[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.

Nonthermal plasma affects viability and morphology of Mycoplasma hominis and Acholeplasma laidlawii.

AIM: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. METHODS AND RESULTS: 10(8) CFU ml(-1) agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10- and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10(5) and 10(3) drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l(-1) antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H2O2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. CONCLUSIONS: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

[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.

[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.

[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.

[A novel method of concentrating bacterial suspensions for transmission electron microscopy]. / Novyi metod kontsentrirovaniia bakterial'nykh suspenzii dlia transmissionnogo élektronno-mikroskopicheskogo analiza.

Presents a new method for making preparations of bacteria and their unculturable forms from suspensions with low concentrations of cells, from 1 x 108 to 1 x 104 per ml. to be examined under transmission electron microscope. The novelty of the method consists in the technique of concentrating bacteria by making a colloid solution consisting of bovine serum albumin and polyethyleneglycol. Associations of protein molecules, polyethyleneglycol, and bacteria, forming in the solution, are sedimented by centrifugation after 48-hour incubation at 4 degrees C. The solidity of the resultant macroscopic sediment is sufficient for studying the cells contained in it under transmission electron microscope.

[Use of the polymerase chain reaction for studying the processes of Salmonella typhimurium cells to an noncultivated state]. / Ispol'zovanie metoda polimeraznoi tsepnoi reaktsii dlia izucheniia protsessa perekhoda kletok Salmonella typhimurium v nekul'tiviruemoe sostoianie.

The possibility to identify noncultivating forms of Salmonella by the polymerase chain reaction (PCR) has been shown. To do it the technique for Salmonella identification was elaborated, based on amplification of a 500 bp fragment of araC gene. Time course of populations of two Salmonella typhimurium strains during prolonged incubation in water was studied by the techniques of serial dilutions on solid nutrient media, acridine orange staining, and PCR. The strains differed in pathogenicity levels and genetic characteristics. Cells of nonvirulent strain were shown to loose gradually in the process of incubation the ability to grow on solid nutrient media and transform into noncultivating forms whose ability to proliferation can be restored under definite conditions. No difference in the dynamics identified by PCR or traditional microbiological techniques was found for population of virulent Salmonella typhimurium incubated in water indicating the possibility of fast degradation of cells having lost the ability to divide.

[State of coronary circulation and results of 24-hour ECG monitoring in patients with refractory angina pectoris]. / Sostoianie koronarnogo krovoobrashcheniia i rezul'taty sutochnogo monitorirovaniia EKG u bol'nykh s refrakternoi k terapii stenokardii.

The paper compares the data of selective coronary angiography and 24-hour monitoring in 39 patients with refractory angina pectoris. Transient ST-segment changes were revealed in 79% of the patients, ST-segment depression occurring in 54%, ST-segment elevation in 46%, T-wave inversion in 38%. A combination of ST-T interval changes and occurrence of ventricular premature contraction was found in a third of the examinees. There was no statistically significant correlation between the extent of major coronary lesion and the mean length of ST-segment displacements.

[Transcutaneous transcatheter use of laser recanalization of coronary arteries in patients with ischemic heart disease]. / Chrezkozhnoe chrezkateternoe primenenie lazernoi rekanalizatsii koronarnykh arterii u bol'nykh ishimicheskoi bolezn'iu serdtsa.

The paper discusses the potential possibility and effectiveness of X-ray endovascular laser recanalization (ELR) of the coronary arteries in order to treat coronary atherosclerosis in patients with coronary heart disease. The intervention was performed in 4 patients (into the anterior interventricular artery in 3 and into the right coronary artery in 1). In 3 of 4 cases, X-ray ELR proved to be successful, in one case the intervention failed due to technological reasons. Recanalization of a completely occluded segment of the coronary artery with a residual stenosis of no more than 40% was observed in two cases. Laser recanalization of profound local coronary stenosis was made in the mid-third of the vessel in one case. It can be stated that X-ray ELR of the coronary artery may extend the scope of X-ray surgical therapeutical tools of the treatment of coronary atherosclerosis. At the same time, accumulation of clinical experience and further improvement of laser and laser catheter engineering are essential in defining the value and possible scope for the application of this method.

[The detection of antigenemia in typhoid patients at different periods in the course of the infection]. / Vyiavlenie antigenemii u bol'nykh briushnym tifom v raznye periody techeniia infektsii.

The enzyme immunoassay of serum samples, obtained from 37 typhoid patients and previously subjected to bacteriological study with negative results, permitted the detection of S. typhi specific antigens and, as a consequence, the rapid diagnosis of the disease. The prevalence of various antigenic components of S. typhi was observed at different stages of infection. The presence of surface O and Vi antigens was characteristic of acute stages of S. typhi infection. At the same time L-form antigens, in rare cases in combination with surface O and Vi antigens, were characteristic of patients at the period following the acute stage of the disease, as well as chronic carriers. This is indicative of the necessity of introducing specific antibodies to the antigenic determinants of S. typhi L forms into the assortment of diagnostic immune preparations.

[The antigenic characteristics of Salmonella typhi L forms]. / Izuchenie nekotorykh antigennykh osobennostei L-form Salmonella typhi.

The study of the possibility of detecting the specific antigens of S. typhi L forms has revealed that out of three destructive methods under study (osmotic lysis, freezing-thawing, sonication) only ultrasonic disintegration has proved to be effective for S. typhi L forms. Three specific fractions capable of interacting only with specific antibodies to S. typhi L forms have been revealed in the course of chromatographic separation of the soluble antigenic complex of S. typhi stable L forms and the subsequent analysis of the fractions thus obtained in the enzyme immunoassay.

[The possibilities of using variants of immunoenzyme analysis for detecting L-form Salmonella typhi bacteria in biological fluids]. / Izuchenie vozmozhnosti ispol'zovaniia razlichnykh variantov immunofermentnogo analiza dlia vyiavleniia L-form briushno-tifoznykh bakterii v biologicheskikh zhidkostiakh.

The possibility of using, on principle, the enzyme immunoassay (EIA) in different modifications for the detection of S. typhi L-forms in biological fluids (blood, urine) was established. The inhibiting variant of EIA showed the highest sensitivity: 1 ng/ml. The direct sandwich variant permitted the quantitative determination of the antigen of S. typhi L-form in the widest range of 20-500 ng/ml. The indirect enzyme immunometric variant permitted the detection of S. typhi L-forms with a sensitivity of 10(5) colony-forming units per ml only in urine.