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Detection of Respiratory Viruses (continued) Clinical disease All of the aforementioned virus groups can cause a full range of respiratory tract infections—from the typical mild common cold, with signs and symptoms of a runny nose and sneezing, to more severe presentations, such as pharyngitis, laryngitis, bronchitis, or pneumonia. The severity of disease varies depending on the level of immunity of the individual, and is generally more severe in immunocompromised patients [for example, those with human immunodeficiency virus (HIV); transplant recipients receiving antirejection drugs; diabetics; and the elderly]. As the immune competency of individuals and their ability to fight infection and cancer begins to decline in the fourth decade of life, the elderly patient is particularly susceptible to virus infections, which are often fatal in these circumstances. Traditional diagnostic methods In clinical practice, a specific virus is often not identified due to the lack of a laboratory test that is sensitive enough to detect it. New viruses have historically accounted for a signifi- cant proportion of infections where no virus could be detected in the absence of tests. Typically, viruses have been identified by their shape and size and classified into families; for example, the coronavirus has a "crown" around its surface made up from a single protein (Fig. 1). Virology laboratories have histori- cally diagnosed only six conventional respiratory viruses using traditional methods. These methods include, first and foremost, virus isolation in cell culture using up to four different cell lines (not all viruses grow in all cell lines). Preformed cell cultures in 15-cm-long (6-in.-long) tubes are inoculated with specimens, placed in a roller drum (where they are constantly rotated for 10 days), and viewed daily under a microscope for virus-in- duced cell damage, indicating the presence of a virus. In theory, culture can be sensitive enough for detection of a single living virus particle. However, at the same time, sensitivity can be lost when specific antibodies in the specimen neutralize the virus, preventing its growth (see table). The second most important detection method has been direct fluorescent antibody (DFA) staining for the presence of virus-infected cells. This method involves collecting epithe- lial cells from a nasopharyngeal swab, fixing them to a glass microscope slide, staining with individual antibodies labeled with a fluorescent tag, and viewing the slide with a fluorescent microscope (Fig. 2). This method has sensitivities ranging from 65% to 90% for the six viruses commonly detected. Sensitiv- ity can be compromised if the specimen is collected too late in the course of infection when the number of cells contain- ing viral proteins is diminished. The third most commonly used method is shell vial culture. This involves inoculating an aliquot of the specimen onto a preformed cell monolayer in a small vial containing a mixture of two susceptible cells, which is then centrifuged to enhance virus attachment and entry. The centrifugation-assisted inoculation of the cells increases the amount of viral proteins produced, allowing staining to be performed at 24–48 h and thus providing a test result to be obtained significantly earlier than the 7–10 days necessary for traditional cell culture. Rapid enzyme-linked immunosorbent assays (ELISAs), in which a monoclonal antibody conjugated to an enzyme is used to rapidly detect and quantify the presence of an antigen in a sample, have been developed as bedside tests for influenza and RSV. + ward ' s science Methods* Advantages Disadvantages Cell culture Low sensitivity Not all viruses are culturable DFA Gold standard method Positivity rates vary by laboratory ELISA Provides point of care result Insensitive NAAT Highly sensitive Expensive M-PCR Detects several viruses Expensive Table—Advantages and disadvantages of various methods for detecting respiratory viruses *Abbreviations: DFA = direct fluorescent antibody; ELISA = enzyme-linked immunosorbent assay; NAAT = nucleic acid amplification test; M-PCR = multiplex polymerase chain reaction. Fig. 2 Direct fluorescent antibody (DFA) staining of an influenza-positive specimen (left) showing the presence of virus-infected cells (green) stained with a fluorescent monoclonal antibody, and a negative specimen (right) showing uninfected cells stained with a red counterstain. (Credit: James B. Mahony)