Serological diagnosis of syphilis. Treponemal tests for syphilis

Treponemal tests for syphilis. General description.

To reliably diagnose syphilis and identify anti-syphilitic antibodies in the patient’s body (in blood serum or cerebrospinal fluid), use special laboratory research technologies - 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 their 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 IgM class can be detected already at the end of the second week of the disease.

7. False positive and false negative results

Positive RV results for syphilis in individuals who do not suffer from this disease are called false positives. The rate of false positive results in healthy individuals is 0.2-0.25%. If the percentage of nonspecific false-positive RT results in healthy people is very small, then in some diseases it can be high.

All nonspecific 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, pinta, oral treponema, leptospira.

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

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

8. Modification of the Wasserman 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 turned out to be more sensitive. A special feature of the method of staging the Wasserman reaction in the cold is the three-phase temperature regimes at which complement fixation occurs. This reaction is also performed with cardiolipin and treponemal antigens.

In addition to the qualitative assessment of RF, there is a method for its quantitative statement 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 that still gives a sharply positive result (4+). Quantitative staging of RV is important in the diagnosis of some forms of syphilis and in monitoring the effectiveness of therapy.

9. Scope of application

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

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

• diagnosis of all forms of syphilis,
• monitoring the effectiveness of treatment,
• examinations of persons who have had sexual contact with patients with syphilis,
• examination 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 sent for abortion.

Currently, by order of the Ministry of Health of the Russian Federation, it is recommended to replace RSCT with more sensitive treponemal methods (ELISA or RPGA).

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)

KSR- This complex of reactions, used for the serodiagnosis of syphilis as a standard method. This complex of reactions includes the Wassermann reaction with cardiolipin antigen (an extract from the bovine heart enriched with lecithin and cholesterol) and treponemal antigen (an ultrasonic-treated suspension of apathogenic cultured treponemes pallidum), as well as a microprecipitation reaction (MPR) with plasma or inactivated serum, which is put with cardiolipin antigen

The CSRs become positive in the middle of the primary period (its division into seronegative and seropositive is precisely determined by the CSR), in the secondary period the CSRs 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 DAC:

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

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

Disadvantages of the DAC:

1) Subjectivity in assessing 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 (FPR) are often observed.

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

RIT, RIBT - Treponema pallidum immobilization reaction

Treponema pallidum immobilization test (TPI) is a classic method that is used to detect specific treponemal antibodies. The RIBT reaction uses pathogenic Treponema pallidum T. pallidum (Nichols strain) grown in a rabbit testicle as an antigen. RIBT is based on the loss of motility of living Treponema pallidum after exposure to antibodies from the patient's blood serum and complement. The results are assessed using dark-field microscopy. Although the RIBT test was introduced into clinical practice as a specific test for syphilis, it is labor-intensive, technically complex, time-consuming and expensive to use.

1. History of the RIBT method

The Treponema pallidum immobilization test (TPI) is actually the first specific test for diagnosing syphilis. This reaction was introduced in 1949 by American researchers R. W. Nelson and M. M. Mayer and was discussed in detail in scientific works in subsequent decades. Unsuccessful attempts to use live treponemes in tests have been made before. Thanks to the fact that Nelson was able to create an environment in which treponemes remained viable for up to 8 days, his research was a success.

2. The principle of the RIBT method

The method is based on the phenomenon of loss of motility by treponema pallidums in the presence of immobilizing antitreponemal antibodies of the test blood serum and complement under anaerobic conditions. The antigen is live pathogenic Treponema pallidum obtained from rabbits artificially infected with syphilis.

3. Setting up the RIBT test

The reaction involves the test serum, complement and antigen. Blood serum of the subject is added to live treponema obtained from rabbit testicular tissue after artificial infection. If there are anti-treponemal antibodies-immobilisins in the serum, treponema pallidums stop moving (immobilized). Immobilisin antibodies are late antitreponemal antibodies.

The reaction is carried out with heat-inactivated sera or with samples of sera dried on wax paper (dry drops). Inactivation of serum by heating is carried out for 30 minutes at a temperature of 56°C. Before taking blood, the person being examined should not receive medications, especially penicillin. Taking medications is discontinued for the period of their possible retention in the body.

Nichols strain bacteria obtained from 7–10-day-old rabbit syphilitic orchitis (inflammation of the testicle) are used as an antigen. The period from the moment of setting up the reaction to recording 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 rejected. Canned complement cannot be used in the immobilization reaction of Treponema pallidum, because it is toxic to microorganisms.

The immobilization reaction uses excess complement. Its quantity largely depends on the survival environment for Treponema pallidum.

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

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

The results of RIBT are assessed after removing the tubes from the thermostat and microanaerostat (i.e. after 18-20 hours of experiment). Using a Pasteur pipette, a drop of the contents of the test tube is applied to a glass slide, which is covered with a coverslip and examined in a dark field microscope (objective 40, eyepiece 10X). Several fields of view are examined in different parts of the preparation, counting the number of mobile and immobile treponemes pallidum in each. The counting begins with the drug from the control and then from the test tube.

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

If there is a lack of complement in the experiment, immobilizing antibodies do not show their activity properly and the treponemes remain mobile. Therefore, after the experiment, residual complement is determined in order to assess whether the motility of Treponema pallidum in the test tubes was due to the lack of complement. For this purpose, a hemolytic system is used - a mixture of a suspension of sheep erythrocytes and diluted hemolytic serum kept in a thermostat.

Residual complement is determined by adding a hemolytic system to each tube the required volume. The tubes are placed in a thermostat at 37° for 45 minutes. In test tubes, hemolysis of red blood cells 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 must be repeated. Repeated blood serum testing is not performed only if 100% immobilization of Treponema pallidum is noted.

4. Accounting for RIBT results

Counting of immobilized treponemas that have lost their mobility is carried out under a microscope using the dark-field microscopy method. The researcher is required to have the skill of assessing the movement of treponemes. He should pay attention to the intensity of movements made by Treponema pallidum. 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 treponemes from movement with fluid flow.

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

X = (M – C)×100/M

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

The immobilization reaction of Treponema pallidum is assessed as

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

The immobilization reaction of Treponema pallidum becomes positive at the end of the primary - beginning of the secondary period of syphilis (from the 7-8th week from the moment of infection or more). However, RIBT is of little use for diagnosing the early stages of syphilis, since antibodies that immobilize Treponema pallidum and are detected in the reaction appear only 3-6 weeks after infection. Immobilisin antibodies belong to the IgG class of immunoglobulins. They appear in the blood later than reagins (anticardiolipin antibodies), later than fluorescein antibodies (detected by RIF and ELISA) and precipitins (detected by bladder cancer).

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

RIBT for a long time was recognized as the most specific test for syphilis. According to the literature, the specificity of RIBT is 99%, 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 application of the method

The scope of application of RIBT is gradually narrowing due to the duration of installation, 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 has decreased significantly in recent years. In the United States, 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 to diagnose 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 results of other serological tests are discrepant.

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 negatived during antisyphilitic therapy. As a result, it is unsuitable for monitoring the progress of antisyphilitic therapy.

False-positive results (FPR) with RIBT are rare and were noted 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. As patients age, the number of false-positive RIBT results increases.

RIBT may be false positive if the test serum contains treponemocidal substances (for example, penicillins, tetracyclines, erythromycin), which cause nonspecific immobilization of Treponema pallidum. This may be a consequence of the patient taking treponemocidal antibiotics, so the examination is not carried out for persons who have received antibiotics within the last month. Blood for RIBT can be tested no earlier than 2 weeks after finishing taking antibiotics and other antisyphilitic drugs.

9. Modifications of the immobilization reaction of Treponema pallidum

In addition to the microanaerostat technique, there is a melange method of RIBT according to N.M. Ovchinnikov. Anaerobic conditions when setting up a 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 allows you to do without a vacuum pump, a cylinder with a mixture of nitrogen and carbon dioxide, or a microanaerostat. A comparative study on a large clinical material yielded results 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 keeping rabbits and conducting testing. This labor-intensive test is currently used mainly for scientific purposes. In the majority foreign countries For almost 40 years, RIBT has been practically used not for diagnostic purposes, but only in research work.

Disadvantages of reaction:

• RIBT requires working with live pathogenic Treponema pallidum of the Nichols strain, which remains infectious to humans despite adaptation to rabbits
• staging 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 assessment
• lack of automation
• it is not possible 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 receiving full treatment.
• the reaction may 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 a rapid diagnostic method for identifying microbial antigens or determining antibodies. Tests based on fluorescent signal detection are considered one of the best tests for syphilis.

1. History of the method

The fluorescent treponemal antibody (FTA) test was first developed in 1957 by Deacon and co-authors (Deacon, Falcone and Harris).

2. 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 are able to 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 pallid Treponema strain Nichols 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 Treponema pallidum, dried and fixed in glass with acetone.

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

3. Method of conducting research using the RIF method

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

To detect antigen-antibody complexes, a luminescent serum representing anti-species (anti-human) immunoglobulins conjugated to FITC is used. The presence of antibodies to treponemes in the serum is determined by the glow of treponemes 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 fluorescence microscope. The results are assessed by the degree of luminescence of the treponemes in the preparation. In the presence of antibodies, the glow of treponemes is visible, but if there were no anti-treponema antibodies in the serum, then the treponemes are not visible. The degree of luminescence of dried pale treponema fixed to glass is indicated in “pluses” (from “–” to “++++”). Negative result - no glow or background level - 1+.

5. During what periods of illness 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 slightly earlier than RV. According to some data, 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 in all stages of infection, from the incubation period to late syphilis. According to WHO, the sensitivity of RIF for primary syphilis is 70-100%, for secondary and late syphilis - 96-100%, specificity - 94-100%. According to TsNIKVI, the sensitivity of RIF for all forms of syphilis is 99.1%.

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

7. Scope of application of the method

RIF is applied:

• as a confirmatory reaction in early, latent syphilis
• when establishing a retrospective diagnosis
• to differentiate latent forms of syphilis and false-positive test results for 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 perform. To perform RIF, it is necessary to have a vivarium or acquire a suspension of pathogenic Treponema pallidum, which limits the possibilities of the reaction. However, in recent years, test systems have begun to appear on the domestic market that allow 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 errors during production, false positive and false negative results

LPRs are rare when RIF is diagnosed (in collagenosis, borreliosis).

RIF is still considered one of the best tests for syphilis, the “gold standard” of serodiagnosis. RIF is easier to set up than RIBT,

Despite its high diagnostic value, the widespread introduction of RIF into everyday practice is hampered by the need to use live T. pallidum, the high cost and duration of the study. Setting up a reaction is labor-intensive. In addition, the assessment of the results of the RIF is subjective.

Advantages of 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) Subjectivity of 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 receiving full treatment. Therefore, these reactions cannot be used to monitor cure.

10. Method modifications

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

• 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 diagnosis) – a highly specific method for the serodiagnosis of syphilis.
• RIF-10(10-fold dilution of the test serum) is 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 is RIF-abs- immunofluorescence reaction with absorption. Before performing the reaction, the patient's serum is depleted with a mixture of non-pathogenic treponemes to eliminate cross-reactions. Group antibodies are removed from the test serum using cultural treponemes 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,
• identification of false-positive results of CSR and bladder cancer, especially in pregnant women and somatic patients with suspected syphilis,
• to establish a retrospective diagnosis of the disease.

RIF-abs is not very informative when 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 the serodiagnosis of syphilis. It is used for arbitration cases, but for a reliable result, a fresh concentrated suspension of T. pallidum strain Nichols from seven-day orchitis in a rabbit is required, which cannot be frozen.

2. In the USSR it was installed in two modifications - RIF-10 And RIF-200, i.e. with 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 high specificity of the reaction, but its sensitivity decreases somewhat. RIF-10 is more sensitive, but more often gives nonspecific positive results than RIF-200, which is highly specific. RIF-10 is more sensitive, RIF-200 and RIF-abs are more specific.

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

3. RIF-ts carried out with liquor. The reaction is performed using whole cerebrospinal fluid to identify specific lesions of the central nervous system.

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

There are 2 known modifications of this reaction:

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

– Russian version of RIF-abs-IgM, characterized in that a sorbent is added to the blood serum being tested that removes IgG antibodies, and RIF-abs is administered with the remaining IgM antibodies.

Main indications for RIF-abs-IgM testing 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 relapse of syphilis, in which RIF-abs-IgM will be negative, and RIF-abs will be positive;

– assessment of the effectiveness of therapy for early acquired or congenital syphilis: after adequate treatment RIF-abs-IgM becomes negative over 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. Consequently, class M antibodies against treponema pallidum can appear in the child’s body 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 turns out to be extremely useful in diagnosing 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.

Setting up a reaction 19S(IgM)-RIF-abs involves preliminary separation using 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 for staging this reaction is complex and labor-intensive, requiring special equipment and specialist training.

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 treponema, sensitized by the serum of a patient with syphilis, in the presence of complement and erythrocytes, adheres to the surface of erythrocytes and, during centrifugation, is carried away with them into the sediment, disappearing from the supernatant.

To set up the reaction, the following ingredients are used: test serum, antigen, complement, donor red blood cells, isotonic sodium chloride solution. A suspension of pale treponema strain Nichols is used as an antigen.

This test was most widely studied in relation to the serodiagnosis of syphilis by domestic and foreign authors in the 50-60s. Evidence regarding the value of RIP as a diagnostic test has been inconsistent. The reaction required maximum accuracy, since inaccurate dispensing of ingredients or an excess or deficiency of the test material in the preparation resulted in unreliable results.

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

G.P. Avdeeva, having used other temperature and time regimes in the production of the antigen, obtained different results when studying RIP. According to her data, 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 industrial production of the antigen for RIP did not allow this test to be more widely studied and introduced into practice.

RPHA passive hemagglutination reaction

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

1. History of the RPGA method

For the first time, the use of RPHA for the diagnosis of syphilis was reported by G. Blumental and W. Bachman (1932). In 1965, an indirect or passive hemagglutination test was proposed to diagnose syphilis. Modification of the reaction using various antigens was reported by Ratlev T. in 1965 - 1967. 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. The principle of the RPGA method

From a prepared homogeneous suspension of erythrocytes “loaded” with antigens, upon addition of the test serum containing antibodies, a precipitate in the form of flakes precipitates. The resulting precipitate consists of red blood cells “glued together” with antibodies, and is called "hemagglutinate". A suspension of red blood cells is prepared in advance and supplied as part of diagnostic test systems.

The process of gluing together red blood cells that have antigens on their surface is called “hemagglutination.” Bonding occurs under the influence of specific antibodies (agglutinins). The reaction is called "passive", because the erythrocytes' own antigens do not react, and the erythrocytes themselves perform exclusively an auxiliary indicator function.

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

In the passive hemagglutination reaction for diagnosing syphilis, sheep or bird erythrocytes coated with Treponema pallidum antigens are used as an antigen. When serum containing specific antibodies is added, red blood cells stick together (agglutination).

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

3. Setting up a passive hemagglutination reaction

RPGA 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 thus obtained is called sensitized erythrocytes. Red blood cell diagnostics are called red blood cells sensitized with an antigen.

To prepare the diagnosticum, erythrocytes from sheep or birds (usually chicken), first treated with formaldehyde and then 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 the ultrasonic antigen of cultured Treponema pallidum can also be used.

Only serum is tested (do not use blood plasma). Hemolyzed and turbid 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, positive and negative controls are used.

Samples of the test blood serum and test red blood cells are added to the wells (wells) 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, the formation of antigen-antibody complexes associated with the surface of the carriers (erythrocytes) occurs. 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 fall down under the influence of gravity, are distributed over the entire surface of the bottom of the hole and form a characteristic picture of an “inverted umbrella”.

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

Immune complexes are not formed if there are no specific antibodies in the sample or when control (intact) red blood cells are added to the reaction. At the same time, red blood cells gradually collect at the lowest point of the bottom of the hole, forming a figure in the form of a compact spot or “button”, sometimes with a slight clearing in the center.

If a person’s blood serum contains anti-erythrocyte antibodies, then an “umbrella” will form in any case - both in reaction with test erythrocytes and with control erythrocytes. In this case, it is recommended to use other medical technologies to identify specific antitreponemal antibodies.

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

4. Accounting for the results of the passive hemagglutination reaction

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

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

The results of the study are assessed using a 4+ system (from “–” to “++++”) based on the size of the film formed. When agglutination occurs, red blood cells are located on the surface of the hole in the form of an “umbrella”, and if the result is negative, the red blood cells freely slide down and accumulate at the bottom in the center of the hole in the form of a “button”.

Generally accepted assessment of RPGA results:

4+ - positive RPGA. Agglutinated red blood cells in the form of an “umbrella” evenly line the entire surface of the hole;

3+ - positive RPGA. Red blood cells line the entire surface of the hole, but some of them “slide” to the center. In this case, a noticeable ring is formed along the periphery of the sediment;

2+ - weakly positive RPHA. Red blood cells form a film on a small area of ​​the lower part of the hole, forming a dense ring of red blood cell sediment with a noticeable clearing in the center;

1+ - indeterminate RPHA, red blood cells form a loose sediment at the bottom of the well with unclear edges and a slight lumen in the center;

(–) - negative RPGA, all red blood cells lie at the bottom of the well in the form of a compact sediment (“buttons” or rings) against a clean surrounding background (without surrounding granular sediment).

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

The reaction results can be recorded automatically using special analyzers. In addition to qualitative research, all test systems provide quantitative analysis with titer determination.

5. During 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 chancre) and remains positive for years after treatment.

With a very high level of antibodies to treponema in the test serum (which is most typical for secondary syphilis), a false negative RPGA result is possible (the so-called “prozone” phenomenon).

Specific agglutinin antibodies are detected in the blood of people who have had syphilis for a long time, so RPGA cannot be recommended for the differential diagnosis of reinfection or 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 bladder cancer or to RPR) method in monitoring the effectiveness of treatment by studying the dynamics of the decrease in antibody titers. A prerequisite for this is the use of the same RPGA test system as during the first (before treatment) examination of the patient, as well as the testing in the same laboratory.

6. Sensitivity and specificity of RPGA

RPGA is considered a highly sensitive and specific test. This reaction is a valuable diagnostic test for all forms of syphilis, but it is especially sensitive in late forms of the disease. Depending on the stage of the disease, the sensitivity of RPGA varies. With primary syphilis, the sensitivity of RPGA is 76% (and higher), with secondary syphilis - up to 100%. For latent early syphilis - 97%, for late syphilis - 94%, with a specificity of 98–100%. More low sensitivity in fresh forms of the disease is explained by the later formation of agglutinins.

According to the State Institution “TsNIKVI Roszdrav”, the sensitivity of RPGA in diagnosing various forms of syphilis was 99.4%. Most researchers note 98-99% specificity for RPHA.

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

7. Scope of application of the RPGA method

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

8. Features, advantages and disadvantages of RPGA

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

The RPGA technique is easy to perform and does not require special equipment: all you need is a hemagglutination plate. The research does not take a long time; the reaction is highly sensitive and specific. Testing of the method in clinical practice has shown that it is extremely simple, cheap and sensitive. Like ELISA, RPGA 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 obtain results – 45 minutes,
• suitable for mass screening (only 25 µl of serum diluted 1:20 is required),
• high degree of standardization,
• presence of internal controls,
• long shelf life,
• acceptable price
• possibility of accounting automation.

The disadvantages of RPGA should also be noted:

• the possibility of nonspecific reactions in the presence of anti-erythrocyte antibodies,
• lack of correlation between titer and stage of syphilis,
• later positivity of the 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,
• possibility of automating the reaction,
• there is no need to work with live Treponema pallidum,
• there is no need for a vivarium.

9. Sources and causes of errors when performing RPGA, false positive and false negative results

The passive hemagglutination test is a relatively simple test; 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. Errors made can lead to the appearance and registration of both false negative and false positive reaction results. False-positive RPGA results may be due to the influence of human and biological factors.

False positive results may be obtained

• when studying blood sera of patients with non-venereal treponematoses,
• due to rheumatoid factor
• due to antibodies cross-reacting with the treponemal antigen, formed during various systemic or 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 the mother's IgG, which complicates the interpretation of the results and the diagnosis of congenital syphilis.

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

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

The most typical technical errors when performing RPGA, leading to unreliable results, include:

• inaccurate dilution of ingredients,
• temperature violation,
• violation of the incubation time of reagents,
• violation of deadlines for applying reagents to the tablet,
• discrepancy between the pH of solutions and the required ones,
• contamination of laboratory glassware.

The following technical issues may also be a source of errors when setting up the RPGA:

• exclusion of control blood sera from the reaction;
• uneven concentration of red blood cells in the diagnosticum due to insufficient mixing before use;
• violation of the terms and conditions of storage of diagnosticum and control red blood cells; use of expired kits;
• use of contaminated test tubes, pipette tips, pipettes, immunological plates, solutions when performing reactions;
• inaccuracies in the initial dilution of the serum sample;
• insufficient care in performing serial double dilutions;
• non-compliance with temperature conditions and incubation time;
• the presence of extraneous vibrations and shaking of the immunological tablet during incubation;
• violation of the procedure for staging RPGA, expressed in refusal to perform the study with control erythrocytes.

The use of blood plasma containing anticoagulants that can cause nonspecific erythrocyte agglutination (RPGA) may lead to results that cannot be interpreted.

The number of false positive and false negative results is lower than with other serological tests. LPRs when staging RPHA are rare and are possible with treponematoses (yaws, bejel, pinta). Also, false-positive results were recorded (less than 1% in total) in drug addicts, in patients with infectious mononucleosis, borreliosis, leprosy, collagenosis, liver cirrhosis, lymphosarcoma, as well as in pregnant women.

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

10. Modifications of the RPGA method

There are micro- and macro-modifications of the RPHA setup; the first one is more often used due to its efficiency, speed of setup and recording of the results.

In addition, an automatic diagnostic complex image analysis, 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 and software complex recognizes the image, processes the data and provides an answer in relative units.

To automate the recording of RPGA results, readers and automatic analyzers are also used.

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

Brief Description of the TPPA Test

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

TPPA is used for serological diagnosis of antibodies to various species and subspecies of pathogenic Treponema. This test can be used to detect antibodies to the pathogens syphilis, pint, bejel and yaws.

The research 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 contained in the patient's blood serum.

TPPA is a confirmatory treponemal test that is useful for both small sample testing and mass screening. TPPA is used abroad as a confirmatory test and to replace the micro-hemagglutination test MHA-TP (microhemagglutination assay for antibodies to T. pallidum).

The sensitivity of the TPPA test is between 85% and 100%, and 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 treponemes (such as T. pallidum endemicum, pertenue, or carateum) may cause false-positive results. There are a number of methods available to remove these antibodies from serum samples before testing.

Principle of TPPA test

TPPA is a method of passive agglutination of gelatin particles in human serum or plasma. Serum containing antibodies to pathogenic treponema reacts with gelatin particles sensitized with the antigen of pale treponema strain Nichols, 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 absent, then 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 this compact “button” for each serum, i.e. no agglutination.

Application of the TPPA test

TRPA is a universal-purpose test that can be equally successfully used both during mandatory preventive examination of population groups for syphilis (screening) and in specialized dermatovenerological 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 the serodiagnosis of syphilis. Other advantages of the test include high reproducibility, as well as simplicity and speed of setting up the reaction.

The TPPA test is used to confirm positive results from nontreponemal syphilis screening tests, such as the VDRL test, and to evaluate patients with negative nontreponemal test results 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 must be carried out in mandatory combination with other indicators and symptoms of the disease.

TPPA test results

The result is assessed according to the “pluses” system - from (–) to (2+). The test results are visible to the naked eye and are interpreted as follows:

Degree of agglutination	Test indicator	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 hole with a slight gap in the center and a smooth outer border	–	Negative
The particles form a “button” in the center of the hole with a smooth outer border	–	Negative

The results are taken into account to determine compliance with the description above. Samples that show an indeterminate result (±) must be retested. If a sample shows an indeterminate result in several TPPA tests, it is recommended to test using other methods.

The results of the analysis should not be viewed 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 tested again. To make a diagnosis, it is necessary to take into account the patient's clinical symptoms, clinical history and other data.

As with the RPHA reaction, TPPA may exhibit a prozone phenomenon and a false negative result if the serum sample contains too high an antibody titer.

ELISA - Enzyme immunoassay

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

The introduction into practice of enzyme-linked immunosorbent assay (ELISA) 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 ability to automate 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 and co-author. (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 assessed 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 ELISA method for diagnosing syphilis was developed by V.N. Bednova, A.V. Babiy and A.V. Kotrovsky (1982, 1983).

2. Principle of the ELISA method

The enzyme-linked immunosorbent assay (ELISA) is similar in reaction mechanism to RIF (the same antibodies are detected). The enzyme immunoassay reaction is classified 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, the indirect version of ELISA is mainly used. The principle of the most commonly used indirect reaction is as follows. On the surface of the wells of a polystyrene tablet, immune complexes are fixed that are formed when the antibodies of a syphilis patient interact with Treponema pallidum antigens. After this, 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. If there are antibodies in it, an antigen-antibody complex is formed on the surface of the carrier. To “manifest” the results of the reaction, anti-human Ig antibodies 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 varying intensity.

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

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

ELISA provides the ability to detect serum Igs of different classes. There are systems on the market that allow you to separately determine IgM and IgG and total antibodies.

Specific antigens used for ELISA can have different origins:

ultra-voice- they are obtained by destroying the bacterial cell T. pallidum by ultrasound or another method;

recombinant- they are obtained by genetic engineering technologies by introducing into the genome of a bacterial cell (for example, E. Coli) a gene responsible for the synthesis of a specific T. pallidum antigen, followed by an increase in the bacterial mass of the producing microorganism, the destruction of these cells, 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. With a normal immune response, this usually does not happen. One or more immunogenic peptides isolated from a protein antigen have a special antigenicity, and most antibodies are formed specifically to them. The most intense immune response develops to them. The most informative ELISA showed the immunodominant regions of Treponema pallidum proteins with molecular weights of 15 kDa, 17 kDa and 47 kDa. A detergent extract or sonicate of pathogenic treponemes of the Nichols strain was used as an antigen.

3. Method of conducting research using the method

The principle of the method is to detect a specific antigen-antibody complex sorbed 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 the substrate, taken into account quantitatively spectrophotometrically.

Antigens used to sensitize the wells of a polystyrene plate can be:

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

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

4. Accounting for results

In ELISA, an enzyme reaction is used to visualize the antigen-antibody reaction ( alkaline phosphatase or horseradish peroxidase) with the substrate, which changes its color. The intensity of the color determines the positivity of the reaction (from “–” to “++++”). ELISA results can be assessed visually using 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 results are assessed spectrophotometrically, this eliminates subjective interpretation.

5. During what periods of illness is it better to use

The timing of ELISA positivity is 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 incubation and remain positive in all periods. ELISA is especially valuable in the early diagnosis of syphilis - positive results for detecting antibodies can be obtained already at the end of the incubation period of the disease (i.e., 4–6 weeks after infection).

6. Sensitivity and specificity

ELISA is a highly sensitive and specific test, which is its advantage. ELISA is close to RIF in terms of reaction mechanism, sensitivity and specificity, because 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 specificity - 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 variant (enzyme-linked immunosorbent assay, ELISA) is one of the most sensitive serological reactions, allowing the detection of 0.0005 μg/ml of antibodies and 0.000005 μg/ml of antigens.

7. Scope of application of the method

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

The ELISA method can be used in a quantitative version 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 Order of the Ministry of Health of the Russian Federation No. 87 of March 26, 2001 “On improving the serological diagnosis of syphilis” (Appendix No. 1 “Staging screening and diagnostic tests for syphilis”). The order plans to replace RSC in the complex of seroreactions (SRC) with ELISA and RPGA.

8. Sources and causes of errors during production, false positive and false negative results

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 source of errors when performing ELISA may be the following points:

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

Do not practice freezing a sample of biological material two or more times; if necessary, repeat the study, use serum from a duplicate test tube;

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

Use of reaction components from other diagnostic kits;

Violation of the timing of reaction stages;

Drying of the wells of the immunological plate at the reaction stages;

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

Failure to comply with the frequency of metrological control of the operating parameters of the devices used (washer and spectrophotometer);

Use of contaminated laboratory glassware when setting up reactions: flasks, graduated cylinders, test tubes, pipettes, pipette tips;

Lack of care in sample dilutions biological material;

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

Errors when transferring study results to the registration log, study protocol or response 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, and the use of internal positive and negative controls make it possible to obtain ELISA test results with high reproducibility.

9. Features, advantages and disadvantages

ELISA is a modern, promising method for the serodiagnosis of syphilis due to its simplicity, reproducibility, and accessibility. Determination of antibodies by ELISA is the perfect way diagnostics suitable for simultaneous examination of a large number of samples. Due to its advantages, it has gained popularity in all countries.

ELISA research technology requires compliance with all recommendations of manufacturers of commercial diagnostic test kits and thoroughness of the research procedure. To conduct research in ELISA, it is necessary to purchase additional special equipment. The staging 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 study a large number of samples of biological material with a high degree of standardization of the study, minimization of the subjective element in evaluating the results, the possibility of storing fixed study protocols, which allows you to archive study data and access 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 capabilities

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 allows you to simultaneously study a flow with a large number of samples of biological material with a high degree of standardization of the study, minimizing the subjective element in assessing the results, the ability to store fixed research protocols, allowing you to archive research data and access them again for retrospective analysis .

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

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

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

~ increased risk of infection of personnel.

10. Method modifications

Along with the classical indirect ELISA, the method trap ELISA. It was proposed in 1989 by O. E. Ijsselmudien et al. to detect IgM 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 plate, and after incubation of the test serum, all IgM bind to the carrier. The presence of antigen-specific Tr. among the associated IgM. pallidum is detected using Tr. pallidum conjugated with the enzyme.

This method allows you to detect antibodies not only of the IgM class, but also of the IgG and IgA classes. To do this, the wells of the plates are sensitized with affinity-purified antibodies of a certain class against human immunoglobulins. In this case, 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 representing the connection of the treponemal antigen with the enzyme.

The use of a trap version of an enzyme-linked immunosorbent assay 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 anti-treponemal IgG are also excluded.

ELISAs have found widespread use abroad as options. enzyme immune assay (EIA) and system ICE Syphilis. In the latter, a mixture of antibodies to IgM and IgG, as well as three recombinant proteins of T. pallidum, is sorbed in the cells of the plate. Positive results are tested in the same test system in duplicate to produce the final result.

In Russia, a number of test systems have been developed for the serodiagnosis of syphilis using the ELISA method 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 a reaction on nitrocellulose strips, ELISA with capillary blood, as well as immunoblotting, or Western Blot, with the simultaneous detection of antibodies to various components of the pathogen.

A modern improved modification of ELISA is immunoblotting.

ICA (immunochemiluminescence assay), ICL (immunochemiluminescence)

With the development of laboratory diagnostics in the context of 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 helps improve the quality of diagnosis. 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 emission of photons during the transition of electronically excited products of oxidative chemical reactions to their original 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 non-isotopic methods, chemiluminescence provides the highest sensitivity. For immunometric methods, the sensitivity of chemiluminescence is orders of magnitude greater than the sensitivity of radioimmunoassay.

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

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

The method uses recombinant lipoproteins obtained by genetic engineering methods as antigens, which are complete analogues of T. pallidum antigens.

ICA method based on results clinical trial has received recognition and is recommended for use in the laboratory diagnosis of syphilis in the Russian Federation as a screening and confirmatory test if appropriate equipment is available in laboratories. 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 become part of the practice of both global and domestic laboratory diagnostics with the introduction of automation, provides the following advantages:

• eliminating the influence of subjective factors at the analytical stage of the study
• personnel safety when performing research on potentially infected biological material
• increasing the speed at which the patient receives results
• standardization of studies and quality control of studies
• delivering reliable, reliable results to patients
• high quality of clinical laboratory research.

In terms of sensitivity, the ICA method is comparable to the radioimmunoassay method, 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 quantitatively determine 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).

Methods for detecting treponeme-specific antibodies based on immunochromatography (ICH) methods are relatively new for use in the Russian Federation. As in the ICA method, recombinant lipoproteins obtained by genetic engineering methods (for example: antigens Tp15, Tp17, Tp47) and the biosynthetic peptide TmpA 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 treponeme-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 for epidemiological indications.

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

PBT (simple rapid bedside tests)

Relatively recently, in the serodiagnosis of syphilis, a direction has begun to develop the so-called simple rapid tests (SRT), or bedside tests (Point-of-care - POC). Simple rapid bedside tests, or immunochromatographic tests, allow rapid determination of the content of treponeme-specific antibodies to the causative agent of syphilis in serum and whole blood samples without the use of special laboratory equipment and can 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 the immunochromatographic determination of antibodies to treponemal antigens of Treponema pallidum and are performed on strips; in this case, both whole blood and serum can be used (Herring A., Ballard R. et al, 2006). The test format allows research to be carried out in the absence of special equipment and without the use of electricity. However, due to the relatively low sensitivity and specificity, these tests are not yet widely used in practice.

Immunoblotting (Western Blot)

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

Immunoblotting is one of the most modern methods for serodiagnosis of syphilis and is actively used abroad. It received its name (“Western blotting”) as a humorous response to the names of the techniques for determining DNA (“Southern blotting”) and RNA (“Northern blotting”).

1. History of the method

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

2. Classic immunoblot

Classic immunoblot(Western Blot Analysis) combines enzyme immunoassay and the use of an electrophoretic method. The antigen proteins 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 dots (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 a medium, positively charged molecules move towards the negatively charged electrode, and negatively charged molecules move towards the positive electrode.

Most globular proteins are assembled in such a way that most of the charged groups, namely hydrophilic ones, are located on the surface of the protein, while uncharged, hydrophobic residues are embedded in the interior of the globule. In an electric field, proteins migrate according to their charge towards the 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, the protein mixture is preincubated in the presence of sodium dodecyl sulfate (SDS) before electrophoresis.

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 polyacrylamide gel (PAGE) is the size of the protein, or rather its molecular weight. This made it possible to use the method of PAGE electrophoresis 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 (sodium dodecyl sulfate polyacrylamide gel electrophoresis method). (1) - control positive result, (2) - control negative result, (3-6) - clinical samples.

In tests for syphilis using this method, Treponema pallidum, previously purified and destroyed into its constituent components, is subjected to electrophoresis in a polyacrylamide or agarose gel. In this case, the antigens included in its composition are separated by molecular weight.

Then, using vertical electrophoresis, the antigens are transferred to a strip of nitrocellulose, which now contains a spectrum of antigenic bands invisible to the eye, characteristic of Treponema pallidum.

When performing an 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 during the incubation process they strongly bind to strictly corresponding (complementary) antigenic bands and form an “antigen-antibody” complex.

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

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

The presence of bands in certain areas of the nitrocellulose plate confirms the presence in the tested serum of antibodies to strictly defined antigens of Treponema pallidum.

The immunodeterminants 15, 5, 17, 44.5, 47 kD determined using this method have diagnostic significance for 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

Line immunoassay (LIA) allows the simultaneous determination of antibodies to Treponema pallidum antigens in human serum or plasma. Antigens are pre-applied to test strips, which are supplied as part of commercial diagnostic kits.

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

The strips use artificial antigens obtained by genetic engineering methods - recombinant proteins or synthetic polypeptide constructs. These are analogs of the surface antigens of Treponema pallidum - TpN15, TpN17, TpN47 and TmpA with molecular weights 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 test sample is incubated in a cuvette, where an indicator strip is also placed. Antibodies to T. pallidum are determined by binding to antigens applied to test strips. If T. pallidum-specific antibodies are present in the sample, they form bonds with distinct antigen lineages. 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 assessed 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 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 causative agent of syphilis, 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 whose operation is based on signal reflection, which eliminates subjective assessment and provides the potential for automation.

5. During what periods of illness is it better to use

Tests using immunoblotting can detect specific antibodies to Treponema pallidum antigens at a very early stage of the disease. The most significant antibodies in the diagnosis of syphilis are antibodies to Treponema pallidum antigens TpN15, TpN17, TmpA and TpN47. These antigens are membrane proteins with 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 later to all TmpA. When taking into account the test results, the serum reactivity to each of them is assessed separately. For example, when a linear blot shows reactivity only to the antigen lines TpN17 and TpN47, this means that the disease is in the early stages. The more antigen lines become reactive, the further the infection progresses. When treating 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 seroresistance. The search for IgG to the same molecules is characterized by a sensitivity of 61.6% for seroresistance 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 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 quantities of less than 1 ng in the test volume.

The sensitivity of the linear blot is also 99.6%, and the specificity ranges 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%) for diagnosing syphilis.

7. Scope of application 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 in cases where other treponemal tests give questionable and contradictory results. Using immunoblotting, you can confirm the diagnosis in patients with positive or indeterminate test results, including those obtained using RPGA or ELISA.

8. Sources and causes of errors during production, false positive and false negative results

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 the synthesis of antibodies, as well as in case of errors at the stage of diagnosis.

The use of modern test systems dictates strict compliance with all requirements for both the material and the execution 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), and non-compliance with the recommendations of the test kit manufacturers. . Errors may also occur during the collection, delivery, storage of serum samples, and during testing. In addition to spoiled samples, other possible reasons include the use of expired test kits, as well as errors in the analysis of study 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 or control sera. Identification of the antibody target is based on the molecular weight of the protein with which the antibodies from the patient's serum react. In a single reaction, antibody binding to multiple antigens can be detected, each of which can be precisely characterized. Due to this, immunoblotting has a number of advantages over other methods for detecting antibodies, the results of which depend on standardization, sensitivity, substrate quality, instability or insolubility of certain antigens

The method is easily reproducible, relatively simple 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 various specificities to the overall response.

The use of highly purified recombinant and peptide antigens minimizes nonspecific serum reactivity. Using the method makes it possible to avoid labor-intensive and subjective methods posing a danger to the researcher (inoculation of G. pallidum strains, work with pathogenic T. pallidum when staging a reaction). This determines the preference of the immunoblotting method over other treponemal tests (RIF and especially RIBT) for diagnosing syphilis.

10. Method modifications

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

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

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

xMAP technology

xMAP is a modern technology that allows for multiparametric studies through the simultaneous detection of multiple analytes in one 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 detection agent (detection antibodies, fluorescent label) is added to the solution. To detect the analyte being determined, a system of two lasers is used, which allows for both a qualitative assessment of the presence of the analyte in the sample (for this, a red laser is used, classifying microspheres by color), and a quantitative assessment of the content of the analyte in the sample (for this, a green laser is used).

The earlier and more accurately syphilis is detected, the simpler the treatment and the higher the likelihood that it will go smoothly for the patient.

The goal for all laboratory tests is the same: to make a diagnosis unambiguously and quickly. But none of the modern high-tech tests for syphilis gives the result unambiguously and with 100% accuracy. Old methods are being improved, new ones are being invented, but still in clinical practice, doctors always have to use a combination of several different tests for syphilis. Doctors cannot rely on the result of any one thing.

There are so many types of tests for syphilis that it is impossible to understand all the abbreviations right away:

The disease was first identified using a laboratory reaction in 1906. This is the merit of the German scientist August Wasserman, after whom the reaction is named. Much time has passed since then, the method is outdated and is not used in practice, but the diagnosis of syphilis is still firmly associated with the analysis of RV.

A person may need to undergo testing for syphilis for a variety of reasons.
The very first reason that comes to mind is when you suspect an infection, and in practice it is not the most common. In this case, it is important to understand that the infection has an incubation period (from the moment of infection until the formation of chancroid) and a primary seronegative period (chancroid in the first three weeks) - during this time the tests will be negative. Therefore, if the concerns are serious, the tests are repeated after a few weeks.

More often, people who do not suspect any infection need to be tested for syphilis. This usually happens when applying for a job (the analysis is included in the medical record) and during periodic medical examinations (medical examinations). It is also mandatory to donate blood for syphilis:

• donors,
• women in the first weeks of pregnancy - twice, when registered at the antenatal clinic and in the maternity hospital a few weeks before birth,
• patients before surgery or any other medical invasive procedures ( FGDS, bronchoscopy, etc.).

At the end of the article we answered the most FAQ people who have been diagnosed with syphilis. There is no time to read details about research methods -.

All types of tests for syphilis

There are 2 main groups of research methods for syphilis: direct and indirect.

• Direct method- this is a study in which the infection itself is looked for in the biomaterial - individual representatives of the pathogen as a whole, or their pieces - DNA.
• Indirect methods(serological reactions) is a test in which they try to detect antibodies to the causative agent of syphilis in the blood. The logic is this: if an immune response is found that is characteristic of some kind of infection, it means that there is an infection itself that caused this immune response.

Direct methods- the most reliable: if the bacterium is “caught red-handed”, then the presence of the disease is considered proven. But Treponema pallidum is difficult to catch, and negative test results do not rule out the presence of infection. It makes sense to carry out these studies only in the presence of rashes and only when early form syphilis - up to two years of illness. Those. It is impossible to determine latent syphilis or its late forms using these methods, therefore in clinical practice they are rarely used and only to confirm other tests.

Direct methods include: Dark-field microscopy, Infection of laboratory animals, PCR .

1. Dark field microscopy ( TPM) - examination of Treponema pallidum under a microscope. The material is taken from chancre or rashes. The method is cheap and fast, and detects syphilis at the very beginning of the primary period, when blood tests for syphilis are still negative. But bacteria, which are present in small quantities in the rash, may easily not be captured by scraping. Plus, Treponema pallidum can be easily confused with other inhabitants of the oral cavity, anal canal, etc.
2. Infecting laboratory animals is a very expensive and painstaking method, used only in research practice.
3. PCR- a relatively new method, they are looking for DNA infections. Any tissue or liquid that may contain treponema pallidum is suitable for research: blood, urine, prostate secretions, ejaculate, scrapings from skin rashes, from the genitourinary tract, oropharynx or conjunctiva. The analysis is very sensitive and specific. But complicated and expensive. It is prescribed in case of questionable results of other tests.

Indirect methods, also known as serological reactions, are the basis for laboratory testing of syphilis. It is these methods that are used for mass screening of the population, for confirming diagnosis and monitoring treatment. Indirect research methods are divided into non-treponemal and treponemal tests.

Non-treponemal tests are noticeably cheaper. To carry them out, they do not use the antigen protein itself, which is specific for syphilitic treponema, but its replacement, the cardiolipin antigen. These tests are highly sensitive but poorly specific. This means that such tests will identify everyone who has syphilis and even more: healthy people can also have false positive results. They are used for mass screening of the population, but in case of a positive result they necessarily require confirmation by more specific tests - treponemal tests. Non-treponemal tests are also very useful in assessing the effectiveness of treatment: when effective treatment the volume of antibodies in the blood decreases, and their titer decreases accordingly (we’ll talk about these titers in more detail a little further). The most reliable result of these non-treponemal tests will be during early syphilis, especially in the secondary period.

Non-treponemal tests include:

• Wasserman reaction( , she is RV, or RSK) - is already outdated and not in use, but due to its strong association with the disease, this is often the name given to any test for screening the population for syphilis. If you see the note “analysis” in the doctor’s referral RV" - don’t be embarrassed, in the laboratory they will probably understand everything correctly and will do it RPR.
• Microprecipitation reaction (MR, she's the same RMP) is a simple and cheap test to detect syphilis. Previously used as the main non-treponemal test, but has now given way to a more convenient and objective RPR-test.
• Rapid plasmareagin test (RPR-test) is a quick, simple and convenient test for mass screening of the population and treatment monitoring. It is the main non-treponemal test used in Russia and abroad.
• TRUST - this is a more modern modification RPR-test. It is otherwise designated as RPR-test with toludine red. In Russia it is used only in a small number of laboratories.
• VDRL - this analysis is similar in reliability of results RMP, and is also inferior RPR. It has not found widespread use in Russia.
• USR-test(or its modification - RST-test) - more advanced VDRL test, however in Russia it is also used extremely rarely.

Treponemal tests carried out with treponemal antigens. They are more specific, and therefore more carefully screen out the healthy from the sick. But their sensitivity is lower, and such tests may miss a sick person, especially at an early stage of the disease. Another feature is that treponemal tests appear later than non-treponemal tests, only three to four weeks after the appearance of chancre. Therefore, they cannot be used as screening tests. The main purpose of treponemal tests is to confirm or refute the results of non-treponemal tests.

Also, the results of treponemal tests will remain positive for several years after successful treatment. Because of this, they are not used to monitor the effectiveness of treatment, and also do not rely on the results of these tests unless they are confirmed by non-treponemal tests.

Treponemal tests include:

• RPGA (or its more modern modification - TRPA,TRNA) - passive hemagglutination reaction. The main treponemal reaction now used abroad and in Russia. A simple and convenient test to determine syphilis antibodies in the body.
• ELISA (anti-Tr. pallidum IgG/IgM) - enzyme immunoassay, also known as ELISA from the English abbreviation. This test can be performed with both cardiolipin antigen and treponemal antigen. It can be used as both screening and confirmatory. In terms of reliability, it is not inferior RPGA and is also the recommended treponemal test to confirm the diagnosis of syphilis.
• Immunoblotting- this is a more expensive advanced ELISA-test. Used only in doubtful cases.
• REEF — immunofluorescence reaction. Technically difficult and expensive analysis. It is secondary and is used to confirm the diagnosis in doubtful cases.
• RIBT (RIT) - reaction of immobilization (immobilization) of Treponema pallidum. This reaction is complex, time consuming and difficult to interpret the result. It is still used in some places, but is gradually fading into the background, giving way to RPGA And ELISA.

Interpretation of serological tests for syphilis:

Algorithm for diagnosing syphilis

Any diagnosis consists of three main pillars of medicine: anamnesis (medical history), clinical manifestations(symptoms) and laboratory examination. If the doctor, based on the patient’s story and an external examination of his body, suspects syphilis, he prescribes a set of tests (or a set of serological reactions - KSR). It must include 1 non-treponemal test ( RMP or RPR) and 1 treponemal test ( RPGA or ELISA). If the results of these tests diverge, an additional alternative treponemal test is performed ( ELISA or RPGA). This is the simplest scheme. In case of doubtful indicators, depending on the situation, the doctor prescribes other diagnostic methods.

Express test for syphilis, or how to determine syphilis at home

There is a test for syphilis that you can do yourself. It can be freely purchased at a pharmacy, the average cost is 200-300 rubles. The principle of determining the disease is similar to non-treponemal RPR. Manufacturers claim high accuracy, but in reality it is low, no more than 70%.

The algorithm for checking is similar to a pregnancy test, only blood is used instead of urine. A drop of blood is applied to the indicator, and within 10-15 minutes the result appears. 1 strip - negative test, 2 strips - positive test.
We do not recommend this diagnostic method. If you have any doubts about syphilis, it is better to immediately consult a doctor, or at least an independent laboratory. It will be a little more expensive and longer, but much more accurate.

Decoding the results for syphilis: pluses, crosses and credits.

The doctor’s further tactics depend on the results of certain tests. The results of screening tests are expressed either in crosses (pluses) or in a separate entry:

4 or 3 crosses - a positive result; further examination for syphilis is necessary using other diagnostic methods.
2 or 1 cross is a questionable result; it is recommended to repeat the result after 10 days.
0 crosses - negative result, syphilis was not detected.

In case of a positive and questionable reaction, additional research taken blood: diluting it from 1:2 to 1:1024 and adding a drop of cardiolipin antigen to each blood titer. The result records the maximum titer at which the reaction occurred: the greater the dilution, the more value titer, the higher the amount of Treponema pallidum in the blood. But the main task of determining the titer is not to calculate the degree of blood contamination, but to monitor the success of treatment: treatment is considered effective if the titer drops 4 times in 4 months. By the end of treatment, nontreponemal tests should be negative.

The highest sensitivity of screening tests is observed in the secondary period of syphilis (100%), slightly less in the primary (86%) and even less in the tertiary (73%).

Important nuances in diagnosing syphilis:

1. When conducting tests, false positive results are possible. They are especially common during screenings. If you have never had syphilis and the tests are positive, you shouldn’t panic right away; you need to do at least one more alternative test.
2. False negative results also occur. If syphilis is suspected, it is better to repeat the test after a few weeks.
3. Cured syphilis remains positive on treponemal tests for several years or life.

The most frequently asked questions about syphilis tests

How to get tested for syphilis for free?

To do this, you need to go to the clinic at your place of residence and visit your local doctor, who will give you a referral for testing. Testing for syphilis is free for all residents RF according to the policy Compulsory medical insurance.

Where can I get tested for syphilis anonymously?

Tests can be done anonymously in any paid laboratory; skin and venous dispensaries often offer this service themselves. It is also possible to test for syphilis at home using rapid tests that are sold in pharmacies. However, it must be remembered that such a test does not give an accurate result, and if you suspect syphilis, you should consult a doctor.

How many days after sexual intercourse can you donate blood for syphilis?

In 1-1.5 months. If infection occurs, the test for syphilis will be positive no earlier than seven to ten days after the appearance of chancroid, or 4-5 weeks after infection. This period may be longer, so if the results are negative, the test should be repeated after 2 weeks.

Where is blood taken for syphilis?

Blood for syphilis is most often taken from a vein, but can also be taken from a finger. It depends on the type of analysis.

Preparation. How to get tested for syphilis?

Before donating blood for syphilis, you should not eat for four hours—blood must be donated on an empty stomach. In addition, you should not drink alcohol 12 hours before the test. This is important because liver damage from alcohol can cause false positive tests.

How long does it take to test for syphilis on average?

The results are usually known the next day. Conducting rapid tests takes no more than 30 minutes.

What test is taken for syphilis and what is it called?

For screening, when there is no suspicion of the disease, either RMP(microprecipitation reaction), or RPR(rapid plasmareagin test). Sometimes such screening tests are called the Wasserman test.

If there are any real suspicions or doubts, they are never limited to one analysis. Simultaneously perform one of any of the screening group ( RMP or RPR) and one of any of the more specific test group ( RPGA or ELISA), then act depending on the results and history of the patient.

Can a syphilis test be wrong?

Maybe! Probability of error different methods depends primarily on the period of illness and the general condition of the body.

Non-treponemal tests are most sensitive at the height of the disease - in the secondary period. Due to their low specificity, they often give false-positive results. This may happen due to fever, flu or other infectious disease, recent vaccination, chronic diseases and a number of other reasons.

Treponemal tests are more sensitive in the later period. They can also give false-positive results, but only if there are pathogenic bacteria similar to Treponema pallidum in the body that cause other diseases: non-venereal treponematoses pinta (rare in Russia) or Lyme disease (transmitted through a tick bite).

False negative test results are possible with all diagnostic methods. They depend on the body's immune response: no response - no reaction to syphilis. This is possible in people infected with HIV, as well as those who are immunocompromised for other reasons. In addition, there is a reverse reaction: hyperproduction of antibodies, the “prozone” effect, in which there are so many antibodies that they prevent each other from reacting with the antigen. The result is a false negative result.

Can general tests show syphilis?

Syphilis cannot be determined either by a general blood test or by a biochemical test. Won't show it general analysis urine or a regular vaginal swab. All tests for syphilis are highly specialized and each has its own name. Using any other tests, it is impossible to calculate whether a person has syphilis or not. But what will other tests show if a person has syphilis? Let's look at each of them:

Complete blood count: shows the main blood cells - red blood cells, white blood cells, platelets. At the end of the primary and at the beginning of the secondary period, a person’s leukocytes may rise and also increase ESR- an indicator of inflammation. These are very non-specific indicators that simply indicate that the body is fighting against bacterial infection. Otherwise, the blood test will correspond to the general condition of the body.

Biochemical blood test: shows the functioning of the liver, kidneys, heart, pancreas and other organs. If syphilis has not yet affected these organs, and they are working properly, the blood test will be normal.

General urine test: shows the functioning of the kidneys and endocrine system, as well as general state body. If acute or chronic diseases these systems are not present - the analysis will be normal.

Vaginal smear: determines whether there is an inflammatory or oncological process, as well as dysbiosis. It is impossible to diagnose syphilis using such a smear.

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Summary. Last place of work: Federal State Institution of Science "Central Research Institute of Epidemiology" of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare. Institute complex problems restoration of human reserve capabilities. ACADEMY OF FAMILY AND PARENTAL CULTURE “CHILDREN’S WORLD” Within the framework of the national program for demographic development of Russia SCHOOL FOR FUTURE PARENTS “COMMUNICATION BEFORE BIRTH” Job title: Senior Researcher. Obstetrician-gynecologist, infectious disease specialist. Education 1988-1995 Moscow Medical Dental Institute named after. Semashko, majoring in general medicine (diploma EV No. 362251) 1995-1997 clinical residency at MMSI named after. Semashko in the specialty “obstetrics and gynecology” with an “excellent” rating. 1995 “Ultrasound diagnostics in obstetrics and gynecology” RMAPO. 2000 “Lasers in clinical medicine” RMAPO. 2000 “Viral and bacterial diseases outside and during pregnancy” NTSAGi P RAMS. 2001 “Breast diseases in the practice of an obstetrician-gynecologist” NCAG and P RAMS. 2001 “Basics of colposcopy. Pathology of the cervix. Modern methods of treatment benign diseases cervix" NCAG and P RAMS. 2002 “HIV – infection and viral hepatitis” RMAPO. 2003 “candidate minimum” exams in the specialty “obstetrics and gynecology” and “infectious diseases”.

Question: Hello, in 2014 I tested for syphilis (anti-Treponema pallidum Ig M - negative; anti-Treponema pallidum total Ig M+ Ig G half. KP 11.2 Titer 1:640; RMP - positive +++ Titer 1:8 ). I took tests in an independent laboratory. With these tests I came to see a venereologist, after several confirmatory tests, a diagnosis was made: “early latent syphilis”, because I did not and do not have any external signs of illness. She underwent inpatient treatment (penicillin intramuscularly for 20 days (kd 80 million units); test data after discharge: rmp_3+;ifa IgM (+)KP=1.4.IgG(+) KP=5.5.rpga 4+;rif abs 3+;rif200 2+;hiv hepatitis -negative smears for STIs -no pathology. Currently I am on serocontrol, because the latest tests gave a weakly positive result (I took an EDS in December 2015) before that, for six months the results were negative and They already wanted to deregister me, the doctor recommended that I come back for tests in 6 months: in June. Wanting to find out if my tests had changed for the better, I took them to an independent laboratory in April 2016 and here is the result: IgM negative, total IgM + IgG positive. CP = 24.939 Titer 1:1280, RMP positive +++ Titer 1:32. Please explain why the results are so bad. They have become much worse than before treatment. Re-infection is excluded: I have not had sexual intercourse since the discovery of the disease.

Doctor's answer: Hello! You need to contact your doctor. Retake the analysis at the KVD.

Medical services in Moscow:

Question: Hello! Help me decipher the CSR analysis: anti-treponema pallidum IgM negative, anti-treponema pallidum total IgM+IgG positive KP 11.2 Titer 1:640, siphilis EDS(RMP) positive +++ Titer 1:8. What does it mean?

Doctor's answer: Hello! You are sick with syphilis, you need to see a dermatovenerologist.

Question: Hello! Tell me, please, in 2010. After donating blood, as a donor, I received weakly positive results for specific syphilis tests (RIF, DAC). RW and MCI remained consistently negative. My husband's tests are all negative. I haven't been sick before. The tests were repeated 6 times (from July to November) - the test results did not change. She was treated in a hospital (21 days, 8 penicillin injections per day intramuscularly).

2011 - RW and MCI for me and my husband are negative.

2012 - I get pregnant, RW and MCI are negative. My husband does too. Professional treatment is not prescribed. In December 2012 I give birth.

The child in the maternity hospital has the results of a blood test (BCT), narrow specialists, X-ray - negative.

2 weeks after giving birth, I take tests from a dermatologist to deregister - RW and MCI - negative, RIF, CSR - weakly positive. Retake tests in March.

At the age of 43 days, the child underwent an MCI test - the result was negative. They didn’t take blood for specific tests because they couldn’t find a crown, so they postponed it until March.

KozhVenDispanser insists on prophylactic treatment for the child; my husband and I wrote a refusal to undergo prophylactic treatment.

You DO NOT want to inject a one and a half month old baby with antibiotics “just in case.”

Please tell me what caused these test results for me?

What is the probability that the child is really sick?

How often will a child have to donate blood and up to what age?

What test results are decisive for deregistration?

When my son goes to kindergarten, will the nurse be aware of my diagnosis and suspicion of the child’s illness? We're not even talking about medical confidentiality...

How to explain to the staff of the children's clinic that the child POSSIBLY has congenital syphilis and what to shout in the corridor when there is a line of parents with children all around: “Oh, it’s you with syphilis, go ahead, now we’ll take a test!” - at least not correctly (here’s a medical secret for you!!!).

How to explain to a doctor in a medical center that the health of a child is, first of all, OUR, PARENTAL, care and responsibility. If, when trying to discuss the POSSIBLE course of the disease and POSSIBLE treatment, the phrases are heard: “Are you a doctor? What difference does it make to you, you still won’t understand! So you refuse to inject him, and he won’t live to see three years with you, mark my words. On the Internet Have you read it? Well, maybe you’ll treat it yourself?”

Thanks in advance for your answer.

Julia

Doctor's answer: Hello! It is difficult to recommend anything in your case. The child needs to be treated only if the tests are positive; they need to be taken every 6 months. Write a complaint to the chief physician or the Ministry of Health against doctors who do not observe medical confidentiality. Take a voice recorder and record all statements addressed to you. You can use the help of a lawyer... Don’t give up, fight for your rights, although in Ukraine it is more difficult than in Russia.

Question: Hello! Pregnancy 23 weeks. Please tell me whether I need to undergo preventive treatment if the following results (three times) were tested for syphilis: CSR A1-, A2-, Microreaction - negative, RGPA3+, RIF3+, ELISA M - not detected, G 3. Never received treatment , third pregnancy. One sexual partner (husband) for 12 years. All his tests are negative. Thank you

Doctor's answer: Hello! There is no need to undergo treatment. Observation required.

Question: I had syphilis 3 years ago. Now I have tested TPHA positive 1:320, IgM negative, IgG 173., RPR positive. Do I need to undergo treatment again? Thanks in advance for your answer

Doctor's answer: Hello. Detection of IgG indicates the presence of immunity to this disease. The activity of the process is indicated by the detection of IgM. Diagnosis of latent syphilis: microprecipitation reaction (MPR) or its analogue RPR/RPR in qualitative and quantitative versions (non-treponemal tests), and in case of a positive result, confirmation in two treponemal tests simultaneously from the following: RPGA, ELISA, RIF, DAC, RIT (before 2006). “Lewis RPR test”, “Lewis RPGA test”, “ICE Syphilis”, “INNO LIA Syphilis Score”. The issue of treatment is decided during a face-to-face consultation. You should consult a venereologist.

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Serological blood tests are extremely important. Currently, a complex of serological reactions is used - usually the classic Wasserman reaction and two sediment reactions are used. Of the sediment reactions, the Kahn, Sachs-Vitebsky and cytocholic reactions are most often used.

The results of serological studies are designated by pluses as follows: + + + + strongly positive, + + + positive, + + and + weakly positive; a negative reaction is indicated by a minus (-).

In addition to the indicated complex of serological reactions, in recent years the immobilization test of Treponema pallidum (Nelson-Mayer test), which is carried out with specific antigen(suspension of Treponema pallidum). This reaction is more specific than the mentioned complex of reactions that are carried out with nonspecific antigens. The Nelson-Mayer test is of particular value in deciding the specificity of the positive Wasserman reaction and sediment reactions in individuals who do not have symptoms of syphilis.

Serological reactions during different periods of syphilis. The diagnostic value of serological reactions in different periods of syphilis is not the same. In the primary period - within 2-3 weeks after the onset of primary syphiloma - serological reactions are usually negative; starting from the 3-4th week they become positive in some patients. As the disease approaches the secondary period, the percentage of positive serological reactions increases, and by the time the disease appears secondary symptoms syphilis they become positive in almost 100% of patients.

In the fresh secondary and recurrent periods of the disease, serological reactions in the blood remain positive in almost 100% of patients.

In the tertiary period of syphilis with active manifestations of the disease, serological reactions can often, in about 15% of patients, be negative, and in the absence of active symptoms of the disease, the number of negative reactions can reach 30-40%.

Serological reactions as a criterion for successful treatment of syphilis. Specific treatment of syphilis must be carried out under the control of serological reactions. Usually, in patients with primary seropositive and secondary fresh periods, serological reactions become negative after 1-2 months of rational treatment of syphilis; in the secondary relapse period they may become negative at the same time or slightly later. If serological reactions do not become negative within the specified time frame, this indicates insufficient effectiveness of treatment. In such cases, it is necessary to increase the effectiveness of therapy, but not by increasing the dosage of drugs, but by changing the treatment method and adding methods of nonspecific therapy.

Strongly positive serological reactions in the absence of clinical symptoms can be considered a symptom of syphilis, provided that they remain strongly positive upon repeated testing and that the study was carried out in a qualified serological laboratory.

Weakly positive serological reactions in the absence of clinical symptoms and other data cannot serve as support for the diagnosis of syphilis; such results should be taken into account only in cases where the patient had syphilis in the past and was treated accordingly. Weakly positive serological reactions may also be important if the patient has a disease of unknown etiology. In these cases, the results of serological reactions must be assessed taking into account other anamnestic, clinical and laboratory data.

Nonspecific serological reactions can be observed in many diseases. Thus, a sharply positive result often occurs with leprosy and relapsing fever. In malaria, tuberculosis, brucellosis and many other infectious diseases, as well as in patients with malignant tumors, the results are usually weakly positive or positive (+ + +) and are rarely strongly positive (+ + + +).

Use before the study alcoholic drinks or fatty foods may cause a nonspecific, weakly positive reaction. Blood for serological studies should be taken on an empty stomach.

In the absence of a serological laboratory, it is recommended to send blood for testing by mail in an envelope in the form of a “dry drop”.

To obtain a dry drop, 5 ml of blood from the cubital vein is drawn into a test tube. The tube with blood is left for 2 hours at room temperature. The resulting blood clot is separated from the walls of the test tube with a calcined and cooled wire, then the blood is placed in a cool place until the next morning. The next day, use a graduated pipette to draw 0.75 ml of serum, which is applied to wax paper or a strip of cellophane. The serum on paper or cellophane is left until the next day at room temperature, protecting it from dust and flies. After this, wax paper or cellophane with the dried serum is rolled up in the form of a pharmaceutical powder, placed in an envelope and, along with an accompanying note, sent by mail to the nearest serological laboratory.

It should be noted that the method of serological reactions using a “dried drop” of blood is less reliable than the usual one.

Diagnosis of syphilis is based on clinical and laboratory data. Among the latter, serological studies are extremely valuable, which are carried out not only to confirm the diagnosis of syphilis, but also to monitor its dynamics under the influence of the therapy.

Serology of syphilis today represents separate area knowledge. Created various reactions, received in different countries state recognition. It is important that the results obtained are correctly interpreted, which can only be achieved with knowledge of the basics of serology.

In our country, for the serological diagnosis of syphilis, in accordance with the methodological recommendations of the Ministry of Health, the following is used:

Precipitation microreaction (MR) with cardiolipin antigen is a screening test for screening the population for syphilis;

The reagin plasma test (RPR), also being non-treponemal, is used as a screening test;

A complex of serological reactions (CSR), which includes the complement fixation reaction (CFR) with treponemal and cardiolipin antigens and MR;

Treponema pallidum immobilization reaction (TRI), in which pathogenic Treponema pallidum of the Nichols strain is used as an antigen;

Immunofluorescence reaction (RIF) (in modifications: RIF-abs, RIF-c and RIF with capillary blood from a finger); pathogenic Treponema pallidum of the Nichols strain is used as an antigen in RIF;

Passive hemagglutination reaction (RPHA) with antigen from cultural or pathogenic Treponema pallidum;

Enzyme-linked immunosorbent assay (ELISA) with antigen from cultural or pathogenic Treponema pallidum.

All of these reactions have different sensitivity and specificity and are recommended for use depending on the task at hand.

During mass preventive examinations of the population for syphilis, MR is used with blood plasma or inactivated blood serum of the subjects. The reaction results are evaluated -

qualitatively as 4+, 3+, 2+ and negative. The advantage of the express method is the speed of obtaining an answer (in 30-40 minutes), the small volume of blood required for the test (2-3 drops), which can be taken from patients from a finger.

Using this express method, individuals who are subject to periodic examinations are examined. medical examinations for sexually transmitted diseases, patients in somatic hospitals, persons placed in special detention centers. If the express method is used in isolation, it is only a screening test. Based on its positive result, a diagnosis of syphilis is not made, and the subjects are referred to a dermatovenerologist for further clinical examination and examination of their blood using any of the other diagnostic tests (DSR, RIBT, RPGA, ELISA or RIF). The express method is not used in pregnant women or donors due to the fact that it often gives false positive results. Average medical workers who have undergone special training, take blood from a finger and perform an MRI. Laboratory doctors are required to take its results into account.

RSCs with treponemal and cardiolipin antigens are used to confirm the diagnosis of syphilis in the presence of active manifestations of the disease, to examine persons who have had sexual contact with a patient with syphilis, to identify latent (latent) syphilis, the effectiveness of therapy, when examining patients in psychiatric and neurological hospitals, donors and pregnant women, including persons referred for abortion.

Blood for research is taken in an amount of 5-7 ml from the ulnar vein with a sterile needle, observing the rules of asepsis. U infants blood can be obtained from the temporal vein or from incisions in the heel. Blood is taken strictly on an empty stomach (5-6 hours after eating) and left in clean, dry tubes for 2-3 hours at room temperature to clot. Testing of DCS and specific reactions is carried out in serological laboratories of skin and venereological institutions, and in rural areas - in laboratories of rural district hospitals.

The dry drop method is very convenient for sending blood for testing to distant laboratories. To do this, the next day after blood collection, the serum is separated from the clot. Using a graduated pipette, take 1 ml of serum and pour it in the form of two separate circles onto a strip of thick paper (waxed or cellophane) measuring approximately 6x8 cm. On the free edge of the paper, write the patient’s last name, first name and patronymic, the date of blood collection and the serial number. The serum is left on paper, protected from direct sunlight and dust.

for 24 hours at room temperature to dry. After this, strips of paper with dried serum are rolled up and sent to the laboratory.

RSC in CSR with cardiolipin antigen is not very sensitive and becomes positive 2-4 weeks after the onset of chancre, and the reagin titer gradually increases and reaches a maximum (1:160-1:320 and above) with secondary fresh syphilis. Then the reagin titer gradually falls and in case of secondary recurrent syphilis it usually does not exceed 1:80-1:20. In patients with tertiary syphilis, these reactions give a positive result only in 70% of cases.

It should be emphasized that CSR is not strictly specific for syphilis and in some cases can give false-positive (nonspecific) results. Such false-positive reactions are observed in patients with leprosy, malaria, sometimes with an autoimmune disease, neoplasms, pneumonia, tuberculosis, liver diseases, when taking medications (sulfonamides, glucosides, valerian, etc.), as well as during pregnancy, menstruation, etc. d. When taking blood for testing for CSR, it is necessary to warn the subject so that 2-3 days before he does not drink alcohol, fatty foods, or take medications. It is not recommended to test blood within the 1st week after vaccination, injury, surgery, during febrile conditions, during the first 2 weeks after birth, in newborns in the first 10 days of life, since physicochemical changes in the blood serum in these conditions may be similar to those observed in patients with syphilis.

False-positive results can also be obtained due to technical errors (incomplete hemolysis, unsterile blood collection, insufficient qualifications of laboratory technicians).

To differentiate false-positive CSR results from true ones, to diagnose latent and late forms of syphilis, if a syphilitic infection is suspected, to establish a retrospective diagnosis of the disease, it is recommended to use specific serological tests (RIBT, RPGA, ELISA or RIF)

To carry out specific serological reactions, blood in an amount of 5-10 ml is also taken from the antecubital vein on an empty stomach. Blood is poured into a dry test tube for testing for RIF and into a sterile test tube for testing for RIBT. Specific serological reactions to syphilis are performed in specialized laboratories of dermatovenerological institutions.

RIF is based on an indirect method for determining fluorescent antibodies. The antigen in this reaction is a suspension of killed cultural Treponema pallidum, fixed to glass slides, onto which the test and anti-species fluorescent serum are applied. The results of RIF are determined under a fluorescent microscope by assessing the glow of the treponemes in the preparation. If the result is positive, the treponemes have a yellowish-green glow, the degree of which is indicated by pluses from 1 to 4; with negative results, the treponemes do not glow.

Another specific reaction to syphilis - RIBT - is based on the phenomenon of immobilization of Treponema pallidum by antigens of the patient's blood serum in the presence of complement. A suspension of live Treponema pallidum obtained from rabbits infected with syphilis is used as an antigen for RIBT. Counting of treponemes that have lost mobility (immobilized) is carried out under a microscope. The results of the reaction are assessed as percentages: from 0 to 20% - negative, from 21 to 30% - doubtful, from 31 to 50% - weakly positive, from 51 to 100% - positive. RIBT becomes positive at the end of the primary period of syphilis and remains so during all periods of this disease, and sometimes even after full anti-syphilitic treatment. In tertiary syphilis, specific lesions of internal organs, the nervous system, and congenital syphilis, when the CSR is often negative, RIBT gives positive results in 98-100% of cases. The diagnosis of latent syphilis must be confirmed by a positive RIBT.

RIBT can also give false-positive results if the test serum contains treponemocidal substances (antibiotics - penicillin, tetracycline) that cause nonspecific (toxic) immobilization of Treponema pallidum. Therefore, you cannot test your blood for this reaction earlier than 2 weeks after you stop taking antibiotics.

Serological studies can also be used to determine damage to the nervous system by syphilis by examining the cerebrospinal fluid of the patient. It is also examined for the presence of protein and enzyme elements, which indicate pathology and help diagnose one or another form of neurosyphilis. Cerebrospinal fluid is obtained by lumbar puncture. Performed aseptically, it does not pose any danger and can be performed by a doctor even on an outpatient basis. Serological studies cerebrospinal fluid indicated in all cases of syphilis