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    Si Ola

    Uploaded by

    Matthew Baluyot

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    © All Rights Reserved
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    Si Ola

    Uploaded by

    Matthew Baluyot
    43 views32 pages

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    SI OLA.pptx

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    43 views32 pages

    Si Ola

    Uploaded by

    Matthew Baluyot

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 32

    Serology and

    Molecular
    Detection of Viral
    Infection
    Epstein-Barr Virus
     Rapid Slide Agglutination Test (Monospot Test)

     Indirect Immunofluorescence Assay (IFA)

     Enzyme –Linked Immunosorbent Assay (ELISA)

     Chemiluminescent Immunoassay (CLIA)

     Flow Cytometry
    Cytomegalovirus

    Viral Culture

    CMV Antigenemia Assay

    CMV DNA or mRNA

    Assays for CMV IgG

    Assays for CMV IgM


    Varicella-Zoster Virus
     Culture of Virus

     Tzanck cells in stained smears

     Direct Immunofluorescence

     Real-time PCR for VZV DNA

     Quantitative real-time PCR

     Fluorescent antibody to membrane antigen (FAMA)

     Enzyme –Linked Immunosorbent Assay (ELISA)


    Other Viral Infections
    Rubella
     Culture of Virus
     Demonstration of viral RNA
     Detection of virus-specific antibodies
     RT-PCR
     IFA
     EIA
     Hemagglutination Inhibition (HI)
     Latex Agglutination
     Immunoassay
     Solid- Phase capture ELISA
    Other Viral Infections
    Rubeola
     IgM capture ELISA
     Rubeola specific IgG (ELISA)
     RT PCR
    Other Viral Infections
    Mumps
     Real-time RT-PCR
     ELISA
     solid phase IgM capture assays
    Other Viral Infections
    Human T-Cell Lymphotropic Viruses (type 1)
    Human T-Cell Lymphotropic Viruses (type 2)
     ELISA
     CLIA
     Particle Agglutination
     Western Blot Assay
     Line Immunoassay (LIA)
     IFA
     PCR
     Human herpesvirus (has carrier state after primary infection)

     Dr. M. Anthony Epstein & Yvonne Barr (1964)

     Most intensively studied human cancer virus

     1984 - entire genome of one EBV strain was completely sequenced

     Drs. Werner and Gertrude Henle

    Established relationship of EBV to several cancers (ex. Burkitt’s


    lymphoma)

     Parasitizes every cell system


    signal transduction
    cell cycle control
    regulation of gene expression
    posttranscriptional RNA processing
    protein modification and stability
    DNA replication.
    SEROLOGY
     Adult: 10-20% with acute IM do not produce IM heterophile antibody

     Pediatric: more than 50% y0unger than 4 years with IM are heterophile negative

     Candidates for EBV serology include those who:


    Do not exhibit classic symptoms of IM
    Heterophile negative
    Immunosuppresed

     Infection with EBV results in the expression of:


    Viral capsid antigen (VCA)
    Early antigen (EA)
    Nuclear antigen (NA)

     With corresponding antibody responses


    SEROLOGY
    Viral Capsid Antigen (VCA)
     Produced by infected B cells
     Can be found in the cytoplasm
     Anti-VCA IgM: detectable early in the course of infection, but low in concentration, disappears w/in 2-4 months
     Anti-VCA IgG: detectable w/in 4-7 days after onset of signs and symptoms, persists for an extended period
    (lifelong)
    SEROLOGY
    Early Antigen (EA)

     A complex of two components:


    Early antigen-diffuse (EA-D): nucleus and cytoplasm of the B cells
    Early antigen-restricted (EA-R): found as mass in the cytoplasm

     Anti-EA-D IgG and anti-EA-R IgG are not consistent indicators of the disease stage
    SEROLOGY
    Epstein-Barr Nuclear Antigen (EBNA)

     Found in the nucleus of all EBV-infected cells


     Does not become available for stimulation until after incubation period of IM
     Antibodies to NA are absent during acute IM
     Anti-EBNA IgG does not appear until convalescent period
     NA gradually increase through convalescence and reach plateau 3-12 months after infection
     Previous exposure to EBV: titer 1:10-1:60
     Nasopharyngeal carcinoma: high
     Burkitt’s lymphoma: barely detectable to very high
    SEROLOGY
    Immunofluoroscence
     Antigen substrate slides containing EBV-infected B cells are incubated w/ patient’s serum

     Addition of fluorescein-conjugated antihuman IgG or IgM

     Disadvantages:
    time-consuming
    difficult to interpret
    prone to interference from other serum components
    SEROLOGY
    Enzyme-linked immunosorbent assay (ELISA)
     May be used to detect antibodies to EBNA
     Uses synthetic peptide antigen
     Sensitivity: 98.9%
     Specificity: 99.0%
    SEROLOGY
    Paul-Bunnell Screening Test
    Principle:
     Hemagglutination test which detects heterophile antibodies in patient serum when mixed with antigen-
    bearing sheep erythrocytes
     Dilutions of inactivated patient serum+ sheep erythrocytes (incubated, centrifuged, macroscopically
    examined for agglutination)
     Positive reactions (preliminary)

    Sources of Error:
     Hepatitis infection and Hodgkin’s disease
     Improperly inactivated serum (causes hemolysis)

    Clinical Applications:
     For presence of heterophile antibodies
     Simple and inexpensive
     Negative results with individuals who do not produce IM heterophile antibodies (EBV serology may be
    indicated)
    SEROLOGY
    Paul-Bunnell Screening Test

    Limitations:
     Only indicates presence or absence of heterophile antibodies
     Doesn’t distinguish between antibodies assoc. with IM or serum sickness, or Forssman antigen
     Heterophile antibody assay lacks sensitivity as diagnosic criterion for IM
     Sheep erythrocytes are less sensitive than from other species
     Patient may take up to 3 months before developing detectable heterophiletiter
    SEROLOGY
    Davidsohn Differential Test
    Principle:
     Classic procedure distinguishes between heterophile antibodies that agglutinate in antigen-bearing erythrocytes
    of sheep

     Differential nature: sheep and beef (ox) erythrocytes bear some common antigens not present on the kidney
    cells of guinea pig. Exposure of patient serum to guinea pig cells (rich in Forssman antigen) produces differential
    absorption.

     Differentiates heterophile types of antibody assoc. with IM or serum sickness.

     Performed only if the preliminary Paul-Bunnell test is positive in a titer 1:56 or greater.

     Serum sickness occurs as result of sensitization to animal serum (usually, horse serum)

     Agglutinins for Sheep Erythrocytes in Human Serum


    SEROLOGY
    Davidsohn Differential Test
    SEROLOGY
    Davidsohn Differential Test

    Reporting results:
     If reduced titers with either beef or guinea pig cells, antibody source can be attributed
    to one of the heterophile antibody types.
     Normal serum: 1:28 with occasional 1:56 result
     In IM: suspicious 1:56 titer and a titer of 1:224 as positive result

    Sources of error:
     Incorrect pipetting/ use of nonactivated serum

    Clinical applications:
     Distinguish between three types of heterophile antibodies

    Limitations:
     Time-consuming
    SEROLOGY
    MonoSlide Test
    Principle:
     The BBLMonoSlide procedure (BD BBLMonoSlide, Becton, Dickinson, Franklin Lakes, NJ) is based on
    agglutination of horse erythrocytes by heterophile antibody present in IM.

     Horse RBCs exhibit antigens directed against both Forssman and IM antibodies, thus, a differential
    absorption of patient serum is necessary to distinguish specific heterophile antibody from Forssman type.

     absorption steps comparable with Davidsohn’s sheep agglutinin test

     Serum/plasma is absorbed with both guinea pig kidney (contains only the Forssman antigen) and beef
    erythrocyte (contains only the antigen associated with IM) stroma

     Therefore, guinea pig kidney absorbs only the Forssman type of heterophile antibody and beef erythrocyte
    absorbs only the heterophile antibody of IM

     Uses disposable card, guinea pig kidney antigen for absorption, and specially treated horse eryhrocytes
    (color enhanced) to ↑ specificity, ↑ sensitivity and ↑ readibility.

     No special equipment needed for reading


    SEROLOGY
    MonoSlide Test

    Reporting Results: Qualitative Method


     Positive: dark clumps against blue-green background, uniformly distributed throughout the test circle
     Negative: no agglutination, may have fine granulariy against brown-tan background or peripheral color development
    assoc. with fine granularity

    Procedure Notes:
     If positive with qualitative method, titration procedure may be performed to provide quantitative indication of the
    level of heterophile antibody

    Sources of Error:
     False-positive results may be caused by:
     Observing agglutination after observation time
     Misinterpreting agglutination
     Simultaneous occurrence of IM and hepatitis has been reported
     Residual heterophile antibody present after clinical symptoms have subsided
    SEROLOGY
    MonoSlide Test
    SEROLOGY
    MonoSlide Test

    Clinical Applications:
     Infectious diseases may mimic symptoms of IM like influenza, rubella, and hepatitis
     Positive test result indicates presence of heterophile antibody specific for IM

    Limitations:
     Diagnosis of IM should be based on results of all clinical and lab findings.
     Some segments of population do not produce detectable heterophile antibody.
     Detectable levels of heterophile antibody may persist for months and even years.

    Note:
     Other forms of rapid testing include Wampole Laboratories Colorcard Mono and Mono-plus
    MOLECULAR
    Quantitative Real Time PCR (qPCR)

     EBV DNA present within malignant cells


     EBV PCR may help identify genomic polymorphisms or deletions
     The assay that many consider to be the gold standard for identifying tumor-associated virus is EBER in
    situ hybridization.
    MOLECULAR
    Quantitative Real Time PCR (qPCR)

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