An Overview: Dr. Mejbah Uddin Ahmed

Diagnosis of Viral Disease

An Overview

Dr. Mejbah Uddin Ahmed

Diagnostic Methods in Virology • Microscopic identification • Detection of Antigen • Detection of Antibody • Detection of viral nucleic acid • Isolation by culture

Microscopy: Light Microscopy: Detects multinucleated giant cells or inclusion bodies. Electron Microscopy: Morphology and shape of virus particles. UV microscopy: For fluorescent antibody staining of virus infected cells.

Immunofluorescense

Positive immunofluorescence test for viral antigen.

Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants:-

Classical Immune electron microscopy (IEM) Sample is treated with specific anti-sera before being put up for EM. Viral particles present will be agglutinated and thus congregate together by the antibody. Solid phase immune electron microscopy (SPIEM) the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.

Problems with Electron Microscopy • Expensive equipment • Expensive maintenance • Require experienced observer • Sensitivity often low

Antigen Detection: Viral antigen can be detected in patients blood or body fluid by immunofluorescence, ELISA, western blot etc.

Detection of Antibody Because of the difficulty of virus culture, antibody can be detected byimmunofluorescence, ELISA, western blot, CFT & other tests.

Detection of Antibody Primary Infection • 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera • Presence of IgM • A single high titre of IgG (or total antibody) - very unreliable Reinfection • fold or more increase in titre of IgG or total antibody between acute and convalescent sera • Absence or slight increase in IgM

Molecular Methods Viral genome or mRNA can be detected in patients blood or tissues by several techniques. • DNA Hybridization,

DNA Sequencing

• PCR,

RTPCR

• DNA or RNA probe

Blotting techniques

• NASBA

Virus culture Viral growth requires cell cultures, because virus can not replicate without living cells. * Cell Culture. * Embryonated Egg. * Animal inoculation: disease or death

Growth of virus on embryonated eggs

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 etc.

Cell Culture * Primary cell culture support the widest range of viruses. * However, it is very expensive and often difficult to obtain a reliable supply. * Continuous cells are the most easy to handle but the range of viruses supported is often limited.

Cell Cultures Viral growth in cell cultures produces cytopathic effect (multinucleated giant cell formation), that can be confirmed by: neutralization, interference, haemadsorption, immunofluorescence, ELISA, RIA, CFT, tests.

Cytopathic Effect

Syncytium formation in cell culture

Problems with cell culture Long period (up to 4 weeks) require. Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen. Susceptible to bacterial contamination. Susceptible to toxic substances which may be present in the specimen. Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

Rapid Culture Techniques

Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. •

The cell sheet is grown on individual cover slips in a plastic bottle.

•

Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days.

•

The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence.

Viruses Isolated by Cell Culture V iru ses readily isolated by cell cu ltu re

L ess frequ en tly isola te d viruses

H erpes Sim plex

V aricella-Z o ster

C ytom egalovirus

M easles

A deno viruses

R ub ella

P olioviruses

R hinoviruses

C ox sackie B viruses

C ox sackie A viruses

E ch oviruses In fluenza P arainfluenza M um ps R espiratory S ync ytial V iru s

Specimens for Routine Tests Clinical Category 1. 2. 3. 4. 5. 6. 7. 8.

Meningitis Encephalitis Paralytic disease Respiratory illness Hepatitis Gastroenteritis Congenital diseases Skin lesions

9. Eye lesions 10.Myocarditis 11.Myositis 12.Glandular fever 13.Post Mortem

Blood + + + + +

Throat swab + + + +

Faeces

CSF

+ + +

+ + +

Other

Brain biopsy Nasopharyngeal aspirate

+ + +

+ + + +

+

Urine, saliva Lesion sample e.g. vesicle fluid, skin scrapping Eye swab Pericardial fluid

+ Autopsy

After use, swabs should be broken into a small bottle containing 2 ml of virus transport medium. Swabs should be sent to the laboratory as soon as possible without freezing. Faeces, CSF, biopsy or autopsy specimens should be put into a dry sterile container.