Diagnosis of viral infections
The diagnosis of viral infections involves identifying the presence of viruses within infected organisms, which are intracellular parasites composed of nucleic acids and a protein coat. Various diagnostic methods are employed, including traditional tests like serology and tissue culture, as well as advanced molecular techniques. Serological tests focus on detecting virus antigens or measuring antibodies produced by the immune response, with methods such as the hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) being commonly used.
Electron microscopy serves as a crucial tool for visualizing viruses and can provide preliminary identification clues for further testing. Although tissue culture was once a primary method for growing viruses for identification, it is becoming less favored due to its time-consuming nature and lower sensitivity compared to other methods. Molecular techniques, including polymerase chain reaction (PCR) and multiplex PCR, have gained popularity for their speed and accuracy in detecting viral genomes. These newer methods are typically more sensitive and specific than traditional approaches, leading to enhanced diagnostic capabilities in the field of virology.
Diagnosis of viral infections
Definition
Viruses are intracellular parasitic organisms that infect the cells of other organisms. Viruses consist of nucleic acids surrounded by a protein coat known as a capsid. The clinical signs presented by a person during a suspected viral infection determine the samples collected for laboratory tests. Traditional tests such as serology and tissue culture, along with electron microscopy, remain the mainstays of viral diagnosis, but molecular methods have become more popular too.
Serology
Virus proteins are known as antigens when they elicit an immune response by the body. This immune response leads to the body’s formation of antibodies. Antibodies, also known as immunoglobulins, comprise five classes (IgM, IgG, IgA, IgD, and IgE), which are based on the structural characteristics and biological activity of the antigen. Serological tests detect virus antigens, measure serum antibody levels (titers), and relate these titers to the clinical state of the affected person.
All serological tests are based on the formation of an antigen-antibody complex. Typically, after infection, the IgM antibody is the first to appear, followed by a much larger rise in IgG antibodies; however, the dynamics of the antibody levels can vary greatly depending on many factors.
Standard or classical serological tests are those that have been in long-time use. The hemagglutination inhibition test determines the presence and quantity of virus antigen or antibody by the clumping of red blood cells. The single radial hemolysis technique determines the amount of virus antibody present in a serum sample by reacting it with red blood cells containing antigen and complement and by measuring the resultant circular zone of hemolysis. The complement fixation test measures the amount of antibody in serum or spinal fluid by the amount of complement consumed in the test medium.
The immunofluorescence test detects virus antigen that binds to a fluorescent-labeled antibody. In neutralization tests, virus and serum are mixed and inoculated into cell culture, eggs, or animals. The loss of resultant infectivity of the virus is called neutralization. The particle agglutination test involves coating the surface of latex particles with antigen (or antibody). A sample containing an antibody (or antigen) is added; resultant agglutination is a positive test.
Newer serological methods have been developed. In radioimmunoassay, either the antigen or the antibody is tagged with a radioactive molecule, and the radioactivity of the resultant complex is measured. In the enzyme-linked immunoabsorbent assay (ELISA), antibodies are attached to a solid support. A sample that contains virus is added; the antigen binds to the antibodies and an antibody-enzyme conjugate is added, which binds to the antigen. Finally, the substrate of the enzyme is added to form a colored complex. The reverse is also possible, starting with antigen bound to the solid support. The Western blot test can detect multiple antibodies directed against a single virus antigen.
Microscopic Examination
Electron microscopy (EM) has filled an essential, long-time role in virus diagnosis, helping to detect new and unusual disease outbreaks. EM requires the isolation of the virus for examination, which can require concentration of sample fluids. However, visualizing the pathogen can provide important preliminary identification clues that can help determine correct follow-up tests. Typically, a negative stain is prepared in which the virus appears clear or light colored against a darker background. Immunoaggregation, or the clumping of virus particles, is sometimes practiced to help visualize virus particles.
EM can be important for studying structural features, as it can help determine the function of various viral components. The findings can lead to the development of methods of treatment or of vaccines. Atomic force microscopy is now being used to improve the visualization of viruses.
Although viruses cannot be visualized by light microscopy, the procedure is used to examine cells or tissues for the effects of viral infections. The presence of inclusion bodies is an example.
Tissue Culture
Growing virus in tissue culture is the traditional means to augment the quantity of virus for identification. Three types of cell cultures are in use: primary cells from adult animals; semi-continuous cells from embryonic tissue; and continuous cells (immortalized tumor cell lines). The blood or tissue sample containing the suspected virus is inoculated into the cell culture, and the presence of growing virus is observed. The growing virus can kill the cells (cytopathic effect) or acquire the ability to stick to red blood cells (hemadsorption). Tissue culture is declining in importance because of the extended time required to obtain results, because of its low sensitivity, and because many viruses will not grow in cell culture.
Molecular Methods
Molecular methods are based on determining the genome, or genetic makeup, of the virus. Virus genomes are made up of nucleic acids, either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Before testing, RNA must be converted to DNA by a process known as reverse transcriptase. To ensure sufficient amounts for detection, the small amounts of viral DNA present in a serum sample must then be amplified by the polymerase chain reaction (PCR) process. The development of what is called real-time PCR, which combines the rapid assay time of PCR amplification with an inbuilt detection system, is considered to be a major advance in virus disease diagnosis.
Another advance is the development of multiplex PCR, which can amplify several regions of DNA simultaneously. This technology can result in considerable savings in time and cost while facilitating the screening and identification of virus species. Another test, Western blotting, can measure antibody to several viral antigens simultaneously.
Finally, nanotechnology has entered the field of virus diagnostics. Its proponents claim that it can provide simple, rapid, and sensitive solutions. Semiconducting nanowires, magnetic nanoparticles, and fluorescent nanoparticles are finding applications in viral diagnoses.
Impact
The newer molecular methods are usually more sensitive and specific than tissue culture for virus diagnosis. However, traditional immunoassay methods, such as ELISA or immunofluorescence, may still have the advantages of speed, convenience, and ease of use, so they continue to be used for early diagnosis.
Bibliography
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