Initial Diagnostics (Screening)
The first stage of the patient's laboratory examination is screening in nature. Its main task is to identify potentially infected blood serum samples for their further in-depth study.
The main method used at this stage is ELISA (Enzyme-Linked Immunosorbent Assay). The purpose of this study is complex: test systems are designed to simultaneously detect specific antibodies to the virus produced by the human immune system, as well as the viral p24 antigen. This combined approach increases the reliability of the primary diagnostic link, but a positive ELISA result is not yet a definitive diagnosis and requires mandatory verification.
Confirmatory Diagnostics (Reference Method)
Samples that test positive during screening are sent for confirmatory diagnostics. Immunoblotting is used as the reference method that finalizes the microbiological diagnosis. This method allows for a detailed separation of viral proteins and the detection of antibodies to specific structural components of the pathogen.
A strict criterion for making a positive diagnosis is the detection in the patient's blood serum of antibodies to at least two viral envelope glycoproteins. These surface proteins are encoded by genes of the env group. The most important rule of the algorithm is that the diagnosis is confirmed based on envelope proteins regardless of whether antibodies to virion core proteins (the gag group) or viral enzymes (the pol group) are present in the patient's serum.
Role of PCR in the Examination Algorithm
Molecular genetic methods, such as polymerase chain reaction (PCR), play an important but strictly auxiliary role in the microbiological algorithm. PCR is categorically not used for initial diagnostics. The main reason for this lies in the extremely high genetic variability of the virus — due to constant mutations, primers may fail to recognize the altered genome, leading to a false-negative result during screening.
PCR begins to be used only after the final confirmation of the diagnosis by serological methods. The method is necessary to solve three clinical tasks:
- Viral load assessment: counting the number of virus copies allows predicting the further course of the disease and assessing the severity of the infectious process.
- Subtype identification: detecting the specific genetic variant of the circulating virus.
- Drug resistance detection: searching for mutations that make the virus resistant to applied medications.
Immunological Monitoring
Since the virus purposefully destroys the patient's immune system, microbiological diagnostics is inextricably linked with immunological monitoring. Physicians need to constantly monitor the degree of preservation of the body's defenses.
The basis of monitoring is the regular counting of key T-lymphocyte subpopulations — cells with CD4 and CD8 receptors. The main marker of increasing immunosuppression is considered to be a drop in the number of CD4 lymphocytes. If this indicator falls below the critical mark of 500 cells/µL, this indicates pronounced immune depression and requires immediate correction of patient management tactics.
Briefly: answers to topical questions
Which specific envelope glycoproteins (env) are detected in HIV immunoblotting?
HIV immunoblotting detects antibodies to specific envelope glycoproteins — products of the env gene: the surface glycoprotein gp120 and the transmembrane glycoprotein gp41. Reliable confirmation of infection is considered to be the detection of antibodies to at least two envelope glycoproteins; antibodies to internal proteins (gag and pol) may be present or absent.
Which viral proteins are encoded by the gag and pol genes in HIV?
The gag gene encodes structural proteins of the inner part of the virion: p24, a capsid protein forming the capsid; p17, a matrix protein; p9 and p6, involved in the release of new viral particles; and p7, involved in genomic RNA packaging.
The pol gene encodes the enzyme complex: reverse transcriptase (p66/p51), integrase (p31–32), protease (p10), and RNase H (p15).
To remember the groups of proteins in immunoblotting: Env (Envelope) — main for diagnosis, Gag (core), Pol (Polymerase — enzymes).
Class and exam questions
Why is PCR not used at the screening stage?
PCR is not used for primary diagnosis due to the high genetic variability of the virus. Mutations can alter the target regions of the genome, making the virus invisible to standard test systems at the initial stage.
What are the criteria for a positive result in immunoblotting?
The diagnosis is considered confirmed if antibodies to at least two viral envelope glycoproteins (env group proteins) are detected in the serum. The presence of antibodies to core proteins (gag) and enzymes (pol) is not mandatory in this case.
Which indicator indicates the development of immunosuppression?
The marker of immunosuppression during immunological monitoring is a decrease in the level of CD4 lymphocytes below the mark of 500 cells/µL.
What is PCR used for after a diagnosis has been made?
PCR is used to assess viral load (which determines the severity of the process and prognosis), identify the viral subtype, and determine the pathogen's drug resistance.
What else to study on this topic
- Structure and functions of viral envelope glycoproteins (env group).
- Characteristics of core proteins (gag) and viral enzymes (pol).
- The p24 antigen: timing of appearance in the blood and diagnostic significance.
- Clinical interpretation of viral load indicators.
- Mechanisms of viral genetic variability and resistance formation.
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