) 422 (747%) 152 (269%)

Hasanjani Roushan et al

(2004)7469 314 (670%) 252 (537%) 357 (761%) .. .. 27 (58%)

Pappas et al (2005)6 100 91 (91%) 44 (44%) .. 26 (26%) 7 (7%) 16 (16%)

Troy et al (2005)36 28 25 (89%) 15 (54%) .. 13 (46%) 8 (29%) 5 (18%)

Andriopoulos et al

(2007)33144 144 (100%) 125 (868%) 138 (958%) 140 (972%) .. 74 (514%)

Giannakopoulos et al

(2006)31

52 42 (81%) 43 (83%) 8 (15%) 7 (13%) .. ..

Mantur et al (2004)32 93 49 (53%) 19 (20%) .. .. .. ..

Tsolia et al (2002)39 39 27 (69%) 27 (69%) 8 (21%) 13 (33%) 11 (28%) 15 (38%)

Data shown as number of patients with symptom (%). ..=not reported. *Constitutional symptoms comprise anorexia, malaise, asthenia, weight loss, etc. Studies in

paediatric population only.

Table :Most common clinical findings in patients with brucellosis


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potential, and an apparent tendency for complicateddisease in human beings.34 Despite major ongoingcontroversies in the taxonomy of Brucella species,35 the

bulk of human disease is caused by two species:B melitensis andB abortus. Clinical differences betweenspecies are diffi cult to determine, since few studies havecompared the clinical manifestations of suffi cient casesof each species.36 The limited data available provideconflicting evidence as to which species results in morecases of chronic or complicated disease.

Because of the protean clinical manifestations ofbrucellosis, the cornerstone of clinical diagnosis lies intaking a detailed history and paying careful attention toepidemiological information. Special attention must beplaced on determining whether ingestion of contaminateddairy products or contact with infected animals hasoccurred. Detailed patient interviews are crucial for thediagnosis of human brucellosis, especially in urban andnon-endemic areas, and in cases of imported brucella, inwhich travellers acquire the disease abroad and becomeill in non-endemic settings. The diagnosis of a patientwith brucellosis should prompt the clinician to considerthe likely mode of transmission, and possibly to initiatescreening of contacts that may have had the sameexposure (eg, ingestion of contaminated dairy productsor exposure to animal source).37

Clinical studies have shown that fever is the mostcommon feature of brucellosis, followed by osteoarticularinvolvement, sweating, and constitutional symptoms(table 1). On physical examination, the most common

findings are hepatomegaly and splenomegaly, whichoccur in about one-third of patients. Lymphadenopathyis seen in about 10% of patients. Osteoarticularmanifestations (sacroiliitis, spondylitis, peripheralarthritis, and osteomyelitis) account for over half of thefocal complications. Genitourinary complications(orchiepididymitis, glomerulonephritis, and renalabscesses) can be found in around 10% of patients.Neurological findings are not as uncommon as they areoften portrayed; one study from Turkey reported that in aseries of 305 patients with brucellosis, 20 (66%) patientspresented with neurological involvement.40Neurologicalfindings can be diverse and could include peripheralneuropathies, chorea, meningoencephalitis, transientischaemic attacks, psychiatric manifestations, and cranial

nerve compromise. Mucocutaneous manifestationsinclude erythematous papular lesions, purpura, dermalcysts, and Stevens-Johnson syndrome. Pulmonary

manifestations, including pleural effusions andpneumonias, can be found in up to 16% of complicatedcases of brucellosis.41Leucocytosis is observed in about9% of patients and if found, focal complications shouldbe excluded (table 2). Leucopenia (11% of patients) andthrombocytopenia (10% of patients) are seen in similarfrequencies. Anaemia is seen more frequently, affecting26% of patients. Common disease findings, however,may vary between different areas and populations.Endocarditiswith the aortic valve being the mostcommonly affected structure and multiple valveinvolvement being common within this subset ofpatients42is the most serious complication, accountingfor most of the 5% total mortality rate of humanbrucellosis.

In brucellosis with focal complications, imagingstudies can provide useful anatomic and topographicconfirmation of suspected lesions to enable adequateplanning of medical and surgical treatment. Advances inthe imaging field have produced mixed results in thediagnosis of brucellosis: attempts at improving bonescintigraphy have failed to produce breakthroughs,43,44whereas improved computed tomography (such asdiffusion/perfusion studies), and magnetic resonanceimaging (such as fluid-attenuated inversion recovery[FLAIR] modalities) have enabled improved sensitivityin detecting both bone and soft-tissue lesions (figure 1).45,46

Digital subtraction angiography and transoesophagealechocardiography have also proved remarkably useful indetecting vascular and valvular insults in neurobrucellosisand brucellar endocarditis.42,45,47

Childhood brucellosis deserves special mention, sincethe condition is easily overlooked in infants.39The mostcommon osteoarticular finding in children ismonoarticular arthritis (usually of the knees and hips),whereas in adults, sacroiliitis is most frequent. Limiteddata support vertical transmission of humanbrucellosis48,49 and transmission via breastmilk.50,51Reports of transmission of human brucellosis via bloodtransfusion have also been published,52,53 and someauthors have even suggested that sexual transmissioncan occur.54Further study is needed to substantiate these

Total number

of patients

Anaemia Leucopenia Leucocytosis Thrombo-

cytopenia

Thrombo-

cytosis

E le va te d CRP E le va te d ESR P osit iv e R F

Kokoglu et al (2006)9 138 46 (333%) 30 (217%) 10 (72%) 27 (196%) .. 72 (522%) 58 (420%) 37 (268%)

Hasanjani Roushan et al (2004)7 469 71 (151%) 14 (30%) 57 (122%) 16 (34%) 5 (11%) 277 (591%) 365 (778%) 40 (85%)

Barroso Garcia et al (2002)30 565 171 (303%) 61 (108%) 34 (60%) .. .. .. 390 (690%) ..

Troy et al (2005)36 28 21 (75%) 23 (82%) .. 5 (18%) .. .. .. ..

Tsolia et al (2002)39* 39 18 (46%) .. 1 (3%) 3 (8%) .. .. .. ..

Data shown as number of patients with laboratory finding (%). ..=not reported. CRP=C-reactive protein. ESR=erythrocyte sedimentation rate. RF=rheumatoid factor. *Studies in paediatric population only.

Table : Most common laboratory findings in patients with brucellosis


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claims, although intrauterine transmission, transmissionduring delivery, and transmission through breastmilkare indeed among the main routes of transmission inthe mammalian reservoirs.

CultureBlood culture is the gold standard in the diagnosis ofbacterial infections, including brucellosis (table 3).

Although the biphasic Ruiz-Castaeda system (figure 2)is the traditional method for the isolation ofBrucellasppfrom clinical samples,6062it has now largely been replacedby automated culture systemssuch as the lysiscentrifugation method58,59with increased sensitivity andreduced culture times.5557,63

The sensitivity of blood culture depends on severalfactors, particularly the phase of the disease and previoususe of antibiotics.64,65 For instance, in acute cases, thesensitivities of the Ruiz-Castaeda method and lysiscentrifugation have been reported as high as 80% and90%, respectively, but as low as 30% and 70%, respectively,in chronic cases.59,64,66Bone marrow cultures may provide ahigher sensitivity, yield faster culture times, and may besuperior to blood cultures when evaluating patients with

previous antibiotic use.55,6467Brucellacan also be culturedfrom pus, tissue samples, and cerebrospinal, pleural, joint,or ascitic fluid.68,69Since brucellosis constitutes one of themost common laboratory-acquired infections, special careshould be taken when using the lysis centrifugationmethod to avoid infection from contaminated aerosols.7073

So far, there is no evidence to suggest that drugresistance has an important part in treatment failure andrelapse.74,75 An early study by Ariza and colleagues76showedthat brucella strains recovered from relapsed patients hadantibiotic sensitivity profiles identical to the originalstrains recovered during the initial infection. ResistantBrucella spp isolates have rarely been reported. Sincebrucelladoes not contain any plasmids and human beingsare end hosts, these factors may contribute to the absenceof any pronounced degree of antibiotic resistance.

SerodiagnosisAgglutination testsIn the absence of culture facilities, the diagnosis ofbrucellosis traditionally relies on serological testing witha variety of agglutination tests such as the Rose Bengaltest, the serum agglutination test, and the antiglobulin orCoombs test. In general, the Rose Bengal test is used asa screening test, and positive results are confirmed by theserum agglutination test.38,77These agglutination tests arebased on the reactivity of antibodies against smooth

lipopolysaccharide. These antibodies tend to persist inpatients long after recovery; therefore, in endemic areas,high background values could occur that may affect thediagnostic value of the test.78Furthermore, the brucellasmooth lipopolysaccharide antigen tends to show cross-reactivity with other Gram-negative bacteria such asYersinia enterocolitica 0:9, Vibrio cholerae, Escherichia coliO:157, and Francisella tularensis, increasing the possibilityof false-positive results.79 The sensitivity of the RoseBengal test is very high, however, and false-negativeresults are rarely observed.38,80The specificity of the assayis also fairly high, and in unexposed populations, false-positive results are rare. Differences in the quality of thereagent used and disagreement in the interpretation ofresults might add to variability of test results.81,82

BA

Figure : Magnetic resonance imaging (MRI) has become a powerful aid in

investigating bone and soft-tissue lesions in brucellosis patients

(A) T1 and (B) T2-weighted MRI images of a 63-year-old male with spinal

osteomyelitis, sacroiliitis, and psoas abscess caused by brucella infection.

Incubation time Requires blind

sub-cultures

Sensitivity

(disease stage)

References

Ruiz-Castaeda 721 days Yes 7080% (acute);

90% (acute);

70% (chronic)

48, 55, 56, 57

Bone marrowculture

47 days Depends onmethod and media

used

97% (acute);90% (subacute);

50% (chronic)

49, 54, 58, 59

Table :Characteristics and sensitivities of culture methods in clinical specimens

BA

Figure : Blood culture is the gold standard in the diagnosis of brucellosis

(A) The lysis centrifugation method has a better sensitivity and provides quicker

results than (B) the biphasic Ruiz-Castaeda method.


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As with other serological tests, the sensitivity and

specificity of the confirmatory agglutination tests forbrucellosis depend on the cut-off value used, and on thebackground level of reactive antibodies in the population.By doing the test on a serial dilution of the samples withresults judged positive above a certain titre, the specificityand positive predictive value of a positive test result couldbe increased by selecting a higher cut-off value for areaswhere brucellosis is endemic. However, by selecting ahigher cut-off value the sensitivity decreases and becausepatients with acute brucellosis and those with persistingand relapsing disease may present with low antibodylevels, the interpretation of test results and diagnosis ofthese patients by serological testing might not bestraightforward. In a recent study, Mantecon and co-

workers83 calculated a sensitivity for the serumagglutination test of 846% at any titre for patients withculture-confirmed acute brucellosis. However, in thesame study it was noted that the sensitivity at thegenerally accepted cut-off value of a serum dilution of1/160 was 647%, while at a cut-off value of 1/320 thesensitivity was just 471%. The latter cut-off value is oftenrecommended when the assay is used in endemic areas.If collection of a follow-up sample is feasible, thesensitivity of the test could be increased by testing pairedserum samples and looking for seroconversion, or a four-fold increase in titre. Demonstration of seroconversionor a significant increase in antibody titre provides strongsupportive evidence for the infection and this may beobserved by testing a follow-up sample collected a fewweeks to several months after the initial diagnosis.

To exclude the possibility of cross-reactive IgMantibodies, the 2-mercaptoethanol test for measuringspecific agglutinating IgG antibodies is sometimes used;results are compared with the serum agglutination testtitre and reactivity in the 2-mercaptoethanol test is takenas evidence for the presence of specific IgG antibodies.However, many patients have low levels of agglutinatingIgG antibodies and results can easily be misinterpreted.Coombs test may be more suitable for confirmation ofbrucellosis in relapsing patients or patients withpersisting disease, but few laboratories have the expertise

and equipment to do this very sensitive but complex anddemanding technique.

ELISAELISA has become increasingly popular as a well-standardised assay for brucellosis. The sensitivity ofELISAs prepared in the laboratory may be high, especiallywhen the detection of specific IgM antibodies iscomplemented with the detection of specific IgGantibodies.83The specificity of ELISA, however, seems tobe less than that of the agglutination tests. Since ELISAfor brucella is based on the detection of antibodies againstsmooth lipopolysaccharide, the cut-off value may needadjustment to optimise specificity when used in endemicareas, and this may influence sensitivity.78Curiously, test

performance of commercial ELISAs, as described in their

package inserts, is based on comparison with commercialELISAs of other brands, and not culture. Manufacturersfurther overlook the fact that cut-off values should beestablished based on local epidemiological conditions.Furthermore, the only study on this topic, by Araj andcolleagues,84used healthy individuals as negative controls,thereby possibly overestimating test specificity, and didnot investigate patients who presented with clinicalsuspicion of brucellosis but had a different final diagnosis,and who may have had cross-reactive antibodies.Commercial ELISAs also have not been evaluated underdifferent epidemiological conditions and results shouldbe interpreted with care. Studies testing the more recentlydeveloped Brucellacapt (Vircell SL, Granada, Spain) assay

in an endemic region in Spain showed that at a cut-offvalue of 1/80, a sensitivity of 98% for culture-confirmedpatients and a specificity of 96% for samples collectedfrom healthy individuals was obtained; however, aspecificity of 63% was found when testing patients whohad conditions other than brucellosis.85At a higher cut-off value, the specificity improved but the sensitivitydropped.

Serological testing with agglutination tests40 andELISA86 has been applied in the diagnosis of centralnervous system brucellosis with varying success, andfurther research is aimed to improve the diagnosis ofthis condition.

Rapid point-of-care assaysThe serum agglutination test, Coombs test, ELISA, andBrucellacapt all require a well-equipped laboratory, afacility often lacking in health centres of resource-poorcountries where the disease is endemic. Rapid tests suchas the fluorescent polarisation immunoassay (FPA) forbrucellosis and the immunochromatographic brucellaIgM/IgG lateral flow assay (LFA; figure 3), a simplifiedversion of ELISA, have great potential as point-of-caretests.87,88The FPA test is done by incubation of a serumsample with brucella O-polysaccharide antigen linked toa fluorescent probe.87 The sensitivity of this test at theselected cut-off value is 96% for culture-confirmed

brucellosis, and the specificity was determined to be98% for samples from healthy blood donors. The LFAuses a drop of blood obtained by fingerprick, does notrequire specific training, is easy to interpret, and can beused at the bedside. The components are stabilised anddo not require refrigeration for transportation orstorage.89The sensitivity and specificity of LFA are high(more than 95%), and the test can be used at all stages ofdisease.

Further studies will be needed to confirm theusefulness of these new point-of-care tests in differentclinical settings in endemic areas, with particularattention to the diagnosis of patients with acute andrelapsing brucellosis. The requirement of specificequipment and reagents might make the FPA test too


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expensive, but a simple test such as the LFA could be

ideal for field testing risk groups during outbreaks.

88

Another useful application for these tests is to screenthe contacts of brucellosispatients.9092

Treatment monitoringCurrently, tests to predict treatment outcome and relapseare not available. The 2-mercaptoethanol test issometimes used to monitor response to treatment. 93 Itwas observed that a low 2-mercaptoethanol titremeasured 12 months after treatment is consistent withcure, whereas a substantial proportion of patientscontinuing to show elevated 2-mercaptoethanol titresare symptomatic. The predictive value of the2-mercaptoethanol test is, however, debated. In view of

the risk of relapse and treatment failure in humanbrucellosis, more accurate markers for predicting theoutcome of treatment are needed. Serological changesare more easily observed in ELISA than in the classicagglutination tests, with relapsing patients showing arise in IgG antibody levels.78 The predictive value of

serum antibodies against smooth lipopolysaccharide as

measured in ELISA for persisting disease seems to below because 12 months after therapy, 25% of curedpatients have specific IgM antibodies and almost90% have specific IgG antibodies. Two-dimensional gelelectrophoresis has identified several immunogenicbrucella proteins. Some of these proteins that areexpressed during infection may well be of diagnosticimportance in evaluating response to treatment.94

Molecular detectionApplications in the diagnosis of brucellosisPCR is a convenient tool for the diagnosis of humanbrucellosis that may improve sensitivity compared withculture.95Several genus-specific PCR systems using primer

pairs that target 16S RNA sequences and the genes ofdifferent outer membrane proteins have been developed.96101Each of these PCR systems produces a discrete DNAproduct, whose length is identical for and specific to allBrucella species. Queipo-Ortuno and co-workers102 found100% sensitivity and 983% specificity using the B4/B5primer pair amplifying a 223-bp fragment of the bcsp31gene, compared with 70% sensitivity for blood culture.Adequate comparisons of the different PCR systems andprimers are still lacking, and results may presumablydepend on the nature of the clinical specimen, the samplepreparation procedure, and the duration and stage ofillness. For example, Zerva and colleagues103reported thatthe sensitivity of the B4/B5 primer pair improved from61% to 94% when serum instead of whole blood sampleswas used. However, using a modified detection system,Vrioni and co-workers104 found no improvement in thedetection rate by testing whole blood samples.

The incorporation of a robust DNA extraction method,such as the diatom-guanidinium isothiocyanate method,which effectively removes inhibitors commonly presentin a variety of clinical specimens, may improve sensitivityand reproducibility.105 Indeed, using a commerciallyavailable sample preparation kit, based on guanidiniumisothiocyanate for sample lysis and DNA extraction, anda solid matrix to bind and isolate the purified DNA, Mitkaand colleagues106 found an almost 100% sensitivity for

each of four PCR systems by testing either serum, buffycoat, or whole blood samples from 200 patients withacute brucellosis of whom 74% were culture-confirmed.

PCR could be particularly useful in patients withspecific complications such as neurobrucellosis, or otherlocalised infections, since serological testing often failsin such patients.107109 However, because these PCRsystems are complex, time consuming, and have a highrisk of contamination, they are less suitable for routinediagnostic purposes; real-time PCR systems have beendeveloped that are faster and less prone to contaminationand are thus more clinically useful.109115 Comparativeanalysis of the various real-time PCRs is needed to assesstheir diagnostic value. However, the high costs of theseassays will restrict their use.

1/1601/40

1/640

1/6401/320

1/20

280

IgM IgG IgM IgG

SAT2ME

Coombs

BA

Figure :The brucella IgM/IgG lateral flow assay, a point-of-care test for the confirmation of brucellosis

The brucella IgM/IgG lateral flow assay is an example of a simple and rapid test for the diagnosis of brucellosis that

can be done at the bedside using a drop of blood colle cted by fingerprick. The results for two serum samples, one

containing (A) specific IgG antibodies and (B) one containing specific IgM antibodies, are shown. Ratios indicate

the serum dilution at which the specified reference tests reacted for the two serum samples. SAT=serumagglutination test. 2ME=2-mercaptoethanol test.


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Applications in the assessment of brucellosis treatment

Relapsing brucellosis is another diagnostic challenge inwhich PCR could prove to be useful.116PCR was recentlyused to assess treatment effi cacy.117 PCR showed thepersistence of low levels of brucella DNA in the serum oftreated patients, with seven (35%) patients testing positive12 years after the end of treatment. Of theseseven patients, four relapsed and three did not. In anotherstudy, follow-up samples collected after treatmentcompletion showed that two out of 20 patients testedPCR positive, and these PCR results were confirmed byculture.118 These results strongly suggest that thebacterium may persist in the blood of treated patients fora long time, likely inside macrophages, which couldaccount for the high treatment failure and relapse

rates.106,119 Indeed, one study has shown that persistenceof brucella DNA correlates with ineffective treatment andrelapse.106Therefore, PCR may be used as an alternativeto culture in the confirmation of brucellosis, to monitortreatment effi cacy, and to diagnose relapsing patients.

Other applications of PCRPCR also appears to be useful in species differentiationand biotyping of isolates. Short nucleotide repeatsequences present at different loci in the brucellagenome show a wide variation in the number of repeatsbetween species and isolates.120,121 PCR amplification ofthese variable repeats is more robust than classic typingmethods for species and biovar identification, and ismore powerful than other molecular tools such as outermembrane protein typing,122,123 IS711 typing,124 or typingby amplified fragment length polymorphism.125 PCRamplification of a set of loci containing these repeats hasallowed the characterisation of individual and uniqueisolates even from within the same region.126,127 Thisapplication could be applied epidemiologically to traceinfections to specific flocks or dairy producers, and theemergence of cases with identical isolates could perhapsbe taken as evidence for the presence of a focus ofintensified transmission. Furthermore, the methodcould prove useful in discriminating reinfection andrelapse.127

TreatmentWHO has not updated its recommended treatmentregimens for brucellosis in more than 20 years,128despitetreatment failure and relapse rates ranging from 46% to24% for the oral regimen and 5% to 8% for the oral/parenteral regimen.129,130The cause for such high failurerates remains unclear. Since antibiotic resistance can bediscounted as a major factor, it would seem logical toassume that patient compliance is an important factor,especially when one considers the length of treatmentand the frequency of adverse reactions. Another possibilityis suggested by the observations of persistent PCRpositivity despite successful or optimum therapy.106,118,119Treatment failure results in increased medical costs,

increased patient suffering, and hence more effective

regimens are urgently needed.Some attempts at predicting relapses have been made,but these require large-scale substantiation to determinetheir true clinical effectiveness. Ariza and colleagues131determined the following independent risk factors forrelapse from a prospective cohort of 530 adults: less-effective antibiotic therapy, positive blood cultures duringinitial disease, disease duration of 10 days or less beforestart of treatment, male sex, and a platelet count of 150x10per L or less.131Solera and colleagues132have proposed amultivariate model to stratify a patients risk of relapsinginto groups of low (45%), medium (32%), and high (67%)probability of relapse within 12 months. The independentpredictors of relapse were temperatures of 383C or

higher, positive blood cultures at baseline, and a durationof symptoms before treatment of less than 10 days.

The WHO oral regimen consists of 200 mg doxycyclineplus 600900 mg rifampicin daily for a minimum of6 weeks, whereas the alternate oral/parenteral schemereplaces rifampicin with 15 mg/kg streptomycin daily forthe first 23 weeks of treatment only. Although theseregimens are based on the results of many clinical andin-vitro studies, much controversy remains regarding theideal treatment. An in-vitro study has shown thatdoxycycline and rifampicin retain adequate activity at apH of 5 (the pH of brucella-destroying phagolysosomes),and that rifampicin increases its activity at low pH.75

A meta-analysis has shown that the classic streptomycinplus tetracycline treatment regimen results in a highercure rate and in fewer relapses than the WHO oralregimen.133The search for an oral-only monotherapy hasso far failed, but has resulted in the evaluation of manycandidates including macrolides and quinolones. A recentstudy evaluating minocycline in combination withrifampicin showed a very low relapse rate.134 Concernsabout treatment failure and relapse, and fear of emergingrifampicin resistance in areas endemic to tuberculosishave led to the pursuit of triple-drug combinations withsome encouraging initial results.135,136 Solera and co-workers137have suggested the addition of gentamycin forthe first 7 or preferably 10 days to the standard oral regimen,

and Mantur and colleagues32did not observe any relapseamong 93 patients treated with a gentamycin-doxycycline-rifampicin triple therapy. Extending the antibiotictreatment also appears to have a positive effect on relapseand failure rates in all treatment regimens. Improvedantibiotic delivery methodsfor example, throughantibiotic-loaded microspherescould shorten duration,decrease toxicities, and improve the effi cacy of treatment;however, more research in this field is still needed. 138

Evaluation of immunomodulation with levamisole plusconventional therapy in the management of chronicbrucellosis has shown mixed results.139141Although initialreports of the addition of interferon alfa-2a to standardtherapy in anergic patients seemed somewhat promising,this has not led to any practical application.142Currently,


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extended treatment with standard drug combinations

should be given to those patients with persisting signsand symptoms of recurrent disease. Additionally, whentreating focal infections, careful attention must be givento the penetration and activity of the drug in the particulartissue involved, and the choice and duration of therapymust be individualised, with prolonged treatment incases with specific complications such as endocarditis orcentral nervous system involvement. The more effectivedoxycycline-streptomycin combination is preferred inpatients with more severe disease, such as spinalinvolvement, and the duration of therapy may beprolonged.143 Abscesses and specific focalised forms ofbrucellosis including endocarditis, cerebral, epidural, orsplenic abscess might require surgical interventions

since these forms are resistant to antibiotics. Finally, thetreatment of brucellosis in children and pregnant andlactating women requires special attention andinsuffi cient data are available to give specificrecommendations.49,128

Special considerations for developing countriesMost of the programmes that have been successful incontrolling brucellosis took place in developed nations,with developing countries continuing to bear the burdenof this disease.65Notably, most cases of human brucellosisin non-endemic developed countries result from dairyproducts imported from endemic areas36,144146 or frompatients who import the disease.144,147Despite the existenceof effective vaccines for cattle (S19) and goats (Rev 1),control efforts in economically poor endemic areas havefailed as a result of inconsistent infrastructure and lackof funding.148,149 A major issue is that control measuresshould continue for a long period of time and becomplemented with a monitoring system that may behard to keep in place once the number of cases begins todecrease. A case in point is the current situation of manyformer Soviet republics, where the collapse of the USSRand its administrative, economic, and public-healthinfrastructure has led to the re-emergence ofbrucellosis.150

Data on the yearly economic impact of brucellosis in

the developing world associated with disease in livestockhave generally been hard to assess, especially in Africa.3In countries such as Argentina and Mexico, whichdepend heavily on the sale of livestock products for bothdomestic and international markets, these annual costsare estimated to be US$60 million and $200 millionrespectively.151,152Studies done in developing countries bythe United Nations highlight the need for effectivecontrol programmes, which have an obvious benefit tothe health of both human beings and livestock. If thecosts of control programmes are shared between thepublic and private sectors and include international aid,they are likely to be profitable and cost effective.153

The economic impact in terms of human disease hasbeen even harder to gauge; not only must the cost of

treatment and diagnosis be considered, but also the cost

in terms of disability-adjusted life years. Regardless ofthe measures used, the economic burden of humanbrucellosis in endemic areas is high and justifieswidespread and sustained control efforts.5,153

Disease control in nomadic and migratory populationshas proven especially challenging. Patients may not haveaccess to medical services, and staff at local health-carecentres may not be able to make or confirm the diagnosis.The diffi culty in obtaining cheap, accurate, and timelydiagnostic tools for human brucellosis is a major problemin urban and rural settings in the developing world. Newdiagnostic methods, particularly point-of-care tests thatdo not require laboratory facilities, will contributesubstantially to early diagnosis and patient management,

and help to create awareness. Adequate brucellosiscontrol will probably have the biggest impact on high-risk shepherding communities who depend on theirherds to satisfy their economic and nutritional needs.Although rural shepherding communities and abattoirand veterinary workers have traditionally been regardedas the main risk groups, it is important to recognise thatlarge urban populations in developing countries are alsoat risk of acquiring the disease by consumingunpasteurised dairy products, as shown by a brucellaantibody prevalence rate of 123% in milk suppliessampled at 219 consumer households in urban Kenya.154

ConclusionsDeveloping countries could benefit from the renewedinterest in brucellosis shown by the scientific community.Molecular detection methods such as PCR amplificationand genotyping will be powerful epidemiological toolsfor confirmation of the disease and for identification ofsources of infection. These methods do not carry the riskof laboratory-acquired infection that culture techniquesdo. Additionally, rapid point-of-care assays will enablefast and accessible diagnostic capabilities even in remoteareas. The search for new and improved treatmentregimens will hopefully provide strategies to effectivelycure even the most complex cases of brucellosis oftenseen in endemic areas.

New insights into the pathogenesis of Brucella spphave not yet led to new developments in the treatment ofpatients as might have been hoped. The fact that brucelladoes not use any of the classic virulence mechanismshas perhaps made investigation in this area slower, butcertainly more exciting. Since brucellosis poses a severehealth threat and restricts export of livestock and animalproducts from endemic areas, it hampers much-neededeconomic development in these areas. Effective vaccinesare currently available and it is important to find meansand resources for their effective use in resource-poorcountries in conjunction with sustained control effortsthat incorporate local farming practices, dietary habits,and traditional beliefs. Brucellosis is routinelyoverlooked, misdiagnosed, or at best diagnosed


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incidentally; therefore, physicians in both endemic andnon-endemic areas must become aware of and considerbrucellosis in their differential diagnosis of febrilediseases with peculiar musculoskeletal or other focalfindings. Clinicians recognition and reporting of the

disease is essential for the allocation of resources andefforts for the development of sustained controlmeasures.

Conflicts of interest

We declare that we have no conflicts of interest.

Acknowledgments

We thank Gavin Jackson for editorial support.

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