Methods of laboratory diagnosis of HIV infection. Guidelines for the diagnosis of HIV infection

Produced in several different ways. It would seem that there is nothing easier than to identify this disease through blood sampling. But it is not so. The diagnosis of HIV can indeed be detected in this way, but further research is carried out by different methods. It largely depends on them what treatment will be prescribed to the patient, and what measures will be taken subsequently. Among the most common and highly effective methods, it is customary to distinguish screening analysis and immune blotting. Each of them should be considered in more detail.

AIDS diagnostics: screening analysis

A screening test or ELISA test method is used at the initial stage of diagnosing HIV infection. When developing this method in the laboratory, the proteins of the virus were artificially created. They react in a special way to antibodies. The latter are produced in the body when cells appear in it, infected with a terrible disease. In this case, laboratory diagnostics of HIV is carried out using artificial enzymes. Interacting with antibodies, they stain in a certain color. A strip with an indicator, after getting blood on it, is placed under a special apparatus with the help of which it is possible to determine whether a person has this disease or not. Modern methods of HIV diagnostics, including ELISA, make it possible to determine the fact of infection or its absence with high accuracy. But, like all equipment, the screening apparatus has an error. That is why, if necessary, the analysis is repeated by the patient.

It is important to know that the ELISA test is one of the earliest methods for determining the presence of an immunodeficiency virus in the body. HIV infection is a diagnosis that can only be detected a few weeks after infection. When infected cells enter the bloodstream, the body's immune system begins to actively resist. Antibodies are produced, which are detected by laboratory research after two or three weeks. Early diagnosis of HIV infection by means of an ELISA test allows detecting antibodies to the immunodeficiency virus in human blood. This is its main difference from other modern methods.

It is worth noting that in some people, antibodies to this disease begin to be produced at a later date. It can take from three to six weeks from the moment of infection to the beginning of this process. That is why medical experts recommend screening tests four to five weeks after unprotected sex or other reason for suspecting an accidental infection.

ELISA methods for laboratory diagnosis of HIV infection have been developed for a long time. Today there are four generations of tests. The most accurate and effective of them are those that were developed last. Laboratory diagnostics of HIV infection and AIDS using the third and fourth generation ELISA tests is carried out using recombinant proteins and peptides. The sensitivity of these tests to antibodies produced by the body is 92 - 93%. We are talking about Russian research methods. Europeans have learned to do such tests with a sensitivity of 99%.

Modern methods of laboratory diagnostics of HIV infection based on ELISA are used not only to detect the presence of a disease. With the help of screening analysis, it is possible to monitor the spread of the immunodeficiency virus, as well as to collect test material before taking blood from donors.

ELISA is a standard method for diagnosing HIV infection: how is it carried out, why does it give a false result?

Diagnosis of HIV and AIDS by ELISA is a standard procedure. The patient's blood is drawn from a vein. For analysis, five milliliters of material is sufficient. Clinical hIV type 2 and type 1 diagnostics are performed at least eight hours after eating. Doctors recommend doing it in the morning on an empty stomach. The results of a complete study are known after two or three days. Express screening is done in less time. This usually takes only a few hours. However, the error in this case increases. An express diagnosis of HIV is necessary in emergency cases, for example, when a patient needs urgent surgery. Such an analysis is necessarily performed before unscheduled surgical interventions, because if a patient has an immunodeficiency virus, doctors observe increased safety measures. Clinical diagnosis of HIV infection in a short period of time is also necessary in the case when donor blood must be taken quickly to save another patient.

There are cases when a person was diagnosed with AIDS or HIV, but as a result he was not confirmed, or vice versa. Why is this happening in the case of the ELISA test? A false negative result may be due to the fact that the blood was not properly prepared for research. Sometimes the reason for this is the incorrect execution of her fence. A false negative result when verifying the diagnosis of HIV infection is not confirmed even in the case when materials for research were taken too early. After all, after infection, at least three weeks should have passed. There are cases when the result of the detection of the immunodeficiency virus is false positive. This is due to the general state of the immune and hormonal systems of the human body. In most cases, the patient has diseases that are the result of a false positive result. We are talking about alcoholic hepatitis, in which the liver produces special enzymes, which can lead to an incorrect diagnosis of HIV. Pregnancy and certain autoimmune diseases, along with multiple myeloma, can also cause false positives. This list could include dialysis patients and those who were vaccinated shortly before the differential diagnosis of HIV.

Speaking about screening analysis, it is important to note that this research method requires confirmation. Doctors, before making a diagnosis of HIV based on it, be sure to send a patient for immune blotting.

The main methods of laboratory diagnosis of HIV infection: immune blotting

AIDS, the diagnosis and treatment of which are closely related, is detected in several ways at once. As mentioned above, one ELISA test is not enough to finally determine the diagnosis. Differential diagnosis of HIV infection in this case is complemented by immune blotting. In modern medicine, it is called the final confirmatory method for making a diagnosis. For research in this case, proteins contained in the patient's blood are used. They are separated in a special gel, after which laboratory workers have the opportunity to study the molecular composition of blood.

The timing of HIV diagnosis with this method is from one to three days. For research, blood is taken from the subject's vein, which is placed on a tester with a gel. The result, which is questioned, may occur during pregnancy, the presence of oncological processes, tuberculosis. A false negative reaction to the test can occur if the study is performed incorrectly and if it was carried out too early. At least three weeks should pass from the moment of infection. After all, the time after which HIV is diagnosed directly depends on the state of immunity.

Other methods of diagnosing AIDS and HIV

Radiological diagnostics for AIDS are used, as a rule, in the terminal or secondary stages. With their help, it is possible to establish the changes that occur in the body due to secondary diseases or opportunistic infections.

Bioresonance HIV diagnostics, which is also called non-linear scanning, is used mainly in private clinics to detect the immunodeficiency virus. Official medicine considers this method to be insufficiently accurate and effective. Perhaps, the improvement of non-linear diagnostics in the future will make it possible to identify this terrible diagnosis already in the incubation period.

Diagnosis of HIV infection: serological method and its features

This is a relatively new technique in which laboratory criteria for diagnosing HIV infection are based on the antigen-antibody principle. In the case of the immunodeficiency virus, a search for antibodies to the virus occurs in the human body. For this, an artificially derived antigen is used, to which antibodies must respond. Specific serological markers of HIV infection cause specific changes in blood composition. It should be noted that this research method is most effective in the acute stage, which ends with seroconversion. It is followed by an asymptomatic latency period, during which it is not easy to find the antigen.

Reference diagnosis of HIV infection: description and features

HIV reference values \u200b\u200bare considered the most accurate in identifying a diagnosis. This research method combines ELISA test and immune blotting. The HIV diagnostic reference laboratory allows the collection of tests and their subsequent processing in two ways at once. This allows you to get the most accurate result within 24 hours. Re-checking the analysis obtained in the reference laboratory is necessary only if the data of the ELISA test and the immune blotting contradict each other. This is a fairly rare occurrence that most often occurs in people with the above-described concomitant diseases.

Diagnostics of HIV is one of the primary tasks facing the employees of the dermatovenerologic dispensary, as well as the employees of the polyclinic.

The disease is characterized by doctors as very insidious. It is characterized by a chronic course and does not respond to full treatment. It is important to detect it in a timely manner in order to take control and prevent uncontrolled spread. What are the features of the human immunodeficiency virus, and how it can be infected, patients are often interested.

What are the ways to diagnose the disease, and what signs suggest infection?

Today you can hear from everywhere how dangerous HIV infection is. However, few explain what this danger is. As a result, patients have an incomplete set of information and, as a result, do not take the threat seriously. But HIV is extremely dangerous. It is classified as a slowly progressive viral disease prone to a chronic course. In this pathology, the immune system is primarily affected.

Doctors draw the attention of patients to the fact that death does not occur from the immunodeficiency virus itself, as such.

A person dies from concomitant infections, the body is no longer capable of providing full protection against which. Also, the cause of death is cancerous tumors, which are not able to fight the reduced immunity.

In fact, the mechanism by which HIV infection affects the immune system is quite complex. As doctors note, patients do not need to understand it thoroughly. It is enough to know that the disease can reduce the level of immunity to critical values. As a result, the body will be unable to defend itself against various external influences, which will lead to death sooner or later.

How does infection occur

It is important to understand that HIV infection today is surrounded by a wide variety of myths.

Patients are very poorly informed about when they can get infected and when health is out of danger.

The first thing to remember is that HIV is very fragile in its environment. This means that a pathogenic microorganism is able to live fully and for a long time only in the human body. He does not tolerate heating over 50 degrees (dies instantly). Also unable to resist drying processes. Not all body fluids contain enough virus for infection to occur.

The greatest danger is posed by:

• blood;
• pre-ejaculate;
• sperm;
• discharge from the female vagina;
• lymph;
• breast milk.

If any of these fluids come into contact with mucous membranes, in which there are microtrauma, or with injured skin, infection occurs.

It is also possible if foreign fluid enters the bloodstream directly. Saliva and tears, contrary to popular belief, are not a threat. Due to the nature of the virus and its low survival rate, it is transmitted in several ways:

• genital tract i.e. with unprotected sexual intercourse, which inevitably entails contact of biological fluids and mucous membranes of the body susceptible to the pathogen;
• parenteral route i.e. transmission of the virus with blood during its transfusion or due to the use of non-sterile instruments for medical purposes;
• vertical path i.e. from mother to child (today, if a woman takes antiretroviral therapy and refuses to breastfeed, the likelihood of infection of the child during childbirth is minimized).

It is important to understand that if micro-trauma or open wounds are required for infection through the skin, then this is not necessary for infection through the mucous membrane. The difference is explained by the fact that the mucous membranes and skin of the human body have a completely different structure. This difference must be taken into account.

What are the signs to suspect HIV

Many patients are interested in the question of what signs are usually suspected of being infected with the human immunodeficiency virus.

• an unreasonable increase in temperature of the systemic type, which cannot be explained by any other infection, and which persists for a long time, despite the measures taken for treatment;
• a strong increase in lymph nodes in size (first of all, the nodes in the groin area suffer, but their involvement throughout the body is also possible);

• a strong decrease in body weight, which cannot be explained by diets, stress, hormonal disruptions and other reasons;
• complaints of stool disorders that have followed the patient for a long time, and it is not possible to find the reason why they appeared;
• a pronounced tendency to the transition of any infectious diseases into chronic forms, and the nature of the pathogen does not matter much, both bacterial and viral pathologies are chronic;
• diseases provoked by opportunistic microflora develop, which does not pose a threat to a person whose immunity is fully functional (for example, mycoplasmosis, ureaplasmosis, candidiasis, etc.).

The clinic of HIV infection is very nonspecific, as doctors say. Because of this, it is often difficult to make a diagnosis. Many patients completely ignore alarming symptoms, preferring not to seek medical help. Even if the disease strongly affects their general well-being.

It is important to understand that HIV infection may not be felt at all for a long time. And when the first signs appear, a person may not even associate them with the possibility of his infection and make attempts to be treated at home.

Diagnostic methods

Laboratory diagnostics of HIV has been developed for a long time and has been successfully used to diagnose this dangerous disease.

The disease cannot be recognized by symptoms alone. Therefore, confirmation of the diagnosis based on laboratory techniques often plays a decisive role.

There are various methods for diagnosing HIV. In Russia, in the first place, preference is given to immune blotting, as well as ELISA reactions. These methods are often used as screening methods, for example, when checking medical personnel.

ELISA systems

Often, patients ask their doctors what method to start a diagnostic search with if they suspect infection with the human immunodeficiency virus.

Any competent doctor will say that preference should be given to enzyme immunoassay. It is this technique that is the first diagnostic stage in Russia.

The principle of ELISA is simple. In the laboratory, doctors created special proteins. They are able to detect and interact with antibodies produced by the body in response to contact with HIV. Then a special indicator enzyme is added to the system, which changes its color. At the final stage, the material is processed using a special apparatus, and the doctor receives the final result.

ELISA is very popular.

First of all, due to the fact that results can be obtained even if no more than a few weeks have passed since the introduction of the pathogen into the body.

It is important to understand that the enzyme immunoassay does not detect the virus itself in the blood, but antibodies to it.

In many people, they may begin to be produced later than two weeks later, which may cause the result to be erroneous. There are several generations of ELISA tests.

The most modern and highly accurate are those that belong to the 3rd and 4th generations. Doctors note that it is best, if there is a choice, to give preference to European reagents, since their accuracy reaches 99%. The time for obtaining ELISA results is on average 2 to 10 days.

Why ELISA can be false

It is important to understand that enzyme immunoassay can give both false positive and false negative results. Although the risk of such a development of events is extremely small.

The patient can get false negative results if the analysis was submitted too early, and antibodies have not yet had time to form in the body.

To exclude such a reaction, patients are recommended to be tested several times at different intervals.

A false positive test occurs in some diseases. For example, patients with:

• alcoholic hepatitis;
• myelomas in large numbers;
• some autoimmune diseases;
• women during pregnancy, etc.

In such diseases, human blood is replenished with antibodies. They can resemble HIV antibodies in structure, which confuses the reagents, provoking a reaction. Of course, in recent years, test systems have become more and more sensitive. However, the problem of false results has not yet been fully resolved.

Immunoblotting

In modern conditions, it is impossible to make a positive HIV diagnosis relying only on ELISA. It is necessary to confirm the results obtained, which is performed using the reaction of immune blotting (immunoblotting, IB).

To perform IB, the laboratory must have special test strips. Viral proteins are applied to them. Before the analysis, the patient's blood taken from the vein is prepared in a special way.

The obtained biological material is added to the gel, in which the proteins are separated by their weight. Then, a previously prepared strip is lowered into the resulting mass.

The strip becomes wet (blotting occurs), stripes are detected on it if there are HIV proteins in the material. If proteins are absent, wetting does not change the appearance of the strip.

There are several interpretations of Western blotting. However, no matter what method a particular hospital or laboratory uses to decode, the probability of correct diagnosis is 99.9%.

Can immunoblotting give incorrect results, patients often wonder? Yes, this is possible, for example, if the patient is sick with tuberculosis, is pregnant, or suffers from oncology.

PCR to help

PCR is another method that can diagnose human immunodeficiency virus in blood and other biological fluids, where its concentration is quite high.

As doctors note, the polymerase chain reaction can give a positive result within 10 days after the first contact of the body with the infection.

It is important to understand that PCR in some cases gives false positive results. This is explained by the fact that the method has a very high sensitivity.

As a result, it often reacts to similar antibodies, indicating completely different pathological processes in the patient's body.

Despite the high sensitivity and low probability of obtaining false results, PCR is not widely used. This is explained by several factors. First, to perform the polymerase chain reaction requires special equipment, the price of which is quite high. Secondly, the personnel working with the equipment must be highly qualified, which can also cause difficulties. Together, these features make PCR an expensive diagnostic method and, as a result, not available to everyone.

Despite the fact that PCR is not a screening method, it is used, for example, to test a newborn for infection with the human immunodeficiency virus.

Express systems for diagnostics

Doctors and scientists have gone to great lengths to create rapid tests to assess HIV infection. As doctors note, when using these systems, it is possible to get a result within 15 minutes after the test was performed.

Rapid HIV tests are based on the principle of immunochromatography. The system usually includes a strip soaked in special reagents.

The patient's task is to apply blood, semen, or any other biological fluid that may contain antibodies to the virus.

If they are found, then two colored stripes will appear on the strip, one of which is control and the other diagnostic. If not, then only the control strip will come to light.

It is important to understand that rapid tests do not give a 100% guarantee that a person is not infected or, on the contrary, infected with HIV. In any case, the results obtained with their help must be confirmed in the laboratory using immunoblotting.

Express test systems are convenient for patients who want to calm themselves at home. However, as doctors note, even if with their help a person received a negative result, if you suspect negative changes in the body, you should still consult a doctor.

Which doctor should you contact if you suspect infection

Many patients wonder which doctor they should go to if they suspect HIV infection. First of all, it is recommended to visit a venereologist. It is this medical professional who specializes in diseases that can be transmitted from person to person through sexual contact.

The venereologist will be able to conduct a competent examination, collect anamnesis and decide which examinations are necessary for the patient to make an accurate diagnosis. At his discretion, he can also refer the patient to the infectious diseases hospital. Especially if he still suspects he has HIV.

Human immunodeficiency virus is a common disease. Anyone who is sexually active can face him.

Knowledge of the characteristics of the spread and diagnosis of this disease in modern realities is vital if the patient wants to maintain his health and longevity. Only a timely visit to the doctor will allow you to take the infection under control and protect yourself from it!

Clinical and laboratory diagnostics of HIV infection has three directions:

1. Establishing the fact of HIV infection, diagnosis of HIV infection.
2. Determination of the stage of the clinical course of the disease and the identification of secondary diseases.
3. Prediction of the progression of the clinical course of the disease, laboratory monitoring of the effectiveness of treatment and side effects of antiretroviral drugs.

1. Establishment of HIV infection, diagnosis of HIV infection

To determine HIV infection, the following specific indicators are used: antibodies to HIV, HIV antigens, HIV RNA and provirus DNA. Antibodies to HIV are determined by enzyme-linked immunosorbent assay (ELISA) or immunoblotting, which is essentially a type of ELISA. Antigens (proteins) of HIV are determined by ELISA. With the help of molecular genetic methods of polymerase chain reaction (PCR) and bDNA, HIV RNA and provirus DNA can be determined. The use of an additional method of hybridization of nucleic acids with specific DNA probes makes it possible to check the specificity of the DNA sequences obtained during PCR. PCR sensitivity is the detection of viral genes in one in five thousand cells.

In primary infection, the following dynamics of HIV markers in the blood of infected people is observed. In the first month, as a result of the activation of the replicative process, there is a sharp increase in the viral load (the content of HIV RNA in the plasma), then, due to the dissemination of the virus and massive infection of target cells in the blood and lymph nodes, it becomes possible to determine the proviral DNA. The fact of detecting provirus DNA integrated into the genome of the target cell is of paramount diagnostic value.

The viral load reflects the intensity of the replicative process in infected cells. During the period of primary infection, the level of viral load is different when infected with different HIV subtypes, but the dynamics of its changes is approximately the same. So, when infected with subtype B, for example, if in the first month after infection the viral load is 700 copies / ml, then in the 2nd month there is a decrease to 600, in the 3rd - up to 100, in the 4th - up to 50 copies / ml. Such dynamics is observed against the background of an increase in the content of specific antibodies to HIV in the blood. The content of proviral DNA in blood mononuclear cells of HIV-infected patients is characterized by relative constancy during the first 6 months with slight fluctuations in some subtypes. Thus, RNA and DNA loads are not identical.

During the incubation stage, for some time, there is no formation of specific antibodies to HIV in an amount sufficient for determination by existing laboratory methods. Before the registration of antibodies, the appearance in the blood of the Nef protein, which represses the replicative process, and the structural protein p24, are observed for a very short time. The p24 antigen can be detected in the blood by the method of enzyme-linked immunosorbent assay as early as 1-2 ped after infection and be determined until the 8th week, then its content sharply decreases. Further, in the clinical course of HIV infection, there is a second rise in the p24 protein content in the blood. It falls on the formation of AIDS. The disappearance of free (not bound by antibodies) p24 core proteins in the blood and the appearance of specific antibodies to HIV proteins mark the onset of seroconversion (Fig. 9.6).

Viremia and antigenemia cause the formation of specific IgM antibodies (anti-p24, anti-gp41, anti-gp120, anti-gp160). Free antibodies of the IgM and IgG classes to the p24 protein can appear starting from the 2nd week, their content increases within 2-4 weeks, reaching a certain level, at which it remains for months (IgM) and years (IgG) (Fig. 9.7).

The appearance of complete seroconversion, when a high level of specific IgG antibodies to the structural proteins of HIV p24, gp41, gp120, gp160 is recorded in the peripheral blood, greatly facilitates the diagnosis of HIV infection. Antibodies to HIV appear in 90-95% of those infected within 3 months after infection, in 5-9% - in the period from 3 to 6 months from the moment of infection and in 0.5-1% - at a later date.

Despite the fact that antibodies to HIV appear last, the main laboratory diagnostic indicator to date is the detection of specific antibodies by ELISA and immunoblotting.

Data presented in table 9.2 [show] and 9.3 [show] , clearly demonstrate the high sensitivity of modern enzyme-linked immunosorbent assay systems for the determination of antibodies to HIV, which is superior to the sensitivity of immunoblotting. In some cases, upon receipt of a primary positive result in ELISA, it can be confirmed in immunoblotting only after 2-3 weeks.

When examining HIV-infected patients (HIV-infected) using immunoblotting test systems from leading companies in the world, antibodies to gp160 and p24 / 25 are detected in all cases, antibodies to other proteins are detected in 38.8-93.3% of cases (Table. 9.4 [show] ).

Difficulties in detecting antibodies in patients with HIV infection may arise during periods of massive viremia and antigenemia, when the existing specific antibodies in the blood are associated with viral particles, and the replicative process outstrips the production of new antiviral antibodies. This situation can arise and disappear during the infectious process.

In patients with an initially weakened immune system, viremia and antigenemia appear earlier and remain at a high level until the outcome of the disease. In such patients, there is a low content of free antibodies to HIV, due to two reasons - insufficient production of antibodies by B-lymphocytes and binding of antibodies by virions and soluble HIV proteins, therefore, to determine infection, test systems with increased sensitivity or modifications of analysis methods that provide for the stage of antibody release are required from immune complexes.

Most often, a decrease in the content of antibodies to HIV for the indicated reasons occurs at the terminal stage, when antibodies to HIV in the serum may not be detected either by enzyme-linked immunosorbent assay or by the method of immunoblotting (Western blot). In addition to the appearance of specific antibodies to HIV, the immune response in the first 4 months is characterized by a decrease in the content of infected CD4 + cells in the blood and an increase in CD8 + cells. Further, the content of cells carrying CD4 and CD8 receptors stabilizes and remains unchanged for some time. The increase in the content of CD8-lymphocytes is a protective reaction, because cell-dependent cytotoxicity is realized by CD8 + lymphocytes, which are aimed at destroying HPV-infected cells. Initially, cytotoxic lymphocytes (CTLs) react to the regulatory protein Nef of the virus, which plays an important role in reducing the viral (RNA) load in the plasma of an HIV-infected person in the first months. Then a response from the CTL is formed to others, incl. structural, HIV proteins, as a result of which, 12 months after infection, the cytotoxic effect increases significantly.

Schemes for laboratory diagnosis of HIV infection

Taking into account the given dynamics of specific markers of HIV infection in practice, it is advisable to adhere to the following laboratory diagnostic schemes in adults (Fig. 9.8-9.10).

The diagrams reflect the three main stages of the primary laboratory diagnosis of HIV infection:

1. Screening.
2. Reference.
3. Expert.

The need for several stages of laboratory diagnostics is primarily due to economic considerations. So, for example, the cost of carrying out one expert study with the help of domestic test systems using the immunoblotting method is up to $ 40, screening (using the ELISA method) - about 0.2, that is, the ratio is 1: 200.

At the first stage (Fig. 9.8), the subjects are tested for antibodies to HIV using a single enzyme-linked immunosorbent assay designed to detect antibodies to both types of virus - HIV-1 and HIV-2.

Manufacturers in the proposed test systems use viral lysate, recombinant proteins, synthetic peptides as an antigenic basis. Each of the listed carriers of HIV antigenic determinants has its own advantages and disadvantages. Therefore, when choosing test kits of approximately equal cost, kits with the highest sensitivity (preferably 100%) should be preferred. Among the test systems of the same cost and sensitivity, it is advisable to focus on those with the maximum specificity.

Based on the virus lysate, the first test systems for laboratory diagnosis of HIV infection were created. In the 1980s, such test systems were characterized by a sensitivity of less than 100% and a low specificity, manifested by a large number (up to 60%) of false-positive results.

During the formation of a virion in a culture of lymphocytes, its membrane is created from the outer membrane and therefore contains antigens of the main histocompatibility complex of classes I and II. This circumstance causes false-positive reactions in the event that antibodies to alloantigens of histocompatibility are present in the blood of patients.

Later, to obtain a virus, it was proposed to use a culture of macrophages, in which viral particles are formed mainly intracellularly by budding not from the outer cell membrane, but from the membranes of the endoplasmic reticulum. This technology has reduced the number of false positives.

One of the best in terms of the most important characteristics - sensitivity and specificity - are recognized enzyme-linked immunosorbent assays, which use a combination of purified viral lysate with synthetic peptides, which are the most antigenically significant portions of the virus proteins, or recombinant proteins.

The sensitivity of the test system also depends on the characteristics of the other components of the kits. Thus, test systems in which conjugates are used that recognize antibodies not only of the IgG class, but also of IgM and IgA, make it possible to detect an earlier phase of seroconversion. The use of test systems seems to be promising, with the help of which it is possible to simultaneously determine both antiviral antibodies and p24 antigen, which makes the laboratory diagnosis of HIV infection even earlier.

The primary positive result must be rechecked by re-examining the sample in the same test system, but preferably in a different batch and by a different laboratory assistant. If a negative result is obtained during a second study, the study is carried out a third time.

After confirmation of a positive result, it is advisable to re-draw blood and test it for antibodies to HIV as primary. Repeated blood sampling helps to prevent errors caused by inaccurate labeling of tubes and filling out referral forms.

Serum serum positive at the screening stage is sent for reference studies performed using two or three highly specific ELISA test systems. In the case of two positive results, an expert study is carried out using the immunoblotting method.

The use of enzyme-linked immunosorbent assays in reference diagnostics, which can be used to differentiate specific antibodies to HIV-1 and HIV-2, facilitates further work and allows you to study a positive sample at the expert stage immediately using the appropriate immunoblotting (HIV-1 or HIV-2) ...

A laboratory expert opinion on HIV infection is made only on the basis of a positive Western blot result. When conducting expert diagnostics, it is necessary to use the nomenclature of genes and gene products of HIV proposed in 1990 by a group of WHO experts (Table 9.5 [show] ).

The specificity of the bands on the immunoblot should be assessed very carefully and carefully, using the results of studies of control sera (positive and negative), which are carried out in parallel with the study of experimental samples, and a sample of the immunoblot with the designation of HIV proteins (attached by the manufacturer to the test system). The interpretation of the results obtained should be carried out in accordance with the instructions attached to the test system. As a rule, the criterion of positivity is the mandatory presence of antibodies to two proteins (precursor, outer or transmembrane) encoded by the env gene, and the possible presence of antibodies to the products of two other structural HIV genes - gag and pol (Table 9.6 [show] ).

In case of obtaining a dubious result, it is necessary to use the list of recommendations for the final clarification of the results of immunoblotting (Table 9.7 [show] ).

According to the recommendations of the Russian Scientific and Methodological Center for the Prevention and Control of AIDS, a positive result is considered if there are antibodies to at least one of the gp41, gp120, gp160 proteins in combination with antibodies to other specific HIV-1 proteins or without them. These recommendations are made on the basis of experience with the sera of children from nosocomial foci, in which antibodies were often determined only to one of the virus envelope proteins.

Most of the patients who were initially examined seropositive in ELISA belong to the phase of persistent generalized lymphadenopathy (PGL) or to the asymptomatic phase. Therefore, on an immunoblot (a nitrocellulose strip on which HIV proteins are immobilized), as a rule, the following combination of antibodies to HIV-1 is determined: antibodies to the env coat proteins gp160, gp120 and gp41, encoded by the env gene, in combination with antibodies to core proteins p24 (protein nucleocapsid encoded by the gag gene) and p31 / 34 (endonuclease encoded by the pol gene).

Positive reactions with only gag and / or po proteins can occur in the early phase of seroconversion, and also indicate an infection with HIV-2 or a non-specific reaction.

In case of obtaining a dubious result, it is possible to use various methodological techniques to clarify the fact of HIV infection.

Depending on the technical capabilities (availability of diagnostic kits and reagents, equipment with special equipment and personnel training), the expert laboratory carries out additional diagnostic studies (Figure 9.10).

In some cases, it is advisable to use molecular genetic methods to determine the genetic sequences of HIV in serum, blood lymphocytes, or lymph node punctures. Verification of the specificity of DNA sequences obtained as a result of PCR can be carried out by the method of hybridization of nucleic acids with specific DNA probes.

Methods of radioimmunoprecipitation (RIP) and indirect immunofluorescence (IFL) can also be used for the final verification of sera with questionable results in immunoblotting.

Detection of HIV RNA in blood plasma by qualitative or quantitative method is not significant for the diagnosis of HIV infection. This result should be confirmed by standard methods, such as immunoblotting, 2-4 months after receiving the primary questionable or negative response.

Isolation of HIV in cell culture is the ultimate truth. However, the method is complicated, expensive, and is performed only in specially equipped research laboratories.

The content of CD4 + - cells in the blood is a nonspecific indicator, however, in disputable cases (ELISA "+", immunoblot "-", the presence of clinical signs of HIV infection / AIDS) it can be used as a guide for making an expert decision. If in the laboratory it is possible to perform only immunoblotting, then you should adhere to the recommendations set out in table. 9.7 and Fig. 9.9.

Persons whose serum expert examination yielded dubious (uncertain) results, with the exception of cases of detecting antibodies only to p17 (HIV-1) or p16 (HIV-2), should be retested within 6 months (after 3 months). In the case of true HIV infection, after 3-6 months, a "positive" trend is observed in the spectrum of antibodies - additional formation of antibodies to other proteins of the virus. A false reaction is characterized by the persistence of a dubious immune blotting pattern for a long time or the disappearance of suspicious bands. If after the specified period the results of repeated immunoblotting are negative or remain doubtful, then in the absence of risk factors, clinical symptoms or other factors associated with HIV infection, the person can be considered seronegative for antibodies to HIV-1 and HIV-2.

False positive results due to the content in the blood of patients of antibodies to alloantigens of histocompatibility, which are part of the HIV envelope, appear on the immunoblot in the form of bands at the level of gp41 and gp31. The reasons for other nonspecific reactions (for example, to p24, which is often found in individuals with autoimmune processes) have not yet been clarified.

Improvement of the technology for the production of enzyme-linked immunosorbent assays made it possible to achieve high sensitivity - up to 99.99%, while the sensitivity of the immunoblotting method is 97%. Therefore, a negative result in immunoblotting with positive results in ELISA may indicate the initial period of seroconversion, characterized by a low level of specific antibodies. Therefore, it is necessary to repeat the study after 1.5-2 months, that is, the period of time required for the completion of seroconversion, for reaching the concentration of specific antibodies in the blood, sufficient for detection by immunoblotting.

A positive result (results) of a study at the reference or only screening stage of laboratory diagnosis of HIV infection, that is, a positive result in any enzyme-linked immunosorbent assay, ultimately not confirmed by expert methods, is interpreted as the presence of cross-reacting antibodies in the blood. Cross-reacting refers to the binding of nonspecific sites by antibodies on HIV proteins or peptides used as an antigenic basis in the test system in which a positive result is obtained.

In the absence of immunodeficiency and clinical signs of HIV infection, such persons are considered seronegative for HIV antibodies and should be removed from the register.

The final diagnosis of HIV infection is established only on the basis of all clinical, epidemiological and laboratory data. Only the attending physician has the right to inform the patient about the diagnosis of HIV infection.

The main method of confirmatory (expert) laboratory diagnosis of HIV infection is immunoblotting. However, given its lower sensitivity compared to ELISA, a number of researchers suggested using a combination of several test systems for the final determination of the presence of specific antibodies to HIV. For example, G. van der Groen et al. proposed an alternative to immunoblotting method for checking positive results of the screening stage of laboratory diagnosis of HIV infection. It involves the study of the material in parallel in three test systems, which are based on different methods for detecting specific antibodies to HIV (several variants of ELISA, agglutination reaction) using antigens of different nature. The authors managed to find such combinations of test systems, the use of which provides 100% sensitivity and specificity when compared with the results obtained in immunoblotting.

The cheapness of this method of expert diagnostics is an undoubted advantage, however, the lack of information about which specific proteins of the virus have antibodies in the patient's blood does not allow assessing the specificity of the reaction in each individual case, as well as tracking changes in the spectrum of antibodies at an early stage of seroconversion.

Laboratory diagnostics of HIV infection in children born to HIV-infected mothers has its own characteristics. From the moment of birth for a long time (up to 15 months), maternal antibodies to HIV can circulate in the blood of such children. Only immunoglobulins of the IgG class penetrate the placental barrier, therefore, the detection of HPV-specific nmmupoglobulins of the IgM and IgA classes in a child allows confirmation of infection, but a negative result cannot indicate the absence of HIV.

Children under 1 month of age do not have HPV replication yet, and the only verification method is PCR. Determination of p24 antigen in children older than 1 month is also a confirmatory method.

The absence of antibodies to HIV in newborns does not mean that the virus has not penetrated the placental barrier. In any case, children of HIV-infected mothers are subject to laboratory diagnostic examination and observation within 36 months from birth.

The results of laboratory tests for markers of HIV infection require careful interpretation and should be considered only in conjunction with data from epidemiological and clinical examinations. On the other hand, it should be noted that despite the high sensitivity of modern methods, negative research results cannot completely exclude the presence of HIV infection. Therefore, a negative test result, for example, by immunoblotting, can only be formulated as the absence of specific antibodies to HIV-1 and HIV-2.

Diagnosis of HIV infection in seronegative patients

The quality of test systems used in laboratory diagnostics of HIV infection is improving every year, their sensitivity is increasing. However, the high variability of HIV can lead to the emergence of new types, antibodies to which may not be recognized by existing test systems. In addition, there are known cases of atypical humoral response of the host's immune system to the virus. Thus, L. Montagnier in 1996 reported about two AIDS patients in whom specific antibodies had not been detected in their blood for several years, the diagnosis was made on the basis of clinical data and laboratory confirmed only by the isolation of HPV-1 in cell culture. In such cases, it is necessary to use the WHO recommendations, according to which the clinical diagnosis of HIV infection in adults and children is possible in the presence of one of the 12 AIDS-indicator diseases:

1. candidiasis of the esophagus, trachea, bronchi, lungs;
2. extrapulmonary cryptococcosis;
3. cryptosporidiosis with diarrhea for more than one month;
4. cytomegalovirus lesion of any organ (except for and in addition to the liver, spleen and lymph nodes in a patient older than 1 month):
5. an infection caused by the herpes simplex virus persisting for more than 1 month in a patient older than 1 month;
6. lymphoma of the brain in a patient younger than 60 years old;
7. lymphocytic interstitial pneumonia in a child under 13 years old;
8. dissiminated infection caused by bacteria of the Micobacterium avium intracellulare group or M. Kansassii;
9. pneumocystis pneumonia;
10. progressive multifocal leukoencephalopathy;
11. toxoplasmosis of the central nervous system in patients older than 1 month.

The presence of one of these diseases makes it possible to diagnose HIV infection in the absence of the possibility of conducting a laboratory blood test for the presence of antibodies to HIV, or even if a seronegative result is obtained.

Source: Medical laboratory diagnostics, programs and algorithms. Ed. prof. Karpishchenko A.I., St. Petersburg, Intermedica, 2001

Only special tests can reveal AIDS. The testing process itself consists of detecting antibodies that are produced by the body in response to the appearance of a virus. Laboratory tests for AIDS include four tests that are used to detect the disease.

The first - ELISA (enzyme-linked immunosorbent assay) is the most common diagnostic method. This analysis can determine the amount of antibodies that have accumulated in the body. This is usually possible only if the virus has entered the bloodstream and three months have passed since that moment. As practice shows, one percent of the results give a false negative or false positive result.

A false negative result is possible if antibodies have not yet developed to the virus, or if AIDS is at the terminal stage, that is, the virus is present in the blood, but there is so much of it that antibodies and CD-4 cells are practically absent. To verify a negative result, you can re-pass the ELISA, but only after 3 months.

In most cases, a false positive result is given by examination of patients suffering from chronic autoimmune, infectious, oncological and other diseases. A false positive result can also be found in pregnant women. Therefore, each positive result is necessarily rechecked, but only on the immunoblot - this is a more sensitive test.

If the immunoblot result is positive, especially after the ELISA result, then the reliability is 99.9%, and this is the maximum accuracy for a medical test (moreover, any).

The second is immunoblot (full Immune Blotting). This test detects the presence of specific antibodies to HIV. In this case, the result is negative, positive, doubtful (it is also called indefinite).

A dubious result may indicate that HIV is present in the bloodstream, for which the body has not yet developed the entire spectrum of antibodies. In this case, antibodies to HIV in the serum appear in 1-3-6 months. one after another, from the moment the first questionable result was received. If the results are confirmed, then this indicates HIV infection in the initial stage.

The uncertain result of this test may indicate the opposite. There is no HIV infection itself, but the human body has antibodies similar to antibodies to HIV. As a rule, a dubious result occurs in pregnant women, in patients with tuberculosis, recipients, cancer patients, as well as in those who have received multiple blood transfusions (i.e. blood transfusions). A patient whose result of this test showed uncertainty is monitored by an infectious disease doctor for six months and is tested in 1-3-6 months.

The third is PCR (polymerase chain reaction). This AIDS diagnosis allows you to determine the DNA and RNA of the virus. This is a fairly sensitive and effective reaction that allows you to study DNA to get a result. In this case, DNA is taken from one cell and examined by multiplying the characteristic DNA sequences

This test applies:

• to determine which virus is in the body (there are HIV 1 and HIV-2);
• to determine the presence or absence of HIV;
• to determine and control viral load;
• to determine the HIV status of a newborn who was born to an HIV-infected mother.

This test has a high sensitivity: it can detect a virus even if about ten days have passed since the supposed moment of infection. Such a high sensitivity of the test reacts to other infections, therefore, it often gives false positive results. The results of this test are not a basis for a definitive diagnosis.

This test requires highly qualified medical personnel and sophisticated laboratory equipment. This is an expensive test and is therefore not used in free HIV testing.

The fourth is an express test. This test is used mainly in emergency cases - during childbirth, for health reasons during surgery. Rapid test results are usually confirmed by routine HIV testing.

/ 16
The worst Best

Testing for HIV infection is subject to:

2. Persons with suspected or confirmed diagnosis: bacterial infection in children under 13 years of age, multiple and recurrent; candidiasis of the esophagus, trachea, bronchi or lungs; cervical invasive cancer; disseminated or extrapulmonary coccidioidomycosis; extrapulmonary cryptococcosis; cryptosporidiosis with diarrhea for 1 month or more; cytomegalovirus lesions of other organs, except for the liver, spleen, lymph nodes in patients older than 1 month; cytomegalovirus retinitis with loss of vision; herpetic infection causing multifocal ulcers that do not heal within 1 month, or bronchitis, pneumonia, esophagitis; disseminated or extrapulmonary histoplasmosis; isosporosis with diarrhea for more than 1 month; tuberculosis widespread or extrapulmonary; pulmonary tuberculosis in adults or adolescents over 13 years of age; extrapulmonary tuberculosis; other disease caused by mycobacteria other than M. tuberculosis disseminated or extrapulmonary; pneumonia caused by pneumocysts; progressive multifocal leukoencephalopathy; salmonella (except Salmonella typhi) septicemia, recurrent; toxoylase-mosaic of the brain in children over 1 month of age; Kaposi's sarcomas; lymphoid interstitial pneumonia in children under 13 years of age; Burkitt's lymphomas; immunoblastic lymphoma; primary brain lymphoma; wasting syndrome, hepatitis B, HBsAg carriage; infectious mononucleosis; recurrent herpes zoster in people over 60 years old; sexually transmitted diseases.

The highly specialized laboratory carries out:

a) determination of antibodies, antigens and immune complexes circulating in the blood; cultivation of the virus, identification of its genomic material and enzymes;

b) assessment of the functions of the cellular link of the immune system. The main role belongs to the methods of serological diagnostics, aimed at determining antibodies, as well as antigens of the pathogen in the blood and other biological fluids of the body.

HIV antibody testing is performed to:

a) safety of blood transfusions and transplants;

b) surveillance, testing in order to monitor the prevalence of HIV infection and study the dynamics of its prevalence in a certain population;

c) diagnostics of HIV infection, i.e. voluntary testing of blood serum of practically healthy people or patients with various clinical signs and symptoms similar to HIV infection or AIDS.

The system for laboratory diagnostics of HIV infection is built on a three-stage principle. The first stage is screening, designed to perform primary blood tests for the presence of antibodies to HIV proteins. The second stage is the reference one - it allows using special methodological techniques to clarify (confirm) the primary positive result obtained at the screening stage. The third ethane - expert, is intended for the final verification of the presence and specificity of HIV infection markers identified at the previous stages of laboratory diagnostics. The need for several stages of laboratory diagnostics is primarily due to economic considerations.

In practice, several tests are used to detect HIV-infected with a sufficient degree of reliability:

ELISA (ELISA) -test (enzyme-linked immunosorbent assay) detection of the first level, is characterized by high sensitivity, although less specific than the following;

Immune blot (Western-blot), a very specific and most used test to differentiate between HIV-1 and HIV-2;

P25 antigenemia test, effective in the initial stages of infection;

Polymerase chain reaction (PCR).

In cases of mass screening of blood samples, it is recommended to test mixtures of sera from a group of subjects, compiled in such a way that the final dilution of each sample does not exceed 1: 100. If the serum-current mixture is positive, each serum of the positive mixture is tested. This method does not lead to a loss of sensitivity in both ELISA and immunoblot, but it reduces labor costs and the cost of the initial examination by 60-80%.

In the primary serodiagnosis of HIV infection, total antibodies are determined using screening screening tests - ELISA and agglutination reactions. At the second (arbitration) stage, a more complex test is used - an immunoblot, which allows not only to confirm or reject the initial conclusion, but also to do this at the level of determining antibodies to individual virus proteins.

Linked immunosorbent assay (ELISA) is the main and most widely used method for the determination of antibodies to HIV. But the disadvantages of using ELISA in serodiagnosis of HIV infection include frequent false positive results. In this regard, the result in ELISA is not a basis for a conclusion about HIV seropositivity of the subject. This is due to insufficient purification of the immunosorbent from ballast proteins; spontaneous binding of serum antibodies to plastic, if its areas not occupied by the immunosorbent are not sufficiently blocked or not blocked by a completely special neutral protein; cross-interaction with HIV proteins of the immunosorbent of various proteins present in the blood of persons with certain, more often autoimmune pathological processes such as multiple sclerosis, SLE, tuberculosis; with frequent donation, infectious and oncological diseases, burns, pregnancy, repeated blood transfusions, organ and tissue transplants, as well as people on hemodialysis; with the presence of rheumatoid factor in the blood, often provoking HIV-false positive reactions; the presence in the blood of the examined people of antibodies to HIV gag proteins and, first of all, to the p24 protein (obviously, antibodies to exo- or endogenous, not yet identified retroviruses are formed). Since anti-p24 is synthesized without fail in the early stages of HIV seroconversion, further immunological observation of persons with antibodies to HIV gag proteins is carried out, as well as their removal from donation.

The sensitivity and specificity of the enzyme-linked immunosorbent assay is constantly increasing. As a result, the fourth generation ELISA is not inferior in its diagnostic capabilities to immune blotting and can be used not only at screening, but also at the confirmatory stage of diagnosing HIV infection [Smolskaya T. T., 1997].

Immunoblotting is the final method of serological diagnostics, which makes it possible to make a final conclusion about HIV-positiveness or negativity of the subject.

There is a clear correlation between the results of the study of sera in immunoblot and ELISA - twice positive in ELISA with different test systems, sera in 97-98% of cases are then HIV-positive in immunoblotting. If the sera were positive in ELISA only in one of the two test systems used, in the immunoblot they are positive in only 4% of cases. In 5% of cases, when conducting confirmatory studies in persons with positive data, ELISA-immunoblot can give "indeterminate" results, and among them, in about 20% of cases, "indeterminate" results cause antibodies to HIV-1 gag proteins (p55, p25, p18 ). The presence of antibodies only to HIV-1 gag proteins is the reason for additional examination of blood serum for HIV-2 infection.

Evaluation of the results of immunoblotting is carried out strictly in accordance with the instructions attached to the test system. In the absence of instructions on the interpretation of the results in the instructions, the WHO criteria should be used.

Upon receipt of positive results of studies at the reference stage of laboratory diagnosis of HIV infection and a negative result of the study by the method of immune blotting, a mandatory repeated expert diagnosis is carried out 6 months after the first examination.

If the results of immunoblotting 12 months after the study of the first sample remain negative or uncertain, then in the absence of risk factors, clinical symptoms or other factors associated with HIV infection, the subject is removed from dispensary observation.

Among serological methods, in case of uncertain results, immunoblot is used as an expert diagnosis. radioimmunoprecipitation (RIP). It is based on the use of virus proteins labeled with radioactive iodine, and precipitates are detected using beta counters. The disadvantages of the method include the high cost of equipment, the need for equipment for these purposes, special rooms.

Persons who have been diagnosed with HIV infection are subject to constant dynamic observation with mandatory laboratory examination every 6 months.

Polymerase chain reaction (PCR) reveals pre-multiplied nucleotide sequences specific to the genome of a given pathogen. Isolated multiplication of a gene or its fragment, called amplification, PCR makes it possible to carry out in vitro using the enzyme thermostable DNA polymerase. In 2-3 hours, PCR allows you to get millions of copies of a specific part of the virus. In HIV infection, from the cellular RNA, including the RNA of the virus, if it has been reproduced in the cell or has been integrated into its genome, a sufficient amount of proviral DNA is obtained by reverse transcription and hybridization with labeled oligonucleotide "probes" for analysis, which is detected and quantified , as well as in relation to belonging to the HIV genome, by radioactive or other label of the probe, establishing the homology of DNA and virus-specific amino acid sequences. PCR sensitivity is the detection of viral genes in one in five thousand cells.

PCR, including quantitative, can be used only to determine the viral load on plasma to resolve the issue of starting drug treatment for a patient or changing antiretroviral drugs. PCR cannot be recommended for the diagnosis of HIV infection, since even the most modern methods of its statement and reagents allow determining the viral load not lower than a certain level - 50 copies / ml. And the complexity of PCR testing and its high cost (about $ 200) negate its widespread use as a method of daily laboratory diagnosis of HIV infection. Thus, PCR remains indispensable only for assessing the viral load on plasma in patients with an already established diagnosis of HIV infection in order to resolve the issue of patient therapy.

The stages of laboratory diagnosis of HIV infection are shown schematically in Fig. 1.

Figure: 1. Stages of laboratory diagnosis of HIV infection

During HIV infection, there is a period of "dark laboratory window" when the amount of antibodies against HIV is insufficient for the sensitivity of test systems. This period ranges from one week to three months from the moment of HIV infection, depending on the sensitivity level of the test system. Given this phenomenon, difficulties arise when examining donated blood from persons who are in the mentioned period of HIV infection. Therefore, in most countries of the world, a system for using blood has been introduced only after it has been stored for 3-6 months in order to carry out a mandatory re-examination for HIV infection of donors of these doses of blood and its components.

The stage of primary manifestations is characterized by the activity of the replicative process. The emerging viremia and antigenemia cause the formation of specific antibodies of the IgM class: anti-p24, anti-gp41, anti-gp120. The p24 antigen in some of the infected can be detected in the blood by ELISA already 2 weeks after infection and determined up to 8 weeks. Further, in the clinical course of HIV infection, there is a second rise in the p24 protein content in the blood, which occurs during the formation of the AIDS stage.

The emergence of complete seroconversion, when a high level of specific IgG antibodies to the structural proteins of HIV gp41, p24, gpl20 is recorded in the peripheral blood, greatly facilitates the diagnosis of HIV infection. Most commercial kits are designed to indicate just such antibodies.

Difficulties in detecting antibodies in patients with HIV infection can arise during periods of massive viremia and antigenemia, when the available specific antibodies in the blood are spent on binding viral particles, and the replicative process outpaces the production of new antiviral antibodies.

In individuals with an initially weakened immune system, viremia and antigenemia appear earlier and remain at a high level until the outcome of the disease. At the same time, such patients have a low content of free antibodies to HIV, due to two reasons - insufficient production of antibodies by B-lymphocytes and binding of virions and soluble HIV proteins by antibodies, therefore, to determine infection, test systems with increased sensitivity or modifications of analysis methods are required. the step of releasing antibodies from immune complexes.

Despite the abundance of specific markers of HIV infection, the most often determined is the presence of total antibodies to HIV proteins. The term "total" means the presence of two classes of antibodies (IgG and IgM) and a wide range of antibodies to various, primarily structural, HIV proteins.

Determination of CD4 cells. The main clinical and laboratory indicator for diagnosing the stage of HIV infection, the degree of destruction of the immune system in patients in everyday life was the determination of the content of CD4 + lymphocytes: a decrease in the level below 200 cells / mm3 is the main criterion for diagnosing AIDS. It is believed that all HIV-infected persons with a CD4 + lymphocyte count of 200 cells / mm3 and below require both antiviral therapy and prevention of Pneumocystis pneumonia. And although 1/3 of HIV-infected with the number of C04 + lymphocytes less than 200 cells / mm3 do not have clinical manifestations, experience has shown that they develop symptoms in the next 2 months, so they are all regarded as patients with AIDS.