High-quality, commercially available viral antibody reagents have led to the development of fluorescent antibody, enzyme immunoassay, latex agglutination, and immunoperoxidase tests that detect viral antigen in patient specimens.

Direct and indirect immunofluorescent methods are used. Direct immunofluorescent testing involves the use of a labeled antiviral antibody; the label is usually fluorescein isothiocyanate (FITC), which is layered over a specimen suspected of containing a homologous virus. The indirect immunofluorescent procedure is a two-step test in which unlabeled antiviral antibody is added to the slide, followed by a labeled (FITC) antiglobulin that binds to the first-step antibody bound to virus in the specimen. Direct immunofluorescence is generally more rapid and specific than indirect immunofluorescence but less sensitive. The increased sensitivity of indirect immunofluorescence results from signal amplification that occurs with the addition of the second antibody. Signal amplification decreases specificity by increasing nonspecific background fluorescence.

Direct immunofluorescence is best suited to situations in which large quantities of virus are suspected or when high-quality, concentrated monoclonal antibodies are used, such as for the detection of RSV in a patient specimen or the identification of viruses growing in cell culture.

Indirect immunofluorescence should be used when lower quantities of virus are suspected, such as detection of respiratory viruses in specimens from adult patients. High-quality monoclonal antibodies improve the sensitivity and specificity of immunofluorescence testing.

Strict criteria for the interpretation of fluorescent pat terns must be used. This includes standard interpretation of fluorescent intensity (Table 1) and recognition of viral inclusion morphology. Nuclear and cytoplasmic staining patterns are typical for influenza virus, adenovirus, and the herpes viruses; cytoplasmic staining is typical for RSV, parainfluenza, and mumps viruses; and staining within multinucleated giant cells is typical of measles virus or the herpes virus (see Figures 1 to 3; also Figure 4). False-positive staining can occur with specimens containing yeasts, certain bacteria, mucus, or leukocytes. Leukocytes, which contain Fc receptors for antibody, also can cause nonspecific binding of antibody conjugates. To verify employees’ ability to interpret FA tests, every laboratory should perform viral culture or some alternative detection method along with immunofluorescence until in-house performance has been established.

Table1. Interpretation of Fluorescence Intensity Using FITC

Fig1. Flowchart for the detection and identification of pedi atric respiratory viruses.

Fig2. Viral inclusions. A, Pap-stained smear showing multinucleated giant cells typical of herpes simplex or varicella-zoster viruses. B, Hematoxylin and eosin (HE)–stained lung tissue containing intranuclear inclusion within enlarged cytomegalovirus (CMV)–infected cells. C, HE-stained lung tissue containing epithelial cells with intranuclear inclusions characteristic of adenovirus. D, HE-stained liver from stillborn fetus showing intranuclear inclusions in erythroblasts (extramedullary hematopoiesis) resulting from parvovirus infection. E, Pap stain of exfoliated cervicovaginal epithelial cells showing perinuclear vacuolization and nuclear enlargement characteristic of human papillomavirus infection. F, HE-stained epidermis filled with molluscum bodies, which are large, eosinophilic, cytoplasmic inclusions resulting from infection with molluscum contagiosum virus. G, HE-stained cells infected with measles virus. H, HE-stained brain tissue showing oval, eosinophilic rabies cytoplasmic inclusion (Negri body). (E and F from Murray PR, Kobayashi GS, Pfaller MA, et al, editors: Medical microbiology, ed 2, St Louis, 1994, Mosby.)

Fig3. Electron micrographs of viruses. A, Rotavirus. B, Adenovirus. C, Norwalk agent virus. D, Coronavirus. Continued

Fig3. cont’d G E, Herpes simplex virus. F, Measles virus. G, Negatively stained preparation of JC virus in brain tissue. (C from Howard BJ, Klaas J, Rubin SJ, et al: Clinical and pathogenic microbiology, St Louis, 1987, Mosby; D and F from US Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control, Atlanta, Ga; G courtesy Dr. Gabriele M. ZuRhein, UNW-Madison, Madison, Wisconsin.)

Fig4. A, RSV-infected RMK (rhesus monkey kidney) cells at 400×, stained with Light Diagnostics RSV MoAb. Fluorescence is seen in the cytoplasm and associated with syncytia. Cytoplasmic staining is often punctuate with small inclusions. B, HSV I: HSV I infected Vero cell control slide 200×. Stained with Pathfinder HSV 1 MoAb DFA assay. Fluorescent staining is cytoplasmic. C, Influenza B infected RMK cells at 400×. Stained with Light Diagnostics Influenza B MoAb. Fluorescence is nuclear, cytoplasmic, or both. Nuclear staining is uniformly bright and the cytoplasmic staining is often punctuate with l large inclusions. D, Herpes Simplex II infected A549 cells at 200×. Stained with Pathfinder HSV II MoAb DFA assay. Fluorescence may stain the cytoplasm, the nucleus, or both depending on the stage of the infection cycle. When infected cells are rounded, staining may appear nuclear due to cytoplasm covering the nucleus. E, HSV II Infected A549 cells at 200×. F, HSV II Infected A549 cells at 400×. Picture shows the multinucleated “giant” cells characteristic of HSV II CPE (cytopathogenic effect).

Fig4. cont’d

The most useful immunofluorescent stains in the clinical virology laboratory are those for RSV, influenza and parainfluenza viruses, adenovirus, HSV, VZV, and CMV. A pool of antibodies can be used to screen a specimen for multiple viruses. A positive screen is tested with each individual reagent to identify the exact virus. Screening pools have been used successfully to detect respiratory viruses in specimens from children. Such pools are less sensitive when used with specimens from adults because of the lower numbers of viral particles in the specimens.

Enzyme immunoassay methods used in clinical virology include solid-phase enzyme-linked immunosorbent assay (solid-phase ELISA) and the membrane-bound enzyme-linked immunosorbent assay (membrane ELISA). Solid-phase ELISA is performed in a small test tube or microtiter tray. Breakaway strips of microtiter wells are available for low-volume test runs (Figure 5). The remaining, unused wells can be saved for future testing. Membrane ELISA tests have been developed for low-volume testing and for cases in which rapid results are needed. They can be performed by individuals with minimum training and usually require less than 30 minutes to complete. The membrane method uses a handheld reaction chamber with a cellulose-like mem brane. Specimen and reagents are applied to the mem brane. After a short incubation period, a chromogenic (color) reaction occurs on the surface of the membrane and is read visually. Built-in controls on the same mem brane provide convenient monitoring of test procedures. Figure 6 illustrates a membrane ELISA used to detect rotavirus. The most used enzyme immunoassays for antigen detection are those for RSV (solid-phase and membrane), rotavirus (solid-phase and membrane), and influenza viruses (membrane).

Fig5. Solid-phase enzyme immunoassay for detection of rotavirus with breakaway strips of microtiter wells for small-batch testing. (Courtesy Children’s Hospital Medical Center of Akron, Akron, Ohio.)

Fig6. Positive- (top) and negative-membrane enzyme linked immunoassays (ELISAs) for detection of rotavirus. The red line in the reaction area on the left represents a positive test result. A red line in the reaction area on the right represents an internal test control ensuring that the test has been carried out correctly. If the test control line is not present, the test is invalid and must be repeated.

Advantages of enzyme immunoassays are the use of relatively stable reagents and results that can be interpreted qualitatively (positive or negative) or quantitatively (titer or degree of positive reaction). It is important to note that enzyme immunoassays frequently have an indeterminate or borderline interpretative category. This result implies that low levels of viral antigen or background interference prevented a clear-cut positive or negative result. Such results usually require testing of a second specimen to avoid interference or to detect a rise in antigen level. ELISAs are sensitive and simple to perform and can be easily automated. However, specimen quality cannot be evaluated; that is, the number of cells cannot be assessed, as can be determined microscopically with fluorescent immunoassays.

Immunoperoxidase staining, and latex agglutination are additional techniques used to detect viral antigen. Immunoperoxidase staining is commonly used to stain histologic sections for virus but is less popular than immunofluorescence staining in clinical virology laboratories. Latex agglutination is an easy and inexpensive method but lacks sensitivity compared with ELISA and fluorescent immunoassays.

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مواضيع ذات صلة

Pathogenesis of SARS-CoV-2 and COVID-19

Viral Serology :General Principles

Virus detection methods: Cell Culture

Virus detection methods: Molecular Detection Using Nucleic Acid Probes and Polymerase Chain Reaction Assays

Arenavirus diseases

Bunyavirus diseases

Bunyavirus Encephalitis viruses

Dengue Virus

Clinical Features of Yellow fever

Virus detection methods: Electron Microscopy

Virus detection methods: Cytology and Histology

Specimen Transport and Storage in viral infections