Diagnosis of viral infections basics

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Diagnosis of viral infections basics

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Diagnosis of viral infections basics

  1. 1. DIAGNOSIS OFVIRAL INFECTIONS basics Dr.T.V.Rao MD Dr.T.V.Rao MD
  2. 2. Koch’s Postulates Applicable in many bacterial diseases1. Organism present only in diseased individuals2. Organism cultivated in pure culture from diseased individual
  3. 3. Koch’s Postulates3. Organism causes disease when injected into healthy individuals4. Organism re-isolated from infected individual from point 3. Figure 1.15
  4. 4. River’s Postulates• T.M. River, 1937• Modified from Koch’s Postulates (proof of bacterial diseases)1. Isolate virus from diseased hosts.2. Cultivation of virus in host cells.3. Proof of filterability.4. Production of a comparable disease when the cultivated virus is used to infect experimental animals.5. Reisolation of the same virus from the infected experimental animal.6. Detection of a specific immune response to the virus.
  5. 5. Methods of StudyMuch more expensive and difficult to study animal viruses than bacteriophages• Cultivation in host cells – Living animal – Embryonated chicken eggs – Cell or tissue culture (= in vitro)
  6. 6. Laboratory Diagnosis of Viral Diseases Different from Bacterial Infections Dr.T.V.Rao MD 6
  7. 7. Viral Diagnostics in the Clinical Laboratory• Over 60% of all infectious disease cases seen by a physician are due to viral infections.• Quality of patient specimens and their transport to the laboratory is important.
  8. 8. Storage and Collection of Biological Specimens for Viral Testing• What types of specimens are collected to diagnose? – Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum – Eye infections: throat and Conjunctival swab/scraping – Gastrointestinal tract infections: stool and rectal swabs – Vesicular rash: vesicle fluid, skin scrapings – Maculopapular rash: throat, stool, and rectal swabs – CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid – Genital infections: vesicle fluid or swab – Urinary tract infections: urine – Blood borne infections: blood
  9. 9. Three General Approaches forLaboratory Diagnosis of Viral Infections• Direct detection –Microscopy or staining• Virus Isolation –PCR• Serology –Antibodies
  10. 10. Why Difficult• In the past Growth of Virus was not rapid.• Diagnosis becomes routine today due to availability of Rapid method.• Important in HBV and HIV infections.• Rubella in pregnant women.• Simple methods – Microscopy and detection of inclusion bodies. Dr.T.V.Rao MD 10
  11. 11. Diagnostic Methods in Virology1. Direct Examination2. Indirect Examination (Virus Isolation)3. Serology Dr.T.V.Rao MD 11
  12. 12. Microscopy• Light Microscopy – elementary bodies• Electron Microscopy Rota viral detection• Florescent Microscopy Direct / Indirect Dr.T.V.Rao MD 12
  13. 13. Direct Detection• Electron Microscopy – Examine specimen for viruses• Immuno-electron microscopy – Labeled antibody• Immunofluorescence – Fluorescent tag bound to Fc region of Ab
  14. 14. Direct Examination1. Antigen Detection immunofluorescence, ELISA etc.2. Electron Microscopy morphology of virus particles immune electron microscopy3. Light Microscopy histological appearance inclusion bodies4. Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR) Dr.T.V.Rao MD 14
  15. 15. Direct Detection• Electron Microscopy – Examine specimen for viruses• Immuno-electron microscopy – Labeled antibody• Immunofluorescence – Fluorescent tag bound to Fc region of Ab
  16. 16. Quantitative Assays• Plaque assays –Lytic viruses only –Steps • Serial dilution of virion- containing solution • Create tissue culture plates • Spread diluted virus • Overlay with agar— prevents diffusion • Count number of plaques Figure 5.8: Plaque assays used to –Each plaque represents 1 quantitate a viral stock. PFU (Plaque Forming Unit) Courtesy of Teri Shors
  17. 17. Indirect Examination1. Cell Culture Cytopathic effect (CPE) haemabsorption immunofluorescence2. Eggs pocks on CAM haemagglutination inclusion bodies3. Animals disease or death Dr.T.V.Rao MD 17
  18. 18. Serology Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.Classical Techniques Newer Techniques1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay Dr.T.V.Rao MD 18
  19. 19. Virus IsolationCell Cultures are most widely used for virus isolation, there are 3types of cell cultures: 1. Primary cells - Monkey Kidney 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCKPrimary cell culture are widely acknowledged as the best cell culturesystems available since they support the widest range of viruses. However,they are very expensive and it is often difficult to obtain a reliable supply.Continuous cells are the most easy to handle but the range of virusessupported is often limited. Dr.T.V.Rao MD 19
  20. 20. Cytopathic Effects• Visible results of viral infection• Cell death by – Multiplying viruses – Inhibition of DNA, RNA or protein synthesis – Effects on permeability of membrane• Cytopathic effects (CPEs) of infected cells can be observed with inverted light microscopes • Rounding/detachment from plastic flask • Syncytia/fusion – Fusion of cells • Shrinkage • Increased refractivity • Aggregation • Loss of adherence • Cell lysis/death• Common observations of CPEs – Inclusion body formation • Intracellular virus parts (replication or assembly) – Hemadsorption assays
  21. 21. Cell CulturesGrowing virus may produce 1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific. 2. Haemabsorption - cells acquire the ability to stick to mammalian red blood cells.Confirmation of the identity of the virus may be carried out usingneutralization, haemabsorption-inhibition or immunofluorescencetests. Dr.T.V.Rao MD 21
  22. 22. Detection of Viral antigen• Use of Immunofluorescence Imunoelectrphoresis Radio immunoassay RIA ELISA Dr.T.V.Rao MD 22
  23. 23. Serology• Since the isolation and identification of viruses is not commonly done in the clinical laboratory, the clinical picture and serology plays a greater role in the diagnosis of viral disease. The major types of antibodies that are assayed for are neutralizing, haemagglutination inhibiting and complement fixing antibodies. Complement fixing antibodies follow the kinetics of IgM and are most useful in indicating a current or recent infection Dr.T.V.Rao MD 23
  24. 24. Serology • The development of antibodies to different components of the virus is used in staging the disease. For example in hepatitis B and HIV infections this approach is used. Dr.T.V.Rao MD 24
  25. 25. Viral Serology • Indirect – Primary and secondary responses to viral infections • IgM (1st exposure) • IgG (2nd exposure)Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.
  26. 26. Serological Diagnosis• Detection of Immunologlublins Ig G. Ig M Ig A• Raise of titers Ist sample later sample (convalescent sample) tested after 10 – 14 days Raise of titer is diagnostic Dr.T.V.Rao MD 26
  27. 27. Viral Serology• Enzyme-Linked Immuno-Sorbant Assays (ELISAs) – Enzyme reacts with substrate to produce colored product – Very sensitive • HIV test – If positive twice, Western Blotting is performed next
  28. 28. ELISA for HIV antibodyMicro plate ELISA for HIV antibody: colored wells indicate reactivity Dr.T.V.Rao MD 28
  29. 29. © Hank Morgan/Science Photo Library/Photo Researchers, Inc.HIV ELISA test.
  30. 30. ELISA ProceduresFigure 5-19a Figure 5-19b Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000.
  31. 31. Western BlotHIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive Dr.T.V.Rao MD 31
  32. 32. (b) (c) Image courtesy of Bio-Rad LaboratoriesFigure 5.21b: The structure of HIV-1. Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.
  33. 33. Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.Figure 5.21a: The basic principles behind the Western blotting procedure.
  34. 34. Virus Isolation– Nucleic acid methods • PCR (DNA), RT-PCR (RNA) • Can be used to detect viruses that are noncultivatable • Rapid identification (e.g. RT-PCR—4 Corners outbreak of hantavirus or FRET in the field) • Can be used to manage patients (e.g. HIV viral load) Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
  35. 35. Polymerase Chain Reaction• Advantages of PCR: – Extremely high sensitivity, may detect down to one viral genome per sample volume – Easy to set up – Fast turnaround time• Disadvantages of PCR – Extremely liable to contamination – High degree of operator skill required – Not easy to set up a quantitative assay. – A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not. Dr.T.V.Rao MD 35• .
  36. 36. Schematic of PCR Dr.T.V.Rao MD 36Each cycle doubles the copy number of the target
  37. 37. Growing Technologies• Molecular methods• Probes• Polymerase chain reaction Can produce Rapid Highly scientific Specific Dr.T.V.Rao MD 37
  38. 38. Regular Methods in Use• Egg inoculation Pox virus, Influenza• Into tissue culture Dr.T.V.Rao MD 38
  39. 39. Advantages of Molecular Methods with• To Increases Sensitivity and Specificity.• With PCR• RT PCR Dr.T.V.Rao MD 39
  40. 40. • Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Professionals in the Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 40

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