Lab Diagnosis of Viral Infections PDF

Lab diagnosis of viral infections Lela NIshnianidze, M.D. Invited lecturer GAU  Lab methods accomplish The laboratory methods accomplish the following results: 1. Description of virus-induced cytopathologic eff...

Lab diagnosis of viral infections Lela NIshnianidze, M.D. Invited lecturer GAU  Lab methods accomplish The laboratory methods accomplish the following results: 1. Description of virus-induced cytopathologic effects (CPEs) on cells 2. Detection of viral particles 3. Isolation and growth of the virus 4. Detection and analysis of viral components (e.g., proteins [antigens], enzymes, genomes) 5. Evaluation of the patient’s immune response to the virus (serology)  Specimen Collection Selection of the appropriate specimen for analysis is often complicated because several viruses may cause the same clinical disease development of meningitis symptoms during the summer suggests an arbovirus, in which case cerebrospinal fluid (CSF) and blood should be collected, or an enterovirus, in which case CSF, a throat swab, and stool specimens should be collected for genome analysis and possible virus isolation inflammation of the active by an infection or an autoimmune response specimen collection tissues of the brain caused A focal encephalitis with a temporal lobe localization preceded by headaches and disorientation suggests herpes simplex virus (HSV) infection, for which CSF can be relatively quickly analyzed for viral deoxyribonucleic acid (DNA) sequences by polymerase chain reaction (PCR) amplification. Specimens should be collected early in the acute phase of infection HSV and varicella-zoster virus (VZV) may not be recoverable from lesions more than 5 days after the onset of symptoms Viruses are best transported and stored on ice and in special media that contain antibiotics and proteins, such as serum albumin or gelatin. Significant losses in infectious titers occur when enveloped viruses (e.g., HSV, VZV, influenza virus) are kept at room temperature or frozen at −20° C. This is not a risk for nonenveloped viruses (e.g., adenoviruses, enteroviruses). specimens  Cytology intranuclear basophilic (owl’s-eye) inclusion bodies found in large cells of tissues with cytomegalovirus (CMV) or in the sediment of urine from patients with the infection are readily identifiable owl's eye inclusion Cowdry type A nuclear inclusions in single cells or in large syncytia (multiple cells fused together) are a characteristic finding in cells infected with HSV or VZV Negri bodies Rabies may be detected through the finding of cytoplasmic Negri bodies (rabies virus inclusions) in brain tissue cowdry bodies  Lab diagnosis Often the cytologic specimens will be examined for the presence of specific viral antigens by immunofluorescence or viral genomes by in situ hybridization or PCR for a rapid, definitive identification  Electron Microscopy not a standard procedure Enteric viruses (e.g., rotavirus) that are produced in abundance and have a characteristic morphology can be detected in stool by these methods.  Viral Isolation and Growth A virus can be grown in tissue culture, embryonated eggs, and experimental animals  cell culture Primary monkey kidney cells are excellent for the recovery of influenza viruses, paramyxoviruses, many enteroviruses, and some adenoviruses. Human fetal diploid cells, which are generally fibroblastic cells, support the growth of a broad spectrum of viruses (e.g., HSV, VZV, CMV, adenoviruses, picornaviruses). HeLa cells, a continuous line of epithelial cells derived from a human cancer, are excellent for the recovery of respiratory syncytial virus, adenoviruses, and HSV. Many clinically significant viruses can be recovered in at least one of these cell cultures  An innovative approach to Viral Detection detection of HSV infection uses genetically modified by immunofluorescence, or by genome tissue culture cells that analysis of the infected cell culture express the β- rubella virus may not cause a CPE galactosidase gene and (cytopathologic effect), but it does prevent (interfere with) the replication of can be stained blue when picornaviruses in a process known as infected with HSV heterologous interference, which can be (enzyme-linked virus- used to detect the rubella virus. inducible system [ELVIS]) Cells infected with the influenza virus, parainfluenza virus, mumps virus, and togavirus express a viral glycoprotein (hemagglutinin) that binds erythrocytes of defined animal species to the infected cell surface (hemadsorption). The virus can then be identified from the specific antibody that blocks the hemagglutination, a process called hemagglutination inhibition (HI).  Detection of Viral Proteins and Genetic Material detection and assay of characteristic enzymes or activities can identify and quantitate specific viruses. the presence of reverse transcriptase in serum or cell culture indicates the presence of a retrovirus or DNA probes, with sequences complementary hepadnavirus. to specific Viral antigens on the cell surface or regions of a viral genome, can be used like within the cell can be detected by antibodies as immunofluorescence and enzyme sensitive and specific tools for detecting a immunoassay (EIA) virus. These probes can detect the virus even in the Virus or antigen released from infected absence of viral replication. cells can be detected by enzyme-linked DNA probe analysis is especially useful for immunosorbent assay (ELISA), detecting slowly replicating or nonproductive radioimmunoassay (RIA), and latex viruses, such as CMV and human agglutination (LA) papillomavirus, or when the viral antigen  Viral Serology Serologic studies are used for the identification of viruses that are difficult to isolate and The detection of virus-specific grow in cell culture, as well as viruses that immunoglobulin (Ig)M antibody, cause diseases of long duration (e.g., EBV, which is present during the first 2 or 3 HBV, HIV) weeks of a primary infection, Serology can be used to identify the virus and its generally indicates a recent primary strain or serotype, whether it is an acute or infection. chronic disease, and determine whether it is a primary infection or a reinfection Seroconversion is indicated by at least a fourfold increase in the antibody titer between the serum obtained during the acute phase of disease and that obtained at least 2 to 3 weeks later during the convalescent phase. Reinfection or recurrence later in life causes an anamnestic (secondary or booster) response. Antibody titers may remain high in patients who suffer frequent recurrence of a disease (e.g., herpesviruses). Viral Serology antibodies to the envelope and capsid antigens of EBV are detected first. Then during convalescence, antibodies to nuclear antigens, such as the EBV nuclear antigen, are detected.  Serologic Test Methods Neutralization and HI The indirect fluorescent antibody test and solid-phase immunoassays, such as latex (hemmaglutination inhibition) tests - agglutination and ELISA, are commonly used to nucleic acids of viruses encode proteins, detect and quantitate viral antigen and such as hemagglutinin, that are antiviral antibody expressed on the surface of the virus. The ELISA test is used to screen the blood supply to exclude individuals who are seropositive for these hemagglutinin proteins of the virus hepatitis B and C viruses and HIV. bind to or clump erythrocytes creating a Western blot analysis has become very important lattice, which settle irregularly in the to confirm seroconversion and hence infection with bottom of the test tube or the microtiter HIV. well. Antibody neutralization of virus inhibits infection and subsequent cytopathologic effects in tissue culture cells. For HI, antibody in serum prevents a standardized amount of virus from binding to and agglutinating erythrocytes. Therefore hemagglutination is inhibited when antibodies are present.  Limitations of Serologic Methods The presence of an antiviral antibody indicates previous infection but is not sufficient to False-positive or false-negative test indicate when the infection occurred. results may confuse the diagnosis. The finding of virus-specific IgM, a patient antibody may be bound with fourfold increase in the antibody viral antigen (as occurs in patients with titer between acute and convalescent hepatitis B) in immune complexes, sera, or specific antibody profiles is thereby preventing antibody detection indicative of recent infection Serologic cross-reactions between different viruses may also confuse the identity of the infecting agent (e.g., parainfluenza and mumps express related antigens). antibody used in the assay may be too specific (many monoclonal antibodies) and may not recognize strains of virus from the same family, giving a false- negative result (e.g., rhinovirus).