Lab diagnosis of viruses


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Lab diagnosis of viruses

  1. 1. Laboratory diagnosis of Viral infections 9/22/2013 Cristi Francis 1
  2. 2. River’s Postulates (Modified from Koch’s postulates) 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. 9/22/2013 Cristi Francis 2
  3. 3. Indications for lab diagnosis of viral infection • If rubella is diagnosed in the first trimester of pregnancy, abortion is recommended • If a baby is borne of an HbsAg positive mother ,abortion is recommended For proper management of certain diseases • for which antiviral chemotherapy is available (herpes viruses) Diagnosis of diseases caused by viruses • For hepatitis B & HIV virus helps to prevent spread of these viruses Screening of blood donors • To initiate appropriate control measures Early detection of epidemics 9/22/2013 Cristi Francis 3
  4. 4. 3 General approaches for Laboratory diagnosis of Viral infections Direct demonstration of virus & its components Isolation of virus Detection of specific antibodies 9/22/2013 Cristi Francis 4
  5. 5. Microscopy •Examine specimen for virusesElectron Microscope •Labeled antibody Immuno-electron microscopy •Fluorescent tag bound to Fc region of AbImmunofluorescence •Histological appearance of affected cells •Inclusion bodiesLight microscope 9/22/2013 Cristi Francis 5
  6. 6. Electronmicrographs Adenovirus Rotavirus (courtesy of Linda Stannard, University of Cape Town, S.A.)
  7. 7. Inclusion bodies • Inclusion bodies are virus-specific intracellular globular masses which are produced during replication of virus in host cells. • They can be demonstrated in virus infected cells under light microscope after fixation & staining • Eg: Negri bodies in rabies . 9/22/2013 Cristi Francis 7
  8. 8. Negri bodies Negri bodies neuron 9/22/2013 Cristi Francis 8
  9. 9. Isolation of virus • Laboratory animals • Fertilized Hen’s Egg Chorioallantoic membrane Allantoic cavity Amniotic cavity Yolk sac • Organ/Tissue/Cell Culture • Growth identified by serological method like neutralization. 9/22/2013 Cristi Francis 9
  10. 10. Regular Methods in Use • Egg inoculation Pox virus, Influenza • Into tissue culture Cristi Francis 109/22/2013
  11. 11. Egg inoculation 9/22/2013 Cristi Francis 11
  12. 12. Cell culture Cell Cultures are most widely used for virus isolation, there are 3 types 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, MDCK Primary cell culture are widely acknowledged as the best cell culture systems 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 viruses supported is often limited. 9/22/2013 12Cristi Francis
  13. 13. Cytopathic Effect (1) Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells. (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town) 9/22/2013 13Cristi Francis
  14. 14. Cytopathic Effect (2) Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom). (courtesy of Linda Stannard, University of Cape Town, S.A.) 9/22/2013 14Cristi Francis
  15. 15. Haemadsorption Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.) 9/22/2013 15Cristi Francis
  16. 16. Tissue culture Tissue culture / Explant culture Fragments of minced tissue can be used as “explants” This method is rarely used nowadays 9/22/2013 Cristi Francis
  17. 17. 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. Cristi Francis9/22/2013
  18. 18. Viral Serology – 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.9/22/2013 18Cristi Francis
  19. 19. 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 Cristi Francis 199/22/2013
  20. 20. Serology Criteria for diagnosing Primary Infection • 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera • Presence of IgM • Seroconversion • A single high titre of IgG (or total antibody) - very unreliable Criteria for diagnosing Reinfection • fold or more increase in titre of IgG or total antibody between acute and convalescent sera • Absence or slight increase in IgM 9/22/2013 Cristi Francis 20
  21. 21. PCR Each cycle doubles the copy number of the target A target DNA sequence can be amplified to the point where it can be readily identified using labelled probes in a hybridisation assay • The technique is used for the diagnosis of infections caused by HIV , HPV , Herpes simplex virus, Hepatitis B & C,Enterovirus,EBV ,Rubella & Rotavirus 9/22/2013 Cristi Francis 21
  22. 22. 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. • . 9/22/2013 Cristi Francis 22
  23. 23. Detection of specific antobodies Micro plate ELISA for HIV antibody: colored wells indicate reactivity Cristi Francis 239/22/2013
  24. 24. ELISA Procedures Figure 5-19a Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000. Figure 5-19b 9/22/2013 24Cristi Francis
  25. 25. ELISA • 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 9/22/2013 25Cristi Francis
  26. 26. Neutralization, hemagglutination, and hemagglutination inhibition assays. In the assay shown, tenfold dilutions of serum were incubated with virus. Aliquots of the mixture were then added to cell cultures or erythrocytes. In the absence of antibody, the virus infected the monolayer (indicated by CPE) and caused hemagglutination (i.e., formed a gel-like suspension of erythrocytes). In the presence of the antibody, infection was blocked (neutralization), and hemagglutination was inhibited, allowing the erythrocytes to pellet. The titer of antibody in the serum was 100. pfu, Plaque-forming units.From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6. Antibody detection Cristi Francis9/22/2013 26
  27. 27. Diagnostic methods for common viruses 9/22/2013 Cristi Francis 27
  28. 28. Thank You for your patient listening 9/22/2013 Cristi Francis 28