[Measurement of genital mycoplasma concentration by using a microbiological and molecular-biological methods].

AIM: Establishment of ratios that would allow to execute recalculation of mycoplasma concentration from CFU/ml and/or CCU/ml into units obtained during PCR analysis--geq/ml. MATERIALS AND METHODS: Pure cultures of Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum were studied by cultural and molecular-biological methods with quantitative evaluation. Studies of initial cultures as well as series of 10-fold dilutions were carried out. 32 experiments in total were carried out. RESULTS: Ratio between geq/ml and CFU/ml for M. hominis was 3.5; geq/ ml and CCU/ml ratio--4.4. Ratio between geq/ml and CCU/ml for U. parvum was 7.1; for U. urealyticum--11.2. CONCLUSION: Ratios between indexes obtained during quantitative study of pure genital micoplasma cultures by using 2 methods were established.

[Etiologic deciphering of community-acquired pneumonia caused by mycoplasma pneumoniae].

AIM: Use of a complex of methods for etiologic deciphering of an acute respiratory infection. MATERIALS AND METHODS: Clinical samples of blood sera, nasopharynx washes and sputum were obtained from 35 patients with acute respiratory disease (ARD). "Difco PPLO Broth" was used for M. pneumoniae cultivation. AHR, IFR, PCR, IFA were used in the study. RESULTS: Results of the study have shown that M. pneumoniae antigens in blood, sera samples were detected in AHR in 32 patients, and specific G and M class antibodies--in 21 and 18 cases, respectively. Simultaneous detection of IgG and IgM was registered in 14 patients. M. pneumoniae cell DNA was detected in 10 of 20 blood sera samples. Circulating immune complexes were isolated from blood sera of 8 patients (4 with pneumonia, 4 with ARD) and M. pneumoniae antigens were detected in them by using direct-IFR. IFR study of sputum and nasopharynx smears has shown that M. pneumoniae antigens were detected in 29 of 35 samples. In 12 of 15 smear samples M. pneumoniae. DNA was detected by PCR. In 10 cases results of antigen detection by IFR as well as DNA in PCR coincided. Results of analysis of all the clinical material have shown that in 33 of 35 patients positive results coincided for 2 or 3 and in some cases 4 of the laboratory study methods used. CONCLUSION: The use of diagnostic test complex significantly increases the accuracy of the study results, and detection of specific antibodies allows to determine disease period.

[Effect of low temperature plasma on various mycoplasma species].

AIM: Study the influence of low temperature (cold) electrolyte plasma (CEP) on survivability of some mycoplasma strains growing in agar as well as mycoplasma that most frequently contaminate transplantable human cell lines of normal and malignant origin with the aim of decontamination. MATERIALS AND METHODS: Mycoplasma hominis, Mycoplasma arginini and Aholeplasma laidlawii grown in agar and mycoplasma that contaminated transplantable human cell lines of normal (MT4) and malignant (HeLa) origin. Plasma source--Plasmatom device that generates CEP at normal atmosphere pressure and environment temperature. Exposure to plasma was carried out with adherence to the same modes for all the variants of biological substrate. The duration of exposure was selected randomly from 15 to 300 seconds. RESULTS: A pronounced bactericidal effect of high doses of CEP on all the tested mycoplasma variants exposed immediately after seeding into agar was shown. However after a passage a residual number of survived colonies was registered. Passage of colonies exposed in grown state even to high doses of CEP also showed survival of a residual number of bacteria in all the tested mycoplasma species. Exposure of M. hominis immediately after seeding to low doses of CEP resulted in formation of unusual mini-colonies identical to those isolated from humans infected by the same mycoplasma. During microbiological seeding into agar of cultural fluid from 2 spontaneously contaminated strains of transplantable human cells and exposed to CEP growth ofmycoplasma was not detected. CONCLUSION: CEP has pronounced bactericidal properties on various mycoplasma strains growing in both agar and contaminating eukaryotic cells. However even at high doses of exposure to CEP an insignificant part of bacterial cells growing in agar still survives. This may indicate a high degree of heterogeneity and adaptation of mycoplasma subjected to even such hard exposure as cold plasma with plasma-chemical mechanism of destruction of biological substrate.

[Generalized mycoplasma infection in patients and carriers].

AIM: Study of possibility of generalization of mycoplasma infection in patients with urogenital pathology. MATERIALS AND METHODS: Among the examined patients 5 males characterized by risky sexual behavior with pronounced symptoms of infection or without those were selected. Patients were examined by a complex of methods for the presence of mycoplasma infection by culture, PCR, DFA, PHA, AHR and by detection of specific immune complexes in blood sera. Scrapes from urogenital tract, blood sera samples, urine, saliva, prostatic fluid were materials for the study. RESULTS: In blood of all patients in ELISA antibodies against Mycoplasma hominis were detected; in PHA they were detected only in 2 individuals. In all the patients in blood CIC were detected including antigens and DNA of one or several mycoplasma species. Sperm of 3 individuals was infected by Ureaplasma spp., 2--M. genitalium. In saliva of 2 individuals M. hominis was detected, 3--U. urealyticum. CONCLUSION: In all the examined patients the infection was shown to have generalized character. This phenomenon presents itself as quite significant because mycoplasma may cause anti-apoptotic and oncogenic effect.

[Circulating immune complexes as a depot of conservation of mycoplasma cell components].

AIM: Study the possibility of prolonged conservation in macroorganism of antigens, mycoplasma cell DNA and live pathogen cells as part of CIC against the background of persisting antigen biostructures. MATERIALS AND METHODS: Aggregate-hemagglutination, direct immunofluorescence reactions and PCR method were used to determine antigens and DNA. Circulating immune complexes from blood sera samples were isolated by M. Digeon et al., mycoplasma isolation from CIC was carried out in SP-4 medium, species identity of the isolated mini-colonies was confirmed by real-time PCR method. RESULTS: In patients with urogenital and respiratory pathology the frequency of detection of Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma pneumoniae in free state was 63.3, 53.1 and 80.82% of cases, respectively. Specific CIC in patients with verified respiratory mycoplasmosis 1 month after the onset of the disease were registered in patients with severe course of the disease, bronchitis and diseases of upper respiratory tract--in 92.5, 74.7 and 25.7% of cases, respectively. In children, bronchial asthma patients the frequency of detection of antigens and DNA of M. pneumoniae cells in free state was 72.6 and 12.33%, as part of CIC--in 60.27 and 43.8% of cases, respectively. Antigens and DNA of M. hominis in blood of this group of patients were detected in 32.9 and 26.02%, as part of CIC--in 53.42 and 52.05% of cases, respectively. During repeated examination of 12 children after etiotropic therapy execution (generally in 1.5 - 6 months) in 75% of cases antigens of both M. pneumoniae and M. hominis were detected in free state and as part of CIC. DNA of cells of these mycoplasma species were detected in 20 and 33%, as part of CIC--in41.6 and 50% of cases, respectively. In 5 patients after 6 months (after 1 year in 1 case) mycoplasma antigens and DNA were identified in CIC or in blood sera. During cultivation of CIC components precipitated from 5 blood samples of patients of this group containing M. hominis DNA, culture of M. hominis mini-colonies were isolated in 4 cases. CONCLUSION: The possibility of prolonged persistence of antigens, DNA and whole mycoplasma cells in both free state and as part of CIC in patients with respiratory and urogenital pathology was shown. CIC are thus a peculiar depot, a place of conservation of not only various mycoplasma cell components, but also live cells.

[Unusual forms of persistence of Mycoplasma hominis in organism of infected humans].

AIM: Study previously unknown forms of persistence of Mycoplasma hominis in host organism. MATERIALS AND METHODS: Culture method was used for detection of mycoplasmas. Identification was carried out by serological, electron microscopy methods, classic PCR and real time PCR; circulating immune complexes (CIC) were isolated by PEG precipitation. RESULTS: Classic micoplasma cultures could not be isolated from blood even once. At the same time "mini-colony" cultures composed of mini-cells that were hardly passaged but sometimes formed continuous layer of the same colonies were isolated from blood serum samples with high frequency. During reseeding for more than 1 year they never acquired classic form. Not only antigens of M. hominis but its DNA were shown to be present in CIC. Viable cells forming "mini-colonies" identical to those isolated from blood sera were isolated from circulating immune complexes. A system of evidence on identity of isolated M. hominis cultures is presented. Cultures had infectivity and an ability to persist in organs of experimentally infected mice. CONCLUSION: The isolated forms are apparently the result of adaptation of mycoplasmas to humoral immunity factors.

[The atypical forms of mycoplasmas persisting in the organism of mycoplasma's infected persons].

The antigens, DNA and RNA of mycoplasmas are preset in the blood serum of persons infected with urogenital mycoplasmas. The planting of patients' tests of blood serum containing antigen M. hominis on the artificial growth mediums resulted in the growth of mini-colonies of mini-cells (20-50 nm). The colonies subcultured hardly but sometimes formed solid bacterial lawn though never acquired "fried-egg" classical mycoplasma form. The proofs of identity of these colonies to M. hominis are presented. The mini-cells possessed infectiousness and ability to persist on a long-run in the internal organs of experimentally infected mice. Apparently, mini-cells are formed under impact of stress factors of the host immune defense and they are one of forms of mycoplasma's persistence in human organism.

[Identification of Mycoplasma in patients with suspected prostate cancer].

AIM: Tests for Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum in males with suspected prostate cancer. MATERIALS AND METHODS: Identification of mycoplasms was performed in prostate tissue samples using universal PCR as well as in serum samples of patients with suspected prostate cancer using ELISA for detection of IgG to M. hominis. Two hundred and fifty samples from each lobe of prostate were obtained from 125 patients with suspected prostate cancer by transrectal polyfocal biopsy. Blood samples were drawn from the same patients for ELISA. RESULTS: Out of 125 patients with suspected prostate cancer, 20.5% were positive for Mycoplasma by PCR. Between studied species, only M. hominis was found in big proportion of analyzed samples. Out of 118 serum samples, 30.5% were positive for IgG to M. hominis in ELISA. CONCLUSION: Fact of presence of Mycoplasma species in tissue of prostate was established in 20.5% pf patients with suspected prostate cancer. Obtained results show that M. hominis is frequently infects prostate tissue and that this infection was more common in patients with high grade prostatic interstitial neoplasia and prostate cancer than in patients with benign changes of prostate tissue or in persons without prostate disease. This allows to suggest that infection with M. hominis could play an important role in development of cancer.

[Mechanisms of interaction between HIV and Mycoplasma arginini in vitro].

AIM: Until now, the problem of effective therapy of HIV-infection is not resolved due to integrative type of interaction of HIV virus with target cell - T-lymphocyte. The study was aimed on search of method of deletion of HIV DNA-provirus from cell's genome. MATERIALS AND METHODS: Non-pathogenic for humans Mycoplasma arginini was used for coinfection of HIV-infected cells in model systems in vitro. RESULTS: Complex of mechanisms was documented leading to: blocking up to 50 - 60% of extracellular virus (according to titration results), cancel of apoptosis in infected cells stained on Hoechst, formation of defective vif(-) virions, which together with arginine-desaminase of M. arginini arrange permissive conditions for activation of cellular APOBEC3G with subsequent disruption of DNA- provirus and blocking of viral infection. As studies of ultrastructure showed, listed events resulted from direct interaction of HIV with mycoplasma. CONCLUSION: The elimination of HIV DNA-provirus is possible by co-infection of T-lymphocytes with M. arginini.

[Persistence of Mycoplasma hominis and Ureaplasma urealyticum in organism of infected animals].

AIM: To study the possibility of existence of antigenemia during urogenital mycoplasmal infections by detection the antigens of agents in blood and viscera of infected animals. MATERIALS AND METHODS: Rabbits and mice were intraperitoneally inoculated with Mycoplasma hominis and Ureaplasma urealyticum, their antigens and DNAs. Samples of blood and visceral organs were studied by several methods: cultural with use of standard media, PCR, RT-PCR, indirect hemagglutination test, and immunofluorescence assay for detection of antibodies. RESULTS: Bacteremia with M. hominis develops during 2 months after inoculation in rabbits and 3 weeks after inoculation in mice. Antigens of M. hominis and U. urealyticum were detected in serum and visceral organs significantly frequently than live cells and DNAs. Prolonged preservation of the antigens in animals' blood and viscera after intraperitoneal administration of "pure" antigens points to the presence of true mycoplasmal antigenemia. Forms of existence of antigens in organism are different-they can represent corpuscular antigens as well as soluble molecular compounds circulating in blood both in free state and in structure of immune complexes. Antigens as well as live cells are preserved in all studied organs. CONCLUSION: Inoculation of rabbits and mice with M. hominis or U. urealyticum resulted in development of generalized infection with persistence of the agent in all studied organs during initial phase of infection and predominant persistence in organs of immunogenesis during later phases.

[Preservation of antigens and DNA of urogenital mycoplasmas in circulating immune complexes].

AIM: To study the time of preservation of antigens and DNA of urogenital mycoplasmas in circulating immune complexes (CIC) in blood of rabbits after single inoculation. MATERIALS AND METHODS: Rabbits were inoculated with Mycoplasma hominis and Ureaplasma urealiticum cell cultures washed in fetal calf serum. Reaction of aggregate-hemagglutination, immunofluorescence assay and PCR were used for detection of mycoplasmas' antigens and DNA. RESULTS: It was shown that DNA and antigens of M. hominis persist in free state and in structure of CIC during 1 month and 3 months respectively. In immunized rabbits antigens and DNA of mycoplasmas were detected in CIC structure even 6 months after the last immunization. Pattern of detection of DNA and antigens of U. urealyticum in blood of inoculated rabbits consists in that both DNA and antigens of the microorganism were detected in structure of CIC in blood samples during 70 days, whereas in free state they were detected only during 35 days. Incomplete elimination of CIC is possibly related to their small size (11S and lower) that allows them to circulate for a long time. CONCLUSION: Prolonged persistence of antigens and DNA of mycoplasmas in CIC structure is a fact that requires refinement of diagnostic criteria used for control of effectiveness of etiotropic therapy.

[Lipid-associated membrane lipopeptides of M. arginini activate NF-kB by interacting with TLR2/1, TLR2/6, and TLR2/CD14].

Various strains of mycoplasmas cause activation of transcriptional factor NF-kB as a result of interaction with different combinations of Toll-like receptors (TLR). It is well known that the MALP-2 protein of M. fermentans activates the NF-kB through interaction with the TLR2/6, lipid-associated membrane lipopeptides (LAMPs) of M. penetrans through the TLR1/2, LAMPs of M. pneumoniae through combinations of Toll-like receptors (TLR2/6 and TLR1/2), and superantigene of M. arthritidis through the TLR2 and TLR4-dependent pathways. In this study, we defined specific Toll-like receptors for LAMPs of M. arginini. For carrying out the research we used cell lines 293-null, 293-hTLR2, 293-hTLR1/2, 293-hTLR2/CD14, 293-hTLR2/6, 293-hTLR4/ CD14-MD2 expressing certain combinations of TLR and their coreceptors. It was shown that LAMPs of M. arginini cause activation of NF-kB interacting with TLR2/1, TLR2/6 and TLR2/ CD14, but not with TLR2 alone or TLR4.

[Serum detection of human urogenital mycoplasma].

The authors compared the detection rate of DNA of urogenital Mycoplasmas (Ureaplasma spp. and M. hominis) in the urogenital tract (UGT) and serum samples by polymerase chain reaction. Testing the smears and serum samples from the same patients (n = 112) showed that Ureaplasma was more frequently detected in the UGT smears than in the serum samples. There was the same trend towards M. hominis although the difference was not so significant. Therefore, the detection of these microorganisms in UGT is of more informative value for diagnostic purposes. Examination of the serum samples for these purposes provides useful information in understanding the mechanisms of generalization of Mycoplasma infections and studying the routes and modes of spreading these bacteria in the organism.

[Mycoplasma M. arginini infection induces constitutive activation of NF-kappaB and inhibits apoptosis in cells expressing toll-like receptors TLR2/6].

NF-kappaB is one of the main transcriptional factors that is responsible for cell survival under stresses. It was shown that various species of mycoplasma and their structural components were able to stimulate NF-kappaB activation as a result of their interaction with specific toll-like receptors on eukaryotic cell surface. Based on these studies, we suggested that activation of NF-kappaB in response to mycoplasmal infection could enhance the resistance of infected cells in response to proapoptotic stimuli. In this study we showed that infection of cells expressing toll-like receptors TLR2/6 with mycoplasma M. arginini leaded to suppression of apoptosis induced by chemotherapeutic agents (cisplatin, 5-fluorouracil, taxol).

[Duration of preservation of viable cells, DNA, and antigens of Mycoplasma hominis and ureaplasmas in human serum at 37 degrees C].

How long the viable cells of M. hominis, Ureaplasma spp., U. urealyticum, U. parvum, and their antigens retained in human serum at 37 degrees C was investigated. M. hominis cells were shown to hold their viability within 12 days with a gradual titer drop, the antigens being also detected within 12 days whereas intracellular and extracellular DNAs were seen within 40 days (an observation time). Under the same conditions, Ureaplasma cells died after 24 hours, their antigens were disrupted following 3 days and intracellular and extracellular DNAs of different species were detectable by polymerase chain reaction (PCR) within 17-40 days. The long preservation of extracellular and dead cell DNAs suggests that diagnostic examination of patients by means of PCR may yield false-positive results.

[Immunologic parameters of monkeys infected by urogenital mycoplasmas].

In monkeys contained in captivity conditions in open-air cages or in group cages human mycoplasmas are often detected: antigens of Mycoplasma hominis in blood serum were revealed in 33.3% of cases, and antibodies to it--in 15.6% of cases. IgM to M. hominis were detected more often than IgG. In 8 monkeys both types of immunoglobulins were detected. Rates of detection of Ureaplasma urealyticum antigens and specific antibodies were 43.1% and 31.1% respectively, and IgG were found more frequently than IgM (in 22 cases both types of immunoglobulins were revealed). High rates of M. hominis and U. urealyticum antigens and antibodies detection in blood serum of both healthy monkeys and monkeys with urogenital tract diseases show prevalence of human mycoplasmas carriage among monkeys contained in captivity conditions.

[Mycoplasmas in non-gonococcal urethritis].

The paper presents data on the etiological role of different types of genital mycoplasmas, such as U. urealyticum (U), M. hominis (Mh), M. genitalium (Mg), in the development of non-gonococcal urethritis (NGU). A hundred and twenty patients with acute and chronic urethritis were examined. A control group comprised clinically healthy males. Polymerase chain reaction and cultural tests more frequently revealed genital Mycoplasmas in patients with acute and chronic urethritis than in the controls, Uu being more common than other mycoplasmas. Uu antibodies were detected only in some part of the infected, more frequently in acute urethritis. Class G antibodies to Mh were equally identified in healthy individuals and patients. Criteria for the involvement of Mycoplasma infection in the development of NGU are presented.

[The mycoplasma infection rate in children with bronchial asthma].

A total of 167 children with bronchial asthma (BA) have been examined for the mycoplasma infection rate. Among investigated patients 62,8% were infected with one or more mycoplasma species. The prolonged persistence in patient body as well as biological properties of mycoplasmas give grounds to consider these agents as a risk factor in the development of the allergy-infection-borne BA and its relapses.

[The comparison of mycoplasma detection methods in the urogenital tract infection].

Six different methods have been employed to detect M. hominis (Mh) and U. urealyticum (Uu) in clinical samples collected from 67 men. The results obtained by PCR and IF test were approximately equal: 13.6 and 13.44%--Mh and 44.4 and 48.8%--Uu, respectively. Mycoplasmas were detected by cultural method less frequently (9.6%--Mh, 32.2%--Uu). The highest infection rates were obtained in the test for blood antigens (40%--Mh and 63%--Uu). At present a commercial diagnosticum to detect mycoplasma antigents in blood is lacking. Sometimes the results of cultural method are positive, while the PCR results are negative. So the optimal scheme based on both PCR and culture has been proposed.

[Detection of mycoplasma antigens and immune complexes in sera of the children with bronchial asthma].

The study was targeted at revealing Mycoplasma pneumoniae (M.p.) and Mycoplasma hominis (M.h.) antigens in blood samples of children with bronchial asthma (BA),both in a free state and those included in circulating immune complexes (CIC). The mycoplasma antigens of one or both species have been detected in one third out of 62 patients with BA. In this group of patients the frequency of detection of specific antibodies twice exceeded that of mycoplasma antigens. Testing paired blood samples of children with BA (n=26) showed, that at receipt in a hospital and a month after primary examination the mycoplasma antigens were detected in 16 and 12 patients, respectively, the association of M.p. and M.h. antigens being more frequent. Data on distribution of antibodies according to immunoglobulin classes testify that basically two (M, G), or four (M, G, A, E) classes were registered in children, the M class antibodies in high percentage of cases (from 36.6 up to 50.0%)being detected in every term of examination. These data indirectly testify that the antigens can be partly included in the CIC structure. The level of the total CIC content in BA children's blood sera one month after hospitalization twice exceeded the value detected at primary examination. Three months later later this parameter decreased not reaching the control value. The differential analysis of the precipitated CIC within the whole period of examination showed that mycoplasma antigens were present in the CIC structure in 87.4 - 65.0% of cases. The data obtained precondition future studies on the role of mycoplasma and M.p. and M.h. antigens included in the CIC in the pathogenesis of BA.