PIF method for chlamydia. The study of the reliability of the results of laboratory studies using PIF and PCR methods in the diagnosis of urogenital chlamydial infection

INTRODUCTION

Chlamydia (Chlamydia) - small gram-negative coccoid bacteria 250-1500 nm in size (0.25-1 microns). They have all the main features of bacteria: they contain two types of nucleic acids (DNA and RNA), ribosomes, muramic acid (a component of the cell wall of gram-negative bacteria), multiply by binary fission and are sensitive to some antibiotics. Family Chlamydiaceae (included in Chlamydiales order) is obligate intracellular bacteria that have two forms of life (elementary and reticular bodies), a similar two-phase development cycle (consisting of alternation of various forms - an elementary body and a reticular body), have a tendency to persistence (or to latent (hidden) existence).

According to the new international classification There are four families and 5 genera of Chlamydia. In each genus, from one to six species are represented, differing from each other in a number of phenotypic characters. Each species not only has its own place in the classification, but also has its own pathogenic potencies that require a special therapeutic approach.

Consider the most significant (pathogenic) types of chlamydia for humans(without diving into this subtle and complex classification). (one) Chlamydia psittaci: causes atypical pneumonia, encephalomyocarditis, arthritis, pyelonephritis, encephalomyocarditis in humans (this chlamydia is an absolute zoonosis, that is, the infection is transmitted to humans from an animal; transmission routes are airborne and airborne). (2) Chlamydia pneumonia(absolute anthroponosis - transmitted to a person exclusively from a sick or infected person, human infection occurs by airborne droplets and airborne dust): causes acute respiratory diseases in adults, in particular bronchitis and a mild form of pneumonia, tending to chronicize the process. (3) Chlamydia trachomatis(it contains 18 antigenic serotypes): occurs only in humans and causes a wide range of diseases, including urogenital diseases, conjunctivitis, some forms of arthritis (chlamydia serotypes that cause damage to the urogenital tract are transmitted from person to person through sexual contact).

The characteristic of chlamydia is the fact that they develop only in a special type of epithelium, in the so-called prismatic (cylindrical) epithelium, therefore, organs covered by this epithelium are affected. Chlamydia pneumonia and Chlamydia psittaci, affecting the epithelium of the respiratory tract, primarily the bronchi, cause chronic, less often acute, bronchitis and pneumonia (“pneumonia”). Chlamydia trachomatis, damaging the mucous membrane of the urethra, cervical canal, fallopian tubes, leads not only to acute inflammatory processes, but also to chronicity with subsequent complications, the most formidable of which is infertility

Numerous clinical and epidemiological studies indicate a widespread chlamydial infection. Today, chlamydial infection has firmly taken second place after pneumococcus in the spectrum of causative agents of pneumonia, which often tend to be chronic and severe with fatal outcomes. 50-80% of cases of reproductive dysfunction in women are due to infectious lesions, among which chlamydial infections are the most common.

Clinical diagnosis of chlamydia is difficult prolonged asymptomatic course, uncharacteristic clinical manifestations, inefficiency of immunochemical and serological methods of analysis, high cost, duration and laboriousness of the microbiological method.

TECHNIQUE FOR COLLECTION OF MATERIAL

Before considering the methods of laboratory diagnosis of chlamydial infection, it is necessary to consider the technique of taking the material, since this procedure is one of the most important stages in the diagnosis of chlamydia - the stage of sampling.

In the study of chlamydia by the method of infection of cell culture before taking the material, patients should not take tetracycline antibiotics for a month; with a cytological technique, including the use of monoclonal fluorescent antibodies, antibiotics should not be used two weeks before the study. Before taking material from the urethra, patients should not urinate for 1-1.5 hours.

When taking material for chlamydia, remember that optimal for the presence and reproduction of chlamydia are certain areas of the cylindrical epithelium of the genitourinary tract (anterior urethra at a depth of 2.5-4 cm in men, the mucous membrane of the cervical canal of the uterus at a depth of 1.5 cm in women). When taking material from the cervix, the key point is the removal of the mucous plug. The correctness of the scraping of the cells of the cervical canal largely depends on the thoroughness of this preparatory procedure. The mucous plug is removed with a cotton swab and tweezers, and then the material is taken with a special cervex brush or voba-brush, which has a number of advantages, since the presence of cells from the entire surface of the cervical canal, the transformation zone, is important to obtain a representative result. Sufficiently informative is the study in men of the sediment of the first portion of morning urine by the polymerase chain reaction (PCR).

Collection of material from girls produced from the mucous membrane of the vestibule of the vagina, in some cases - from the posterior fornix of the vagina through the hymenal rings. The object of research for the presence of chlamydia can be swabs from the mucous membrane of the eyes, nasopharynx, sputum, bronchoalveolar swab.

LABORATORY DIAGNOSIS

Cytoscopic methods. With the cytoscopic method, simultaneously with the search for cytoplasmic Halbershedter-Prowachek inclusion cells, the number of leukocytes is taken into account as an indicator of inflammation, as well as Additional Information about the presence of concomitant bacterial microflora, yeast-like fungi, Trichomonas, etc. The cytoscopic method is widely available, but is effective only in acute forms of infection, much less effective and informative in chronic forms diseases.

Immunomorphological methods. These methods are based on the detection of antigenic substances of chlamydia in the epithelium and other tissues by treating preparations with antibodies. The antibodies of the diagnostic antichlamydial serum are connected to any label - luminescent (FITC-antibodies) or enzymatic (enzyme-labeled antibodies).

Direct immunofluorescence(PIF). This method involves the direct detection of chlamydia antigens. With fluorescent microscopy, chlamydial inclusions are determined as formations in the epithelial cell with green or yellow-green fluorescence against a brown-orange background of the cell cytoplasm. Inclusions can have a granular, homogeneous or mixed structure. The diagnostic information content of the PIF is due to the fact that with its help not only corpuscular, but also soluble antigens of chlamydia are detected.

Indirect immunofluorescence method. The indirect method of immunofluorescence is used in cases where there is no FITC-conjugate of anti-chlamydial antibodies. In these cases, a preparation prepared from clinical samples prepared in the same way as for PIF is first treated with anti-chlamydia antibodies obtained by chlamydia immunization of sheep, rabbits, mice or other animals, and then with a second serum specific to the animal species that was immunized with chlamydia. Antibodies of the second serum are conjugated with FITC.

ELISA methods. These methods are based on the detection of soluble chlamydia antigen in test samples. Most often in clinical practice, reagent kits based on the method of solid-phase ELISA analysis are used to determine chlamydia antigens. The solid phase is coated with chlamydial monoclonal antibodies of established specificity. Amplification is achieved using polymer conjugation technology, resulting in fixation, in which each bound site of the antigen has a polymer complex with a high molecular weight fragment. In addition, amplification is performed at a labeled reproduction step using proprietary enzyme-amplification technology. Visually positive samples are stained yellow-orange. The color intensity is proportional to the amount of chlamydia antigen. The exact result of the study is determined using special devices for ELISA.

Chlamydia isolation in McCoy cell culture. One of the best, but at the same time the most time-consuming, is the method of diagnosing chlamydia by isolating the pathogen in a cell culture treated with various antimetabolites (the “gold standard”). For this purpose, a sensitive cell culture treated with cycloheximide is usually used. The sensitivity of the cultural method compared to PCR is 70-80%, but at the same time it surpasses molecular biological diagnostic methods in specificity.

The culture method is a reference method in evaluating the effectiveness of antibacterial treatment. In the study of bioassays by PCR after a course of chemotherapy, in some cases, "false positive from a clinical point of view" results can be obtained. This is due to the fact that it is impossible to unambiguously assess the viability and pathogenicity of a microbial cell based on the detection of a fragment of its genome, using only the data of molecular biological methods. In this case, when examining clinical material using culture seeding, microbial cells that have lost these clinically important properties will not grow in cell culture.

Diagnosis by polymerase chain reaction(PCR). Extremely high rates of sensitivity and specificity of PCR make this technique largely revolutionary in laboratory diagnostics, but, despite this, special attention should be paid, no doubt, to the issues of correct interpretation of the results obtained when using it. The main targets in the detection of chlamydia trachomatis by PCR are the nucleotide sequence of the species-specific cryptic plasmid, the genome sequence of the main inner membrane protein, and ribosomal genes.

Compared to widely used immunological tests, PCR diagnostics has a number of advantages:(1) high and adjustable specificity, due only to the nucleotide sequence used in this diagnostic system; (2) high sensitivity, which makes it possible to diagnose not only acute, but also latent infections (it is possible to detect even single bacteria or viruses); (3) the chemical similarity of all nucleic acids allowing the development of universal procedures for the detection of various infectious agents; (4) the ability to identify the pathogen within 4.5-5 hours.

note . Currently, there is no laboratory method to avoid both false positive and false negative results. When diagnosing chlamydia, complex laboratory diagnostics (PIF, ELISA, cultural method, PCR, detection of antibody titers to pathogen antigens) is necessary, which allows to identify the pathogen, determine the stage of the disease, and justify the need for prescribing antibacterial drugs. The study of the immune status and the reasonable use of immunomodulators will improve the effectiveness of treatment in the long term after infection.

A well-chosen mutual fund is the key to successful investment. Unfortunately, in this difficult matter there is no universal advice that would suit every investor. But there are criteria, the analysis of which will help not to make a mistake with the choice of a mutual fund.

Analysis and evaluation of existing mutual funds according to various criteria, the most important of which are:

verification of the foundation's license;

the time of the fund's operation on the market;

profitability of the mutual fund;

coefficients characterizing the effectiveness of management of mutual funds;

price net assets fund;

the amount of costs.

Let's consider each of these criteria in more detail.

Foundation License Check

The first step when choosing a mutual fund is to clarify whether the management company license to carry out activities for the management of mutual funds. Investors should be wary of both the lack of a license from the management company and the expired license. In this case, it is likely that fraudulent schemes will be used. According to the law "On Investment Funds", only the presence of a license entitles the management company to manage mutual funds and use the words "management company" and "share fund" in its name.

Fund operating time on the market

When choosing a mutual fund in without fail it is necessary to take into account its time on the market. Despite the fact that the collective investment industry has existed in Russia for only 10 years, for a mutual fund, 5 years of work on the market is very good indicator. About 50 mutual funds can boast of such a period of work, including: Dobrynya Nikitich, Pyotr Stolypin, Pallada - second-tier stock fund, Lukoil Fund promising investments", Monomakh Energia", Alfa Capital, Solid Invest, etc. It is not recommended to invest in mutual funds that have been operating on the market for less than two years, since the short period of existence of the mutual fund will cause difficulties in objectively assessing its results. As a rule the longer the fund exists, the more information and statistical data about its work will be available.The only type of mutual funds for which you can not take into account the time of work on the market are index mutual funds.Due to their specifics (in their structure, they almost completely repeat one or other index) index mutual funds are completely dependent on the state stock market and the economy as a whole. And the time of work here is completely irrelevant. You should not make the classic mistake of novice shareholders and buy shares of a newly formed mutual fund, guided by its high yield. According to statistics, most newly formed funds show good results in the first year of operation, but very few can repeat them in the future. Experts tend to blame the growth of fund assets for everything, because the portfolio becomes large and clumsy over time, and it becomes more and more difficult to manage it.

Return on a mutual fund

The yield of a mutual fund is exactly the criterion that novice investors are so greedy for. It seems that since the fund performed well this year, why shouldn't it do just as well this year? Alas! Harsh statistics prove that no leading fund has become a leader for several years in a row. Was in the top ten - yes, but not the leader. The return on the fund is expressed as a percentage increase in the value of the share. Naturally, when analyzing and evaluating mutual funds, there is nowhere without profitability, but you cannot elevate it to the rank of an absolute criterion and make decisions based only on the profitability of funds. After all, as it is written everywhere in small print: "The return shown by the fund in the past is not a guarantee that the same return will be shown in the future." Moreover, one cannot unconditionally trust advertising that promises hundreds of percent per annum. Don't forget that the last few years have been years of growth, and the high yields of mutual funds are in most cases not only the result of the work of managing managers. A competent investor will compare the dynamics of the value of a share of a mutual fund with the dynamics of the RTS and MICEX indices and will be able to assess whether the managers were able to beat the market or they systematically lost to the market. In fact, there are not many mutual funds that beat the market, so if the fund goes head-to-head with the market, this is already not bad. Profitability can become a criterion for choosing a mutual fund only when it is a stable profitability, that is, for a long time (at least 2-3 years), the fund shows a constant profitability. Finally, we should not forget that return and risk go hand in hand. Having been seduced by high profitability, you need to understand that in the event of a downturn in the market, you can lose a significant part of your capital. So profitability is profitability, but you should not forget about the coefficients either.

Net asset value (NAV)

The value of the net assets of a mutual fund is the amount of money of the shareholders that are under the management of the fund. On the one hand, a large NAV makes the fund's portfolio bulky and unwieldy, and on the other hand, it allows for larger investments. So we follow the principle - the more NAV, the better the mutual fund. In addition, a large NAV, as a rule, indicates the popularity of the mutual fund and its good reputation.

In any case, the amount of net assets is not a decisive criterion when choosing a mutual fund.

The amount of costs

The Federal Law "On Investment Funds" establishes that the expenses of the management company for the management mutual fund may not exceed 10% average annual cost net assets of the fund. That is, for the remuneration of the management company, for the services of the depositary, appraiser, auditor and others, the shareholder should not be charged more than 10% of the value of the shares he owns. Discounts and surcharges are also limited. The maximum amount of the premium cannot be more than 1.5% of the share value, and maximum size discounts - more than 3%. Against the backdrop of tougher competition, many management companies are trying to minimize their costs in order to attract investors. Naturally, the lower the costs of the mutual fund, the greater the income of the shareholder. But on the other hand, you should not be tempted by small costs, especially if we are talking about a start-up management company. It is better not to save a couple of percent and entrust your money to a reliable and reputable management company.

Chlamydia cannot be diagnosed by symptoms alone. Laboratory tests help to accurately determine this disease. Almost every laboratory offers several tests for chlamydia. But how not to get confused in numerous methods and accurately determine chlamydia? We talk about each of them and understand what are the pros and cons.

How can and how not to diagnose chlamydia?

Each method has its own characteristics, and some cannot be called effective at all. So let's find out what is suitable for the definition of chlamydia, and what should not be taken seriously.

polymerase chain reaction ( PCR)

This is one of the most reliable tests for chlamydia. It detects bacteria with almost 100% accuracy.

By the presence of antibodies, as well as by their quantity, one can understand whether a person has chlamydia and what stage of chlamydia (acute or chronic) he is sick with. Also ELISA helps to determine whether the treatment was successful, if the analysis is taken as a control.

It is important to remember that this method is very unreliable. If there is a choice, it is better to use more accurate methods.

Smear microscopy (aka bacterioscopy) according to Gram

This method cannot detect chlamydia at all - so it is very important to distinguish it from others so as not to make a meaningless analysis.

A smear microscopy is an analysis of human secretions (vaginal mucus, semen, and others) under a microscope using another stain - according to the Hans Gram method. It is he who is carried out by all paid laboratories called "general smear", "microscopy", or "bacterioscopy".

Gram stain allows you to see different bacteria that are invisible under a microscope without stain. It also helps to count leukocytes - a type of human immune cells. But alas: the Gram method cannot detect chlamydia, because they simply do not stain in this way.

The analysis is suitable only as an additional one - to assess the general condition of a particular organ, or if other diseases are suspected.

Chlamydia cannot be established by Gram smear microscopy!

Mini-test (rapid test for chlamydia)

This method is completely useless. It is worth knowing about it, just so as not to throw money away.

The analysis can be bought at a pharmacy and carried out independently at home. According to the mechanism of action, it is very similar to a pregnancy test: a few drops of urine or mucus from the vagina are applied to the hole, then the result is evaluated by the number of strips.

The instructions say that if a person has chlamydia, the test will show two stripes.

However, a negative test result does not guarantee that the disease does not really exist. Positive, however, is also unreliable. In fact, the analysis does not report anything useful. Therefore, if you suspect chlamydia, it is wiser to take more accurate tests.

As a result, consider comparison table all tests for chlamydia:

Analysis for chlamydia is considered one of the most unreliable diagnostic methods because the bacterium during the study period can hide in a cell and not be detected. Therefore, doctors, in order to confirm the presence or absence of the disease, prescribe several tests.

Chlamydia is caused by the bacterium Chlamydia trachomatis, which lives in vaginal fluid or semen. Infection usually occurs sexually, and therefore is very common and extremely dangerous. It affects women's health especially hard: chlamydia easily destroys the reproductive organs.

Chlamydia is considered a "silent" disease, as the bacterium may not show itself with sharp symptoms or even not let you know about its presence. Therefore, a person often needs a long time to understand whether he is ill or not. During this time, the bacterium is able to spread throughout the body and harm it.

Symptoms of chlamydia usually appear within one to three weeks after infection. It can be:

• purulent discharge from the genitals;
• itching, cutting pains during urination;
• vitreous discharge from the urethra;
• adhesion of the sponges of the urethra;
• feeling of weakness;
• symptoms of poisoning;
• slight increase in body temperature.

In 60% of cases, the symptoms of chlamydia do not make themselves felt at all and appear only in the form of complications. It can be prostatitis, endometritis and other diseases that lead to infertility.

Chlamydia in women

Complications of chlamydia are more common in women than in men. If the disease is advanced, chlamydia can enter the uterus, destroy the fallopian tubes and surrounding tissues. If a woman is pregnant, the health of not only the mother, but also the child is at risk. Chlamydia in the blood during pregnancy can cause infection of the baby and its premature birth.

An ectopic pregnancy can also be a complication. It leads to miscarriage or is the cause of mandatory abortion, because the fetus outside the uterus is not able to develop normally and ruptures the fallopian tubes as it grows.

An analysis for chlamydia in women involves passing:

• general analysis blood (shows an increased number of eosinophils);
• blood for chlamydia by ELISA (enzymatic immunoassay);
• a smear from the vagina (by studying the PIF or PCR method);
• smear from the cervix (PIF and PCR);
• scraping of the external opening of the urethra (PIF and PCR).

If the blood for chlamydia by ELISA and smear showed positive results during pregnancy, it is necessary to do an examination of the amniotic fluid. This will allow you to find out if the infection has passed to the fetus.

Bacteria in the baby's body

Chlamydia is a pathogen that can infect the body of not only an adult, but also a child. And, although chlamydia is considered a sexually transmitted disease, which is transmitted mainly through sexual contact, chlamydia can enter the child's body in other ways.

A baby can easily become infected with the bacterium Chlamydia trachomatus not only in the womb or during childbirth, but also by airborne droplets: while his immunity is being formed, the body is extremely vulnerable. The bacterium has an extremely negative effect on a crumb that has become infected through the air: chlamydia affects the conjunctiva of the eyes, the respiratory system, and the mucous membranes of the child.

The same symptoms can occur in a child who has contracted chlamydia in the mother's body. Also, he may develop diseases of the genitals, lungs, hearing. In severe cases, blindness may succumb.

A baby who becomes infected in the birth canal during delivery may develop an eye infection or pneumonia. This is explained by the fact that the baby, moving through the birth canal, comes into contact with maternal blood and secretions. According to statistics, half of children develop conjunctivitis during the first three months, and 5-20% develop pneumonia.

Symptoms of conjunctivitis are manifested in the fact that the child has swelling of the tissues around one or both eyes, then purulent discharge appears. The course of treatment includes erythromycin. Pneumonia makes itself felt with a sharp cough, often without fever. In this case, the child is prescribed an x-ray: with a positive result, infiltrates are visible in the lung tissue.

In men, chlamydia can infect the circulatory system, respiratory, musculoskeletal system, vision. Most often, the genitourinary system of men becomes the object of attack of chlamydia.

Diseases of the genitourinary system of men include chlamydial prostatitis, inflammation of the urethra and cystitis. Also, the bacterium is the cause of potency disorders in men, arthritis, infertility, diseases of the circulatory system. Therefore, every man is obliged to consult a venereologist if he has symptoms that signal chlamydia.

With an exacerbation of the disease, an increase in temperature is possible, a decrease in working capacity in men. The mucous membranes begin to secrete mucus and pus of a transparent color, itching and burning during urination. In half of the cases, men may not observe pronounced signs of this infection, and therefore do not pay attention to it. In this case, the situation is dangerous because they will be passive carriers of chlamydia, representing a danger to others, especially women.

PCR and PIF

The main tests for chlamydia are:

• PCR - polymerase chain reaction (studies the liquid medium of the body - blood, mucus, urine, etc.);
• DIF - direct immunofluorescence method (studies scrapings);
• ELISA - enzyme immunoassay for chlamydia.

Sometimes a swab is taken from patients for chlamydia from the urethra and examined by chromatography. The advantage of this method is that it can be used even independently at home. Among the disadvantages is low reliability, since bacteria, having penetrated tissue cells, may not find themselves in the mucous environment of the body.

According to scientific data, PCR for chlamydia shows the highest sensitivity in the analysis of chlamydia: its reliability is about 95%. It should be borne in mind that PCR on chlamydia can also give a false positive result when DNA of dead bacteria is detected.

The second most reliable is the mutual fund, the correctness of which is 70%. The method of direct immunofluorescence (DIF) is able to determine both the chronic stage of chlamydia and the exacerbation phase. A feature of the PIF is that this method allows you to determine both the pathogens themselves and the antigens produced by the immune system.

PIF reliably diagnoses the presence of chlamydia even when the symptoms of their manifestations change during the development of the disease. The high sensitivity of the PIF method is explained by the fact that specific monoclonal antibodies are used during the analysis. Taking scrapings for PIF is no different from taking scrapings for other microscopic analyses. One of the disadvantages of the PIF method is that it does not detect chlamydia during the incubation period.

Features of enzyme immunoassay

The least sensitive was the analysis of chlamydia by ELISA, when venous blood can be studied. This method studies the presence in the studied sample of antibodies that the body produces in response to infection with chlamydia. Antibodies-immunoglobulins, which are produced as an immune response, belong to the classes IgG, IgA, IgM. Their number fluctuates throughout the duration of the disease, and this change towards an increase in the number of antibodies often covers the beginning of recovery.

Blood on chlamydia by ELISA allows not only to determine the presence of infection, but also to find out the stage of the disease, acute or chronic. Immediately after infection, the immune system begins to produce antibodies. If the ELISA shows an increased amount of IgM antibodies, this indicates that chlamydia is in an acute stage. After one to two weeks, a blood test for chlamydia ELISA shows the presence of IgA antibodies. IgG antibodies may appear in ELISA results 3-4 weeks after infection.

The interpretation of the test results, which is carried out by the ELISA method, is done on the basis of the norms of antibody concentration in titers. With an exacerbation of the disease, blood for chlamydia by ELISA shows a sharp increase in the concentrations of IgM and IgA antibodies. Based on these data, it is possible to predict the course of the disease for a period of up to two months.

The excess of the average values ​​of antibody concentrations in the ELISA results are:

With an exacerbation of the chronic stage of the disease, the greatest increase in ELISA shows in IgG antibodies - up to 50,000, while the rest of the numbers remain unchanged. When recovering, the ELISA shows a drop in IgG to 100-200, while the values ​​of IgA and IgM do not change further.

Features of therapy

Fortunately, these days, chlamydia is very treatable and the biggest hurdle is timely diagnosis. In order to detect the disease in time, at least once a year, you need to take a general blood test. If eosinophils and some other types of white blood cells are abnormal, other tests should be done to determine the cause. It will be useful for women to periodically visit a gynecologist and take a smear for analysis.

When chlamydia is detected, treatment includes a course of antibiotics from the tetracycline group: they cope well with the causative agents of this disease. If there is an allergy, the doctor prescribes another treatment. Since chlamydia is a sexually transmitted disease, it is not superfluous to protect yourself from infection during sex.

Chlamydia trachomatis is an intracellular microorganism that causes a number of diseases of the genitourinary system and eyes. Variants (serovars) of Chlamydia trachomatis are divided into "eye", "...

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Study Description

Preparation for the study:

• Before taking material for analysis, it is necessary to exclude antibiotics for a week, refrain from sexual contact for 3 days and not urinate for 1 hour
• In women, swabs are taken from the cervix, vagina, and urethra
• During menstruation and within 3 days after the sampling of the material is not carried out