The meaning of the numbers 1 128 is syphilis. Basic methods for diagnosing syphilis and decoding indicators

Syphilis is classified as a sexually transmitted disease, the main "culprit" of which is pale treponema. The specified bacterium can enter the body after sexual contact, or by household means.

Diagnostic measures aimed at identifying this disease are complex. Test results can be affected by various antibiotics, pregnancy, and other factors that will be described in the article.

When an analysis for syphilis is prescribed - indications for diagnosis

Some patients, coming for examination to a gynecologist or andrologist, do not provide objective information about the quality of their sexual life.

Perhaps the reason is the usual embarrassment, or maybe the reason for this is the lack of information in the field of sexually transmitted diseases.

The doctor can send for examination even if syphilis does not manifest itself in any way, and the patient is 100% sure that he could not become infected with this disease. The fact is that the considered pathology can be transmitted through everyday contact or be asymptomatic.

Testing for syphilis is ordered if:

• You need to be registered during pregnancy.
• The patient wishes to donate blood as a donor.
• There is a prospect to take a certain position (soldier, health worker, cook, etc.), which requires passing a special medical commission.
• The person is in prison.
• There was a sexual intercourse with a patient with syphilis.
• The mother of a newborn baby has syphilis.
• The patient showed signs of this disease. Often these are rashes in the genital area.
• The first analysis confirmed the presence of the disease in question.

Regular blood testing is done in the presence of syphilis. This is necessary to control the quality of therapeutic measures.

After therapy, the patient also takes blood for research.

How to get tested for syphilis

For research manipulations often use blood from a vein. In certain situations, the laboratory assistant can take the right sample for diagnosis from the finger, or from the spinal cord.

The interval from the moment of delivery to the receipt of results may be different: from one day to two weeks. Everything will be determined by the type of testing.

In preparing for the delivery of a blood test to identify the ailment in question, the following recommendations should be followed:

• Fatty foods a day before the test should be excluded from the diet. It will provoke opalescence of the blood serum, which will distort the results obtained.
• Avoid food for at least 8 hours before testing for syphilis.
• Alcohol, nicotine may interfere with the evaluation of the reaction. Experts advise not to drink drinks containing alcohol 24 hours before the test, and you should wait with cigarettes at least an hour before the test.
• If the patient is taking antibiotics, the specified analysis should be carried out at least a week after the end of treatment.

Methods for submitting material for research and decoding indicators

Today, none of the methods for diagnosing this disease is able to guarantee the accuracy of the information obtained. In any case, there are errors, and they can reach 10%.

In this regard, apply complex of research methods.

Serological analysis - non-specific and specific tests

This type of diagnosis is indicated for limited symptoms of the disease or its complete absence.

There are two types of serodiagnosis:

1.Non-specific tests

They are relevant when you need to test a large group of people for syphilis, but this technique is not suitable when you need to confirm the diagnosis.

Testing is not difficult, but the final assessment should be given by the doctor.

These types of diagnostics include the following tests:

A) Precipitation microreaction (MR)

A similar study is indicative after one month after infection. Blood from a finger is subject to examination, but sometimes cerebrospinal fluid can be used.

Positive test result ( antibodies in titer vary from 1:2 to 1:320 ) does not yet mean that the patient has syphilis: it is possible to finally confirm the diagnosis by passing additional tests.

A negative reaction can be the result of two options:

• The patient does not have syphilis.
• Syphilis is, but - at the initial stage of development.

B) Wasserman reaction ( P.B. RW)

The material for testing here is the same as in the above analysis.

This method of examination is able to provide objective information at least 6 weeks after infection. It is possible to speak about the presence of the indicated venereal pathology if the antibody titers are 1:2 - 1:800.

The results of analyzes in RV are evaluated by the following mathematical signs:

• « — » No syphilis.
• « + "or" ++ "- a weakly positive reaction is stated.
• « +++ ' is a positive reaction.
• « ++++ » - the patient has a sharply positive reaction to syphilis.

2. Specific tests

There are many different assays for this type of examination that target specific antibodies. They do not appear in the blood immediately, but about a month after infection and can remain there for several years (if untreated).

The doctor must reasonably choose one or another type of analysis, know about each of them in detail, navigate the results obtained, and be able to differentiate the diagnosis after receiving the answer.

The most common types of specific tests are:

A) Immunofluorescence reaction (RIF)

It is relevant in the very early stages of syphilis, but the optimal period for testing is 6-8 weeks after infection.

For the study, capillary / venous blood is needed.

• Pregnancy, connective tissue defects can cause a false reaction, which is assessed by the sign " — «.
• Positive results are expressed as pluses (" + ") from one to four.

B) Passive agglutination reaction (RPHA)

During this test, a small amount of blood is taken from a finger/vein, which is then mixed with ram/rooster red blood cells. In the presence of the causative agent of this disease in the bloodstream, microbodies stick together, followed by their subsidence.

This type of testing is highly sensitive: it can confirm a positive reaction to syphilis for a long time after treatment.

Mononucleosis and errors in the structure of the connective tissue can also be the cause of a false positive reaction.

It takes a maximum of 1 hour to receive a response, and patients can check themselves 4 weeks after infection: at earlier times, antibodies will not be produced in sufficient volume.

You can judge how long the infection has been in the blood by the titers:

• If their value does not exceed 1:320, then the infection has occurred recently.
• The higher the titers, the longer the treponema is in the body.

C) Enzyme immunoassay (ELISA)

One of the most reliable methods for diagnosing this disease, which began to be used at the end of the last century.

It is very indicative already 21 days after infection, and a positive result by 98-99% will indicate the presence of syphilis.

ELISA is often used after non-specific tests, or in combination with some specific tests.

ELISA test by detecting one or another group of antibodies in the blood ( IgA, IgM, IgG) makes it possible to determine the stage of the disease:

• If the blood sample containsIgA but absentIgM,IgG: no more than 14 days have passed from the moment pale treponema entered the body.
• If identifiedIgA,IgM, but noIgG: infection occurred about 28 days ago.
• The presence in the blood of all the above antibodies indicates that more than a month has passed since infection.
• If the blood reaction to the presenceIgA is negative andIgM,IgG positive: a huge period of time has passed since the moment of infection or the treatment of the disease was successful.

D) Treponema pallidum immobilization reaction (RIBT)

One of the most popular methods for diagnosing syphilis.

It makes no sense to use it in the early stages of infection, but after the 12th week, the results of the RIBT test are 99% reliable.

This diagnostic method is used for suspected neurosyphilis, syphilis of internal organs, or in combination with non-specific tests.

When taking durant antibiotics, the patient must wait at least 25 days after the end of therapy. Water-soluble antibiotics require less time to be excreted from the body: 7-8 days.

Blood is taken from a vein, on an empty stomach, and the results are interpreted as a percentage of immobilization:

• If the level of immobilization does not exceed 20%, the test for syphilis is considered negative.
• When exceeding 50%, the reaction to the indicated pathology is positive.

In other cases, a repeat study is prescribed.

D) Immunoblotting

One of the newest methods of research, which is turned to when other tests give a questionable result.

With this diagnostic manipulation, it is possible to detect even the minimum amount of antibodies in the blood: it has almost 100% accuracy.

Not all clinics carry out such testing: it is not cheap.

Laboratory analysis

The cost of the analysis under consideration is very low, and you can find out about the result in 30 minutes.

1. To carry out such a study, a sample is taken from a patient from ulcerative / erosive defects located in the genital area. Microscopic examination of the taken sample is often carried out in the laboratory.

The affected areas are initially wiped with saline. This will help protect the damaged area from the ingress of harmful microorganisms.

2. Next, using a special loop, irritate the surface several minutes until a clear white liquid appears. You should be careful with this manipulation: it is impossible for blood impurities to get into the sample taken.

3. The extracted liquid is transferred to a transparent glass. Sometimes it is mixed with saline.

A positive reaction can be said when typical treponemas are detected, which will have at least 8 curls. If the result is negative, the procedure is repeated (sometimes several times).

Syphilis trna is a specific test for the detection of the IgG antigen. Usually, this study is used after a positive result in RPR or to evaluate the effectiveness of the selected treatment. Syphilis trna is a highly sensitive test that can show a positive result up to 20 years after successful therapy, and in some cases, residual traces of the disease can persist for the rest of your life.

The test for syphilis tpha (passive hemagglutination reaction) was proposed by T. Rathlev in 1965. The essence of the method was the reaction of gluing and precipitation of erythrocytes, the surface of which in syphilis contains antigens to pale treponema, which is observed only if there are antitreponemal antibodies in the blood.

Trna analysis is recommended to be carried out one month after the expected date of infection. Before the collection itself, you should refrain from eating for at least 8 hours. If the results of the tran test indicate the absence of infection, but the patient has symptoms, early symptoms of infection, or had sexual intercourse with a person who was found to be infected with spirochetes in the past, then it is recommended to conduct another control study with an interval of 2 weeks. Unlike a false-negative tpha result, when using this method for detecting the IgG antigen, which can be caused by too early a study, a false-positive result for pale treponema can be observed in three cases:

• Infectious mononucleosis;
• Lepre's disease;
• Systemic lesions of connective tissues.

To date, the tpha test has been used as the most effective specific serological test to confirm the positive result of other tests for syphilis or, if necessary, to evaluate the effectiveness of treatment.

The results of studies of the presence of the IgG antigen are not limited to only two marks "negative" or "positive", the results of the study also indicate the antibody titer, which the attending physician focuses on during the patient's stay in the hospital to assess the effectiveness of the therapeutic effect. By the value of the titers, one can draw a conclusion about the current period of infection and the success of therapeutic measures. Thus, the titers characteristic of the first stage of the disease range from 1:80 to 1:320. With the transition of the disease to the secondary stage, the analysis for syphilis tpha will show a noticeable increase in titers to 1:5120, followed by a decrease in the latent period to 1:80. After successful therapy, titers may drop significantly, but will remain positive for many years after discharge.

Sometimes, with a positive study of tpha, the titers are so insignificant that the diagnosis of syphilis raises serious doubts. In this case, a note is made about the doubtfulness of the data obtained and a retake is recommended in 1-2 weeks.

Usually, tests for syphilis trna are taken in the morning for a short period of time, which is associated with the requirement for the study, among which the need to deliver blood samples to the laboratory no later than 2 hours after its collection is indicated. In this case, the samples must be transported at temperatures up to +8°C.

The test material for tpha is blood serum donated by a person on an empty stomach. If all the necessary conditions are met during blood sampling and subsequent analysis, then the accuracy of the data obtained will be 76% for primary syphiloma, 100% for the secondary form of the disease, and 94 for the late stage of the disease. Detection of pale treponema during periods of asymptomatic course of the disease is about 97%. And the level of specificity, reaching up to 99%, makes it possible to almost completely exclude variants of false positive results, facilitating the differential diagnosis of patients.

Treponemal tests for syphilis. General description.

In order to reliably diagnose syphilis and identify anti-syphilitic antibodies in the patient's body (in the blood serum or cerebrospinal fluid), special laboratory research technologies are used - the so-called serological methods.

When conducting diagnostic tests for syphilis, various serological reactions are used: agglutination, precipitation, immunofluorescence, complement fixation, enzyme immunoassay, etc. All these serological reactions are based on the interaction of antigens and antibodies.

Specific serological tests are called treponemal because these tests use treponema pallidum or its antigens, that is, antigens of treponemal origin. The purpose of treponemal tests is to identify specific antibodies to the antigenic structures of the causative agent of syphilis, that is, antibodies directed specifically against the T. Pallidum bacteria themselves, and not against body tissues damaged by treponema. Specific antitreponemal antibodies of the IgM class can be detected as early as the end of the second week of illness.

7. False positive and false negative results

A positive PB test for syphilis in people who do not have the disease is called a false positive. The frequency of false positive results in healthy individuals is 0.2-0.25%. If the percentage of nonspecific false positive results of RV in healthy people is very low, then in some diseases it can be high.

All non-specific results of serological reactions can be divided into the following main groups:

1. Diseases caused by the presence of common antigens in similar pathogens (spirochetes): relapsing fever, yaws, bejel, pint, oral treponema, leptospira.

2. Positive reactions due to changes in lipid metabolism and changes in serum globulins. These include positive results in pregnant women with gout, impaired lipid composition as a result of poisoning with lead, phosphorus, after taking sodium salicylate, digitalis, etc. These reactions should also include positive reactions in certain infectious diseases (typhus, malaria, pneumonia , leprosy, endocarditis, collagenosis, myocardial infarction, concussion, cancer, cirrhosis of the liver, etc.)

3. Technical errors of conduct. Incorrect choice of complement dose, non-compliance with the conditions and terms of storage of reagents, exclusion of control samples of blood serum from the reaction, use of contaminated test tubes and instruments.

8. Modification of the Wassermann reaction

There are modifications of the Wasserman reaction in qualitative and quantitative versions, in the cold, with cerebrospinal fluid.

Modification of RV in the cold appeared to be more sensitive. A feature of the methodology for setting up the Wasserman reaction in the cold is the three-phase temperature regimes at which complement binding proceeds. This reaction is also put with cardiolipin and treponemal antigen.

In addition to the qualitative assessment of RW, there is a method for its quantitative setting with various dilutions of blood serum (1:10, 1:20, 1:80, 1:160, 1:320). The reagin titer is determined by the maximum dilution, which still gives a sharply positive result (4+). Quantitative formulation of RV is important in the diagnosis of some forms of syphilis and in monitoring the effectiveness of therapy.

9. Scope

In Russia, RSKt is part of the complex of standard serological tests for syphilis (SSR).

The Wasserman reaction with treponemal and cardiolipin antigen (RSKt) is used to

• diagnosis of all forms of syphilis,
• control over the effectiveness of treatment,
• examinations of persons who have had sexual contact with a patient with syphilis,
• examinations of persons with clinical and anamnestic suspicion of syphilis
• during preventive examination for syphilis of patients in psychiatric and neurological hospitals, donors and pregnant women, including persons referred for artificial termination of pregnancy.

At present, by order of the Ministry of Health of the Russian Federation, it is recommended to replace RSKT with more sensitive treponemal methods (ELISA or RPHA).

Abroad, the Wasserman reaction with treponemal antigen has not been used in clinical laboratory practice for a long time and is not included in the list of standard tests recommended by the World Health Organization.

Complex of classical serological reactions (CSR)

DAC- it reaction complex used for the serodiagnosis of syphilis as a standard method. This complex of reactions includes the Wasserman reaction with cardiolipin antigen (an extract from the heart of a bull enriched with lecithin and cholesterol) and treponemal antigen (a suspension of apathogenic cultural pale treponemas treated with ultrasound), as well as a microprecipitation reaction (RMP) with plasma or inactivated serum, which is placed with cardiolipin antigen

CSR become positive in the middle of the primary period (its division into seronegative and seropositive is precisely determined by CSR), in the secondary period CSR are positive in 98-100% of patients, and in the tertiary period - only in 60-70%. That is, as the duration of the disease increases, the positivity of the CSR gradually decreases.

Advantages of the KSR:

1) Cheapness, simplicity and speed of setting. This is especially true for the microprecipitation reaction: RMP is currently the main screening (screening) method;

2) Non-treponemal tests are convenient to use to monitor the cure of syphilis.

Disadvantages of the CSR:

1) Subjectivity of evaluation of the results of reactions ("by eye");

2) Low sensitivity in late forms of syphilis;

3) Lack of specificity compared to more modern tests. When they are carried out, false positive reactions (LPR) are often noted.

LPR may be due to cross-reactivity between the pale spirochete and other microbes, lipid and protein metabolism disorders, cell membrane instability, and the formation of autoantibodies. LPR are noted in acute (malaria, infectious mononucleosis, etc.) and chronic (tuberculosis, leprosy, hepatitis, borreliosis, etc.) infections, myocardial infarction, liver cirrhosis, collagenoses (especially in SLE), oncopathology, vaccination, drug use, abuse of alcohol and fatty foods. False-positive may be CSR in the last weeks of pregnancy, after childbirth, and in some women during menstruation. False-negative CSR results may be associated with HIV infection.

RIT, RIBT - Immobilization reaction of pale treponema

Treponema pallidum immobilization test (TPI; Treponema pallidum immobilization test, TPI) is a classic method that serves to detect specific treponemal antibodies. The RIBT reaction uses pathogenic treponema pallidum T. pallidum (Nichols strain) grown in rabbit testicles as an antigen. RIBT is based on the loss of mobility of live pale treponemas after exposure to antibodies from the patient's blood serum and complement. The results are evaluated by dark field microscopy. Despite the fact that the RIBT test was introduced into clinical practice as a specific test for syphilis, it is laborious, technically complex, time consuming and expensive to use.

1. History of the RIBT method

Treponema pallidum immobilization test (TPRT) is actually the first specific test for the diagnosis of syphilis. This reaction was presented in 1949 by the American researchers R. W. Nelson and M. M. Mayer and discussed in detail in scientific papers in subsequent decades. Unsuccessful attempts to use live treponemas in tests have been made before. Due to the fact that Nelson managed to create an environment in which treponemas remained viable for up to 8 days, his research was crowned with success.

2. Principle of the RIBT method

The method is based on the phenomenon of loss of mobility by pale treponemas in the presence of immobilizing antitreponemal antibodies of the studied blood serum and complement under anaerobic conditions. The antigen is live pathogenic pale treponema obtained from rabbits artificially infected with syphilis.

3. Setting up the RIBT test

The test serum, complement and antigen participate in the reaction. The blood serum of the subject is added to live treponemes obtained from rabbit testicle tissues after artificial infection. In the presence of anti-treponemal antibodies-immobilizins in the serum, pale treponemas stop moving (immobilized). Immobilisin antibodies are late antitreponemal antibodies.

The reaction is performed with heat-inactivated sera or with samples of sera dried on waxed paper (dry drops). Serum inactivation by heating is carried out for 30 minutes at a temperature of 56°C. Before taking blood, the subject should not receive medication, especially penicillin preparations. Reception of drugs is canceled for the period of their possible delay in the body.

As an antigen, bacteria of the Nichols strain, obtained from a 7–10-day rabbit syphilitic orchitis (testicular inflammation), are used. The period from the moment the reaction was set up to the registration of its results lasts 18-20 hours, therefore, a survival environment is necessary to maintain the viability and good mobility of microorganisms.

RIBT uses guinea pig complement. To obtain complement, blood must be taken under sterile conditions from several guinea pigs.

In case of bacterial contamination, the complement is discarded. Cannot be used in the reaction of immobilization of pale treponema preserved complement, tk. it is toxic to microorganisms.

In the immobilization reaction, an excess of complement is used. Its amount is largely dependent on the survival environment for pale treponema.

RIBT is placed in sterile boxes, previously irradiated with a bactericidal quartz lamp for 45-60 minutes. Each blood serum is examined in two test tubes: experienced and control. In both test tubes, the test serum and antigen are added in the required quantities. Active complement is poured into the test tube, and the same amount of inactivated guinea pig blood serum is poured into the control tube. After filling, the contents of the tubes are mixed by gentle shaking.

RIBT proceeds under anaerobic conditions. Test tubes with ingredients are placed in a microaerostat, from which atmospheric air is sucked off by a vacuum pump and a gas mixture is injected from a cylinder (95 parts of nitrogen and 5 parts of carbon dioxide). The microanaerostat with test tubes is placed in a thermostat (35°C) for 18-20 hours.

The evaluation of the results of RIBT is carried out after removing the test tubes from the thermostat and microanaerostat (ie, after 18-20 hours of experience). With a Pasteur pipette, a drop of the contents of the test tube is applied to a glass slide, which is covered with a cover slip and examined in the dark field of a microscope (objective 40, eyepiece 10X). Several fields of view are examined in different parts of the preparation, counting the number of mobile and immobile pale treponemas in each. Counting starts with the drug from the control, and then from the test tube.

When setting up the reaction, 5 control studies are used: with obviously positive and negative blood sera, with active and inactivated complement and survival medium for pale treponema. Control negative blood serum is used to judge the degree of mobility of pale treponema in this experiment. Control positive blood serum - to assess the degree of immobilizing activity under the conditions of this experiment. The study of active and inactivated complement and the environment is carried out to determine their effect on the mobility of pale treponema.

With a lack of complement in the experiment, immobilizing antibodies do not show their activity properly and treponemas remain mobile. Therefore, after the experiment, the determination of residual complement is carried out in order to assess whether the mobility of pale treponemas in the test tubes was due to the lack of complement. For this, a hemolytic system is used - a mixture of a suspension of ram erythrocytes and diluted hemolytic serum kept in a thermostat.

Residual complement is determined by adding the hemolytic system in the required volume to each tube. The tubes are placed in a thermostat at 37° for 45 minutes. In experimental test tubes, hemolysis of erythrocytes should occur, in control tubes there should be a delay in hemolysis. The absence of hemolysis in the test tubes indicates an insufficient amount of complement, in these cases the study should be repeated. Repeated examination of blood serum is not performed only if 100% immobilization of pale treponemas is noted.

4. Accounting for the results of RIBT

The immobilized treponemas are counted under a microscope using dark-field microscopy. The researcher is required to have the skill of assessing the movement of treponemas. He should pay attention to the intensity of movements made by pale treponema. In this bacterium, it is not always possible to observe wave-like contractions and flexion movements, sometimes only rotational ones. You should also be able to distinguish active movements of treponemas from movement with fluid flow.

To evaluate the results of the reaction, the percentage of immobilization of pale treponemas is calculated, i.e. the ratio of mobile and immobile treponemas in the experiment (with active complement) and control (with inactive complement) according to the formula:

X \u003d (M - C) × 100 / M

where M is the number of mobile treponemas in the control; C - the number of mobile treponemas in the experiment; X - % immobilization. In practical work, the percentage of immobilization is determined according to a pre-compiled table using the above formula.

The reaction of immobilization of pale treponema is estimated as

• positive during immobilization 51 - 100% treponem,
• weakly positive: 31 - 50% immobile treponemas,
• doubtful: 21 - 30% immobile treponemas,
• negative: 0 - 20% immobile treponemas.

The reaction of immobilization of pale treponema becomes positive at the end of the primary - the beginning of the secondary period of syphilis (from the 7-8th week from the moment of infection and more). At the same time, RIBT is of little use for diagnosing the early stages of syphilis, since antibodies that immobilize pale treponema and are determined in the reaction appear only 3-6 weeks after infection. Antibodies-immobilisins belong to the class of immunoglobulins IgG. They appear in the blood later than reagins (anticardiolipin antibodies), later than fluorescein antibodies (RIF and ELISA are detected) and precipitins (RMP are detected).

In the future, RIBT remains positive. There is a high sensitivity of the reaction in late forms of syphilis. With secondary, late syphilis, neurosyphilis, congenital syphilis, a positive RIBT result is recorded in 95-100% of cases. With tertiary syphilis, with specific lesions of internal organs, the nervous system, when RV is often negative, RIBT gives positive results in 98-100% of cases.

RIBT has long been recognized as the most specific test for syphilis. According to the literature, the specificity of RIBT is 99%, the sensitivity ranges from 79 to 94%. According to TsNIKVI, the sensitivity of RIBT (in total, for all stages of syphilis) is 87.7%.

7. Scope of the method

The scope of RIBT is gradually narrowing due to the duration of setting, high cost and labor intensity. RIBT is a rather complex and costly analysis that requires highly qualified personnel and the presence of a vivarium. In this regard, the use of this method in recent years has been significantly reduced. In the US, this test is currently used only in research laboratories.

Based on the complexity and high cost of RIF and RIBT, it makes sense to use them for the diagnosis of late and latent forms of syphilis. RIBT retains its position as a "reaction-arbiter" in the differential diagnosis of early latent forms of syphilis and false positive results. This reaction may be useful in diagnosing neurosyphilis and when other serologic tests are inconsistent.

RIBT becomes positive much later than RIF and RV. Therefore, it is not used to diagnose infectious forms of syphilis.

RIBT, like RIF, is very slowly negative in the process of antisyphilitic therapy. As a result, it is unsuitable for monitoring the progress of antisyphilitic therapy.

False-positive results (FPR) in RIBT are rare and have been observed mainly in a number of treponematoses (yaws, pinta, bejel), which are not found in Russia, as well as in leprosy, sarcoidosis, SLE, tuberculosis, cirrhosis of the liver and some other rare diseases of a non-syphilitic nature. With the age of patients, the number of false-positive results of RIBT increases.

RIBT may be false positive if the test serum contains treponemicidal substances (for example, penicillins, tetracyclines, erythromycin) that cause nonspecific immobilization of pale treponema. This may be due to the patient taking treponemocidal antibiotics, so the examination is not carried out in persons who have received antibiotics within the last month. Blood for RIBT can be examined no earlier than 2 weeks after the end of antibiotics and other antisyphilitic drugs.

9. Modifications of the reaction of immobilization of pale treponemas

In addition to the microanaerostatic technique, there is a melange setting of RIBT according to N.M. Ovchinnikov. Anaerobic conditions during the formulation of the reaction are created by placing the reacting mixture in a melanger (leukocyte mixer), both ends of which are closed with a rubber ring. The melange reaction technique makes it possible to dispense with a vacuum pump, a cylinder with a mixture of nitrogen and carbon dioxide, and a microanaerostat. In a comparative study on a large clinical material, results were obtained that are not inferior to the classical anaerostatic technique.

10. Features, advantages and disadvantages of RIBT

RIBT is a technically complex and expensive diagnostic method. The technology requires significant funds for the maintenance of rabbits and testing. This time-consuming test is currently used mainly for scientific purposes. In most foreign countries, for almost 40 years, RIBT has been practically used not for diagnostic purposes, but only in research work.

Reaction Disadvantages:

• RIBT requires work with live pathogenic treponema pallidum of the Nichols strain, which remains infectious to humans despite adaptation to rabbits
• the reaction is complex, time-consuming and expensive
• a vivarium is required
• highly qualified personnel are required to set up the reaction, record the results and maintain the vivarium
• subjectivity of results evaluation
• lack of automation
• there is no way to standardize this serological method.
• the reaction is not applicable against the background of ongoing antisyphilitic therapy
• inability to use to control cure. RIBT in patients with syphilis can remain positive for many years (and even for life), despite the received full treatment.
• the reaction can give false positive results in patients with malignant tumors, diabetes, leprosy, autoimmune diseases, pneumonia, severe cardiovascular pathology.

The advantages of RIBT are:

1) Sufficiently high sensitivity;

2) High specificity.

RIF (Immunofluorescence reaction)

Immunofluorescence reaction (RIF) is an express diagnostic method for detecting microbial antigens or detecting antibodies. Tests based on the detection of a fluorescent signal are considered among the best tests for syphilis.

1. History of the method

Fluorescent treponemal antibody (FTA) was first developed in 1957 by Deacon et al. (Deacon, Falcone and Harris).

2. The principle of the method

The RIF method is based on the fact that tissue antigens or microbes treated with immune sera with antibodies labeled with fluorochromes can glow in the UV rays of a fluorescent microscope. Bacteria in a smear, treated with such a luminescent serum, glow along the periphery of the cell in the form of a green border

As an antigen in RIF, a suspension of live pathogenic pale treponemas of the Nichols strain from rabbit orchitis is used, which is dried on a glass slide and fixed with acetone. The patient's blood serum is added to the pale treponemas dried and fixed with acetone to the glass.

After washing, the preparation is treated with serum containing antibodies against human immunoglobulins labeled with fluorescein. Once again, the preparation is washed and viewed under a fluorescent microscope. If the test serum contains anti-treponemal fluorescein antibodies, a yellow-green glow of treponemas will be noted.

3. The method of conducting research by the RIF method

The antigen fixed on the glass slide (pathogenic treponema pallidum) is processed by the test serum. After washing, the preparation is treated with fluorescent anti-human immunoglobulin serum labeled with fluorochrome. In this case, the resulting fluorescent complex (anti-human globulin + fluorescein thioisocyanate) binds to human globulin on the surface of pale treponema, providing a glow of pale treponema under a fluorescent microscope.

To detect antigen-antibody complexes, luminescent serum is used, representing anti-species (anti-human) immunoglobulins conjugated with FITC. The presence of antibodies to treponemas in the serum is determined by the glow of treponemas when examined under a fluorescent microscope. The test is carried out in qualitative and semi-quantitative versions.

4. Accounting for results

Visualization of the RIF results is carried out using a fluorescent microscope. The results are evaluated by the degree of luminescence of treponemas in the preparation. In the presence of antibodies, the glow of treponemas is visible, but if there were no antitreponemal antibodies in the serum, then treponemas are not visible. The degree of luminescence of dried pale treponema fixed to the glass is indicated in “pluses” (from “–” to “++++”). Negative result - no glow or background level - 1+.

5. At what periods of the disease is it better to use

The immunofluorescence reaction (RIF) is quite sensitive at all stages of infection, from the end of the incubation period to late syphilis. The primary period of syphilis in the classical course begins 3-4 weeks after infection. RIF becomes positive in the first days of the primary period or even at the end of the incubation period, from the 3rd week after infection. The results of RIF remain positive in all periods, including in late forms.

RIF becomes positive somewhat earlier than RW. According to some reports, positive RIF occurs in 80% of patients with primary seronegative syphilis. In the secondary period, RIF is positive in almost 100% of cases. It is always positive in latent syphilis and gives 95-100% positive results in late forms of the disease and congenital syphilis.

6. Sensitivity and specificity

Immunofluorescence reaction (RIF) is a group of methods with high sensitivity and specificity. RIF is sensitive at all stages of infection, from the period of incubation to late syphilis. According to WHO, the sensitivity of RIF in primary syphilis is 70-100%, in secondary and late syphilis - 96-100%, specificity - 94-100%. According to TsNIKVI, the sensitivity of RIF in all forms of syphilis is 99.1%.

The specificity of RIF can be increased by pre-treatment of the test serum with a sorbent - ultrasonified treponemal antigen that binds group antibodies (RIF-abs).

7. Scope of the method

RIF applies:

• as a confirmatory reaction in early, latent syphilis
• for retrospective diagnosis
• for differentiation of the latent forms of syphilis and false-positive results of researches on syphilis.
• as a confirmatory test for neurosyphilis.

RIF is widely used as a confirmatory test, but it is not intended for routine use or screening because it is technically difficult to set up. To perform RIF, it is necessary to have a vivarium or purchase a suspension of pathogenic pale treponemas, which limits the possibility of a reaction. However, in recent years, test systems have begun to appear on the domestic market, allowing the reaction to be carried out in the absence of a vivarium and its own laboratory strain of pathogenic treponema pallidum.

8. Sources and causes of staging errors, false positives and false negatives

LPR when setting RIF are rare (with collagenoses, borreliosis).

RIF is still considered one of the best tests for syphilis, the "gold standard" of serodiagnosis. RIF in comparison with RIBT is easier to set up,

Despite the high diagnostic value, the need to use live T. pallidum, the high cost and duration of the study hinder the widespread introduction of RIF into everyday practice. Setting up the reaction is laborious. In addition, the evaluation of the results of the RIF is subjective.

Advantages of the RIF and RIBT are:

1) High sensitivity (especially for RIF);

2) High specificity (especially for RIBT).

Disadvantages of RIF and RIBT:

1) Technical complexity, high cost of methods.

2) Subjective evaluation of results, lack of automation;

3) RIF and RIBT in patients with syphilis can remain positive for many years (and even for life), despite the received full treatment. Therefore, these reactions cannot be used to control cure.

10. Method modifications

In practice, for the serodiagnosis of syphilis, several modifications of the immunofluorescence reaction are used and have been used:

• RIF-abs- the most sensitive method of serodiagnosis of syphilis, it becomes positive earlier than other reactions (from the 3rd week from infection);
• RIF-200(the patient's serum is diluted 200 times upon presentation) is a highly specific method for the serodiagnosis of syphilis.
• RIF-10(10 times dilution of the test serum) - a more sensitive method than RIF-200.
• RIF-ts carried out with liquor.
• RIF-abs-IgM- detection of early antitreponemal antibodies of the IgM class.

1. The most widespread modification RIF-abs- immunofluorescence reaction with absorption. Before setting up the reaction, the serum of the subject is depleted with a mixture of non-pathogenic treponemas to exclude cross-reactions. Group antibodies are removed from the studied serum using cultural treponemas destroyed by ultrasound, which significantly increases the specificity of the reaction. Since the test serum is used in a 1:5 dilution, RIF-abs is highly sensitive.

The main indications for the use of RIF-abs in clinical practice are:

• diagnosis of latent and late forms of syphilis,
• detection of false-positive results of CSR and RMP, especially in pregnant and somatic patients with suspected syphilis,
• to establish a retrospective diagnosis of the disease.

RIF-abs is not very informative in assessing the results of treatment: in 85% of patients who received adequate antisyphilitic therapy, positive results of RIF persist for many years.

This reaction is called the "gold standard" for syphilis serodiagnosis. It is used for arbitration cases, but a fresh concentrated suspension of T. pallidum strain Nichols from 7-day-old orchitis in a rabbit is required for a reliable result, which should not be frozen.

2. In the USSR it was installed in two versions - RIF-10 and RIF-200, i.e. with a dilution of the test serum by 10 and 200 times. RIF-200 - the test serum is diluted 200 times to reduce the number of false positive results. This provides a high specificity of the reaction, but its sensitivity drops somewhat. RIF-10 is more sensitive, but more often gives non-specific positive results, than RIF-200, which is characterized by high specificity. RIF-10 is more sensitive, RIF-200 and RIF-abs are more specific.

The sensitivity of RIF-200 and RIF-abs is estimated at 84–99%, and the specificity is 97–99%.

3. RIF-ts carried out with liquor. The reaction is set using whole cerebrospinal fluid to identify specific CNS lesions.

4. Reaction RIF-abs-IgM proposed for the detection of early antitreponemal antibodies of the IgM class. This reaction can be used to diagnose congenital syphilis, early forms of syphilis, and to differentiate between cases of reinfection and serorelapse.

Two modifications of this reaction are known:

– FTA-ABS-IgM, based on the use of an anti-IgM conjugate (fluorescein-labeled antibodies to human IgM) in the second phase of the reaction instead of anti-human fluorescent globulin;

- the Russian version of RIF-abs-IgM, characterized in that a sorbent is added to the test blood serum, removing IgG antibodies, and RIF-abs is placed with the remaining IgM antibodies.

Main indications to the formulation of RIF-abs-IgM are:

- serodiagnosis of congenital syphilis in the absence of manifest manifestations of congenital syphilis on the skin and mucous membranes in the child;

- differential diagnosis of reinfection and clinical-serological or serological recurrence of syphilis, in which RIF-abs-IgM will be negative, and RIF-abs - positive;

- evaluation of the effectiveness of therapy for early acquired or congenital syphilis: after adequate treatment, RIF-abs-IgM becomes negative within the next 3-6 months.

This reaction can be used to detect congenital syphilis. It is known that large IgM molecules cannot pass through a healthy placenta. Therefore, class M antibodies against treponema pallidum may appear in the body of the child either due to a violation of the barrier function of the placenta, or they are produced by the body of a child with syphilis. Antibodies of the IgM class appear in the blood of a patient with syphilis already in the first weeks of the disease, and antibodies of the IgG class appear later. Separate determination of antibodies of both classes is extremely useful in the diagnosis of congenital syphilis in children, since the presence of IgM class antibodies in a child in the first month of life will indicate that they are formed by the body of a child with syphilis, while the detection of only IgG antibodies will indicate maternal the origin of the latter.

Statement of the reaction 19S(IgM)-RIF-abs involves preliminary separation by gel filtration of larger 19S IgM molecules from

fractions of smaller 7S IgG molecules. Further study in the RIF-abs reaction of blood serum containing only the 19S IgM fraction,

eliminates all possible sources of errors. But the technique of setting up this reaction is complex and time-consuming, requires special equipment and training of specialists.

Immune adhesion reaction (RIP, TPIA - Treponema pallida immunoadherence).

This reaction is based on the use of a phenomenon described by Rieckenberg in 1912. RIP is based on the fact that virulent tissue treponemas, sensitized by the serum of a patient with syphilis, in the presence of complement and erythrocytes, adhere to the surface of erythrocytes and, during centrifugation, are carried away with them into the sediment, disappearing from the supernatant.

The following ingredients are used to set up the reaction: test serum, antigen, complement, donor erythrocytes, isotonic sodium chloride solution. As an antigen, a suspension of pale treponema of the Nichols strain is used.

Most widely in relation to the serodiagnosis of syphilis, this test was studied by domestic and foreign authors in the 50-60s. Data on the value of RIP as a diagnostic test have been conflicting. The reaction required maximum accuracy, since with inaccurate pouring of ingredients, with an excess or deficiency of the test material in the preparation, unreliable results were obtained.

In Russia, extensive research was carried out by L.V. Sazonova, who obtained similar results in RIP and RIT using a freshly prepared suspension of pathogenic pale treponemas of the Nichols strain. However, the use of an antigen heated or preserved with phenol sharply distorted the results of the reaction and made the antigen unstable. Recommend this test to replace RIT L.V. Sazonova considered it impossible.

G.P. Avdeeva, using other temperature and time regimes in the preparation of the antigen, obtained different results in the study of RIP. According to her, the sensitivity of this reaction is higher than the sensitivity of KCP and RIT, but somewhat inferior to RIF, and the specificity of RIP, RIT and RIF is close.

However, the lack of production release of the antigen for RIP did not allow a wider study of this test and its introduction into practice.

RPHA reaction of passive hemagglutination

Passive hemagglutination reaction (RPHA) is a common serological test that is firmly rooted in laboratory practice. has a fairly high level of efficiency in the study.

1. History of the RPHA method

For the first time, G.Blumental and W.Bachman (1932) reported on the use of RPHA for the diagnosis of syphilis. In 1965, an indirect or passive hemagglutination reaction was proposed for the diagnosis of syphilis. Reaction modification using different antigens was reported by T. Ratlev in 1965 - 1967. The micromodification of RPGA was proposed by Sokh R.M. and co-authors in 1969. The first commercial test system was developed by Japanese scientists Tomisava et. al. in 1969

2. Principle of the RPHA method

From a prepared homogeneous suspension of erythrocytes "loaded" with antigens, when the test serum containing antibodies is added, a precipitate in the form of flakes precipitates. The resulting precipitate consists of erythrocytes "glued" by antibodies, and is called "hemagglutinate". A suspension of erythrocytes is prepared in advance and supplied as part of diagnostic test systems.

The process of gluing red blood cells, on the surface of which antigens are present, is called "hemagglutination". Bonding occurs under the action of specific antibodies (agglutinins). The reaction is called "passive", because own antigens of erythrocytes do not react, and the erythrocytes themselves perform an exclusively auxiliary indicator function.

The passive (indirect) hemagglutination reaction is a type of agglutination reaction in which erythrocytes (from the Greek háima - blood) are used as carriers of antigens, and not other particles. In general, in the agglutination reaction under the action of antibodies, microbes or other cells stick together and precipitate - not necessarily erythrocytes, but, for example, latex particles, bacteria or other antigen-bearing corpuscular particles.

In the passive hemagglutination reaction for the diagnosis of syphilis, ram or bird erythrocytes coated with pale treponema antigens are used as an antigen. When serum containing specific antibodies is added, erythrocytes stick together (agglutination).

The reaction of RPHA is referred to as immunological methods, because. it is based on the specific interaction of the antigen of pathogenic treponema pallidum with an antibody. In accordance with the "lattice theory" agglutination is the result of "cross-linking" of surface antigen molecules with antibody molecules (immunoglobulins).

3. Statement of the reaction of passive hemagglutination

RPHA is placed in plastic tablets or in test tubes with dilutions of the patient's blood serum, to which an erythrocyte diagnosticum is added.

The process of combining an antigen with erythrocytes is called sensitization, and the artificial corpuscular antigen obtained in this way is called sensitized erythrocytes. Erythrocyte diagnosticums are called erythrocytes sensitized by an antigen.

To prepare the diagnosticum, ram or bird erythrocytes (usually chicken) treated first with formalin and then with tannin are used, which are sensitized with ultra-sound antigen of pathogenic treponema pallidum (Nichols strain) or recombinant treponema pallidum proteins (TpN15, TpN17, TpN47). Sheep erythrocytes sensitized with ultrasonified antigen of cultured treponema pallidum can also be used.

Serum only (do not use plasma). Hemolyzed and cloudy samples are not suitable. Non-sensitized erythrocytes serve as a negative control (to exclude the presence of anti-erythrocyte antibodies). In each series of productions use positive and negative controls.

Samples of the studied blood serum and test erythrocytes are introduced into the wells (cells) of the immunological tablet. If the patient's blood serum contains specific anti-treponemal antibodies, then when the test serum is added to the well with the antigen, antigen-antibody complexes are formed that are associated with the surface of the carriers (erythrocytes). Visually, this is manifested by the gluing of red blood cells, i.e., hemagglutination, which is visible to the naked eye. Immune complexes "antibody-antigen-erythrocyte", which gradually descend under the influence of gravity, are distributed over the entire surface of the bottom of the well and form a characteristic picture of an "inverted umbrella".

Depending on the amount of antibodies contained in the test sample, the “inverted umbrella” image varies from the maximum, occupying the entire surface of the bottom of the well, to a small area in the central, lowest part of it (with enlightenment in the center and the formation of a more intense ring of settled erythrocytes along the periphery).

Immune complexes are not formed if there are no specific antibodies in the sample or when control (intact) erythrocytes are added to the reaction. At the same time, erythrocytes gradually gather at the lowest point of the well bottom, forming a figure in the form of a compact spot or "button", sometimes with a slight enlightenment in the center.

If the human blood serum contains anti-erythrocyte antibodies, then the "umbrella" will form in any case - both in reaction with test erythrocytes and with control erythrocytes. In this case, other medical technologies are recommended to detect specific antitreponemal antibodies.

The phenomenon of prozone (the impossibility of reaction due to excess antibodies) is possible, which is eliminated by dilution of the serum.

4. Accounting for the results of the passive hemagglutination reaction

The results of RPGA are taken into account visually after 60-120 minutes when setting up the micromethod and after 2–4 hours or the next day when setting up the macrovariant. When using larger (nucleated) bird erythrocytes, a clearer picture is obtained, and the results are recorded at an earlier date.

It is possible to determine the titer (high titer TPHA ≥ 1:2 560).

The results of the study are evaluated according to the 4+ system (from "-" to "++++") according to the size of the formed film. When agglutination occurs, erythrocytes are located on the surface of the hole in the form of an "umbrella", and with a negative result, erythrocytes freely slide down and accumulate on the bottom in the center of the hole in the form of a "button".

The generally accepted assessment of the results of RPGA:

4+ - positive RPHA. Agglutinated erythrocytes in the form of an "umbrella" evenly line the entire surface of the hole;

3+ - positive RPHA. Erythrocytes line the entire surface of the hole, but some of them "slip" to the center. At the same time, a noticeable ring is formed along the periphery of the deposit;

2+ - weakly positive RPHA. Erythrocytes form a film on a small area of ​​the lower part of the well, forming a dense ring of erythrocyte sediment with a noticeable enlightenment in the center;

1+ - indeterminate RPHA, erythrocytes form a loose sediment at the bottom of the well with fuzzy edges and a slight lumen in the center;

(–) - negative RPHA, all erythrocytes lie at the bottom of the well in the form of a compact sediment ("buttons" or ringlets) against a clean surrounding background (without surrounding granular sedimentation).

In foreign practice, the results of RPGA are also evaluated as reactive (in the case of agglutinate formation), weakly reactive (if the formations are insignificant) and non-reactive (if agglutination is not observed).

Accounting for the results of the reaction can be done automatically using special analyzers. In addition to a qualitative study, all test systems provide for quantitative analysis with titer determination.

5. At what periods of the disease is it better to use RPHA

RPHA becomes positive in the middle of the primary period (7-8 weeks from the moment of infection, 3-4 weeks after the appearance of a hard chancre) and remains positive for years after treatment.

With a very high level of antibodies to treponema in the studied serum (which is most characteristic of secondary syphilis), a false-negative result of TPHA is possible (the so-called "prozone" phenomenon).

Specific agglutinin antibodies are detected in the blood of people who have recovered from syphilis for a long time, so RPGA cannot be recommended for the differential diagnosis of reinfection or for determining the severity of the infectious process.

RPGA is not used to control cure, because. may remain positive many years after recovery. At the same time, it can be used as an additional (to RMP or RPR) method in monitoring the effectiveness of the treatment, investigating the dynamics of the decrease in antibody titers. A prerequisite for this is the use of the same RPHA test system as in the first (before treatment) examination of the patient, as well as the study in the same laboratory.

6. Sensitivity and specificity of RPHA

RPHA is considered a highly sensitive and specific test. This reaction is a valuable diagnostic test in all forms of syphilis, but it is especially sensitive in advanced forms of the disease. The sensitivity of RPHA varies depending on the stage of the disease. With primary syphilis, the sensitivity of RPHA is 76% (and higher), with secondary syphilis - up to 100%. With latent early - 97%, with late syphilis - 94%, with a specificity of 98-100%. Lower sensitivity in fresh forms of the disease is due to the later formation of agglutinins.

According to the State Institution "TsNIKVI Roszdrav", the sensitivity of RPHA in the diagnosis of various forms of syphilis was 99.4%. Most researchers note 98-99% specificity of RPHA.

In terms of sensitivity and specificity, RPHA is not inferior, and in late forms and congenital syphilis, it even surpasses RIF and RIBT.

7. Scope of application of the RPGA method

TPHA can be used as both a screening and confirmatory test; can be used in a semi-quantitative variant with the calculation of antibody titer. A quantitative method for setting up RPHA, a micromethod, as well as an automated microhemagglutination reaction have been developed.

8. Features, advantages and disadvantages of RPHA

According to the literature, RPGA has consistently taken a leading position in clinical practice in most countries of the world. TPHA is the most widely used test in STI clinics abroad.

The RPGA technique is easy to perform, does not require special equipment: only a hemagglutination plate is needed to set it up. The study does not take a long time; the reaction is highly sensitive and specific. Approbation of the method in clinical practice has shown that it is extremely simple, cheap and sensitive. Like ELISA, RPHA is simple to perform, does not require highly qualified personnel and special equipment, and its automation is possible.

Advantages of the RPGA test:

• easy to set up and interpret,
• does not require special equipment,
• time to get the result - 45 minutes,
• suitable for mass screening (only 25 µl of serum is needed at a dilution of 1:20),
• high degree of standardization,
• the presence of internal controls,
• long shelf life
• acceptable price
• the possibility of automating accounting.

It should also be noted the disadvantages of RPGA:

• the possibility of non-specific reactions in the presence of anti-erythrocyte antibodies,
• lack of correlation of titer and stage of syphilis,
• later positive reaction in the early stages of syphilis,
• the possibility of false positive reactions in persons who have taken alcohol, drug addicts,
• sensitivity to vibration and temperature in the laboratory.

The advantages of RPGA compared to RIBT and RIF are:

• use of industrial test systems,
• the possibility of automating the reaction,
• there is no need to work with live pale treponema,
• eliminates the need for a vivarium.

9. Sources and causes of errors in the setting of RPHA, false positive and false negative results

The reaction of passive hemagglutination is a relatively simple study; when performing it, it is necessary to follow all the recommendations of the diagnosticum manufacturers and the rules of work in the clinical diagnostic laboratory. The mistakes made can lead to the appearance and registration of both false-negative and false-positive results of the reaction. False-positive results of RPGA may be due to the influence of the human factor and biological factors.

False positive results can be obtained

• in the study of blood sera of patients with non-venereal treponematoses,
• due to rheumatoid factor
• due to antibodies cross-reacting with the treponemal antigen, which are formed during various systemic or drug-induced and drug-induced metabolic disorders,
• due to abnormal levels of immunoglobulins;
• in newborns - due to the formation in the body of the fetus or child of IgM antibodies to mother's IgG, which complicates the interpretation of the results and the diagnosis of congenital syphilis.

Errors caused by the influence of the factor of human participation on the study:

• contaminated microplates
• incorrect pipetting
• vibration in the laboratory
• the air temperature in the laboratory is outside the temperature range: 18–25 degrees

The most typical technical errors in the setting of RPGA, leading to unreliable results, include:

• inaccurate dilution of ingredients,
• temperature violation,
• violation of the time of incubation of reagents,
• violation of the terms of applying reagents to the tablet,
• inconsistency of the pH of the solutions with the required ones,
• contamination of laboratory glassware.

The following technical points can also be a source of errors when setting up RPGA:

• exclusion from the formulation of the reaction of control blood sera;
• uneven concentration of erythrocytes in the diagnosticum due to insufficient mixing before use;
• violation of the terms and conditions of storage of the diagnosticum and control erythrocytes; use of expired kits;
• the use of contaminated test tubes, pipette tips, pipettes, immunological plates, solutions when setting up reactions;
• inaccuracies in the initial dilution of a blood serum sample;
• insufficient thoroughness in performing consecutive double dilutions;
• non-compliance with the temperature regime and incubation time;
• the presence of extraneous vibrations and shaking of the immunological plate during incubation;
• violation of the method of setting RPGA, expressed in the refusal to perform the study with control erythrocytes.

The use of blood plasma containing anticoagulants capable of causing non-specific erythrocyte agglutination (RPHA) may lead to results that cannot be interpreted.

The number of false positive and false negative results is less than with other serological tests. LPR in the setting of RPHA are rare and possible with treponematoses (yaws, bejel, pint). Also, false-positive results were recorded (less than 1% in total) in drug addicts, in patients with infectious mononucleosis, borreliosis, leprosy, with collagenoses, liver cirrhosis, lymphosarcoma, and also in pregnant women.

False-negative results of the reaction may be due to competition between IgM and IgG antibodies. False-negative results are also possible in HIV-infected patients.

10. Modifications of the RPHA method

There are micro and macro modifications of the RPGA setting, the first one is more often used because of the economy, speed of setting and taking into account the results.

In addition, an automatic diagnostic complex for image analysis was developed, which made it possible to perform a quantitative automatic assessment of the results and eliminate subjectivity in the interpretation of the data obtained. The hardware-software complex recognizes the image, processes the data and gives the answer in relative units.

Readers and automatic analyzers are also used to automate the recording of RPGA results.

TPPA (Treponema pallidum particle agglutination) - an agglutination test of artificial particles to detect antibodies to Treponema pallidum

Brief description of the TPPA test

Currently, a modification of the passive hemagglutination method - TPRA (Treponema pallidum particle agglutination) is also used to diagnose syphilis, in which the pale treponema antigen is fixed on gelatin particles. Since artificial polymer particles do not have their own antigens that determine biological activity, then kits for syphilis serodiagnosis based on them have reason to be considered more advanced. The use of biologically inert artificial particles minimizes non-specific agglutination commonly seen with other carriers.

TPPA is used for the serological diagnosis of antibodies to various types and subspecies of pathogenic treponemas. This test can be used to detect antibodies to the causative agents of syphilis, pint, bejel, and yaws.

The study procedure is very simple and does not require special equipment - standard "U"-shaped microplates are used. The test is based on the agglutination of gelatin particles sensitized with T. pallidum antigens, antibodies found in the patient's blood serum.

TPPA is a confirmatory treponemal test that can be used conveniently for both small numbers of specimens and mass screening. TPPA is used abroad as a confirmatory test and to replace the microhemagglutination test MHA-TP (microhemagglutination assay for antibodies to T. pallidum).

The sensitivity of the TPPA test is between 85% and 100%, while the specificity is between 98% and 100%. The sensitivity of TPPA for primary syphilis is 88%, for secondary and late latent syphilis 98%-100%.

If TPPA is used to diagnose syphilis, antibodies to other types of treponema (such as T. pallidum endemicum, pertenue, or carateum) may cause false positive results. There are a number of methods to remove these antibodies from serum samples before starting the test.

Principle of the TPPA test

TPPA is a passive agglutination method for gelatin particles in human serum or plasma. Serum containing antibodies to pathogenic treponema reacts with gelatin particles sensitized with the antigen of pale treponema of the Nichols strain, subjected to ultrasound. As a result of the reaction, a smooth film of agglutinated gelatin particles is formed in the well of the microtiter plate.

If antibodies are not present, the particles settle to the bottom of the well of the plate, forming a compact "button" of non-agglutinated particles. Control wells with non-sensitized gelatin particles should also show such a compact "button" for each serum, i.e. no agglutination.

Application of the TPPA test

TPPA is a universal test that can be equally successfully used both in the mandatory preventive examination of population groups for syphilis (screening), and in specialized dermatovenereological institutions. The advantage of the TPPA test is its high sensitivity, which is not inferior to classical tests, which until recently were the "gold standard" for syphilis serodiagnosis. Other advantages of the test include high reproducibility, as well as the simplicity and speed of setting up the reaction.

The TPPA test is used to confirm positive results of non-treponemal screening tests for syphilis, such as the VDRL test, and to evaluate patients with negative non-treponemal tests but who have signs or symptoms suggestive of late syphilis. The use of TPPA as the sole screening test for syphilis is not recommended.

In addition, the TPPA agglutination test can be used to examine cerebrospinal fluid samples in the diagnosis of neurosyphilis. In this case, as with other serological tests, the interpretation of the results should be carried out with the obligatory combination with other indicators and symptoms of the disease.

TPPA test results

The result is evaluated according to the system of "pluses" - from (-) to (2+). The test results are visible to the naked eye and are interpreted as follows:

Degree of agglutination	Test score	Interpretation
Agglutinated particles evenly line the bottom of the plate well	2+	Positive
Significant large ring with uneven outer margins and peripheral agglutination	1+	Positive
The particles form a compact ring with a gap in the center and smooth smooth external borders	±	weakly positive
The particles form a compact ring in the center of the well with a slight gap in the center and a smooth outer boundary.	–	Negative
Particles form a "button" in the center of the hole with a smooth outer boundary	–	Negative

Results are recorded to determine compliance with the description above. Samples showing an indeterminate result (±) should be retested. In the event that a sample shows an indeterminate result in several TPPA tests, it is recommended to conduct a study using other methods.

The results of the analysis should not be considered in isolation. For example, at an early stage of infection, the number of antibodies is still too small, as a result of which TPPA and many other methods lack sensitivity. Therefore, if syphilis is suspected, even if the test results are negative, the samples must be re-examined. To make a diagnosis, it is necessary to take into account the clinical symptoms that the patient has, the clinical history and other data.

As in the TPHA test, the prozone phenomenon and a false negative result can be observed for TPPA if the serum sample contains too high an antibody titer.

ELISA - ELISA

Enzyme immunoassay (ELISA; The enzyme linked immunosorbent assay, ELISA) is one of the many methods for the serological diagnosis of infectious diseases. Enzyme immunoassay (ELISA) in the serodiagnosis of syphilis is a test for antibodies of classes M, G and A (IgM, IgG, IgA) against antigens of pale treponema. It is possible to perform ELISA with cerebrospinal fluid.

The introduction of enzyme-linked immunosorbent assay (ELISA) into practice instead of the Wasserman reaction and other cardiolipin tests has significantly improved the quality of laboratory diagnosis of syphilis. A significant advantage of this method is the possibility of automating the research process, which reduces the influence of the human factor.

1. History of the ELISA method

The basic principles of enzyme immunoassay on the surface of a solid-phase carrier were developed by E. Engvar et al. (1971), B.Van Weeman and A.Schuurs (1971). The enzyme immunoassay they developed was first proposed for the diagnosis of syphilis in 1975 by J. Veldkamp and A. Visser, who appreciated the potential of this automated test. ELISA began to be widely used in the diagnosis of syphilis in the 1980s, when diagnostic tests were developed and certified and testing methods were standardized. In the USSR, the technique of ELISA for the diagnosis of syphilis was developed by V.N. Bednova, A.V. Babiy and A.V. Kotrovsky (1982, 1983).

2. Principle of the ELISA method

Enzyme-linked immunosorbent assay (ELISA) according to the reaction mechanism is close to RIF (the same antibodies are detected). The enzyme immunoassay reaction is referred to as an immunological reaction based on the highly specific interaction of treponema pallidum antigens with the antibodies of a patient with syphilis.

In syphilidological practice, an indirect variant of ELISA is mainly used. The principle of the most commonly used indirect reaction variant is as follows. On the surface of the wells of a polystyrene plate, immune complexes are fixed, which are formed during the interaction of antibodies of a patient with syphilis with antigens of pale treponema. After that, they are detected in a color reaction using specific conjugates and appropriate substrate-chromogenic additives.

The procedure for performing the test is as follows: the patient's serum is placed on a solid-phase carrier with an antigen attached to it. In the presence of antibodies in it, an antigen-antibody complex is formed on the surface of the carrier. To "manifest" the results of the reaction, anti-species antibodies to human Ig conjugated with enzyme markers are used. In the case of a positive reaction, the enzyme attached to the antigen-antibody complex decomposes the substrate added to the system, resulting in the development of color staining of different intensity.

In the reaction, the determination of the complex of AG and AT adsorbed on the solid phase is carried out using antiglobulin antibodies labeled with the enzyme, according to the color reaction of the enzyme with the substrate.

In the reaction, the determination of the complex of antigens and antibodies adsorbed on the solid phase is carried out using enzyme-labeled antiglobulin antibodies.

ELISA presents the possibility of detecting serum Ig of different classes. There are systems on the market that allow you to determine separately IgM and IgG and total antibodies.

Specific antigens used for ELISA may have a different origin:

ultra-voiced- they are obtained as a result of the destruction of the bacterial cell T.pallidum by ultrasound or other method;

recombinant- they are obtained by genetic engineering technologies by introducing into the genome of a bacterial cell (for example, Escherichia coli E.Coli) the gene responsible for the synthesis of a certain T.pallidum antigen, followed by the growth of the bacterial mass of the producing microorganism, the destruction of these cells, the isolation and purification of the antigen;

peptide- obtained as a result of sequential chemical synthesis of antigenic epitopes of T.pallidum proteins.

The body is able to form antibodies to almost any part of the antigen molecule. In a normal immune response, this usually does not occur. One or more immunogenic peptides isolated from a protein antigen have a special antigenicity, and most antibodies are formed specifically to them. They develop the most intense immune response. The most informative in ELISA showed immunodominant regions of proteins of pale treponema with a molecular weight of 15 kD, 17 kD and 47 kD. A detergent extract or sonicate of pathogenic treponemas of the Nichols strain was used as an antigen.

3. The method of conducting research by the method

The principle of the method is to identify a specific antigen-antibody complex adsorbed on the solid phase (the surface of the wells of a plastic plate) with antiglobulin antibodies labeled with an enzyme (peroxidase) using a color reaction with a substrate, which is quantified spectrophotometrically.

The antigens used to sensitize the wells of the polystyrene plate can be:

• lysate- obtained as a result of the destruction of pale treponema by ultrasound;
• peptide- obtained as a result of chemical synthesis of fragments of proteins of pale treponema and having antigenic reactivity similar to the original proteins of the pathogen;
• recombinant- obtained using genetic engineering methods, carrying antigenic determinants identical to pale treponema.

In syphilidological practice, an indirect variant of ELISA is usually used.

4. Accounting for results

In ELISA, to visualize the antigen-antibody reaction, an enzyme reaction (alkaline phosphatase or horseradish peroxidase) with a substrate is used, which changes its color. The intensity of the color determines the positivity of the reaction (from "-" to "++++"). The results of ELISA can be assessed visually on a 4-point system or instrumentally in the form of digital indicators of optical density obtained on special readers (such as Multiscan) at a wavelength of 492 nm. Because the evaluation of the results is carried out spectrophotometrically, this excludes subjective interpretation.

5. At what periods of the disease is it better to use

Terms of ELISA positivity - from the 4th week from infection. The results of ELISA (as well as RIF) become positive in the first days of the primary period or at the end of the incubation and remain positive in all periods. ELISA is especially valuable in the early diagnosis of syphilis - positive antibody results can be obtained already at the end of the incubation period of the disease (ie, 4-6 weeks after infection).

6. Sensitivity and specificity

ELISA is a highly sensitive and specific test, which is its advantage. ELISA in terms of reaction mechanism, sensitivity and specificity is close to RIF, tk. the same antibodies take part in both reactions. Most researchers note high specificity and sensitivity at all stages of the disease. The sensitivity of various ELISA variants, according to G. A. Dmitriev, reaches 98–100%, and the specificity is 96–100%. According to TsNIKVI, the sensitivity of ELISA for syphilis is 99.1% (Order No. 87 M3 of the Russian Federation).

The solid-phase ELISA (enzyme-linked immunosorbent assay, ELISA) variant is one of the most sensitive serological tests, allowing detection of 0.0005 µg/ml of antibodies and 0.000005 µg/ml of antigens.

7. Scope of the method

The method is recommended to be used when examining pregnant women, contact persons, donors and representatives of risk groups. ELISA can be used both as a screening and confirmatory test. In a relatively short time of its history, the ELISA has evolved from an indicative test into a confirmatory one. In the diagnosis of syphilis, the test is also used as a confirmatory test for samples showing positive results using various non-treponemal tests and TPHA.

The ELISA method can be used quantitatively with the calculation of the index of positivity, or reactivity, which allows you to evaluate the level of antibodies in the sample.

Currently, in Russia, the use of enzyme immunoassay for the diagnosis of syphilis is regulated by the Order of the Ministry of Health of the Russian Federation No. 87 dated March 26, 2001 "On improving the serological diagnosis of syphilis" (Appendix No. 1 "Setting up screening and diagnostic tests for syphilis"). In the order, it is planned to replace RSK in the complex of seroreactions (SSR) with ELISA and RPHA.

8. Sources and causes of staging errors, false positives and false negatives

Enzyme immunoassay is a complex multi-stage study, in which it is necessary to strictly follow all the recommendations of the manufacturers of diagnostic kits, the rules for adjusting and configuring the equipment used in the study.

The following points can be the source of errors when setting up an ELISA:

A study in the reaction of hyperlipidemic, hemolyzed blood serum or samples with signs of bacterial growth;

Do not practice double or more freezing of a sample of biological material; if necessary, re-examination, use serum from a duplicate tube;

Violation of the terms and conditions of storage of the immunosorbent and washing solution; use of expired test kits;

Application of reaction components from other diagnostic kits;

Violation of the time of the reaction stages;

Drying the wells of the immunological plate during the reaction steps;

Reuse of plastic dishes (plastic trays) for the preparation of a mixture of chromogen and substrate;

Non-compliance with the frequency of metrological control of the operation parameters of the devices used (washer and spectrophotometer);

The use of contaminated laboratory glassware when setting up reactions: flasks, measuring cylinders, test tubes, pipettes, pipette tips;

Insufficient thoroughness in performing dilutions of samples of biological material;

Errors when introducing a sample of biological material into the corresponding well of the tablet;

Errors when transferring study results to the registration log, study protocol or answer form.

Careful implementation of the recommendations of the instructions for the use of diagnostic test kits, regular verification of the accuracy of pipette dispensers and automatic devices, the use of internal positive and negative controls allow obtaining highly reproducible ELISA results.

9. Features, advantages and disadvantages

ELISA refers to modern, promising methods of syphilis serodiagnosis due to its simplicity, reproducibility, and availability. The detection of antibodies by ELISA is an ideal diagnostic method suitable for the simultaneous examination of a large number of samples. Due to its advantages, it has gained popularity in all countries.

The technology of ELISA research provides for compliance with all recommendations of manufacturers of commercial diagnostic test kits and the thoroughness of the research procedure. To conduct research in the ELISA, it is necessary to purchase additional special equipment. The setting technique takes a longer time compared to RMP, RPR and RPGA.

The presence of automation of a number of stages or the entire process as a whole allows you to simultaneously examine a large number of samples of biological material with a high degree of standardization of the study, minimizing the subjective element in the evaluation of results, the possibility of storing fixed study protocols, which makes it possible to archive study data and refer to them again for retrospective analysis.

Advantages:

• Enables differentiated and total determination of IgM and IgG antibodies to the causative agent of syphilis.
• high sensitivity and specificity approaching 100%,
• reproducibility
• automation possibilities

The advantages of ELISA include high specificity (96-100%) and sensitivity (98-100%) noted by most researchers.

The presence of automation of a number of stages or the entire process as a whole makes it possible to simultaneously examine a stream with a large number of samples of biological material with a high degree of standardization of the study, minimizing the subjective element in the evaluation of results, the possibility of storing fixed study protocols that allow archiving study data and accessing them again for retrospective analysis .

Significant disadvantages of manual methods, including the determination of antibodies to T. pallidum antigens by ELISA, are:

~ possible errors of personnel during the study (oversight, negligence, violation of technology);

~ the impossibility of complete standardization of the research process with a manual version of the ELISA;

~ increased risk of personnel infection.

10. Method modifications

Along with the classical indirect ELISA, the method is also known. trapping ELISA. It was proposed in 1989 by O. E. Ijsselmudien et al. to detect IgM class antibodies to Tr antigens. pallidum. The method has high specificity and sensitivity.

Purified antibodies to class M human immunoglobulins are sorbed on the surface of the wells of the tablet, and after incubation of the test serum, all IgM bind to the carrier. The presence among the associated IgM specific for antigens Tr. pallidum is detected using Tr antigens. pallidum conjugated with an enzyme.

This method allows you to detect antibodies not only of the IgM class, but also of the IgG, IgA classes. To do this, the wells of the plates are sensitized with affinity purified antibodies of a certain class against human immunoglobulins. At the same time, antibodies of this class are caught from the serum, and the detection of treponemal-specific antibodies is carried out by combining them with a conjugate, which is a combination of treponemal antigen with an enzyme.

The use of a trapped ELISA reduces the risk of false positive reactions mediated by rheumatoid blood factor, cross-reactions between immunoglobulins of classes M and G. When examining newborns, in this case, false positive test results associated with the detection of IgM directed against maternal antitreponemal IgG are also excluded.

As ELISA variants abroad are widely used enzyme immune assay (EIA) and system ICE Syphilis. In the latter, a mixture of antibodies to IgM and IgG, as well as three recombinant T. pallidum proteins, was adsorbed in the wells of the plate. Positive results are tested in the same test system in two repetitions to give the final result.

In Russia, a number of test systems have been developed for the serodiagnosis of syphilis by ELISA with domestic reagents. These systems have high specificity and sensitivity.

In addition to the above, there are also other ELISA options, including those that allow direct detection of the pathogen in the blood (direct ELISA), dot-ELISA with the reaction on nitrocellulose strips, ELISA with capillary blood, as well as immunoblotting, or Western Blot, with simultaneous detection of antibodies to various components of the pathogen.

A modern improved modification of ELISA is immunoblotting.

ICA (immunochemiluminescent analysis), ICL (immunochemiluminescence)

With the development of laboratory diagnostics in the context of the modernization of healthcare in the Russian Federation, automation of laboratory research has entered the practice of laboratories, which makes it possible to standardize the analytical stages of research. This improves the quality of diagnostics. With the introduction of automation, new high-tech methods with high sensitivity and specificity began to be used, such as chemiluminescence immunoassay (CLIA)

Chemiluminescence is the process of photon emission during the transition of electronically excited products of oxidative chemical reactions to the initial energy state. During the chemiluminescence reaction, a significant amount of energy is released, and the quantum yield of the emitted light is quite high. Of all the non-isotopic methods, chemiluminescence provides the highest sensitivity. For immunometric methods, the sensitivity of chemiluminescence is orders of magnitude higher than that of radioimmunoassay.

The method of immunochemiluminescence (ICL) has now found its application in the diagnosis of tumor markers, autoimmune diseases, diabetes, cardiac markers, hormones (thyroid, adrenal, female and male sex hormones), torch infections, viral hepatitis, herpes group viruses.

Based on the method of immunochemiluminescence, a number of highly sensitive and specific (98-100%) test systems for the diagnosis of syphilis have been developed, which are mainly used abroad.

In the method, recombinant lipoproteins obtained by genetic engineering methods are used as antigens, which are complete analogues of T.pallidum antigens.

Based on the results of a clinical study, the ICA method has been recognized and recommended for use in the laboratory diagnosis of syphilis in the Russian Federation as a screening and confirmatory test if the laboratories have the appropriate equipment. In 2012, ICA was included as a screening and confirmatory test for the diagnosis of syphilis in the Clinical Guidelines for the Management of Patients with Sexually Transmitted Infections and Urogenital Infections.

Thus, the use of high-tech ICA, which has entered the practice of both world and domestic laboratory diagnostics with the introduction of automation, provides the following advantages:

• exclusion of the influence of subjective factors at the analytical stage of the study
• safety of personnel when performing studies of potentially infected biological material
• increasing the speed of obtaining results by the patient
• research standardization and research quality control
• delivery of reliable reliable results to patients
• high quality clinical laboratory research.

In terms of sensitivity, the ICA method is comparable to the method of radioimmunoassay, and in terms of ease of execution, safety for personnel, and many other parameters, it surpasses it.

The ICA method, which has high sensitivity and specificity (98–100%), makes it possible to quantify the level of antibodies to the causative agent of syphilis, and can be used to confirm syphilitic infection and screening. Limitations of use: cannot be used to monitor the effectiveness of therapy, may give a false positive result.

Immunochromatography (ICH).

Relatively new for use in the Russian Federation are methods for detecting treponema-specific antibodies based on immunochromatography (ICH) methods. As in the ICA method, recombinant lipoproteins obtained by genetic engineering methods (for example: Tp15, Tp17, Tp47 antigens) and the biosynthetic TmpA peptide are used as antigens. The listed antigens are also used in various combinations as part of immunosorbents in ELISA and immunoblotting.

The ICG method allows you to quickly determine the content of treponema-specific antibodies to the causative agent of syphilis in serum and whole blood samples without the use of special laboratory equipment and is used in the provision of primary health care, including epidemiological indications.

Limitations of use: cannot be used to monitor the effectiveness of therapy, may give a false positive result.

PBT (simple rapid tests at the bedside)

Relatively recently, in the serodiagnosis of syphilis, a direction began to develop for the development of so-called simple rapid tests (PBT), or tests at the patient's bedside (Point-of-care - ROC). Simple rapid bedside tests, or immunochromatographic tests, make it possible to quickly determine the content of treponema-specific antibodies to the causative agent of syphilis in serum and whole blood samples without the use of special laboratory equipment and be used in primary health care, including for epidemiological indications. Limitations of use: cannot be used to monitor the effectiveness of therapy, may give a false positive result.

These tests are based on immunochromatographic detection of antibodies to treponemal antigens of pale treponema and are performed on strips; in this case, both whole blood and serum can be used (Herring A., Ballard R. et al, 2006). The format of the tests allows you to conduct research in the absence of special equipment and without the use of electricity. However, due to the relatively low sensitivity and specificity, these tests have not yet found their wide application in practice.

Immunoblotting (Western Blot)

In recent years, research methods have appeared aimed at detecting and analyzing the content of antibodies. for each antigen pale treponema separately. One such method is the method of immune blotting (or blotting, immunoblotting, Western blot), which is used to determine IgG or IgM antibodies to pale treponema. The method of immunoblotting is a modification of enzyme immunoassay - a variant of ELISA.

Immunoblotting is one of the most modern methods of syphilis serodiagnosis and is actively used abroad. It got its name ("Western blotting") as a humorous response to the names of DNA detection techniques ("Southern Blotting") and RNA ("Northern Blotting").

1. History of the method

Immunoblotting (Western blot) is used to determine IgG or IgM antibodies to pale treponema antigens (Herremans M. et al., 2007).

2. Classic immunoblot

Classic immunoblot(Western Blot Analysis) combines enzyme immunoassay and the use of an electrophoretic method. Protein antigens of the causative agent of syphilis are preliminarily separated by electrophoresis and transferred to a carrier - a nitrocellulose membrane (strip). This transfer is called blotting. Then this carrier with separated points (blots) is treated with the test serum and antibodies to IgG or IgM labeled with enzymes or radioactive substances. The method involves the use of natural or recombinant treponema proteins.

electrophoresis is the separation of charged compounds based on their mobility in an electrophoretic field. When a potential difference is applied in some medium, positively charged molecules move towards a negatively charged electrode, and negatively charged molecules move towards a positive electrode.

Most globular proteins are assembled in such a way that most of the charged groups, namely the hydrophilic ones, are located on the surface of the protein, while the uncharged, hydrophobic residues are immersed inside the globule. In an electric field, in accordance with their charge, proteins migrate towards an oppositely charged electrode.

Electrophoretic separation of proteins is carried out in a synthetic gel medium based on polyacrylamide. To separate proteins according to their molecular weight, before electrophoresis, the protein mixture is preincubated in the presence of sodium dodecyl sulfate (SDS).

Detailed studies by foreign scientists Weber and Osborn (1969) showed that after such treatment, the only factor that can affect the mobility of proteins in a polyacrylamide gel (PAAG) is the size of the protein, or rather its molecular weight. This made it possible to apply the method of electrophoresis in SDS-PAGE in the presence of SDS for a fairly accurate determination of the molecular weight of proteins and peptides. In foreign literature, this method was called SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis).

Reactivity profile in the classic WB test with natural antigens (polyacrylamide gel electrophoresis method with sodium dodecyl sulfate). (1) - control positive result, (2) - control negative result, (3-6) - clinical samples.

In tests for syphilis using this method, previously purified and destroyed to its constituent components, pale treponema is subjected to electrophoresis in a polyacrylamide or agarose gel. At the same time, the antigens included in its composition are separated by molecular weight.

Then, by vertical electrophoresis, the antigens are transferred to a strip of nitrocellulose, which now contains an invisible spectrum of antigenic bands characteristic of pale treponema.

During the analysis, the test material (serum, patient's blood plasma, etc.) is applied to a nitrocellulose strip (strip). If there are specific antibodies in the sample, then in the process of incubation they strongly bind to the antigenic bands that strictly correspond (complementary) to them and form an “antigen-antibody” complex.

After the removal of unbound immunoglobulins during the washing of the strip, incubation with a conjugate - enzyme-labeled antibodies to human immunoglobulins (antibodies to human IgG or IgM) follows, during which antibodies labeled with the enzyme are attached to the existing antigen-antibody complexes on the membrane surface.

After the removal of the unbound conjugate during incubation with the substrate solution, the enzyme interacts with the substrate, resulting in a color reaction - staining of the membrane sections (in the form of bands) where individual antigens of pale treponema are located, antibodies to which were in the test serum. The intensity of staining depends on the amount of bound antibodies from the serum. The result of the reaction is evaluated visually.

The presence of bands in certain areas of the nitrocellulose plate confirms the presence in the studied serum of antibodies to strictly defined antigens of pale treponema.

The immunodeterminants 15, 5, 17, 44.5, 47 kD determined using this method have diagnostic significance in syphilis. Ig G immunoblotting is similar in sensitivity and specificity to RIF with absorption (RIF abs). Ig M-immunoblotting can serve as a diagnostic test for congenital syphilis.

3. Immunoblot in linear immunoassay format

Linear immunoassay (line immunoassay, LIA) allows you to simultaneously determine antibodies to pale treponema antigens in human serum or plasma. Antigens are pre-applied to test strips (strips), which are supplied as part of commercial diagnostic kits.

The strips are made of nitrocellulose membrane, polyamide membrane with a plastic backing or nylon membrane with a plastic backing. During manufacture, several discrete antigens are placed on the test strip as separate antigenic lines. In addition to these syphilis antigens, four more control lines are applied to each lane: streptavidin and controls (3+, 1+ and ± cut line).

For the strips, artificial antigens obtained by genetic engineering are used - recombinant proteins or synthetic polypeptide constructs. These are analogues of the surface antigens of pale treponema - TpN15, TpN17, TpN47 and TmpA with a molecular weight of 15, 17, 47 kDa and 44.5 (TmpA), where kDa=kiloDaltons. With their help, serum antibodies to various immunodominant components of the pathogen are determined.

The incubation of the test sample takes place in a cuvette, where the indicator strip is also placed. Antibodies to T. pallidum are determined by binding to antigens printed on the test strips. If there are T. pallidum-specific antibodies in the sample, they form bonds with individual antigen lines. When the antibodies contained in the test serum interact with discrete T. pallidum antigens placed on the test strip, an antigen-antibody complex is formed in the form of visually detectable color bands.

When taking into account the results of immunoblotting, the reactivity of the serum to each of the antigens is evaluated separately. A total positive response is issued when a result is obtained simultaneously with two or three of the presented antigens.

Research procedures are carried out manually or on a special analyzer, with the interpretation of the results using a specialized computer program for infectious diseases.

Due to the high degree of purification of recombinant antigens and the simultaneous detection of antibodies to the most immunogenic determinants of the syphilis pathogen, the method has high sensitivity and specificity.

4. Accounting for results

To read the intensity of staining of antigenic bands on strips, photometers have been developed, the operation of which is based on signal reflection, which eliminates subjective assessment and provides a potential opportunity for automation.

5. At what periods of the disease is it better to use

Tests using the Western blot method can detect specific antibodies to Treponema pallidum antigens at a very early stage of the disease. The most significant in the diagnosis of syphilis are antibodies to antigens of pale treponema TpN15, TpN17, TmpA and TpN47. These antigens are membrane proteins having molecular weights of 15, 17, 44.5 and 47 kDa, respectively.

When treponema pallidum is introduced into the human body, antisyphilitic antibodies appear in this order: first, an immune response to TpN17 antigens develops, then to TpN47, after TpN15 and after all TmpA. When taking into account the test results, the reactivity of the serum to each of them separately is evaluated. For example, when in a linear blot there is reactivity only to the TpN17 and TpN47 antigenic line, this means that the disease is at an early stage. The more antigenic lines become reactive, the further the infection progresses. In the treatment of syphilis, the pattern of reactivity in this test changes.

Determination of IgM to proteins with a molecular weight of 15.17, 41, 47 kDa makes it possible to identify 29.2% of patients with secondary syphilis, 12.5% ​​with early latent syphilis and 8.0% with seroresistant. The search for IgG to the same molecules is characterized by a sensitivity of 61.6% for seroresistant and 100% for secondary and early latent syphilis.

6. Sensitivity and specificity

According to the literature, the sensitivity and specificity of the test are very high - 99.6-100% and 99.3-99.5%, respectively. In the classical method, this is achieved through the electrophoretic separation of proteins, glyco- and lipoproteins and the maximum specificity of detecting immune sera or monoclonal antibodies. Under optimal conditions, immunoblotting can detect antigen in amounts of less than 1 ng in the test volume.

The sensitivity of the linear blot is also 99.6% and the specificity is between 99.3% and 99.5%.

According to experts, immunoblotting with recombinant highly specific antigens is similar in sensitivity and specificity to RIF abs.

According to foreign literature data and the results of a study at TsNIKVI, the immunoblotting method has high sensitivity (98.8-100%) and specificity (97.1-100%) in the diagnosis of syphilis.

7. Scope of the method

Immunoblotting (immunoblot) is a highly specific and highly sensitive reference method. High sensitivity and specificity allow this method to be considered as a confirmatory test for syphilis. The method can be used to confirm the diagnosis, as well as if other treponemal tests give questionable and conflicting results. Western blotting can confirm the diagnosis in patients with positive or indeterminate test results, including those obtained using TPHA or ELISA.

8. Sources and causes of staging errors, false positives and false negatives

False positive results are very rare. False-negative results are also quite rare and are observed in patients with immunodeficiencies - more often in HIV-infected people and with the effect of a diagnostic "window" due to a delay in antibody synthesis, as well as errors at the staging stage.

The use of modern test systems dictates the exact observance of all the requirements for both the material and the performance of the reaction. Basically, the source of errors is a violation of the order and technology of the study (especially for the classic Western blot), non-compliance with the recommendations of test kit manufacturers. . Errors can also be made in the collection, delivery, storage of blood serum samples, as well as in the performance of tests. In addition to tainted samples, other possible causes include the use of expired test kits and errors in the analysis of test results.

9. Features, advantages and disadvantages

The uniqueness of the immunoblot lies in its high information content and reliability of the results.

The test does not require highly purified antigens, reference and control sera. The identification of an antibody target is based on the molecular weight of the protein with which antibodies from the patient's serum react. In one reaction, the binding of antibodies to several antigens can be detected, each of which can be accurately characterized. Due to this, immunoblotting has a number of advantages over other methods for detecting antibodies, the results of which depend on standardization, sensitivity, quality of the substrate, instability or insolubility of certain antigens.

The method is easily reproducible, relatively easy to perform and interpret the results, makes it possible to determine the spectrum of antibodies to several T. pallidum antigens at once and evaluate the contribution of antibodies of different specificity to the overall reaction.

The use of highly purified recombinant and peptide antigens minimizes the nonspecific reactivity of sera. The use of the method makes it possible to avoid time-consuming and subjective methods that are dangerous for the researcher (transplantation of strains of G. pallidum, work with pathogenic T. pallidum in the formulation of the reaction). This determines the preference for the method of immunoblotting over other treponemal tests (RIF and especially RIBT) for the diagnosis of syphilis.

10. Method modifications

There are several commercial test systems based on this method. Some of them are in the format western blot, consist of strips on which protein components of T. pallidum are transferred, previously separated by polyacrylamide gel electrophoresis.

Others are in the format linear blot, consist of strips on which recombinant and synthetic polypeptides are adsorbed in the form of discrete lines - analogues of surface antigens of T. pallidum, TpN15, TpN17, TpN47 and TmpA.

Western blot results are more difficult to interpret because the strips show a wide variety of antibodies, many of which are group specific. In contrast, the interpretation of linear blot results is straightforward; in addition, additional control lines printed on the strip allow for semi-quantitative assessment of the level of antibodies to the four antigens of T. pallidum.

xMAP technology

xMAP is a state-of-the-art technology that makes it possible to carry out multiparametric studies by simultaneously detecting multiple analytes in a single biological sample. Colored microspheres coated with capture reagents (oligonucleotides, antibodies, antigens) are used as the solid phase.

The test sample is added to the solution containing the microspheres. The analytes detected in the sample are bound to the corresponding microsphere, after which a detecting agent (detecting antibodies, fluorescent label) is added to the solution. To detect the analyzed analyte, a system of two lasers is used, which allows both a qualitative assessment of the presence of an analyte in a sample (for this, a red laser is used that classifies microspheres by color) and a quantitative assessment of the analyte content in a sample (for this, a green laser is used).

The study is carried out automatically on analyzers such as Bio-Plex 200 (bio-rad), Bio-Plex2200 (bio-rad), Luminex100 (luminex), Luminex200 (luminex) equipped with software.

The performance of xMAP technology significantly exceeds the performance of classical enzyme-linked immunosorbent assay (ELISA) with a significantly smaller amount of biomaterial.

xMAP technology, which has a high analytical sensitivity, is currently used to solve a wide range of clinical problems. With its help, in one sample of biological material, simultaneous detection of known oncomarkers, cardiac markers, markers of the acute phase and diabetes mellitus, a wide range of cytokines, chemokines, and growth factors is carried out. Simultaneous detection of multiple analytes in one biological sample can significantly reduce the time of patient examination. To date, a number of test systems have been developed for the detection of antibodies to STI pathogens, in particular syphilitic infection, using the xMAP method.

MEDICAL CENTER"On Samotechnaya"

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Syphilis is an infectious disease that can be transmitted through sexual contact. The causative agent of the disease is a bacterium such as pale treponema (spirochete), which affects the internal organs, mucous membranes and skin.

To detect the disease, blood tests are used, and in some cases, cerebrospinal fluid. The results are indicated by pluses or crosses are used in an amount from 1 to 4.

Syphilis four crosses is considered the most dangerous stage for a person. The interpretation of the tests and the diagnosis is determined solely by the doctor.

Four stages of the disease and their characteristics

The definition of a sexually transmitted disease is carried out by studying the blood for the presence of treponema.

This method of recognizing syphilis using a serological reaction is the most common of many tests.

The immunologist created a special system for characterizing the disease, in which crosses indicate the amount of antibodies. It is important to know that they are not in the disease itself, but there are treponemas, ulcers, and a syphilitic rash.

An increase in antibody titer indicates the active reproduction of the pathogen, and crosses are contained in any analysis with a positive assessment of the presence of antibodies. Consider the stages of the disease and their features.

Syphilis one cross

If there are crosses, syphilis is positive, but there are doubts even when observing antibodies in the blood to fight the disease.

Therefore, doctors call such a test result doubtful. Often the result of the test can mean another disease.

A result of 1+ means that little time has passed since the infection stage. Plus may be present after complete treatment, when antibodies have survived.

Syphilis two crosses

Two crosses mean a positive result, which indicates the presence of treponema in the blood.

An increase in titer indicates a low concentration in the blood. So, you need to examine the bacterium to validate the conclusion 2 plus before starting therapy.

Syphilis three crosses

A blood test with a score of three crosses indicates a positive result and cannot be refuted. A repeated study of the blood only confirms the diagnosis of the 3rd cross, which is typical for the disease at the II stage of development.

Syphilis four crosses

The most unfavorable conclusion is the result of 4 crosses. But this does not mean at all that the disease cannot be cured.

This stage is characterized by a noticeable rash, hair loss, fever. The number of antibodies is at a high level, so the conclusion is beyond doubt.

How is the examination carried out?

Recognition of syphilis is carried out in two stages, starting with the examination of the patient, ending with the study of blood for antibodies.

The doctor examines the patient, while already determining the likelihood of the presence of the disease:

• detection of ulcers on the genitals or in the oral cavity;
• dermatological rashes, seals;
• baldness in the head.

The doctor clarifies information from the patient, based on questions about the presence of suspicious sexual intercourse or treatment of a sexually transmitted disease.

Examinations in the laboratory

Today, a study on the detection of syphilis 4 cross can be taken in many ways, the most famous are presented below:

• RPR is a test that determines antibodies in the blood to phospholipids of the cytoplasmic membrane;
• RIF (immunofluorescence reaction) is a more sensitive reaction, as it shows a positive result already at the first stage in 80% of patients;
• RW (method of the German immunologist Wassermann) is a fast and reliable research method that allows you to conduct an examination and prescribe effective pharmaceuticals;
• enzyme immunoassay of blood;
• the reaction is based on the phenomenon of immobilization of bacteria by antibodies such as immobilisins;
• passive hemagglutination shows the presence and amount of antibodies.

To date, syphilis can be treated at any stage. But it is much easier to tolerate treatment at the first manifestations of the disease, when the infection has not affected the entire body.

The term of treatment and medications are prescribed by a venereologist based on the individual characteristics of the human body and the stage of the lesion.

Do not forget that the best prevention of syphilis is a close relationship with a regular partner, in whose health you are completely sure.

RPHA analysis is a technique that is one of the most important among other blood tests. With the help of it, you can determine the development of unpleasant diseases of the intestinal-infectious nature. But often the analysis is used to detect syphilis. The study has a high accuracy and reliability.

RPHA stands for passive hemagglutination reaction.

RPHA is based on the phenomenon of agglutination of erythrocytes, in which antigens - RPHA reagents - are adsorbed on the surface when the blood serum of a patient with syphilis is added to them.

After the serum of a syphilis-infected person is combined with the RPHA reagent, agglutination (attachment or gluing) of erythrocytes occurs and they precipitate. The degree of adhesion depends directly on the accumulation and density of antibodies in the blood serum, for this reason, RPGA helps not only to detect the presence of antibodies, but also to identify their number.

The result is divided into primary and secondary infection, expressed in the manifestation of various antibodies. In the primary, IgM is activated, in the secondary, IgG. The accuracy of the study to the second phase becomes even more specific. If in the first phase it is about 96%, then in the second it can reach up to 99%.

The analysis is good as an independent study, but it is better to use it as a confirmatory method after a positive non-specific study.

Due to its high accuracy, this analysis is increasingly being used to confirm or refute the onset of a patient's syphilis lesion.

Also, for greater reliability, after RPHA, another confirmatory test can be performed, for example, a “treponemal” enzyme immunoassay.

In general, diagnostics is always carried out in a complex way, not using only 1 type of examination, but by combining various complementary methods.

Ask your question to the doctor of clinical laboratory diagnostics

Anna Poniaeva. She graduated from the Nizhny Novgorod Medical Academy (2007-2014) and residency in clinical laboratory diagnostics (2014-2016).

A small minus or, rather, a feature of the analysis is its sensitivity even after the cure of the disease. The result is kept positive throughout the life of a person who has had syphilis. For this reason, RPHA is not used to diagnose early or late disease. It is also impossible to use this analysis to determine the effectiveness of the treatment.

With any positive result, there is a suspicion of infection. But occasionally a false positive reaction can occur. True, its quantitative presence usually does not exceed 3% and is caused by a number of non-infectious reasons.