Diagnostic principles and sample collection
Globally, laboratory diagnosis of influenza develops in three main directions:
- Isolation and subsequent identification of the virus itself.
- Indication of viral antigens or RNA molecules directly in cells.
- Serological testing aimed at detecting specific antibodies.
For successful pathogen detection, it is critically important to collect clinical material correctly. The optimal period for collecting nasopharyngeal discharge (in the form of swabs or washes) and nasal mucosa impression smears is the first three days from the onset of the disease. It is during this period that the concentration of viral particles on the mucous membranes reaches its maximum.
Rapid diagnostics and molecular genetic method
If a physician needs to quickly obtain preliminary data, rapid diagnostic methods are used. Their main target is structural viral antigens in the collected biomaterial. To visualize and detect them, laboratories use the immunofluorescence assay (IFA, both direct and indirect variants) and enzyme-linked immunosorbent assay (ELISA).
The molecular genetic method aims to directly identify the genetic apparatus of the pathogen. Since the genome of this infectious agent is represented by an RNA strand, the primary search tool is reverse transcription polymerase chain reaction (RT-PCR). This approach allows viral RNA to be detected in clinical samples with the highest precision.
Virological method (“Gold standard”)
Classic virus isolation is considered the reference but most complex approach. It is strictly divided into three sequential stages:
- Cultivation (amplification). Living biological systems are required for virus propagation. Developing chick embryos, laboratory animals, or cell cultures are used in laboratory practice. The latter include primary cell cultures of monkey kidneys (rhesus macaques) or dogs.
- Indication. At this stage, the very fact of virus propagation in the system is confirmed. In cell cultures, the presence of the pathogen is indicated by the development of a cytopathic effect (CPE), the formation of specific plaques, or a color change in the medium during a color test. When using animals, it is judged by their death or characteristic clinical and pathomorphological changes. The fact of reproduction is also confirmed by the properties of the virus itself using hemagglutination (HA) and hemadsorption assays.
- Identification. The final step is determining the specific species and type of the isolated agent. This procedure is based on a detailed study of its antigenic structure. To this end, complement fixation tests (CFT), hemagglutination inhibition assays (HI), neutralization tests, and ELISA are performed.
Serological method
Unlike genetic or virological screening, the serological method evaluates not the pathogen itself, but the patient's immune system response.
The fundamental principle of the method is the study of paired blood sera. The first blood sample is taken at the very beginning of the illness, and the second 10–14 days later. A strict criterion for confirming the diagnosis is at least a fourfold increase in antibody titer in the second serum compared to the first.
Various reactions are used to assess antibody levels: HI, CFT, ELISA, and neutralization assay (NT). Due to the need to wait a long time for the second serum sample, this method is unsuitable for early disease detection. It has predominantly retrospective significance, making it possible to reliably confirm the diagnosis after a past episode of infection.
Briefly: answers to topical questions
Which surface glycoproteins of the influenza virus are the main antigens for determining its subtype?
The main surface antigens of the influenza virus that determine its subtype are hemagglutinin and neuraminidase.
- Hemagglutinin (HA) is responsible for virus adsorption and fusion with the cell.
- Neuraminidase (NA) removes sialic acid residues from hemagglutinin, which is necessary for the manifestation of infectious properties.
These protective antigens vary independently of each other. A total of 15 HA subtypes and 9 NA subtypes are known, of which H1, H2, H3, and N1, N2 are stably pathogenic for humans.
Which laboratory animal species are most sensitive to the influenza virus and are used for its isolation?
The sources indicate that laboratory animals are used to isolate the influenza virus, but specific most sensitive species are not named. In 1933, the human influenza virus was first isolated after infecting ferrets with nasopharyngeal washes. Virus propagation in infected animals is detected by their death or clinical and pathomorphological changes.
What is the mechanism of the hemadsorption assay used to indicate influenza virus?
The hemadsorption assay is based on changes in the surface properties of infected cells: influenza virus-infected cells acquire the ability to adsorb, i.e., attach, erythrocytes to their membrane. The method allows the virus to be detected even in the absence of a visible cytopathic effect. To detect viruses released from cells into the culture fluid, the hemagglutination assay can also be used.
The rule of "fours" for serology: the diagnosis is confirmed only with a fourfold increase in antibody titer in paired sera taken at an interval of about 14 days.
Class and exam questions
Why is the serological method not used for emergency diagnostics?
Because it requires paired sera with a collection interval of 10–14 days. This method is intended for retrospective confirmation of the diagnosis when the patient is already recovering.
How is the fact of virus propagation proven in cell culture?
This is assessed at the indication stage by the development of a cytopathic effect, the appearance of plaques, changes in the color test, as well as by hemagglutination (HA) and hemadsorption assays.
What biological material is best to collect for searching viral RNA?
It is optimal to use nasopharyngeal discharge (wash or swab) and nasal mucosa impression smears collected in the first 3 days from the onset of the disease.
What else to study on this topic
- Mechanism of performing RT-PCR
- Principles of reading results of hemagglutination (HA) and hemadsorption assays
- Features of virus cultivation in chick embryos
- Antigenic structure of viruses and its role at the identification stage
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