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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