Diagnostic Virology Flashcards by Sarah Rehl

Q

HSV Test Specificity, Cost Time

A

Glycoprotein G ELISA= cheapest, 1-2 weeks
Genital Culture= $90, 24 hrs- fastest
Western blot= most expensive, 2 wks

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Q

Respiratory syncytial virus (RSV)

A

is a very common virus that leads to mild, cold-like symptoms in adults and older healthy children

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Q

RSV vs. Flu

A

institute droplet precautions

RSV= order respiratory viral DFA (direct fluorescent antibody)

Influenza= order influenza A/ H1N1 PCR

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Q

why test for viral infections?

A

Some viruses are susceptible to antiviral compounds.

Patient treatment may depend upon virus.

Public health measures can be instituted.

Surveillance can determine significance and prevalence of disease.

Surveillance can identify new viruses or significance of “old” viruses

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Q

Diagnostic Methods in Virology

A

Direct Examination - visualization
Indirect Examination - virus isolation
Serology – antibody reactions

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Q

Direct Examination (visualization)

A

Antigen Detection -immunofluorescence, ELISA etc.
Electron Microscopy-morphology of virus particles
- immune electron microscopy
Light Microscopy -histological appearance
- inclusion bodies
Viral Genome Detection- hybridization with specific nucleic acid probes & polymerase chain reaction (PCR)

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Q

Immunoelectron Microscopy Technique

A

direct examination

Antibody binding to virion forming clumps or aggregates followed by visualization

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Q

Morphology of Virus Particles- Errors

A

Morphology based identification can be misdiagnosed in:

Parainfluenzavirus and Paramyxovirus (Both are enveloped with peplomeres)
Coronavirus virions (SARS) confused with morphology of Paramyxovirus

delayed treatment and public health measures

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Q

Symptomology: Respiratory

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: Nasal or throat swab; sputum
Virus: influenza

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Q

Symptomology: Eye

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: Corneal or conjunctiva swab
Virus: adenovirus

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Q

Symptomology: Central Nervous System

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: CNS- cerebrospinal fluid
Virus: herpes

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Q

Symptomology: Skin

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: skin lesion; vesicle swab/scraping, punch biopsy
Virus: herpes; papilloma

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Q

Symptomology: Genital disease

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: Mucosal swab or scraping
Virus: herpes; HIV

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Q

Symptomology: Enteric Disease

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: Fecal Swab; Urine Sample
Virus: Fecal= norovirus; urine= cytomegalovirus (genus of herpes)

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Q

Symptomology: General Infection

Collecting Viral samples for Laboratory Diagnosis

A

Specimen: Blood sample; throat swab; fecal swab
Virus: blood= HIV & West Nile

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Q

Symptomology: Autoposy/ Biopsy

Collecting Viral samples for Laboratory Diagnosis

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A

Specimen: tissue from diseased organs
Virus: rabies

Q

Indirect Examination (virus isolation)

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A

look for CPE, disease or antibodies

Cell Culture cytopathic effect (CPE)
hemadsorption (HA – Influenza)
immunofluorescence
Eggs pocks on CAM (chorioallantoic membrane)
hemagglutination
inclusion bodies
Animals disease or death

Q

Serology (antibody interactions)

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A

looking for increase in IgM or titer over time

Detection of rising titers of antibody between acute and convalescent stages of infection
or
the detection of IgM in primary infection.

Q

What is most widely used for virus isolation?

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A

Cell cultures

Q

Types of cell cultures

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A

Primary cells (**the best cell culture systems available since they support the widest range of viruses, but very expensive & often difficult to obtain a reliable supply)
- Monkey Kidney, foreskin
Semi-continuous cells
- Human embryonic kidney and skin fibroblasts
Continuous cells (*most easy to handle but range of viruses supported is often limited)
- HeLa, Vero, Hep2 (human laryngeal carcinoma cells contain human papillomavirus, HeLa contaminant ), LLC-MK2 (rhesus monkey kidney), MDCK (Madin Darby Canine Kidney)

Q

Growing a virus in cell cultures may produce…

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A

Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific.
Hemadsorption - cells acquire the ability to stick to mammalian red blood cells.

Q

Confirmation of the identity of a virus (in a cell culture)

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A

Use:
neutralization
hemadsorption-inhibition
immunofluorescence tests

Q

Diagnostic test for genital and oral herpes

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A

Viral culture needs to be examined

Q

Immunofluorescence

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A

labeled second antibody reacts to the first labeled antibody

antibody binds to to intracellular antigen; fluorescein-labeled anti-Ig binds; fluoresces by UV microscopy

DFA (high sensitivity and specificity)= diagnostic method for rabies

Enzyme Immunoassay: EIA ELISA

diagnostic test that detects the virus Need anti-virus antibody (binds specimen), specimen (virus), enzyme linked to antibody & substrate antibody binds to antigen; enzyme labeled anti-Ig binds to antibody; substrate color changes

Classic Molecular Techniques

ex: Dot-blot, Southern blot, in-situ hybridization - depend on the use of specific DNA/RNA probes for hybridization - specificity of the reaction depends on the conditions used for hybridization - more tedious and expensive than conventional techniques

Southern Blot of *32P-HPV-8 to HPV-5C | what is detected in this test?

determines type of HPV based on size Specimen is cut with different specific enzymes, electrophoresed, transferred to membrane, radioactive HPV hybridized to other HPV strains, different bands show depending on restriction enzyme add sizes of restriction fragments to determine total size

Polymerase Chain Reaction (PCR)

detects DNA template amplifies DNA needs ds template, denature to bring out DNA, add primers, make lots of copies (~30 cycles)

Serological Procedures

detects antibody- antigen interaction - enzyme immunoassay - radioimmunoassay - western blot - latex particle agglutination - Virus neutralization - Hemmagglutination inhibition - Immunofluorescence - Immunodiffusion - Complement fixation

Radioimmunoassay

antibody binds to antigen; radiolabeled anti-Ig binds to antibody RIA's robustness + historical use as golden standard + consistently reliable results + low reagent cost per test= RIA methods are again becoming popular

Western Blot

virus disrupted, proteins separated by gel electrophoresis and transferred (blotted) onto nylon membrane; antiserum binds to viral proteins; labeled anti-Ig binds to particular bands; revealed by EIA or autoradiography

Hemagglutination Inhibition Test

Tests for how much antibody is in serum antibody binds to virus that agglutinate RBCs antibody titer= serum dilution that hemagglutination is inhibited components: microtiter plate, test serum, virus, red blood cells

Immunoblotting

used to confirm HIV positive blood -Western blot tests antibodies for several proteins: glycoprotein gp120 and p24 core 1. protein separation 2. protein transfer 3. antigen detection (using labeled antibody)

Radioimmunoassay or Enzyme Immunoassay

Detects antibody (determine if you had a recent infection)

what is the gold standard diagnostic method?

virus isolation

Lab hazards during diagnostic procedures

aerosol, ingestion, skin penetration

RT-PCR and Internal Standar RNA

internal standards of a different template help determine original concentration of the HIV RNA copies real time quantitative PCR (qCR)

Real Time qPCR Probes

(A) TaqMan probes Exonuclease activity of Taq pol removes probe dye (B) FRET --Fluorescent resonance energy transfer Energy transfer between two adjacent probes releases fluorescent light (C) Molecular Beacons Quenching of fluorophores until hybridization occurs

What virus causes desquamation of | the ciliated tracheal cells?

Influenza & rhinovirus