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

MICROBIAL LANDSCAPE OF MICROFLORA OF A PHARYNX AT PATIENTS WITH TONSILLIT’S PATHOLOGY

The microbial landscape of microflora of a pharynx at patients with tonsillit’s pathology were studied. Materials and methods. 79 patients from GBUZ “Research Institute of Clinical Otorhinolaryngology” (78.5% patients with various forms of chronic tonsillitis and 21.5% of patients without chronic tonsillitis (control group). Among patients of the main group with chronic tonsillitis, at 60 (96.8%) patients there was a diagnosis chronic tonsillitis a toxical-allergic form of 1 degree (TAF I), at 1 (1.6%) the patient — chronic tonsillitis a toxical-allergic form II of degree (TAF II) and at 1 (1.6%) the patient — chronic tonsillitis. Identification of microorganisms carried out on cultural-morphological and biochemical properties. Specific identification of the hardly cultivated microorganisms and Corynebacterium was carried out by MALDI-TOFF MS of BioMerieux VITEK MS MALDI-TOF (“bioMerieux”, France). Identification of the allocated Corynebacterium was carried out by amplification of a gene rpoB and the subsequent direct sequencing. Results. The majority (98.7%) of the allocated microorganisms, treated 27 types and were Gram-positive. It is revealed 159 strains of 29 species of microorganisms, from them 41.4% of strains belonged to the Streptococcus, 19.7% — Staphylococcus, 36.9% — Corynebacterium. Among Streptococcus — 55.4% of S. viridans, 38.4% — S. pyogenes and 3.1% — of S. pneumoniae и S. oralis; Staphylococcus — 64.5% of S. aureus, 32.2% of S. epidermidis and 3.3% of S. hominis. 18 types of Corynebacterium — С. tuberculostearicum (17.2% strains), C. рseudodiphtheriticum (15.5% strains) and C. aurimucosum (18.9% strains) are revealed. At 44.3% of the surveyed patients the microflora is presented by a monoculture and at 55.7% associations are revealed. Conclusion. The microflora at patients with tonsillit’s pathology is characterized. At the expressed pathological process in a microbiota of a pharynx the monoculture while existence of associations testifies to less expressed pathological process prevails

AN ACCELERATED METHOD OF DIPHTHERIA GENE DIAGNOSTICS BASED ON ISOTHERMAL AMPLIFICATION TO DETECT DNA OF THE CAUSATIVE AGENT

Aim. Development of an accelerated method of gene diagnostics of diphtheria based on isothermal amplification for detection of DNA of the causative agent. Materials and methods. The study was carried out in typical collection strains from GKPM-Obolensk, as well as in 135 strains of C. diphtheriae isolated from bacteriological laboratories of the regions of Russian Federation and sent to the Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology Strain isolation was carried out in accordance with Ml 4.2.698-98 and 4.2.3065-13. Chromosomal DNA was isolated by standard heating method, as well as using 3 commercial kits. Detection of the amplification results was carried out in horizontal electrophoresis in 1.5% agarose gel. Results. The developed method of gene diagnostics was established to allow detection of DNA of toxigenic C. diphtheriae strains of 2 biovars, as well as DNA of non-toxigenic tox-gene bearing strains (NTTB) of C. diphtheriae mitis biovar with mechanisms of lack of expression of diphtheria toxin gene due to the presence of deletion or mobile genetic IS element in the tox gene. Non-toxigenic tox-gene bearing C. diphtheriae strain with the mechanism of lack of diphtheria toxin gene expression due to the presence of transposon in the tox gene are identified as non-toxigenic. Evaluation of the analytical sensitivity in comparative studies using 3 commercial kits for FNA isolation has shown that sensitivity reached 4.5x 101 GE/ml using Ribo-prep kit. H igh specificity of the developed method is shown, it was evaluated in 18 strains of 16 other members of the Corynebacterium genus and 20 typical strains of microorganisms isolated from oropharynx or causing infections of the respiratory tract. Approbation of the developed method was carried out in model experiments in imitators of clinical samples by infection of single-use sterile dry tampons by consecutive dilutions of the bacterial cultures (with parallel seeding into dense nutrient media) and was 103 GE/ml. Conclusion. The developed method of accelerated gene diagnostics of the diphtheria infection has a high diagnostic effectiveness, specificity and sensitivity, allows to detect 103 - 4.5x10 GE/ml C. diphtheriae in clinical material with simultaneous verification of toxigenic and non-toxigenic strains

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

OPTIMIZATION OF A METHOD OF ISOTHERMAL AMPLIFICATION (LAMP) FOR DIAGNOSIS OF WHOOPING COUGH

Aim. Optimization of the accelerated whooping cough method of isothermal amplification for DNA Bordetela pertussis. Materials and methods. The research was conducted on 35 standard collection strains and 169 strains of Bordetella allocated in bacteriological laboratories of territorial subjects of the Russian Federation. The research included 329 clinical samples received from patients with whooping cough and the persons, contact with them, hospitalized in IDCH No. 1 DZM. Chromosomal DNA was extracted with a standard method of boiling from strains, from clinical samples by means of commercial sets. Identification of causative agents of whooping cough were performed with use of the АмплиСенс® Bordetella multi-FL. Results. We performed optimization method of a diagnostics of whooping cough by LAMP with detection by means of an electrophoresis and with naked-eye inspection under normal light is developed. The developed method allows to detect a DNA of B.pertussis within 4 - 5 hours in clinical material. The analytical sensitivity was 102 GE/ml. Assessment of validity showed that the developed method possesses 99,6% sensitivity and 98,7% specificity; predictive value positiveness and negative result was 99,6% and 98,7%, respectively; the index of accuracy (diagnostic efficiency) - 99,4%; the likelihood ratio of positive and negative result - 76,6 and 0,004, respectively. Assessment of analytical reliability in 100% of cases showed convergence and reproducibility of a technique. Conclusion. Diagnostic test on DNA of B.pertussis identification by LAMP method will allow to increase efficiency of laboratory diagnosis of whooping cough

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