[The kit of reagents for polymerase chain reaction diagnostic of infections caused by B. pertussis, B. parapertussis and B. bronchiseptica].

The effective treatment of whooping cough directly depends of early diagnostics. The polymerase chain reaction diagnostic is the most perspective diagnostic technique. The kit of reagents is developed to diagnose whooping cough, parapertussis and bronchosepticosis with polymerase chain reaction. The evaluation of its analytical characteristics was carried out. The sensitivity made 1 x 103 of genome equivalents per 1 ml of sample (the sorption technique of DNA extraction was applied) and 5 x 102 of genome equivalents per 1 ml (the precipitation technique of DNA extraction was used). The specificity of test in the framework of analyzed panel of strains and isolates of microorganisms made 100%. The diagnostic sensitivity of analysis exceeded the sensitivity of bacteriological analysis up to 20 times. The application of this kit of reagents permits to detect and to differentiate DNA of agent of whooping cough, parapertussis during one working day already at the beginning of catarrhal period of disease and up to 18th day from the moment of cough appearance. In perspective, this process creates an opportunity to apply timely the specific therapy. The specter of agents of acute respiratory diseases brining on acute prolonged cough in children who were directed to bacteriological analysis to confirm whooping cough is investigated.

[Laboratory diagnostics in evaluation of acute respiratory viral infection morbidity in 2010 - 2011 epidemic season].

AIM: Study of etiological structure of ARVI and evaluation of acute respiratory virus infection morbidity in 2010 - 2011 epidemic season taking into account the data of laboratory diagnostics by method of polymerase chain reaction with hybridization-fluorescent detection. MATERIALS AND METHODS: By using reagent kits produced by Central Research Institute of Epidemiology for the detection of primary causative agents of influenza and ARVI 129 children and 94 adult patients monitored in an outpatient setting as well as 103 children hospitalized due to ARI were examined. RESULTS: Etiological structure of ARVI was studied; proportion of influenza and other actual causative agents of ARVI in monthly dynamics were established. During epidemic rise of influenza (January-March 2011) the proportion of influenza A viruses was 24% (peak in January--31%), the proportion of influenza B viruses--5%, rhinoviruses--9%, metapneumovirus was detected in 6% of cases, parainfluenza viruses (1 - 4 type) and adenovirses--4% each, coronaviruses--in 3%, respiratory syncytial virus--in 2%, bocavirus--in 1% of the studied samples. In influenza structure A/H1N1pdm2009 virus, its proportion was 70%, influenza virus B (26.9%), influenza virus A/H3N2 (2.6%) predominated. Indexes for monthly morbidity caused by each of the ARVI causative agents were calculated. CONCLUSION: The proposed approach allowed to evaluate ARVI morbidity taking into account laboratory-confirmed etiological factors. A 5 time increase in ARI morbidity in adults in February 2011 was shown to be mostly due to an increase in influenza A morbidity as well as involvement of influenza B virus, metapneumoviruses, coronaviruses, parainfluenza viruses and rhinoviruses into the epidemic process. Increase of morbidity of children by 1.4 times was also seen during activization of influenza viruses and metapneumovirus. The analysis of monitoring results allowed to prognose increase of respiratory-syncytial viral infection epidemic activity from September 2011 to February 2012.

[Molecular characterization of pandemic influenza viruses A/H1N1(sw2009) isolated in Russia in 2009-2010].

AIM: Assessment of genetic diversity of influenza virus A/H1N1 (sw2009) variants circulated in Russia, study of virus' pathogenicity in humans and potential resistance to antiviral drugs. MATERIALS AND METHODS: Sequencing of PCR-fragments of genome of influenza viruses isolated from clinical and autopsy samples of 436 patients. Four full genome sequences of influenza viruses A/H1N1 (sw2009) were obtained. Phylogenetic analysis was performed. RESULTS: High degree of homology (98.9-100%) was found among influenza A/H1N1(sw2009) viruses in HA and NA genes as well as in their aminoacid sequences (1.3 and 1.4% respectively). Differences in other proteins did not exceed 1.1%. Diversity was found in position 222 of receptor-binding locus of HA and single amino acid polymorphism--in several internal proteins. Known mutations determining resistance to Tamiflu and Arbidol were not detected. All viruses were resistant to remantadine. Molecular markers of high pathogenicity were not found. CONCLUSION: High homology of influenza viruses determines low level of antigenic differences although in populations of viruses there are variants with different levels of adaptation to human organism and different affinity to receptors of upper and lower respiratory tract that can determine their different transmissibility.

[Pandemic influenza A/H1N1 (sw2009) in Russia: epidemiology, diagnosis, clinical picture, and treatment].

AIM: To study the epidemiological and clinical features of the 2009-2010 pandemic influenza in Russia. SUBJECTS AND METHODS: Materials from 874 patients, including postmortem samples from 287 subjects, were examined applying the AmpliSens Influenza virus A/H1-swine-FL PCR kit designed and produced by the Central Research Institute of Epidemiology. The clinical and postmortem characteristics of 68 patients who had died from influenza A/H1N1 (sw2009) were analyzed in detail. RESULTS: The cause of deaths was primary virus pneumonia in most cases. The major manifestation of viral pathogenicity was impaired microcirculation leading to hemorrhage. No mutations conferring resistance to oseltamivir and arbidol were found. All A/H1N1swl viruses had genetic markers of remantadin resistance. CONCLUSION: The reagent kits developed by the Central Research Institute of Epidemiology proved to be effective. It is necessary to set up PCR laboratories that differentially diagnose influenza and acute respiratory viral infections in health care facilities in order to make early laboratory diagnosis of influenza and to timely perform its specific therapy.

[The characteristics of epidemic influenza A and B virus strains circulating in Russia during the 2007-2008 season].

In 2007-2008 in Russia, the epidemic upsurge of influenza morbidity was caused by the active circulation of influenza A(H1N1, A(H3N2), and B viruses. The center for Ecology and Epidemiology of Influenza studied 334 epidemic strains. The results of a comparative study of the svirus specificity of commercial test systems (AmpliSens Influenza virus A/B and AmpliSens Influenza virus A/H5N1) for the polymerase chain reaction diagnosis and virological assays, including virus isolation, revealed their high correlation, which confirms that they may be expensively used to monitor the circulation of influenza viruses in the Russian Federation. All the strains were isolated in the MDCK cell culture. Influenza A(H1N1) viruses (n = 127) were antigenic variants of the reference strains A/Solomon Islands/3/06 and A/Brisbane/59107. Influenza A(H3N2) viruses (n = 49) were antigenic variants of the reference strains A/Wisconsin/67/05 and A/Brisbane/10/08. One hundred and fifty seven Influenza B strains were drift variants of the reference strains B/Florida/4/06 and B/Shanghai/361/02 of lineage B/Yamagata/16/88 and one strain, a variant of Malaysia/2506/04 related to lineage B/victoria/2/87. The isolates interacted actively with human 0(I) blood group erythrocytes and much more weakly with chicken ones. All study influenza A(H1N1) viruses (n = 74) preserved their sensitivity to rimantadine while 24 (77%) of the 31 study influenza A(H3N2) virus strains were resistant. A study of the time course of changes in the generation of antibodies in the donor sera obtained in Moscow and the Moscow Region in different periods of the epidemic process revealed an increase in antibodies to the reference influenza A and B virus strains circulating in this period.

[A new mutation c.422C>G (p.S141C) in homo- and heterozygous forms of the human leptin gene].

Mutation g.15409C>G, c.422C>G (p.S141C) in homo- and heterozygous forms of the human LEP gene was identified among some patients of the high mountain village of Karaul, Ashkhabad oblast, Turkmenistan, some of which suffer from adiposity. It causes the substitution S120C in the secreted leptin. The mature leptin molecule (146 aa) has only two Cys residues (C96 and C146) forming an S-S bridge, which is important for the hormone function. A third mutation, C120, in the molecule might disturb the correct formation of the S-S bond and could alter the leptin activity.

[Application of molecular genetic methods during Legionnaires' disease outbreak in town Verkhnyaya Pyshma].

The aim of the study was to perform molecular genetic analysis based on multi-locus sequence typing in order to identify source of Legionnaires' disease outbreak in town Verkhnyaya Pyshma in July 2007 and genetic profile of the causative agent. Sequence-based typing protocol recommended by European Working Group on Legionella infection (EWGLI) was used. It was not possible to obtain satisfactory results of Fla gene sequencing for all samples. Obtained allelic profiles of other genes were typical for L. pneumophila. Allelic profiles of L. pneumophila isolated from patients were identical and matched with L. pneumophila DNA detected in water from hot water supply of domestic building, but differed from cooling tower's isolates and isolates from showerhead in apartment of one patient. Identity of 5 genes of L. pneumophila isolated from autopsy samples and from hot water of central hot water supply of domestic building confirms aspiration route of infection through hot water contaminated by the microorganism. L. pneumophila detected in water from cooling tower, showerhead in apartment of one patient, and from drainage canal of hot water supply station belonged to other allelic variants and, therefore, are not related with the outbreak.

[Development and use of the assay for Legionella pneumophila detection based on fluorescent real-time/endpoint polymerase-chain reaction].

The aim of the work was to develop a PCR-based assay for detection of L. pneumophila and L. micdadei in environmental samples as well as in clinical samples from low respiratory tract and to assess its analytic characteristics. The assay was used during investigation of the outbreak developed in July 2007 in town Verkhnyaya Pyshma (Sverdlovsk region). Polymerase-chain reaction (PCR)with fluorescent detection,sequencing and cloning of DNA fragments were used. Developed assay based on the PCR with fluorescent real-time/ endpointdetection is able to detect L. pneumophila in clinical and environmental samples and to quantify amount of bacterial DNA in water. Specificity of analysis (100%) was assessed using the panel of bacterial strains and samples from healthy individuals. Analytic sensitivity of assay and quantitation limit was 1000 GU in 1 ml. Sensitivity of the assay of artificially contaminated biological samples was 1000 bacteria in 1 ml. During outbreak investigation L. pneumophila DNAwas detected in 4 lung samples from 4 fatal cases, from 1 of 2 sputum samples, 1 of 2 bronchoalveolar lavage samples with X-ray confirmed pneumonia. Legionella's DNA was found in samples from cooling towers, central hot water supply as well as from showerheads in apartments of 3 patients. Fountain and drinking water samples were PCR-negative. Specificity of PCR-positive results was confirmed by sequencing. Use of the assay during outbreak in- vestigation allowed to confirm the diagnosis in fatal cases and quickly identify the possible source of infection.

[Isolation and molecular characterization of the influenza virus A/H5N1 strains isolated during outbreak of avian influenza among birds in the European part of Russia in 2005: strain with ozeltamivir-resistance mutation was found].

Isolation and characterization of the influenza virus A/H5N1 strains, isolated from chicken in the Yandovka village (Tula Region) and from wild swan near the orifice of the Volga River that died during an outbreak of avian flu in autumn 2005, were carried out. Genetic and phylogenetic analyses were performed. The goals of the analysis were to determine possible geographical origin of the strain, genetic similarity of isolated strains to earlier sequenced isolates, epidemic potential, existence of pathogenicity markers, and resistance to antiviral drugs. It was shown that the isolated influenza virus belonged to highly pathogenic variants of China origin by a reassortment of viruses genotypes Z and V circulated in poultry and wild birds. A number of molecular markers of pathogenicity to gallinaceous birds and mammals were found out. Mutations in the hemagglutinin gene promoting potentially high rate of replication in humans as well as mutations causing the resistance to amantadine/rimantadine were not found. The strain isolated from wild swan had the mutation causing resistance to tamiflu/ozeltamivir.

[Molecular and genetic study of the role of hormones, receptors, and enzymes in regulation of reproduction, lipid metabolism, and other human physiological functions].

Prevalence of uterine progesterone receptors over estrogen ones, high uterine cAMP level, and low uterine prostaglandin level are necessary conditions of normal pregnancy. In cases of spontaneous and antiprogestin RU486-induced abortions, estrogen receptors prevail over progesterone ones, cAMP level decreases, and prostaglandin concentration in decidual tissue increases. Porcine and bovine beta-lipotropines were the first proteins, whose correct amino acid sequence was first determined in Russia. Several research centers carried out collaborative studies of the nucleotide sequences of human and animal proopiomelanocortin (lipotropin precursor) and prolactin cDNA. Researchers constructed genetic engineering producers of human pre-proinsulin and somatostatin, identified structural genes expressed in pancreatic beta-cells, studied antigenic properties of glutamic acid decarboxylase (GAD), which determine insulin-dependent diabetes, and identified the cholesterase determinant. They revealed mutations in the genes of proopiomelanocortin and melanocortin receptors (MC4-P), which inhibit leptin regulation of appetite and are associated with human obesity.

[Development and evaluation of the kit for detection of SARS-associated Coronavirus RNA]. / Razrabotka i aprobtsiia test-sistemy dlia vyiavleniia RNK virusa vizyvaiushchego tiagezhelyi ostryi resperativnyi sindrom.

AIM: To develop a diagnostic kit for detection of SARS (severe acute respiratory syndrome)-related coronavirus RNA based on reverse transcription and polymerase chain reaction and to estimate its specificity and sensitivity. MATERIAL AND METHODS: 68 virus and bacterial cultures, 240 clinical samples from people without SARS symptoms and also 22 RNA samples from patients with SARS symptoms received during the epidemic in Beijing were used. RESULTS: The specificity of the kit was determined using animal coronaviruses and other bacterial and viral strains, causing acute respiratory and intestinal infections, and was shown to be 100%. The sensitivity of the kit in different clinical samples was 2.2 x 10(3) genome equivalents of recombinant SARS RNA in 1 ml of the specimen. The kit was evaluated in the Institute of Microbiology and Epidemiology of Beijing (China) using SARS-cov viral suspension and clinical samples from patients with suspected SARS. It was shown that kit was able to detect 10 TCID/50 ml of SARS-Cov virus. Testing of clinical samples from patients with suspected SARS showed that diagnostic sensitivity of the kit was 95%. Detection of the SARS-Cov RNA was more effective in feces compared to sputum 990 and 40%, respectively). CONCLUSION: The kit "AmpliSens SARS" for qualitative detection of SARS-related coronavirus RNA by reverse transcription and polymerase chain reaction (PCR) in nasopharyngeal wash/aspirates, naso/oropharyngeal swabs, plasma, and extract from feces has been developed in the Central Research Institute for Epidemiology of the RF Ministry of Health. The kit contains reagents for RNA isolation and purification, cDNA synthesis by reverse transcription of RNA, for PCR and for electrophoretic analysis of amplified products. The kit also contains recombinant positive and internal control samples allowing to control efficiency of analysis and showed good analytical and diagnostic characteristics.

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b]. / Kharakteristika moskovskikh shtammov Haemophilus influenzae tipa b metodom mul'tilokusnogo sekvenirovaniia-tipirovaniia.

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

[Screening of mutations in genes of pro-opiomelanocortin in patients with constitutional exogenous obesity]. / Skrining mutatsii v gene proopiomelanokortina u bol'nykh konstutsional'no-ékzogennym ozhireniem.

Proopiomelanocortin (POMC) is a precursor of ACTH, beta- and gamma-liportopins, alpha-, beta- and gamma-MSH, beta-endorphin. alpha-, beta- and gamma-MSH are synthesized by hypothalamus neurons, and leptin stimulates their synthesis. These hormones regulate food consumption and energy metabolism by via melanocortin receptors (MC3-R and MC4-R) in hypothalamus. Screening mutations in the coding region of human POMC has been carried out with PCR, SSCP and DNA sequencing and the association study of these mutations and human obesity has been performed. Group of patients with the exogenous obesity (BMI 37.8 +/- 6.8 kg/m2) consisted of 228 persons (173 women and 55 men). 145 blood donors (67 women and 78 men) without obesity (BMI J25 kg/m2, 23.1 +/- 2.2 kg/m2) and 170 women without apparent obesity at the beginning of the study were included in the control group. 8 polymorph sites: insertions; missense and silent mutations have been identified in the coding region of POMC. Among them 1) two heterozygous mutations: the insertion of 6 b.p. (GGGCCC) in codon 176 inducing the insertion of two amino acid residues (Arg-Ala) in POMC and nonsense mutation (G-7316-T) in codon 180 of gamma-LTH coding region of the same DNA chain were identified in 4 women (5.8%) out of 69 patients with morbid obesity (BMI 40-53 kg/m2). These mutations were not found in control (n = 315). 2) The new heterozygous mutation T-7130-C (Phe118Leu) in active site of alpha-MSH has been identified in POMC gene of a woman suffering with obesity since the early childhood. 3) Mutation A-7341-G (Glu188Gly) seemed to have a protective effect because it was revealed more frequently in control (3.9%) than in obese patients (0.66%). The results of genetic study of two pedigrees suggested the dominant influence of the first two mutations (1 and 2) on woman obesity.

[Study of the association between constitutional exogenous obesity and polymorphism of the apolipoprotein B gene]. / Issledovanie sviazi konstitutsional'no-ékzogennogo ozhireniia s polimorfizmom v lokuse gena apolipoproteina B.

An attempt was made to associate the insertion-deletion (Ins/Del) polymorphism of the apolipoprotein B gene (apoB) with obesity and to identify alleles and genotypes predisposing to this disorder. The apoB Ins/Del allele frequencies observed in the Russian population were similar to those in West European populations and significantly differed from frequencies reported for Asian populations. Patients with obesity did not differ from healthy individuals in allele and genotype frequencies regardless of whether total or sex-stratified samples were compared. Estimation of relative risk for individuals with genotype Ins/Ins did not reveal a significant association between obesity and this genotype. Thus, constitutional exogenous obesity did not prove to be associated with the Ins/Del polymorphism of the apoB gene in the Russian population.

[Pilot experience of studying calicivirus infection in Moscow children]. / Pervyi opyt izucheniia kalitsivirusnoi infektsii u detei v Moskve.

An RT-PCR-based assay for detecting calicivirus RNA was developed. Samples of feces of 124 children patients with acute gastroenteritis were tested for the presence of NLV-1, NLV-2, and SLV. Calicivirus infection was detected in 6.4% of cases of children with acute gastroenteritis. All isolates were non-homogeneous Norwalk like Viruses 2 genotype with maximum nucleotide homology to the strains, isolated in UK and Japan. The use of the RT-PCR assay developed in our laboratory allowed the etiology of 22.2% of cases of acute gastroenteritis of uncertain etiology to be identified.