Virological diagnostics
The virological diagnosis (or virus diagnostics ) is used for investigation of virological disease. There are various ways of doing this. In the early stages of infection , antigen detection (i.e. detection of individual proteins of the virus ) or genome detection (i.e. detection of the genetic makeup of the virus) is carried out first. At an advanced stage of the disease, antibody detection (i.e. the detection of specific antibodies of the infected person against the virus) can then also be carried out. In the early phase, the detection of antibodies of the immunoglobulin class M (IgM) and later the detection of antibodies of the immunoglobulin class G (IgG) is appropriate. In some viral infections, the detection of antibodies of immunoglobulin class A (IgA) may also be appropriate. However, proof is generally only possible after the diagnostic gap has been identified .
Stage diagnostics
In the case of a large number of virological pathogens, the sequence of the aforementioned examinations is carried out according to a more or less predetermined pattern, which is referred to as stage diagnostics. The step-by-step diagnosis will be explained using the example of a hepatitis C (HCV) infection.
- The attending physician suspects an HCV infection
- In the first step, the patient's blood is examined for antibodies against the HCV using a blood test .
- If no antibodies can be detected, it can be assumed that the patient has not had an HCV infection. However, it must be taken into account that the patient is in the early phase of an infection and no antibodies have yet been formed against the virus.
- Each time antibodies against the virus are detected, the result is validated in a different test (confirmatory test). If antibodies against the virus are also detected in this test, it can be assumed that the patient has or had an HCV infection.
- To further clarify the infection, a PCR (as direct pathogen detection and to determine the viral load and the associated infectivity of the patient) and a typing of the virus (determination of the subtype of the virus) are carried out. These two tests provide important information for planning and monitoring a therapy attempt.
A virus isolation is always appropriate when very little viral antigen is detectable in the patient material or if a new virus to be characterized. For this purpose, the patient material is placed on cell cultures , provided a cell culture model for the virus in question exists. The viruses contained in the material then infect these cells and multiply. For some viruses, enrichment by ultracentrifugation is necessary.
Antigen (Ag) or pathogen detection
Plaque assay
If an infection occurs, changes in the morphology of the cells ( cytopathic effect , CPE) can be observed with some viruses . This can be quantified in a plaque assay . However, only some viruses cause a cytopathic effect ( e.g. herpes simplex viruses , chickenpox viruses , influenza viruses , human adenoviruses , picornaviruses , enteroviruses , measles viruses , mumps viruses and rubella viruses ). Some other viruses do not cause CPE or there are no cells that are infected by these viruses in vitro . The cells showing a CPE or the culture media supernatant of these cells can then be used for further diagnostic tests.
One advantage of the plaque assay over PCR is that the direct effect of a virus can be detected, since only functional viruses can multiply and cause plaques. This is why the faster method is usually followed by detection with cell culture (see also PCR ).
Electron microscopy
Viruses that are present in high quantities in the patient's material can be viewed in the transmission electron microscope after fixation and contrasting (with uranyl acetate , osmium tetroxide or tungstophosphoric acid ) . It is very fast, but the initial purchase of an electron microscope is very expensive at € 200,000 to € 500,000. The determination depends on the sample material, the processing, the magnetic focusing and the quality of the images generated and is therefore carried out by trained persons. The differentiation of virus subtypes is only possible with the help of antibodies that have been previously marked with colloidal gold ( immunogold staining ) due to their identical shape .
Ag ELISA
In the case of infections with adenoviruses or rotaviruses, the patient's stool can serve as the starting material for an Ag-ELISA (ELISA: enzyme-linked immunosorbent assay ). Large numbers of these viruses are excreted in the stool. In this process, the viral proteins are bound by marked virus-specific antibodies . The antigen can then be indirectly characterized via the marking (e.g. fluorescent dyes, enzymes). In the case of viruses that cause respiratory symptoms, such tests can be carried out from smear materials or comparable materials. Enriched samples (see virus isolation) can also serve as starting material for this test.
Western blot
Viral proteins can be detected in Western Blot with antibodies that recognize continuous epitopes . Discontinuous epitopes renature mostly incompletely after SDS-PAGE .
Immunofluorescence test
In the immunofluorescence test, viral proteins are detected by binding fluorescence- labeled antibodies in a fixed cell culture with a fluorescence microscope, provided the epitope is not masked by denaturation or formylation during fixation or an antibody against fixed epitopes exists.
Immunoperoxidase test
The indirect immunoperoxidase assay (IPA) works in the same way as the IFT, only the virus is detected using a reporter enzyme ( horseradish peroxidase ) instead of fluorescent labeling .
Genome Evidence
Polymerase chain reaction
The polymerase chain reaction (English Polymerase Chain Reaction, PCR) is used to detect genome equivalents of the pathogen. For viruses that have an RNA genome, a step is necessary before detection by means of PCR , which transcribes the RNA into DNA . The enzyme that catalyzes this process is reverse transcriptase (RT). The transcription of the RNA into DNA and the subsequent amplification of the genome via PCR is summarized under the abbreviation RT-PCR. The PCR or RT-PCR is established for almost all human pathogenic viruses. It is used when other detection methods do not provide a positive result, but there is an urgent suspicion of a viral infection (see above), to determine genome equivalents for prognostic purposes, for therapy monitoring or if no other methods are available. For a direct proof of infectivity, proof on cell culture usually follows, since in the PCR it is not possible to differentiate between viral particles and infectious viruses. However, this is crucial for precise quantification , but not for qualitative evidence.
DNA sequencing
The DNA sequence can be identified by DNA sequencing , sometimes also in high throughput . At the level of the nucleic acids there are sometimes hardly any matches, which is why a slightly changed virus is not noticeable in the conventional test procedures. Completely unknown viruses can be identified with high-throughput genome sequencing just a few days after infection. Your genome is read from the host cell's genetic pool without the need for a clue as to which sequence to look for. Up to 1.6 million bases are identified simultaneously on a plate. First, all of the host cell's mRNA is sequenced - up to 200 million bases a day. Then the host sequences known from genome research are masked out using a special computer program until only the unknown sequences remain. Parts of it correspond to the genetic information of the virus. Known as well as strongly changed or unknown infectious agents can be identified in clinical samples.
In situ hybridization
The in-situ hybridization is a method in which the genome in tissue sections by hybridization with radioactively or digoxigenin -labeled DNA fragments with inverted complementary is detected sequence. This method is mainly used for the determination and typing of human papilloma viruses (HPV).
Southern blot and Northern blot
The viral genome can be detected by hybridization with radioactively or digoxigenin- labeled DNA fragments with an inverse complementary sequence. In the case of DNA viruses this can be done by Southern blot and in the case of RNA viruses by Northern blot .
Antibody detection
The detection of antibodies that are directed against viral pathogens only provides a limited indication of an acute infection. The detection of antibodies of class G (IgG) is an indication of a previous infection or vaccination . The detection of IgM antibodies can be an indication of an acute infection, but class M AKs can still be detected in many viruses even after the acute phase. In order to be able to make a statement about the stage of the infection, two samples must be examined every few days. An increase in the antibody concentration or an increase in titer can be viewed as an indication of a fresh or recurring infection. Methods for antigen (Ag), genome or pathogen detection should also be used for clarification in many cases.
Hemagglutination Inhibition
The hemagglutination inhibition test (HHT or HIT) is only used for viruses whose surface proteins are able to bind erythrocytes . This phenomenon is based on the binding of the viral proteins to sugar structures on the surfaces of erythrocytes. These erythrocytes bound in this way form a network that no longer sinks to the bottom of the reaction vessel. In the HHT, the human serum to be tested is now added to the system made up of surface proteins from the virus and erythrocytes . Antibodies that are directed against the virus bind to the surface proteins of the viruses and hemagglutination is prevented. For quantification , the serum to be tested is introduced into the test in dilution stages. This allows the titer to be determined which is just sufficient to prevent hemagglutination. In routine diagnostics, this test is used to determine the rubella titer.
Neutralization test
With the help of neutralization tests (NT) antibodies are determined from patient sera that actually have a protective effect. They are able to neutralize the infection of the cells by the virus. It should be noted that especially with viruses, the cellular immune response plays a decisive role, since if the viruses have penetrated the cell, the humoral immune response is only of limited importance in neutralizing the viral infection. Neutralization tests are used for picornaviruses and the cytomegalovirus .
Complement fixation reaction
The complement fixation reaction (KBR) essentially only recognizes IgG1, IgG3 and IgM antibodies. For the diagnosis of an acute infection, two consecutive serum samples (5–10 days apart) are therefore necessary, in which an increased CFR titer can be determined in the case of an acute infection. The KBR is a very old method that is currently being replaced by newer methods. u. is replaced. It can be used for almost all viral infections. Mainly, however, it is used in virological AK diagnosis of influenza A / B, parainfluenza, RSV, VZV, measles, mumps and adenoviruses.
Immunofluorescence test
The starting material for the immunofluorescence test (IFT) are mostly virus-infected cells. These were fixed on glass slides and can be incubated with the patient's serum. The unbound antibodies are removed by washing and the cells are incubated with a second antibody. This antibody is marked with a fluorochrome . This dye emits light of a certain wavelength when irradiated with UV light of a different wavelength. The result can be made visible in the fluorescence microscope . Both IgG antibodies and IgM antibodies can be detected. The IFT is used for patient antibodies against many viruses. The most common use of the IFT is for infections with influenza viruses and the Epstein-Barr virus .
Immunoperoxidase test
The indirect immunoperoxidase assay (IPA) works in the same way as the IFT, except that the antibodies are detected using a reporter enzyme ( horseradish peroxidase ) instead of fluorescent labeling .
ELISA
The enzyme-linked immunosorbent assay ( ELISA ) is the test that is used for almost all viral pathogens. From an economic point of view, the ELISA is the most widespread from an economic point of view through the use of partially or fully automatic machines that process the tests. Otherwise, the restrictions mentioned in the introduction apply.
Western blot
The Western Blot is mainly used for the diagnosis of HIV 1 and 2, rubella, CMV, EBV, Hanta viruses, HTLV and HCV. It is used to confirm the results obtained in the ELISA test or to deal with other diagnostic questions. It offers the possibility to detect antibodies against several viral proteins at the same time.
Immunoprecipitates
The double immunodiffusion and the Gruber-Widal reaction are used less often due to the comparatively high detection limits . However, they are among the earliest antibodies to be detected.
See also
literature
- Otto A. Haller, Thomas Mertens (Hrsg.): Diagnostics and therapy of viral diseases. Guidelines of the Society for Virology. Urban & Fischer, Munich et al. 1999, ISBN 3-437-21970-7 .
- Dietrich Falke (Ed.): Virology at the bedside. Clinic, diagnostics, therapy. Springer, Berlin et al. 1998, ISBN 3-540-64261-7 .
- David M. Knipe, Peter M. Howley (Eds.): Fields' Virology. 2 volumes. 5th edition. Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia PA et al. 2007, ISBN 978-0-7817-6060-7 .