[PERSISTENCE OF MYCOPLASMA DURING UROLITHIASIS].

AIM: Study the frequency of detection of mycoplasma and ureaplasma in clinical material from urolithiasis patients. MATERIALS AND METHODS: Clinical material samples (blood sera, urine, uroliths) from 31 urolithiasis patients were obtained during operations of urolith-removal. Cultural method, LAR and PCR were used in the study. RESULTS: The study of clinical material from 31 patients by PCR has shown, that in 25 individuals. (80.6%) DNA of mycoplasma and ureaplasma was detected, and mycoplasma DNA was more frequently detected in uroliths and less--in-blood sera. Mycoplasma hominis DNA was detected in clinical material of a significantly largerninmber of patients. 23 cultures were isolated from 8 patients by a cultural method, that were identified by PCR as M. hominis. All the isolates have grown as "mini colonies". Even after multiple passages in agar medium, reversion of "mini-colonies" into colonies with a classic morphology was not obtained. CONCLUSION: A high frequency of detection of mycoplasma and ureaplasma in clinical material of patients with urolithiasis was established. The isolated M. hominis cultures have only grown as "mini-colonies". The phenomenon discovered could give evidence on high variability of mycoplasma and a possibility of existence of previously unknown form of their persistence in human organism.

[MICROBIAL COMMUNITIES ON KIDNEY STONES].

The clinical material obtained surgically in patients with kidney stone disease (KSD) was tested for content of the stone microflora using PCR and standard microbiological methods. It was demonstrated that about 50% of stones in patients with KSD were infected with various infection agents as observed using standard microbiological and molecular genetic methods. The percentage of detection of the Mycoplasma hominis using cultural method is lower than the percentage detected using PCR, which is due to difficult isolation and cultivation, as well as DNA fragments of mycoplasma observed after antibiotic therapy. Studies based on modern microscopy methods showed that microorganisms on the surface of the kidney stone formed multispecies biofilms.

[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.

[Dynamics of change of ureaplasma laboratory strain titers and quantity of their DNA in transport medium at varying temperature].

AIM: Study of preservation dynamics of ureaplasma laboratory strain live cultures and their DNA in transport medium at varying temperature. MATERIALS AND METHODS: The study was carried out in laboratory strains Ureaplasma urealyticum serotype 8 and Ureaplasma parvum serotype 1. The quantity of live ureaplasmas was determined by method of tenfold dilutions in liquid medium. The growth of ureaplasmas was registered by changes in the color of the cultivation medium due to its alkalization by metabolism products and expressed in CCU/ml. DNA quantity in samples was determined by real time PCR performed by using Florocenosis-micoplasmas-FL test system produced by ILS. RESULTS: Live ureaplasmas wer shown to be preserved in transport medium at 4 degrees C for 12 - 29 days, at 18 - 22 degrees C--for 9 - 20 days and at 37 degrees C--for only 2 days. In samples incubated at 37 degrees C the quantity of live ureaplasmas increased and then sharply decreased to 0, at lower temperature titers of the cells decreased smoothly. The quantity of ureaplasma DNA in the process of their incubation did not change significantly. CONCLUSION: Fundamental differences in the duration of survival of U. urealyticum strain and U. parvum strain in transport medium at varying temperature were not detected. Based on the studies performed a practical conclusion can be drawn that in cases of emergency when clinical material transportation is necessary its storage in transport medium for several days is acceptable.

[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.

[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.

[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.

[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.

[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.

[Drug sensitivity of Ureaplasma urealyticum, persisting in patients with chronic inflammatory diseases of the urogenital tract]. / Chuvstvitel'nost' k lekarstvennym preparatam Ureaplasma urealyticum, persistiruiushchikh v organizme bol'nykh pri khronicheskikh vospalitel'nykh zabolevaniiakh urogenital'nogo trakta.

A total of 65 U. urealyticum cultures isolated from patients with chronic inflammatory diseases of the urogenital tract after their prolonged persistence of in the human body, were studied for sensitivity to medicinal preparations of different groups: tetracyclines (tetracycline and doxycycline), macrolids (erythomycin, clarithromycin, midecamycin, josamycin), quinolon (pefloxacin), amino glycoside (gentamicin), lincoamides (lincomycin, clindamycin). The majority of isolated U. urealyticun were highly sensitive to josamycin, clacide, doxycycline (89.2, 84.6, 76/9% respectively), and somewhat lesser number of these organisms were highly sensitive to midecamycin and pefloxacin (51.3 and 44.4% respectively). Among U. urealyticum strains circulating in the Moscow region some strains which persisted in patients with chronic inflammatory diseases of the urogenital tract for a long time were found to be resistant to erythromycin (23.1%), tetracycline (19.5%), and in very rare cases (1.6%) they were found to multiple drug resistance to all preparations under study. In view of the varying sensitivity of the clinical isolates of U. urealyticum to medicines and the presence of resistant forms in their population, the sensitivity of the isolated U. urealyticum should be determined in vitro prior to drug therapy.

[Detection of antigens with the aid of the aggregate-hemagglutination reaction of different serotypes of Ureaplasma urealyticum in the blood serum of patients with inflammatory diseases of the urogenital tract and disorders of reproductive function]. / Obnaruzhennye s pomoshch'iu reaktsii agregat-gemaggliutinatsii antigenov razlichnykh biovarov Ureaplasma urealyticum v syvorotke krovi patsientov s vospalitel'nymi zabolevaniiami urogenital'nogo trakta i narusheniiami reproduktivnoi funktsii.

A total of 110 samples of the blood serum, obtained from persons with inflammatory diseases of the urogenital tract and with disorders of the reproductive function were examined by the aggregate-hemagglutination reaction for antigens of the 3d and 8th serotypes, U.u., belonging, respectively, to biovars Parvo and T-960. Antigens U.u. of the 3d, 8th or of both serotypes were detected in 75 (68.18%) of the studies samples. There were no antigens U.u. of the 3d or 8th serotypes in 35 (31.82%) samples. Antigens of biovars Parvo and T-960 were detected at an approximately identical frequency rate. Besides, the mentioned antigens were found in almost one half of blood sera positive to the U.u. samples (48%). No significant differences between the concentrations of antigens Parvo and T-960 were detected in the blood serum samples in case both of them were found in one sample or only one ureaplasma serovar was found in a sample.

[Application of different methods in the diagnosis of respiratory mycoplasmosis]. / Opyt primeneniia razlichnykh metodov diagnostiki respiratornogo mikoplazmoza.

A complex of methods for the detection of Mycoplasma pneumoniae and Mycoplasma hominis in children and adults with respiratory diseases (acute, chronic and obstructive bronchitis, pneumonia, recurring croup, bronchial asthma), as well as in children frequently having acute respiratory diseases, has been worked out and tested. Both infective agents are frequently detected in the above mentioned pathological processes as monoinfection or mixed infection. Mycoplasma antigens are capable of prolonged (up to 1 year) persistence in the patient body in spite of etiotropic therapy and the presence of specific antibodies. The method of the preliminary treatment of specimens for the polymerase chain reaction is proposed: the specimens are subjected to prolonged deproteinization, which makes it possible to detect M. pneumoniae in some cases of chronic infection when it cannot be detected by routine methods.

[The use of polymerase chain reaction for detection of tetracycline resistance urogenital mycoplasma ]. / Primenenie polimeraznoi tsepnoi reaktsii dlia vyiavleniia teratsiklinustochivykh shtammov urogenital'nykh mikoplazm.

PCR was used for detecting tetracyclin resistance genes tet-M and tet-O in Mollicutes. The tet determinants were detected in U. urealyticum and M. hominis genomes in 60 and 57.14%, respectively. PCR was used to detect urogenital mycoplasma in patients with different urogenital infections. The rate of detection of U. urealyticum was 10.71-25.1% in males and 23.08-61.91% in females, for M. hominis these values were 6.25-11.45% and 3.85-22.99%, and for M. genitalium 6.25-29.64% and 4.76-30.0%, respectively. Hence, the detection rate of tet determinants in mycoplasma by PCR was high (7-60%) in all groups of patients.

[Mechanisms of urogenital mycoplasma and the methods for their detection]. / Mekhanizmy persistentsii urogenital'nykh mikoplazm i metody ikh vyiavleniia.

The mechanisms which determine the prolonged persistence of mycoplasmas in the infected body are presented. The comparative evaluation of the methods used for the detection of mycoplasmas, their antigens and antibodies to them in different biological substrates has been made. As shown in this study, the most informative methods, similar in sensitivity are agregate hemagglutination and immunofluorescence tests, polymerase chain reaction. In the immunofluorescence test commercially licensed test systems produced by the Gamaleya Research Institute are used. The methods and test systems have been approved in the examination of large groups of patients with urogenital pathology. The studies have shown that exact data on the presence of infection in patients can be obtained by examination with the use of several methods.

[Detection of tetracycline- and erythromycin-resistant urogenital Mycoplasma strains using PCR]. / Vyiavlenie tetratsiklin- i éritromitsinustoichivykh shtammov urogenital'nykh mikoplasm s pomoshch'iu PTsR.

The use of PCR made it possible to obtain data on the occurrence of genital mycoplasms in subjects with different pathological states of their urogenital system. The detection rate of U.urealyticum was 10.7-25.1% in males and 23.0-61.9% in females. The detection rate of Mycoplasma hominis was 6.2-11.4% in males and 3.8-22.9% in females. The detection rate of M.genitalium was 6.2-29.6% in males and 4.7-30.0% in females. The study carried out by means of amplification test systems for the detection of genes controlling resistance to tetracycline (tet-M and tet-O) and to erythromycin (erm) demonstrated that U.urealyticum contained the tet-determinant in 60% of cases and M.hominis, in 57.14% of cases. As revealed with the use of PCR techniques, the occurrence of erythromycin-resistant Ureaplasma strains was not high and was equal to 2.1%.

[Urogenital mycoplasmosis and Mycoplasma carriage during pregnancy and in inflammatory processes of the genitalia in workers in the electronics industry]. / Urogenital'nyi mikoplazmoz i mikoplazmonositel'stvo pri beremennosti i vospalitel'nykh protsessakh genitalii u rabotnits élektronnoi promyshlennosti.

To find out the spread of urogenital Mycoplasma carriership urogenital mycoplasmosis (UGM) among women living and working under similar conditions and making up risk groups with respect to these infections, pregnant women, gynecological patients and clinically healthy women were specially surveyed. As revealed in this survey, UGM and Mycoplasma carriership were found in clinically healthy female workers significantly more often than in other similar groups of the same region. In the group of pregnant women the occurrence of Mycoplasma carriership and UGM reached 90%. In cases of sterility the facts of asymptomatic Mycoplasma carriership and UGM were registered.

[The laboratory diagnosis of mycoplasmosis and ureaplasmosis in urological and gynecological patients]. / Laboratornaia diagnostika mikoplazmoza i ureaplazmoza u urologicherskikh i ginekologicheskikh bol'nykh.

The survey of 630 patients with urogenital pathology, habitual miscarriage and sterility revealed that they were mostly (91-100%) infected with M. hominis and/or U.urealyticum. This fact indicates the necessity of organizing the epidemiological control of these infections. It is expedient to use the complex of laboratory methods for diagnosing these infections through the effectiveness of such methods may vary in different nosological forms. Thus, colpitis and nonspecific urethritis were shown to be most effectively diagnosed by the serological methods.