Diagnostic Principles and Specimen Collection
Globally, laboratory diagnostics of influenza develop 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 finding specific antibodies.
To successfully detect the pathogen, it is critical to collect clinical material correctly. The optimal period for taking nasopharyngeal discharge (as swabs or washings) and smear-imprints from the nasal mucosa 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 Methods
If a physician needs preliminary data quickly, 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 detect the genetic apparatus of the pathogen. Since the genome of this infectious agent is represented by an RNA chain, the main search tool is the polymerase chain reaction (PCR) with a reverse transcription step. This approach allows viral RNA to be detected in clinical samples with high 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 chicken 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 actual fact of viral replication 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 (color test). When using animals, indication 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 assays (HA) and hemadsorption.
- 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. For this purpose, 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 reaction.
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 rise in the antibody titer in the second serum compared to the first.
Various reactions are used to assess antibody levels: HI, CFT, ELISA, and neutralization tests. Due to the need to wait for the second serum sample, this method is unsuitable for early disease detection. It holds predominantly retrospective significance, allowing reliable confirmation of the diagnosis after the infectious episode has passed.