Viral Diagnostic

8/14/2019 Viral Diagnostic

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Why Perform Viral DiagnosticTests?

Can aid the physician with further therapeuticmeasures and interventions

Results have prognostic value

Availability of rapid methods and antiviralchemotherapeutic agents

Early and specific viral diagnosis may permit

earlier institution of preventive public healthmeasures


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Approaches to Viral Diagnosis

Cytologic studies

Intracellular viral inclusions, giant cells, syncytia

Direct examination by electron microscopy

Especially helpful for non-cultivable agents of humanviral gastroenteritis

Isolation of viruses in tissue culture, avian hostsystems, or animals

Demonstration of viral components directly in clinicalspecimens or in host-cell systems by immunologic ormolecular methods


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Owls Eye Inclusion of Cytomegalovirus

(CMV) in Lung Tissue


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Electron Micrograph of HerpesSimplex Virus type 1 (HSV-1)


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General Guidelines for Viral

Specimen Collection Best chance for isolation of viruses exists

when the specimen is collected as early inthe clinical course as possible

Respiratory viruses shed for 5-7 days, withhighest titers during the prodromal period

HSV and VZV may not be recoverable assoon as 5 days after onset of symptoms

Collect 7-10 ml of clotted blood (red toptube) during the acute phase of the illness


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Time Course of Viral Infection


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Swabs for Collection of Virology

Specimens

Swab material should be made of sterile Rayon, orDacron

DO NOT USE CALCIUM ALGINATE SWABS FOR

VIROLOGY SPECIMENS!

Toxic to enveloped viruses (e.g., HSV)

False-positive direct fluorescent antibody tests

DO NOT USE SWABS WITH WOODEN SHAFTS!

Formaldehyde in wood may inhibit recovery ofviruses


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Viral Transport Medium (VTM)

Buffered saline or other isotonic solution with aprotein stabilizer

Stabilizers

Gelatin, whole serum, bovine serum albumin,bacteriologic broths (e.g., brain-heart infusion),tissue culture medium (e.g., HBBS) with serum

Antibiotics

Penicillin or vancomycin

Streptomycin or gentamicin

Amphotericin B

pH of 7.2-7.4


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Identification of Viruses in Culture

Type of cell line, time to detection and nature of thecytopathic effect (CPE)

Confirmatory identification

Neutralization Hemagglutination/hemadsorption inhibition

Direct and indirect fluorescent antibody tests

Immunoperoxidase testing

Enzyme immunoassay

In situhybridization


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Identification by Neutralization


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Identification by Direct FluorescentAntibody (DFA) Staining

Employs monoclonal antibodies conjugated to fluoroisothiocyanate (FITC, fluorescein)

Rapid

Can be used to identify CMV, HSV, and other virusesin shell vials after 24-48 hours


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Characteristic CPE of RSV Observed in A549Cell Line (l), Cells Removed from Monolayer (r)


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Patient with Herpetic Vesicles on Lower Lip (l) &Direct Scraping of Cells from Unroofed LesionPlaced on Slide (r)


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Other Methods for Direct Detection ofViruses in Clinical Specimens

Enzyme immunoassay

RSV and influenza A in nasopharyngeal aspirates

Rotavirus and adenovirus types 40/41 in stool

specimens Electron microscopy/immune electron microscopy

Non-cultivable agents of gastroenteritis (e.g.,Norwalk agent, caliciviruses, astroviruses)

Nucleic acid amplification techniques

PCR, in situ hybridization


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EIA for Detection of RotavirusAntigen in Stool Specimens


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Immunoelectron Micrograph ofNorovirus (i.e.Norwalk-like Viruses) Also called small, round-

structured viruses about 27nm in diameter

Cause nausea, diarrhea,comiting, and cramping and

constitutional symptoms Transmitted by close contact

with infected people, fomitescontaminated with the virus,and eating contaminatedfood or drinkingcontaminated liquids

Cause of several outbreaksof gastroenteritis on cruiseships in recent years


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Rapid Tests for Influenza

Several available

Performed onnasopharyngealaspirate specimens

Vary significantly insensitivity/specificity


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In Situ Hybridization Assay


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Direct Detection by PolymeraseChain Reaction


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Methods for Viral AntibodyDetection

Complement fixation

Hemagglutination inhibition

Enzyme immunoassay Indirect fluorescent antibody assay

Latex agglutination assay

Western immunoblot IgM capture assay


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Complement Fixation Test


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Hemagglutination Inhibition (HAI)


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Hemagglutination Inhibition Assay for Detectionof Abs in Acute and Convalescent Serum

Highest dilution of serum where hemagglutination of RBCs bythe added virus occurs = endpoint

Acute phase = serum dilution of 1:10 (titer = 10)

Convalescent phase = serum dilution of 1:160 (titer = 160)

Greater than or equal to a 4-fold increase in titer is serumdilution of >= 1:80


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Hemagglutination Inhibition (HAI) Assay forDetection of Anti-Influenza A Abs in Acute andConvalescent Sera

Highest dilution of acuteserum specimenproducinghemagglutination

inhibition is 1:10 (titer =10)

Highest dilution ofconvalescent serumproducinghemagglutination-inhibition is 1:320 (titer= 320)


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Enzyme Immunoassay for Detection ofSpecific Viral Antibodies


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Indirect Fluorescent Antibody (ITA) Testfor Detection of Specific Viral Antibodies


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Latex Agglutination Tests for ViralAntibodies


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Western Immunoblot Procedure


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Structure of Human ImmunodeficiencyVirus type 1 (HIV-1)


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Western Immunoblot for HIV-1Antibodies Blots shows antibodies directed against

gp160/120

p66 p51

gp41

p31 p24

P17 Positive blot criteria

1. gp160/120 and p24

2. gp41 and p24 3. gp160/120 and gp41

No bands = Negative

Any other combination =indeterminant


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IgM Capture Assay for Detection ofSpecific IgM Antibodies

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Viral Diagnostic