Laboratory Diagnosis of Brucellosis

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Laboratory Diagnosis of Brucellosis:

Laboratory Diagnosis of Brucellosis By Mohammad Reza Faryabi MSc of Immunology

Criteria  :

Criteria   Definitive Criteria Isolation of  Brucella  species from a sterile sit  Seroconversion  or significant increase in  Brucella   Ab  level in acute & convalescent sera  Suggestive Criteria A single high  titre  of specific  Brucella   Ab (CDC: Brucella total Ab titer of greater than or equal to 1:160 by standard tube agglutination test (SAT) or Brucella microagglutination test (BMAT) in one or more serum specimens obtained after onset of symptoms)   Detection of Brucella DNA in a clinical specimen by PCR assay

Brucella Species:

Brucella Species B. Abortus B. Melitensis B. Suis B. Canis B. Ovis B. Neotomae B. Pinnipedalis B. Ceti B. Microti B. Inopinata

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Classification of methods - 1 Direct Methods Bacteriological Diagnosis Immunohistochemistry Molecular Methods Indirect Methods Serologic Tests Rapid slide agglutination test Rose bengal plate test Standard agglutination tube (SAT) test Indirect Coombs test 2-mercaptoethanol (2ME) Complement fixation (CF) ELISAs

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Classification of methods - 2 Screening tests Rapid slide agglutination test Rose bengal plate test Agar gel immunodiffusion test Complementary or confirmatory tests Indirect Coombs 2-mercaptoethanol (2ME) Complement fixation (CF) ELISAs Fluorescence polarization assay Monitoring /epidemiological surveillance tests Milk ring test

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Bacteriological Diagnosis Gold standard Isolation of the organism (Culture) Requires Biosafety level-3 (BSL-3) Non automated Biphasic Ruiz–Castaneda bottles Automatic Bactec (BD Diagnostics, Sparks, MD, USA) BacTAlert (BioMerieux, Durham, NC, USA)

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Immunohistochemistry Used in studies of pathogenesis and diagnosis of brucellosis To detect Brucella Ags in formalin-fixed, paraffin-embedded tissues of organs from aborted bovine fetuses An advantage of this technique is that it does not require viable bacteria

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Molecular Methods Rapid detection Can be used on isolates & clinical specimens For diagnosis and epidemiologic studies For identification of species and biotypes of Brucella spp Differentiation between virulent and vaccine strains Sensitivity ranging from 50% to 100% Specificity between 60% to 98%

Laboratory Diagnosis/Tests:

Laboratory Diagnosis/Tests Types of Molecular Methods Multiplex polymerase chain reaction typing Real-time PCR High resolution melt Restriction fragment length polymorphism based approaches (RFLP) Single nucleotide polymorphisms typing (SNPs) Tandem repeat based typing

Serologic Tests:

Serologic Tests Characterization of humoral immune response to Brucella Initial production of IgM followed afterward by the production of IgG

Serologic Tests:

Serologic Tests Useful major Ags for diagnosis of brucellosis Smooth (S) lipopolysaccharide (LPS) of the outer membrane Internal proteins

Serologic Tests:

Serologic Tests Detected Abs: Abs reacting Ags S-LPS Most of control & eradication programs rely on serologic methods cross reactions Abs against smooth Brucella species (e.g., B. abortus , B.melitensis , and B. suis) cross react with Ag preparations from B. abortus Abs against rough Brucella species (e.g., B. ovis and B. canis ) cross react with Ag preparations from B. ovis

Serologic Tests:

Serologic Tests Rapid slide agglutination test Rose bengal plate test Standard agglutination tube (SAT) Indirect Coombs test Immunocapture-agglutination test (Brucellacapt) 2-mercaptoethanol (2ME) Complement fixation (CF) ELISAs Agar Gel immunodiffusion Ttest Lateral flow assay

Standard agglutination tube (SAT):

Standard agglutination tube (SAT) Developed by Wright et al. in 1897 The first developed serological test for diagnosis of brucellosis The most frequently used method for diagnosing human brucellosis Based on bacterial Ag agglutination, particularly by IgM under neutral pH Low specificity

Microplate Agglutination Test (MAT):

Microplate Agglutination Test (MAT) Variant of the SAT or enzyme-linked immunosorbent assay ( ELISA ) Able to process a large number of samples U bottom well strips coated with anti human immunoglobulin’s After addition and dilution of serum , the Ag is added and strips are incubated for 24 h until agglutination takes place This assay allows the detection of both agglutinating and incomplete Abs

Indirect Coombs Test:

Indirect Coombs Test Most suitable & sensitive for confirmation of relapsing patients with persistent disease An extension of the SAT Remains positive longer than other agglutination tests Used for detection of Incomplete , blocking or nonagglutinating IgG For complicated and chronic cases Misses about 7% of cases compared with ELISA

Immunocapture-Agglutination Test (Brucellacapt):

Immunocapture-Agglutination Test (Brucellacapt) Detect agglutinating & nonagglutinating Abs with high sensitivity Determines blocking Abs at diagnosis & during follow up for patients Based on the sandwich ELISA Covered microwell with Coombs Abs against human origin IgG, IgA & IgM

Immunocapture-Agglutination Test (Brucellacapt):

Immunocapture-Agglutination Test (Brucellacapt) Diagnose disease in patients with longstanding evolution of brucellosis that is not detected by SAT The interpretation is often difficult in endemic areas More complex, expensive

Agar Gel immunodiffusion Ttest:

Agar Gel immunodiffusion Ttest Advantages Based on precipitation of the Ag-Ab complex Often used for the diagnosis of B. ovis infection Sensitivity levels are comparable to complement fixation Disadvantages Marked decrease in sensitivity in chronic infections High variability of the quality of commercially available Ags Sensitivity: 50 to 92.7% Specificity: 94.3 and 100%

Lateral flow assay:

Lateral flow assay A simplified version of the ELISA High sensitivity & specificity for Brucella IgM and IgG Uses a drop of blood obtained by finger prick Can be done as a bedside procedure A rapid & simple diagnostic test

Brucellin allergic skin test:

Brucellin allergic skin test An allergic test ( delayed type hypersensitivity reaction) Detects the specific cellular immune response induced by Brucella spp. Injection of brucellergene , a protein extract of a rough strain of Brucella spp is followed by a local inflammatory response in a sensitized animal Highly efficient in discriminating between true brucellosis cases & false positive serological reactions Highly specific but its weak sensitivity A good test for herds but not for individual

Safety :

Safety Brucella species Are easily aerosolized Have a low infectious dose Cited at levels between 10 & 100 microorganisms Have a prolonged incubation period Have potential to induce a broad range of clinical manifestations Generate challenges for prompt diagnosis One of more frequent source of infection : laboratory-acquired brucellosis, as a result of aerosols generated during the manipulation of cultures Serological Monitoring After Laboratory Exposure ( CDC ) 0, 6, 12, 18 & 24 weeks post exposure

Summary:

Summary Brucellosis Criteria Classifications of diagnosis tests Types of serologic tests Laboratory Notification Flowchart of Brucellosis Safety

References:

References Minda Asfaw Geresu and Gezahegne Mamo Kassa , A Review on Diagnostic Methods of Brucellosis. Geresu and Kassa , J Veterinar Sci Techno 2016, 7:3. DOI: 10.4172/2157-7579.1000323 Sang- Hee Park, Yoo-Hoon Lee, et al, Application of the Microagglutination Test for Serologic Diagnosis of Human Brucellosis. Osong Public Health Res Perspect 2012 3(1), 19e23 doi:10.1016/j.phrp.2012.01.003 BRUCELLOSIS REFERENCE GUIDE: EXPOSURES, TESTING, AND PREVENTION. Centers for Disease Control and Prevention , National Center for Emerging and Zoonotic Infectious Diseases Massoud Hajia , PhD, Fatemeh Fallah , PhD, et al, Comparison of Methods for Diagnosing Brucellosis. Lab Medicine, 2013, Volume 44, Number 1. DOI: 10.1309/LM4J9MWOBIPA6RBN Fernando Padilla Poester *,1, Klaus Nielsen, et al, Diagnosis of Brucellosis. Veterinary Science Journal, 2010, 4, 46-60. María P. Jime´nez de Bagu¨e´s, , et al, Different Responses of Macrophages to Smooth and Rough Brucella spp.: Relationship to Virulence. INFECTION AND IMMUNITY, Apr. 2004, p. 2429–2433. DOI: 10.1128/IAI.72.4.2429–2433.2004 M. Concepcio´n Go´mez,1* Jose´ A. Nieto, et al, Evaluation of Seven Tests for Diagnosis of Human Brucellosis in an Area Where the Disease Is Endemic. CLINICAL AND VACCINE IMMUNOLOGY, June 2008, p. 1031–1033. doi:10.1128/CVI.00424-07 Laboratory Notification Flowcharts. NSW Department of Health, March 2006, version 1.3 Marcos Mancilla , Smooth to Rough Dissociation in Brucella : The Missing Link to Virulence. Front. Cell. Infect. Microbiol ., 2016, https://doi.org/10.3389/fcimb.2015.00098 محمدرضا شیرزادی، محمد زینلی و دیگران، راهنمای تشخیص و درمان بروسلوزیس. وزارت بهداشت، درمان و آموزش پزشکی، معاونت بهداشت، مرکز مدیریت بیماریهای واگیر، اداره مبارزه با بیماریهای قابل انتقال بین انسان و حیوان، 1392 دکتر سید عباس حسینی تقوی، روشهای آزمایشگاهی تشخیص تب مالت ( سرولوژی). وزارت بهداشت درمان و آموزش پزشكي، اداره كل آزمايشگاه مرجع سلامت.

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