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Pathology (113)
PATH 3610 (113)
Rob Foster (35)
Lecture 3

Lecture 3 notes

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PATH 3610
Rob Foster

LECTURE 3 – DIAGNOSIS Requirements for diagnostic methods - Standardized test and reagents of good quality - Miniaturized assays to conserve reagents and reduce costs - Speed - Simplicity - Cost-effectiveness - Automated (high tech instruments) - Computerized analysis and printouts: objectivity, reporting, record keeping, billing - Specialized laboratories Sensitivity vs specificity Sensitivity - If a person has a disease, how often will the test be positive (true positive rate)? Put another way, if the test is highly sensitive and the test result is negative you can be nearly certain that they don’t have disease. Specificity - If a person does not have the disease how often will the test be negative (true negative rate)? In other terms, if the test result for a highly specific test is positive you can be nearly certain that they actually have the disease. Methods of viral diagnosis  Detecting active infection: o Viral culture o Histopathology o Electron microscopy o Detection of viral antigens o Detection of viral nucleic acid  Assessing virus-specific immune response: o Serologic testing Measures of viral infectivity Different viral detection methods (pros and cons) The methods can be broadly divided into 3 categories: 1. Those that measure virus infectivity 2. Those that examine viral serology 3. Those that rely on molecular methods Approaches to detection of viruses: I. Virus isolation II. Direct detection of virus III. Indirect detection of virus I. Virus isolation - In animals - In embryonated eggs o Virus replication is evidenced by: death of embryo (or lesions), lesions/pocks on chorio-allantoic membrane (CAM) – each pock formed by one infectious unit, morphology and color of the pock is often characteristic of a particular group of viruses - In cultured cells o Recognition of virus growth in cell culture: cytophatic effects (rounding, clumping, fusion, syncytium, inclusion body) Advantages – disadvantages • Virus isolation – gold standard  a single infectious particle could be sufficient • Only method that can detect unexpected viruses • Supply of particles for further characterization (especially important for unknown viruses) • Supply of particles for archiving • Slow • Expensive • Technically demanding, only in specialized laboratories • Some agents cannot be propagated II. Direct detection of virus Plaque Assay - Lytic viruses only - Each plaque represents 1 PFU (plaque forming unit) Indicator cells which contain a virus-inducible promoter - Genetically engineered cell lines - Promoters usually attached to detectable gene (β-galactosidase) Transformation assay - Used to determine the titers or the biological activity of non-plaque forming viruses that have the ability to cause cellular growth transformation - Transformed cells show other cytophatic effects – e.g. loss of contact inhibition - Rather than count plaques, count foci  focus is when cells pile up (tumor formation) - FFU = focus forming unit Endpoint dilution assay - Viruses that do not lyse the cell membrane - Add serially diluted aliquots od virus to cultured cells and scored for the presence of viral replication  analyze cells or cell culture fluids for the presence of viral proteins or enzymes; or probe with fluorescently labeled antibodies against a viral antigens, and use fluorescence microscopy to count and quantify the number of foci. 1. Direct visualization of virion particles (EM) – advantages and disadvantages • Morphological, staining characteristics of the virus  direct identification • Fast, simples, cheap •
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