Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men.

OBJECTIVES: Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. METHODS: We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. RESULTS: In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. CONCLUSIONS: We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

[A comparative analysis of allele frequencies of rs1801133 and rs1801131 of MTHFR in patients with stroke and healthy people from the Moscow region]. / Sravnitel'nyi analiz chastot allel'nykh variantov rs1801133 i rs1801131 gena MTHFR v vyborke patsientov, perenesshikh ishemicheskii insul't, i u zdorovogo naseleniia v Moskovskom regione.

AIM: To study genetic characteristics of the population of the Moscow region and analyze the association of rs1801133 and rs1801131 of MTHFR with the risk of ischemic stroke (IS). MATERIAL AND METHODS: A sample of 170 and 115 patients with atherothrombotic and cardioembolic subtypes of IS and 360 residents of the Moscow region without IS were examined. MTHFR alleles were determined by a multiplex real-time polymerase chain reaction. RESULTS AND CONCLUSION: No association between the frequencies of MTHFR alleles and the risk of ischemic stroke was found. The comparison of allele frequencies with those in Caucasian populations published in the dbSNP (NCBI) and 1000 Genomes Project databases revealed significant differences for rs1801133 from the EUR 1000 Genomes Project. The allele frequency data for MTHFR could increase the accuracy and reliability of the individual risk calculation for multifactorial diseases in the Russian population.

[Features of hemorrhagic fever with renal syndrome with diabetes mellitus].

AIM: Diabetes mellitus (DM) is known to be a risk factor in adverse outcomes and complications in many infectious diseases. In the combination of hemorrhagic fever with renal syndrome (HFRS) and DM there are mutually exclusive pathogenetic States - hyperosmolarity, characteristic of DM is layered on the reduction of osmotic blood pressure in HFRS. Under these conditions, the effect of one disease (DM) on the clinical manifestations of another (HFRS) is not obvious. The aim of the work is to find out the clinical features during hfps in patients with DM. MATERIALS AND METHODS: The study is based on the results of the retrospective studies "case - control" - studied the information contained in medical records of patient (form 003/u), who suffered HFRS in 2006-2018. The Selection of cards produced randomly. In the end, there were formed two groups: the first - 981 patient who suffered HFRS and had no signs of diabetes; the second, 33 patients who suffered HFRS on the background of previously existing (28 people), or the first identified (5) diabetes. RESULTS: With a combination of HFRS and DM, a mild course of the disease is 2.5 times more common, there are no severe forms. Among this group of patients, complications are almost 10 times less common, less likely to develop infectious - toxic shock, acute kidney damage (class F by RIFLE), pulmonary edema. The combination of DM and HFRS is manifested by less high and prolonged fever, less high levels of urea and creatinine in the blood. CONCLUSION: HFRS in combination with DM is easier, apparently, high osmolarity of the blood is a stabilizing factor during the disease.

Serodiagnosis of Borrelia miyamotoi disease by measuring antibodies against GlpQ and variable major proteins.

OBJECTIVES: Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls. METHODS: We assessed seroreactivity against GlpQ and four Vmps in a well-described, longitudinal cohort of sera from BMD patients (n=182), healthy blood donors (n=136) and controls (n=68). All samples were tested by ELISA and positive sera were tested by western blot, and antibody dynamics and diagnostic value were assessed. RESULTS: IgM antibodies against GlpQ and Vmps peaked between 11 and 20 days, and IgG between 21 and 50 days, after disease onset. Various combinations of GlpQ and Vmps increased sensitivity and/or specificity compared to single antigens. Notably, the GlpQ or variable large protein (Vlp)-15/16 combination yielded a sensitivity of 94.7% (95% CI: 75.4-99.7) 11-20 days after disease onset and a specificity of 96.6% (92.7-98.4) for IgM. A specificity of 100% (97.8-100) for IgM, and 98.3% for IgG (95.2-100), was found when positivity was defined as reactivity to GlpQ and any Vmp, with maximum sensitivities of 79% (56.7-91.5) for IgM and 86.7% (62.1-97.6) for IgG. CONCLUSIONS: We clearly demonstrate here the diagnostic potential of these seromarkers. Our findings will facilitate future epidemiological and clinical studies on BMD and lead to the development of a serologic test to be used in clinical practice.

[Clinical presentation of Ixodes tick-borne borreliosis caused by Borrelia miyamotoi in the context of an immune response to the pathogen]. / Klinicheskie proiavleniia iksodovogo kleshchevogo borrelioza, vyzvannogo Borrelia miyamotoi, v kontekste immunnogo otveta na vozbuditel'.

Ixodes tick-borne borreliosis caused by Borrelia miyamotoi (ITBB-BM) is a previously unknown infectious disease discovered in Russia. AIM: The present study continues the investigation of the clinical features of ITBB-BM in the context of an immune system-pathogen interaction. SUBJECTS AND METHODS: The study enrolled 117 patients with ITBB-BM and a comparison group of 71 patients with Lyme disease (LD) that is ITBB with erythema migrans. All the patients were treated at the New Hospital, Yekateringburg. More than 100 clinical, epidemiological and laboratory parameters were obtained from each patient's medical history and included in the general database. A subset of patients hospitalized in 2015 and 2016 underwent additional laboratory examinations. Namely, the levels of B. miyamotoi-specific IgM and IgG antibodies were measured by the protein microarray containing GlpQ protein and four variable major proteins (VMPs): Vlp15/16, Vlp18, Vsp1, and Vlp5. The blood concentration of Borrelia was estimated by quantitative real-time PCR. RESULTS: In contrast to LD, first of all (p<0.001) the following clinical features were typical for ITBB-BM: the absence of erythema migrans (in 95% of patients), fever (93%), fatigue (96%), headache (82%), chill (41%), nausea (28%), lymphopenia (56%), thrombocytopenia (46%), the abnormal levels of alanine aminotransferase (54%) and C-reactive protein (98%), proteinuria (61%). Given the set of these indicators, the course of ITBB-BM was more severe in approximately 70% of patients. At admission, only 13% and 38% of patients had antibodies to GlpQ and VMPs, respectively; at discharge, antibodies to GlpQ and VMPs were detected in 88% of patients. There was no statistically significant association of the antibody response with individual clinical manifestations and laboratory parameters of the disease. However, patients with more severe ITBB-BM produced less IgM antibodies to VMPs and GlpQ at the time of discharge. CONCLUSION: ITBB-BM is a moderate systemic disease accompanied by the production of specific antibodies in virtually all patients.

Development and optimization of an in vitro cultivation protocol allows for isolation of Borrelia miyamotoi from patients with hard tick-borne relapsing fever.

OBJECTIVES: Borrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients. METHODS: Modified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro. Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF. RESULTS: Dipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 µg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g, suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF. CONCLUSIONS: Direct culture from K2-EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers.

[Complex assessment of the contribution of genetic factors to the risk of ischemic stroke]. / Kompleksnaia otsenka vklada geneticheskikh faktorov v razvitie ishemicheskogo insul'ta.

AIM: To develop a method of the complex assessment of genetic risk for ischemic stroke (IS) and evaluate its effectiveness. MATERIAL AND METHODS: Genotyping of 182 patients with atherothrombotic and cardioembolic subtypes of IS and 360 healthy individuals of 48 single nucleotide polymorphic loci (SNP) associated with the risk of II and its subtypes was performed. RESULTS AND CONCLUSION: In each group of SNPs, composite indicators of genetic risk of IS in groups of patients and healthy controls were identified. Differences between the calculated values of the genetic risk in these groups were significant (p <0,05). The quality of the binary classification validated by ROC-analysis confirmed the predictive potential of the proposed method of risk calculation for determining the genetic predisposition to the development of IS.

[Follow-up of patients with Ixodes tick-borne borrelioses caused by Borrelia miyamotoi or Borrelia burgdorferi sensu lato]. / Katamnez bol'nykh iksodovymi kleshchevymi borreliozami, vyzvannymi Borrelia miyamotoi ili Borrelia burgdorferi sensu lato.

Ixodes tick-borne borrelioses (ITBB) are caused by two different spirochetes: Borrelia from the group of Borrelia burgdorferi sensu lato, the agents of the classic Lyme borreliosis (LB), and Borrelia miyamotoi that belongs to the group of Borrelia causing tick-borne relapsing fevers. ITBB caused by B. miyamotoi (BM-ITBB) is a previously unknown infectious disease discovered in Russia. It is known that the LB sequelae may reduce the long-term life guality of convalescents. AIM: To study the follow-up of those who have recovered from new BM-ITBB infection in comparison with persons who have had LB. SUBJECTS AND METHODS: The investigation enrolled 41 patients with BM-ITBB and 41 patients with LB who were treated at the Republican Infectious Diseases Hospital of Udmurtia. Within a year after the disease, they were followed up through clinical and instrumental examination of cardiac performance, expanded biochemical analysis of blood and urine, which could; estimate kidney and liver functions, and psychological questioning. RESULTS: Asthenic syndrome and complaints about and objective signs of cardiac dysfunctions persisted supraventricular extrasystoles, left ventricular diastolic dysfunction, and elevated and/or unstable systolic blood pressure were detected in 20-30% of the convalescents for a long time. Kidney dysfunctions were manifested in albuminuria and the decrease of glomerular filtration rate. A year following the disease, 10-20% patients had persistently elevated concentrations of alanine aminotransferase, aspartate aminotransferase, and C-reactive protein and had higher levels of total cholesterol and low-density lipoproteins. The pathological consequences of ITBB were polymorphic and varied in different patients; in general, only 68% of them showed health improvement. CONCLUSION: We assume that a significant role in the pathogenesis of BM-ITBB and LB is played by vascular endothelial damage possibly associated with the inflammatory and autoimmune aspects of an immune response in Borrelia infection. The consequences of this damage may persist and even intensify during a year, which provokes chronic dysfunction of the heart, kidney, or liver in a number of convalescents.

[Relapsing (recurrent) disease caused by Borrelia miyamotoi].

AIM: To clarify the clinical, laboratory, and epidemiological characteristics of relapsing Ixodes tick-borne borreliosis (ITB) caused by Borrelia miyamotoi. SUBJECTS AND METHODS: Retrospective clinical observation was made in 79 inpatients of the Republican Infectious Diseases Hospital (Udmurt Republic), who had been diagnosed with B. miyamotoi-caused disease verified by real-time polymerase chain reaction. The latter and enzyme immunoassay ruled out possible vector-borne coinfections (ITB caused by B. burgdorferi sensu lato; tick-borne encephalitis; anaplasmosis; and ehrlichiosis). RESULTS: The recurrent course of the disease was observed in 8 (10%) of the 79 patients. The relapsing fever curve was noted in 6 of the 8 patients; 4 patients had 2 episodes of fever and 2 patients had 3 episodes; the wave-like continuous type of fever cannot enable one to estimate the specific number of episodes in 2 more cases. Relapses occurred in all the 8 patients before antibiotic treatment. Febrile syndrome (weakness, headache, chill, fever, sweating, dizziness, nausea, vomiting, myalgia, and arthralgia) was leading in patients with relapses. These patients were less frequently observed to have signs of organ dysfunctions than those with one episode of fever. The values of clinical and biochemical blood tests and urinalyses were normal and near-normal in the majority of patients on hospital admission. CONCLUSION: Relapsing B. miyamotoi infection cases detected in the directed study proved to be unrecognized by practical health authorities during the first and sometimes second episodes of fever. This indicates that the prevalence of this disease is essentially underestimated and there is a need to increase physicians' alertness and awareness and to introduce adequate diagnostic methods.

[The blood coagulation system and microcirculatory disorders in ixodid tick-borne borreliosis caused by Borrelia miyamotoi].

AIM: To study blood coagulation and microcirculatory disorders as a possible cause of transient dysfunctions of organs (the kidney, liver, heart, lung, etc.) in patients with ixodid tick-borne borreliosis caused by Borrelia miyamotoi (Bmt). SUBJECTS AND METHODS; Twenty-four patients with Lyme disease (LD) and 28 Bmt patients treated at Izhevsk City Hospital (Udmurtia) were examined in the study. Platelet counts and the presence of D-dimers were determined; activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen and antithrombin III levels, and Factor XIIa-dependent fibrin clot lysis time were measured. Slit lamp microscopy of the conjunctiva was. also carried out. Results. Platelet counts'were less than 150,000 per pL of blood in 43% of the Bmt patients. All the Bmt patients had at least one abnormal coagulation parameter of the eight ones that were tested; 64% of them had marked coagulation disorders with three or more abnormal laboratory findings. In contrast, all the eight parameters were normal in 71% of the LD patients. The other seven LD patients had only one or two abnormal coagulation parameters (p < 0.001 in comparison with Bmt patients). Microscopic examination of eye capillary blood flow revealed pathological findings that included aggregates of erythrocytes and obstructed and/or sinuous capillaries in 22 (79%) of the Bmt patients, but none of the LD patients. A total of 14 Bmt patients had both coagulation and microcirculatory abnormalities. Eleven of them also had transient signs of organ dysfunction. CONCLUSION: As far as Borrelia secrete no known toxins, we hypothesized that uncovered disorders of blood coagulation and microcirculation in Bmt patients may contribute to organ dysfunction.

High seroprevalence of Borrelia miyamotoi antibodies in forestry workers and individuals suspected of human granulocytic anaplasmosis in the Netherlands.

Substantial exposure to Borrelia miyamotoi occurs through bites from Ixodes ricinus ticks in the Netherlands, which also transmit Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. Direct evidence for B. miyamotoi infection in European populations is scarce. A flu-like illness with high fever, resembling human granulocytic anaplasmosis, has been attributed to B. miyamotoi infections in relatively small groups. Borrelia miyamotoi infections associated with chronic meningoencephalitis have also been described in case reports. Assuming that an IgG antibody response against B. miyamotoi antigens reflects (endured) infection, the seroprevalence in different risk groups was examined. Sera from nine out of ten confirmed B. miyamotoi infections from Russia were found to be positive with the recombinant antigen used, and no significant cross-reactivity was observed in secondary syphilis patients. The seroprevalence in blood donors was set at 2.0% (95% CI 0.4-5.7%). Elevated seroprevalences in individuals with serologically confirmed, 7.4% (2.0-17.9%), or unconfirmed, 8.6% (1.8-23%), Lyme neuroborreliosis were not significantly different from those in blood donors. The prevalence of anti-B. miyamotoi antibodies among forestry workers was 10% (5.3-16.8%) and in patients with serologically unconfirmed but suspected human granulocytic anaplasmosis was 14.6% (9.0-21.8%); these were significantly higher compared with the seroprevalence in blood donors. Our findings indicate that infections with B. miyamotoi occur in tick-exposed individuals in the Netherlands. In addition, B. miyamotoi infections should be considered in patients reporting tick bites and febrile illness with unresolved aetiology in the Netherlands, and other countries where I. ricinus ticks are endemic.

[Real-time pcr procedure for determination of Streptococcus pneumoniae serotypes].

AIM: Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes. MATERIALS AND METHODS: S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No. 2, Moscow, were studied with the aim of testing the possibility of practical use of RT-PCR. RESULTS: Primers and probes were selected for the determination of 16 vaccine and/or frequently encountered serotypes distributed among 4 reaction mixtures also including a pair of primers and a probe for cpsA gene detection that is present in all the capsule pneumococci (internal control). The procedure was tested on a collection of 108 pneumococci strains gathered in Research Institute of Antimicrobial Therapy and serotyped earlier by specific PCR with electrophoretic detection and serologically by using Pneumotest-Latex kit. The sensitivity and specificity of the RT-PCR was 100%. RT-PCR procedure allowed to determine pneumococcus serotype in 79% of CSF clinical samples containing S. pneumoniae DNA. Serotype 3 and 23F were detected most frequently (13%, each). CONCLUSION: RT-PCR application does not assume causative agent seeding stage, significantly reduces analysis execution time and increases sensitivity of the study. The developed procedure will allow to begin addressing the important problem--clarification of spectra and frequency of occurrence of pneumococci serotypes circulating on the territory of Russia.

[A method for determination of Neisseria meningitidis serogroup A, B, C and W by real-time PCR].

AIM: Development and testing of a real-time PCR method for detection of Neisseria meningitiis serogroup A, B, C and W DNA. MATERIALS AND METHODS: Reference strains and 187 samples of cerebrospinal fluid (CSF) from meningocci meningitis patients were used in the study. Multiplex PCR was carried out in an instrument with 5 channels of fluorescent detection: RESULTS: Analysis of specific serogroup loci of the genome and design of oligonucleotides for the detection of DNA of all the capsule meningococci and 4 serogroups in particular was carried out. PCR conditions were optimized; specificity was shown and analytical sensitivity was evaluated using reference strains. DNA of the following serogroups was detected during study of clinical CSF samples: A--in 103 samples (55%), B--in 45 (24%), C--in 30 (16%), W--in 5 (3%). Only DNA of meningococci capstle gene ctrA was found in 4 samples; presumably, they contained DNA of other serogroups. Multilocus sequence-typing and detection of antigenic determinants of PorA and FetA genes for 27 DNA samples of group A menincococci as well as DNA of 5 group W meningococci and 4 ungroupable was carried out. CONCLUSION: The method proposed allows to carry out serogrouping of no less than 95% of strains or DNA samples isolated from CSF of meningococci infection patients. Combined with other recommended non-cultural methods of genotyping, it may be useful for complex characteristics of pathogenic meningococci.

[The application of decision trees for constructing an algorithm for the differential diagnosis of zoonotic infections].

AIM: To attempt to construct an algorithm using the routine epidemiological, clinical and laboratory data for the differential diagnosis of ixodid tick-borne borreliosis (ITBB) caused by Borrelia miyamotoi (BM-ITBB) and other zoonotic infections that are endemic in Russia. SUBJECTS AND METHODS: The investigation enrolled the adult patients treated at the Republican Hospital for Infectious Diseases (Izhevsk) in 2010-2012 with diagnoses of BM-ITBB (n = 71), Lyme disease (n = 38), tick-borne encephalitis (TBE) (n = 25), and hemorrhagic fever with renal syndrome (n = 27). The Decision Tree procedure in IBM SPSS Statistics was used to analyze more than 65 variables characterizing a disease case. RESULTS: The final decision tree had 7 dichotomous fissions in accordance with the values of several indices (presence of erythema migrans, tick bite, goat's milk consumption, sweating, vertigo, nausea, abdominal pain, as well as blood concentrations of platelets, alanine aminotransferase, and count, and urea) and formed 8 terminal nodes. The proposed algorithm provides correct classification in 95% of disease cases. CONCLUSION: ITBB caused by B. miyamotoi can be successfully discriminated from other widespread zoonotic infections. Thus, practitioners have an additional opportunity to detect and diagnose the "new" infection BM-ITBB.

[Clinical presentation of "new" tick-borne borreliosis caused by Borrelia miyamotoi].

AIM: The objective of this study was to confirm the role of B. miyamotoi in the etiology of ITBB-WOEM in Udmurtia and to investigate in detail the clinical presentation of this "new" disease. MATERIALS AND METHODS: 50 adult patients with ITBB-WOEM treated in Republic Hospital for Infectious Diseases, Udmurtia, in 2010-2011 had PCR-confirmed infection by B. miyamotoi. The laboratory evidence of co-infection by other pathogens, including tick-borne encephalitis virus, B. burgdorferi sensu lato, A. phagocytophilum, E. chaffeensis, and E. muris, were absent. RESULTS: All patients had a tick bite from 10 to 18 days before the acute disease onset. The main clinical signs were high fever, fatigue, headache, chill, and sweat. Clinical, biochemical, and instrumental investigations also showed the signs of functional impairment of various organs: the liver (in about half of the patients), kidney (in 10 patients), heart (6 patients), etc. In contrast, acute ITBB with erythema migrans was usually a localized infection without a pronounced intoxication syndrome and impairments of the organs. CONCLUSION: ITBB-WOEM caused by B. miyamotoi is a systemic disease that is clinically closer to relapsing fevers transmitted by argasid ticks than to Lyme borreliosis. The number of B. miyamotoi infections in Russia may be comparable with that of Lyme disease cases, so the investigations of epidemiology, clinical presentation and therapy of this "new" disease are urgently requested.

[Genetic characteristics of Neisseria meningitidis strains obtained from healthy carriers during meningococcal infection outbreaks].

AIM: Genetic and antigenic characterization of Neisseria meningitidis strains isolated during meningococcal infection outbreaks from individuals in contact with patients with generalized form of meningococcal infection. MATERIALS AND METHODS: Strains obtained in 2007 - 2009 in Moscow during examination of individuals that were in contact with patients during meningococcal infection outbreaks were analyzed. Multilocus sequence typing, genetic subtyping and typing of VR fragment (FetA) techniques were used. RESULTS: Data regarding investigated strains were submitted to the database at http://pubmlst.org/neisseria/. Previously undescribed sequence types were found in 12 strains, sequence-type could not be determined in 2 strains, 2 strains lacked VR fragment (FetA). Serogroup A meningococci had "P1.5-2,10: F3-5" antigenic profile and belonged to ST-75 and ST-3349 sequence-type, these data does not support the emergence of epidemically significant strains in the territory under surveillance. All typed serogroup C strains and 1 serogroup B strain are of "ST-41/44 complex/Lineage 3" clonal complex. Subtypes of serogroup C meningococci strains match subtypes of strains that cause generalized forms of infection, while serogroup B strains isolated from the carriers and strains isolated from the patients had different antigenic profiles. Ungrouppable strains had notably higher level of genetic and antigenic diversity: only 6 of 16 strains (37.5%) could be sequence-typed using earlier data, all these strains are of clonal complex "ST-53 complex" that consists mostly of strains isolated from the carriers. CONCLUSION. Ratio of meningococci population circulating in Moscow and subpopulation capable of causing generalized form of meningococcal infection (GFMI) is different for meningococci of various serogroups. Ungrouppable strains isolated from the carriers are highly different from strains causing GFMI.

[Genotyping of West Nile fever virus strains circulating in southern Russia as an epidemiological investigation method: principles and results].

AIM: Characteristic of West Nile fever (WNF) virus strains circulating in southern Russia. MATERIALS AND METHODS: WNF RNA was amplified directly from clinical samples, mosquitoes and bird tissues by PCR, nucleotides were sequenced directly and analyzed comparatively. RESULTS: Related but different genovariants of WNF lineage 1a--"Volgograd" and "Astrakhan"--circulated during WNF outbreaks of 1999 and 2000-2003 in Volgograd and Astrakhan regions. In 2005 "Volgograd" WNF variant emerged in Astrakhan region and along with "Astrakhan" variant caused a new morbidity increase. In 2004 in sera of 2 WNF patients from Rostov region WNF lineage 2 RNA was detected, this was the first WNF clinical case caused by WNF lineage 2 outside of Africa. WNF outbreak in Volgograd region in 2007 was caused by this unique WNF lineage that may preliminary be called Russian. Finally, during a major WNF outbreak in 2010 in Volgograd and Rostov regions in clinical samples only russian genovariant WNF lineage 2 RNA was detected again. CONCLUSION: After emergence of a certain WNF genovariant the virus is capable of persisting in natural foci in southern Russia. A near disappearance of one of the WNF clones by substitution or displacement with another maybe possible. Determination of genetic characteristics of WNF strains circulating in Russia is an important element of WNF epidemiological surveillance and control of this disease.

[Usage of real time polymerase chain reaction for diagnostics of different tick-borne infections].

AIM: To create and test the complex of polymerase chain reaction-based methods for detection of pathogens vectored by ticks in clinical and environmental samples. MATERIALS AND METHODS: Real time PCR methods with hybridization-fluorescent detection were developed for detection of tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophillum, Erlichia muris/E. chaffeensis, and B. miyamotoi. First four methods were combined in one assay in multiprime format. Efficacy of the assay was assessed by testing of blood samples from patients with tickborreliosis (166 patients), tick-born encephalitis (22 patients) and mixed infection tick-borne encephalitis + borreliosis (21 patients) from Sverdlovsk region. RESULTS: It was shown that using PCR-based assay for testing the blood samples obtained during admission, it was possible to determine the etiology of disease in 39% of patients, whereas on the basis of serological data diagnosis, as a rule, is made not earlier than on 2nd week of therapy. False-positive results of PCR diagnostics were not observed. Infections caused by Anaplasma or Erlichia were not observed. It was shown that > 50% of cases of tick borreliosis without erythema were caused by B. miyamotoi, whereas B. burgdorferi sensu lato predominated as a causative agent of erythemic form of borreliosis. CONCLUSION: Proposed complex of methods is useful for rapid diagnostics of tick-borne infections including previously unknown infection caused by B. miyamotoi.

[Genetic characteristics of Haemophilus influenzae type b isolated from children with bacterial meningitis in Moscow in 2007-2009].

AIM: Genetic characterization of 37 strains and CSF samples containing DNA of Haemophilus influenzae type b isolated in Moscow during 2007-2009. MATERIALS AND METHODS: Multilocus sequence-typing method was used and also variant of method for capsule type determination was approbated. RESULTS: Ten sequence types, of which 7 were described in previous studies and 3 were revealed for the first time during this work, were detected in studied sample. ST-6 and ST-92 were the most frequently detected--9 strains (24%) of Hib belonging to each sequence type were revealed. All detected sequence types, except one, belong to clonal complex "ST-6" ("A1/A2"). Obtained data were compared with results of typing of Hib strains isolated in Moscow in 1999-2001. Genetic changes in studied population of Hib are characterized by decreased proportion of Hib belonging to ST-6 (from 54% to 24%) and increased number of sequence types belonging to clonal complex "ST-6" differing from ST-6 on more than one locus of allelic profile (from 2 types [2 strains, 5.4%] to 5 types [9 strains, 24%]). CONCLUSION: In 2007-2009, number of Hib strains with sequence type ST-95 (7 strains, 19%), which is typical for strains circulating in Russia, is markedly increased. Capsule type I was detected in 32 (86.5%) of studied strains, whereas capsule type II--in 5 (13.5%) of studied strains. Capsule type II was detected only in Hib strains with ST-80 sequence type.

[Genetic characteristic of Haemophilus influenzae type B strains isolated in Russian regions].

AIM: Genotyping of Hib strains isolated in regions of Russia as well as characterization of genetic relations of typed strains with strains isolated in other areas. MATERIALS AND METHODS: Genetic characterization of 31 strains of Hib isolated in Russian regions during 2005-2008 was performed by multilocus sequence typing. RESULTS: Studied strains belonged to 11 variants of sequence types, 6 of which were described in previous studies, whereas other 5 were isolated for the first time during this study. The most common isolated strains were ST-92 (13 strains or 42%) and ST-6 (6 strains or 19%). Typed strains were distributed to two clonal complexes. Clonal complex "A1/A2" ("ST-6") incorporates all typed strains except ST-93 strain belonging to clonal complex "B1b" ("ST-93"). The majority of studied strains (19 or 61%) had difference from "central" sequence type of clonal complex, A1/A2 ("ST-6") on not more than one allele. CONCLUSION: Clonal structure of isolated strains is analogous to the one observed in Moscow and foreign strains.