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Viral Diagnostics

Overview. Clinical virology lab can provide significant benefit to patient careTraditionally epidemiologic and academic roleCurrent rapid assays impact on therapeutic and public health decisions. Change largely due to molecular methods. Impact of PCR on virology. Recent identification of several

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Viral Diagnostics

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    1. Viral Diagnostics Jonathan Gubbay Ontario Agency for Health Protection and Promotion Public Health Laboratory-Toronto

    2. Overview Clinical virology lab can provide significant benefit to patient care Traditionally epidemiologic and academic role Current rapid assays impact on therapeutic and public health decisions. Change largely due to molecular methods

    3. Impact of PCR on virology Recent identification of several respiratory viruses Metapneumovirus Multiple coronaviruses: SARS, 229E, NL63, OC43, HKU1. Human bocavirus Polyomaviruses KI, WU

    4. Why Expanding Role for Diagnostic Virology Lab Increased pool of immunocompromised Increasing antiviral agents Results in increasing demand for rapid methods, viral load testing, antiviral susceptibility, genotyping.

    5. Methods in use in virology. Detecting Active Infection: Electron Microscopy Viral culture Detection of viral antigens Detection of viral nucleic acid. Histopathology Assessing virus-specific immune response Serologic testing (won’t cover today)

    6. Specimen choice and collection Specimen quality limits test quality Pathogen detection depends on: Appropriate collection site. Proper timing of specimen collection. Effective and timely processing of sufficient specimen.

    7. Specimen storage and transport Keep specimens other than blood at 4şC If delay >24hrs, freeze at 70şC or below. Avoid any storage at -20şC: greater loss in infectivity Nonenveloped viruses (adenovirus, enteroviruses) more stable than enveloped (e.g. RSV, VZV, CMV).

    8. Viral Transport Medium Salt solution – ensures proper ionic concentrations Buffer - maintains pH Protein - for virus stability Antibiotics or antifungals – to prevent contamination

    9. Cell Culture Viruses are obligate intracellular organisms – require living cells for virus isolation Advantages: Relatively sensitive and specific Can detect many different viruses Provides a viral isolate for further characterization (serotyping, genotyping, susceptibility)

    10. Cell culture –limitations Certain viruses don’t grow or grow slowly Other techniques for detecting viral infection more cost effective Successful culture depends on viability of virus in specimen

    11. Standard Cell Cultures Originally used animals and embryonated eggs. Monolayer cell culture techniques (1933) Roller tube cell cultures (1940)

    12. Standard Cell Cultures Primary cells 1-2 passages Diploid (semicontinuous) cells 20-50 passages Heteroploid cells. Indefinite passages

    13. Cytopathic Effect Monitor tube cultures daily initially Monitor for 10-21 days Compare to uninoculated controls from same batch Rounding, refractile cells, syncytium formation, cell destruction

    14. Cell culture – clues to virus causing CPE Type of specimen Cell line displaying CPE Type of CPE

    15. Hemadsorbing viruses Orthomyxoviruses (influenza) and some paramyxoviruses (parainfluenza, measles, mumps) Insert viral glycoproteins (haemaglutinin) into host cell membrane. Promotes attachment of RBC of certain species (e.g guinea pig) to cell membrane.

    16. Interference Rubella virus growing in monkey kidney cells inhibits infection with echovirus 2.

    17. Adenovirus CPE

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