FEATURES OF GENOTYPiNG VARIABILITY OF STRAINS OF BORDETELLA PERTUSSIS ALLOCATED FROM PATIENTS WITH WHOOPING COUGH IN RUSSIA

On the basis of studying of features of structure of 7 genes determining major factors of pathogenicity of the causative agent of whooping cough - ptxA, ptxB, ptxC, ptxD, ptxE, ptxP and prn, features of genotyping variability of strains of B.pertussis allocated from patients with whooping cough in Russia are shown. Dynamics of formation of population of strains of the causative agent of whooping cough is tracked and is established that population of strains of B.pertussis is formed by clonal expansion of strains with, new genetic structure of major factors of pathogenicity - pertussis toxin and. pertactin. The structure of population of strains of B.pertussis causing a disease by whooping cough, at the present stage of epidemic process of a pertussis infection - domination. of strains with new «non-vaccine» alleles genes - ptxA1 (97,7 %), ptxC2 (87,4 %), prn2 (89,5 %) and. ptxP3 (93,3 %), in 2,2 % cases with ptxB2 allele and. in 1,3 % cases with. prn9 allele circulate

Characterization of toxigenic Corynebacterium diphtheriae strains isolated in Russia

The aim of the study was to characterize toxigenic strains of Corynebacterium diphtheriae by examining 12 toxigenic strains of C. diphtheriae isolated in Russia between January, 2017 to June, 2019. The morphological, toxigenic and biochemical properties of C. diphtheriae was studied. Genotyping of C. diphtheriae strains was performed using MLST and dtxR gene sequencing with subsequent phylogenetic analysis. Results. Toxigenic strains of C. diphtheriae were isolated in the Novosibirsk, Samara and Chelyabinsk Regions, the Khanty-Mansi Autonomous Okrug — Yugra as well as the Republic of Northern Ossetia — Alania. Among these strains, 5 were isolated from diphtheria patients (moderate disease found in one case, mild course — remaining patients) and 7 strains were isolated from bacterial carriers. In two cases C. diphtheriae from diphtheria patients were identified as ST25 sequence type, gravis variant; in one case — ST8 type, gravis variant; two cases — ST67 sequence type, mitis variant. In asymptomatic carriers of tox-positive C. diphtheriae strains they belonged to ST25 sequence type, gravis variant — in two cases, ST67 type, mitis variant — in four cases. A sequencing type was not identified in one case. All sequence types were widespread globally being presented by a large number of isolates in the PubMLST and characterized by a substantial amount of derivative sequence types. At the same time, they belonged to different clonal complexes and differed markedly from each other contributing to their reliable difference as assessed by MLST. Study of gene dtxR sequence diversity showed that all allelic variants were typical for the representatives of these sequence types. New alleles of gene dtxR were not revealed in strains examined. It was shown that non-synonymous substitution C440T leading to A147V amino acid substitution was found solely in one allele distributed in ST8, ST185, ST195 and ST451 types suggesting at late mutation. In contrast, the polymorphism C640A resulting in the amino acid substitution L214I was found not only in the same allele, but also in the basal tree branches indicating that isoleucine was in the ancestral sequence of the protein

Определение противококлюшных антител у школьников с длительным кашлем

Objective: to assess anti-pertussis immunity in schoolchildren aged 7–17 who complained of a prolonged cough during the 11-year follow-up period.  Materials and methods. The study included 1046 patients aged 7 to 17 years who applied to the Consultative and Diagnostic Center of the G.N. Gabrichevsky Research Institute of Epidemiology and Microbiology with complaints of prolonged cough in the period from 2010 to 2020. Blood serums were examined in ELISA with the determination of IgM, IgG, IgA antibodies using RIDASCREEN test system (Germany).  Results. An active infection with the detection of IgM and/ or IgA, IgG antibodies above threshold levels was detected in 51,3% of children with prolonged cough, while annually in a fairly high percentage throughout the follow-up period. Active pertussis infection, established based on the detection of IgM, IgG, IgA antibodies above thresholds in blood serum samples, prevailed in children 12–15 years old, accounting for more than 60% in children with prolonged cough. Antipertussis immunity as a result of childhood vaccination or previous disease was detected in 16.1-20.2% of people in the period 2010–2014 and in 12,8-20,9% in 2015–2020.  Conclusion. The results obtained by us on the study of anti-pertussis immunity in schoolchildren confirm the presence of active latent circulation of the pathogen whooping cough among children of this age cohort and, therefore, the presence of unaccounted for cases of the disease. This confirms the importance of timely diagnosis of pertussis, isolation of children for the period of active infection and justifies the need for the widespread introduction of a second revaccination against pertussis. Цель: оценка противококлюшного иммунитета у школьников 7–17 лет, обратившихся с жалобами на длительный кашель, в течение 11-летнего периода наблюдения.  Материалы и методы. В исследование включено 1046 пациентов в возрасте от 7 до 17 лет, обратившихся в консультативно-диагностический центр Московского научно-исследовательского института эпидемиологии и микробиологии им. Г.Н. Габричевского с жалобами на длительный кашель в период с 2010 по 2020 г. Сыворотки крови исследовали в ИФА с определением IgM, IgG, IgA антител с помощью тест-системы «RIDASCRЕЕN» (Германия). Результаты. Активная инфекция с выявлением антител классов IgM и (или) IgA, IgG выше пороговых уровней выявлена у 51,3% детей с длительным кашлем, при этом ежегодно в достаточно высоком проценте на протяжении всего периода наблюдения. Активная коклюшная инфекция, установленная на основании выявления в образцах сывороток крови антител IgM, IgG, IgA выше пороговых значений, преобладала у детей 12–15 лет, составляя выше 60% у детей с длительным кашлем. Противококлюшный иммунитет в результате проведенной в детстве вакцинации или перенесенного заболевания выявили у 16,1–20,2% лиц в период 2010–2014 гг. и у 12,8–20,9% – в 2015–2020 гг.  Заключение. Полученные нами результаты по изучению противококлюшного иммунитета у школьников подтверждают наличие активной скрытой циркуляции возбудителя коклюша среди школьников и, следовательно, наличие недоучтенных случаев заболевания. Это подтверждает важность своевременной диагностики коклюша, изоляции детей на период активной инфекции и обосновывает необходимость повсеместного введения второй ревакцинации против коклюша.

Выявление Bordetella holmesii среди больных, госпитализированных в стационар с подозрением на коклюш или коклюшеподобные заболевания

Purpose. To reveal and estimate prevalence of B. holmesii among the patients hospitalized with suspicion pertussis and pertussis-like illnesses.Materials and methods. 424 clinical samples received from patients with of pertussis and pertussis-like illnesses in GBUZ IKB № 1 DZM in 2017–2018 are investigated. Identification of fragments of a genome of Bordetella was carried out in PCR-RT with “Amplisens® Bordetella multi-FL”. For identification of fragments of a genome of B. holmesii used PCR-RT with primers of IS481, IS1001 and hIS1001.Results. The research included 424 patients, from them 56,1% of children aged till 1 year, 41,3% of children – are more senior than 1 year and 2,6% of adults. When using test system 60,4% of the samples containing DNA of B. pertussis are revealed; 1,9% of samples – DNA of B. parapertussis; in 34,9% of samples it is received negative and in 2,8% – doubtful results. The research of 424 samples in PCR-RT by means of IS481, IS1001 and hIS1001 primers showed that 61,1% of samples contained DNA of B. pertussis; 0,7% of samples – DNA of B. parapertussis and 3,8% of samples – DNA of B. holmesii. In 143 samples the result was negative. From 16 DNA of B. holmesii – positive samples, 9 samples were negative in test system earlier, in 2 samples – the doubtful result, 1 sample was earlier identified as DNA of B. parapertussis and in 4 samples DNA of B. pertussis and B. holmesii are found.Conclusion. The research demonstrates circulation of B. holmesii in the territory of Russia that is confirmed by identification of positive samples in 3,8% of cases among the sick children and adults hospitalized in a hospital with suspicion of pertussis and pertussis-like illnesses. For increase in efficiency of laboratory confirmation of the clinical diagnosis of pertussis and pertussis-like illnesses the genodiagnostic of a pertussis is recommended to improve taking into account identification DNA of B. holmesii.Цель: выявить и оценить распространенность B. holmesii среди больных, госпитализированных в стационар с подозрением на коклюш и коклюшеподобные заболевания.Материалы и методы. Исследовано 424 пробы клинического материала от больных с подозрением на коклюш и коклюшеподобные заболевания, госпитализированных в Инфекционную клиническую больницу № 1 в 2017– 2018 гг. Выявление фрагментов генома бордетелл осуществляли в ПЦР-РВ с «АмплиСенс® Bordetella multi-FL». Для идентификации фрагментов генома B. holmesii использовали ПЦР-РВ с праймерами IS481, IS1001 и hIS1001. Результаты. В исследование включено 424 пациента, из них 57,6% детей до 1 года, 42,3% детей старше 1 года и 2,6% взрослых. При использовании тестсистемы обнаружено 60,4% образцов, содержащих ДНК B. pertussis; 1,9% образцов – ДНК B. parapertussis; в 34,9% образцов получен отрицательный и в 2,8% – сомнительный результаты. Исследование 424 образцов в ПЦР-РВ с помощью IS481, IS1001 и hIS1001 праймеров показало, что 61,1% образцов содержали ДНК B. pertussis; 0,7% образцов – ДНК В. parapertussis и 3,8% образцов – ДНК B. holmesii. В 143 образцах результат был отрицательным. Из 16 ДНК B. holmesii, 9 образцов ранее были отрицательными, в 2 образцах – сомнительный результат и 1 образец был ранее идентифицирован как ДНК B. parapertussis, в 4 образцах обнаружена ДНК B. pertussis и B. holmesii.Заключение. Исследование свидетельствует о циркуляции B. holmesii на территории России, что под тверждается выявлением положительных образцов в 3,8% случаев среди больных детей и взрослых, госпитализированных в стационар с подозрением на коклюш и коклюшеподобные заболевания. Для повышения эффективности лабораторного подтверждения клинического диагноза коклюша и коклюшеподобных заболеваний рекомендуется совершенствовать генодиагностику коклюшной инфекции с учетом идентификации ДНК B. holmesii

MICROBIOCENOSIS OF SKIN IN BROMHIDROSIS PATIENTS

Aim. Evaluate the composition of microorganisms of skin microbiocenosis of axilla in brom-hidrosis patients. Materials and methods. 23 patients were examined (11 - 17 years) under the observation at Pirogov CCDC of the National Medical-Surgery Centre. Identification was carried out using biochemical test-systems BioMerieux VITEK MS MALDI-TOF («bioMerieux», France) and 16SrRNA genesequencing with consequent juxtaposition with EMBL/NCBI. Medium and high degree of skin seeding with microbiota was present in most of the patients with bromhidrosis (52.2 and 43.5%). 137 strains belonging to 5 genera of microorganisms were identified - Corynebacterium, Staphylococcus, Moraxella, Micrococcus, Candida and Bacillus spp. Coiynehacte-rium genus strains (8 species) and Staphylococcus genus (5 species) prevailed in microbiocenosis (89.1%). C. tuberculostearicum strains dominated among Corynebacterium, and S. hominis - Staphylococcus. Conclusion. In most of the cases (82.6%) in patients microbiocenosis of skin of axilla was presented by consortiums of microorganisms with prevalence of Corynebacterium and Staphylococcus microorganisms

Identification of Bordetella holmesii among the patients hospitalized with suspicion of pertussis and pertussis-like illnesses

Purpose. To reveal and estimate prevalence of B. holmesii among the patients hospitalized with suspicion pertussis and pertussis-like illnesses.Materials and methods. 424 clinical samples received from patients with of pertussis and pertussis-like illnesses in GBUZ IKB № 1 DZM in 2017–2018 are investigated. Identification of fragments of a genome of Bordetella was carried out in PCR-RT with “Amplisens® Bordetella multi-FL”. For identification of fragments of a genome of B. holmesii used PCR-RT with primers of IS481, IS1001 and hIS1001.Results. The research included 424 patients, from them 56,1% of children aged till 1 year, 41,3% of children – are more senior than 1 year and 2,6% of adults. When using test system 60,4% of the samples containing DNA of B. pertussis are revealed; 1,9% of samples – DNA of B. parapertussis; in 34,9% of samples it is received negative and in 2,8% – doubtful results. The research of 424 samples in PCR-RT by means of IS481, IS1001 and hIS1001 primers showed that 61,1% of samples contained DNA of B. pertussis; 0,7% of samples – DNA of B. parapertussis and 3,8% of samples – DNA of B. holmesii. In 143 samples the result was negative. From 16 DNA of B. holmesii – positive samples, 9 samples were negative in test system earlier, in 2 samples – the doubtful result, 1 sample was earlier identified as DNA of B. parapertussis and in 4 samples DNA of B. pertussis and B. holmesii are found.Conclusion. The research demonstrates circulation of B. holmesii in the territory of Russia that is confirmed by identification of positive samples in 3,8% of cases among the sick children and adults hospitalized in a hospital with suspicion of pertussis and pertussis-like illnesses. For increase in efficiency of laboratory confirmation of the clinical diagnosis of pertussis and pertussis-like illnesses the genodiagnostic of a pertussis is recommended to improve taking into account identification DNA of B. holmesii

Противококлюшные антитела классов IgМ, IgG и IgA у беременных в I–II–III триместрах

Purpose: to investigate the level anti-pertussis IgM, IgG, IgA in pregnant women in the I — II — III trimesters. Materials and methods. A total of 288 pregnant women aged. 17 — 43 years (in the I — II — III trimesters) were examined. A serums samples tested for IgM, IgG, IgA against pertussis by ELISA of RIDASCREEN (Germany). Results. Anti-pertussis IgG concentration below the low limit of detection occurred in 75,7% of pregnant women. The majority of seropositive among pregnant women was in the age group 26 — 35 years (82,9%). The proportion of seronegative among pregnant women increased. with increasing pregnancy periods, as well as depending on age. In III trimester IgG were detected in 18,2% of pregnant women. The most seronegative among pregnant women were in the III trimester and at the age of over 36 years old. The detection of IgM, IgG and IgA made it possible to detect the active infection process in 11 (3,9%) pregnant women by elevated level of IgA. High IgA level only in combination with IgM was in serum sample from 1 pregnant woman, high IgA level in combination with IgG at negative values of IgM was in serum samples from 8 pregnant woman. Only IgA in serum sample from 2 pregnant women (I and III trimesters of pregnancy) were detected. This is probably due to the presence of whooping cough or mucosal contamination with B.pertussis (persisting IgA). Conclusion. The serological studies have shown the need to develop algorithms for protecting newborns — from, the moment pregnant women are registered, to the onset of childbirth. These algorithms will provide information about on the presence of whooping cough and will help prevent infection of the newborn.Цель: определение наличия противококлюшных антител классов IgM, IgG, IgA у беременных в I—II— III триместрах беременности.Материалы и методы. В исследование включено 288 пациенток (17—43 лет), находящихся в I—II—III триместрах беременности. Сыворотки крови исследовали в ИФА с определением IgM, IgG, IgA антител с помощью тест-системы ««RIDASCREEN» (Германия).Результаты. При определении только IgG антител 24,3% беременных оказались серопозитивными. Максимальный удельный вес серопозитивных беременных был отмечен в возрастной группе 26—35 лет. (82,9%). Удельный вес серонегативных лиц имел тенденцию к возрастанию с увеличением сроков беременности, а также в зависимости от возраста. В III триместре антитела класса IgG были определены у 18,2% лиц. Больше всего серонегативных лиц было выявлено в III триместре у женщин старше 36 лет. Одновременное определение антител классов IgM, IgG и IgA позволило выявить активный процесс инфицирования у 11 (3,9%) беременных по повышенным уровням антител класса IgA, в том числе у 1 беременной в сочетании только с IgM антителами, у 8 — в сочетании с IgG антителами при отрицательных значениях IgM антител. У 2 беременных, находившихся в I и III триместрах беременности, выявлены только IgA антитела, что, возможно, связано с перенесенной коклюшной инфекцией или контаминацией слизистых оболочек возбудителем (определяются так называемые персистирующие IgA-антитела).Заключение. Проведенные нами серологические исследования показали необходимость разработки алгоритмов защиты новорожденных, которые следует начинать задолго до рождения — от момента постановки беременных на учет до наступления родов. Данные алгоритмы позволят получить своевременную информацию о вероятности инфицированности возбудителем коклюша и тем самым будут способствовать предупреждению инфицирования новорожденного