[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.

[Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens].

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.

[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.

[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.

[Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens]. / Immunolobiologicheskie svoistva monoklonal'nykh antitel k antigenam Mycoplasma hominis.

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.

[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.

[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.

[Monoclonal antibodies to Mycoplasma pneumoniae antigens]. / Monoklonal'nye antitela k antigenam Mycoplasma pneumoniae]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.

[The differentiation of the antigens making up the circulating immune complexes]. / Differentsiatsiia antigenov v sostave tsirkuliruiushchikh immunnykh kompleksov.

A simple method for the detection and analysis of circulating immune complexes (CIC) in specimens of biological fluids is proposed. The method was approved in the examination of patients with chronic infections caused by mycoplasmas and Streptococcus pyogenes L-forms. The method made it possible to diagnose infectious diseases accompanied by the formation of immune complexes and to study the dynamics of the processes of the accumulation and elimination of CIC in the course of the disease. Thus, the detection rate of specific antigens (Ag) incorporated into CIC in patients with mycoplasmal pneumonia exceeded 90 %. In children aged up to 1 year this rate decreased to 40 %. The diagnostic value of the determination of specific Ag incorporated into CIC was shown in streptococcal infections caused by S.pyogenes L-forms, viz. in frequently relapsing erysipelas, as well as in subacute rheumatism and in infectious allergic myocarditis.