Sample material and rapid diagnostics
The initial and most crucial stage of diagnostics is the correct collection of clinical material. When viral respiratory infections are suspected, respiratory tract secretions are sent for analysis: washes and impression smears from the nose and throat, as well as nasopharyngeal mucus.
To obtain results as quickly as possible in modern clinical practice, rapid diagnostic methods are used. Their main goal is the prompt detection of pathogen traces in the patient's body:
- Detection of viral antigens. Performed directly in the affected cells of the respiratory tract. For this purpose, the immunofluorescence assay (IFA, including direct and indirect modifications) and enzyme-linked immunosorbent assay (ELISA) are used.
- Detection of genetic material. Performed using the polymerase chain reaction (PCR). This method is critically important and especially relevant when searching for fastidious viruses that are difficult or impossible to grow using standard laboratory methods.
Virological method (cultivation)
The classical virological method involves isolating the pathogen. Cell cultures serve as the nutrient medium, which are infected with the patient's respiratory tract secretions.
After the material is introduced, the process is divided into two consecutive stages:
1. Virus indication This is the stage of primary detection of the presence of any virus in the culture. Successful reproduction of the pathogen is indicated by the following phenomena:
- The appearance of a cytopathic effect (CPE) — visible cell changes or cell death.
- A positive hemagglutination (HA) and hemadsorption reaction (characteristic of viruses with specific hemagglutinating properties).
- The formation of specific intracellular inclusions. The location of the inclusions can suggest the type of virus: for example, intranuclear inclusions are typical of adenoviruses, while cytoplasmic perinuclear inclusions are typical of reoviruses.
- Using the plaque assay and the 'color test'.
2. Virus identification Once the presence of a virus has been proven, its species must be determined. Identification is carried out by studying the antigenic structure of the pathogen. A wide range of serological reactions is used for this: CFT, IHA, ELISA, HIFA, and the neutralization test (NT).
Serological method
Serological diagnostics is based on assessing the humoral immune response of the body to the introduction of the pathogen. The main principle of the method is the study of the patient's paired sera.
Key features of the serological approach:
- Timing of collection: the first serum is taken at the onset of the disease, the second — with an interval of 10–14 days.
- Diagnostic criterion: the diagnosis is considered confirmed only if there is a 4-fold or greater increase in the titer of specific antibodies.
- Reactions used: neutralization test (NT), complement fixation test (CFT), indirect hemagglutination assay (IHA), and hemagglutination inhibition assay (HIFA).
It is important to note the clinical significance of this method. Since the average duration of uncomplicated ARVI is only 5–7 days, the serological method takes longer than the disease itself (due to the need to wait for antibody production). Therefore, it serves primarily for retrospective diagnostics (confirming a past infection) and conducting a detailed epidemiological analysis in the population.
Three stages of diagnostics: Rapid — looking for virus 'parts' (PCR, ELISA); Virology — 'growing' the virus in cells; Serology — looking for the body's response (antibodies in paired sera).
Class and exam questions
Why is the serological method used primarily for retrospective diagnostics?
The duration of clinical manifestations of the disease is usually 5–7 days, which is significantly shorter than the time required for full antibody production. Reliable analysis requires paired sera with an interval of 10–14 days, by which time the patient has already recovered.
What is the absolute diagnostic criterion when examining paired sera?
Reliable confirmation of a past infection is a 4-fold or greater increase in the titer of specific antibodies in the second serum compared to the first.
What does the indication stage involve in the virological method?
Indication is the detection of the very fact of virus replication in a cell culture. It is judged by the cytopathic effect, the formation of inclusions, the plaque assay, as well as hemagglutination and hemadsorption reactions.
Which method is best suited for diagnosing fastidious viruses?
For pathogens that grow poorly in cell cultures, it is optimal to use PCR, as this method allows for the direct detection of the virus's genetic material.
What else to study on this topic
- Mechanisms of cytopathic effect (CPE) formation in cell cultures
- Principles of setting up hemagglutination (HA) and hemadsorption reactions
- Morphological differences between intranuclear and cytoplasmic inclusions
- Methodology for performing the 'color test' and plaque assay
- Algorithm for setting up the complement fixation test (CFT) for identification
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