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This study performed an epidemiological survey of Neisseria meningitidis strains isolated from patients and from asymptomatic carriers. Altogether, 74 N. meningitidis strains (46 invasive and 28 non-invasive) were isolated between February 2011 and May 2018 in different regions of the Republic of Belarus. Serogenotyping was carried out by real-time PCR. Minimum inhibitory concentrations (MICs) of antibiotics were determined by broth microdilution and results were interpreted in accordance with EUCAST. The serogroups of N. meningitidis were determined as follows: serogroup B – 65%, C – 11%, W – 9%, A – 5%, Y – 4%, and Z and NG – 3% each. The MIC50 and MIC90 for benzylpenicillin (0.032/0.064–0.125 mg/L), ampicillin (0.032/0.125 mg/L), amoxicillin (0.125/0.25 mg/L), cefotaxime (0.016/0.016 mg/L), ceftriaxone (0.002/0.016 mg/L), ciprofloxacin (0.004/0.008 mg/L), chloramphenicol (1/1 mg/L), meropenem (0.008/0.008–0.016 mg/L), tetracycline (0.25/0.5 mg/L), and rifampicin (0.016/0.25 mg/L) were established. Strains with intermediate susceptibility for benzylpenicillin (12.3%), ampicillin (6.8%), and amoxicillin (24.7%) have been identified. In this study, we report the first rifampicin-resistant N. meningitidis in Belarus.

Corynebacterium ulcerans is an important zoonotic pathogen which is causing diphtheria-like disease in humans globally. In this study, the genomes of three recently isolated C. ulcerans strains, 4940, 2590 and BR-AD 2649, respectively from an asymptomatic carrier, a patient with pharyngitis and a canine host, were sequenced to investigate their virulence potential. A comparative analysis was performed including the published genome sequences of 16 other C. ulcerans isolates. C. ulcerans strains belong to two lineages; 13 strains are grouped together in lineage 1, and six strains comprise lineage 2. Consistent with the zoonotic nature of C. ulcerans infections, isolates from both the human and canine hosts clustered in both the lineages. Most of the strains possessed spaDEF and spaBC gene clusters along with the virulence genes cpp, pld, cwlH, nanH, rpfI, tspA and vsp1. The gene encoding Shiga-like toxin was only present in one strain, and 11 strains carried the tox gene encoding the diphtheria-like toxin. However, none of strains 4940, 2590 and BR-AD 2649 carried any toxin genes. These strains varied in the number of prophages in their genomes, which suggests that they play an important role in introducing diversity in C. ulcerans. The pan-genomic analyses revealed a variation in the number of membrane-associated and secreted proteins that may contribute to the variation in pathogenicity among different strains. Keywords: Corynebacterium ulcerans, diphtheria, nontoxigenic, sore throat, toxigenic, vaccine, virulence, zoonotic

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Modern medicine has achieved much progress in the field of medical and surgical repair of a variety of disorders. It is especially true for the early stages of treatments. In long-term period, however, instead of recovery, we frequently observe progressive regression, which completely annuls all our efforts. Moreover, the algorithm used to correct acute process may sometimes be detrimental to organs and tissues. Such situation is also frequent in the treatment of atherosclerotic descending aortic aneurysms (DAA). This is because the successful medical and even surgical repair to prevent aneurysm rupture in long-term period may trigger some detrimental processes in other regions thus producing negative results of the treatment. The present article doesn’t aim to prove some provisions in DAA clinical picture, atherosclerosis and inflammatory states. But we have faced some systematicity at these clinical implications which are absolutely not specified in literature. We have managed to find an explanation for this thing making a scrupulous analysis of nonsurgical sources. Comparing them with our observations, we have found out that aseptic and septic inflammation of connective tissue, probably, is a key component in the formation of DAA disregarding of which correction of dyslipidemia may lead to negative results. We are looking for in-depth research and discussion.

Within the last few years, there has been a strong trend to rethink the issue of management of atherosclerotic descending thoracic and abdominal aortic aneurysms (AAAs). When etiopathogenetic associations among changes observed during the progression of the disease were not fully described, surgeons had successfully applied, although traumatic, but a rather radical method to rescue from the rupture threat. As we gained experience and knowledge about long-term outcomes, mostly concerned mortality, we realized that surgery could not be the main tactical approach to AAAs treatment due to its frequent inefficiency and failure to guarantee that the disease would be suppressed including co-morbidities, polymorphic processes and clinical manifestations. It all required more sparing treatment strategies. The situation gave rise to a more argumentative and sparing medical-and-surgical approach to treatment based on a more in-depth understanding of the etiopathogenesis of the disease whereas surgery would remain of prime importance when appropriate. The following has been developed to improve treatment outcomes for AAA: 1) Multifactorial determination of indications for surgical correction with outlining the area of relative and absolute risk of aneurysm rupture; 2) Method of conservative treatment aimed to attain and maintain optimal blood pressure, target levels of cholesterol and low-density lipoproteins, as well as reduce oxidative and inflammatory processes in aorta, strengthen its wall, stabilize the disease and control co-morbidities. A four-year follow-up of patients using this developed technology has yielded more preferred results suggesting the need for narrowing indications for surgery to treat AAAs. Another advantage of the sparing approach to treat AAA is economic, due to fewer operations and implantations of stent-grafts, considering the fact that medical treatment should be used in operated subjects, too.

Traditionally Nocardia asteroids was considered the predominant species of causing no-cardiosis. The improved identification of isolates using molecular techniques have shown that the genus exhibits considerable taxonomic complexity and phenotypic base identification can be ambiguous. The aim of this study was to assess the species distribution of Nocardia strains mostly recovered from patients suspected of having tuberculosis, during three years period (2009-2012). The clinical isolates were identified to species level using conventional tests and genotypic methods using single and multi locus sequence analysis (MLSA) of 16S rRNA, gyrB and secA1 genes. Nocardiosis was diagnosed in 46 patients. The most frequent underlying condition were organ transplantation (6 patients; 13%), cancer (6 patients; 13%), human immunodeficiency virus (HIV) (6 patients; 13%), non-infectious chronic lung disease (5 patients; 10.8%) and tuberculosis (4 patients; 8.7%). Nocardia species was recovered from 46 different clinical specimens, the most common of which was bronchoalveolar lavage (BAL) (43.5%). Eleven different Nocardia species were identified: N. asteroides (n = 12), N. cyriacigeorgica (n = 9), N. farcinica (n = 7), N. wallacei (n = 6), N. carnea (n = 3), N. otitidiscaviarum (n = 3), N. abscessus (n = 1), N. arthritidis (n = 1), N. kruczakiae (n = 1), N. nova (n = 2) and N. veterana (n = 1). In conclusion, infection caused by Nocardia species appears to be more common than generally appreciated. The current study provides further evidence that Nocardia species are capable of causing a wide range of human diseases in healthy and immunocompromised patients. MLSA is a reliable method for accurate species identification of Nocardia isolates and would be more feasible for routine use in clinical laboratories.

The aim of the study was to investigate behavior of resistant Mycobacterium tuberculosis (MTB) isolates under a high dose of ofloxacin and its morphological changes. 19 extensively drug resistant (XDR) clinical isolates of MTB were grown on Löwenstein–Jensen medium containing progressively increasing concentrations of ofloxacin (2, 4, 8, 16, 32 mg/L). Ultra-structure analyses of resistant isolates grown on ofloxacin were conducted by transmission electron microscopy (TEM). Fixation was carried out by 4% glutaraldehyde in 0.1 M sodium cacodylate buffer on 300 mesh carbon formvar copper grid. The samples were negatively stained with uranium acetate suspension. All 19 XDR MTB isolates were grown and formed colonies successfully on 2, 4, 8 mg/L, seven isolates on 16 mg/L, and four isolates on 32 mg/L ofloxacin. Morphological changes and unusual forms were detected in 8, 16 and 32 mg/L ofloxacin at 43%, 76.5% and 81% of cells, respectively. Swollen form (protoplast like), ghost-like cell, degraded forms, and in a few cases, detached cytoplasm from cell wall were clearly detected in high drug concentrations in comparison to control. Changes in morphology were increased with increasing ofloxacin concentrations (p < 0.05). Some XDR isolates could be successfully grown on high doses of ofloxacin (32 mg/L), but with changes in morphology. It was concluded that several magnitudes of the drug doses could not prevent growth of drug resistant forms.

In recent years, the increasing number of cardiologists and cardiac surgeons tend to think that surgical treatment of patients with atherosclerotic aneurisms does not fully comply with contemporary ideas of what the disease is. Some data show that early operations in the presence of this pathology are associated with an unreasonably high mortality. Additionally, the use of intra-aortic stents and grafts cannot principally affect the therapeutic efficacy. Therefore, more attention is paid to the development of conservative therapeutic approaches leaving surgery defeated without surgical treatment. Two groups of patients with similar descending thoracic aortic atherosclerotic aneurisms (DTAAA) and abdominal aortic aneurisms (AAA) were retro- and prospectively studied over a 2-year period. Control group (Comparison group), (63 patients) received common surgical treatment from 2009 to 2010 whereas Main group (121 subjects) received multifaceted medical treatment to remove inflammatory reactions, strengthen aortic wall and control its dilation from 2011 to 2012. Operative treatment was used only in case of potential aneurism rupture. The comparison of the two groups of subjects showed that 2-year all-cause mortality in control group was 20.6% while in the main group it amounted to 9.1% due to the similar incidence of aneurism ruptures and deaths associated with concomitant diseases. It suggests that the odds ratio (OR) of survival when using attenuated therapeutic approach to treating atherosclerotic aneurisms is 2.6-fold higher compared to conventional surgical approach. One of the principal factors contributing to a higher mortality when using traditional surgical approach was the presence of polyorgan pathology that required constant medical correction irrespective of therapeutic option (surgical or medical) used. Another important factor is aortic aneurism wall frailty. The development of mechanisms that would allow its strengthening is considered a principal challenge of cutting-edge medicine that should be based on studies of triggers, molecular genetic bases of aortic wall immune-depending inflammatory formation, the production of pro-inflammatory cytokines, metalloproteinase activity that damages elastin and collagen fibers.

summary Partial sequences of KatG and GyrA genes have been obtained from multi and extensively drug-resistant (MDR and XDR) clinical isolates of Mycobacterium tuberculosis. Nonsynonymous (DN) and synonymous (DS) distances between those sequences have been calculated by Kumar method. Results revealed that DN is significantly higher than DS between some pairs of partial GyrA sequences. We found out that DN is higher than DS in many other partial and complete sequences of KatG and GyrA coding regions deposited in GenBank. The cause of the DN > DS situation is in several nonsynonymous substitutions occurrence (which may be associated with drug-resistance or not) in the absence of synonymous substitutions. Low rates of synonymous mutations occurrence is a consequence of the strong mutational GC-pressure. Due to the high saturation of third codon positions by guanine and cytosine (78.81 � 0.17% for all the genes from M. tuberculosis H37Rv genome), the probability to be synonymous for the nucleotide mutation of preferable (AT to GC) direction is low. Fixation of a single nonsynonymous mutation leading to drugresistance is a consequence of Darwinian selection. This clear example of Darwinian selection on the molecular level can be confirmed by selection test (DN > DS) only in case of DN and DS calculation in pairs of sequences possessing at least two additional nonsynonymous mutations which may be neutral or excessive.

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 (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 25–65 years, 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 (18–60 years) and 188 women of working age (18–55 years) participated. Males were significantly more likely to have MDR-TB than females. All cases with XDR-TB were older than 14 years 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.

Objective: This is the new comparative geogenetic molecular evolution research of M. tuberculosis in Iran and Belarus. Thus, we researched the genetic patterns of samples collected in the first survey of anti-tuberculosis drug-resistance by gene coding of RNA polymerase as part of the international project of on tuberculosis. Method: DNA extraction and amplification of rpoB gene was performed. All PCR products of gene were sequenced using the Amersham auto sequencer. For analysing phenogram has been demonstrated by method UPGMA and Neighbour- Joining. Clinical isolates (70/473) were analyzed by using sequencing gene rpoB and genotyped by program DNAMAN and MEGA. Results: The all data were compared with the international database of national center for biotechnology information web- site. Multi drug resistant of tuberculosis patient (MDR-TB) was 92% in never treated and 8% in previously treated. Muta- tions in rpoB gene and katG genes were showed in 95% and 84% of the MDR isolates, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. The other isolates are divided in Iran into 2 groups: group A - similar to the Eastern strains (China, Taiwan) and group B - strains of another genotype. And 3 groups in Belarus: group A - Strains of the first group are more similar to the standard European and Eastern ones China and Taiwan) which diverged in the last 10 years (Genetic evolu- tion rate), i.e. they are relatively new ones, and that is confirmed by the mutations, group B - Strains of the second group diverged earlier; they are older than the strains of the first group (16 years old- time and rate of evolution) and group C - Strains of the third group are similar to European strains and only circulate in Brest region. They are grouped separately on the phenogram and became prevalent in Iran (they are called Iranian residential strains and also is genetic analogy between group A from Iran and Belarusian isolates. Conclusion: This research gives a first result on genetic evolution of the M. tuberculosis strains distributing in the Iran and Belarus during the first survey of anti-tuberculosis drug-resistance and is homologies between groups A from Iran with group A from Belarus. It may aid in the creation of a national database that will be a valuable support for further studies. Keyword: Rate of Genetic Evolution, M. tuberculosis, Iranian and Belarusian isolates.

Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.

The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Iran. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG gene amplification, and DNA sequencing analysis were performed. Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG gene. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC--ACC), 316 (GGC--AGC), 309 (GGT--GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC ≥ 5-10 µg/mL). Furthermore, it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively. In this study, 62% (n= 21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC--ACC), 316 (GGC--GTT), 309 (GGT--GGT), and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5-10 µg/mL).

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.

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% ( n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).

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. Arjomandzadegan M1, Nazari R2, Zolfaghari MR2, Taherahmadi M2, Sadrnia M3, Titov LP4, Ahmadi A5, Shojapoor M6. • 1Infectious Diseases Research Center and Department of Microbiology, Arak University of Medical Sciences, Arak, Iran. mmatinam81@yahoo.com; arjomandzadegan@arakmu.ac.irn. • 2Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran. • 3Department of Biology, Payame Noor University, Tehran, Iran. • 4Research Institute for Epidemiology and Microbiology, Minsk, Belarus. • 5Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran. • 6Research Center of Molecular Medicine, Arak University of Medical Sciences, Arak, Iran. Abstract 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 37 Keywords: Ethambutol, Mycobacterium tuberculosis, PCR-RFLP, susceptibility

Background Gonorrhoea and widely spread antimicrobial resistance (AMR) in its etiological agent Neisseria gonorrhoeae are major public health concerns worldwide. Gonococcal AMR surveillance nationally and internationally, to identify emerging resistance and inform treatment guidelines, is imperative for public health purposes. In 2009, AMR surveillance was initiated in Belarus, Eastern Europe because no gonococcal AMR data had been available for at least two decades. Herein, the prevalence and trends of gonococcal AMR and molecular epidemiological characteristics of N. gonorrhoeae strains from 2010 to 2013 in Belarus, are described. Methods N. gonorrhoeae isolates (n=193) obtained in the Mogilev (n=142), Minsk (n=36) and Vitebsk (n=15) regions of Belarus in 2010 (n=72), 2011 (n=6), 2012 (n=75) and 2013 (n=40) were analyzed in regards to AMR using the Etest method and for molecular epidemiology with N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Results During 2010–2013, the proportions of resistant N. gonorrhoeae isolates were as follows: tetracycline 36%, ciprofloxacin 28%, penicillin G 9%, azithromycin 5%, and cefixime 0.5%. Only one (0.5%) β-lactamase producing isolate was detected. No isolates resistant to ceftriaxone and spectinomycin were identified. Overall, the resistance levels to tetracycline, ciprofloxacin and penicillin G were relatively stable. Interestingly, the level of resistance to azithromycin declined from 12% in 2010 to 0% in 2013 (P

One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with Bst EII and Pvu II revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C . diphtheriae . This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

Mycobacterium tuberculosis growth rate is closely coupled to rRNA transcription which is regulated through carD gene. The aim of this study was to determine the sequence of carD gene in drug susceptible and resistant clinical isolates of M. tuberculosis and designing of a PCR assay based on carD sequence for rapid detection of this bacterium.Specific primers for amplification of carD gene were carefully designed, so that whole sequence of gene could be amplified; therefore primers were positioned at the upstream (promoter of this gene and ispD gene) and downstream (in ispD gene). DNA from 41 clinical isolates of M. tuberculosis with different pattern of drug resistance was used in the study. PCR conditions and annealing temperature were designed by means of online programs. PCR products were sequenced by ABI system.PCR product of carD gene was a 524 bp fragment. This method could detect all resistant and susceptible strains of M. tuberculosis. The size of amplified fragment was similar in all investigated samples. Sequence analysis showed that there was similar sequence in all of our isolates therefore probably this gene is considered to be conservative. Translation of nucleotide mode to amino acids was showed that TRCF domain in N-terminal of protein CarD was found to be fully conservative.This is the first study on the sequence of carD gene in clinical isolates of M. tuberculosis. This conservative gene is recommended for use as a target for designing of suitable inhibitors as anti-tuberculosis drug because its importance for life of MTB. In the other hand, a PCR detection method based on detection of carD gene was recommended for rapid detection in routine test.

In this study, we evaluated a novel macroarray-based spoligotyping method for Corynebacterium diphtheriae strain typing. A total of 20 C. diphtheriae biotype gravis toxigenic isolates collected in Belarus from suspected foci of diphtheria infection (diphtheria cases, carriers, or contacts) were subjected to DNA fingerprinting. All strains had an identical ribotyping profile that was identified as ribotype 'Rossija' by comparison with the international ribotype database at the Institut Pasteur of Paris. A spoligotyping method based on simultaneous reverse-hybridization analysis of two CRISPR (clustered, regularly interspaced short palindromic repeats) loci differentiated these strains into three spoligotypes. Comparison of the spoligotyping results with the epidemiological linkage network helped us to resolve suspected links in the chains of transmission. To conclude, the C. diphtheriae spoligotyping method demonstrated its utility in the field study, in particular, underlining the importance of the use of both CRISPR loci. The generated discrete data can be presented in digital binary format and be easily exchanged between laboratories and stored in local and global databases.

Meningococcal infections are major causes of death in children globally. In Belarus, the incidence of cases and fatality rate of meningococcal infections are low and comparable to the levels in other European countries.In the present study, the molecular and epidemiological traits of Neisseria meningitidis strains circulating in Belarus were characterized and compared to isolates from other European countries.Twenty N. meningitidis strains isolated from patients (n = 13) and healthy contacts (n = 7) during 2006–2012 in Belarus were selected for multilocus sequence typing (MLST), genosubtyping and FetA typing. TheSTs of the Belarusian strains were phylogenetically compared to the STs of 110 selected strains from 22 other European countries.Overall, eleven different genosubtypes were observed, there were seven variants of variable region of the fet Agene detected. The majority of the STs (95%) found in Belarus were novel and allthose were submitted to the Neisseria MLST database for assignment. Several newly discovered alleles of fumC (allele 451) and gdh (allele 560 and 621) appeared to be descendants of alleles which are widespread in Europe, and single aroE alleles (602 and 603) occurred as a result of separate evolution.N. meningitidis strains circulating in Belarus are heterogeneous and include sequence types, possibly, locally evolved in Belarus as well as representatives of widespread European hyperinvasive clonal complexes.

Identification of Mycobacterium species is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA to discriminate many closely related species and the unreliability of phenotypic testing. We investigated a collection of nontuberculous mycobacteria (NTM) strains isolated from suspected tuberculosis patients at Tuberculosis Reference Centre (Ahvaz, Iran) and Masoud Laboratory (Tehran, Iran) during 2008–2012 to evaluate the species spectrum of NTM isolates. Based on phenotypic tests, the isolates were identified up to species or complex level; however they were heterogonous by hsp65-PCR restriction fragment length polymorphism analysis (PRA) method. Representative isolates from each hsp65-PRA pattern, were subjected to identification using single locus and multi locus sequence analysis (MLSA) based on 16S rRNA, rpoB, hsp65 and 16S–23S internal transcribes spacer (ITS) fragments to determine their taxonomic affiliations. All 92 NTM isolates from different clinical specimens were considered as etiological agents causing disease according to American Thoracic Society (ATS) guideline. Phenotypic evaluation alone assigned 66 (72%) isolates to a species or complex level and consequently 76 (82%) isolates showed previously reported hsp65-PRA patterns. Although sequence base identification using single locus such as 16S rRNA, rpoB, hsp65 or ITS identified the isolates up to species level, MLSA correctly identified 16 different species of NTM from clinical isolates. In summary, four-locus MLSA is a reliable method for elucidating taxonomic data and reliable species identification of Mycobacterium isolates and therefore, would be more feasible for routine use in Tuberculosis (TB) reference laboratory.

All members of the Mycobacterium tuberculosis complex were assigned to one of the three principle genetic groups based on KatG463/GyrA95 polymorphism.A total of 202 isolates of M. tuberculosis consisting of 50 susceptible, 121 MDR (multidrug resistant) and 31 XDR (extensively drug resistant) isolated from culture-confirmed tuberculosis patients in different regions of Belarus and Iran (Tehran and Markazi province). Isolates were screened by sequencing and polymerase chain reaction restriction fragment length polymorphism (RFLP) assay, and were further divided into three principal genetic groups (PGG), based on Sreevatsan's pattern as polymorphisms in KatG463/GyrA95 codons.Among the 104 isolates, characterized as MDR from Belarus, 57 (54.8 ± 4.8%), 30 (28.8 ± 4.43%), 17 (16.3 ± 3.6), belonged to PGG 1, 2, and 3, respectively (p0.05). Thirty one XDR isolates from Belarus had a similar pattern as 15 (48.4%), 12 (38.7%), 4 (12.9%) PGG 1, 2, and 3, respectively. From Iranian samples, Markazi isolates (susceptible to drugs) had a pattern as 12 (36.5%), 15 (45.5%), 3 (6%), and Tehran samples were (selected MDR): 9 (53%), 6 (35.2%), 2 (11.8%) (PGG 1, 2, and 3, respectively). In a study of tuberculosis patients, who were in prison, no relation was found between PGG and resistance to isoniazid, but most of the identified isolates belonged to PGG 1 (45.5 ± 10.9%) (p0.05). Overall, the group 1 isolates showed more frequency in MDR and XDR rather than susceptible strains, and there aren't any relations to geographic region.

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 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.

Identification ofMycobacterium tuberculosisandM. bovisis 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 ofM. tuberculosisandM. bovis.As a BLAST and BLASTP search we selected regulatory genewhiB7that encodes multi-drug resistance in this bacterium. Thirty clinical isolates ofM. tuberculosiswere sequenced and all the mutations in genewhiB7were detected. The best set of several pairs of primers was selected and used in comparison byrpoBgene for differentiation ofM. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenicMycobacteriumisolates and 30 clinical isolates ofM. tuberculosis.It was proved that only clinical isolates ofM. tuberculosisandM. bovishave positive bands of 667 bpwhiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products ofwhiB7gene were observed for ten positive sputum samples (preliminarily approved to be positive forM. tuberculosisby commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoBgene 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 ofM. tuberculosisandM. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.

Corynebacterium diphtheriae the causative pathogen of human diphtheria infects the nasopharynx or skin. Although diphtheria has been extensively studied, little is known about the two key aspects of C. diphtheriae invasiveness: colonization and invasion. The role of adhesive properties in establishing the infection of C. diphtheriae strains, independent of toxin production, still needs to be clarified. In this study, we describe a novel gene involved in adherence to epithelial cells. Transformation of C. diphtheriae 225, biotype gravis, ribotype St-Petersburg by EZ:TN(KAN-2)Tnp Transposome was undertaken. A C. diphtheriae 225 Tn5 insertion library of 2800 mutants was created. Five hundred and eighty five transformants were qualitatively screened for reduced adherence to HEp-2 cells by an adherence assay. One mutant strain consistently exhibiting 15.2% of the wild-type adherence was isolated. The DNA flanking the transposon was identified by inverse PCR and subsequent sequencing. The disrupted gene was 94% identical to the C. diphtheriae DIP1621 gene that belongs to unclassified genes. In conclusion, the disruption of the C. diphtheriae DIP1621 gene led to decreased adherence to epithelial cells; its exact function remains to be established.

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2.1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogenous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile -associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Iran. 91 sputum were collected from suspected tuberculosis patients, 34 Rif-r isolates (87%) were identified as M. tuberculosis. Polymerase chain reaction (PCR) amplification and DNA sequencing methods were performed. 411 bp fragments of rpoB gene were sequenced and mutations in 81 bp regions were analyzed. 60 mutations and 13 micro deletions were identified in 29 RIF-r MBT (85%). Among 60 mutations, 6 silent and 54 missense were identified. Missense mutations produced 23 types of amino acid substitutions. In 5 RIF-r MBT isolates (15%) no mutations were found in the core region of the rpoB gene. All silent mutations were localized in codon 507. Most frequent mutations detected from Iranian strains were in codons 523 and 526. Five alleles in codon 526 and 3 alleles in triplets in each codons 507, 508, 513 were found. 6 (19%) strains harboured single mutations 6 (18%) placed in codons 526, 510 while the rest of isolates 23 (69%) had multiple mutations: Double 11 (34%), triple 7 (22%), and quartile mutations 1 (3%) and 4 (12%) of strains harboured 5 mutations respectively.

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. Mutations in different codons causing resistance to isoniazide in the gene of catalase peroxides (katG) in Belarusian strains was determined. 42 strains of Rif-r and Inhr(MDR) were isolated in different regions of Belarus. Culture susceptibility testing of all 42 strains revealed resistance to streptomycin (90%), 16 strains (43%) were resistant to etambutol. DNA Extraction, Standard PCR identification and katG gene amplification were performed. The most affected codons of katG gene were 315(95%), 316(16.2%), and 309(14.5%). Four types of mutations were identified in codon 315: AGC--ACC (n=36)32.4%, AGC--AGG (n=1) 0.9%, AGC--AAC (n=2) 1.8%, AGC--GGC (n=1) 0.9%. One type of mutation was found in codon 316: GGC--AGC (n=18)16.2%, four types of mutations were detected in codon 309: GGT--GGT (n=7)6.3%, GGT--GCT (n=4)3.6%, GGT--GTC (n=3)2.7%, GGT--GGG (n=1)0.9%. Mutations in codon 309 make up 34%, in codon 316 (37%) and other types of mutations 29% of all detected mutations. In 2 isolated strains mutation were identified in codons 463, 35 and, in codons 454, 357 respectively and 2 isolates, there were not found any mutations. Concluding, all INH-r MBT had resistance-associated nucleotide changes mostly in codons 315.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis collected from patients in southern endemic region of Iran. Drug susceptibility testing was determined by using the BACTEC system and CDC standard conventional proportional method. All 28 isolates were resistant to isoniazid (100%), rifampicine (14%) streptomycin (82%) and 8 (28%) was resistant to ethambutol. Mono-resistance to isoniazid was observed in four isolates (14%). In 21 isolates three types of mutations were found in codon 315: AGC → ACC (Ser → Thr) (61%), AGC → AGG (Ser → Arg) (3.5%) and AGC → AAC (Ser → Asn) (11%). Moreover, one type of mutation was seen in codons 311 (32%), 299 (28.5%), and 322 (21.5%). Of 28 isolates 12 (46%) found with one mutation, 7 (27%) had two, 5 (19%) had three and 2 (8%) revealed to have four mutations in katG gene. In nine isolates single mutation was observed in codon 311 (GAC → TAC). This study indicates presence of multiple mutations in codon 315 and 299 among multiple drug-resistant (MDR) strains of M. tuberculosis collected from patients sputum having secondary infections in adjacent southern endemic border of Iran.

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.

Background The reemergence of epidemic diphtheria in Belarus in 1990s has provided us with important information on the biology of the disease and the diversity of the causative agent Corynebacterium diphtheriae. Molecular investigations were conducted with the aim to analyze the genetic variability of C diphtheriae during the post-epidemic period. Methods The biotype and toxigenicity status of 3513 C. diphtheriae strains isolated from all areas in Belarus during a declining period of diphtheria morbidity (1996–2005) was undertaken. Of these, 384 strains were isolated from diphtheria cases, 1968 from tonsillitis patients, 426 from contacts and 735 from healthy carriers. Four hundred and thirty two selected strains were ribotyped. Results The C diphtheriae gravis biotype, which was prevalent during 1996–2000, was "replaced" by the mitis biotype during 2001–2005. The distribution of toxigenic C. diphtheriae strains also decreased from 47.1% (1996) to 5.8% (2005). Changes in the distribution of the epidemic ribotypes Sankt-Peterburg and Rossija were also observed. During 2001–2005 the proportion of the Sankt-Peterburg ribotype decreased from 24.3% to 2.3%, in contrast to the Rossija ribotype, that increased from 25.1% to 49.1%. The circulation of other toxigenic ribotypes (Otchakov, Lyon, Bangladesh), which were prevalent during the period of high diphtheria incidence, also decreased. But at the same time, the proportion of non-toxigenic strains with the Cluj and Rossija ribotypes dramatically increased and accounted for 49.3% and 30.1%, respectively. Conclusion The decrease in morbidity correlated with the dramatic decrease in the isolation of the gravis biotype and Sankt Peterburg ribotype, and the prevalence of the Rossija ribotype along with other rare ribotypes associated with non-toxigenic strains (Cluj and Rossija, in particular).

Between January and June 2006, 87 measles cases were notified in Belarus. Measles cases were reported in five of the seven regions.

OBJECTIVE. Autoantibodies against C1q (anti-C1q) have been found in a number of autoimmune and renal diseases. They are best described in adult patients with systemic lupus erythematosus, where a strong correlation between the occurrence of anti-C1q and severe lupus nephritis (LN) has been observed. However, the role of anti-C1q in children with systemic lupus erythematosus has not yet been determined. Furthermore, the clinical importance of anti-C1q in other forms of glomerulonephritis remains to be elucidated. The aim of this study was to investigate anti-C1q in children with different forms of glomerulonephritis including LN.METHODS. We prospectively investigated 112 children with different forms of newly diagnosed glomerulonephritis for the presence of anti-C1q by an enzyme-linked immunosorbent assay and compared them with healthy controls. Associations between anti-C1q and disease manifestations at the time of the measurements and during follow-up were investigated.RESULTS. Twenty-one of 112 patients were positive for anti-C1q compared with 0 of 40 healthy controls. Anti-C1q was associated with activity in LN and with disease severity in patients with acute poststreptococcal glomerulonephritis (APSGN). In LN, 7 of 12 patients were found to be anti-C1q positive. Six of these 7 had active disease at the time of the serum sampling compared with 1 of 5 of the anti-C1q-negative children. In children with APSGN, 8 of 24 were positive for anti-C1q. Anti-C1q-positive APSGN patients had significantly higher proteinuria and more often hypertension than those without anti-C1q. All 4 patients in which APSGN did not resolve spontaneously were anti-C1q positive.CONCLUSIONS. Anti-C1q is associated with active LN in children. In addition, children with anti-C1q-positive APSGN have more severe disease than those who are anti-C1q negative. These data suggest APSGN is another disease in which anti-C1q has a pathogenic role.

To assess the antibiotic policies in Central Eastern European (CEE) countries, a questionnaire on the prevalence of resistance, antibiotic consumption data for ambulatory and hospital care and antibiotic policies, was mailed to national representatives. Data on antibiotic resistance and consumption of antibiotics at national levels are limited and vary considerably among countries. The importance of surveillance data in altering perceptions of the prevalence of resistance is shown by the comparison of surveillance data and interview data. Interview data without surveillance data produced the widest range of estimates of the prevalence of resistance in streptococcus pneumonia -5% in Lithuania and 82% in Belarus. The average consumption of antibiotics in ambulatory care in eight CEE countries in 2001 was 19.35 defined daily doses (DDD)/1000 inhabitants per day, (range 13.1 - 24.8 DDD) and in hospitals in six CEE countries was 2.2 DDD/1000 inhabitants per day (range 1.3-4.5). Over the counter sales of antibiotics are available in some countries. Antibiotic policy interventions do not exist or only apply to specific problems or interventions. Better implementation of antibiotic interventions and education on antibiotic use should be a high priority in this region. An effective strategy requires close co-operation, consultations and partnership at national and international level in particular, via existing international organisations.

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.

Studies on the immediate and long-term effects of radiation on the B-system immunity of children who were affected by radiation after the Chernobyl disaster (from 1986-1992) are summarized in this paper. Complete clinical and immunological examination of more than 6000 children have been carried out. The dynamics of the immune system, with ongoing reactions of cell proliferation and differentiation, gene amplification, transcription, translation, biosynthesis and switching production of isotypes and subclasses of immunoglobulins, as well as specific and nonspecific (natural) antibodies, make it highly susceptible to the action of radiation in addition to other ecological factors. B-system of immunity (B-cel level, concentration of immunoglobulins-M, G, A, E; subclasses of IgG (IgG1-IgG4) in the serum and saliva, and the level of nonspecific heterophilic autoantibodies (RF, antithyroglobulin) were investigated in children of differing ages and sex living in the territories of the Republic of Belarus contaminated with radionuclides. Research showed decreased levels of B-cell and IgM and IgG isotopes 40-50 days after the disaster and increased levels of IgA immunoglobulins at that time. Long-term effects of low doses of radiation showed increased concentrations of IgM and IgG, correlating changes in the B-system of immunity with the level of 137Cs contamination in the territory of residence and also with the amount of 137Cs found in the children.

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.

Background The potential use of microorganisms as agents of biological warfare (BW) is a growing concern. Lassa virus, a member of the Arenavirus class of Hemorrhagic fever (HF) viruses has emerged as a worldwide concern among public health officials. The purpose of the present study was to further elucidate the antiviral activity spectrum of stampidine, a novel nucleoside analog with potent anti-viral activity against the immunodeficiency viruses HIV-1, HIV-2, and FIV, by examining its effects on survival of mice challenged with Lassa virus. Methods We examined the therapeutic effect of Stampidine in CBA mice inoculated with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or nontoxic doses of stampidine administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Results The probability of survival following the Lassa challenge was significantly improved for stampidine treated mice (Kaplan Meier, Chi-squared = 11.7, df = 2, Log-Rank p-value = 0.003). Conclusion Therefore, stampidine shows clinical potential as a new agent for treatment of viral hemorrhagic fevers caused by Lassa virus.

Molecular detection of fluoroquinolone resistance-associated gyrA mutations in ofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Iran and Belarus • Mohammad Arjomandzadegana, , , , Leonid Titovb, Parissa Farniac, Parviz Owliad, Reza Ranjbare, Fatemeh Sheikholeslamic, Larissa Surkovaf • a Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran • b Research Institute for Epidemiology and Microbiology, Minsk, Belarus • c Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran • d Molecular Microbiology Research Center, Shahed University, Tehran, Iran • e Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran • f Institute for Pulmonology and Phthisiology, Minsk, Belarus Abstract Objective/background: Detection of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene could determine resistance to fluoroquinolone antituberculosis drugs. The aim of this study was to detect mutations in QRDRs. Methods: From 184 clinical isolates of Mycobacterium tuberculosis, ofloxacin resistance was proven in 42 isolates using the proportion method. The molecular basis of resistance to ofloxacin were investigated by the determination of mutations in the QRDR region of the gyrA gene. Extracted DNA fragments of 194 bp from the gyrA gene were amplified and an automatic DNA sequencer was used for the sequencing process. Results: Molecular genetic analysis of 42 resistant M. tuberculosis strains demonstrated that they belong to Principal Genetic Group (PGG) 1 in 19 cases (45.2 ± 10.9%), to PGG2 in 15 cases (35.7 ± 10.5%), and to PGG3 in eight cases (19.0 ± 8.4%). Isolates from PGG1 were dominant among resistant isolates (P < .05). It was found that 24 (57%) resistant isolates carried mutations at codon 94 with five different amino acid changes: D94A (n = 11), D94G (n = 3), D94T (n = 4), D94A (n = 4), and D94Y (n = 2). The remaining 18 (43%) isolates had mutations in codon A90V (GCG → GTG) and S91P (TCG → CCG). Five isolates had two mutations in codons 90 and 94. There was no difference between mutations at these two codons in resistant isolates of the two countries (P < .001). There was no polymorphism observed in codon 95 in any of the ofloxacin-susceptible isolates. Conclusion: It was concluded that the determination of nucleotide sequences of QRDRs can be used as a molecular test for the rapid detection of ofloxacin resistance. Furthermore, frequencies in gyrA codons in Belarus and Iran were similar, therefore it is not of geographical concern for the two countries. Keywords • gyrA; Mutation; Mycobacterium tuberculosis; Ofloxacin; Resistance