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Laboratory Diagnosis of
Viral Infection
Dr. Sshrutkirti Gupta
• Collection of samples
• Transport
• Cultivation
• Methods of diagnosis
Following Points will discussed today
Importance of diagnosis of viral
infection
• Rubella diagnosed in 1st trimester-abortion is recommended.
• If baby born to HBV Surface Ag positive mother, immunization at
birth is mandatory.
• Diagnosis of diseases caused by viruses for which antiviral
treatment is available (eg. Herpes)
• Screening of HIV ,HBV helps in preventing transmission of the
disease
• Early detection of epidemics like influenza,
helps to initiate appropriate control measures.
Sample collection
• Respiratory tract- Nasal washing, throat
swab, nasal swab, sputum
(adeno, influenza, enterovirus, orthromyxo, paramyxo,
HSV, Rubella)
• Gastrointestinal tract-stool, rectal swab
(Reovirus, Rotavirus, Adenovirus, norwalk calcivirus)
• Maculopapular Rash-Throat swab, rectal
swab
(Adeno, entero, Rubella, measles)
• Vesicular rash-Vesicle fluid, scraping or swab
Coxsackie echo, HSV, VZV
• Central Nervous system-
Picorna- Stool ,
Arbovirus- rarely cultured
Rabies- Tissue, saliva, brain biopsy
HSV, CMV, Mumps, Measles- CSF, Biopsy
• Urinary Tract Infection-
Adenovirus, CMV-urine
• Blood born infections
HIV,HTLV,HBV,HCV,HDV-Blood
….Cont Sample collection
Methods of diagnosis
• Direct microscopy- virus
• inclusion bodies
• Isolation of virus/cultivation
• Antigen detection
• Antibody detection
• Nucleic acid detection
Microscopy
• A-Electron microscopy
• B- Fluorescent microscopy
• C- Light microscopy
Electron Microscopy
• Demonstration of viral particles in clinical samples
• Mainly for those whose cultivation is difficult
• Immunoelectron microscopy – more sensitivity
Rabies virus
Lab diagnosis-viruses.ppt
Direct demonstration-EM
Sample
• Faeces
• Vesicular fluid
• CSF
• Urine
Virus
Rotavirus, HAV, Adeno,
Norwalk, astrovirus.
Herpes simplex, VZ
Enterovirus, VZ
CMV
EM structure of Flu virus
Microscopy
Fluorescent microscopy
Rabies
Paramyxovirus,
Orthomyxoviruses,
Adenovirus
Herpesvirus
Lab diagnosis-viruses.ppt
Light microscopy
Inclusion bodies
• Rabies- negri bodies
• Vaccinia (Guarnieri bodies)
• Molluscum contagiosum (Molluscum bodies)
• Cowdry TypeA- Herpes, Yellow fever
• Cowdry type B-Adeno, Polio
Stains- Giemsa, Eosin-methylene blue,
Haematoxylin
Intracytoplasmic inclusion
bodies
Lab diagnosis-viruses.ppt
Cultivation
• By inoculation in to animals
• By inoculation into embryonated egg
• By inoculation into cell culture
• Sample process to remove bacterial
contamination
Animal inoculation
• Suckling mice- Arbovirus, Coxsackie
• Different routes- Intracerebral,
subcutaneous, intraperitoneal, intranasal
• Observe for signs of disease, death
• Tissue tested for presence of virus-
neutralisation test (antisera),
demonstrate inclusion bodies
Embryonated Egg inoculation
Egg Innoculation
Site
• Chorioallantoic
Membrane
• Allantoic cavity
• Amniotic cavity
• Yolk sac
Virus
• Pox (pocks)-variola or
vaccinia
• Influenza, yellow fever
• Influenza
• Chlamydiae, rickettsiae
Tissue culture
• Organ culture
• Explant culture- rarely used
• Cell culture
Organ culture
• Bits of organ maintained in tissue
culture growth medium
• Used mainly for highly specified
parasites of certain organ
eg- corona virus- Tracheal ring culture
Cell Culture
• Primary cell culture
• Diploid cell culture
• Continuous cell line
Primary cell culture
Limited growth up to 5-50 divisions
Use- primary isolation, vaccine production
Examples of cell lines
1. Rhesus monkey kidney cell culture
2. Human amnion cell culture
3.Chick embryo fibroblast cell culture
Diploid cell strain
• cells of single type –contains same no. of
chromosomes as in the parent cell
• Limited growth 50 cell division
• Developed from fibroblast
• WI-38-human embryonic lung cell strain
Rabies virus vaccine
HL-8 (Rhesus embryo cell strain)
…Cont Cell culture
Continuous cell line
• Single type capable of unlimited growth
eg-
1. HeLa (Human carcinoma of cervix cell line)
2. HEP-2(human epithelioma of larynx cell line)
3. Mckoy (human synovial carcinoma cell line)
4. BHK-21 ( Baby hamster kidney cell line)
Detection of growth in cell
culture
• 1 Cytopathic effect
• 2 Haemadsorption
• 3 Interference
• 4 Transformation
• 5 Immunoflurescence
• 6 Electron microscopy
• 7 Detection of enzymes
Cytopathic effects
a.Syncytium formation- measles, RSV
neighbouring cell fused together to form
multinucleated giant cell.
b. Cell necrosis & lysis- Enterovirus
c. Roundening of cells- Picornavirus
viral replication leads to neclear pyknosis ,
rounding, refratility, degeneration
d. Discrete focal degneration- Herpes
Lab diagnosis-viruses.ppt
Herpes
Cell necrosis by enterovirus
Rounding of cells-polio virus
Rounding of cells- poliovirus
Lab diagnosis-viruses.ppt
Lab diagnosis-viruses.ppt
Lab diagnosis-viruses.ppt
…Cont Cytopathic effects
• Haemadsorption- Influenza,
parainfluenza,Toga –express
Haemagglutinin on infected cells
• RBC suspension layered on cell layer
• RBC adsorb on infected cells
Antigen detection
• Immunoflurescence
• Serological tests
Antibody detection
• Serological tests
Lab diagnosis-viruses.ppt
Newer techniques
• Molecular
• DNA PCR
• RNA PCR

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Lab diagnosis-viruses.ppt

  • 1. Laboratory Diagnosis of Viral Infection Dr. Sshrutkirti Gupta
  • 2. • Collection of samples • Transport • Cultivation • Methods of diagnosis Following Points will discussed today
  • 3. Importance of diagnosis of viral infection • Rubella diagnosed in 1st trimester-abortion is recommended. • If baby born to HBV Surface Ag positive mother, immunization at birth is mandatory. • Diagnosis of diseases caused by viruses for which antiviral treatment is available (eg. Herpes) • Screening of HIV ,HBV helps in preventing transmission of the disease • Early detection of epidemics like influenza, helps to initiate appropriate control measures.
  • 4. Sample collection • Respiratory tract- Nasal washing, throat swab, nasal swab, sputum (adeno, influenza, enterovirus, orthromyxo, paramyxo, HSV, Rubella) • Gastrointestinal tract-stool, rectal swab (Reovirus, Rotavirus, Adenovirus, norwalk calcivirus) • Maculopapular Rash-Throat swab, rectal swab (Adeno, entero, Rubella, measles)
  • 5. • Vesicular rash-Vesicle fluid, scraping or swab Coxsackie echo, HSV, VZV • Central Nervous system- Picorna- Stool , Arbovirus- rarely cultured Rabies- Tissue, saliva, brain biopsy HSV, CMV, Mumps, Measles- CSF, Biopsy • Urinary Tract Infection- Adenovirus, CMV-urine • Blood born infections HIV,HTLV,HBV,HCV,HDV-Blood ….Cont Sample collection
  • 6. Methods of diagnosis • Direct microscopy- virus • inclusion bodies • Isolation of virus/cultivation • Antigen detection • Antibody detection • Nucleic acid detection
  • 7. Microscopy • A-Electron microscopy • B- Fluorescent microscopy • C- Light microscopy Electron Microscopy • Demonstration of viral particles in clinical samples • Mainly for those whose cultivation is difficult • Immunoelectron microscopy – more sensitivity Rabies virus
  • 9. Direct demonstration-EM Sample • Faeces • Vesicular fluid • CSF • Urine Virus Rotavirus, HAV, Adeno, Norwalk, astrovirus. Herpes simplex, VZ Enterovirus, VZ CMV
  • 10. EM structure of Flu virus
  • 13. Light microscopy Inclusion bodies • Rabies- negri bodies • Vaccinia (Guarnieri bodies) • Molluscum contagiosum (Molluscum bodies) • Cowdry TypeA- Herpes, Yellow fever • Cowdry type B-Adeno, Polio Stains- Giemsa, Eosin-methylene blue, Haematoxylin
  • 16. Cultivation • By inoculation in to animals • By inoculation into embryonated egg • By inoculation into cell culture • Sample process to remove bacterial contamination
  • 17. Animal inoculation • Suckling mice- Arbovirus, Coxsackie • Different routes- Intracerebral, subcutaneous, intraperitoneal, intranasal • Observe for signs of disease, death • Tissue tested for presence of virus- neutralisation test (antisera), demonstrate inclusion bodies
  • 19. Egg Innoculation Site • Chorioallantoic Membrane • Allantoic cavity • Amniotic cavity • Yolk sac Virus • Pox (pocks)-variola or vaccinia • Influenza, yellow fever • Influenza • Chlamydiae, rickettsiae
  • 20. Tissue culture • Organ culture • Explant culture- rarely used • Cell culture
  • 21. Organ culture • Bits of organ maintained in tissue culture growth medium • Used mainly for highly specified parasites of certain organ eg- corona virus- Tracheal ring culture
  • 22. Cell Culture • Primary cell culture • Diploid cell culture • Continuous cell line
  • 23. Primary cell culture Limited growth up to 5-50 divisions Use- primary isolation, vaccine production Examples of cell lines 1. Rhesus monkey kidney cell culture 2. Human amnion cell culture 3.Chick embryo fibroblast cell culture
  • 24. Diploid cell strain • cells of single type –contains same no. of chromosomes as in the parent cell • Limited growth 50 cell division • Developed from fibroblast • WI-38-human embryonic lung cell strain Rabies virus vaccine HL-8 (Rhesus embryo cell strain)
  • 25. …Cont Cell culture Continuous cell line • Single type capable of unlimited growth eg- 1. HeLa (Human carcinoma of cervix cell line) 2. HEP-2(human epithelioma of larynx cell line) 3. Mckoy (human synovial carcinoma cell line) 4. BHK-21 ( Baby hamster kidney cell line)
  • 26. Detection of growth in cell culture • 1 Cytopathic effect • 2 Haemadsorption • 3 Interference • 4 Transformation • 5 Immunoflurescence • 6 Electron microscopy • 7 Detection of enzymes
  • 27. Cytopathic effects a.Syncytium formation- measles, RSV neighbouring cell fused together to form multinucleated giant cell. b. Cell necrosis & lysis- Enterovirus c. Roundening of cells- Picornavirus viral replication leads to neclear pyknosis , rounding, refratility, degeneration d. Discrete focal degneration- Herpes
  • 30. Cell necrosis by enterovirus
  • 32. Rounding of cells- poliovirus
  • 36. …Cont Cytopathic effects • Haemadsorption- Influenza, parainfluenza,Toga –express Haemagglutinin on infected cells • RBC suspension layered on cell layer • RBC adsorb on infected cells
  • 37. Antigen detection • Immunoflurescence • Serological tests Antibody detection • Serological tests
  • 39. Newer techniques • Molecular • DNA PCR • RNA PCR