Viral Diagnosis V3'1

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Viral Diagnosis (V3.1)
Viral infections common but laboratory
investigations rare

• Lack of appropriate therapy
• Self limiting
• Clinical or general diagnosis.
• Bacterial disease is excluded
• Many lab tests are slow / difficult / costly
• Examples

.. reserved for those situations where they make
a significant contribution to management of the
patient.
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Overview of approaches (V3.2)

• Morphological
• Direct / indirect visual demonstration (EM, IF,
  histo or cyto). Specific but not sensitive.
• Immuno-serology
• Viral Ag or viral Ab, widely used, Ab levels can
  be hard to interpret and retrospective,
• Isolation
• ? Gold std. Slow and technical. Sens and spec.
• Nucleic acid
• ?Gold std, especially if PCR first. Sens and spec

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Morphological (V3.3a)
Viral infections common but laboratory
investigations rare

• Electron microscopy.
• Expensive equip / preparation
• High titre reqd
• Limited use, good if virus shape and location are
  specific.

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Morphological (V3.3b)

• Cytology / Histology
• Viral inclusions or cellular changes
• Nuclear or cytoplasmic
• Mostly cases of happenstance

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Morphological (V3.3v)

• Immunological staining
• Label the Ab (rgt) with fluoresceine or
  peroxidase. Direct and indirect.
• Can stain viral particles, or expressed proteins.

RSV
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Immunoserology Ag detection (V3.4a)

• ELISA
• Capture and detect.
• Solid phase, automated, quantifiable.
• Membrane bound single test

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Immunoserology Ag detection (V3.4b)

• Latex agglutination
• Simple, stable, now even without needing to add
  rgt.
• Rotavirus, Adenovirus popular

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Immunoserology Ab detection (V3.5)

• Used for diagnosis and immune status
• IgM - early, transient, not placental.
• IgG - for life.
• Positive IgM
• Recent / current infection?
• Paired sera
• Acute and convalescent samples.
• Rise in titre of IgG
• Single high titre.

PROBLEMS ? Immunosuppressed / re-activation or
re-infection / cause and effect
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Virus Isolation / Cell culture (V3.6a)

• Living cells as reagents
• Amplification, ?gold std, very sens and spec
• Good all purpose bait
• Cell lines
• Primary, semi-continuous, continuous
• Culture medium of..
• Nutrients, buffers and.?.....?

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Virus Isolation / Cell culture (V3.6b)

• Inoculate and wait.....
• for cytopathic effect (CPE)
• Changes in the nature of the cells that occurs as
  a result of viral growth.
• CPE a virus is growing
• False positives
• Viral contamination, cell contamination, toxins,
  aged cells
• Always include a non inoculated control.

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CPE and identification (V3.6c)

• Compare CPE to colonial morphology and cell lines
  to selective media.
• Can stain with immuno-fluorescent stain or with a
  cyto stain.
• Can do Ab neutralization.
• Can do nucleic acid detection.

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Limitations of cell culture (V3.6d)

• Requires equipment and operator skill.
• Ongoing costs even without doing any tests.
• Issues with transport and viability.

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Nucleic acid detection (V3.7)

• Initially done directly on specimens using
  labeled probes - poor sensitivity.
• PCR has revolutionized this approach.
• Limitless applications
• Limited only by commercial availability.
• Large labs design and validate in house
  applications.

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Lab Context (V3.8)

• Sometimes disease specific..or..
• Rash / febrile / lyphadenopathy request viral
  studies.
• Serology profile
• General isolation
• Specimens are often not from the affected organ
  or site, but often the site of shedding.
• Urine, saliva, throat swabs, blood, faeces.