Hybridization-fluorescence Detection Research Articles

Etiology of acute intestinal infections of viral character in the Altai territory

BACKGROUND: The Incidence of acute intestinal infections (AII) is generally maintained at a high level. Significant progress made in the field of laboratory diagnostics allowed us to proceed to a detailed study of the etiological structure of AII, and as a result, it was found that in recent years the role of pathogens of a viral nature has significantly increased, but a detailed study of the characteristics of these infections requires further research. AIMS: To study the structure and clinical and laboratory features of intestinal infections of viral etiology in adult hospitalized patients in the Altai Territory. MATERIAL AND METHODS: From 2017 to 2020, a single-stage study was conducted of 67 patients of an infectious hospital with AII of viral etiology, aged from 18 to 76 years, hospitalized in the infectious departments of the City Hospital No. 5, Barnaul. In order to determine the structure of AII, the polymerase chain reaction (PCR) method with hybridization-fluorescence detection AmpliSens AII screen-FL, bacteriological and serological methods were used. Taking into account the results of laboratory examination (molecular-genetic, bacteriological and serological), all patients were divided into 3 groups: group 1 ― 45 patients with viral monoinfection (67.2%), group 2 ― 9 patients with viral mixed infection (13.4%) and group 3 ― 13 patients with combined viral ― bacterial etiology (19.4%). Data processing and graphical representation were performed using computer programs Statistica 10.0 (Russian version) (Russia), Excel 2010 (Windows 10). RESULTS: As a result of the study, it was found that among the AII of viral etiology, 67.2% had monoviral AII and 32.8% had mixed AII-mixed viral (13.4%) and viral-bacterial etiology (19.4%). Among monoviral AII dominated norovirus infection (in 52,9%). Noted that the differences of clinical and laboratory parameters of the monoviral and mixed-viral infections have not been found, and there were no differences in groups of monoviral infections: rotavirus, adenovirus and norovirus, which is consistent with earlier data. As well as co-infection of salmonellosis, in combination with one and/or several viruses, more often occurred with febrile syndrome, watery diarrhea and abdominal pain. CONCLUSION: Thus, in adult patients in the conditions of an infectious hospital, mono-and mixed-virus infection (in combination with two or more viruses or bacteria) was detected in the structure of viral AII by PCR. There was no statistically significant difference in clinical and laboratory parameters in the study groups due to the insufficient sample size and the feasibility of conducting further studies using a similar protocol with a larger number of patients.

Effectiveness and safety of «Nifuroxazide-ECO» in therapy of escherichioses in children

In conditions of increasing antibiotic resistance and widespread antibiotic-associated diarrhea caused by Clostridium difficile, it is necessary to choose a rational drug for the empirical treatment of escherichioses, which has a wide range of antibacterial activity and does not lead to the development of serious side effects and disruption of the gut microbiota.The aim of the study is to assess the effectiveness and safety of the use of the drug “Nifuroxazid-ECO” in the integrated therapy of escherichioses in children.Patients and methods: 50 patients aged 1 month to 18 years were selected for work by random sampling, hospitalized in the intestinal department of the Pediatric Research and Clinical Center for Infectious Diseases of Russia with non-severe clinical forms of escherichioses. All patients underwent routine clinical, biochemical, instrumental examination. DNA detection of the pathogen was carried out by molecular methods in fecal samples using a set of reagents to detect and differentiate the DNA of diaregenic E. coli in environmental objects and clinical material by polymerase chain reaction (PCR) with hybridization-fluorescent detection. Patients received the drug “Nifuroxazid-ECO”at age dosage, every 6–8 hours for 5–7 days.Results: Against the background of the therapy, a significant majority of patients showed clinical improvement. The average duration of hospitalization was 4.8 days, the duration of preservation of diarrhoeal syndrome was 3.4 days. The drug showed good tolerability. There were no cases of antibiotic-associated diarrhea, re-hospitalization at an early date after treatment, serious adverse reactions.Conclusion: Studies have shown that the drug “Nifuroxazid- ECO” is a safe and effective modern drug which can be successfully used in the therapy of non-severe escherichioses in children.

ТЕХНОЛОГИЯ REAL-TIME ПЦР-ГЕНОТИПИРОВАНИЯ BOS TAURUS ПО ВАРИАНТАМ АЛЛЕЛЕЙ А И B ГЕНА КАППА-КАЗЕИНА

The main goal of the study was to develop and test an effective technology for cattle genotyping by the CSN3 gene based on real-time PCR with hybridization-fluorescence detection. There was developed a method for real-time PCR for cattle genotyping by A and B alleles of the CSN3 gene in the format of hybridization-fluorescence detection, involving the use of two 5/-fluorescence-labeled forward allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher at the 3/-end of the oligonucleotide. As a result of practical studies aimed at testing the developed method, we obtained the technical result provided by the proposed technology, expressed in the effective identification of the desired genotypes due to correct interpretation of these curves of increasing fluorescence intensity, the results reliability of which was also confirmed by the well-known PCR-RFLP analysis technique for Bos taurus genotyping for similar allelic variants of the kappa-casein gene.

Molecular detection and genotyping of β-human papillomavirus and it's association with epithelial skin neoplasms.

Epidemiological and molecular biological data suggest that β-human papillomavirus (HPV) can cause epithelial skin tumors, but the relationship remains unclear. A new approach to the diagnosis of HPV is based on the measurement of viral consistence. We examined 52 immune-compromised and immune-competent patients in order to identify the association of epithelial neoplasms with β-HPV. Determination of HPV was performed by polymerase chain reaction with hybridization-fluorescence detection in real-time. Amplification and detection were carried out with "Rotor-Gene" 3000 ("Corbett Research," Australia). To quantify the beta HPV genus, we used recombinant plasmid positive controls as well as control plasmid of β-globin fragments taken from human genes (Central Scientific Institute of Epidemiology Rospotrebnadzor). We have found that β-HPV DNA predominated in fibroepithelial polyps (64%) and in the apparently healthy skin (54%) of immune-compromised patients versus 47% in the skin of healthy donors. Mixed infection was detected in fibroepithelial polyps of 57% in the immune-compromised patients. Viral consistence in the fibroepithelial polyps was higher than in the apparently normal donors' skin. The high detection of HPV DNA was found in fibroepithelial polyps and in the apparently healthy skin of immune-compromised patients whereas a high level of HPV DNA was only found in fibroepithelial polyps.

Genetic characteristics of antimicrobial resistance mechanisms in Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii isolated from blood and cerebrospinal fluid of children

Objective. To assess antimicrobial susceptibility, presence of resistance genes and determine the phenotypic groups of K. pneumoniae, P. aeruginosa and A. baumannii isolated from blood and cerebrospinal fluid of children with nosocomial infections in intensive care units from 2014 to 2020. Materials and Methods. Minimum inhibitory concentrations of antibiotics were determined using the serial broth microdilution method. The identification of genes encoding the production of carbapenemases was carried out using hybridization fluorescence detection. Results. A total of 63 isolates of K. рneumoniae, 23 isolates of P. aeruginosa and 14 isolates of A. baumannii were tested in this study. K. pneumoniae was detected in 10.3%. P. aeruginosa was isolated at a frequency of 3.5%. A. baumannii accounted for 2.3%. The proportion of carbapenemresistant K. pneumoniae strains to meropenem and imipenem was 33% and 37%, respectively, of all isolates. Resistance to colistin and polymyxin in K. pneumoniae isolates was 33% and 24%, respectively. The production of carbapenemases OXA-48 was detected in 25 (89%) isolates. The presence of NDM, VIM, KPC carbapenemases was not detected. Among P. aeruginosa, 65% were resistant to meropenem, and 74% to imipenem. The highest activity against P. aeruginosa in vitro was exhibited by polymyxins. There were no strains that were insensitive to colistin. The detection rate of metallo-β-lactamases (MBL) in P. aeruginosa strains was 48%. Only VIM-type MBLs were identified. No other types of MBL have been found. A. baumannii was non-susceptible to meropenem in 64% and to imipenem in 71%. The highest in vitro activity against A. baumannii was shown by polymyxin. Rate of colistin resistance was 29%. The OXA-40 and OXA-23 genes were detected in 5 and 3 isolates, respectively. Conclusions. There were high resistance rates to most antimicrobials among K. pneumoniae, P. aeruginosa и A. baumannii isolated from blood and cerebrospinal fluid in children with nosocomial infections. The increase in carbapenem resistance rates was also observed. Carbapenem resistance was due to OXA48 carbapenemases in K. pneumoniae, VIM-type MBLs in P. aeruginosa, and OXA-40 and OXA-23 carbapenemases in A. baumannii.

Analysis of gmo contents as a component in risk-oriented surveillance over food products safety

Control over use of genetically modified products is a vital task within a risk-oriented model for surveillance over food safety products all over the world including the Russian Federation. Our research goal was to examine domestically manufactured food products in order to determine whether they contained certain regulatory sequences typical for genetically modified organisms. We applied polymerase chain reaction with hybridization-fluorescent detection in real time mode to examine 77 food products samples; the task was to determine whether they contained DNA enhancer (E-35S) and promoter (P-35S) of S35 sequence belonging to cauliflower mosaic virus, terminator of nopaline synthase gene from Agrobacterium tumefaciens (T-NOS), 35S enhancer (E-FMV) and promoter (P-FMV) of Figwort mosaic virus, as well as vegetable DNA inducing soya DNA. When analyzing the extracted DNA, we didn’t detect transgenic elements in any samples; however, there were vegetable components reveled in them including 68.8% samples with soya DNA. We established that some sausages were falsified as they contained vegetable elements. In 15.6% cases data on a product structure turned out to be false because soya DNA was not listed on consumer package. Our research on determining soya DNA and transgenic elements in food products indicates that soya ingredients have been added into food products in spite of their absence in relevant documents as recipe components. All the obtained results taken into account, we assume it is necessary to improve control procedures for detecting genetically modified and vegetable components used as ingredients in food products as their falsification can make for changes not only in their consumer properties but also damage consumers’ health.

Анализ содержания компонентов генетически модифицированных организмов как элемент риск-ориентированного надзора за безопасностью пищевых продуктов

Control over use of genetically modified products is a vital task within a risk-oriented model for surveillance over food safety products all over the world including the Russian Federation. Our research goal was to examine domestically manufactured food products in order to determine whether they contained certain regulatory sequences typical for genetically modified organisms. We applied polymerase chain reaction with hybridization-fluorescent detection in real time mode to examine 77 food products samples; the task was to determine whether they contained DNA enhancer (E-35S) and promoter (P-35S) of S35 sequence belonging to cauliflower mosaic virus, terminator of nopaline synthase gene from Agrobacterium tumefaciens (T-NOS), 35S enhancer (E-FMV) and promoter (P-FMV) of Figwort mosaic virus, as well as vegetable DNA inducing soya DNA. When analyzing the extracted DNA, we didn’t detect transgenic elements in any samples; however, there were vegetable components reveled in them including 68.8% samples with soya DNA. We established that some sausages were falsified as they contained vegetable elements. In 15.6% cases data on a product structure turned out to be false because soya DNA was not listed on consumer package. Our research on determining soya DNA and transgenic elements in food products indicates that soya ingredients have been added into food products in spite of their absence in relevant documents as recipe components. All the obtained results taken into account, we assume it is necessary to improve control procedures for detecting genetically modified and vegetable components used as ingredients in food products as their falsification can make for changes not only in their consumer properties but also damage consumers’ health.

Characteristics of CoVID-19 in children: the first experience in the hospital of st. Petersburg

Objective:to identify the clinical, laboratory and epidemiological features of the new coronavirus (CV) infection inthe provision of specialized medical care to children in themegalopolis of the Russian Federation.Methods:674 cases of hospitalization of patients frombirth to 17 years old inclusive with confirmed COVID-19 inthe period from March 26 to June 26, 2020 in a children’smultidisciplinary hospital in St. Petersburg. Diagnostics ofSARS-COV-2 in upper respiratory tract (URT) smears wascarried out by PCR (a set of reagents for detecting RNA ofcoronavirus 2019-nCoV by PCR with hybridization-fluorescence detection “Vector-PCRRV-2019-nCoV-RG”). Patientsunderwent 4 (3; 5) repeated examinations depending on thediagnosis of the referral, as well as the duration of the convalescent virus carriage. The analysis of the severity of thecourse of the disease, the main clinical manifestations andtheir relationship with the development of pneumonia, as wellas the epidemiological features of COVID-19 in children. Theduration of inpatient treatment, outcomes and the need forintensive care are described. Changes in a number of laboratory parameters on analyzers made in the USA were assessed:a clinical blood test on a hematological one - Coulter UniCel(Beckman Coulter), a biochemical blood test on a biochemical one - Uni Cel DxC (Beckman Coulter), a coagulogram ona hemostasis analyzer (Instrumentation Laboratory).Results:Overall, there was a favorable course ofCOVID-19 in children. Intensive therapy was required only in3.6% of cases with a total mortality rate of 0.15%, Kawasakilike syndrome was recorded in 0.3% of cases. In 1/3 of patients, prolonged viral shedding from the upper respiratorytract was detected. In children, intrafamilial infection fromadults was in the lead; schoolchildren accounted for half ofall hospitalizations. A distinctive feature of the new infectionwas mild clinical symptoms with fever and catarrhal symptoms up to 4/5 of cases, gastrointestinal symptoms - in every third patient. There were no significant differences in the severity of the disease by age. Pneumonia, diagnosed in ¾cases by computed tomography, complicated the course in13.1% of cases. The defeat of the lungs was accompanied byfever and dry cough, and in a more severe course: desaturation, chest pains, a feeling of insufficiency of inspiration. Theage peaks of the incidence of pneumonia were revealed: at4, 9, 12 years old and at the age of 17 years, the maximum(in 1/3 of cases). Laboratory changes were insignificant andquickly reversible.Conclusion:the course of COVID-19 in children in themegalopolis of Russia is comparable with foreign information. However, taking into account the experience of “Spanish ‘flu”, it is possible that in pediatric practice the numberof severe forms and unfavorable outcomes may change inthe near future, especially due to the difficulty of diagnosingKawasaki-like syndrome and the need for a multidisciplinaryapproach to the treatment of such patients. Currently, themost vulnerable to the new CV are children with severe oncological, neurological and cardiovascular pathology, whohave a rapid decompensation of the underlying diseaseagainst the background of COVID-19.

Immunomorphological features of cervical dysplasia depending on the progression of papillomavirus infection in women with infertility.

Background. With the development and improvement of modern methods of immunohistochemical examination, new possibilities for studying the molecular-biological features of cervical dysplasia (CD/CIN) emerged, opening new stages of its morphogenesis. Taking into account the demographic situation in Ukraine, and the fact that incidence rate of squamous cell intraepithelial cervical lesions and infertility have a tendency to increase, it becomes apparent the need for further investigation of CIN associated with the (HPV) infection. Objective. The purpose of the study was to determine the immunohistochemical features of cervical dysplasia in women with infertility, depending on the progression of papillomavirus infection. Methods. Specific quantitative diagnostics of real-time polymerase-chain reaction with hybridization-fluorescence detection was used to detect papillomavirus infection. Immunohistochemical studies used primary monoclonal antibodies to Ki-67, p63, VEGF, p16ink4a, estrogen (ER) and progesterone receptors (PR), E-cadherin. The results obtained during the study were subjected to statistical processing by methods of variational statistics. Results. Immunomorphological features of cervical dysplasia depending on the progression of papillomavirus infection have been established in women with infertility, and the reliable influence of HPV WRC loading on the level of expression of molecular-biological markers depending on the severity of cervical dysplasia has been demonstrated. Conclusion.Therefore, the use of Ki-67, p63, pl6ink4a, VEGF, E-cadherin markers is the most optimal for the implementation of the diagnostic process and for determining the course of CIN, since the change in their expression most often corresponded to the severity of CIN.

Identification and molecular-genetic characteristics of the hepatitis B virus among HIV-infected patients in Arkhangelsk.

To estimate the prevalence and characterize the hepatitis B virus among HIV-infected patients with virological failure of antiretroviral therapy in Arkhangelsk. HBV markers determinations (HBsAg, anti-HBs IgG, antiHBcor IgG, DNA HBV) were performed in isolates from blood plasma samples 64 HIV-infected patients with virological failure of antiretroviral therapy (viral load >50 IU / ml after 6 months of antiretroviral therapy or an increase in viral load after primary suppression of viral replication). For the detection of the hepatitis B virus, nucleic acids were isolated using the commercial kit «AmplePrime Ribo-prep». The virus presence analysis was performing by the polymerase chain reaction (PCR) method with hybridization-fluorescence detection in "real time" using the commercial set of «AmpliSens® HBV-FL». In the future, we used the method developed by the Saint-Petersburg Pasteur Institute, which allows detecting HBV in biological material with a low viral load. HBsAg-negative (occult) HBV was detect in 28 (43.8%) HIVinfected patients. Only HBV genotype D was detected, and the HBV subgenotype D1 prevailed (39.3%) compared with the HBV subgenotype D2 (32.1%) and D3 (28.6%). Serological markers in 42.8% of patients with HBV DNA were founding. Two HBV isolates with drug resistance mutations in the polymerase gene, leaded to amino acid substitutions (L180M, M204V) associated with the resistance development to lamivudine, entecavir, telbivudine and tenofovir were identifying. The occult (HBsAg-negative) HBV high prevalence among HIV-infected patients suggests the need to use molecular-biological diagnostic methods to identify HBV, as well as to analyze the HBV drug resistance mutation before starting antiretroviral therapy for HIV.

NEW APPROACHES IN THE TREATMENT OF CHRONIC VIRAL EPSTEIN-BARR INFECTION

Introduction. Epstein-Barr virus (EBV) causes recurrent infectious mononucleosis-like symptoms. Today poisons of insects and animals were shown to be rich sources of antimicrobial substances (peptides) and contain a wide range of active biological compounds. Antimicrobial peptides play an important role in the immune response of the host’s innate immunity to pathogenic microorganisms. Based on antimicrobial peptides in Russia, an antiviral drug allokin-alpha has been developed. The active ingredient of the drug is cytokine-like peptide alloferon. The purpose of the study is to evaluate the effect of allokin-alpha therapy on the amount of EBV DNA in saliva samples and clinical complaints in patients with chronic Epstein-Barr viral infection (EBI). Material and methods. 59 chronic EBI patients (45 women and 14 men; mean age 32.52 ± 1.75 years) were examined. Patients were subjected to quantitative determination of Epstein-Barr virus DNA in saliva samples by the method of polymerase chain reaction (PCR) with real-time hybridization-fluorescence detection. The analytical sensitivity of the test system is 400 copies/ml. The patients were divided into two groups: 26 patients who received allokin-alpha therapy (9 injections subcutaneously with 1.0 mg every other day) were included in the 1st group; the 2nd group included 33 patients who received Valtrex (500 mg 2 times/day, orally) for 2 months. Results. After treatment with allocin-alpha, negative results of PCR were obtained in 59.67% of patients. After two months of Valtrex therapy, negative PCR results were obtained in only 27.27% of patients. The correlation analysis revealed a significant effect of the initial number of copies of EBV DNA on the severity of clinical complaints in the general group of EBV patients. Discussion. Allokin-alpha improves the recognition of virus-infected cells and helps to suppress viral replication. Conclusion. Allokin-alpha therapy can be recommended for the treatment of chronic EBV infection in a dose of 1 mg subcutaneously every other day with a course dose of at least 9 injections.

ACUTE INTESTINAL INFECTIONS IN AMBULATORY PRACTICE

There is presented the structure of acute intestinal infections (AIIs) at the outpatient stage of medical care in 135 outpatients (70 men and 65 women, aged from 15 to 55 years) visited the Health Center of Barnaul. In all patients, the mild course of gastroenteritis predominated (the stool frequency did not exceed 4-5 times a day, body temperature - 37.2 0C). The investigation of biological material from patients (feces) was carried out by polymerase chain reaction (PCR) with hybridization-fluorescent detection “AmpliSens® AII screen-FL”. The results showed the high efficiency of the test system used, as in 118 out of 135 samples (87%) there was found genetic material of different etiology, 66.1% were of the viral origin, among which rotaviruses and noroviruses prevailed, 25.4% of samples had bacterial origin (Salmonella prevailed) and 19.5% of mixed virus-viral or bacterial-viral etiology. The work showed both the high sensitivity and specificity of the PCR method in the etiological diagnosis of AII. Among the examined patients, AII of viral origin prevailed (66.1%).

DISTRIBUTION OF P21 G1026A AND P21 G369C ALLELIC POLYMORPHISMS IN PATIENTS WITH GASTRIC CANCER WITH DIFFERENT CLINICAL AND MORPHOLOGICAL CHARACTERISTICS OF THE TUMOR

The variants distribution of polymorphic sites -1026G>A and -369G>C of the p21 gene in gastric cancer according to tumor histological type (intestinal or diffuse gastric cancer) and the tumor stage (with or without metastases) was investigated. DNA samples isolated from the morphologically intact stomach tissue obtained from the resection margin from 200 gastric cancer patients via standard methods, were studied for polymorphisms -1026G>A and -369G>C of the p21 gene by real-time polymerase chain reaction with hybridization-fluorescence detection using the appropriate pairs of oligonucleotide primers and probes. The carriage of allele A and genotype AA of the polymorphism -1026G>A, as well as allele C and genotype CC of the polymorphism -369G>C of the p21 gene, were predominantly associated with diffuse histological type of gastric cancer. In the cohort of patients with metastatic gastric cancer, the frequencies of allele C, genotypes GC and CC of the polymorphism -369G>C, and allele A, genotype AA of the polymorphism -1026G>A of the p21 gene, were statistically higher compared to the corresponding parameters of patients with non-metastatic gastric cancer.

Research of prevalence of polymorphism of the TLR 9 type gene in patients with infectious mononucleosis, caused by Epstein-Barr virus

There was studied the prevalence of -1486 T/C polymorphism of TLR-9 gene in 52 patients with infectious mononucleosis (IM), caused by Epstein-Barr virus. Three main genotypes of - 1486 T/C of TLR-9 gene: TT, TC, CC were identified based on obtained results. The study of the frequency of appearance of separate genotypes in patients with IM revealed the dominance of CC and TT genotypes compared with the control group. The study of the distribution frequency of -1486 T/C-polymorphism of TLR-9 gene for different genotypes demonstrated the specificity of CC genotype changes in patients with IM and the absence of such changes for TT and TC genotypes.Aim of research. The study of the frequency of - 1486 T/C polymorphism of TLR-9 gene in patients with IM, caused by Epstein-Barr virus.Materials and methods. The study for determining -1486 T / C polymorphism of TLR-9 gene was realized in 52 patients with IM. Among them women - 31 (59,6 %), men - 21 (40,4 %) in the age diapason from 18 to 34 years. The control group for studying the prevalence of - 1486 T/Cpolymorphism of TLR-9 gene consisted of 40 healthy donors. The mean age 24,2 ± 2,4 years, from 18 to 44 years.For revealing DNA of EBV by the method of the reverse transcription of PCR with hybridization-fluorescent detection of amplification products, sets of reagents Amplisens (Russia) were used. The polymorphic zone - 1486 T / C, rs187084 of TLR9 gene was used by PCR amplification in real time by determining the length of restrictive fragments of PCR using Ncol restrictive fragment and oligonucleotide primers.Results. The analysis of results of - 1486 T/C polymorphism of TLR-9 gene allowed to identify three main genotypes - TT, TC, CC. The study of the frequency of separate genotypes detection allowed to establish the prevalence of CC and TT genotypes compared with the heterozygotic TC genotype. The study of the distribution frequency of -1486 T/C-polymorphism of TLR-9 gene for different genotypes demonstrated the specificity of CC genotype changes in patients with IM and the absence of such changes for TT and TC genotypes.Our research established the correlation between - 1486 TLR-9 C/Cpolymorphism with IM that proves the important role of mediated TLR signalization in the pathogenesis of EBV infection. The study of genes polymorphism among receptors that take part in recognizing a virus is necessary for determining the genetic background, connected with the infection risk, disease course and possible IM results. It allows to reveal risk groups among patients and to carry out the timely therapy.Conclusions: 1. It was proved, that - 1486 T/C polymorphism of TLR-9 gene is detected reliably more often in patients with IM than in the control group.2. The distribution of -1486 Т / С polymorphism of TLR-9 allowed to reveal the association of CC genotype with manifest forms of IM

POLYMORPHISM ARG72PRO OF THE P53 GENE AND THE RISK OF HUMAN PAPILLOMAVIRUS ASSOCIATED CERVICAL CANCER IN WOMEN FROM KYRGYZ REPUBLIC

Aim. Allele frequency and genotype distribution in the cervical cancer group were compared with those of the control group to determine whether polymorphism Arg72Pro of Р53 gene elevates the susceptibility of Kyrgyz women to cervical cancer (CC). Materials and Methods. A total of 102 women (mean age 53,5±10 years) of Kyrgyz nationality with cervical cancer were recruited into the study and 102 healthy women were used as the control group (mean age 46,5±8,5 years). The diagnosis of cervical cancer was confirmed histologically. 88 % (90/102) of women with cervical cancer were human papillomavirus (HPV) positive. 17 % (15/90) specimens were positive for HPV type 16, 48 % (43/90) were positive for HPV type 18 and 35 % (32/90) were positive both HPV - 16/18. Presence of human papillomavirus DNA types 16 and 18 was analyzed by polymerase chain reaction with hybridization-fluorescence detection. The Arg72Pro polymorphisms of the P53 gene were determined by the PCR-RFLP method. Results. No significant difference was found between genotype distributions in the cervical cancer patients and the control group (χ2=1,24; р=0,54). However, the Arg72Arg genotype and Arg72 allele was significantly more frequent in women with cervical cancers infected with HPV than in the control group (χ2=7,25; р=0,027 for для genotype, χ2=6,83; р=0,009 for allele). Women who are HIV positive and having the Arg72 allele had 1,94 fold (OR=1,94 [1,20-3,15]; р=0,009) higher risk of developing CC compared with subjects carrying neither of these alleles. HPV positive women carrying the genotype Arg72Arg had 1,85 fold (OR=1,85 [1,03-3,32]; р=0,027) higher risk of CC. Conclusion. The Arg72Arg genotype of р53 gene in HIV positive women from Kyrgyz Republic may represent a potential risk factor for the development of cervical cancer.

SUBSPECIES DIFFERENTIATION OF YERSINIA PESTIS STRAINS BY PCR WITH HYBRIDIZATION-FLUORESCENT DETECTION

Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time. DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.3.1, Mega 5.0 and BLAST algorithm based on comparison of full-genome sequenc- es of Y.pestis strains. Primers and TAqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Ypestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Ypestis strains of various subspecies.

Seborrheic keratosis: clinical characteristics and an association with the β-genus human papillomavirus

Background : Seborrheic keratosis is a common benign tumor of unclear etiology . Aim : To study associations between clinical characteristics of seborrheic keratosis and its course with quantitative parameters of the β-genus human papillomavirus (β-HPV). Materials and methods : The main study group included 91 patients with seborrheic keratosis (29 male and 62 female), aged 40 to 75 years (mean ± SD, 59.2 ± 6.4 years). The control group included 30 healthy individuals persons aged 40 to 70 years (mean 57.6 ± 4.1 years). The diagnosis of seborrheic keratosis was confirmed by dermoscopy and pathomorphological assessment, with 100% of patients having the acanthotic type of seborrheic keratosis. HPV was identified by polymerase chain reaction with hybridization fluorescent detection in real time mode with use of three oligonucleotide systems for detection of HPV genus β1 (subtypes 5, 8, 12, 14, 19, 21, 25, 36, 47); β2 (subtypes 9, 15, 17, 22, 23, 37, 38, 80), and β3 (subtypes 49, 75, 76). Results: The most frequent clinical type of seborrheic keratosis was the maculopapular one (61/91 patients, 67%) localized mainly on head, neck, anterior and posterior trunk. β-HPV was found in 88.8% biopsy samples taken from the seborrheic keratosis areas in 24 of 27 patients and in 66.6% biopsy samples taken from the normal skin areas of the same patients (р = 0.04). This is significantly more frequent that the HPV contamination among healthy individuals, which was 28.7% (р = 0.02). Mixed association of β-HPV was also more frequent in the HPV-positive patients (21/24, or 83.3%; compared to the control group, р = 0,00001). The maximal (significant) viral load was found in the subgroup of seborrheic keratosis patients with the number of proliferative lesions of 10 and more (4.08 ± 0.3 lg/105 tо 5.7 ± 0,3 lg/105). Conclusion: The maculopapular type of seborrheic keratosis is characterized by mixed HPV β1, β2, β3 infection found in 77.7% of cases irrespective of the number of proliferative lesions. It could be hypothesized that multiple seborrheic keratosis (more than 10 lesions) develops in simultaneous presence of HPV β1, β2, β3 and is associated with a higher probability of new seborrheic keratosis lesions on intact skin.

OPTIMIZATION OF THE TECHNOLOGICAL SCHEME FOR THE PRODUCTION OF PREPARATIONS FOR GENE DIAGNOSTICS OF PARTICULARLY DANGEROUS INFECTIOUS DISEASES USING POLYMERASE CHAIN REACTION WITH HYBRIDIZATION-FLUORESCENT REGISTRATION OF RESULTS

Objective of the study was to optimize technological scheme for the production of preparation for gene diagnostics of plague with hybridization-fluorescent registration (HFR) of results, which is associated with the assessment and selection of synthesis conditions and method of oligonucleotide probe purification included into the kit and carrying fluorophore (6-carboxyfluorescein) and quencher (Black Hole Quencher-1) tags, as well as to evaluate their properties and implement this new technological line into manufacturing. Materials and methods . The object of investigation was the reagent panel “Gene Yersinia pestis Identification-HFR” and its constituent elements, probes and primers, providing for hmsH gene amplification. Synthesis of the primers and probes was carried out in 8-well DNA synthesizer ASM-800 (Bioset, Russia), using solid phosphite amid method. For the experiments the probes obtained were purified either by means of electrophoresis in 20 % polyacrylamide gel (size 20×20) or (size 8×10) with further purification through RP-cartridges in semi-automated mode in OPS-201 system and manually, or on RP-cartridges only in manual mode; or applying highperformance liquid chromatography (HPLC). Specific activity of the produced probes was tested by polymerase chain reaction using the DNA isolated from bacterial suspensions of Yersinia pestis C-624 strain, in concentrations up to 1·10 3 – 1·10 6 mc/ml. Results and conclusions . Optimized has been synthesis technology; selected has been the method for oligonucleotide probe purification, carrying phluorophore-6-carboxyphluorescin and Black Hole Quencher-1. Demonstrated is viability of applying the stated oligonucleotides or HPLC, or electrophoresis in 20 % polyacrylamide gel with 7 M urea and further purification through RP-cartridges in manual mode for purification purposes. Introduction of the optimized technological scheme in manufacturing of preparations for gene indication and identification of particularly dangerous pathogens with hybridization-fluorescent detection will allow for the reduction of lead time by 50 % and original cost – by 66 %.

Molecular genotypic detection of beta human papillomavirus DNA in the diagnosis of their association with certain skin epithelial neoplasias in immunocompromised and immunocompetent patients in the mode Real-Time

Epidemiological and molecular biological data suggest that the beta human papillomavirus (HPV) can cause the development of a number of epithelial skin tumors, but this interconnection is currently not fully understood. Besides detection of beta HPV, it is necessary to measure and quantify the viral load, it is a new approach in the diagnosis of HPV infection. 52 patients were examined to identify the association of epithelial neoplasias beta human papilloma virus in immunocompromised and immunocompetent patients in the department of chronic hemodialysis and kidney transplantation of Moscow Regional Research and Clinical Institute. HPV detection was performed by polymerase chain reaction (PCR) with hybridization-fluorescence detection in real time (Real-Time). Amplification and detection were performed on the tool Rotor-Gene 3000 (“Corbett Research”, Australia). For quantification of beta HPV recombinant plasmid positive controls, as well as control plasmidfragment offi-globin human gene (FSIS CRIE Rospotrebnadzor) were used. In fibroepithelial polyp and in apparently healthy skin of immunosuppressive patients - renal transplant recipients (RPT) with a high frequency was detected beta HPV DNA - 64 and 54%, respectively, compared to healthy skin (47% of cases). In fibroepithelial polyp in 57% of immunosuppressive RPT mixed infection was revealed. Viral load in the fibroepithelial polyp was higher than that as in apparently normal skin and in donors skin. The high detection ofHPVDNA in RTP, as in the fibroepithelial polyp and in apparently healthy skin was found. Immunocompetent patients had high HPV DNA detection only in fibroepithelial polyp compared with apparently healthy skin.

Burkholderia mallei genotyping based on different region analysis

The development of methods for glanders agent genotyping is urgent due to the high pathogenicity of this microbe, lack of effective preventive measures, and threat of the use of Burkholderia mallei as a biological weapon. In this study, we propose a scheme for genotyping of B. mallei strains based on different region analysis (DFR). The choice of variable loci specific for different strains of the glanders agent was performed by analyzing the annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for nine selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. DFR analysis was assessed in 14 B. mallei strains. The strain genetic profiles revealed that developed DFR-typing was characterized by a high discrimination power (Hunter–Gaston index was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.