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LABORATORY DIAGNOSIS OF VIRAL INFECTIONS. In developing countries, virological specimens will need to be transferred from district laboratories to regional or central virology laboratories. Diagnostic Methods in Virology 1. Direct Examination 2. Indirect Examination (Virus Isolation) 3. Serology 1-Direct Examination This is now becoming a widely used-and fast- method of virus diagnosis. Virus or viruses' antigen is detected in lesions, fluid, tissues, or excretions from the patient and a result can be obtained within an hour or two of receipt of specimen. 1. Serology (Antigen Detection) Immunofluorescence, ELISA etc. 2. Electron Microscopy: a- Morphology of virus particles b- Immune electron microscopy 3. Light Microscopy a- Histological appearance b- Inclusion bodies 4. Viral Genome Detection a. Hybridization. c. Polymerase Chain Reaction (PCR) Electron Microscopy •106 virus particles per ml required for visualization, 50,000 - 60,000 magnification normally used. •Viruses may be detected in the following specimens. 1. Faeces Rotavirus, Adenovirus 2. Vesicle Fluid HSV, VZV 3. Skin scrapings papillomavirus. Immune Electron Microscopy The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants:- a. Classical Immune electron microscopy (IEM) 1. The sample is treated with specific anti-sera before being put up for EM. 2. Viral particles present will be agglutinated and thus congregate together by the antibody. b. Solid phase immune electron microscopy (SPIEM) 1. The grid is coated with specific anti-sera. 2.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. 2-Indirect Examination 1.Cell Culture 2. Eggs 3. Animals cytopathic effect (CPE) haemabsorption immunofluorescence Haemagglutination Inclusion bodies disease or death Virus Isolation Cell Cultures are most widely used for virus isolation. there are 3 types of cell cultures: 1-Primary cell cultures: are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. 2-Semi-continuous cells. 3-Continuous cells : are the most easy to handle but the range of viruses supported is often limited. Growing virus may produce: 1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or nonspecific. 2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells. Problems with cell culture •Long period (up to 4 weeks) required for result. •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 CMV DEAFF test is the best example, whereby •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. 3- Serology Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection. It is far the most widely used way to diagnose virus infection. Criteria for diagnosing Primary Infection: -4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera. Criteria for diagnosing Reinfection: -fold or more increase in titre of IgG or total antibody between acute and convalescent sera. - Presence of IgM - Absence or slight increase in IgM Serological events following primary infection and reinfection. Note that in reinfection, IgM may be absent or only present transiently at a low level. Tests used in serology: The old but well-tried and reliable technique of complement fixation test is now being replaced by more sensitive assays, especially those which detect virus-specific IgM. But, note, the complement fixation test is still an indispensable technique in virus laboratory. Below are some of the most widely used tests: 1. Enzyme linked immunosorbent assay (ELSA): Now widely used and often available as commercially produced kits, because of its: •Sensitivity. •Low cost compared with other serological techniques. •Because it is possible to test large numbers of specimens at the same time. At present ELISA is used mainly to diagnose infections causes by: •Rotaviruses. •Hepatitis B virus and hepatitis C surface antigen. Virus + patient's serum .Add enzyme-labelled anti-human IgM antiserum Incubate Stop reaction Add substrate Measure reaction by colour intensity in optical density reader Calculate as positive or negative reaction by comparison with controls Anti-human IgM (or anti IgG) antibodies is used to detect specific IgM (or IgG) in the serum under test. Labeled anti-human antibody is used to detect the virus antibody: the label is an enzyme which reacts with a suitable substrate (the most used is alkaline phosphatase: paranitrophenyl phosphate) to produce a visible colour change.