CC BY
11
II. ДИАГНОСТИКА И ЛЕЧЕНИЕ
APPLICATION OF DIRECT IMMUNOFLUORESCENCE REACTION IN SEROLOGICAL DIAGNOSIS OF UROGENITAL CHLAMYDIAL INFECTION
Nurakhova A.D.1, Abdilova G.B.2, Zhakupkalieva Zh.Z.3
1Kazakh Medical University of Continuing Education, Almaty, Kazakhstan 2National Scientific Center of Surgery named after A.N. Syzganov, Almaty, Kazakhstan 3Almaty Regional Center for the Prevention and Control of AIDS, Almaty, Kazakhstan
Abstract
The paper analyzes the results of direct immunofluorescence studies of biological material for the presence of Chlamydia trachomatis, carried out in a private diagnostic laboratory in Almaty during the second half of 2019. We have shown that the direct immunofluorescence method can be used for the diagnosis of urogenital chlamydia. This method allows diagnosing chlamydial antigens, which excludes cross-reactions. However, the implementation of this diagnostic method requires high professionalism from the performer.
АВТОРЛАР ТУРАЛЫ
Нурахова Алма Дандыбаевна -
Казак, медициналык узд!кс!з б!л!м беру университетам клиникалык зертханалык диагностика кафедрасы, доцент, медицина Fылымдарыньщ кандидаты, доцент.
Абдилова Гульнур Бекмурзаевна -
А.Н. Сы^анов атындагы УлттыкFылыми хирургия орталы^ыныц клиникалык диагностикалык зертханасыныц мецгеруш1с1
Жакыпкалиева Жанна Заркы/нбещызы - зертханашы дэрйвр, Алматы облыстыкЖИтС-тыц алдын алу жэне 0Fан карсы курес орталы^ы.
Туйш сездер
трихомонас инфекциясы, хла-мидиалды инфекция, жанама иммунофлуоресценттк реакция, иммунофлуоресценцияньщ ткелей реакциясы
Применение реакции прямой иммунофлюоресценции в серологической диагностике урогенитальной хламидийной инфекции
Нурахова А.Д.1, Абдилова Г.Б.2, Жакупкалиева Ж.З.3
1Казахский медицинский университет непрерывного образования, г. Алматы, Казахстан Национальный научный центр хирургии им. А.Н.Сызганова, г. Алматы, Казахстан 3Алматинский областной центр по профилактике и борьбе со СПИД, г. Алматы, Казахстан
Аннотация
Мы сообщаем о случае диагностики смешанной инфекции, передаваемой половым путем. В выше указанном случае затрагивается вопрос диагностики урогенитальных инфекций, вызванных смешанной флорой. Были сделаны выводы о необходимости комплексного подхода к диагностике и, в целом, ведению больных хроническим воспалительными заболеваниями органов урогенитального тракта. Соблюдение принципов междисциплинарной интеграции при осуществлении диагностических и лечебных технологий будет позволять адекватной реабилитации урологического, гинекологического и репродуктивного здоровья пациентов.
МРНТИ 76.29.50 UDC 616-093
ABOUT THEАUTHORS
Nurakhova Alma Dandybaevna - Associate Professor, Department of Clinical Laboratory Diagnostics, KazMUNO, candidate of medical sciences, assistant professor.
Abdilova Gulnur Bekmurzaevna - head of the clinical diagnostic laboratory of the National Scientific Center of Surgery named after A.N.Syzganova.
Zhakupkalieva Zhanna Zarkynbekovna
- laboratory doctor, Almaty Regional Center for the Prevention and Control of AIDS.
Keywords
serological diagnostics, chlamydial infection, direct immunofluorescence method
Урогенитальды хламидиялык инфекцияньщ серологиялык диагностикасында ткелей иммунофлюоресценция реакциясын колдану
Нурахова А.Д.1, Абдилова Г.Б.2, Жакыпкалиева Ж.З.3
'Казак, медициналык, Yздiксiз 6miM беру университету Алматы каласы, Казахстан
2А.Н.Сыз?анов атында?ы Улттык fbrnb^ хирургия орталы^ы, Алматы каласы, Казахстан
3Алматы облыстык, ЖИТС-тык алдын алу жэне о?ан карсы 1^рес орталы^ы, Алматы каласы, Казахстан
Ацдатпа
Б1з аралас жыныстык инфекция диагнозы туралы хабарлап отырамыз. Жогарыда керсетлген жагдайда, аралас флорадан туындаган урогенитальды инфекцияларды диагностикалау туралы мэселе кетершед1. Диагнозды кешенд1 турде кабылдау кажеттш1п туралы жэне несеп-жыныс жолдарыныц созылмалы кабыну аурулары бар наукастарды баскару туралы корытынды жасалды. Диагностикалык жэне емдеу технологияларын енпзуде пэн аралык интеграция кагидаларын сактап, наукастардыц урологиялык гинекологиялык жэне репродуктивт денсаулыгын калпына келт1руге мумюнд1к беред 'г
ОБ АВТОРАХ
Нурахова Алма Дандыбаевна - доцент кафедры клинической лабораторной диагностики Казахского медицинского университета непрерывного образования КазМУнО, кандидат медицинских наук.
Абдилова Гулнур Бекмурзаевна -
заведующая клинико-диагностической лабораторией Национального научного центра хирургии им. А.Н.Сызганова.
Жакупкалиева Жанна Заркынбековна
- врач-лаборант, Алматинский областной центр по профилактике и борьбе со СПИД.
Ключевые слова
трихомонадная инфекция, хламидийная инфекция, реакция непрямой иммуноф-люресценции, реакция прямой иммунофлюоресценции.
Introduction
Chlamydia trachomatis infection is of great social and medical importance to the healthcare system today. This is due to the fact that urogenital chlamydial infection is quite widespread, often causes the development of complications and adversely affects the reproductive health of people. According to the WHO, more than 100 million people are diagnosed annually suffering from the above pathology.
Chlamydia trachomatis affects the cells of the cylindrical and transitional epithelium of the urogenital organs, rectum, posterior pharyngeal wall, conjunctiva, synovial membrane of the joints, as well as epithelial and epithelioid cells of various organs, reticuloendothelial cells, leukocytes, monocytes, macrophages. This pathogen determines the development of infectious and inflammatory diseases of the genitourinary organs of women and men. Most often, urogenital chlamydia occurs without vivid clinical symptoms, which significantly complicates the diagnosis and favors the spread of the disease and the early formation of complications. The predominant clinical forms of chlamydial infection in women are urethritis and cervicitis, in turn, the most common complications are pelvic inflammatory disease. Moreover, after a number of cases of pelvic inflammatory disease, the risk of developing infertility can reach 75% [1]. There is also a certain relationship between the infection caused by Chlamydia trachomatis and the formation of malignant neoplasms. There is a possibility that this pathogen is a bacterial cofactor that contributes to the progression of neoplastic processes.
In men, chlamydial infection most often manifests itself in the form of urethritis and prostatitis, as a result of which complications such as epi-didymitis and orchiepididymitis are usually formed. The latter can lead to the development of infertility. Chlamydia trachomatis can cause autoimmune reactions in the body that lead to Reiter's disease and immunological infertility. A characteristic feature of urogenital chlamydia is the defeat of the genitourinary organs and at the same time other organs and systems of the body, due to the generalization of the infection. At the same time, an erased, chronic course of the disease is observed, accompanied by the incommensurability of morphological lesions in the diseased tissues with the clinical picture, inadequacy of the body's immune response. This leads to the formation of secondary foci of infection.
Materials and methods
The article analyzes the results of direct im-munofluorescence (DIF) studies of biological material for the presence of Chlamydia trachomatis, performed in a private diagnostic laboratory in Al-
maty during the second half of 2019. The sampling of biological material was made from the mucous membranes of the urethra, paraurethral passages (when its expressed), the vagina, and the cervical canal. To obtain material from mucous membranes, a disposable probe was used with a cotton swab with increased adsorption. The material was collected by rotating the swab. Immediately after taking the material, a smear-imprint was prepared by touching the surface of the well of the glass slide. The prepared smear was air dried. The dried smear was fixed in 96% ethanol for 5 minutes. The smear was fixed no more than 5 minutes after taking the material. The material under study should contain as many epithelial cells as possible and the minimum amount of mucus and exudate. Scrapings from the vagina, cervix, cervical canal were taken after removing the mucous plug. In this case, one day before taking the material, it is necessary to perform food or medication provocation. The sample is taken early in the morning before urination or 2-3 hours after the last urination.
A total of 600 studies were performed for the presence of Chlamydia trachomatis. Used diagnostic kits "ChlamyScan" LLC "Agrobiomed", Russia. The smears were viewed in a luminescent microscope "Mikromed", Russia, with an immersion objective (100x) using a special non-fluorescent immersion oil, using a system of filters providing excitation light with a wavelength not exceeding 490 nm and emission with an average wavelength of 520 nm.
The results were recorded immediately after the preparation was mounted. The results of the reaction, depending on the degree of color and brightness of specific fluorescence of elementary and reticular bodies of chlamydia, located in the form of cytoplasmic inclusions or extracellularly were assessed visually using a 4-cross system. The result was considered positive if a bright green glow was recorded in the smear in the form of a point (for elementary bodies (EB) of chlamydia) or an oval (for reticular bodies of chlamydia) for at least three crosses, which stands out against the background of epithelial cells stained in maroon or red-orange color. The result was considered negative if there was no specific glow in the smear with the obligatory presence of at least 50 epithelial cells in the smear. A dubious reaction was considered in the presence of single chlamydial bodies with a staining intensity of two crosses or in the absence of epithelial cells in the field of view. In this case, the analysis was repeated.
Research results
During the second half of 2019, the diagnostic laboratory performed 600 DIF analyzes for the pres-
18
ВЕСТНИК ХИРУРГИИ КАЗАХСТАНА № 3-2020
ence of Chlamydia trachomatis. Of the total number of studies, 410 were performed by men and 190 by women (Fig. 1). The age of patients ranges from 17 to 67 years. Generally, people aged 32 to 55 are more likely to apply. Of the 600 analyzes for Chlamydia trachomatis, 552 (92%) were negative and 48 (8%) were positive.
There are two main strategies for urogenital chlamydial infection: diagnostic screening to identify symptoms of C. trachomatis infection and screening at risk. For microbiological diagnosis of urogenital chlamydia, cultural, microscopic, sero-logical and molecular genetic methods are used. The "gold standard" in the laboratory diagnosis of urogenital chlamydia is the culture method. The culture method is the most time consuming and expensive, long-term study (314 days), requiring compliance with strict rules for the transportation of a clinical sample and temperature conditions, and the presence of highly qualified medical personnel. In terms of specificity (100%), this method is a reference, but its sensitivity can vary from 33 to 85% [2]. To detect genital chlamydia, it is recommended to use cultures from both the urethra and the cervical canal, which allows increasing (by 5%) the excretion of the microbe [1]. In case of inactive chlamydia, when metabolic processes are suspended in non-developing chlamydia, false-negative results can be obtained in cell culture. This is associated with the difficulties of diagnosing chronic persistent chlamydial infection. In chronic ascending infection caused by C. trachomatis, as well as when taking material from a patient with chlamydia with a low number of viable pathogens, the proportion of detecting chlamydia is low.
The microscopic method for diagnosing urogenital chlamydia is the simplest and most affordable. It gives a general idea of the cytological picture, morphology of cells from the lesion, the presence of microbial contamination, fungi and protozoa. Specific morphological signs allow determining the
presence of chlamydia in the preparation. The most common methods are staining preparations according to Romanovsky-Giemsa, May-Grunwald-Giem-sa, Macchiavello, Papanicolaou, Lugol's solution. The sensitivity of the method is 1015%, the specificity is 10-30% [3]. The method is not applicable for screening studies. The diagnostic significance of the microscopic method is rather low: in men it is possible to detect chlamydia with it only in 10-15%, and in women - up to 30-40% of cases (in scrapings from the cervical canal) [4]. With the correct collection of material from patients (from the mucous membranes of the vagina and cheeks) and with the corresponding high qualifications of the doctor, the diagnostic value of light microscopy for the detection of chlamydia is not inferior to the direct reaction of immunofluorescence (DIF) (Fig. 2) [5] and is superior to ELISA.
Serological methods make it possible to detect in biological material (urine, sputum, scraping from the urethra, cervical canal, oropharynx, ejaculate, prostate juice, blood serum, leucocon-centrate, peripheral blood smears, articular fluid) the presence of genus- or species-specific Ag - RIF, or specific antibodies (Ab) of classes IgA, IgM, IgG - ELISA.
The reaction of immunofluorescence (RIF) is based on the detection of chlamydial antigens in the epithelium and other tissues by specific monoclonal or polyclonal antibodies to various chlamydial antigens. There are two types of RIF - direct (DIF) and indirect (RIIF). In the first case, the specific Ab is labeled with flurochrome, and the reaction proceeds in one stage. In the second case, the specific antibodies do not have a label, and labeled antiglobu-lin antibodies are used to identify the antiglobulin antibodies formed on the complex. The RIF result is assessed visually under a fluorescent microscope. It is positive if the drug contains epithelial cells and it is possible to detect at least 5-10 bright green fluorescent elementary bodies (EB).
450 „„„
410
400 350 300 250
200 ■ 190 ,0 ■ ■
100
: I I
Men Women
Gender data
Figure 1.
Distribution of patients by gender
Figure 2.
Scheme of the direct immunofluorescence reaction [5]
Discussion
DIF and RIIF are widely used to diagnose urogenital chlamydia, but their further use is constrained by the fact that their specificity and sensitivity are in the range of 85-99% and 50-90%, respectively [2]. This is due to the fact that specific Ab are produced by different firms and differ in their quality. In addition to strict conditions for the quality of the test systems themselves, preparation of patients for research, the quality of taking material for research, its further processing and storage are important for obtaining reliable results. Highly qualified specialists in fluorescence microscopy are required. Only if it is performed correctly by an experienced laboratory assistant can a PIF be very sensitive and highly specific [6, 7]. DIF is advisable to use for diagnostics in high-risk groups for urogenital chlamydia, especially in patients with clinical manifestations of STIs, and is not justified for detecting Ag Cl.trachomatis in low-risk groups, including those subject to screening [8]. In a comparative study of PCR diagnosticum ("Ampli-cor", Roche) and DIF for detecting Cl.trachomatis in urethral and cervical samples, mainly with a low content of EB, it was found that the sensitivity of PIF is 10 times higher than that of PCR. The diagnostic informative value of a mutual fund is associated with its ability to detect not only corpuscular, but also soluble chlamydial AGs. It is necessary to pay special attention to the qualitative assessment of the research results. The parameters of the method are less dependent on changes in the tinctorial properties of chlamydia during the infection, especially during etiotropic therapy [9].
Serodiagnosis of chlamydial infection has not been sufficiently successful to date, since the disease is caused by a low immunogenic pathogen. Anti-chlamydial antibodies persist for a long time, therefore, even a healthy population may have a background anti-chlamydial antibodies titer. With prolonged and complicated chlamydial infection, serodiagnostic methods can be used.
An important link in the complex of clinical and laboratory measures for urogenital chlamydia is
the assessment of the effectiveness of the therapy - the use of laboratory tests to establish the cure criterion. The main reliable method for establishing the effectiveness of the treatment performed is the culture method (14 days after the end of antibiotic therapy), the use of PCR is not recommended for the purpose of monitoring treatment. Adequate and accessible ways to control the cure of chlamydial infection are PIF and PCR, performed, respectively, not earlier than 2 weeks and 30-40 days after the end of therapy. The combination of various diagnostic techniques significantly increases the efficiency and reliability of monitoring the therapy of urogenital chlamydia.
Currently, there is not only a unified algorithm for examining a patient with suspected urogenital chlamydial infection, especially a widespread form of urogenital chlamydial infection with a protracted, recurrent course of the disease, but also a common opinion on the interpretation of the results obtained. For reliable verification of the pathogen in generalized chlamydial infection, it is necessary to expand the list of investigated clinical samples from patients not only from the urogenital tract, but also from other organs and systems. An important laboratory diagnostic criterion is the detection of chlamydia in the blood, which makes it possible to objectively diagnose the generalized form of urogenital chla-mydial infection [10]. The development of modern, inexpensive, highly sensitive and specific methods of laboratory diagnosis of urogenital chlamydia remains relevant and is of great importance for medical science and practical health care [11].
At the moment, I use two main methods to diagnose chlamydia: PCR (smear or scraping for chlamydia) and ELISA (serological diagnosis - antibodies to chlamydia). The main problem is that antibodies to chlamydia are found in a fairly large number of people. And it often happens that people who have found "chlamydia in the blood" do not have any symptoms of urogenital diseases. So where does an IgG (antibody) titer to chlamydia come from in a healthy person? This question interested researchers from the University of Tennessee (USA). They found [12] that antibodies to chlamydia have cross-reactions. That is, antibodies to Chla-mydia trachomatis are very similar to antibodies to Chlamydia pneumoniae (respiratory chlamydia). Among women with a titer of antibodies to respiratory chlamydia, 81% were found to have antibodies to Chlamydia trachomatis. And vice versa - in the presence of a titer of antibodies to Chlamydia trachomatis, 85% showed a titer to respiratory chlamydia. Chlamydia pneumoniae usually causes upper respiratory tract disease (bronchitis, pharyngitis). And any person in his life has suffered from various respiratory diseases many times. This ex-
20
ВЕСТНИК ХИРУРГИИ КАЗАХСТАНА № 3-2020
plains why many healthy people (who have never suffered from urogenital chlamydia) suddenly show a titer to chlamydia.
Today, almost all world national guidelines do not recommend using the ELISA method for the diagnosis of chlamydia. Probably the above studies are a sufficient argument for this. However, in our country, the study of the level of antibodies to Chlamydia trachomatis is used quite widely. And many people continue to find chlamydia in the blood. Moreover, in some cases, a "blood test for chlamydia" is the only diagnostic method. And some patients have been treating chlamydia for years, the diagnosis of which is made only by the presence of an antibody titer in the blood. And there are quite a lot of such people, given that antibodies to chlamydia are found in almost half of healthy people.
First of all, it is necessary to know about the existence of polyclonal activation syndrome and suspect it in time. To make a diagnosis of various infections, it is not enough just to detect the level of antibodies. In addition, any infection, as a rule, has its own characteristic clinical picture, without which it is not always acceptable to make a diagnosis. It is also desirable to identify the pathogen itself - the PCR method identifies the pathogen only if it is actually present. With a nonspecific increase in the level of antibodies, PCR gives a negative result. It is necessary to actively identify diseases that cause polyclonal activation syndrome (the most common of which is chronic staphylococcal pharyngitis).
References
1. Microbiology & Microbial infections. Topley & Wilsons. - N. Y.: Edward Arnold (Publishers) Ltd., 2005. - Bacteriology. - Vol. 2, Part. VI. - Chapter 78. - P. 2006-2022.
2. Stary A. European Guideline for management of Chlamydia infection // Int. J. STD&AIDS. - 2001. - Vol. 12, N 3. - P. 31-33.
3. Sexually transmitted infections / Ed. V. A. Akovby-an, V. I. Prokhorenkov, E. V. Sokolovsky. - M .: Publishing house "Media Sphere", 2007. - 744 p.
4. Medical laboratory diagnostics: Handbook // Ed. prof. A.I. Karpishchenko. - SPb: Intermedica, 1997 .-- 304 p.
5. APA Style (2020). Available at: stock-image-mechanism-of-indirect-immunofluorescence-test-computer-illustration-164842776 (accessed 1 August 2020).
6. Taylor-Robinson D., Renton A. What tests for diagnosing sexually transmitted infections should be used in industrialized countries // STDs. - 1998. -No. 5. - P. 23-26.
7. Ostergaard L., Moller J.K. Use of PCR and direct immunofluorescence microscopy for confirmation of results obtained by Syva Micro Trak Chlamydia
This polyclonal activation is caused by superantigens. Superantigens are a special group of antigens (found in both bacteria and viruses) that can cause the activation of several different clones of lymphocytes - polyclonal activation. This activation is not specific - that is, lymphocyte clones that are not related to the superantigen undergo activation. If a B-lymphocyte is activated, it begins to synthesize antibodies. When several different clones of B-lymphocytes are stimulated at once, the synthesis of a variety of antibodies is activated. Thus, antibodies to Chlamydia trachomatis can also be found in healthy people. The presence of antibodies can be due to both a previously transferred disease ("serological scar"), and cross-reactions with respiratory chlamydia. Less commonly, the cause of false positive ELISA results is polyclonal activation syndrome. A feature of this syndrome is the detection of acute phase antibodies immediately to a large number of pathogens. Considering all of the above, it should be emphasized that UIF allows diagnosing chlamydial antigens, which excludes cross-reactions, therefore, the method can be recommended for diagnosing urogenital chlamydia.
Conclusions
1. The method of direct immunofluorescence can be used to diagnose urogenital chlamydia.
2. Using the method of direct immunofluores-cence requires high professionalism of the performer.
enzyme immunoassay // J. Clin. Microbiol. - 1995. - Vol. 33, N 10. - P. 2620-2623.
8. Schachter J. DFA, EIA, PCR, LCR and other technologies: what tests should be used for diagnosis of chlamydia infections? // Immunol. Invest. -1997. - Vol. 26, N 1-2. - P. 157-161.
9. Metelskaya V.A., Aleshkin V.A., Zverev V.V. and others. Modern methods of laboratory diagnosis of chlamydia // ZhMEI - 2008. - № 4. - P. 111-117.
10. Lobzin Yu.V., Lyashenko Yu.I., Poznyak A.L. Chlamydial infections. - SPb: LLC "FOLIANT Publishing House". - 2003 .-- 400 p.
11. Akyshbaeva K.S., Esenaliev M.K., Khandilla Z.M. The relevance of urogenital chlamydia among groups of HIV-infected individuals in Kazakhstan // International Journal of Experimental Education. -2017. - No. 3-2. - P. 193-194.
12. Martin D.C., Khare V.K., Miller B.E., Batzer F.R. Association of positive Chlamydia trachomatis and Chlamydia pneumoniae immunoglobulin-gamma titers with increasing age // Journal of the American Association of Gynecologic Laparoscopists -1997. - Vol. 4. - # 5. - P. 583-586. doi: 10.1016/ s1074-3804(05)80092-1.
