Rapid diagnostics and molecular methods
These approaches allow for the fastest possible detection of the pathogen in clinical material (nasopharyngeal washings, imprint smears, sputum, or blood).
- Molecular genetic method: aimed at the direct detection of genetic material. The main tool is the polymerase chain reaction (PCR). Reverse transcription PCR is used to detect RNA-containing viruses (e.g., influenza virus). This method is particularly relevant for viruses that are difficult to cultivate.
- Rapid diagnostics: based on searching for viral antigens directly in infected cells. For this purpose, the indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) are used.
Virological method
Considered the "gold standard" of diagnostics. This is the classical and most reliable approach, comprising three consecutive stages:
- Cultivation (amplification): viruses do not grow on artificial nutrient media, so they are propagated in living systems. Chicken embryos, laboratory animals, or cell cultures (e.g., primary cultures of monkey, dog, or rhesus macaque kidneys) are used.
- Indication (detection): confirmation that the virus has successfully replicated. In animals, this is evaluated by death or clinical and pathomorphological changes. In cell cultures, evaluation is based on the cytopathic effect (CPE), plaque formation, or the results of the "color test". The properties of the viruses themselves—their ability to induce hemagglutination (HA) and hemadsorption reactions—are also utilized. Sometimes, specific inclusions are found inside cells: intranuclear (adenoviruses), perinuclear cytoplasmic (reoviruses), or Guarnieri bodies (variola virus).
- Identification (typings): determination of the exact species of the pathogen based on its antigenic structure. Complement fixation tests (CFT), hemagglutination inhibition assays (HI), ELISA, and virus neutralization tests (VNT) are applied.
Serological method
The method is based on searching for specific antibodies in the patient's blood using HI, CFT, ELISA, indirect hemagglutination assays (IHA), and neutralization tests (NT).
- Principle: examination of paired sera. The first blood sample is collected at the onset of the disease, and the second 10–14 days later.
- Diagnostic criterion: a reliable confirmation of infection is considered to be a fourfold or greater increase in the antibody titer in the second serum sample.
- Clinical significance: the method is most commonly used for retrospective diagnostics (confirming the diagnosis post-factum) and epidemiological analysis. This is because the acute phase of many diseases lasts 5–7 days, and the patient recovers before the body has time to produce a high level of antibodies.
Diagnostic and prevention features
The choice of methods depends on the properties of the specific pathogen:
- Respiratory infections: for viruses with hemagglutinating properties (e.g., parainfluenza), the hemadsorption phenomenon serves as a crucial indication criterion. Post-infection immunity in such diseases is often fragile and type-specific, making reinfection possible.
- Particularly dangerous infections (PDIs): working with the variola virus requires the strictest containment protocols. Electron microscopy, immunofluorescence assay (IFA), and the search for Guarnieri bodies are used for indication. Specific prophylaxis for smallpox was historically carried out using a robust live vaccine (derived from calf skin scrapings or chicken embryos). The vaccine was administered intradermally, via a jet injector, or as oral tablets (developed by A.A. Vorobyov). Thanks to vaccination, smallpox was globally eradicated, and mandatory vaccinations were discontinued in 1980.
To remember the stages of the virological method, use the "Three I's" rule: Isolation (viral cultivation), Indication (detection of its replication), and Identification (determining the exact species by antigens).
Class and exam questions
Why is the serological method more often used for retrospective diagnostics?
The duration of many viral infections (e.g., ARVI) is 5–7 days, which is significantly shorter than the time required to generate antibodies (10–14 days). Therefore, laboratory confirmation of the diagnosis occurs after the patient has already recovered.
What are paired sera and why are they needed?
These are two patient blood samples taken at an interval of 10–14 days. They are necessary to monitor the dynamics of the immune response: the diagnosis is confirmed only with a fourfold (or greater) increase in the antibody titer in the second serum.
How to detect a virus in cell culture if it is not visible under light microscopy?
The presence of the virus (indication) is determined by indirect signs: cell destruction (cytopathic effect), plaque formation, a change in the color of the nutrient medium ("color test"), or the ability of infected cells to agglutinate erythrocytes (hemadsorption).
What else to study on this topic
- Mechanisms of hemagglutination (HA) and hemadsorption reactions
- Types of cytopathic effect and intracellular inclusions
- Features of clinical material collection in respiratory infections
- Principles of working in laboratories dealing with particularly dangerous infections (PDIs)
- History of creation and types of vaccines against smallpox
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