1 بسم الله الرحمن الرحيم diagnosis of brucellosis shahid beheshti university of...

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بسم الله الرحمن الرحيمبسم الله الرحمن الرحيمDiagnosis of Diagnosis of BrucellosisBrucellosis

Shahid Beheshti University of Shahid Beheshti University of medical sciencesmedical sciences

20072007

By: Hatami H. MD. MPHBy: Hatami H. MD. MPH

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Diagnosis is based on :Diagnosis is based on :

• Clinical manifestationsClinical manifestations

• History of contactHistory of contact

• Laboratory changesLaboratory changes

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Differential diagnosisDifferential diagnosis

• Enteric feverEnteric fever

• Miliary TBMiliary TB

• Infectious mononucleosisInfectious mononucleosis

• ToxoplasmosisToxoplasmosis

• Acute rheumatic feverAcute rheumatic fever

• Malaria and relapsing f.Malaria and relapsing f.

• LeishmaniasisLeishmaniasis

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Clinical manifestationsClinical manifestations The clinical Suspicion that the patient has The clinical Suspicion that the patient has

brucellosis should be higher in :brucellosis should be higher in :

• Abattoir workersAbattoir workers

• VeterinariansVeterinarians

• Others exposed to infected Others exposed to infected tissues or animal productstissues or animal products

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Laboratory changes :Laboratory changes :

• C.B.CC.B.C

• Bacteriologic evidenceBacteriologic evidence

• Radiographic changesRadiographic changes

• Serologic testsSerologic tests

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Laboratory changes Laboratory changes C.B.CC.B.C

• GranulopeniaGranulopenia in acute brucellosis in acute brucellosis

• LymphocytosisLymphocytosis in chronic brucellosis in chronic brucellosis

• AtypicalAtypical lymphocytosis lymphocytosis

• AnemiaAnemia due to hypersplenism due to hypersplenism

• ThrombocytopeniaThrombocytopenia causing hemorrhage causing hemorrhage

• Moderate elevation of Moderate elevation of ESRESR

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Bacteriological Bacteriological DiagnosisDiagnosis

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Laboratory changes Laboratory changes Bacteriologic testsBacteriologic tests

• Efforts to grow Br. Abortus from blood, Efforts to grow Br. Abortus from blood, usually failusually fail (30% pos.) (30% pos.)

• When the illness is due to When the illness is due to Br. SuisBr. Suis or Br. or Br. MelitensisMelitensis blood culture usually succeeded blood culture usually succeeded (85% pos.)(85% pos.)

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• Taking Taking bone marrowbone marrow is more rewarding is more rewarding

• Brucella can be isolated from liver taken Brucella can be isolated from liver taken by by biopsybiopsy

• Human Human semensemen may be positive may be positive

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• Blood culture positivity decreases with Blood culture positivity decreases with increasing increasing duration of illnessduration of illness

• Blood or bone marrow cultures should be Blood or bone marrow cultures should be incubated for at least incubated for at least 6 weeks6 weeks

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• Blood culture processed in Blood culture processed in radiometricradiometric detection systems may yield positive detection systems may yield positive cultures in less than cultures in less than 10 days10 days

• The culture of brucella organisms is The culture of brucella organisms is potentially potentially hazardoushazardous to laboratory to laboratory personalpersonal

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Serological Serological DiagnosisDiagnosis

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Laboratory changes Laboratory changes Serologic TestsSerologic Tests

• Tube AgglutinationTube Agglutination• 2ME Agglutination2ME Agglutination• Coombs’ testCoombs’ test• Complement fixationComplement fixation• RadioimmunoassayRadioimmunoassay• ELISAELISA• Rapid AgglutinationRapid Agglutination• Rose bengal testRose bengal test

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Laboratory changes Laboratory changes PCR TestsPCR Tests

• IN KUWAITIN KUWAIT

• CASES = 50CASES = 50

• The sensitivity was 100%The sensitivity was 100%

• The specificity of the test The specificity of the test waswas 98.3% t98.3% t

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Serologic TestsSerologic TestsIgMIgM in acute brucellosis in acute brucellosis

• Rises firstRises first

• Is the only antibody for the first weeksIs the only antibody for the first weeks

• Peaks at about 3 monthsPeaks at about 3 months

• Drop off after 3 monthsDrop off after 3 months

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Serologic TestsSerologic TestsIgGIgG in brucellosis in brucellosis

• Begins to rise in the second weekBegins to rise in the second week

• Remain elevated for > 1 year in Remain elevated for > 1 year in treated patients falls by 6 monthstreated patients falls by 6 months

• In unrecovered patients persists for a In unrecovered patients persists for a long timelong time

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• Serologic tests are based on rising and Serologic tests are based on rising and falling of falling of IgMIgM and and IgGIgG

• During During re-infectionre-infection or or exacerbationexacerbation, , antibody titers become elevated antibody titers become elevated

• In In relapsesrelapses, IgG may be the only , IgG may be the only antibody which risesantibody which rises

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STASTA

2ME2ME

COOMBSCOOMBS

C.F.C.F.

RISRIS

ELISAELISA

IgM + IgGIgM + IgG

IgGIgG

1) If STA is negative and disease 1) If STA is negative and disease is chronic then only IgGis chronic then only IgG

2) If STA is positive, IgM + IgG2) If STA is positive, IgM + IgG

IgGIgG

IgG & IgM separately IgG & IgM separately

IgG & IgM separatelyIgG & IgM separately

Tests Antibodies which can be detected Tests Antibodies which can be detected

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Standard tube agglutination testStandard tube agglutination test

• Is the most Is the most frequentlyfrequently utilized test utilized test

• Measuring antibodies to brucella Measuring antibodies to brucella abortus antigenabortus antigen

• A A fourfoldfourfold or greater rise in titer to or greater rise in titer to 1:160 or higher is significant1:160 or higher is significant

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• Titer of Titer of => 1:160=> 1:160 is suggestive if there is suggestive if there is symptoms consistent with is symptoms consistent with brucellosisbrucellosis

• Acute brucellosisAcute brucellosis is most likely to be is most likely to be associated with a titer above 1:160associated with a titer above 1:160

• A great majority of patients will have A great majority of patients will have titers of 1:160 to 1:320titers of 1:160 to 1:320

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• By 3 weeksBy 3 weeks of illness over 97% of of illness over 97% of patients demonstrate serologic patients demonstrate serologic evidence of infectionevidence of infection

• This test detects antibody to Br. This test detects antibody to Br. AbortusAbortus, Br. , Br. MelitensisMelitensis and Br. and Br. SuisSuis but not to Br. but not to Br. CanisCanis

• Significant STA titers can Significant STA titers can persistpersist for for up to 2 years in 5-7% of casesup to 2 years in 5-7% of cases

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• Individuals with Individuals with subclinicalsubclinical infection infection may demonstrate significant STA may demonstrate significant STA titerstiters

• A A diagnosisdiagnosis of brucellosis can not be of brucellosis can not be established on the titers aloneestablished on the titers alone

• STA test STA test can not differentiatecan not differentiate persisting active infection from persisting active infection from treated brucellosistreated brucellosis

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• In In chronicchronic localized brucellosis STA localized brucellosis STA titers may appear titers may appear absentabsent or or lowlow

• It is not useful in It is not useful in differentiatingdifferentiating relapsing infection from other febrile relapsing infection from other febrile illnesses in patients with past brucella illnesses in patients with past brucella infectionsinfections

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Standard tube agglutination testStandard tube agglutination testFalse positive resultsFalse positive results

• F. tularensisF. tularensis

• Y. enterocoliticaY. enterocolitica

• V. choleraV. cholera

• SalmonellaSalmonella

• Vaccine against F. Y. V & S.Vaccine against F. Y. V & S.

• Brucella skin testingBrucella skin testing

• Stenotrophomonas maltophilaStenotrophomonas maltophila

• E. coli O157E. coli O157

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Standard tube agglutination testStandard tube agglutination testCross reactionsCross reactions

• HeterologousHeterologous titers are lower than titers are lower than homologoushomologous

• Heterologous antibodies can be Heterologous antibodies can be removed by absorptionremoved by absorption

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Standard tube agglutination testStandard tube agglutination testFalse negative resultsFalse negative results

• AgammaglobulinemiaAgammaglobulinemia

• First week of diseaseFirst week of disease

• Disease due to Br. CanisDisease due to Br. Canis

• Chronic brucellosisChronic brucellosis

• Prozone phenomenonProzone phenomenon

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False-negative results in STA testFalse-negative results in STA testBrucella Canis infectionBrucella Canis infection

• The antigen used in the STA test does The antigen used in the STA test does not reacts with brucella not reacts with brucella CanisCanis

• If Br. Canis infection is suspected If Br. Canis infection is suspected serologic tests specific for Br. Canis serologic tests specific for Br. Canis must be requestedmust be requested

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False-negative results in STA testFalse-negative results in STA testChronic brucellosisChronic brucellosis

• STA testSTA test may be positive in low titer may be positive in low titer or may be negativeor may be negative

• CoombsCoombs test and test and C.F.C.F. tests may be tests may be positivepositive

• A positive coombs test and A positive coombs test and CFCF test at test at 1:161:16 in such cases is strung evidence of in such cases is strung evidence of continuing infectioncontinuing infection

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False-negative results in STA testFalse-negative results in STA testProzone & postzone phenomenonProzone & postzone phenomenon

واكنشواكنش آنتي آنتي باديبادي

آنتي آنتي ژنژن

به درستي صورت مي گيردبه درستي صورت مي گيرد

واكنش پروزون )با رقيق واكنش پروزون )با رقيق كردن آنتي بادي اصالح كردن آنتي بادي اصالح

مي شود(مي شود(

پست زون )با رقيق كردن پست زون )با رقيق كردن آنتيژن، رفع مي شود(آنتيژن، رفع مي شود(

مناسبمناسب

زيادزياد




زيادزياد

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False-negative results in STA testFalse-negative results in STA testProzone phenomenonProzone phenomenon

• The Prozone phenomenon appears to The Prozone phenomenon appears to be related to the presence of IgG or be related to the presence of IgG or IgA (IgA (Blocking antibodiesBlocking antibodies))

• It can be eliminated if It can be eliminated if dilutionsdilutions are are carried out to at least 1:1280carried out to at least 1:1280

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Serologic TestsSerologic Tests2-Mercaptoethanol test (2ME)2-Mercaptoethanol test (2ME)

• The The STASTA test measures both test measures both IgMIgM and and IgGIgG

• The addition of The addition of 2ME2ME to the STA test to the STA test results in the destruction of results in the destruction of disulfidedisulfide bonds of IgMbonds of IgM

• 2ME test result is the detection of only 2ME test result is the detection of only IgGIgG

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• With prompt and adequate With prompt and adequate therapytherapy IgGIgG usually become undetectable usually become undetectable within within 5-12 months5-12 months

• Those patients who develop Those patients who develop persistentpersistent infection usually maintain elevated infection usually maintain elevated IgG agglutininsIgG agglutinins

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• 2ME test will be 2ME test will be negativenegative if STA test is if STA test is really negativereally negative

• 2ME test is 2ME test is less sensitiveless sensitive than STA than STA testtest

• The The prognosisprognosis of acute brucellosis of acute brucellosis may be predicted from the fall of 2MEmay be predicted from the fall of 2ME

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Serologic TestsSerologic Testsسرمي آزمون هاي سرمي تفسير آزمون هاي تفسير

چرا در كشور ايران، چرا در كشور ايران، 1:1601:160عيارهاي كمتر از عيارهاي كمتر از

تست رايت را به شرط تست رايت را به شرط وجود عالئم باليني وجود عالئم باليني

منطبق بر بروسلوز حاد منطبق بر بروسلوز حاد يا تحت حاد، يا تحت حاد،

باارزش تلقي مي كنيم؟باارزش تلقي مي كنيم؟

چرا در كشور ايران، چرا در كشور ايران، 1:1601:160عيارهاي كمتر از عيارهاي كمتر از

تست رايت را به شرط تست رايت را به شرط وجود عالئم باليني وجود عالئم باليني

منطبق بر بروسلوز حاد منطبق بر بروسلوز حاد يا تحت حاد، يا تحت حاد،

باارزش تلقي مي كنيم؟باارزش تلقي مي كنيم؟

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Serologic TestsSerologic TestsA and M antigensA and M antigens

• Antigen Antigen AA and and MM are are commoncommon to the to the three main brucella speciesthree main brucella species

• In the In the Br. AbortusBr. Abortus, there is more A , there is more A antigen than M antigenantigen than M antigen

• In the In the Br. MelitensisBr. Melitensis there is more M there is more M antigen than A antigenantigen than A antigen

AA MM MMAABABA BMBM

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• Specific antigenSpecific antigen may show higher may show higher agglutinin titers in patients infected agglutinin titers in patients infected with brucella other than Br. Abortuswith brucella other than Br. Abortus

• Ideally in all countries prepared Ideally in all countries prepared antigen for serological testing should antigen for serological testing should consist of consist of predominant speciespredominant species

AA MM MMAABABA BMBM

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• In IRANIn IRAN We have human brucellosis We have human brucellosis nearly always due to Br. Melitensis nearly always due to Br. Melitensis but use but use Br. Abortus antigenBr. Abortus antigen

• Br. Abortus antigen in our Br. Abortus antigen in our laboratories shows laboratories shows lower agglutininlower agglutinin titerstiters

AA MM MMAABABA BMBM

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• In IRAN We must accept titers lower In IRAN We must accept titers lower than 1:160 if there is signs and than 1:160 if there is signs and symptoms compatible with brucellosissymptoms compatible with brucellosis

• In IRAN We must accept titers lower In IRAN We must accept titers lower than 1:160 if there is signs and than 1:160 if there is signs and symptoms compatible with brucellosissymptoms compatible with brucellosis

AA MM MMAABABA BMBM

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Comparison of serological tests using Comparison of serological tests using Br. Abortus and Br. Melitensis antigenBr. Abortus and Br. Melitensis antigen

TestsTestsDue to Br. AbortusDue to Br. Abortus Due to Br. MelitensisDue to Br. Melitensis

A antigenA antigen M antigenM antigen A antigenA antigen M antigenM antigen

STASTA

CoombsCoombs

1:6401:640

1:12801:1280

1:3201:320

1:6401:640

1:6401:640

1:51201:5120

1:12801:1280

1:102401:10240

Ideally in all countries prepared antigen for serological Ideally in all countries prepared antigen for serological testing should consist of predominant speciestesting should consist of predominant speciesIdeally in all countries prepared antigen for serological Ideally in all countries prepared antigen for serological testing should consist of predominant speciestesting should consist of predominant species

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Serologic TestsSerologic TestsCoombs testCoombs test


• Serum may contain brucella Serum may contain brucella antibodies which do not produce antibodies which do not produce agglutinationagglutination

• Non agglutinating antibodies are Non agglutinating antibodies are called called incompleteincomplete antibodies antibodies

• Incomplete antibodies can be detected Incomplete antibodies can be detected by addition of rabbit by addition of rabbit anti-human anti-human globulinglobulin

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• If STA is negativeIf STA is negative and there are and there are symptoms and signs compatible with symptoms and signs compatible with chronic brucellosis, then combs titer chronic brucellosis, then combs titer of of => 1:40=> 1:40 should be considered should be considered positivepositive

• Coombs test is not recommended Coombs test is not recommended when STA test is positivewhen STA test is positive

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Serologic TestsSerologic TestsComplement fixation testComplement fixation test

• The CF test measures IgG antibodiesThe CF test measures IgG antibodies

• Titers of => 1:16 should be considered Titers of => 1:16 should be considered positivepositive

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Serologic TestsSerologic TestsRadioimmunoassay testRadioimmunoassay test

• RIA test determines anti-brucella IgM RIA test determines anti-brucella IgM and IgGand IgG

• Avoids the difficulties with blocking Avoids the difficulties with blocking or non-agglutinating antibodiesor non-agglutinating antibodies

• Can differentiate between chronic and Can differentiate between chronic and acute brucellosisacute brucellosis

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Serologic TestsSerologic TestsELISA testELISA test

• ELISA test can distinguish acute cases ELISA test can distinguish acute cases from chronic casesfrom chronic cases

• In ELISA test cross reaction can occur In ELISA test cross reaction can occur with yersiniosiswith yersiniosis

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Serologic TestsSerologic TestsRose Bengal plate testRose Bengal plate test

• Is an Is an agglutination testagglutination test in which the in which the brucella cells are bound to a dyebrucella cells are bound to a dye

• Is Is quickquick and and easyeasy to read to read

• It is a useful It is a useful screeningscreening test test

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Brucella skin testBrucella skin test(Brucellin)(Brucellin)

• Demonstrate Demonstrate delayed hypersensitivity delayed hypersensitivity

• The antigen is a filtrate of a culture of The antigen is a filtrate of a culture of brucella organisms or purified extractbrucella organisms or purified extract

• Injects Injects intradermallyintradermally

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Brucella skin testBrucella skin test(Brucellin)(Brucellin)

• The test is positive if local The test is positive if local rednessredness with with indurationinduration is present after is present after 24-4824-48 hourshours

• Antigen can provoke an antibody Antigen can provoke an antibody response or a response or a significant risesignificant rise in a pre- in a pre-existing response existing response

Brucellin test, should not be performedBrucellin test, should not be performed

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Interpretation of STA Interpretation of STA and 2ME testsand 2ME tests

STA => 1:160STA => 1:160

IgMIgM IgM + IgGIgM + IgG IgGIgG

Addition of 2MEAddition of 2ME

NegativeNegative 1:801:80 1:1601:160

1) Cured infection1) Cured infection

2) Contact with ag.2) Contact with ag.

3) First week of bru.3) First week of bru.

1) Acute1) Acute

2) Subacute2) Subacute

3) Exacerbation3) Exacerbation

Late chronicLate chronic

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1 بسم الله الرحمن الرحيم diagnosis of brucellosis shahid beheshti university of...

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