- Email: [email protected]
Lab diagnosis of brucellosis
Accepted Manuscript Title: LAB DIAGNOSIS OF BRUCELLOSIS Author: Bhaskar shenoy Anupam Jaiswal Anuradha Vinod PII: DOI: Reference:
S2212-8328(16)30006-6 http://dx.doi.org/doi:10.1016/j.pid.2016.03.006 PID 167
To appear in: Received date: Revised date: Accepted date:
10-1-2015 18-2-2016 17-3-2016
Please cite this article as: Bhaskar shenoyAnupam JaiswalAnuradha Vinod LAB DIAGNOSIS OF BRUCELLOSIS (2016), http://dx.doi.org/10.1016/j.pid.2016.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
LAB DIAGNOSIS OF BRUCELLOSIS Dr.Bhaskar shenoy* Dr.Anupam Jaiswal**Dr.Anuradha Vinod*** *Consultant & Chief,Division of infectious diseases, ** Specialist Pediatric ER.*** Consultant paediatrician, Department of Pediatrics, Manipal Hospital,Bangalore-560017.
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Email:*[email protected]
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Abstract: Brucellosis is a widespread zoonotic disease posing serious public health problems especially in developing countries. The diagnosis of brucellosis is a greater challenge as it requires a high index of suspicion , clinical and laboratory evidence of diagnosis. Increased travel across the globe to endemic areas for work and pleasure over the last few years has lead to increased diagnostic challenges in non-endemic countries. Laboratory diagnosis of brucellosis is essential for diagnosis and hence effective treatment as it includes atleast two drug regimen for a prolonged period unlike many other infections. Among the various tests available, culture of the organism from the bone marrow, blood and other tissues is the gold standard for diagnosis in brucellosis. However, it is an invasive, time consuming and at times less sensitive. These drawbacks have prompted the use of alternate methods to rapidly and accurately diagnose brucellosis and aid in early intervention or therapy. The alternate methods predominantly include serological tests like ELISA , SAT , etc and molecular tests with varying advantages and drawbacks suited to various clinical situations. These tests help not only in diagnosis but also in followup of disease activity / response to therapy. This article reviews the various culture methods , serological tests and newer diagnostic methods available in making a laboratory diagnosis of brucellosis along with their advantages and drawbacks.
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Keywords Brucellosis, serological tests ,blood culture, brucella markers, lysis centrifugation,ELISA. Introduction: Brucellosis is a re-emerging widespread zoonosis. It is transmitted by contact with fluids from infected animals (sheep, cattle, goats, pigs and other animals) or derived food products such as unpasteurized milk and cheese. Brucellosis is the most common zoonosis in the world accounting for more than five lakh cases occurring annually1. It is particularly a problem in developing countries with high morbidity and is an important cause of economic loss. The reported incidence of brucella in endemic areas varies between < 0.01 to >200 per 100000 population. The low levels of incidence reported in known endemic areas may reflect low or absent levels of surveillance and reporting. However, the true incidence of brucellosis is unknown in most developing countries. Even though brucellosis is less common in pediatric population than adults, places where Brucella melitensis is endemic, pediatric cases are seen. Clinical features: The incubation period varies from 1 – 4 weeks (occasionally several months). Brucellosis is a systemic disease with a broad clinical spectrum, ranging from asymptomatic disease to severe
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Abstract: Brucellosis is a widespread zoonotic disease posing serious public health problem especially indeveloping countries. The diagnosis of brucellosis is a greater challenge as it requires a highindex of suspicion, clinical and laboratory evidence of diagnosis. Increased travel across theglobe to endemic areas for work and pleasure over the last few years has led to increaseddiagnostic challenges in nonendemic countries. Laboratory diagnosis of brucellosis is essential for diagnosis and effective treatment as itinvolves two drugs or more for a prolonged period unlike many other infections. Amongthe various tests available, culture of the organism from the bone marrow, blood and othertissues is the gold standard for diagnosis in brucellosis. However, it is invasive, timeconsuming and at times less sensitive. These drawbacks have prompted the use of alternatemethods to rapidly and accurately diagnose brucellosis and aid in early intervention or therapy. The alternate methods predominantly include serological tests like ELISA, Standard Agglutination Test (SAT) andmolecular tests with varying advantages and drawbacks suited to various clinical situations.These tests help not only in diagnosis but also in followup of disease activity / response totherapy. This article reviews the various culture methods, serological tests and newer diagnosticmethods available in making a laboratory diagnosis of brucellosis along with their advantagesand drawbacks.
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Keywords Brucellosis, Serological test, Blood culture, Brucella markers, Lysis centrifugation, ELISA
Introduction: Brucellosis is a re-emerging widespread zoonosis. It is transmitted by contact with fluids frominfected animals (sheep, cattle, goats, pigs and other animals) or derived food products such asunpasteurized milk and cheese. Brucellosis is the most common zoonosis in the worldaccounting for more than five lakh cases occurring annually1. It is particularly a problem indeveloping countries with high morbidity and is an important cause of economic loss. Thereported incidence of Brucellain endemic areas varies between < 0.01 to >200 per 100000population. The low levels of incidence reported in known endemic areas may reflect low orabsent levels of surveillance and reporting. However, the true incidence of brucellosis isunknown in most developing countries. Even though brucellosis is less common
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in pediatricpopulation than adults, places where Brucella melitensis is endemic, pediatric cases are seen.
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Clinical features:
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The incubation period varies from 1 – 4 weeks (occasionally several months). Brucellosis is asystemic disease with a broad clinical spectrum, ranging from asymptomatic disease to severeand / or fatal illness. Fever, (mostly fever of undetermined origin) is the most common clinicalpresentation, arthralgia is also common. Physical signs include hepatosplenomegaly, generalizedlymphadenopathy and arthritis (in some cases). Neurobrucellosis presents with manifestationslike altered sensorium, seizures, increased intracranial pressure meningitis and encephalitis.
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Diagnosis:
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The diagnosis of brucellosis is difficult on clinical grounds alone and hence invariably based onmicrobiological and serological laboratory tests. A high index of clinical suspicion that is basedon epidemiological information along with travel and occupational history (and exposure toanimals and exotic food) is critical in suspecting brucellosis clinically.
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Lab diagnosis: The routine laboratory tests are nonspecific and include normal to low leukocyte countswith occasional pancytopenia. Minor abnormalities in liver enzymes are common2.In Neurobrucellosis, the cerebrospinal fluid shows pleocytosis (10 – 200 WBC) withmononuclear cells predominantly. The protein levels are elevated and hypoglycorrhachia is also acommon finding. When findings consistent with aseptic meningitis are found; an elevated levelof ADA in CSF suggests Brucellameningitis3.The synovial fluid in Brucellaarthritis presents similar to reactive arthritis (due to salmonella andYersinia) with a WBC count not exceeding 15,000 cells/microliter with lymphocyticpredominance.
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Brucella cultures: The ideal method in making a diagnosis of brucellosis would be culturing the organism fromblood, bone marrow, liver biopsy specimen and / or other body fluids or tissues4. Blood culture in brucellosis: Isolation of brucellae from blood cultures is restricted by the slow growth of this intra-cellularorganism and by the previous use of antimicrobials. Successful diagnosis of brucellosisnecessitates a careful selection of the best suitable culture method and validation of itsperformance. The sensitivity of culturing Brucellaspecies from blood varies from 15% to 70%5. There are three main types of culturing techniques:
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The traditional Ruiz-Castaneda method, The automated systems The yield-optimizing methods such as the lysis centrifugation technique.
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The traditional methods take longer time and extension of culture for days to weeks is usuallyneeded to improve the yield rate. The use of various automated culture systems has improvedthe yield rate and has significantly decreased the period of time to obtain a positive culture. Thevital automated system can detect Brucellae in 36% to 48% of blood cultures within 7 to 10days6. The Bactec 9240 and 9120 automated systems can yield positive cultures in up to 95% ofcases within 4 to 7 days. The Bact / Alert automated culture system yield is about 51% within 4to 5 days. The lysis centrifugation method has high sensitivity in confirming chronic brucellosisand comparable to automated systems in acute cases7. Overall the BACTEC 9000 series is moresensitive and faster than Lysis centrifugation method8.
Bone Marrow Culture The bone marrow culture is the ideal method and the gold standard in diagnosing Brucellosis. Ithas the advantage that the sensitivity does not reduce with prior use of antibiotics9. While thetime to detection is 7 – 21 days with blood culture, in comparison bone marrow cultures takelesser time and are much more sensitive especially in chronic cases9.In patients with brucellosis, bone marrow biopsies may show specific granulomatous changesand cultures of bone marrow aspirates may become positive thus helping in establishing thediagnosis. Bone marrow examination and Brucella cultures of the marrow aspirate areindicated under the following circumstances:
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1. In patients with fever of unknown origin (FUO), particularly in areas those are endemic for brucellosis, 2. In patients stronglysuspected to have brucellosis on clinical groundsbut have negative serology, 3. In patients having unexplained articular or haematological involvement who live inendemic areas and 4. In areas where advanced facilities such as automated culture systemsor polymerase chain reaction (PCR) are not readily available.
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Serological test
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Despite having a plethora of serological tests for the diagnosis of brucellosis, none of thesetests are 100% reliable or perfect. So, serological test results should always be considered orinterpreted in conjunction with patient history, clinical manifestations and other laboratoryfindings. The serological tests detect antibodies to the antigens of brucella in blood. Theantigens include S-LPS (smooth lipopolysaccharide) and cytosolic protein. The serologicaltests like SAT, ELISA detect antibodies against the S-LPS antigen. There is cross reactivitywith other bacteria like Yersinia enterocolitica O:9, Escherichia coli O:157, Francisellatularensis, Salmonella urbana O:30, Vibrio choleraeand others. The cytosolic antibodiesdetection may cross react with Ochrobactrum (a bacteriaclosely related to brucella). It helpsdistinguish infection by brucella and the other gram negative bacteria mentioned above thatcross react with S-LPS. Antibodies to cytosolic protein antigens of Brucella have been studiedby counterimmunoelectrophoresis (CIEP), ELISA and western blotting. a) Serum agglutination testing (SAT) It is the serological test with most widely published data and maximum experience. It wasdeveloped in 1897 by Wright and colleagues. It is easy to perform, does not need expensiveequipment or training. SAT is considered as a standard test against which all other serologicaltests are compared. It measures total quantity of agglutinating antibodies (IgM and IgG). Thespecific IgG quantity is determined by 2-mercaptoethanol (2-ME). The positive titres consistof values of 1:80 in nonendemic and 1:160 in endemic areas10. B.canis does not produceantibodies that cross react with standard brucella antigens which can make the SAT negative. In a situation where there is clinical suspicion, the B.canis serology should be specificallyrequested or alternative method used.
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Negative, false positive or low SAT titers may beencountered in: (1) healthy people who previously suffered the disease, (2) patients during the early stage of infection, (3) patients suffering from chronic brucellosis or relapse and (4) Organisms implicated in cross reactivity of S-LPS detection.
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b) Rose Bengal test (RBT)
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The RBT is a rapid, slide-type agglutination assay performed with a stained B.abortussuspension at a low pH (3.6-3.7) and plain serum. Its simplicity made it an ideal screening testfor small laboratories with limited resources.The overall sensitivity is >99%11. RBT is an agglutination test that is based on reactivity ofantibodies against smooth lipopolysaccharide (LPS) and hence the associated drawbacks ofcross reactivity. As sensitivity is high, false negative results are rarely encountered. To increasespecificity, the test may be applied to a serial dilution [1:2 through 1:64] of the serum samples.The RBT is not of much use in endemic areas or in chronic/relapsing brucellosis. The presentWorld Health Organization (WHO) guidelines recommend the confirmation of the RBT by other assays such as serum agglutination tests12.
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c) Enzyme-linked immunosorbent assay (ELISA) It is another standard test used apart from SAT in the serological diagnosis of Brucellosis.ELISA has become popular as a standard assay for the diagnosis of brucellosis, serologically. Itmeasures IgG, IgA and IgM antibodies and this allows a better interpretation of the clinicalsituation. In the acute stage of illness there is an increase in IgM antibodies followed byincrease in IgG antibodies. Chronic brucellosis shows predominant IgG antibodies withabsence of IgM .Rising level of IgM antibody in paired sera is taken as evidence of recentinfection. The presence of IgM antibodies in a single serum sample also indicates recentinfection. For case detection and an accurate diagnosis of suspected cases, the combination ofELISA IgM and IgG tests should be used. This combination of laboratory tests has been shownto be the most efficient (having equal sensitivity and specificity to SAT) technique in thedetection and diagnosis of brucellosis13. ELISA uses antigen derived from Brucella abortusstrain W99.For follow-up and monitoring of
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prognosis, ELISA Ig M and 2-ME are more promising14. ELISA anti-LPS and anticytoplasmic protein are helpful in combination.
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ELISA is an excellent method for screening large populations for Brucella antibodies and fordifferentiation between acute and chronic phases of the disease15. It is the test of choice forcomplicated, local or chronic cases particularly when other tests are negative while the case isunder high clinical suspicion. It can reveal total and individual specific immunoglobulins (IgG,IgA and IgM) within 4-6 hours with high sensitivity and specificity.Patients with brucellosishave significantly higher ELISA titres in all classes of Ig than controls but the sensitivity andspecificity within each Ig class varied with the titre considered. At a titre of > 1600 the brucellaIgG had asensitivity and specificity of 98 % for patients with acute or chronic brucellosis; thisdecreased with lower reciprocal titres. The brucella IgMtitre of > 400 had a sensitivity of 98% and a specificity of 98 % for patients with acute brucellosis. The brucella IgA titre of > 200showed a sensitivity of 98 % and a specificityof 99 % for patients with either acute or chronicbrucellosis.This study indicates that brucella ELISA is a rapid, sensitive and specific assay,provides a profile of Ig classes in the diagnosis of acute and chronic brucellosis, is useful formass screening and could be considered the method of choice for the serological diagnosis ofbrucellosis.Euro immune microplate ELISA contains solid phase containing boundantigens , the calibration is performed in relative units (RU/ml) or, if an international referenceserum exists, in international units (IU/ml).The IgG value that indicates positivity in thismethod is 16 RU/ml and IgM IS 0.8 ISR21. d) The Microagglutination test (MAT) It is a variant of the SAT or ELISA. It is essentially a miniaturized format of SATperformed in microtiter plates and is recommended for the serodiagnosis of brucellosis.The results ofMAT have shown good agreement with those of ELISA. It is less timeconsuming, requires less volumes of serum and reagents (antigen and serum) than SATand can test multiple samples at the same time. It has good performance in the diagnosisof acutebrucellosis, but has high false-negative rates in complicated and chronic cases.It is also liable to false positive findings because of cross-reactions16. e) Coombs test
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This is one of the most suitable and sensitive test for confirmation of relapsing patientswith persistent disease12. Coombs test is used for detection of incomplete, blocking ornon-agglutinating IgG. It is time consuming, technically difficult, requires skilledpersonnel and not routinely performed in clinical laboratories. It is good for complicatedand chronic cases but misses about 7% of cases compared with ELISA12.
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f) Dipstick assay
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The IgM dipstick assay is a good test to detect IgM antibodies to S-LPS in brucellosis ofless than 3 months. IgM dipstick assay offers higher sensitivity and easier manipulationthan IgM ELISA to detect IgM antibodies to Brucella species. The combined results ofSAT and IgM dipstick assays can provide an indication of the stage of disease for thosepatients in whom the onset of clinical manifestations are not known17 . g) Immunocapture agglutination test; Brucella Capt (BCAP)
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Immunocapture agglutination for anti-Brucella (BCAP) assay has been developed to detectagglutinating and non-agglutinating antibodies with high sensitivity. It is based on thesandwich ELISA system, where a microwell is covered with Coombs antibodies against humanorigin IgG, IgA and IgM antibodies. This Brucella agglutination test occurs in a microwell andis performed with Coombs antibodies and determines the three antibodies that form againstbrucellosis. It has been suggested as a possible substitute for Coombs test and a better markerfor disease activity. It is easier to carry out in 24 hours without a second step necessary as inCoombs test. In comparison with other tests: it is more complex, expensive and slow. It couldhelp to diagnose disease in patients with long standing evolution of brucellosis that is notdetected by SAT and so be used as a second level serological test12,18. h) Lateral flow assay (LFA)
An immunochromatographic Brucella IgM / IgG lateral flow assay is a simplified version ofthe ELISA test and has a great potential as a rapid point-of-care assay. The test has highsensitivity and specificity for Brucella IgM and IgG. It uses a drop of blood obtained by fingerprick and can be done as a bedside procedure. So it is a rapid and a simple diagnostic test that isalso easy to interpret. i) Rapid slide agglutination test (RSAT)
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Molecular tests in the diagnosis of brucellosis
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Infection with B.canis can be missed in patients with routine tests for brucellosis since they donot include B.canis investigation. The RSAT is a suitable screening test for the diagnosis ofhuman brucellosis. A supplementary technique, such as ELISA, could be performed on allpositive RSAT samples that were negative by B. abortus antigen could ensure diagnosticspecificity and confirm the diagnosis19. It is recommended to use MAT and 2-ME/RSAT tocheck sera of all patients, who have clinical suspicion of brucellosis but are negative forbrucellosis using a smooth Brucella antigen19.
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The gold standard for the diagnosis of brucellosis remains the isolation of Brucella speciesfrom samples such as blood or bone marrow. However, PCR-based testing methods are fasterand more sensitive than the traditional methods used in the diagnosis of brucellosis. Theyidentify nucleic acid fragments from the bacteria, thus becoming more useful and practical. Asearly as 10 days after inoculation , PCR can detect brucella and thus aid in early diagnosis andtreatment reducing morbidity. PCR assays targeting various targets (omp43, omp31, the 16SrRNAgene,etc) have been developed to detect Brucella species20. The brucella 16S rRNA genesequencing is useful for genus identification. Species identification can be done by ribotypingamplified fragment length polymorphism analysis, DNA sequencing of omp2 and omp25, andPCR assays targeting species-specific insertions of IS711 or IS650 elements20.PCR RFLP can be used for identification of various Brucella species22 Diagnostic specificity of PCR may reach 100% whilst sensitivity is about 80%. Together withserological assays, PCR can be applied for the diagnosis of new cases of brucellosis,asymptomatic but highly exposed individuals as well as relapsed cases regardless of duration ortype of the disease without relying on blood cultures, particularly in chronic cases. The routineuse of PCR is not yet done as there is need for standardization of methods, infrastructure,equipment, expertise, and a better understanding of the clinical significance of the results.
New Brucella markers The flow cytometry finding of a remarkable upregulation of activation markers on CD4+ andCD8+ cells in seronegative patients with brucellosis can be utilized as a novel diagnostic testfor detection of brucellosis in seronegative individuals. Type
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IV secretion system is animportant virulence factor for intra-cellular survival and persistence of Brucella species in thehost. The Vit B12 component of B.abortus encodes a type IV secretion system that maybe auseful serodiagnostic marker for brucellosis21.
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Conclusion:
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In conclusion, infection by brucella is predominantly by B.melitensis followed by other species. Routine cultures take a longer time and yields relatively low results hence, we recommend the use of automated and semi-automated methods for culture (BACTEC) for acute brucellosis.In chronic brucellosis, the use of lysis centrifugation method to culture blood seems a good choice.The invasiveness and difficulty involved in obtaining a bone marrow limits its use in culture. However, it is the investigation of choice in chronic brucellosis and is indicated in any patient who is facing a diagnostic challenge with a suspicion of brucellosis. Of thevarious serological tests available, B.canis is not tested by serological tests and requires special methods to diagnose while microagglutination test helps identify other species. We recommend use ofRose Bengal Test, MAT or BCAP for easy, rapid screening with further conformation needed from better serological tests like SAT or ELISA. ELISA has shown promising consistency in detection of brucella while having various advantages over other serological tests like reproducibility, best serological results in endemic area and can be automated when samples are large in number. For follow up, we recommend 2 ME or IgG ELISA to look for response to therapy. We prefer the 2 ME as it is easily reproducible and cheap. The molecular testing (PCR) is not a test for routine purpose due to lack of availability and cost reasons. However, when faced with a diagnostic challenge and/or local infection (joint/CSF with negative serological tests - since PCR can be done on any tissue)when available,PCR is a suitable test in difficult situations only.
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BIBLIOGRAPHY 1. Pappas G, Papadimitriou P, akritidis N, Christou L and Tsianos E V The new global map of human brucellosis; Lancet. Infect.Dis ; 2006 , 6, 91–99.
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2. Al-EissaYA ,Assuhaimi SA , al-Fawaz IM et al. Pancytopenia in children with brucellosis : clinical manifestations and bone marrow findings. ActaHaematol 1993;89:132.
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3. López-Cortés LF, Cruz-Ruiz M, Gómez-Mateos J, et al. Adenosine deaminase activity inthe CSF of patients with aseptic meningitis: utility in the diagnosis of tuberculousmeningitis or neurobrucellosis. Clin Infect Dis 1995; 20:525.
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4. Young EJ. Brucellosis: current epidemiology, diagnosis, and management. CurrClin TopInfect Dis 1995; 15:115.
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5. Pappas G, Akritidis N, Bosilkovski M, Tsianos E. Brucellosis. N Engl J Med 2005;352:2325.
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6. AliskanH ; The value of culture and serological methods in the diagnosis of humanbrucellosis. MikrobiyolBul 42: 185-195 ; 2008. 7. Mantur BG, Mangalgi SS. Evaluation of conventional castaneda and lysis centrifugationblood culture techniques for diagnosis of human brucellosis. J ClinMicrobiol 2004;42:4327 8. Yagupsky P, Peled N, Press J, et al. Comparison of BACTEC 9240 Peds Plus mediumand isolator 1.5 microbial tube for detection of Brucella melitensis from blood cultures.J ClinMicrobiol 1997; 35:1382. 9. Gotuzzo E, Carrillo C, Guerra J, Llosa L ; An evaluation of diagnostic methods forbrucellosis-- the value of bone marrow culture. J Infect Dis 1986; 153:122
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10. Al Dahouk S, Tomaso H, Nöckler K, et al. Laboratory-based diagnosis of brucellosis—areview of the literature. Part II: serological tests for brucellosis. Clin Lab 2003; 49:577
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11. Serra J, Viñas M. Laboratory diagnosis of brucellosis in a rural endemic area in northeastern Spain. IntMicrobiol 2004; 7:53. 12. Diaz R, Casanova A, Ariza J, MoriyonI ; The Rose Bengal test in human brucellosis: aneglected test for the diagnosis of a neglected disease. PLoSNegl Trop Dis 5: 1-7 ;2011.
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13. Memish ZA, Almuneef M, Mah MW, et al. Comparison of the Brucella StandardAgglutination Test with the ELISA IgG and IgM in patients with Brucella bacteremia.DiagnMicrobiol Infect Dis 2002; 44:129.
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14. Buchanan TM, Faber LC. 2-mercaptoethanol Brucella agglutination test: Usefulness forpredicting recovery from brucellosis. JOURNAL OF CLINICAL MICROBIOLOGY, June 1980, p. 691-693.
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15. Agasthya AS, Isloor S, KrishnamsettyP ;Seroprevalence study of human brucellosis byconventional tests and indigenous indirect enzyme-linked immunosorbent assay.Scientific World J 1-5 ; 2012.
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16. ArajGF ; Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents36: 12-17; 2010. 17. Meyer and Shaw ;Bergey’s manual of systematic bacteriology. (1stedn) Krieg NR andHolt JG, The Williams & Wilkins Co., Baltimore, USA ; 1984. 18. Ozdemir M, Feyzioğlu B, Kurtoğlu MG, Dogan M, Dagi HT, et al ; A comparison ofimmunocapture agglutination and ELISA methods in serological diagnosis ofbrucellosis. Int J Med Sci 8: 428-432 ; 2011. 19. Lucero NE, Escobar GI, Ayala SM, Jacob N ; Diagnosis of human brucellosis caused byBrucella canis. J Med Microbiol 54: 457-461; 2005. 20. Gee JE, De BK, Levett PN, et al. Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates. J ClinMicrobiol 2004; 42:3649.
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21. Sun Y-H, Rolan HG, den Hartigh AB, Sondervan D, TsolisRM ; Brucella abortus VirB12 is expressed during infection but is not an essential component of the type IVsecretion system. Infect Immun 73: 6048-6054 ; 2005. 22Al Dahouk S1, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC. Neubauer H : Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP).Crit Rev Microbiol. 2005;31(4):191-6 .
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S2212-8328(16)30006-6 http://dx.doi.org/doi:10.1016/j.pid.2016.03.006 PID 167
To appear in: Received date: Revised date: Accepted date:
10-1-2015 18-2-2016 17-3-2016
Please cite this article as: Bhaskar shenoyAnupam JaiswalAnuradha Vinod LAB DIAGNOSIS OF BRUCELLOSIS (2016), http://dx.doi.org/10.1016/j.pid.2016.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
LAB DIAGNOSIS OF BRUCELLOSIS Dr.Bhaskar shenoy* Dr.Anupam Jaiswal**Dr.Anuradha Vinod*** *Consultant & Chief,Division of infectious diseases, ** Specialist Pediatric ER.*** Consultant paediatrician, Department of Pediatrics, Manipal Hospital,Bangalore-560017.
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Email:*[email protected]
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Abstract: Brucellosis is a widespread zoonotic disease posing serious public health problems especially in developing countries. The diagnosis of brucellosis is a greater challenge as it requires a high index of suspicion , clinical and laboratory evidence of diagnosis. Increased travel across the globe to endemic areas for work and pleasure over the last few years has lead to increased diagnostic challenges in non-endemic countries. Laboratory diagnosis of brucellosis is essential for diagnosis and hence effective treatment as it includes atleast two drug regimen for a prolonged period unlike many other infections. Among the various tests available, culture of the organism from the bone marrow, blood and other tissues is the gold standard for diagnosis in brucellosis. However, it is an invasive, time consuming and at times less sensitive. These drawbacks have prompted the use of alternate methods to rapidly and accurately diagnose brucellosis and aid in early intervention or therapy. The alternate methods predominantly include serological tests like ELISA , SAT , etc and molecular tests with varying advantages and drawbacks suited to various clinical situations. These tests help not only in diagnosis but also in followup of disease activity / response to therapy. This article reviews the various culture methods , serological tests and newer diagnostic methods available in making a laboratory diagnosis of brucellosis along with their advantages and drawbacks.
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Keywords Brucellosis, serological tests ,blood culture, brucella markers, lysis centrifugation,ELISA. Introduction: Brucellosis is a re-emerging widespread zoonosis. It is transmitted by contact with fluids from infected animals (sheep, cattle, goats, pigs and other animals) or derived food products such as unpasteurized milk and cheese. Brucellosis is the most common zoonosis in the world accounting for more than five lakh cases occurring annually1. It is particularly a problem in developing countries with high morbidity and is an important cause of economic loss. The reported incidence of brucella in endemic areas varies between < 0.01 to >200 per 100000 population. The low levels of incidence reported in known endemic areas may reflect low or absent levels of surveillance and reporting. However, the true incidence of brucellosis is unknown in most developing countries. Even though brucellosis is less common in pediatric population than adults, places where Brucella melitensis is endemic, pediatric cases are seen. Clinical features: The incubation period varies from 1 – 4 weeks (occasionally several months). Brucellosis is a systemic disease with a broad clinical spectrum, ranging from asymptomatic disease to severe
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Abstract: Brucellosis is a widespread zoonotic disease posing serious public health problem especially indeveloping countries. The diagnosis of brucellosis is a greater challenge as it requires a highindex of suspicion, clinical and laboratory evidence of diagnosis. Increased travel across theglobe to endemic areas for work and pleasure over the last few years has led to increaseddiagnostic challenges in nonendemic countries. Laboratory diagnosis of brucellosis is essential for diagnosis and effective treatment as itinvolves two drugs or more for a prolonged period unlike many other infections. Amongthe various tests available, culture of the organism from the bone marrow, blood and othertissues is the gold standard for diagnosis in brucellosis. However, it is invasive, timeconsuming and at times less sensitive. These drawbacks have prompted the use of alternatemethods to rapidly and accurately diagnose brucellosis and aid in early intervention or therapy. The alternate methods predominantly include serological tests like ELISA, Standard Agglutination Test (SAT) andmolecular tests with varying advantages and drawbacks suited to various clinical situations.These tests help not only in diagnosis but also in followup of disease activity / response totherapy. This article reviews the various culture methods, serological tests and newer diagnosticmethods available in making a laboratory diagnosis of brucellosis along with their advantagesand drawbacks.
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Keywords Brucellosis, Serological test, Blood culture, Brucella markers, Lysis centrifugation, ELISA
Introduction: Brucellosis is a re-emerging widespread zoonosis. It is transmitted by contact with fluids frominfected animals (sheep, cattle, goats, pigs and other animals) or derived food products such asunpasteurized milk and cheese. Brucellosis is the most common zoonosis in the worldaccounting for more than five lakh cases occurring annually1. It is particularly a problem indeveloping countries with high morbidity and is an important cause of economic loss. Thereported incidence of Brucellain endemic areas varies between < 0.01 to >200 per 100000population. The low levels of incidence reported in known endemic areas may reflect low orabsent levels of surveillance and reporting. However, the true incidence of brucellosis isunknown in most developing countries. Even though brucellosis is less common
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in pediatricpopulation than adults, places where Brucella melitensis is endemic, pediatric cases are seen.
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Clinical features:
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The incubation period varies from 1 – 4 weeks (occasionally several months). Brucellosis is asystemic disease with a broad clinical spectrum, ranging from asymptomatic disease to severeand / or fatal illness. Fever, (mostly fever of undetermined origin) is the most common clinicalpresentation, arthralgia is also common. Physical signs include hepatosplenomegaly, generalizedlymphadenopathy and arthritis (in some cases). Neurobrucellosis presents with manifestationslike altered sensorium, seizures, increased intracranial pressure meningitis and encephalitis.
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Diagnosis:
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The diagnosis of brucellosis is difficult on clinical grounds alone and hence invariably based onmicrobiological and serological laboratory tests. A high index of clinical suspicion that is basedon epidemiological information along with travel and occupational history (and exposure toanimals and exotic food) is critical in suspecting brucellosis clinically.
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Lab diagnosis: The routine laboratory tests are nonspecific and include normal to low leukocyte countswith occasional pancytopenia. Minor abnormalities in liver enzymes are common2.In Neurobrucellosis, the cerebrospinal fluid shows pleocytosis (10 – 200 WBC) withmononuclear cells predominantly. The protein levels are elevated and hypoglycorrhachia is also acommon finding. When findings consistent with aseptic meningitis are found; an elevated levelof ADA in CSF suggests Brucellameningitis3.The synovial fluid in Brucellaarthritis presents similar to reactive arthritis (due to salmonella andYersinia) with a WBC count not exceeding 15,000 cells/microliter with lymphocyticpredominance.
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Brucella cultures: The ideal method in making a diagnosis of brucellosis would be culturing the organism fromblood, bone marrow, liver biopsy specimen and / or other body fluids or tissues4. Blood culture in brucellosis: Isolation of brucellae from blood cultures is restricted by the slow growth of this intra-cellularorganism and by the previous use of antimicrobials. Successful diagnosis of brucellosisnecessitates a careful selection of the best suitable culture method and validation of itsperformance. The sensitivity of culturing Brucellaspecies from blood varies from 15% to 70%5. There are three main types of culturing techniques:
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The traditional Ruiz-Castaneda method, The automated systems The yield-optimizing methods such as the lysis centrifugation technique.
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The traditional methods take longer time and extension of culture for days to weeks is usuallyneeded to improve the yield rate. The use of various automated culture systems has improvedthe yield rate and has significantly decreased the period of time to obtain a positive culture. Thevital automated system can detect Brucellae in 36% to 48% of blood cultures within 7 to 10days6. The Bactec 9240 and 9120 automated systems can yield positive cultures in up to 95% ofcases within 4 to 7 days. The Bact / Alert automated culture system yield is about 51% within 4to 5 days. The lysis centrifugation method has high sensitivity in confirming chronic brucellosisand comparable to automated systems in acute cases7. Overall the BACTEC 9000 series is moresensitive and faster than Lysis centrifugation method8.
Bone Marrow Culture The bone marrow culture is the ideal method and the gold standard in diagnosing Brucellosis. Ithas the advantage that the sensitivity does not reduce with prior use of antibiotics9. While thetime to detection is 7 – 21 days with blood culture, in comparison bone marrow cultures takelesser time and are much more sensitive especially in chronic cases9.In patients with brucellosis, bone marrow biopsies may show specific granulomatous changesand cultures of bone marrow aspirates may become positive thus helping in establishing thediagnosis. Bone marrow examination and Brucella cultures of the marrow aspirate areindicated under the following circumstances:
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1. In patients with fever of unknown origin (FUO), particularly in areas those are endemic for brucellosis, 2. In patients stronglysuspected to have brucellosis on clinical groundsbut have negative serology, 3. In patients having unexplained articular or haematological involvement who live inendemic areas and 4. In areas where advanced facilities such as automated culture systemsor polymerase chain reaction (PCR) are not readily available.
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Serological test
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Despite having a plethora of serological tests for the diagnosis of brucellosis, none of thesetests are 100% reliable or perfect. So, serological test results should always be considered orinterpreted in conjunction with patient history, clinical manifestations and other laboratoryfindings. The serological tests detect antibodies to the antigens of brucella in blood. Theantigens include S-LPS (smooth lipopolysaccharide) and cytosolic protein. The serologicaltests like SAT, ELISA detect antibodies against the S-LPS antigen. There is cross reactivitywith other bacteria like Yersinia enterocolitica O:9, Escherichia coli O:157, Francisellatularensis, Salmonella urbana O:30, Vibrio choleraeand others. The cytosolic antibodiesdetection may cross react with Ochrobactrum (a bacteriaclosely related to brucella). It helpsdistinguish infection by brucella and the other gram negative bacteria mentioned above thatcross react with S-LPS. Antibodies to cytosolic protein antigens of Brucella have been studiedby counterimmunoelectrophoresis (CIEP), ELISA and western blotting. a) Serum agglutination testing (SAT) It is the serological test with most widely published data and maximum experience. It wasdeveloped in 1897 by Wright and colleagues. It is easy to perform, does not need expensiveequipment or training. SAT is considered as a standard test against which all other serologicaltests are compared. It measures total quantity of agglutinating antibodies (IgM and IgG). Thespecific IgG quantity is determined by 2-mercaptoethanol (2-ME). The positive titres consistof values of 1:80 in nonendemic and 1:160 in endemic areas10. B.canis does not produceantibodies that cross react with standard brucella antigens which can make the SAT negative. In a situation where there is clinical suspicion, the B.canis serology should be specificallyrequested or alternative method used.
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Negative, false positive or low SAT titers may beencountered in: (1) healthy people who previously suffered the disease, (2) patients during the early stage of infection, (3) patients suffering from chronic brucellosis or relapse and (4) Organisms implicated in cross reactivity of S-LPS detection.
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b) Rose Bengal test (RBT)
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The RBT is a rapid, slide-type agglutination assay performed with a stained B.abortussuspension at a low pH (3.6-3.7) and plain serum. Its simplicity made it an ideal screening testfor small laboratories with limited resources.The overall sensitivity is >99%11. RBT is an agglutination test that is based on reactivity ofantibodies against smooth lipopolysaccharide (LPS) and hence the associated drawbacks ofcross reactivity. As sensitivity is high, false negative results are rarely encountered. To increasespecificity, the test may be applied to a serial dilution [1:2 through 1:64] of the serum samples.The RBT is not of much use in endemic areas or in chronic/relapsing brucellosis. The presentWorld Health Organization (WHO) guidelines recommend the confirmation of the RBT by other assays such as serum agglutination tests12.
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c) Enzyme-linked immunosorbent assay (ELISA) It is another standard test used apart from SAT in the serological diagnosis of Brucellosis.ELISA has become popular as a standard assay for the diagnosis of brucellosis, serologically. Itmeasures IgG, IgA and IgM antibodies and this allows a better interpretation of the clinicalsituation. In the acute stage of illness there is an increase in IgM antibodies followed byincrease in IgG antibodies. Chronic brucellosis shows predominant IgG antibodies withabsence of IgM .Rising level of IgM antibody in paired sera is taken as evidence of recentinfection. The presence of IgM antibodies in a single serum sample also indicates recentinfection. For case detection and an accurate diagnosis of suspected cases, the combination ofELISA IgM and IgG tests should be used. This combination of laboratory tests has been shownto be the most efficient (having equal sensitivity and specificity to SAT) technique in thedetection and diagnosis of brucellosis13. ELISA uses antigen derived from Brucella abortusstrain W99.For follow-up and monitoring of
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prognosis, ELISA Ig M and 2-ME are more promising14. ELISA anti-LPS and anticytoplasmic protein are helpful in combination.
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ELISA is an excellent method for screening large populations for Brucella antibodies and fordifferentiation between acute and chronic phases of the disease15. It is the test of choice forcomplicated, local or chronic cases particularly when other tests are negative while the case isunder high clinical suspicion. It can reveal total and individual specific immunoglobulins (IgG,IgA and IgM) within 4-6 hours with high sensitivity and specificity.Patients with brucellosishave significantly higher ELISA titres in all classes of Ig than controls but the sensitivity andspecificity within each Ig class varied with the titre considered. At a titre of > 1600 the brucellaIgG had asensitivity and specificity of 98 % for patients with acute or chronic brucellosis; thisdecreased with lower reciprocal titres. The brucella IgMtitre of > 400 had a sensitivity of 98% and a specificity of 98 % for patients with acute brucellosis. The brucella IgA titre of > 200showed a sensitivity of 98 % and a specificityof 99 % for patients with either acute or chronicbrucellosis.This study indicates that brucella ELISA is a rapid, sensitive and specific assay,provides a profile of Ig classes in the diagnosis of acute and chronic brucellosis, is useful formass screening and could be considered the method of choice for the serological diagnosis ofbrucellosis.Euro immune microplate ELISA contains solid phase containing boundantigens , the calibration is performed in relative units (RU/ml) or, if an international referenceserum exists, in international units (IU/ml).The IgG value that indicates positivity in thismethod is 16 RU/ml and IgM IS 0.8 ISR21. d) The Microagglutination test (MAT) It is a variant of the SAT or ELISA. It is essentially a miniaturized format of SATperformed in microtiter plates and is recommended for the serodiagnosis of brucellosis.The results ofMAT have shown good agreement with those of ELISA. It is less timeconsuming, requires less volumes of serum and reagents (antigen and serum) than SATand can test multiple samples at the same time. It has good performance in the diagnosisof acutebrucellosis, but has high false-negative rates in complicated and chronic cases.It is also liable to false positive findings because of cross-reactions16. e) Coombs test
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This is one of the most suitable and sensitive test for confirmation of relapsing patientswith persistent disease12. Coombs test is used for detection of incomplete, blocking ornon-agglutinating IgG. It is time consuming, technically difficult, requires skilledpersonnel and not routinely performed in clinical laboratories. It is good for complicatedand chronic cases but misses about 7% of cases compared with ELISA12.
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f) Dipstick assay
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The IgM dipstick assay is a good test to detect IgM antibodies to S-LPS in brucellosis ofless than 3 months. IgM dipstick assay offers higher sensitivity and easier manipulationthan IgM ELISA to detect IgM antibodies to Brucella species. The combined results ofSAT and IgM dipstick assays can provide an indication of the stage of disease for thosepatients in whom the onset of clinical manifestations are not known17 . g) Immunocapture agglutination test; Brucella Capt (BCAP)
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Immunocapture agglutination for anti-Brucella (BCAP) assay has been developed to detectagglutinating and non-agglutinating antibodies with high sensitivity. It is based on thesandwich ELISA system, where a microwell is covered with Coombs antibodies against humanorigin IgG, IgA and IgM antibodies. This Brucella agglutination test occurs in a microwell andis performed with Coombs antibodies and determines the three antibodies that form againstbrucellosis. It has been suggested as a possible substitute for Coombs test and a better markerfor disease activity. It is easier to carry out in 24 hours without a second step necessary as inCoombs test. In comparison with other tests: it is more complex, expensive and slow. It couldhelp to diagnose disease in patients with long standing evolution of brucellosis that is notdetected by SAT and so be used as a second level serological test12,18. h) Lateral flow assay (LFA)
An immunochromatographic Brucella IgM / IgG lateral flow assay is a simplified version ofthe ELISA test and has a great potential as a rapid point-of-care assay. The test has highsensitivity and specificity for Brucella IgM and IgG. It uses a drop of blood obtained by fingerprick and can be done as a bedside procedure. So it is a rapid and a simple diagnostic test that isalso easy to interpret. i) Rapid slide agglutination test (RSAT)
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Molecular tests in the diagnosis of brucellosis
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Infection with B.canis can be missed in patients with routine tests for brucellosis since they donot include B.canis investigation. The RSAT is a suitable screening test for the diagnosis ofhuman brucellosis. A supplementary technique, such as ELISA, could be performed on allpositive RSAT samples that were negative by B. abortus antigen could ensure diagnosticspecificity and confirm the diagnosis19. It is recommended to use MAT and 2-ME/RSAT tocheck sera of all patients, who have clinical suspicion of brucellosis but are negative forbrucellosis using a smooth Brucella antigen19.
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The gold standard for the diagnosis of brucellosis remains the isolation of Brucella speciesfrom samples such as blood or bone marrow. However, PCR-based testing methods are fasterand more sensitive than the traditional methods used in the diagnosis of brucellosis. Theyidentify nucleic acid fragments from the bacteria, thus becoming more useful and practical. Asearly as 10 days after inoculation , PCR can detect brucella and thus aid in early diagnosis andtreatment reducing morbidity. PCR assays targeting various targets (omp43, omp31, the 16SrRNAgene,etc) have been developed to detect Brucella species20. The brucella 16S rRNA genesequencing is useful for genus identification. Species identification can be done by ribotypingamplified fragment length polymorphism analysis, DNA sequencing of omp2 and omp25, andPCR assays targeting species-specific insertions of IS711 or IS650 elements20.PCR RFLP can be used for identification of various Brucella species22 Diagnostic specificity of PCR may reach 100% whilst sensitivity is about 80%. Together withserological assays, PCR can be applied for the diagnosis of new cases of brucellosis,asymptomatic but highly exposed individuals as well as relapsed cases regardless of duration ortype of the disease without relying on blood cultures, particularly in chronic cases. The routineuse of PCR is not yet done as there is need for standardization of methods, infrastructure,equipment, expertise, and a better understanding of the clinical significance of the results.
New Brucella markers The flow cytometry finding of a remarkable upregulation of activation markers on CD4+ andCD8+ cells in seronegative patients with brucellosis can be utilized as a novel diagnostic testfor detection of brucellosis in seronegative individuals. Type
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IV secretion system is animportant virulence factor for intra-cellular survival and persistence of Brucella species in thehost. The Vit B12 component of B.abortus encodes a type IV secretion system that maybe auseful serodiagnostic marker for brucellosis21.
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Conclusion:
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In conclusion, infection by brucella is predominantly by B.melitensis followed by other species. Routine cultures take a longer time and yields relatively low results hence, we recommend the use of automated and semi-automated methods for culture (BACTEC) for acute brucellosis.In chronic brucellosis, the use of lysis centrifugation method to culture blood seems a good choice.The invasiveness and difficulty involved in obtaining a bone marrow limits its use in culture. However, it is the investigation of choice in chronic brucellosis and is indicated in any patient who is facing a diagnostic challenge with a suspicion of brucellosis. Of thevarious serological tests available, B.canis is not tested by serological tests and requires special methods to diagnose while microagglutination test helps identify other species. We recommend use ofRose Bengal Test, MAT or BCAP for easy, rapid screening with further conformation needed from better serological tests like SAT or ELISA. ELISA has shown promising consistency in detection of brucella while having various advantages over other serological tests like reproducibility, best serological results in endemic area and can be automated when samples are large in number. For follow up, we recommend 2 ME or IgG ELISA to look for response to therapy. We prefer the 2 ME as it is easily reproducible and cheap. The molecular testing (PCR) is not a test for routine purpose due to lack of availability and cost reasons. However, when faced with a diagnostic challenge and/or local infection (joint/CSF with negative serological tests - since PCR can be done on any tissue)when available,PCR is a suitable test in difficult situations only.
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2. Al-EissaYA ,Assuhaimi SA , al-Fawaz IM et al. Pancytopenia in children with brucellosis : clinical manifestations and bone marrow findings. ActaHaematol 1993;89:132.
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3. López-Cortés LF, Cruz-Ruiz M, Gómez-Mateos J, et al. Adenosine deaminase activity inthe CSF of patients with aseptic meningitis: utility in the diagnosis of tuberculousmeningitis or neurobrucellosis. Clin Infect Dis 1995; 20:525.
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6. AliskanH ; The value of culture and serological methods in the diagnosis of humanbrucellosis. MikrobiyolBul 42: 185-195 ; 2008. 7. Mantur BG, Mangalgi SS. Evaluation of conventional castaneda and lysis centrifugationblood culture techniques for diagnosis of human brucellosis. J ClinMicrobiol 2004;42:4327 8. Yagupsky P, Peled N, Press J, et al. Comparison of BACTEC 9240 Peds Plus mediumand isolator 1.5 microbial tube for detection of Brucella melitensis from blood cultures.J ClinMicrobiol 1997; 35:1382. 9. Gotuzzo E, Carrillo C, Guerra J, Llosa L ; An evaluation of diagnostic methods forbrucellosis-- the value of bone marrow culture. J Infect Dis 1986; 153:122
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10. Al Dahouk S, Tomaso H, Nöckler K, et al. Laboratory-based diagnosis of brucellosis—areview of the literature. Part II: serological tests for brucellosis. Clin Lab 2003; 49:577
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16. ArajGF ; Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents36: 12-17; 2010. 17. Meyer and Shaw ;Bergey’s manual of systematic bacteriology. (1stedn) Krieg NR andHolt JG, The Williams & Wilkins Co., Baltimore, USA ; 1984. 18. Ozdemir M, Feyzioğlu B, Kurtoğlu MG, Dogan M, Dagi HT, et al ; A comparison ofimmunocapture agglutination and ELISA methods in serological diagnosis ofbrucellosis. Int J Med Sci 8: 428-432 ; 2011. 19. Lucero NE, Escobar GI, Ayala SM, Jacob N ; Diagnosis of human brucellosis caused byBrucella canis. J Med Microbiol 54: 457-461; 2005. 20. Gee JE, De BK, Levett PN, et al. Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates. J ClinMicrobiol 2004; 42:3649.
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21. Sun Y-H, Rolan HG, den Hartigh AB, Sondervan D, TsolisRM ; Brucella abortus VirB12 is expressed during infection but is not an essential component of the type IVsecretion system. Infect Immun 73: 6048-6054 ; 2005. 22Al Dahouk S1, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC. Neubauer H : Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP).Crit Rev Microbiol. 2005;31(4):191-6 .
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