The using of the polymerase chain reaction for the detection of resistance genes in gram-negative bacteria in routine practice in a pediatric hospital.

Objective - assessment of RT-PCR for the detection of carbapenem-resistance genes in gram-negative bacteria. A total, 499 strains of gram-negative microorganisms isolated in two pediatric hospitals in 2019-2020 were studied. Species identification was performed using MALDI-ToF mass-spectrometry (Bruker Daltonics, Germany). Meropenem and imipenem minimal inhibitory concentration (MIC) was determined by E-test method (BioMerieux, France). The presence of acquired carbapenemase genes of IMP, NDM, VIM, KPC, OXA-48, OXA-23, OXA-40, OXA-58-groups was determined by RT-PCR. Klebsiella pneumoniae (34%), Escherichia coli (4%), Serratia marcescens (6%) and other members of Enterobacterales (6%), also gram-negative non-glucose-fermenting bacteria Acinetobacter baumannii (14%), Pseudomonas aeruginosa (36%) were found among selected strains. Carbapenemase production was found in 385 isolates (77%). The main mechanism determining carbapenem resistance in P. aeruginosa was the production of blaVIM (100%). A. baumanii strains harbored OXA-23 (55%) and OXA-40 (45%) carbapenemases. The major determinant of carbapenem resistance in K. pneumoniae isolates was OXA-48 carbapenemase, detected in 63% strains, 13% of the strains possessed blaNDM-group, 16% isolates had a combination of blaNDM-group and blaOXA-48-like. Carbapenemase of KPC-group was found in 8% K. pneumoniae strains. OXA-48 carbapenemase prevailed (95%) among S. marcescens strains. Most of E. coli isolates harbored metallo-beta-lactamase NDM (89%). Other members of Enterobacterales most often had OXA-48 carbapenemase (57%), 39% of the isolates carried blaNDM-group. In one strain, a combination of blaNDM-group and blaOXA-48-like was discovered. RT-PCR is a fast and reliable method for the detection of acquired carbapenemases and can be recommended for routine use in bacteriological laboratories.

[The use of physical factors in the postoperative period with destructive appendicitis in children: the current state of the problem]. / Primenenie fizicheskikh faktorov v posleoperatsionnom periode pri destruktivnom appenditsite u detei: sovremennoe sostoyanie problemy.

Acute appendicitis is the most common childhood surgical disease. With the use of laparoscopic appendectomy for destructive appendicitis in children, the number of postoperative complications significantly decreased. However, in recent years there has been no tendency towards their further decline. The use of non-drug exposure, which has both local and systemic anti-inflammatory effects, can lead to further improvement in the treatment results of the most severe forms of the disease. As a result of a literature review, it was found that there is very little information about the use of physical factors in the postoperative period with destructive appendicitis in children. The effectiveness of the physical factors of systemic exposure (intravenous laser irradiation of blood, the use of ozone) was noted, but currently there are no recommendations on the application of local exposure physiotherapy techniques. Thus, the study of the effectiveness of the use of physical factors in the postoperative period with destructive appendicitis in children at the present stage remains relevant.

[APOΕ gene polymorphism and markers of brain damage in the outcomes of severe traumatic brain injury in children]. / Polimorfizm gena APOΕ i markery povrezhdeniya mozga v iskhodakh tyazheloi cherepno-mozgovoi travmy u detei.

OBJECTIVE: To compare apolipoprotein E (APOE) genotypes with outcomes and levels of neuromarkers in children with severe traumatic brain injury (TBI). MATERIAL AND METHODS: APOE polymorphisms were genotyped in 69 children with severe TBI. The following markers of brain damage were identified: neuron-specific enolase (NSE), glial protein S100b, content of autoantibodies (aAB) to glutamate receptors (to the NR2 subunit of NMDA receptors), aAB to S100b and brain-derived neurotrophic factor (BDNF). RESULTS AND CONCLUSION: There was no association between APOE 3/3, 3/4, 3/2 genotypes and outcomes assessed by the Glasgow Outcome Scale (GOS). The greatest number of favorable outcomes was noted in the group of APOE 3/3 genotype carriers (60%). The ratio of favorable outcomes to unfavorable outcomes was equal (50%:50%) in groups with APOE 3/4 and APOE 3/2 genotypes. An association between APOE polymorphism and BDNF was found: there were normal BDNF levels in the APOE 3/3 group and reduced levels in the APOE 3/2 group. The correlation between neuromarkers and GOS scores was shown for BDNF and aAB to S100b. In children with favorable TBI outcomes, normal BDNF levels and a lower level of aAB to S100b were observed. Regardless of APOE genotypes, almost all children with severe TBI (95%) showed a significant increase in aAB to glutamate receptors in the remote period and most children had an increase in aAB to S100b in the blood. This fact can be explained by the presence of cerebral hypoxia, activation of autoimmune processes and increased BBB permeability, which may be enhanced by increased NO content and intensification of oxidative processes in children with severe TBI.

Emergence of a ST307 clone carrying a novel insertion element MITEKpn1 in the mgrB gene among carbapenem-resistant Klebsiella pneumoniae from Moscow, Russia.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a major nosocomial pathogen with only a few antimicrobial agents, including colistin, remaining active. However, the emergence of colistin-resistant (Col-R) isolates is compromising the activity of colistin. In this study, a collection of 159 CRKP recovered from three hospitals in Moscow (Russian Federation) was examined. The isolates demonstrated resistance to cephalosporins (100%), ciprofloxacin (92.5%), fosfomycin (90.1%), netilmicin (81.1%), gentamicin (84.3%) and amikacin (49.7%). The rate of colistin resistance (MIC > 2 mg/L by broth microdilution) was 44.7%; moreover, 6.7% of isolates were tigecycline-resistant. Among 18 sequence types (STs) discovered, isolates of five lineages including ST307 (n = 46; 28.9%), ST395 (n = 40; 25.2%), ST377 (n = 17; 10.7%), ST48 (n = 17; 10.7%) and ST23 (n = 16; 10.1%) dominated. Carriage of a blaOXA-48-like carbapenemase gene was detected in 146 CRKP (91.8%); 11 (6.9%) and 2 (1.3%) isolates harboured blaNDM-1 and blaKPC-3, respectively. Among 71 Col-R isolates, colistin MICs ranged from 4 mg/L to >1024 mg/L (MIC50/90, 2/512 mg/L). All Col-R isolates were mcr-1-negative. In 19 (26.8%) Col-R isolates, inactivation of mgrB by insertion sequences IS1A, IS1R, ISKpn14 and ISKpn26 and a novel miniature inverted-repeat transposable element (MITE) Kpn1 was observed. Carriage of MITEKpn1 was restricted to six ST307 isolates and affected mgrB at nucleotide position 75. mgrB deletion was observed in four (5.6%) Col-R isolates. Moreover, PmrA and/or PmrB were altered in three (4.5%) Col-R isolates with wild-type mgrB. Thus, blaOXA-48-like-carrying Col-R ST307 K. pneumoniae is emerging as a dominant clone in Moscow.

[The comparison of methods for determination of colistin susceptibility in carbapenem-resistant Klebsiella pneumoniae.]

In recent years, because of carbapenemase spreading in Klebsiella pneumoniae strains, the antibiotic of reserve group, colistin, is increasingly prescribed. In vitro testing of colistin susceptibility in everyday practice has a number of difficulties due to the cationic properties of molecule and weak diffusion into agar. Therefore it is recommended to use the reference Broth Microdilution Method (BMD) for determination of the Minimum Inhibitory Concentration (MIC) for colistin. The purpose of the study was to determine susceptibility to colistin in 119 carbapenem-resistant K. pneumoniae (CRKp) which were isolated from the patients at three hospitals in Moscow in 2012-2016 by the broth microdilution method (BMD) and to compare these data with the ones obtained by epsilometer test (E-test) and VITEK 2 Compact. The proportion of resistant isolates (MIC>2 mg/L) was 52%, 39%, 35% respectively. Both commercial methods demonstrated a high level of the very major error (VME) that was 26% for the E-test method and 34% for the VITEK 2 Compact. The values of categorical agreement and essential agreement (CA, EA) were less than 90%. A single major error (ME) was detected for the VITEK 2 Compact. In conclusion, results of both commercial tests for determination of MIC for colistin showed differences with the results of the reference BMD. It is necessary for clinical laboratories to be aware about this discrepancy and to use E-tests and VITEK 2 Compact with caution to determine colistin susceptibility.

[Changes in serum levels of the key factors of angiogenesis in different outcomes of combined injuries in children]. / Izmeneniia kontsentratsii v syvorotke krovi kliuchevykh faktorov angiogeneza pri razlichnykh iskhodakh sochetannoi travmy u detei.

AIM: To study the changes in the key angiogenic factors VEGF-A and angiogenin (ANG) in children with different outcomes of combined injuries. MATERIAL AND METHODS: Contents of VEGF-A and ANG in blood serum were determined by enzyme immunoassay. The study included 40 patients, 21 boys and 19 girls. Patients were divided into 4 groups according to the outcome of injury: 1 - the recovery or mild residual symptoms; 2 - disabled; 3 - vegetable state; 4 - death. Patients were examined at different times after injury: 1-6 days, 7-11 days, 12-19 and 20-33 days. RESULTS AND CONCLUSION: In the first days after injury, the content of VEGF-A in patients of 1-3 groups was at the level of the reference group, moreover, in patients of the 3rd group it was close to the top edge. In group 4, the content of VEGF-A was maximal in the first days after injury and then gradually decreased to the point of death. In groups 1 and 3, the level of VEGF-A increased significantly starting from the 2nd week while in group 2 this indicator was slightly increased approaching later (up to 33 days of observation) to the upper values in the reference group. In the 3rd group, the content of VEGF-A reached the plateau on the 19th day after injury and was higher than the reference data, but lower than in patients of the 1st group. No correlation between the changes in ANG content at different times after combined injuries and outcome was found. There was a trend towards decreased levels of ANG, especially after 3-4 weeks after injury. The data obtained are important for the control over processes of vascular and tissue reparation after injury and for searching for effective ways of treatment of altered angiogenesis in such patients.

[Antibiotic Resistance and Its Molecular Mechanisms in Carbapenem-Nonsusceptible Klebsiellapneumoniae Isolated in Pediatric ICUs in Moscow.]

Klebsiellapneumoniae is a significant pathogen associated with hospital infections. Its was isolated in intensive care units (ICU) at two pediatric hospitals in Moscow in 2012-2014 from 41% (387/935) of the patients. The rate of carbapenem-nonsusceptibility (Carba-NS) amounted to 25% for imipenem and 27% for meropenem. For further analyses, 67 isolates were selected, including 57 Carba-NS and 10 Carba-susceptible (Carba-S). Among the isolates, 100% was nonsusceptible to the III-IV generation cephalosporins, 50-84% was resistant to aminoglycosides. The rate of nonsusceptibility to ciprofloxacin and phosphomycin exceeded 90%. All the tested Carba-S Kpneumoniae isolates were susceptible to tigecycline, whereas 25% of the Carba-NS isolates was tigecycline-NS. The prevalence of the colistin-NS isolates was the same in Carba-S (20%) and Carba-NS (26%) bacteria. The blamrx_ gene was carried by 100% of the Carba-S isolates, combining with the blaTEM gene in 60% of the isolates. In 89% of the Carba-NS isolates the OXA-48 carbapenemase was detected, which was combined with CTX-M and/or TEM in all but 1 isolate. Thus, over the last decade, the rate of Carba-NS among nosocomial Kpneuynoniae increased and the OXA-48 carbapenemase was shown to be dominating in the mechanism of Carba-NS in the pediatric ICUs in Moscow.

[Biomarkers in children with traumatic brain injury].

OBJECTIVE: to study the content of biomarkers of diagnostic and prognostic value in the assessment of traumatic brain injury (TBI) severity in children. MATERIAL AND METHODS: Authors determined the levels of glial protein S100B, neuronspecific enolase (NSE), autoantibodies (aAb) to glutamate receptors and natural autoantibodies (nAb) to S100B and brain-derived neurotrophic factor (BDNF) in serum/plasma of children with different outcomes of TBI. All parameters were analyzed in the 1-3rd, 7-8th, 14-15th and 20-23rd days after TBI, and, in some cases of severe brain injury and long stay patients in hospital, in 11-12 months after TBI. The severity and outcome of TBI were assessed according the Glasgow coma scale (GCS) and the Glasgow outcome scale (GOS), respectively. RESULTS AND CONCLUSION: The content of NSE and S100B increased immediately after TBI regardless of TBI severity, but in cases with favorable outcome it dropped to a normal level in the first 3 days. The maximum levels of S100B protein and NSE were observed in children with fatal TBI, and higher values were observed throughout the post-traumatic period. The low levels of aAb to NR2-subtype of glutamate receptors that were similar to controls and the high level of nAb to S100B in the first days after severe TBI indicate the failure of compensatory-adaptive immunological mechanisms and the high permeability of the brain-blood barrier, which were poor prognostic signs for children with severe TBI. Mild and severe TBI with full recovery were accompanied by higher values of ВDNF in the 1st day followed by a decline to the 3rd day. The level of BDNF in the 1st day of TBI was the lowest and subsequently continued to decline in patients with severe TBI with fatal outcome.

[Peripheral Blood Lymphocytes Mitochondrial Function in Children with Traumatic Brain Injury].

BACKGRAUND: It is known that mitochondria play an important role in the mechanisms of brain cells damage and death following traumatic brain injury (TBI). However, the relationship between the severity of brain damage following TBI and mitochondrial dysfunction are not well defined. AIM: to study activities of NADN- and succinate dehydrogenases, a key enzyme of mitochondrial oxidative phosphorylation in children with TBI of varying severity and different outcomes; to detect ATP content in lymphocytes; the level of NOx and 3-nitrotyrosine in serum and plasma. Methods: all parameters were determined in the dynamics of one month following TBI, and in some cases up to the death ofpatients. The severity of TBI was scored by Glasgow Coma Scale (GCS), the outcome of TBI-Glasgow Outcome Scale (GOS). Based on the clinical examination children with TBI were divided into 3 groups: (1) mild TBI; (2) severe TBI and (3) severe TBI with fatal outcome. RESULTS: we found that activity of dehydrogenases is significantly reduced only in patients with the poor neurologic outcome. The greatest decrease in these parameters was observed in patients with severe traumatic brain injury and fatal outcome. A direct correlation was found between the indices of dehydrogenases activity and A TP content in lymphocytes (r = 0.97, p = 0.005). The levels of NOx metabolites and 3-nitrotyrosine were significantly increased in children with severe TBI. CONCLUSION: obtained results suggest that mitochondrial dysfunction, impaired cerebral energy metabolism and oxidative stress contribute to cell death in the brain and thus represent therapeutic targets for the treatment of TBI.

[Monitoring and antibiotic resistance profile of tracheal aspirate microbiota in ICU children with severe craniocerebral trauma].

Nosocomial infections and their rational antibiotic treatment represent a major challenge for the healthcare nowadays. In this context, gramnegative bacteria including Pseudomonas aeruginosa, Acinetobacter baumanii and Enterobacteriaceae spp. are etiologically important and characterized by a significant level of antibiotic resistance. To examine dynamics of the respiratory tract colonization by hospital flora, tracheal aspirates obtained at three time points from 69 children with severe craniocerebral trauma during their stay in ICU were analysed. Colonization was observed on the 4th day of the ICU stay with predomination of K. pneumoniae (45%) and A. baumanii (27-37%). P. aeruginosa was detected after the 8th day of the ICU stay with the isolation rate of 33%. Substantial proportions of P. aeruginosa (61%), A. baumanii (78%) and K. pneumoniae (25%) were resistant to carbapenems. In 65 carbapemen resistant isolates, the presence of carbapenemases was examined using PCRs. OXA-48 carbapenemase was detected in 11 out of 14 (78%) K. pneumoniae isolates. Among the A. baumanii isolates, 30/31 (97%) carried OXA-40 and 1/31 (3%) had OXA-23 carbapenemases. None of the examined A. baumanii and K. pneumoniae isolates produced metallo-betalactamases (MBL). In contrast, all 20 carbapenem resistant P. aeruginosa isolates produced a MBL, and in 12 out of 20 (60%) of theme VIM-2 was detected. Thus, gramnegative nosocomial microflora rapidly colonizes ICU patients and has a high level of resistance to antibiotics, including carbapenems.

[Autoantibodies to α7-subunit of neuronal acetylcholine receptor in children with traumatic brain injury].

An objective of the study was to search for new biologically significant markers of brain damage. Levels of blood serum autoantibodies (aAB) to different fragments of α7-subunit of acetylcholine receptor (ACR) were studied in children with traumatic brain injury of different severity. The more severe was trauma, the higher was the level of aAB to fragments of α7-subunit of ACR in the first week after trauma. The data obtained suggest that α7-subunits of ACR and aAB to them are involved in the pathogenesis of traumatic brain lesions and, probably, play a significant role in the course of post traumatic period.

[S100B protein and autoantibodies to S100B protein in diagnostics of brain damage in craniocerebral trauma in children].

Levels of antibodies AB (AB) to S100B and S100B protein were studied in the blood serum of children with different severity and outcomes of traumatic brain injury (TBI) from the 1st to 15-75th days after TBI. Severity and outcomes were assessed using the Glasgow Coma Scale (GCS). Patients were stratified by outcomes into the following groups: complete recovery (group 1), moderate disability (group 2), high disability (group 3), vegetative state (group 4) and fatal outcome (group 5). In patients of groups 1-3, the changes of S100B in the blood serum didn't depend on the severity of brain's damage; the significant increase of S100B protein levels in the 1st day was accompanied by the decrease to the normal range in the following 2-3 days. On the contrary, the levels of nAB in these groups increased starting from 3-5 days corresponding to the severity of brain's damage. The development of vegetative state was accompanied by low S100B and high AB to S100B levels in the blood serum. The maximal level of S100B protein and increased levels of AB were observed in patients with fatal outcome. In most patients with combined TBI, the levels of both parameters were higher compared to those with separate TBI.

Effect of cell therapy on recovery of cognitive functions in rats during the delayed period after brain injury.

We studied the effect of systemic transplantation of human stem cells from various tissues on cognitive functions of the brain in rats during the delayed period after experimental brain injury. Stem cells were shown to increase the efficacy of medical treatment with metabolic and symptomatic drugs for recovery of cognitive functions. They accelerated the formation of the conditioned defense response. Fetal neural stem cells had a stronger effect on some parameters of cognitive function 2 months after brain injury. The efficacy of bone marrow mesenchymal stem cells from adult humans or fetuses was higher 3 months after brain injury.

Morphofunctional study of the therapeutic efficacy of human mesenchymal and neural stem cells in rats with diffuse brain injury.

We studied the effect of transplantation of human stem cells from various tissues on reparative processes in the brain of rats with closed craniocerebral injury. Combined treatment with standard drugs and systemic administration of xenogeneic stem cells had a neuroprotective effect. The morphology of neurons rapidly returned to normal after administration of fetal neural stem cells. Fetal mesenchymal stem cells produced a prolonged effect on proliferative activity of progenitor cells in the subventricular zone of neurogenesis. Adult mesenchymal stem cells had a strong effect on recovery of the vascular bed in ischemic regions.

[The enteral feeding during the treatment of appendicular peritonitis in children]. / Enteral'noe pitanie pri lechenii peritonita appendikuliarnogo proiskhozhdeniia u detei.

A total of 65 patients, aged 3 to 14, with different forms of appendicular peritonitis, in whom the intestinal insufficiency syndrome (IIS), stage 2, was diagnosed, were investigated. 35 patients were in the main group, and 30 patients were in the control group; the subjects of the latter group had a similar form of peritonitis, i.e. IIS, stage 2, it was confirmed clinically and by ultrasound examinations. The laparoscopic operations were carried out in all patients. The patients of both groups received postoperatively a similar therapy, i.e. the antibacterial, metabolic and infusive ones, as well as vitamins and parenteral feeding (daily caloric values--50-120 kcal/kg, protein--2-3 g/kg/day) according to a child's age and a clinical course of peritonitis. All children of the main group received postoperatively an early enteral therapy (EET), which involved 4 stages. Stage 1: introduction of the salt solution with added glutamine through the naso-gastric probe by increasing volumes and with respect to an individual tolerability of a patient; stage 2: introduction of semi-element mixtures; stage 3: introduction of 10% solutions of polymer balanced mixture; stage 4: introduction of 20% solutions of polymer mixtures. Ultrasound scanning and clinical methods were used to assess the efficiency of the recovery of intestinal peristalsis. The conducted investigations showed that the mean duration of the gastric-type EET amounted to 3-4 days, and the IIS was, on the average, arrested by days 6 or 7. The duration of infusive therapy and parenteral feeding went down, in the main group, by 2 days. A trend towards an increase of the erythrocyte level was noted in these patients. No differences were registered in the values of concentrations of total protein and albumin in the blood serum of patients in both groups from the 1st to 7th postoperative days, however, the infusions of plasma and albumin were made by 2 times more often in the main group as compared to the control one. The EET technique preserved the gastric mucous tunic intact, which cut the postoperative complications 2-fold, and consequently, it reduced the stay of patients in hospital on the average by 8 days. A conclusion was made, on the basis of the conducted study, that EET is a pathophysiologically substantiated treatment method for IIS, stage 2, in children with appendicular peritonitis; EET ensures a rapid recovery of gastric-tract functions, it reduces the frequency rate of postoperative complications and cuts the length of both the infusive therapy and of the patients' stay in hospital.

[Respiratory chlamydial infection in children with a complicated course of acute respiratory viral infections]. / Respiratornaia khlamidiinaia infektsiia u detei s oslozhnennym techeniem ostrykh respiratornykh virusnykh infektsii.

The role and influence of Chlamydia trachomatis on the development and course of acute respiratory virus infections (ARVI) in 130 children admitted to the hospital of the Research Institute of Children's Infections (St. Petersburg) was studied. The occurrence of respiratory Chlamydia infections in ARVI patients with an unfavorable premorbid background was 35-36%. The disease took its course simultaneously with the mixed infection of the respiratory tract with viruses, pneumococci and their associations with staphylococci and Haemophilus influenzae. Chlamydia infection in children had no influence on the character of the clinical manifestation of ARVI with the exception of the obstruction syndrome which was constantly observed in children with ARVI (p < 0.001).