Real-time PCR validation to estimate the number of rickettsias in the biological material under study.

Using the example of the clinical strain of R. sibirica «Bayevo 105/87¼, the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents "RealBest DNA Rickettsia species (kit1)" was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.

Modification of isothermal test for detecting DNA of pathogens of tick-borne borreliosis.

Early diagnosis of tick-borne borreliosis determines the indications for etiotropic therapy, and the detection of borrelia in a tick that has bitten you serves as the basis for antibiotic prophylaxis. To determine the causative agent of borreliosis, PCR methods are most widely used, which requires special conditions for organizing the work of laboratories and the use of expensive equipment. In addition, the procedure for isolating bacterial DNA and subsequent amplification takes several hours of working time. At the same time, methods for detecting borrelia in the isothermal LAMP-reaction are described, which makes it possible to significantly speed up the diagnosis, does not require complex equipment and highly qualified personnel. It is also known that LAMP in some cases allows analysis without prior extraction of nucleic acids. The purpose was a development of a modified test for isothermal detection of DNA of borreliosis pathogens for an accelerated result and the possibility of excluding the stage of nucleic acid extraction. We used 40 samples of Borrelia DNA and 11 Ixodes persulcatus ticks. To shorten the detection time for Borrelia, the previously described LAMP method was modified by the introduction of additional loop primers. The copy number of the positive DNA sample of the borrelia plasmid was estimated using digital PCR. The results of the LAMP reaction were compared with those of the commercial PRC-RT test. The additional use of loop primers approximately halved the detection time for Borrelia DNA without affecting the comparative diagnostic efficiency. The analytical sensitivity limit of the modified LAMP method was 4 copies/µl or 21 molecules of the plasmid standard added to the reaction. In comparative testing with RT-PCR, the sensitivity of the LAMP method is 90%, and the specificity is 100%. The possibility of detecting borrelia in ticks without the stage of DNA extraction has been demonstrated for the first time. A modified isothermal method for the detection of pathogens of tick-borne borreliosis has been developed, which allows analysis within 20-30 minutes, including in ticks without preliminary DNA extraction.

[Possibilities of serological verification of siberian tick borne type with use of test system for identification of antibodies to Rickettsia conorii.]

The real epidemiological impact of Spotted Fever Group rickettsioses including Siberian tick-borne typhus (STT) in Russia is not sufficiently studied. One of the reasons is the actual absence of either certified domestic diagnostic kits or the evidence for using foreign test kits for laboratory verification of this group of tick-borne infections in medical practice. Objective of our study was to study the diagnostic accuracy of the ELISA test system based on Rickettsia conorii antigens for serological verification of STT. The ROC analysis was performed and operational characteristics (sensitivity, specificity, accuracy, likelihood ratio of positive and negative results) of the STT serological verification test to identify IgM to rickettsia at different times from the onset of the disease using a test system to detect antibodies to Rickettsia conorii were calculated based on the results of a survey of two groups of patients comparable by gender and age (34 patients with pathognomonic signs of STT and 76 clinically healthy people). It was found that the detection of IgM antibodies to rickettsia using the Rickettsia conorii IgM/IgG ELISA test system (Vircell) allows the disease to be verified 10-14 days after the onset of clinical symptoms in 72% (56-88%) of STT patients. We recommend the interpretation of results of the test system "Rickettsia conorii ELISA IgM/IgG" for serological verification of STT which differ from the manufacturer's recommendations regarding verification of Mediterranean fever caused by R. conorii in the following way: the diagnosis of STT should be considered laboratory confirmed when the index of IgM antibodies (IAT) exceeds 8.0; if the IAT is less than 5.0 then a repeated examination of the patient after 10-14 days will be necessary; if the IAT is in the range of 5.0-8.0 then the sample should be re-examined and / or the patient should be examined after 10-14 days. The use of the test system "Rickettsia conorii ELISA IgM / IgG" is promising for laboratory diagnosis and seroepidemiological studies of Spotted Fever Group rickettsioses in Russia.

[Clinical and laboratory diagnosis of tick-borne rickettsioses in areas of low risk of infection Rickettsia sibirica.]

For the first time, a comparative description of clinical and laboratory parameters in patients with clinical signs of tick-borne rickettsiosis in a natural focus with a low risk of infection with R. sibirica and circulation of R. raoultii is given, depending on the results of serological verification of the diagnosis. Established almost complete coincidence of clinical and laboratory parameters in patients with tick-borne rickettsiosis, regardless of the presence of antibodies to R. sibirica and / or R. raoultii. It was shown that even the complex use of complement fixation test, indirect immunofluorescent test and ELISA for the detection of antibodies to R. sibirica does not allow verification of the diagnosis in a third part of patients with pathognomonic signs of tickborne rickettsiosis. In seropostive patients, antibodies to R. sibirica prevailed, which makes it impossible to differentiate cases of tick-borne rickettsiosis of different species etiology. The possible reasons of the phenomenon of «seronegativeness¼ of patients with tick-borne rickettsiosis and approaches to its study are discussed. It is concluded that with the existing diversity and insufficient knowledge of rickettsiae circulating in natural foci, and the current state of laboratory diagnostics, the basis for the diagnosis of «Tick-borne rickettsiosis¼ should be the clinical and epidemiological signs of this infectious disease, which must be recorded in the established order even no serological verification. For such cases, ICD-10 has two encoding options: A 79.9 - Rickettsiosis, unspecified (infection caused by rickettsia, no other indication) and A 77.9 - Spotted fever, unspecified (tick-borne fever, no other indication) [mkb-10.com].

[Evaluation of the effectiveness of the serological methods for the identification of antibodies in patients with tissue ricketsiosis on the territories of a different risk of Rickettsia sibirica infection.]

Cases of tick-borne rickettsiosis in Siberia and the Far East are associated with R. sibirica, the causative agent of Siberian tick typhus (STT). In connection with a sharp reduction in the nomenclature of diagnostic products and an increase in the spectrum of species of founding rickettsiae on the territory of Russia, new approaches to the laboratory verification of diagnoses are needed. We present an evaluation of the effectiveness of serological research methods (complement fixation test, indirect immunofluorescence, and ELISA) in patients with tick-borne rickettsioses in areas of different risk of infection with R. sibirica. Patients were diagnosed with STT from the highly endemic territory of the Altai Republic and from the Naziayevsky district of the Omsk region, where natural foci of rickettsioses of the spotted fever group was detected with the circulation of two species of pathogenic rickettsia, R. sibirica and R. raoultii. As a control group, samples of sera from epidemic seasons from clinically healthy people in Omsk were used. To verify the diagnosis of Siberian tick typhus, the use of serological methods is most appropriate, of which the most sensitive is ELISA, which allows detecting antibodies at an earlier time. In the ELISA for confirmation of the diagnosis, the first serum can be examined only on IgM. Investigation of the 2nd serum should be performed in ELISA for the presence of IgM and IgG antibodies with R. sibirica antigen. Reaction of indirect immunofluorescence (RNIF) for the study of paired sera should be conducted with specific antigens of rickettsia circulating in this focus. In laboratories not equipped for setting ELISA, it was recommended to use CFT. When the titer increases in two or more times and IgM and IgG are detected in the second serum, taking into account clinical manifestations, the diagnosis of "Siberian tick typhus" can be considered confirmed.

[The molecular biological methods of express-detection of pathogens in ticks taken way from patients as a basis of preventive therapy of tick transmissible infections.]

The complex molecular biologic study of ticks, took off from people in the Omskii oblast during Spring-Summer periods in 2014-2016 established their infection with agents of tick's transmissive infections and testifies a significant risk of infection of population. The analysis of ticks I. persulcatus established that rate of detection of DNA of borrelia made up to 59,5%, rickettsia - 30.6%, anaplasmae and erlichiae - 5.9%. The analysis of ticks D. reticulatus by polymerase chain reaction detected DNA of rickettsia in 20.5%, borrelia - in 6.6%, RNA of tick-borne encephalitis virus - in 3.6%. No DNA of anaplasmae and erlichiae was detected. During sequencing of tests positive to rickettsia the most often Rickettsia raoultii was identified. The approaches to expressdiagnostic and preventive therapy of tick-born transmissive infections were substantiated with consideration for mixt-infection of agents and pathogenic characteristics of developing nosologies. The algorithms of laboratory analysis regulated by the Sanitary Regulations 3.1.33-10-15 "Prevention of infections transmitted by ticks" require a broader implementation.

[THE LABORATORY DIAGNOSTIC OF HUMAN TRANSMISSIBLE INFECTIONS IN COMBINED HOT SPOTS].

The occurrence of common disease carriers of various pathogens condition wide prevalence of combined hot spots of tick infections. The comprehensive approach to laboratory diagnostic of these infections is substantiated taking into account specter of main pathogens transmitted by ticks.

[The problems of laboratory diagnostic of rickettsiosis of group spotted tick-bite fever in Russia].

The article presents analysis of modern techniques of laboratory diagnostic of rickettsiosis of spotted tick-bite fever group. Owing to drastic shortage of list of produced preparations and increasing of specter of detected types of rickettsia in Russia the new approaches to laboratory verification of diagnoses are needed. To detect antibodies to rickettsia of spotted tick-bite fever group can be recommended such techniques as reaction of indirect immune fluorescence and immune enzyme assay with antigens of corresponding types of rickettsia. The most acceptable techniques for detecting and identifying rickettsia of spotted tick-bite fever group are polymerase chain reaction restricting analysis and polymerase chain reaction sequence analysis. The biological methods of analysis are needed to study pathogenic types of rickettsia.

[ON THE ROLE OF RICKETTSIA RAOULTII IN THE EPIDEMIOLOGY OF RICKETTSIOSES IN RUSSIA].

The paper analyzes data on the prevalence and possible epidemiological importance of Rickettsia raoultii. The possible risk of infection in the population was assessed from the results of a serological study of patients after tick suction and from those of molecular biological determination of contamination of removed carriers. The place of Rickettsia raoultii among other rickettsia causing rickettsioses of a tick-borne spotted fever group is assessed in Russia.

[Efficiency of use of immunoglobulin preparations for the postexposure prevention of tick-borne encephalitis in Russia (a review of semi-centennial experience)].

Along with regenerated interest in passive immunization during flavivirus infections, there is an increase in the number of reports with negative opinions as to the expediency of postexposure immunoglobulin prophylaxis (IGP) of tick-borne encephalitis (TBE). Analysis of the publications (since 1959) on this problem has indicated that more than half of them, including all papers with negative opinions as to IGP, contain the results of studies whose design are unsuitable for the solution of the problem associated with the efficiency of medical interventions in the context of evidence-based medicine. Analysis of the results of cohort studies has established that the timely single administration of an immunoglobulin (IG) preparation against TBE in a dose of 0.05 ml/kg body weight ensures protection on average in 79% of cases. Increasing the single dose to 0.1 ml/kg, as re-administration of IG, provides no additional protective effect. The preventive efficacy of IG preparations against TBE varies under the influence of factors, such as the infectious virus dose inoculated from tick suckling, the age of a victim, the antiviral activity of preparations with a antihemagglutinin titer of 1:20 with high infectious virus doses and the absence of reliable rapid tools for its determination on tick suckling, it is necessary to use IG with a titer of at least 1:80 and, desirably, more active in children, for postexposure prophylaxis of TBE.

[Taxonometry, ecology and evolutional relation of rickettsia].

Ecology of Rickettsia is main direction of researches of RAMS academician I. V. Tarasevich and her team. Characteristics of alpha 1-Protobacteriae--parasites of eukaryotic cells--given in the work. The accent to Rickettsia characteristics in relation of their evolution relationship with eukaryote mitochondria is made. Main directions of researches in Q-fever, Tsutsugamushi fever, rickettsioses of tick-borne spotted fever (TSF) group, performed in our country under direction of RAMS academician I. V. Tarasevich, are represented. Results of study with the use of tick experimental model, monoclonal antibodies and genetical methods of TSF group Rickettsia, revealed in Siberia and Far East (focal territories of tick-born rickettsioses) and also in territories with the absence of this infection in Russia and Kazakhstan, are given.

[Ureaplasma urealyticum and Ureaplasma parvum in etiology of urogenital mixt-infections].

The complex study of 104 vaginal samples from patients with urogenital uroplasmosis was carried out. U. parvum were detected in 67.3% patients, U. urealyticum--in 12.5% and in 20.1% cases--two species were registered at the same time. Isolation of clinical significant concentration of both ureaplasma (> 10(4) CFU/ ml) was detected in about 50% of cases. Expression of inflammation of vaginal mucus depended on the level of concentration of infection agents. U. parvum were associated with bacterial vaginosis, while in urogenital candidosis U. parvum was detected rarer than U. urealyticum. The dominant numbers of clinical ureaplasma were high sensitive to "new" macrolides and chinolons, however the high percent of isolates were resistant to erytromicin and doxiciclin.

[Detection of a new species of Rickettsiae in the ticks of Ixodes persulcatus in Russia].

Genetic analyses (polymerase chain reaction and nucleotide sequence test) have been used to describe a new species of Rickettsiae in the ticks Ipersukarus in Russia. This species is named as Rzckettsia tarasevichiae after Academician of the Russian Academy of Medical Sciences Irma Vladimirovna Tarasevich, Head of the Laboratory of Rickettsial Ecology, N.F.Gamaleya Research Institute of Epidemiology and Microbiology. A total of 317 Ipersukatus imagoes were collected in the regions of the Urals, Western Siberia, Eastern Siberia, and Far East in the spring of 2001 and 2002. The authors detected R. tarasevichiae in 25.5 +/- 2.4% (87/317) of the examined I. persulcarus ticks, from 3.8% in the Novosibirsk Region (Western Siberia) to 56.8% in the Primorye Territory (the Far East). This is the first detection of the Rickettsiae in the ticks of the genus Ixodes in Russia. Conceivably, the spread of this Rickettsia species coincides with the area of I. persulcatus that inhabits the biotopes of southern taiga and mixed forests in Eurasia from the western frontier of Russia to the Far East. The nucleotide sequences of the fragments of gene 165 of pRNA and citrate synthctase of this species of Rickettsiae have been declared at the GenBank under numbers AF503168 and AF503167, respectively. Its most phylogenetically close species of Rickettsiae R. canadenss (69% homology in terms of the gene of citrate synthetase and 98% homology in terms of gene 165 of pRNA) which is responsible for cases of acute cerebral vasculitis in Northern America. This requires further studies of a possible role of R. tarasevichiae in the pattern of tick-borne infections recorded in the area of I. persulcatus.

[New evidence for the detection of ehrlichia and anaplasma in ixodes ticks in Russia and Kazakhstan]. / Novye dannye o vyiavlenii érlikhii i anaplazm v iksodovykh kleshchakh v Rossii i Kazakhstane.

Polymerase chain reaction and sequence analysis were used to screen Ehrlichia and Anaplasma DNA in 900 specimens of Ixodes ticks of four genera (Dermacetor, Haemaphysalis, Ixodes, and Hyalomma) collected on 14 administrative territories of Russia and Kazakhstan. Anaplasma phagocytophilia DNA was detected and identified in Ixodes persulcatus ticks gathered in the Altai and Primorye Territories. Ehrlichia muris DNA was genotyped in the I. persulcatus ticks collected in the Tyumen, Omsk, and Novosibirsk Regions and the Altai Territory. Examining the Haemaphysalis concinna collected in the Primorye Territory revealed A. bovis DNA. The I. persulcatus ticks gathered in the Omsk Region were found to have "Ehrlichia-like "Schotti variant" DNA, Studying the ticks of the genera Dermatocentor and Hyalomma gathered in Russia and Kazakhstan failed to detect the representatives of the genera Anaplasma and Ehrlichia. The detection of A. phagocytophilia DNA in the I. persulcatus ticks in the Altai and Primorye territories and the serological verification of cases of human granulocytic anaplasmosis among patients with tick-borne infections in these territories lead to the conclusion that there are natural loci of this disease in West Siberia and the Far East of Russia.

[Detection of Rickettsia hulinii in ticks of the Haemaphysalis concinna species in Russia]. / Obnaruzhenie Rickettsia hulini v kleshchakh Haemaphysalis concinna na territorii Rossii.

The presence of Rickettsia hulinii was detected in ticks of the Haemaphysalis concinna species in the Asiatic part of Russia. It was the first detection of these rickettsiae outside the territory of the Chinese People's Republic. This tick is spread over a broken area and may occur in different regions of Eurasia. At present at least 3 rickettsial species of the tick-borne spotted fever group were found in Ixodes ticks on the territory of Eastern Siberia: R. sibirica, known to be the etiological agent of tick-borne rickettsiosis, R. hulinii with proved pathogenicity for laboratory animals and R.DnS14 with no data available on its pathogenicity.

[Possible types of tick-borne diseases and the predictive role of history data in their diagnosis (parasitological aspects of the problem)]. / Vozmozhnye varianty zabolevanii kleshchevymi infektsiiami i prognosticheskaia rol' anamneza v ikh diagnostike (parazitologicheskie aspekty problemy).

The paper discusses the proven and probable role of different tick-borne pathogens and their combinations in the occurrence of zoonotic diseases. The variants of coexistence of various combinations of 7 proved disease agents (3 species of Borrelia, 2 species of Ehrlichia, Babesia, and TBE virus) with different, if might to be, disease agents, such as rickettsiae of two groups, which do not belong to the Borrelia burgdorferi sensu lato group spirochetes, are analyzed. The difference of their development and the time of transmission after of the vector Ixodes persulcatus Schulze attachment are considered. Based on the their own hypothesis that one tick specimen is able to be the vector of only three human pathogenic agents simultaneously, the authors examined the possible number of combinations of pathogens, which might be obtained by a patient after 1 tick attachment. Proper history data are recommended as a tool for making a correct diagnosis, wherein the time of attachment has to be a main factor. For example, it should be taken into account that TBE virus might be transmitted only some minutes after attachment by not only a female tick, but also a male one. Mixed infection of virus plus Borrelia might develop only following some hours of attachment (most probably after 18-24 hours). The very similar situation has to be considered to lead to virus-Borrelia-Ehrlichia (or Rickettsia) coinfection. Any combination with Babesia involvement may appear not earlier than 24-36 hours after mixed infected tick attachment (or even later). It is necessary to consider a possible role of apathogenic microorganisms which are present in the most of vector specimens in nature and which might be injected with infected tick saliva may play a possible role in the manifestation of disease and in the prediction of their possible influence.

[Genotyping of spotted tick fever rickettsiae, detected in Russia and Kazakhstan]. / Genotipirovanie rikketsii gruppy kleshchevoi piatnistoi likhoradki, vyiavlennykh v Rossii i Kazakhstane.

R. slovaca was first detected in the ticks D. marginatus gathered in the Stavropol Territory and the Voronezh Region (European Russia). The recently discovered rickettsial genotype DnS14 was first found in the ticks D. silvarum from Buryatia and D. niveus from the Karaganda Region (Central Kazakhstan). The rickettsial genotype RpA4 was most common in the ticks of the genus Dermacentor in Russia and Central Kazakhstan. An analysis of the spread of rickettsias of the STF group shows their close ecological relation to definite types of Ixodes. The rickettsias R. slovaca and RpA4 co-exist in the ticks D. marginatus and D. reticulatus (the western part of a Dermacentor area in Eurasia) and DnS14 and R. sibirica do in D. nuttalli and D. silvarum (the eastern part of the area). D. marginatus and D. reticulatus in the areas characterized by the most specific saturation of a Dermacentor area (the south of West Siberia) are carriers and reservoir of R. sibirica. The rickettsial genotype DnS28 may be now considered to be environmentally associated with one species of ticks--D. nuttalli. At least 6 genotypes of STF rickettsias--R. sibirica, R. astrahan fever (R. conorii), R. slovaca, RpA4, DnS14, DnS28--has been currently identified in Russia and Kazakhstan.