Diagnosis of Viral infections Clinical Symptoms History Signs

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 Culture Maintaining cells in culture; Provide suitable temperature, p. H, 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: He. La (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 He. La cells 7 days postinfection with human adenovirus showing pronounced cell enlargement, rounding, and distinctive “grapelike" 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 culture Definitive diagnosis to the virus grown in cell culture: Complement fixation. Hemagglutination inhibition. Neutralization. Radioimmunoassay. Immuno-electron microscopy.

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

Electron Micrograph Stool: Rotavirus

Electron Micrograph Stool: Adenovirus

Electron Micrograph Vesicular fluid Herpesvirus

Electron Micrograph Tissue specimen: Ebola virus

Electron Micrograph Brain (animals) Rabies

Antigen detection Diagnosis of viral infections by detecting viral specific antigens. Examples: Sandwich ELISA. Hepatitis B Surface antigens. HIV p 24 antigens. Immunofuorescence

Antibody detection Using Serological tests for: Screening for viral infections. Diagnosis of acute infection: Sero-conversion. Significant rising titer. Detection of specific Ig. M. ELISA Main problems: False positive e. g. past infection. False negatives e. g. infection in immunosuppressed, Compliment Fixation

Nucleic Acid detection Most 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. RT – PCR. Real time – PCR NASBA. b. DNA. Other.

Cytopathology studying 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.

Cytopathology Tzanck’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 precancerous 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 of tissue obtained from a corpse to recognize pathologic features of certain viral infections that may be present. E. g. Negri bodies: Rabies. Ebola. eosinophilic cytoplasmic inclusion (mass of nucleocapsids) pathognomonic for rabies.