Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.

INTRODUCTION: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. MATERIALS AND METHODS: From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. RESULTS: Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). CONCLUSION: Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.

[Polymorphism of the genes IL-1RA and TNF-alpha in patients with gastritis and duodenal ulcer associated with Helicobacter pylori].

The goal of this work was to determine the rate of the polymorphism of the genes-antagonists of receptor IL-1 (IL-1RA) and TNF-alpha in patients with gastritis and duodenal ulcer associated with Helicobacter pylori. The receptors were tested for the clinical manifestation of the disease. A total of 126 patients with different gastroduodenal pathology and H. pylori in autopsy were tested. The results of this work demonstrated a correlation between the risk of duodenal ulcer and allele A of gene TNF-alpha in position 308 in the patients. The analysis of the gene-IL-1RA polymorphism demonstrated statistically significant difference between the patients in the frequency of the genotype 2/l. The results of this work showed that parallel typing of the genes of H. pylori in virulence was required for characterization of the bacteria-patient association. The correlation between the results of the typing with polymorphism of genes of cytokines in patient autopsy was also required.

[A new approach to postexposure treatment of rabies by complex of immuno- and chemotherapy in belarus].

A method for preventive treatment of rabies with a complex of immuno- and chemotherapeutics was developed. Rifampicin was used a an etiotropic drug. In the experiments on laboratory animals infected with fixed and street strains of rabies virus it was shown to prolong the incubation period and to increase the survival rate. The protective mechanisms of rifampicin against rabies should be associated with inhibition of RNA transcription, as well as immunomodulating function of macrophages, dendritic cells, B- and T-cells. Since 1992, after the approval of the Ministry of Health of Belarus rifampicin is used in complex with antirabic vaccine for postexposure treatment of rabies in people after severe bites by infected animals (wolves, foxes, dogs). For an 18-year period (1992-2009) of integrated application of chemo- and immunotherapy in Belarus there was not registered any case of hydrophobia in people even after the heaviest wolf bites, incompatible with life (penetrating injuries of the skull, scalping, multiple bites).

A study on demographic characteristics of drug resistant Mycobacterium tuberculosis isolates in Belarus.

OBJECTIVE: A descriptive study of drug-resistance patterns by age group and among culture-positive pulmonary tuberculosis (TB) patients referred to the Research Institute for Pulmonology and Phthisiology of Belarus between January 2007 and January 2008. METHODS: Drug susceptibility tests were performed for first- and second-line anti-tuberculosis drugs. Patients were clustered into five resistance categories: mono-resistant (Mono); multi-drug resistant (MDR); all first-line drug resistance (MDR+ES); and extensively drug resistant (XDR). The patients were divided into primary and secondary and into six groups based on age in years (<15, 15-24, 25-44, 45-54, 55-65, and >65). RESULTS: An analysis was undertaken of information gathered from 934TB patients, of whom 660 were men (70.67±1.5%) and 274 were women (29.33±1.5%) (p<0.001). In the age group 25-65years, men outnumbered women between 2.7 and 9.0 times higher. Cases of secondary TB totaled 414 (52.02±1.77%), and primary cases totaled 382 (47.98±1.77%) (p>0.05); 756 of the patients were of working age, and 170 were of non-working age, of whom 570 men of working age (18-60years) and 188 women of working age (18-55years) participated. Males were significantly more likely to have MDR-TB than females. All cases with XDR-TB were older than 14years old. CONCLUSION: As Belarus is a high-burden MDR-TB country and treatment of drug-resistant TB is long and complicated, the findings of this study provided useful information to deliver effective community-based disease control measures and a proposed plane for the effective management of drug-resistant TB at the national level.

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis.

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.

Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7.

Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.

[Impact of a magnetic field on blood separation kinetics in patients with joint diseases].

The paper gives the results of experiments on phase separation of blood in the constant magnetic field that allows the structure of blood to be regulated, without changing its cellular and chemical composition. Blood deposition kinetic relationships were obtained for patients with joint diseases of various etiology (osteoarthritis, osteoarthrosis deformans, endoprosthesis instability, contusions, and joint wounds). They correlate with the severity of an inflammatory process in the joint and its adjacent tissues, with a patient's resistance to the development of pathology, and with red blood cell mobility in the biophysical field of a living organism. Analysis of relationships gives information on concentrations in plasma and hence synovial fluid (the basis of which is blood dialysate) in the liquid-crystalline phospholipid and cholesterol phase that determines the lubricity of synovial fluid and a low friction in the joints. The method may be used for the primary evaluation of efficacy of drugs for joint treatment, which is made in vitro on the blood taken from the patients rather than on the latter.

Study of promoter and structural gene sequence of whiB7 in MDR and XDR forms of Mycobacterium tuberculosis.

Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCR for sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets of primers proved that there were no differences between sequence of susceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design of an inhibitor for the WhiB7 protein in all clinical forms in further studies.

High level association of mutation in KatG315 with MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus.

The mutation in KatG315 is found in the majority of isoniazid resistant strains worldwide, especially in areas with a high incidence of tuberculosis. A total of 138 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis consisting of 108 MDR (multidrug resistant) and 30 XDR (extensively drug resistant) isolated from patients in different regions of Belarus from 2007 to 2008 were screened by a PCR restriction fragment length polymorphism (RFLP) assay and sequencing. As a result, 97.8% prevalence of the KatG315 mutation was detected in all isolates from patients either actually or previously treated with tuberculosis. This mutation was not found in any of 9 INH-susceptible isolates and 2 standard strains of H37Rv and Academia included in the study. All isolates that contained the mutation in KatG315 were classified as MDR and XDR by a culture-based susceptibility testing method. Among the 30 XDR isolates, 15 (50%), 12 (40%), and 3 (10%) were classified into principal genetic groups (PGG) 1, 2, and 3, respectively. It is concluded that INH-resistant MTB were associated with the mutated KatG315 phenotype. The simplicity of the assay, with 100% specificity, permits its implementation in routine practice at clinical microbiology laboratories for first and fast screening of cultures. This method has potential application for rapid diagnosis of INH resistance due to KatG315 mutation.

[Synthesis of olean-18(19)-ene derivatives from betulin].

The rare olean-18(19)-ene triterpenoids moradiol and moronic acid were synthesized from betulin, and their antiviral properties were investigated.

[Biological and immunogenic characteristics of Chlamydia trachomatis strain MT-2A (serovar D) and its use for development of experimental inactivated vaccine].

Biological characteristics of C. trachomatis author's strain MT-2A (serovar D) is presented. Stages of development on its basis the experimental formalin-inactivated vaccine against Chlamydia were described. Humoral and cellular immune response to the vaccine administered on 3-dose immunization schedule in conjunction with polyoxidonium as adjuvant was studied. Significant immunological efficacy of the vaccine was shown. T- and B-cell immune responses were characterized. Titer of IgG antibodies against Chlamydia in blood serum after 3rd dose of the vaccine was 10,880+/-1,817.76. Assessment of T-cell response showed that reaction of delayed hypersensitivity with formation of granuloma presented in 60% of animals. Proportion of immunoblasts in reaction of blast-transformation was 29.3+/-2.8%. Perspectives of further studies of the developed corpuscular vaccine against Chlamydia are discussed.

Detection of mutation in isoniazid-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC-->ACC (n=36) 85%, AGC-->AGG (n=1) 2.3%, AGC-->AAC (n=2) 4.7%, AGC-->GGC (n=1) 2.3%. One type of mutation was found in codon 316: GGC-->AGC (n=18) 41.4%, four types of mutations were detected in codon 309: GGT-->GGT (n=7) 16.1%, GGT-->GCT (n=4) 9.2%, GGT-->GTC (n=3)6.9%, GGT-->GGG (n=1) 2.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315.

[Point mutations in tox promoter/operator and diphtheria toxin repressor (DTXR) gene associated with the level of toxin production by Corynebacterium diphtheriae strains isolated in Belarus].

DNA fragments 129 bp in length containing promoter region of the tox gene from 81 toxigenic strains Corynebacterium diphtheriae were analyzed using the SSCP (single strand conformational polymorphism). We found that only two strains had mutations; the strains also had highest levels of toxin production (over 5120 Vero CD50/ml). Other strains were characterized either as high-level toxin-producing (640-5120 Vero CD50/ml, 41 strains) or low-level toxin-producing (40-320 Vero CD50/ml, 38 strains). Nucleotide sequence analysis revealed single T to C mutations at positions -54 and -184 within -232 - +85 region of tox operon. The first mutation at the -184 position was mapped outside the tox promoter/operator, whereas the second substitution at the -54 position modified the 9-base-pair interrupted palindromic sequence of the tox promoter/operator from ATAATTAGG in the wild-type bacteriophage (to ACAATTAGG in strains with enhanced level of toxin production. Nucleotide sequence analysis of -76 - +681 region of diphtheria toxin repressor (dtxR) gene from 15 strains of C. diphtheriae revealed two missense mutations resulting in amino acid substitutions A 147 V; and L 214 I in the C-terminal region of the DtxR protein. Seven of these strains were identified as high-level toxin-producing and 4 strains, as low-level toxin-producing. In addition, one low-level toxin-producing strain was shown to contain a missense mutation leading to amino acid substitution I 221 T. Three strains, including two highest-level toxin producing strains contained no nucleotide substitutions, as well as the C7(-) strain. The 10 strains belonging to the Sankt-Peterburg and Rossija epidemic ribotypes as well as NCTC 13129 strain (etiologic agent of the diphtheria epidemic outbreak in the Eastern Europe) was shown to contain two mutations A 147 V and L 214 I in the C-terminal region of the DtxR protein.

[The molecular epidemiology of HIV-1 in Belarus in (1996-2004): a predominance of subtype A variants and circulation of subtype B variants].

To study the molecular epidemiology of HIV-1 in Belarus, the genetic sequences of HIV-1 variants were obtained from 50 infected persons, which represented the main stages, risk groups, and geographic areas of the epidemic. The env and gag sequences were studied for HIV-1 variants from 31 persons, the env sequences were for HIV-1 variants from 18 persons, and the gag sequence was for HIV-1 variant from 1 person. Phylogenetic analysis indicated that the sequences of HIV-1 variants from 46 persons were homogenic and evolutionally closely related to IDU-A strains specific for other epidemics in the former Soviet Union are dominating in the epidemic in Belarus. Circulation of epidemiologically unrelated subtype B viruses was also established.

[Photodynamic inhibition of infection caused by herpes simplex virus type 1 in the cultured cells, by using 5-aminolevulinic acid-induced porphyrins].

On simulating infection caused by different herpes simplex virus type 1 (HSV-1) variants responsive and unresponsive to the drugs acyclovir and phosphonoacetic acid in the cultured Vero and C6 cells has revealed the higher ability of target cells to accumulate 5-aminolevulenic acid (ALA)-induced endogenous porphyrins, which determines the selectivity of their photo damages. Optimal conditions have been defined for all the studied HSV-1 variants to show a virus-inhibiting effect upon photodynamic exposure of infected and ALA-treated cell cultures.

[Changes in vaginal microbiocenosis in patients with chronic pelvic inflammatory diseases following antibiotic therapy].

The species composition of the vaginal microorganisms in healthy women and in patients with chronic pelvic inflammatory diseases before and after treatment in a gynecological hospital was studied. The study revealed that antibiotic therapy did not lead to complete clinical convalescence. During bacteriological investigation of patients changes in vaginal microbiocenosis, manifested by a decreased number of microbial species, an increased proportion of Escherichia coli, the occurrence of Staphylococcus aureus, a decreased number of Lactobacillus ssp., were observed. Antibiotic therapy aggravated the dysbiotic microbial picture of the vagina.

[Specific clinical, epidemiological patterns and laboratory diagnostics of enterovirus infection in the Republic of Belarus].

The clinical and epidemiological patterns as well as the results of the laboratory verification of the outbreak of enterovirus infection (EVI) in Minsk during the period of summer-autumn, 2000, are presented. During this outbreak a variety of clinical forms were observed, the serous meningitis being prevalent (57.5%). Practically simultaneous occurrence of infection on the territory of all administrative districts of the city, the predominant involvement of children aged up to 14 years into the outbreak, a high proportion of simultaneous casualities in the multiple foci. A number of circulating enteroviruses (EV)--ECHO 30, ECHO 6 of three serotypes and Coxsackie B5--were simultaneously isolated from clinical material. EV of the same serotypes were isolated from tap drinking water, and neutralizing antibodies to these serotypes were often detected in the patients blood sera. Infectious EV were also present in samples of bottled water and in water reservoirs used for bathing. The routes of EV transmission and the improvement of EVI control are discussed.

Serotype-specific mucosal immune response and subsequent poliovirus replication in vaccinated children.

A total of 32 unimmunized children (median age 5 months) living in an orphanage in Minsk, Belarus, were vaccinated with three doses of oral poliovirus vaccine (OPV) with a 60-day interval between the doses. Blood samples were drawn before the immunizations and 45-50 days after each vaccine dose. Excretion of the vaccine viruses was followed by examining fecal specimens collected weekly after each vaccine dose. All children seroconverted by the second dose of OPV at the latest to all three serotypes of poliovirus but differences were seen regarding the intestinal responses. The strongest responses in both neutralizing antibodies and virus-binding IgA in fecal suspensions were seen toward poliovirus Type 2. Consistent with this, relatively little poliovirus Type 2 excretion was seen after the second and third doses of OPV as compared to that of poliovirus Type 1 or Type 3. The delayed development of functional intestinal immunity against the latter serotypes was associated with relatively weaker intestinal antibody responses compared to poliovirus Type 2. In the case of poliovirus Type 3, about 10% of children were still excreting the vaccine virus 9 weeks after administering the third dose. These results are consistent with epidemiological observations of the serotype-specific efficacy of OPV immunizations. The proportion of specimens showing nonpolio enteric viruses gradually increased through the 6-month study period. This may reflect the possibility that after the first dose of OPV, intensive replication of the vaccine viruses may out compete the nonpolio viruses in the intestines of the vaccinees or, alternatively, mask nonpolio viruses during the cell culture isolation procedure.