Цель. Оценка уровня популяционного иммунитета к SARS-CоV-2 среди населения Гродненской области Республики Беларусь на втором году пандемии COVID-19. Материалы и методы. Оценка популяционного иммунитета к SARS-CоV-2 среди населения Гродненской области проведена в рамках трехэтапного продольного стратифицированного по возрасту сероэпидемиологическогоисследования, включавшего анкетирование и изучение серопревалентностик RBD-фрагменту S-белка SARS-CoV-2. Определение суммарных (IgM и IgG) антител к SARS-CoV-2 осуществляли с помощью качественной иммуноферментной тест-системы Wantai SARS-CoV-2 Total Ab (Beijing Wantai Biological Pharmacy Enterprise, Пекин, Китай). Результаты. Спустя год с момента регистрации первого случая COVID-19 коллективный иммунитет населения Гродненской области составил 58,8%. Доля серопозитивных участников увеличилась на последующих этапах. На первом и третьем этапах статистически значимых различий в уровне серопревалентности как среди детского и взрослого населения, так и среди различных возрастных групп населения не выявлено. На втором этапе исследования наименьшая доля серопозитивных лиц была в возрастной группе 60–69 лет, а наибольшая – в возрастной группе 30–39 лет. Не установлено статистически значимых различий в уровне серопревалентности среди профессиональных групп, а также среди лиц с различным социальным поведением. Заключение. С учетом развития эпидемиологической ситуации, появления новых вариантов вируса SARS-CоV-2, а также продолжающейся вакцинации населения целесообразно дальнейшее изучение динамики формирования популяционного иммунитета. Purpose. Evaluation of the level of population immunity to SARS-CoV-2 among the population of the Grodno region of the Republic of Belarus in the second year of the COVID-19 pandemic. Materials and methods. Evaluation of population immunity to SARS-CoV-2 among the population of the Grodno region was carried out within the framework of a 3-stage longitudinal stratified by age seroepidemiological study, which included questionnaires and a study of the seroprevalence to the RBD fragment of the SARS-CoV-2 S protein among the population. Determination of total (IgM and IgG) antibodies to SARS-CoV-2 was carried out using the qualitative enzyme immunoassay kit Wantai SARS-CoV-2 Total Ab (Beijing Wantai Biological Pharmacy Enterprise, Beijing, China). Results. A year after the registration of the first case of COVID-19, the population immunity of the population of the Grodno region was 58.8%. The proportion of seropositive participants increased in subsequent stages. At the first and third stages, there were no statistically significant differences in the level of seroprevalence both among the child and adult population, and among different age groups of the population. In the second phase of the study, the smallest proportion of seropositive individuals was in the 60–69 age group, and the highest in the 30–39 age group. There were no statistically significant differences in the level of seroprevalence among occupational groups, as well as among persons with different social behaviors. Conclusion. Taking into account the development of the epidemiological situation, the emergence of new variants of the SARS-CoV-2 coronavirus, as well as the ongoing vaccination of the population, it is advisable to further study the dynamics of the formation of population immunity.

Serological tests for the detection of specific antibodies to SARS‐CoV‐2 are an important tool both for confirming the diagnosis and for the epidemiological investigations. Comparative analysis of ELISA test kits for qualitative determination (yes/no) of IgG antibodies to SARS-CoV-2 available for purchase in the Republic of Belarus was done. The study included four commercial kits of various manufacturers: Vector-Best, Russia; N.F.Gamalei Research Center, Russia; Algimed, Belarus; Wuhan Fine Biotech, China. All test kits showed high quality in detection of specific IgG. The kits produced by Vector-Best, Russia, and Wuhan Fine Biotech, China had the highest sensitivity (100.0%); the kits produced by Algimed, Belarus, had the greatest specificity (100.0%). The dependence between the optical density of the samples and the concentration of IgG to SARS-CoV-2 was evaluated. The results showed that it is not possible to make a conclusion about the amount of antibodies based on the optical density of the sample in the test kits designed for their qualitative determination.

Цель. Оценка уровня популяционного иммунитета к SARS-CоV-2 среди населения Гомельской области Республики Беларусь на втором году пандемии COVID-19.Материалы и методы. Оценка популяционного иммунитета к SARS-CоV-2 среди населения Гомельской области проведена в рамках 3-этапного продольного стратифицированного по возрасту сероэпидемиологического исследования, включавшего анкетирование и изучение серопревалентности к RBD фрагменту S белка SARS-CoV-2. Определение суммарных (IgM и IgG) антител к SARS-CoV-2 осуществляли с помощью качественной иммуноферментной тест-системы Wantai SARS-CoV-2 Total Ab (Beijing Wantai Biological Pharmacy Enterprise, Пекин, Китай).Результаты. Уже на первом этапе исследования уровень серопревалентности населения Гомельской области Республики Беларусь к RBD фрагменту S белка SARS-CoV-2 составил 66,4%. Доля серопозитивных участников увеличилась на последующих этапах. Если на первом этапе статистически значимых различий в уровне серопревалентности как среди детского и взрослого населения, так и среди различных возрастных групп населения не выявлено, то на втором и третьем этапах исследования уровень серопревалентности среди взрослого населения был достоверно выше, чем среди детского. Также выявлены значимые различия в уровне серопревалентности среди различных возрастных групп детского и взрослого населения. Определенным фактором риска в распространении коронавирусной инфекции является принадлежность к профессиональным группам. При оценке влияния на уровень серопревалентности факторов риска, связанных с образом жизни, а также с использованием общественного транспорта, статистически значимых различий не выявлено.Заключение. С учетом развития эпидемиологической ситуации, появления новых вариантов вируса SARS-CоV-2, а также продолжающейся вакцинации населения целесообразно дальнейшее изучение динамики формирования популяционного иммунитета. Purpose. Evaluation of the level of population immunity to SARS-CoV-2 among the population of the Gomel region of the Republic of Belarus in the second year of the COVID-19 pandemic.Materials and methods. Evaluation of population immunity to SARS-CoV-2 among the population of the Gomel region was carried out within the framework of a 3-stage longitudinal stratified by age seroepidemiological study, which included questionnaires and a study of the seroprevalence to the RBD fragment of the SARS-CoV-2 S protein among the population. Determination of total (IgM and IgG) antibodies to SARS-CoV-2 was carried out using the qualitative enzyme immunoassay kit Wantai SARS-CoV-2 Total Ab (Beijing Wantai Biological Pharmacy Enterprise, Beijing, China).Results. Already at the first stage of the study, the level of seroprevalence of the population of the Gomel region of the Republic of Belarus to the RBD fragment of the SARS-CoV-2 S protein was 66.4%. The proportion of seropositive participants increased in subsequent stages. If at the first stage there were no statistically significant differences in the level of seroprevalence both among the child and adult population, and among different age groups of the population, then at the second and third stages of the study, the level of seroprevalence among the adult population was significantly higher than among the children’s. Significant differences in the level of seroprevalence among different age groups of children and adults were also revealed. A certain risk factor in the spread of coronavirus infection is belonging to professional groups. When assessing the effect on the level of seroprevalence of risk factors associated with lifestyle, as well as with the use of public transport, no statistically significant differences were revealed.Conclusion. Taking into account the development of the epidemiological situation, the emergence of new variants of the SARS-CoV-2 coronavirus, as well as the ongoing vaccination of the population, it is advisable to further study the dynamics of the formation of population immunity.

Species-specific differentiation of Bordetella pathogens in nasopharyngeal swabs of patients with suspected whooping cough was carried out using developed by us real-time PCR: duplex TaqMan PCR aimed for detecting B. pertussis and B. parapertussis, with primers targeting IS481, IS1001, BP0026 and GAPDH as the human internal control and two singleplex TaqMan PCR aimed for detecting B. holmesii and B. bronchiseptica with primers targeting hIS1001 and flaA specific genes. Among the 3130 throat swab analyzed, B. pertussis, B. parapertussis and B. holmesii were detected in 29.5 %, 0.9 % and 0.4 % of samples respectively. The developed algorithm proved to be a useful molecular-genetic diagnostic tool for whooping cough, established cocirculation of different species of Bordetella genus and demonstrated the predominance of B. pertussis, low rate of B. parapertussis and B. holmesii and the absence of B. bronchiseptica in the circulation in the Republic of Belarus.

Study results of IgG to diphtheria and tetanus in 785 residents aged from 1 to 76 years old from different regions of the Republic of Belarus (in 2017) in long-term (since 1996) immunization schedule: at 3, 4, 5 and at 18 months old – DTP vaccine, at 6 years old – DT, at 11 years old – Diphtheria toxoid, at 16 years old, 26 years old and every following 10 years –Td or Diphtheria toxoid are presented. The antibody concentration was measured by Virion/Serion kits (Germany) and evaluated in accordance with the international standard: less than 0.01 IU/ml – individual is susceptible, 0.01–0.09 IU/ml – levels of antitoxin giving some degree of protection, 0.1 – < 1 IU/ml – protective level of circulating antitoxin, ≥ 1.0 IU/ml – a level of antitoxin giving long-term protection. It was shown that the proportion of immune individuals against diphtheria and tetanus (with antibodies ≥ 0.01 IU/ml) was 96.7% (CI 95.4 ÷ 97.9) and 99.5% (CI 99.0 ÷ 100,0), respectively, and was quite high in all the population age groups – from 87.7 to 100% for diphtheria and from 96.5 to 100% for tetanus. In seropositive individuals IgG were presented mainly in protective and highly protective (≥ 0.1 IU/ml) titers: for diphtheria 93.7% – in 1–14 years old; 88.7% – in 15–19 years old; 78.4% – in 20–76 years old and for tetanus 100.0% – in 1–14 years old; 100.0% – in 15–19 years old; 99.3% – in 20–76 years old. Comparison of the current and previous studies results (in 1989–1994 – during the outbreak of diphtheria, in 1998–2001 – after the mass immunization campaign, in 2004 – in the context of continuous single cases of diphtheria registration in adults) had shown that the data of 2017 demonstrated the highest population immunity level to diphtheria and to tetanus in the last 30 years of observation.

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.

Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 – 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains.

In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.

The SF23 peptide corresponding to the receptor binding fragment of diphtheria toxin (residues 508-530) has been synthesized. This fragment forming a protruding beta hairpin has been chosen because it is the less mutable B-cell epitope. Affine chromatography and ELISA show that antibodies from the sera of persons infected by toxigenic Corynebacterium diphtheriae and those immunized by diphtheria toxoid are able to bind the synthetic SF23 peptide. There are antibodies recognizing the SF23 peptide in the serum of horses hyperimmunized with diphtheria toxoid. Analysis of circular dichroism spectra show formation of beta hairpin by the peptide. Taken together, the results showed that the structure of the less mutable epitope of C diphtheriae toxin was reproduced by the short SF23 peptide. Since antibodies against that epitope should block its interactions with cellular receptor (heparin-binding epidermal growth factor), the SF23 peptide can be considered as a promising candidate for synthetic vaccine development. Fluorescence quenching studies showed the existence of chloride and phosphate binding sites on the SF23 molecule. Phosphate containing adjuvants (aluminum hydroxyphosphate or aluminum hydroxyphosphate sulfate) are recommended to increase the SF23 immunogenic properties. (C) 2014 Elsevier Ltd. All rights reserved.

Background and Objective: Rapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening.Materials and Methods: Targets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella parapertussis in clinical samples. A total of 121 human clinical specimens obtained within 2012-2013 were used to evaluate the multiplex real-time PCR assay. Clinical specimens were also tested for culture and conventional PCR. Sensitivity and specificity for culture, conventional PCR, and multiplex real-time PCR were measured in comparison with a clinical standard for B. pertussis infection.Results: The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for IS481, IS1001 and 10 genomic equivalents per reaction for BP0026 target. When the B. pertussis assays were compared with a clinical standard for B. pertussis infection, sensitivity was 5, 59 and 89% the specificity was 100, 100 and 100% for culture, conventional PCR, and multiplex real-time PCR, respectively.Conclusions: Developed multiplex real-time PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections which is suitable for implementation in a routine laboratory diagnostics.

Иммуноструктура к коклюшу среди детского населения г. Минска / Е. Г. Фисенко [и др.] // Медицинский журнал. - 2012. - № 3. - С. 128-131.

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.

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.

DNA fragments 129 bp in length containing the promoter region of the tox gene from 81 toxigenic Corynebacterium diphtheriae strains were analyzed using single strand conformational polymorphism (SSCP). We found that only two strains carried mutations; these strains also showed the highest levels of the toxin production (over 5120 VERO CD50/ml). Other strains were characterized either as high (640–5120 VERO CD50/ml, 41 strains) or low (40–320 VERO CD50/ml, 38 strains) toxin producers. The nucleotide sequence analysis revealed single T to C mutations at positions −54 and −184 within the −232 to +85 region of the tox operon. The first mutation at position −184 was mapped outside of the tox promoter/operator, whereas the second substitution at position −54 modified the 9-bp interrupted palindromic sequence of the tox promoter/operator from ATAATTAGG in the wild-type bacteriophage β to ACAATTAGG in strains with an enhanced level of the toxin production. The nucleotide sequence analysis of region from −76 to +681 of the diphtheria toxin repressor (dtxR) gene from 15 strains of C. diphtheriae revealed two missense mutations resulting in amino acid substitutions A147V and L214I in the C-terminal (the third) region of the DtxR protein. Seven of these strains were identified as high toxin producers; four strains, as low toxin producers. In addition, one low-level toxin-producing strain was shown to contain a missense mutation leading to the amino acid substitution I221T. Three strains—two strains exhibiting the highest level of the toxin production and the nontoxigenic C7(-) strain—contained no nucleotide substitutions. The ten strains belonging to the Sankt-Peterburg and Rossija epidemic ribotypes, as well as the NCTC 13129 strain (the etiologic agent of the diphtheria epidemic outbreak in the Eastern Europe), contained two mutations, A147V and L214I, in the C-terminal region of the diphtheria toxin repressor (DtxR).

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).

Valentina Kolodkina, Leonid Titov, Tatyana Sharapa, Francine Grimont, Patrick AD Grimont, Androulla Efstratiou 1 Belarusian Research Institute for Epidemiology and Microbiology, Minsk, Belarus; 2 Centre National de Reference pour Corynebacterium diphtheriae, Unite de Biodiversite des Bacteries Pathogenes Emergentes INSERM U 389, Institute Pasteur, Paris, France; 3 HPA/WHO Streptococcus and Diphtheria Reference Unit, Respiratory and Systemic Infection Laboratory, Health Protection Agency Centre for Infections, London, UK;

ABSTRACT 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 BstEII and PvuII 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.

The outbreak of diphtheria in the Republic of Belarus developed in the presence of a sharp deficiency of immunity among the population aged 35 years and over. In connection with a higher initial immunity level resulting from vaccination made in one injection the minimum antibody level (> or = 0.01 I.U./ml) was found in 79.7% of the examinees among the population under 35 years. For the age group of 35-60 years vaccination in one and two injections proved to be insufficiently effective (the minimum protective antibody titer was found, respectively, in 64.3% and 63.3% of the vaccinees). To achieve the full protection of this group of the population, vaccination in three injections was necessary; after that the minimum protective antibody titer was found in 90.7%, the protective antibody titer (0.1 I.U./ml) was found in 53.5% and the highly protective antibody titer (1.0 I.U./ml) was found in 11.6% of the examinees. The low level of highly protected persons among vaccinees receiving three injections was due to a low dose of diphtheria toxoid (2 Lf) in adsorbed DT-m vaccine with reduced antigen content.