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Introduction to Diagnostic Methods
Introduction to Diagnostic Methods: In this lecture educator explains about Laboratory Diagnosis of Viruses. Diagnostic virology is rapidly moving into the mainstream of clinical medicine as a result of the convergence of several independent developments. Historically, diagnostic virology has had to justify its use. The reasons have been that traditional viral diagnostic techniques, especially culture, are slow, expensive, and often peripheral to clinical decision-making, particularly when no therapeutic agents are available.
Common approaches to the diagnosis: There are five approaches to the diagnosis of viral diseases by the use of clinical specimens:
- Identification of the virus in cell culture
- Microscopic identification directly in the specimen
- Serologic procedures to detect a rise in antibody titer or the presence of IgM antibody
- Detection of viral antigens in blood or body fluids
- Detection of viral nucleic acids in blood or the patient’s cells
Identification in cell culture: The growth of viruses requires cell cultures because viruses replicate only in living cells, not on cell-free media the way most bacteria can. Because many viruses are inactivated at room temperature, it is important to inoculate the specimen into the cell culture as soon as possible; brief transport or storage at 4°C is acceptable. Virus growth in cell culture frequently produces a characteristic cytopathic effect (CPE) that can provide a presumptive identification. CPE is a change in the appearance of the virus-infected cells. This change can be in such features as size, shape, and the fusion of cells to form multinucleated giant cells (syncytia). CPE is usually a manifestation of virus-infected cells that are dying or dead. The time taken for the CPE to appear and the type of cell in which the virus produces the CPE are important clues in the presumptive identification. If the virus does not produce a CPE, its presence can be detected by several other techniques:
- Hemadsorption (i.e., attachment of erythrocytes to the surface of virus infected cells). This technique is limited to viruses with a hemagglutinin protein on their envelope, such as mumps, parainfluenza, and influenza viruses.
- Interference with the formation of a CPE by a second virus. For example, rubella virus, which does not cause a CPE, can be detected by interference with the formation of a CPE by certain enteroviruses, such as echovirus or Coxsackie virus.
- A decrease in acid production by infected, dying cells. This can be detected visually by a color change in the phenol red (a pH indicator) in the culture medium. The indicator remains red (alkaline) in the presence of virus-infected cells but turns yellow in the presence of metabolizing normal cells as a result of the acid produced. This technique can be used to detect certain enteroviruses.
Classical Detection Techniques
A definitive identification of the virus grown in cell culture is made by using known antibody in one of several tests. Complement fixation, hemagglutination inhibition, and neutralization of the CPE are the most frequently used tests. Other procedures such as fluorescent antibody, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy are also used in special instances.
Complement fixation: If the antigen (the unknown virus in the culture fluid) and the known antibody are homologous, complement will be fixed (bound) to the antigen–antibody complex. This makes it unavailable to lyse the “indicator” system, which is composed of sensitized red blood cells.
Hemagglutination: If the virus and antibody are homologous, the virus is blocked from attaching to the erythrocytes and no hemagglutination occurs. Only viruses that agglutinate red blood cells can be identified by this method.
Neutralization: If the virus and antibody are homologous, the antibody bound to the surface of the virus blocks its entry into the cell. This neutralizes viral infectivity because it prevents viral replication and subsequent CPE formation or animal infection.
Fluorescent antibody assay: If the virus-infected cells and the fluorescein-tagged antibody are homologous, the typical apple-green color of fluorescein is seen in the cells by ultraviolet (UV) microscopy.
Newer Detection Techniques
Radioimmunoassay: If the virus and the antibody are homologous, there is less antibody remaining to bind to the known radio labelled virus.
ELISA: In the ELISA test to identify a virus, known antibody is bound to a surface. If the virus is present in the patient’s specimen, it will bind to the antibody. A sample of the antibody linked to an enzyme is added, which will attach to the bound virus. The substrate of the enzyme is added, and the amount of the bound enzyme is determined.
Immunoelectron microscopy: If the antibody is homologous to the virus, aggregates of virus–antibody complexes are seen in the electron microscope.
Microscopic and Serologic Methods
Microscopic identification: Viruses can be detected and identified by direct microscopic examination of clinical specimens such as biopsy material or skin lesions. Three different procedures can be used.
- Light microscopy can reveal characteristic inclusion bodies or multinucleated giant cells. The Tzanck smear, which shows herpesvirus-induced multinucleated giant cells in vesicular skin lesions, is a good example.
- UV microscopy is used for fluorescent antibody staining of the virus in infected cells.
- Electron microscopy detects virus particles, which can be characterized by their size and morphology.
Serologic procedures: A rise in the titer of antibody to the virus can be used to diagnose current infection. Seroconversion is the term used to describe the finding of antibody to a virus (or any microbe) in a patient’s serum when the patient previously had no antibody. Stated another way, the patient’s serum has converted from antibody-negative to antibody-positive. A serum sample is obtained as soon as a viral etiology is suspected (acute phase), and a second sample is obtained 10 to 14 days later (convalescent-phase). If the antibody titer in the convalescent-phase serum sample is at least fourfold higher than the titer in the acute-phase serum sample, the patient is considered to be infected. For example, if the titer in the acute-phase serum sample is 1/4 and the titer in the convalescent-phase serum sample is 1/16 or greater, the patient has had a significant rise in antibody titer and has been recently infected. If, however, the titer in the convalescent-phase serum sample is 1/8, this is not a significant rise and should not be interpreted as a sign of recent infection. It is important to realize that an antibody titer on a single sample does not distinguish between a previous infection and a current one. The antibody titer can be determined by many of the immunologic tests mentioned previously. These serologic diagnoses are usually made retrospectively because the disease has frequently run its course by the time the results are obtained.
IgM antibody test: In certain viral diseases, the presence of IgM antibody is used to diagnose current infection. For example, the presence of IgM antibody to core antigen indicates infection by hepatitis B virus. Other nonspecific serologic tests are available. For example, the heterophil antibody test (Monospot) can be used to diagnose infectious mononucleosis. In the heterophile test, human serum is reacted with horse or sheep red blood cells. If the heterophile antibody is present (i.e., if the patient has been infected with Epstein– Barr virus), then agglutination of the red cells occurs.
Detection of Viral Antigens and Nucleic Acids
Detection of p24 antigen: Viral antigens can be detected in the patient’s blood or body fluids by various tests, but most often by an ELISA. Tests for the p24 antigen of human immunodeficiency virus (HIV) and the surface antigen of hepatitis B virus are common examples of this approach.
Detection of viral nucleic acid: Viral nucleic acids (i.e., either the viral genome or viral mRNA) can be detected in the patient’s blood or tissues with complementary DNA or RNA (cDNA or cRNA) as a probe. If only small amounts of viral nucleic acids are present in the patient, the polymerase chain reaction can be used to amplify the viral nucleic acids. Assays for the RNA of HIV and hepatitis C virus and the DNA of hepatitis B virus in the patient’s blood (viral load) are commonly used to monitor the course of the disease and to evaluate the patient’s prognosis.