[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.

[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.

[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.

[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.

[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.

[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 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.

[Influence of environmental factors on the expression of Mycoplasma pneumoniae gene responsible for the synthesis of protein P1 adhesion]. / Vliianie faktorov vneshnei sredy na ékspressiiu gena Mycoplasma pneumoniae, determiniruiushchego sintez belka adgezii P1.

The effect of different physical and chemical factors on the process of transition of persisting mycoplasmas into active virulent state was studied to find out conditions promoting the development of acute infection in the course of chronic infection. The activity of the gene coding the synthesis of protein P1, the main pathogenicity factor, was evaluated in the reverse transcriptase test--PCR under conditions of heat and cold shocks, oxidation stress, varying osmotic pressure. An increased osmotic pressure, heat and cold shocks were shown to induce the transcription activation of the gene coding the synthesis of P1 in the avirulent strain, and consequent restoration of its adhesive properties indicative of virulence. M. pneumoniae avirulent strain was characterized by greater resistance to oxidation stress and to a rise in osmotic pressure, this property requiring further study. Quite probably, M. pneumoniae DNA-binding proteins earlier detected in persisting and avirulent cultures by taking part in the compactization of DNA contribute to the adaptation of mycoplasmas to different stress influences. There are grounds to suggest that M. pneumoniae possess mechanisms regulating the expression of genes, in particular the gene coding the main pathogenicity factor, under the influence of different environmental factors.

[Correlation between activity of the phosphoenol-pyruvate-dependable phosphotransferase system (PTS) and synthesis of adhesion P1 protein in Mycoplasma pneumoniae]. / Vzaimosviaz' mezhdu aktivnost'iu fosfoenolpiruvatzavisimoi fosfotransferaznoi sistemy i sintezom belka adgezii P1 u Mycoplasma pneumoniae.

Three isogenous strains M. pneumoniae, i.e. virulent FH, avirulent FH400 and a revertant with a restored virulence (FHR) and isolated from an avirulent strain, were studied. The mechanism of hemadsorption and the ability to cause an infection in Syrian hamsters were found to be damaged in the avirulent strain. The detection of a specific mRNA by the RT-PCR method showed, apart from the loss of the main adhesin (protein P1), a lack of general components of the phosphoenol-pyruvat-dependable phosphotranspherase system (PTS), i.e. enzyme 1 and protein HPr. The recovery of virulence by passing an attenuated strain through animals with induced immunodeficiency correlated with the recovery of the activity of a gene encoding the P1 adhesion protein and with the onset of the PTS function activity. An analysis of published data was made use of to try to detect a correlation between the functional PTS activity in cell and virulence of M. pneumoniae.

[Differential activity of genes of Mycoplasma pneumoniae in acute and chronic forms of experimental infection]. / Differentsial'naia aktivnost' genov u Mycoplasma pneumoniae pri ostrykh i khronicheskikh formakh éksperimental'noi infektsii.

The complex of investigations was carried out with a view to study the mechanisms making it possible for mycoplasmas to switch off the expression of definite genes in persistence. The main method used in these investigations was the method of molecular portrayal, or RNA fingerprinting, with the use of arbitrary primers. The comparative analysis of gene expression in M. pneumoniae strains having different virulence revealed that in the avirulent strain subcultured under in vitro conditions the expression of considerable number of genes was inhibited. In the body of an experimental animal the expression of many genes, including the gene coding the synthesis of the main pathogenicity factor, adhesion protein, switched on.

[Creation of a controlled model of respiratory mycoplasmosis]. / Sozdanie upravliaemoi modeli respiratornogo mikoplazmoza.

To create the controlled model of respiratory mecoplasmosis, laboratory animals with induced immunodefeciency were infected with M. pneumoniae strain having different degrees of virulence. Immunodeficient state was induced in susceptible animals (hamsters) by the injection of cyclophosphamide. The infection of immunodeficient animals with a virulent strain induced the development of severe manifest pneumonia with 50% mortality rate. The infection of the animals with induced immunodeficiency with the avirulent strain attenuated in the process of 10-year subculturing in acellular media led to the development of moderate pneumonia characterized by the proliferation of the infective agent in pulmonary tissues and the presence of pathomorphological changes. The simultaneous infection of control hamsters with the same strain did not induce the development of infection. The infection of the experimental animals with the avirulent strain in the presence of induced immunodeficiency resulted in the partial restoration of the virulent properties of the strain, which was manifested by the activation of the capacity of the infective agent for colonization and proliferation in body tissues of the animals.

[The isolation and partial characterization of the cell wall proteins of Listeria monocytogenes]. / Vydelenie i chastichnaia kharakteristika belkov keltochnoi stenki Listeria monocytogenes.

The preparative scheme for the purification of proteins with molecular weights of 39 and 79 kD, obtained from L. monocytogenes membrane fractions, has been developed. This technology included the cultivation of bacteria in heart-brain broth, isolation of bacterial membranes, the extraction of their components with Triton-X-100 and chromatography on Superose columns. The purified proteins have been shown to form structures with a molecular weight of 500-100 kD and pl 4.7 in water solutions.

Induction of the immune response to Legionella pneumophila cytolysin by monoclonal anti-idiotypic antibodies.

A panel of mAb (IgG1, IgG3, IgM) against Legionella pneumophila cytolysin (CL)-protease of 37 kDa was obtained. Subtyping of L. pneumophila strains of serogroup 1 by using mAb against CL (mAb-CL) was carried out. The results of comparative analysis of the specificity of mAb-CL and the panel of mAb kindly provided by Dr. J. M. Barbaree (Centers for Disease Control, Atlanta, GA) allowed us to recommend mAb-CL to be used as a diagnostic tool to reveal the pathogenicity of L. pneumophila strains of serogroup 1. Hybridomas were also raised in a syngenic system which produced anti-idiotypic mAb (mAb2) against anti-CL mAb B6/1. The Ab2 belonged to Ab2 gamma type: 1) Ab2 reacted with B6/1 Id only, 2) Ab2 inhibited the interaction of B6/1 Ab1 with CL, and 3) CL inhibited the reaction of Ab2 with Ab1. The use of Ab2 allowed us to show that B6/1 Id is expressed in 4 to 32% of serum antibodies during the primary and secondary immune responses of BALB/c mice to CL. Ab2 induced the production of anti-anti-idiotypic antibodies (Ab3) in BALB/c mice, and some of them reacted with CL. Thus, we have demonstrated the possibility of inducing an antibody response to CL (one of the main L. pneumophila pathogenic factors) in intact syngenic mice with anti-idiotypic antibodies.

[The typing of Legionella pneumophila strains by using monoclonal antibodies to the cytolysin]. / Tipirovanie shtammov Legionella pneumophila s ispol'zovaniem monoklonal'nykh antitel k tsitolizinu.

Studies on the typing of L. pneumophila strains of serogroup 1, isolated from patients and environmental objects, have been made with the use of monoclonal antibodies (McAb) to cytolysin. The results of the comparison of the specificity of our McAb with that of a commercial set McAb obtained from the USA make it possible to recommend preparations based on McAb to cytolysin for the detection of L. pneumophila pathogenic strains of serovar 1. The use of FITC- and peroxidase-labeled McAb to cytolysin permits the reduction of the time necessary for the diagnosis of Legionella infections and the detection of the antigen in a dose of 10 ng/ml.