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12growth by more than 0,5 score by EDSS scale is 1,83 times (95% CI 0,92-3,82) higher than in patients who were treated by the standard protocols than in patients after the SC transplantation.
2. The risk of active focuses appearance after performing ATMSC is by 4,0 times (95% CI 1,027-15,625) lower than using the standard therapy.
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P ELVIC INFLAMMATORY DISEASES IN WOMEN OF REPRODUCTIVE AGE: THE EMPHASIS ON THE ETIOLOGICAL FACTORS AND THE SEARCH FOR NEW DIAGNOSTIC APPROACHES
1Nuradilova D.M., 1M. D. Kaliyeva L. K., 2M. D. Kalimoldayeva S. B.c
1Asfendiyarov Kazakh National Medical University, Department of obstetrics and gynecology No.2, Almaty city,
Republic of Kazakhstan 2State-ownedpublic enterprise with right of economic jurisdiction "Regional diagnostics center", Clinical Diagnostics Laboratory,
Almaty city, Republic of Kazakhstan
Abstract.
The main purpose of the study was to identify risk factors and develop the principles of diagnosis of pelvic inflammatory diseases, based on the detection of pathogens, sexually transmitted diseases.
Aim of the study. The study of risk factors and the development of the principles of diagnosis ofpelvic inflammatory diseases in women of reproductive age.
Material and methods. 112 women aged 20 to 43 years old suffering from pelvic inflammatory diseases were examined with the use of complex laboratory methods (ELISA, PCR, cell culture, immunological phenotyping).
Results. It was found that the most significant risk factors in women with pelvic inflammatory diseases were early onset of sexual activity, number of sexual partners over the four medical abortion history. The most common infections of the genital tract in women with pelvic inflammatory diseases
were candidiasis (59.8%), chlamydia was diagnosed by ELISA at 21.3% of women, trichomoniasis in -30.3% of cases, mycoplasmosis - 12.3%, ureaplasmosis - at 28.4% of women; Bacterial vaginosis was more likely to be determined by ELISA (19.3%). Immunological examination revealed changes in phagocytic response and reducing the amount of mature T-lymphocytes (CD3 +).
Conclusion. The incidence of infections, sexually transmitted infections among women suffering from pelvic inflammatory diseases, was quite high, with more than half of the surveyed noted mixed protozoal, bacterial and fungal infections. Laboratory diagnosis of infections, sexually transmitted diseases should be integrated using a variety of research methods: ELISA, PCR, culture method. Violation of immunological reactivity promotes long torpid course of diseases of the pelvic organs, and calls for the use in treatment regimens along with preparations etiotropic immunomodulators.
Key words: pelvic inflammatory diseases, sexually transmitted infections, women of reproductive age, laboratory diagnostics of sexually transmitted infections.
Introduction. The problem of pelvic inflammatory diseases in women in recent decades continues to be one of the most important in medicine. This is due, firstly, to their widespread - the incidence of pelvic inflammatory diseases (PID) is not only not declining, but growing. Thus, according to WHO experts, the risk of PID among women in the 15-19 age group is from 1 to 8%. In 20-30% of cases PID are the cause of hospitalization [1]. In the world annually about 350 million women develop PID. Most of these women develop chronic inflammatory diseases of the uterus, which in 15% of cases lead to ectopic pregnancy, and in 40-85% of cases can lead to infertility [2]. Second, there were change in the clinical picture and outcomes of PID, many of which currently have erased oligosymptomatic, but at the same time lead to complications that have severe consequences up to the need for surgical treatment of internal organ, pathology of pregnancy, reproductive disorders, fetal infection and others.
It is connected first of all with changing of the character of pathogenic flora of genital tracts of modern women. If earlier E. coli, staphylococci, bacteroides, clostridia, peptostreptococcus and others used to be considered main causative agents of PID [3], nowadays most of researchers consider sexually transmitted infectious matters to be the main causative agents of chronic inflammatory process of reproductive organs [3-6].
The incidence of sexually transmitted infections (STI), takes a threatening nature. For example, in Europe the incidence of sexually transmitted infections, has increased almost 3 times since the 60s of the last century, with a peak incidence between the ages of 18 and 30 years old. For example, 70% of women suffering from salpingitis, under 25 years, of which the majority (75%) - did not give birth. Keep in mind that even a single episode of acute PID in 5-18% of cases can lead to infertility, and a threefold aggravation salpingoophoritis increases this figure to 80% [7]. Infertility in the presence of Chlamydia in the genital tract occur in 50% of cases, gonococci - 30-40%, Ureaplasma - 30%, Trichomonas - 45-50% of cases. Ectopic pregnancy develops in 9-30% of cases in women with urogenital chlamydiosis, and in 40% of cases - in the presence of gonococcal infection [8-11].
STI cause severe obstetric pathology (developing pregnancy, habitual miscarriage, premature rupture of membranes, postpartum endometritis, chorioamnionitis, placenta, intrauterine infection and fetal malformations, intrauterine fetal death), unsuccessful attempts of in vitro fertilization [12-14].
Chronic inflammation of the genital tract in women, caused by STI is also a risk factor for onco-gynecological pathology. Thus, the relationship of cervical cancer with certain strains of human papilloma virus was recognized as unconditional.
The continuing increase in the incidence of STI is related primarily to changes in standards of sexual behavior, changes in the rules of morality, promiscuity, prostitution and early onset of sexual activity.
Along with microbial factor in the spread of STI are important, and triggers, which include physiological (menstruation, childbirth), artificial (abortion, intrauterine contraception, hysteroscopy, hysterosalpingography, gynecological surgery, in vitro fertilization), the weakening of the barrier mechanism of the cervix and the general condition the body (anemia, obesity, diabetes, and others.).
One can not exclude the influence of social and behavioral factors affecting the health of women (chronic stress, economic hardship, chronic alcoholism, smoking, drug addiction), non-traditional forms of sex (oral-genital, anal, etc.).
The aim of study was to investigate the risk factors and the development of the principles of diagnosis of pelvic inflammatory diseases in women of reproductive age.
Materials and methods. The study involved 112 women with PID in age from 20 to 43 years. General clinical examination included clinical history, physical examination results. To identify risk factors and subjective symptoms conducted a survey among examined women.
Swabs from the genital tract were taken from all the patients for microscopic examination. For the diagnosis of urogenital chlamydiosis using ELISA ("Vector-Best", Novosibirsk), polymerase chain reaction ("AmpliSens", Moscow). Trichomoniasis, bacterial vaginosis was diagnosed by ELISA ("Vector-Best", Novosibirsk) and PCR ("AmpliSens", Moscow). To determine Ureaplasma urealyticum, Mycoplasma hominis, using rapid culture method the test systems "Mycoplasma Duo» («Bio-Rad», France, USA), allowing to identify U. urealyticum by its ability to metabolize urea and M. hominis - for ability to metabolize arginine. The fence was made with the gynecological swab (scrapings from the urethra and cervix). The swab was immersed in a transport medium and thoroughly rinsed.
Given the high prevalence of antibiotic-resistant strains of U. urealyticum and M. hominis, pripolozhitelnyh test results carried out additional analysis of "Mycoplasma SIR» («Bio-Rad», France, USA), which allows to determine the sensitivity of the urogenital mycoplasma to antibiotics 8 groups of fluoroquinolones, macrolides, tetracyclines .
There have been a complex immunological examination of all women with PID (main group, n = 112) and healthy female donors of similar age (control group, n = 50), includes the definition of the following indicators: phagocytic activity of neutrophils, phagocytic number and the index of completeness of phagocytosis, concentration of circulating immune complexes, the study of lymphocyte subpopulations (flow cytometry, BD «FACS Canto II», Belgium) to determine the concentration of immunoglobulin A, M, G (nephelometry, «Cobaslntegra», «Rosh diagnostics») in serum.
Statistical processing was performed with the help of application software package STATISTICA 5.0.
Results and discussion. In the age group 20 to 25 years were 60 women (54%), aged between 26 and 35 - 35 (31%), from 36 to 40 years - 17 (15%). Thus, the largest group were women aged 20 to 25 years.
By social status, most of the main group of patients were employees - 85 (75.9%), workers -20 (17.8%), housewives - 7 (6.25%).
According to anamnesis, early onset of sexual activity (from 13 to 16 years) noted 23 patients (20.5%), sexual activity began from 16 to 18 years, 25 women (22.3%) over 18 years - 64 (57.2%). With the onset of sexual activity one sexual partner was in the cases of 23 women with pelvic inflammatory disease (20.5%), the number of sexual partners over four - 89 women (79.5%). Among the surveyed sex life in marriage it was in the cases of 64 women (57.2%), of whom 11 (17%) -remarriage. Outside of marriage 48 women (42.8%) planned pregnancy.
Menstrual function started in the cases of 110 women surveyed (98%) aged 11-14 years; in the cases of 2 women (2%) - 15-17 years. Births had a history of 20 women (17.8%), medical abortion - in 25 (22.3%), spontaneous abortions up to 12 weeks of pregnancy - in the cases of 24 women (21.4%), ectopic (tubal) pregnancy - in 17 (15.2% ).
Complaints for periodic dragging pain in the abdomen imposed on 85 women (75.9%) experienced discomfort in the urethra 36 (32.1%), frequent urination noted 32 women (28.6%), copious bothered 29 (25.9%), women (25.9%).
In gynecological status marked pathology of the cervix (cervicitis, erosion) in 92 women (82.1%), chronic salpingo-oophoritis in 101 (90.1%), endometritis in 36 (32.1%), ovarian dysfunction in 24 women (21.4%).
The results of microscopic examination of smears of discharge of the genital tract of women with PID are shown in table 1.
Table 1 The results of microscopic examination of gynecological smears of women with PID (n = 112)
The number of leukocytes in the field of view Urethra Vagina Cervix
5-10 0 22 (19.6%) 0
15-20 0 34 (30.3%) 30 (26.7%)
25-30 0 27 (24.1%) 49 (43.7%)
50-100 0 9 (8.0%) 33 (29.5%)
"Clue cells" 8 (7.1%) 22 (19.6%) 21 (18.7%)
Leptotryx 0 21 (18.7%) 0
C.albicans 0 67 (59.8%) 0
In the cases of all the 112 women who participated in the survey, it was revealed a violation of microflora, and in most cases it was a mixed infection. For example, bacterial vaginosis (gardnerellosis) combined with candidiasis in 31.2% of the women surveyed, 8.8% observed in the combination of Candida albicans and Leptotryx.
As can be seen from the above, the microscopic examination of smears informative for most pathogens classical STI. This is, primarily, due to fluctuations in pH of the medium and the effects of antibiotic treatment, which resulted in the microorganisms lose their typical morphological features. Thus, the typical forms of urogenital Trichomonas vaginalis (pear and amoeboid) nowadays appear quite rare. Thus, BV Klimenko et al. in the discharge from the urethra in men and vaginal discharge in women observed rounded formations without fixed flagella and undulating membrane in appearance indistinguishable from the host epithelial cells [15].
The pathogen such as Chlamydia trachomatis is virtually impossible to identify due to its intracellular existence. Urogenital mycoplasmas are also unavailable for diagnostics by means of microscopic examination, as they do not have the typical cell wall.
The incidence of STI among women suffering from pelvic inflammatory diseases is shown in figure.
70 60 50 40 30 20 10 0
59.8
128.4 303
I i I i I
Fig. 1. Morbidity rate of STI among women suffering from PID
The most common infection among women diagnosed with PID was candidiasis (59.8%). Chlamydia was diagnosed by ELISA at 21.3% of women at the same time the results of the PCR analysis in this group were positive in only 17.4% of women. A similar situation was observed in the diagnosis of trichomoniasis: at a time when the diagnostic titers of antibodies to Trichomonas vaginalis were detected in 30.3% of patients, PCR analysis revealed the presence of the pathogen in only 4.6% of cases. Bacterial vaginosis is also significantly more likely to be determined by ELISA (19.3%). Microscopically gardnerella detected in 17.9% of cases, the PCR technique - only in 10.3% of cases.
Reducing the effectiveness of STI detection by PCR, apparently due to the location in the submucosal layer of pathogens and the spread of infection by ascending.
Attention is drawn to the high proportion of mixed infections - from 54.6% of the women surveyed.
Set for cultivation, identification and quantification of urogenital mycoplasmas "Mycoplasma Duo" and the set for the determination of sensitivity to antibiotics can be considered the gold standard in the diagnosis of urogenital mycoplasma. M. hominis as a result of this test was found in 12.3% of cases, U.urealyticum met more often - in 28.4% of cases. The most effective in the treatment of urogenital mycoplasma according to the test "Mycoplasma SIR" were drugs doxycycline (90.4%), tetracycline (86.2%), ofloxacin (82.6%). Most strains U.urealyticum and M. hominis were resistant to erythromycin and clindamycin exposed.
In table 2 there are indices of phagocytal reaction by patients with PID and persons from control group.
Table 2 Phagocytosis indices in women with PID
Group of examined n PAN,% PN DPP
Patients with PID 112 36.8412.31%""" 3.21±0.20" 0.74±0.03%**
Control group 50 65.30±4.80 6.10±0.85% 1.41±0.06
*p<0.05; **p<0.01
As can be seen from table 2 phagocytic activity of neutrophils (PAN) was significantly decreased for women with PID, that is, the proportion of neutrophils participating in phagocytosis, was 36.84 ± 2.31%, at a time when apparently healthy women, the figure was 65.30 ± 4.80% (p <0.01).
The indicator such as is phagocytic number (PN), i.e. the average number of microbes absorbed by one of neutrophils was also reduced in patients of the main group. Significant changes undergoes not only the absorption phase of phagocytosis, which is characterized by above mentioned parameters (PAN and PN), and digestive phase of phagocytosis (DPP). In women with chronic inflammatory diseases of genitals DPP was less than 1, i.e. the phagocytic reaction was incomplete, which created conditions for the intracellular persistence of infectious agents. The degree of significance of differences with indicators of control group was high (p <0.01).
Table 3 is a subpopulation of peripheral blood lymphocytes of patients with PID and control
group.
Table 3 Indicators immunological phenotyping of patients with PID
The group surveyed n CD3+thousand cell/mcl CD4+ thousand cell/mcl CD8+thousand cell/mcl CD16+thousand cell/mcl
Patients with PID 112 1130.40±80.61%* 516.24±46.14** 391.54±20.16** 314.45±48.90
Control group 50 1681.36±90.26 981.62±61.83 631.85±82.60 291.27±31.50
*p<0.05; **p<0.001
Comparative analysis of the absolute number of mature T lymphocytes (CD3 +) showed a statistically significant reduction of the number of patients with PID (1130.40 ± 80.61 thousand cell/mcl) compared with control group (1681.36 ± 90.26 thousand cell/mcl). Reducing the number of mature T cells in women with PID were implemented mainly due to the population, which had a helper phenotype (CD4 +), the absolute number of persons whom the core group was significantly lower than the control group (p <0.001).
Humoral immunity study included determination of circulating immune complexes (CIC) and the concentration of serum immunoglobulin classes A, M, G. Results of studies performed in patients and control groups are presented in table 4.
Table 4 Indices of humoral immunity by women with PID
The group surveyed n CIC items IgA, mg/dl IgM, mg/dl IgG, mg/dl
Patients with PID 112 3.85±0.04** 286.32±4.40" 156.30±3.11" 1461.70±26.10
Control group 50 1.02±0.03 161.24±6.20 141.28±5.60 1380.74±37.81
*p<0.05; **p<0.01
In healthy women CIC level was 1.02 ± 0.03 units. In women with chronic pelvic inflammatory diseases, the figure was significantly higher (3.85 ± 0.04 units.), which reflects the identified defects have phagocytosis, a tool for the elimination of the CIC, and the activity of the inflammatory response. Patients with PID was also noted a significant increase in the concentration of serum IgA compared with that of the control group (p <0.05). The content of serum IgG in both groups were almost identical, while IgA and IgM concentrations were significantly higher in women with PID.
Conclusion. Thus, the most significant risk factors for pelvic inflammatory disease in women of reproductive age have an early onset of sexual activity, number of sexual partners over the four medical abortion history. The incidence of STI among women suffering from inflammatory diseases of the pelvic organs, was quite high, with more than half of the surveyed noted mixed protozoal, bacterial and fungal infections. In most cases, microscopic examination of gynecological smears
reflects only the degree of inflammatory activity, but is does not allow to clearly identify the type of pathogen that may be associated with a change in pH of the medium, the consequences of irrational antibiotic therapy, exposure to other damaging factors.
Laboratory diagnosis of STI should be integrated using a variety of research methods: ELISA, PCR, culture method.
In view of widespread antibiotic- resistant strains of STI, if possible, you should use the tests to determine the sensitivity of microorganisms to antibiotics.
Women with PID are characterized by the following changes in immunological reactivity: violation of the absorption and digestive phases of phagocytosis, promoting the intracellular persistence of STI; depression reaction cell type - reducing the number of mature T-helper lymphocytes with phenotype, high CIC and insignificant overproduction of IgA and IgM. These violations of immunological reactivity contribute to long torpid course of pelvic inflammatory diseases in women and necessitate the use in treatment regimens along with preparations etiotropic immunomodulators.
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