Diagnosis of Viral infections

Clinical:Symptoms (History).Signs (Physical Examination).

Investigation:Non laboratory: (e.g X-rays, CT scan, MRI, US)Laboratory:

Non Virological. (CBC, LFT, …)Virological.

Laboratory Diagnosis of viral infections

Viral Isolation.

Electron Microscopy.

Antigen detection.

Antibody detection.

Nucleic acid detection.

Cytology.

Histopathology.

Viral Isolation

Methods Used;Cell Culture (tissue ,organ culture).

Embryonated egg.

Animal inoculation.

Conventional Cell Culture

Viruses are cultivated in cell cultures derived from animal tissues.

Conventional Cell CultureMaintaining cells in culture;

Provide suitable temperature, pH, glucose concentration.

Provide essential nutrients for the cell medium(amino acids, growth factors ..etc).

Addition of antibiotics and antifungal.

Conventional Cell Culture

Cell lines: population

of cells obtained from multicellular organism (animal) which can be maintained to keep undergoing division.

Human cell lines examples:

HeLa (cervical cancer). Human neuroblastoma cells,

Primate cell lines example:

Vero (African green monkey kidney epithelial cell line initiated 1962).

Cytopathic Effect (CPE)

Observable morphologic changes in the cell line which can be tested by light microscope or other techniques.

Cytopathic Effect (CPE)

Giemsa-stained HeLa cells 7 days postinfection with human adenovirus showing pronounced cell enlargement, rounding, and distinctive “grape-like" clusters (ballooning).

Cytopathic Effect (CPE)

Giemsa-stained Vero cells (from an African green monkey kidney) 30 hours postinfection with herpes simplex virus-1 showing cell rounding and clustering

Cytopathic Effect (CPE)

The small syncytia, or multinucleated giant cells, result from fusion of cell membranes formed by Vaccinia virus

Cytopathic Effect (CPE)

rounding, bridging, cell lysis, and syncytium formation with respiratory syncitial virus

Shell Vial Culture (SVC)Mixture of conventional cell culture and antigen detection (IF) method (CMV).

To obtain rapid results (24 – 48 hrs)

Cell Culture: Haemadsorption Phenomenon

Ability of the virus (influenza) in the clinicall specimen to adhere and clump erythrocyte due to expression of haemagglutinin on the cell surface.

Identification can then be confirmed by haemagglutination inhibition, haemadsorption inhibition, or, immunofluorescence.

Cell culture: Interference Phenomenon

Seen Rubella virus: do not produce CPE.

Make the infected cell resistant to normally infected viruses

Cell cultureDefinitive diagnosis to the virus grown in cell culture:

Complement fixation. Hemagglutination inhibition.Neutralization. Radioimmunoassay.Immuno-electron microscopy.

Electron MicroscopePrinciple:

Production of “Electron beam” focuses on the image.Electrons have Wave length 100,000 time shorter than Photons Illuminationof the specimen.Production of magnified image (10 millions times more than light microscope.

Electron MicrographUses negative stain by contrasting the specimen with an optically opaque fluid. Characterization of virus morphology by using different specimens:

Electron MicrographStool:

Rotavirus

Electron MicrographStool:

Adenovirus

Electron MicrographVesicular fluid

Herpesvirus

Electron MicrographTissue specimen:

Ebola virus

Electron MicrographBrain (animals)

Rabies

Antigen detection Diagnosis of viral infections by detecting viral specific antigens.Examples:

Hepatitis B Surface antigens.HIV p24 antigens.

Immunofuorescence

Sandwich ELISA.

Antibody detectionUsing Serological tests for:

Screening for viral infections.Diagnosis of acute infection:

Sero-conversion.Significant rising titer.Detection of specific IgM.

Main problems:False positive e.g. past infection.False negatives e.g. infection in immunosuppressed,

ELISA

Compliment Fixation

Nucleic Acid detectionMost sensitive and most specific method for:

Diagnosis of viral infection e,g. herpes encephalitis.Demonstration of the disease progression e.g. HIV.Genotyping of virus e.g. Hepatitis C.

E.g.PCR.

PCR.RT – PCR.Real time – PCR

NASBA.bDNA.Other.

Cytopathologystudying and diagnosing diseases on the cellular level.

Some cytopathologic features are highly associated with certain viral infections: e.g.

Tzanck’s smear test.Pap smear test.

CytopathologyTzanck’s Smear:

Scrapping the Herpetic ulcer or vesicle.Looking for “multinucleated Giant cells”.Diagnostic for Herpes Simplex virus infection and chicken pox

Cytopathology

Pap smear test:Obtaining a smear from Cervical Canal.Stained by Papanicolaou stain.Screening test detect pre-cancerous changes called cervical intraepithelial neoplasia (CIN) caused by Human papilloma virus.

Immunohistochemistry (IHC)

Process of detecting viral antigens within cells of a tissue section (biopsy). IHC uses the principle of antibodies binding specifically to antigens in biological tissues.

E.g. Immunoperoxidase, immunofluorescence

Spinal cord: Immunoperoxidase-staining of WN viral antigen in glial cells

Postmortem examination

examination of tissue obtained from a corpse to recognize pathologic features of certain viral infections that may be present. E.g.

Rabies.

Ebola.

Negri bodies:eosinophilic cytoplasmic inclusion (mass of nucleocapsids) pathognomonic for rabies.