[Non-gonococcal urethritis in men].

This literature review is dedicated to urethritis which is one of the most common disorders of urogenital tract in men. The current views in its etiology as well as problems in diagnosis with the main being the frequent inability to isolate etiological factor of inflammation it the urethra are described. The analysis of literature suggests a possible role of bacteria, which are associated with bacterial vaginosis in women, in the development of the urethritis in men. However, the frequency of such urethritis and causative role of specific pathogens has not been studied yet. Meanwhile, the exact determination of the causes of inflammation has direct influence on the choice of appropriate etiologic treatment and can increase its efficiency.

[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.

[The comparison of tests for qualitative evaluation of Ureaplasma parvum, Mycoplasma hominis: "Mycoplasma duo", "Ureaplasma microtest", "Mycoplasma microtest" and "AmpliSens-Florocenosis-mycoplasma-FL"].

The genital mycoplasma is an opportunistic bacteria and its detection is to be implemented in qualitative format. The study was organized to compare reagents kits "Mycoplasma Duo", "Ureaplasma Microtest", "Mycoplasma microtest" and "AmpliSens-Florocenosis-Mycoplasma-FL". The study resulted in high indicators of diagnostic sensitivity and diagnostic specificity for all kits. At that, the lowest indicators were registered under application of "Mycoplasma Duo" kit. The study reveled correlation of qualitative values detected by using cultural analysis and polymerase chain reaction. The reproducibility of qualitative values of cultural method occurred significantly lower in comparison with "AmpliSens-Florocenosis-Mycoplasma-FL " kit.

[Results of assessing the quality of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum DNA by polymerase chain reaction in 2006-2007)].

Analyzing the results of detection of C. trachomatis, M. hominis, and U. urealyticum DNA by the participants of the Federal External Quality Assessment System in 2006-2007 indicated that the share of the fluorescent-probe amplification technique both in the real-time mode and by the end-point tended to increase. The above tendency coincided with the other--the higher proportion of laboratories detecting M. hominis and U. urealyticum at low concentrations (as low as 10(3) copies/ml) although no direct caused-and-effect relation could be established between these two tendencies. In 2006-2007, the section participants demonstrated a sufficiently high specificity (94-98%) in the detection of C. trachomatis, M. hominis, and U. urealyticum DNA while the sensitivity of studies left much to be desired--the proportion of correct results at the concentration of DNA of the above microorganisms of 10(3) copies/ml was as high as 73-82%. The considerable share of laboratories that fail to detect microorganisms at low concentrations suggests that it is necessary to continue work to enhance the sensitivity of this important type of laboratory studies.

[PCR studies in laboratory diagnosis: problems of quality assurance].

The determination of DNA and RNA of causative agents by the nucleic acid amplification techniques (NAT) presented in the Russian laboratory diagnosis of infections solely by polymerase chain reaction (PCR) have currently found the widest application in world practice and, in some cases, is a basic procedure for detecting an infectious agent, particularly in the diagnosis of infections caused by human immunodeficiency virus, hepatitis C virus, hepatitis B virus, cytomegalovirus, Chlamydia trachomatis, Neisseria gonorrhoeae, and others. The paper presents the data available in the literature on the main reasons for false positive and false negative results in PCR studies, international and national programs for the outside quality control (OQC) of detection of causative agents for various diseases, by applying NAT. It is emphasized that the regular participation in OQC programs is likely to be useful in improving the quality of PCR studies.

[Esomeprasol and rabeprasol effects on esophageal acidification in patients with gastroesophageal reflux disease, intensively metabolizing inhibitors of proton pump].

AIM: To compare action of different esomeprasol and rabeprasol doses on esophageal acidification in patients with gastroesophageal reflux disease (GERD). MATERIAL AND METHODS: A cross-over study was made in 27 GERD patients (12 males and 15 females). It included esophagoduodenoscopy with biopsy for detection of H-pylori and 24-h pH-metry of esophageal content. Rabeprasol and esomeprasol efficacy was compared for daily doses 20 mg of each (stage I) and rabeprasol 20 mg and esomeprasol 40 mg (stage II). RESULTS: All the patients proved H-pylori positive. Before the treatment mean daily time of marked esophageal acidification was 29-36%. At stage I significant differences between the drugs were absent. At stage If esomeprasol was more effective from the first day of therapy both in relation to time to effect and its intensity. CONCLUSION: Esomeprasol provided a fast and noticeable correction of esophageal acidification. This relieves symptoms of GERD and accelerates esophageal mucosa epithelization.

[Development of "Amplisens-HCV-genotype" reagent set for identification of hepatitis C virus genotypes 1a, 1b, 2a and 3a]. / Razrabotka nabora reagentov "Amplisens HCV-genotip" dlia opredeleniia subtipov 1a, 1b, 2a, 3a virusa gepatita C.

Multiple alignments of 119 nucleotide sequences of isolates of hepatitis C virus (HCV) were carried out to choose the type-specific primers for the 5'-ultra-core fragment of viral genome for the purpose of detecting the HCV 1a, 1b, 2a, and 3a subtypes. A PCR kit of reagents was designed for the amplification of cDNA HCV with selected type-specific primers and for making the electrophoresis in agarous gel. The kit comprises the positive control samples, i.e. HCV genome fragments, subtypes 1a, 1b, 2a and 3a, cloned in the plasmid vector. 440 cDNAHCV samples were simultaneously tested by using the worked out reagents' set and according to the method of Ohno et al. The results were found to be concordant in 336 cases, and were discordant in 4 samples. A sequencing of the PCR products and phylogenetic analysis showed that 1 sample belonged to subtype 4a, 2 samples belonged to subtypes 2k and 1 sample--to subtype 31.

[Detection of Treponema pallidum DNA and RNA in clinical material from patients with syphilis at different stages]. / Vyiavlenie DNK i PNK Treponema pallidum v klinicheskom materiale u patsientov s razlichnymi stadiiami sifilisa.

The results of the development and approval of methods for the detection of T. pallidum DNA and 16S rRNA in clinical material (blood plasma, serous exudates) are presented. T. pallidum DNA was detected with the use of primers to the gene coding protein with a moleculat weight of 47 kD and T. pallidum RNA, with the primers to gene 16S rRNA. The isolation, reverse transcription and amplification of DNA and RNA was carried out in the presence of inner DNA and RNA control respectively. The analytical sensitivity of the developed method was 400 DNA copies per ml. The characteristics of analytical and diagnostic specificity were 100%. The specimens of blood plasma, taken from 292 patients with syphilis at different stages before specific antibacterial therapy, were tested by the PCR. The detection rate of T. pallidum DNA and RNA in blood plasma was, respectively, 91% and 100% in primary seropositive syphilis, 68% and 79% in secondary early syphilis, 19% and 26% in latent unverified syphilis. In secondary relapsing syphilis T. pallidum DNA and RNA were detected in 92% and in latent early syphilis, in 14% of patients. T. pallidum nucleic acids were detected in 1 patient at the seronegative period of primary syphilis. No positive result was obtained in the PCR analysis in any of the patients with diagnosed seroresistance, latent late syphilis and tertiary syphilis. In the study of material taken from chancres of 11 syphilis patients the data obtained by dark-field microscopy and the PCR analysis completely coincided.

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b]. / Kharakteristika moskovskikh shtammov Haemophilus influenzae tipa b metodom mul'tilokusnogo sekvenirovaniia-tipirovaniia.

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

[Effectiveness of lamivudine in the treatment of chronic HBeAg-negative viral hepatitis B: results of continuous therapy for 18 months]. / Effektivnost' lamivudina v lechenii HBeAg-negativnogo khronicheskogo virusnogo gepatita B: rezul'taty nepreryvnoi terapii v techenie 18 mes.

The efficacy of monotherapy with one of the analogues of synthetic nucleosides (lamivudine) was studied in 22 patients with chronic viral hepatitis B (CVHB). The specific feature of the selected group was that there was no serum HBeAg, which is usually regarded as a sign of viral mutation. The time course of changes in the activity of blood and hepatic inflammatory processes and viral replication were studied at equal intervals. There was a direct correlation between the suppression of viral replication and the regression of activity indices, such as ALT, serological markers, DNA of viral hepatitis B, and the morphological constituents of the Knodell index) during lamivudine therapy. However, there were no substantial changes in the parameters of fibrosis throughout the treatment. Therapy resistance was revealed in 3 of the 22 patients with CVHV. Its basis was the mutation of hepatitis B virus in the YMDD-motive, which was detected in 2 of the 3 patients.

[Comparative effectiveness of the antisecretory action of rabeprazole and esomeprazole in people with rapid metabolism of proton pump inhibitors]. / Sravnitel'naia éffektivnost' antisekretornogo deistviia rabeprazola i ézomeprazola u lits, bystro metaboliziruiushchikh ingibitory protonnogo nasosa.

AIM: To compare antisecretory effects of Rabeprazole and Esomeprazole in proton pump inhibitors extensive metabolizers in an open, randomized, two-way crossover study. METHODS: Sixteen GERD H. pylori-positive patients (8 men, mean age 49.6 and 8 women, mean age 49.3) with the homozygous extensive metabolizer genotype of CYP2C19 determined by polymerase chain reaction-restriction fragment length polymorphism analysis received Rabeprazole 20 mg or Esomeprazole 20 mg daily on days 1-6, with a 14-day wash-out period. Intragastric pH was recorded continuously on days 0, 1, 5 and 7. RESULTS: On days 1 and 5 no differences were found between Rabeprazole 20 mg and Esomeprazole 20 mg in 24-hours median pH (day 1: 5.9 versus 5.0; day 5: 6.45 versus 6.3) or in percent of time with pH 4 (day 1: 57.8% versus 50.5%; day 5: 81.4% versus 81.2%). On day 1, mean percent of time pH 4 were significantly greater after Esomeprazole 20 mg 52.6% (95% CI: 23.6-68.2) than Rabeprazole 20 mg 33.0% (95% CI: 15.3-48.2) during 0-6 h (p = 0.02). On day 7 (24 later the last dose), 24-hours median pH was higher after Esomeprazole 20 mg than Rabeprazole 20 mg (2.7 versus 5.05; p = 0.02). CONCLUSIONS: Rabeprazole 20 mg and Esomeprazole 20 mg are equally effective in increasing gastric pH in H. pylori-positive PPI extensive metabolizers on days 1 and 5. Esomeprazole 20 mg is more effective than Rabeprazole 20 mg in maintaining pH 4 during the first 6 hours on the first day and increasing of intragastric pH on the day 24 hours later the last dose.

[Stability of hepatitis C virus RNA in human blood plasma in a panel of control samples]. / Stabil'nost' RNK virusa gepatita C v plazme krovi cheloveka v paneli kontrol'nykh obraztsov.

The stability of hepatitis C virus (HPC) RNA concentration in 5 human plasma samples after storage at +22 degrees C for two months, at -20 degrees C, and +4 degrees C for six months after 10 freezing-unfreezing cycles was evaluated. In this study, the concentration of HCV RNA in the samples was stable after six months of storage at -20 degrees C. The concentration of HCV RNA decreased on the average of 92% after 2-month storage at +22 degrees C. After six months of storage at +4 degrees C and after 10 freezing-unfreezing cycles, that decreased by 28 and 42%, respectively. Based on their own findings, the authors developed a HCV-RNA panel containing 5 positive human plasma samples with RNA levels of 103-105 IU/ml. The panel may be recommended both for the standardization of PCR kits and for the intra- and interlaboratory quality control of PCR laboratories.

[Hematological syndromes in patients with chronic hepatitis C]. / Gematologicheskie sindromy u bol'nykh khronicheskim gepatitom C.

AIM: To examine hemopoiesis and to estimate proinflammatory cytokines in blood serum from 42 patients with chronic viral hepatitis (CVH) associated with 1-3 lineage cytopenia in the blood. MATERIAL AND METHODS: 42 patients with diagnostically complicated hematological picture suggestive of hemoblastosis were examined using standard tests, bone marrow puncture and trepanobiopsy, laparoscopy with biopsy of the liver and spleen, transcutaneous puncture biopsy of the liver under ultrasonic control, explorative laparotomy with splenectomy and liver biopsy and, on demand, laboratory tests for hemolysis, presence of antithrombocytic and antileukocytic antibodies, iron metabolism, karyological analysis of bone marrow cells was also made. RESULTS: The majority of the patients were diagnosed to have chronic hepatitis C with low activity of hepatic inflammation and frequent (55%) absence of the diagnostic antibodies to hepatitis C virus in the serum. In 86% of cases blood cytopenia reflected uneffective hemopoiesis and in 14% of cases hemopoiesis hypoplasia was found. CONCLUSION: Uneffective hemopoiesis, high content of immune response cells-effectors in the bone marrow and high concentration of TNF in the serum indirectly evidence for a pathogenetic relationship of chronic HCV infection with cytopenic hematological syndromes.

[Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones]. / Rol' mutatsii v A-sub''edinitse topoizomerazy IV Acholeplasma laidlawii PG-8B v formirovanii razistentnosti k ftorkhinolonam.

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.

[Analysis of regions determining resistence to fluoroquinolones in genes gyrA and parC in clinical isolates of Mycoplasma hominis]. / Analiz raionov, opredeliaiushchikh rezistentnost' k ftorkhinolonam, genov gyrA i parC v klinicheskikh izoliatakh Mycoplasma hominis.

Fifteen strains of M. hominis isolated from patients with urogenital inflammations were analyzed. Variations in the quinolone resistance-determining regions (QRDR) have been found in fluoroquinolone-resistant M. hominis clinical isolates in comparison with the reference PG21 strain. In one isolate, parC had Asn substitute at position 91.

[Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones]. / Rol' mutatsii v parC i gyrA v formirovanii ustoichivosti Mycoplasma hominis k ftorkhinolonam.

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.

[Formation of M. hominis and A. laidlawii resistance to fluoroquinolones]. / O formirovanii rezistentnosti k ftorkhinolonam u M. hominis i A. laidlawii.

Mycoplasma hominis and Acholeplasma laidlawii cultures resistant to antibacterial fluoroquinolone drugs ciprofloxacin (Cpf), ofloxacin (Ofl), and lomefloxacin (Lmf) were prepared by selection in liquid nutrient medium with ascending concentrations of Cpf. Resistant mycoplasma clones contained point mutations in the gyrase. A gene region determining quinolone resistance (QRDR gyrA): M. hominis contained C-->T transition resulting in substitution of Ser(83) for Leu and A. laidlawii G-->A resulting in substitution of Asp (91) for Asn. The phenomena of mutation formation during mycoplasma culturing in the presence of fluoroquinolones is studied. In the presence of Cpf in culture medium in concentrations of up to 10 micrograms/ml (for M. hominis) and 1 microgram/ml (for A. laidlawii) the mycoplasma populations contained cells with both altered and wild genotype. Culturing in the presence of higher Cpf concentrations resulted in elimination of cells nonmutant for QRDR gyrA. Besides in vitro studies, we analyzed clinical strains of M. hominis in the presence of different Cpf concentrations. M. hominis clones resistant to Cpf varying in genotypes were detected. These data permit a conclusion that the mechanism of fluoroquinolone resistance formation in mycoplasma includes several stages.

[Use of a method of single-stranded DNA fragment conformation polymorphism (SSCP) for detecting resistance of Mycoplasm hominis to fluoroquinolones]. / Ispol'zovanie metoda konformatsionnogo polimorfizma odnotsepochechnykh fragmentov DNK (SSCP) dlia vyiavleniia rezistentnosti Mycoplasma hominis k ftorkhinolonam.

Single-strand conformation polymorphism (SSCP) method was used for nucleotide sequence variation analysis of the gyrase A subunit quinolone resistance determining region (gyrA QRDR) in a laboratory and clinical strains of M. hominis. The couple of primers selected for this region amplified specific product in clinical material. M. hominis cultures growing in the presence of different concentrations of ciprofloxacin were studied by the SSCP method. Ser(83) to Leu mutation described previously was detected in the presence of quinolone in concentrations of at least 10 mcg/ml. In addition, 11 clinical samples were tested. In all cases the results of SSCP were confirmed by direct sequencing of the region. In 2 cases the sequences of gyrA QRDR in clinical strains were the same as in the laboratory strain. A Ser(83)-Leu mutation was identified in 1 clinical sample, while in others nucleotide substitutes did not lead to changes in amino acid sequences. These data demonstrate high informative value of the SSCP method for evaluating nucleotide variation in gyrA QRDR and quinolone resistance of M. hominis.