Laboratory Diagnosis of Viral Infections. G. Jamjoom 2005

Transcription

1 Laboratory Diagnosis of Viral Infections G. Jamjoom 2005

2 Five Main Techniques: Virus Culture and Isolation Serology Rapid Detection of Viral Antigens Detection of Viral Nucleic Acid Electron Microscopy

3 Virus Culture and Isolation Which cell lines How many cell lines

4 Virus Culture and Isolation Cytopathic Effects (CPE)

5 Cell Lines Primary Continuous

6 Examples of Commonly Used Cell Lines : Primary Cell Lines : - monkey kidney cells - human foreskin fibroblasts

7 Examples of Commonly Used Cell Lines : Continuous Cell Lines : - VERO - HEP-2 -MDCK

8

9 tissue culture cells epithelial epithelioid fibroblastic slides from CDC

10 epithelial cells - adenovirus uninfected early infection late infection slides from CDC

11 fibroblastic cells - poliovirus slides from CDC uninfected early infection late infection

12 Diluted 10 fold Diluted 100 fold Diluted 1000 fold

13 PLAQUE ASSAY

14 PLAQUE FORMING UNIT P.F.U. pfu

15 Cytopathic Effects : Rounding Detachment (plaques) Clumping Ballooning (Giant Cell) Fusion (Syncytium Formation) Inclusion Formation

16 Cytopathic Effect of HSV in cell culture: ( Note the ballooning of cells).

17 Cytopathic Effect of CMV (Courtesy of Linda Stannard, University of Cape Town, S.A.)

18 Cytopathic Effect of VZV

19 ADENOVIRAL INCLUSION BODIES

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22 Virus Culture and Isolation Shell Vial

23 DEAFF test for CMV (Virology Laboratory, New-Yale Haven Hospital)

24 Rapid Detection of Viral Antigens

25 CMV pp65 antigenaemia test (Virology Laboratory, New-Yale Haven Hospital)

26 Positive immunofluorescence test for HSV antigen in epithelial cell.

27 Serology : Hemagglutination Inhibition

28 hemagglutination

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31 Serology : No rise Acute Sample Convalescent Sample

32 Serology : Two-fold rise (not significant) Acute Sample Convalescent Sample

33 Serology : Four-fold rise (significant) Acute Sample Convalescent Sample

34 Serology : Hemadsorption

35 MURRAY, 2nd Ed., Fig 53.5 Hemadsorption - influenza virus infected cells

36 Serology : Immunofluorescence

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38 Serology : Enzyme Linked Immuno Sorbent Assay (ELISA) (EIA)

39 HIV/AIDS:Diagnosis and monitoring Serological tests for HIV infection-2 ELISA The viral proteins can be derived from: Gp160 Gp120 Gp24 Gp17/18 Synthetic peptides corresponding to viral proteins are also commonly used in ELISA

40 ELISA

41 Hepatitis A Infection Typical Serological Course Symptoms Total anti- HAV Titre ALT Fecal HAV IgM anti-hav Months after exposure 2 4

42 Acute Hepatitis B Virus Infection with Recovery Typical Serologic Course Symptoms HBeAg anti-hbe Titre Total anti-hbc HBsAg IgM anti-hbc anti-hbs Weeks after Exposure

43 Progression to Chronic Hepatitis B Virus Infection Typical Serologic Course Acute (6 months) HBeAg HBsAg Chronic (Years) anti-hbe Titre Total anti-hbc IgM anti-hbc Years Weeks after

44 Titer Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection Symptoms +/- anti- HCV HCV RNA ALT Normal Years Months Time after Exposure

45 Serology : Western Blot

46 The Western Blot Confirmatory Test (HIV)

47 How to prepare strip for the Western blot

48 Western blot for HIV antibody There are different criteria for the interpretation of HIV Western blot results e.g. CDC, WHO, American Red Cross. The most important antibodies are those against the envelope glycoproteins gp120, gp160, and gp41 p24 antibody is usually present but may be absent in the later stages of HIV infection

49 Detection of Viral Nucleic Acid Polymerase Chain Reaction (PCR)

50 The Polymerase Chain Reaction (PCR)

51 DNA Sequencing

52 Thermal Cyclers for PCR

53 DNA sequencing

54 Electron Microscopy : Used to be very important before virus isolation and identification was developed Still important with viruses that cannot be cultured and with new viruses:

55 Electron Microscopy : Examples : - Adenovirus - Papillomaviruses - Enterovirus - Rotavirus

56 Hepatitis B Virus

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58 Hepatitis A Virus

59 HIV budding and maturation Hsiung, GD et al., Diagnostic Virology 1994 p204 (D. Medina)

60 Erskine Palmer, CDC ROTAVIRUS

61 smallpox virus cytoplasmic assembly and maturation F. A. Murphy, School of Veterinary Medicine, University of California, Davis.

62 Adenovirus

63 Influenza Virus

64 Rotavirus

65 Rift Valley Fever Virus

66 Papilloma virus

67 Enterovirus

68 Paramyxovirus

69 Parvovirus

70 Titer HBV - HDV Superinfection Typical Serologic Course Jaundice Symptoms ALT Total anti-hdv HBsAg HDV RNA IgM anti-hdv Time after Exposure

71 Titer Hepatitis E Virus Infection Typical Serologic Course Symptoms ALT IgG anti-hev IgM anti-hev Virus in stool Weeks after Exposure