Laboratory Diagnosis

Virological diagnosis is based either on demonstration of the virus or its components (antigens or genome) or on demonstration of a specific antibody response. In some infections antibodies are detectable at the onset of clinical disease (e.g. poliomyelitis, hepatitis B (anti-HBc)), or the antibody appearance may be delayed by days (rubella), weeks or months (hepatitis C, HIV infection). Whenever an early diagnosis is important for the institution of antiviral therapy or some other interference measures, the possible use of methods that demonstrate the virus should be considered.

The virus can be demonstrated directly by electron microscopy (gastroenteritis viruses, orfvirus). Alternatively, infectious virus may be demonstrated after inoculation of cell cultures (enteroviruses, adenoviruses, herpes simplex virus, cytomegalovirus), embryonated eggs (influenzaviruses) or laboratory animals (coxsackievirus). Clinicians should carefully follow the instructions issued by their local laboratories with regard to sampling and transportation, especially if infectivity has to be maintained.

Viral genomes can be demonstrated by various nucleic acid hybridization techniques, either in situ or in tissue extracts (slot blot, Southern blot, in situ hybridization) using labelled DNA or RNA probes, or by methods that include amplification of the viral nucleic acid such as polymerase chain reaction (PCR) and ligase chain reaction (LCR). Both PCR and LCR are extremely sensitive, requiring strict precautions in the laboratory to avoid contamination. The gene technology methods are of particular importance for rapid diagnosis of infections that are accessible to antiviral treatment (herpes simplex encephalitis, CMV infection), for diagnosis of infection with viruses that cannot be cultivated (human papillomaviruses) or viruses that grow slowly in culture (enteroviruses), as well as in clinical situations where a definite diagnosis cannot be made by other means (possible HIV infection and hepatitis B or C in newborns and infants).

Several virus antigen tests are available for rapid diagnosis of virus infections. Methods most commonly used are immunofluorescence or immunoperoxidase for respiratory viruses, ELISA for HBsAg, HIV and rotavirus, latex agglutination for rotavirus, and reverse passive haemagglutina-tion for HBsAg. Immunofluorescence and immunoperoxidase procedures depend on the sampling and preservation of infected cells, requiring rapid transport of cooled material. Alternatively, preparation of the slide has to be made locally. Blood (serum) and faeces can be sent in the usual way.

Antibody examinations are mostly performed with serum. Anticoagulants added to whole blood may interfere with complement activity and enzyme functions, and should be avoided. In certain situations (SSPE, herpes simplex encephalitis) antibody titration is performed on cerebrospinal fluid. Acute infection is diagnosed by demonstrating a rise in titre, seroconversion or specific IgM (or IgA). A rise in titre may be seen both in primary infections and in reinfection or after reactivation. A positive IgM test usually indicates a primary infection, but lower concentrations of specific IgM are found in reactivations (CMV infections and zoster) and reinfections (rubella). A variety of methods (complement fixation (CF), haemagglutination inhibition (HI), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF)) are available for demonstration of antibodies, and the choice of test will depend on the virus and whether the clinical problem is the immune status or diagnosing an acute infection. Blood samples for demonstration of seroconversion or titre rise (paired sera) are taken 1-3 weeks apart, depending on the time of exposure or onset of symptoms.

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