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LABORATORY DIAGNOSTICS OF TISSUE INFECTIONS WITH NATURAL FOCUS (TICK-BORNE RICKETTSIOSIS, IXODIC TICKBORNE BORRELIOSIS)
1 Altai State Medical University, Barnaul
2 "Central Research Institute of Epidemiology" of The Federal Service on Customers' Rights Protection and Human Well-being Surveillance, Moscow
O.V. Beskhlebova1, V.M. Granitov1, V.G. Dedkov2
The article presents an assessment of diagnostic efficiency of two modern methods of laboratory diagnostics of tickborne infections - serological and molecular biological. A comparative study of information content of EIA and PCR in diagnostics of tick-borne ricketsiosis (TBR) showed that serological method had the highest frequency (90,0 %) in confirming of clinical diagnosis TBR. The diagnostic value of PCR in the detection of DNA of Rickettsia in skin biopsy specimens was 81,5%. The effectiveness of this method in the detection of DNA of Rickettsia in serum was 11,8%, in whole blood - 5,5%. Immunochip method for the diagnosis of ixodic tick-borne borreliosis showed the maximum diagnostic value on the third week of the disease. The study of paired sera, taken with an interval of 7-10 days, increased its diagnostic efficiency by 30,6%. The study by PCR, using serum and blood from patients, had a low diagnostic value and cannot be recommended as a routine method for diagnosis of ixodic tick-borne borreliosis
Key words: tick-borne rickettsiosis, ixodic tick-borne borreliosis, enzyme-linked immunosorbent assay, polimerase chain reaction, an immunochip.
Since the beginning of the 21st century, a high incidence of tick-borne infections with natural foci has been observed in the territories of various regions of the Russian Federation [1, 2].
According to the Federal Service for Supervision of Consumer Rights Protection and Human Wellbeing in 2016, in Russia, the structure of incidence of tick-borne infections is dominated by ixodic tick-borne borreliosis (ITB) with a morbidity rate of 4.18 per 100,000 population. Further, tickborne encephalitis (TE) and Siberian tick-borne typhus (STT) are 1.39 and 1.06 per 100,000 population, respectively. The incidence of "new" tickborne infections in the territory of the Russian Federation is low (human granulocytic anaplasmosis (HGA) - 0.04, human monocytic ehrlichiosis (HME) - 0.01 per 100 thousand population).
By the present day, more than 50 subjects of Russia are endemic for a number of tick-borne infections [3]. The presence of regional features of the prevalence and clinical picture of these diseases dictates the need for their comprehensive study in each of the subjects of Russia. Despite the fact that there are a number of works that reflect the clinical picture and laboratory diagnosis of this group of diseases in the context of regional pathology, the principal issues concerning the prevalence and structure of tick-borne infections in the country remain relevant [3]. To address these issues, it is necessary, first of all, to improve the quality of diagnosis of tick-borne natural focal infections.
Diagnostics of STT in endemic regions is based, as a rule, on clinical and epidemiological data, and in typical cases does not cause difficulties [4]. However, the possibility of an atypical course 50
of the disease, the presence of conjugated areas and the marked similarity of clinical manifestations with other rickettsia of the group of tick-borne spotted fevers (TSF) dictates the need for laboratory verification of the diagnosis.
Currently, in the territory of the Russian Federation, laboratory diagnosis of STT is carried out on the basis of approved methodological documents [5], where only serological examination methods based on the detection of R. sibirica antigen are regulated. Unfortunately, they are unsuitable for the species-specific verification of other rickettsia (for example, R. heilongjiangensis), since the rickettsia of the TSF group are characterized by cross-sectional serum reactions [6; 7; 8; 9].
In the presence of pathognomonic syndrome -migrating erythema, as well as a characteristic epi-demiological anamnesis (stay on an endemic territory, the fact of tick sucking, seasonal compliance), the diagnosis of an ITB is unquestionable [10; 11]. Nevertheless, the widespread use of non-erythritic forms of ITB and the emergence of clinical manifestations of ITB in any sequence and combination dictates the need for using laboratory diagnostic methods. Despite the variety of laboratory methods, in Russia there are currently no unified algorithms and criteria for laboratory diagnosis of ITB at different stages of the disease, which undoubtedly requires standardization and improvement of the verification of the diagnosis of ITB.
Research objective
To determine the features of laboratory diagnostics of tick-borne natural focal infections (Siberian tick-borne typhus, ixodic tick-borne borreliosis).
Research tasks
1. Verify the diagnosis of tick-borne infections by laboratory methods with the simultaneous study of several biological materials from patients.
2. To evaluate the diagnostic efficacy of serolog-ical and molecular biological methods in the diagnosis of STT and ITB.
3. Conduct a comparative analysis of the diagnostic significance of these research methods.
Materials and methods
During 2013-2016, there were examined 213 patients hospitalized in the infectious department of FSBHI "City Hospital No. 5" in Barnaul, Altai Krai with various diseases transmitted by ixodic ticks. The study was approved by the local ethics committee of the the Altai State Medical University of the Russian Ministry of Health.
In addition to clinical and laboratory tests, all patients underwent molecular biological and serological studies.
Verifying methods of laboratory diagnostics were performed on the basis of the "Central Research Institute of Epidemiology" of The Federal Service on Customers' Rights Protection and Human Well-being Surveillance (FBSI CRIE), Moscow.
Material for molecular biological methods was whole venous blood, paired blood serum of patients, collected on the first day of admission to hospital (4.7 + 0.2 day of the disease) and in dynamics after 7-10 days.
Biopsies (crusts) from the place of primary affect, in cases when their preparation was possible, were taken with sterile tweezers on the 8 ± 0,3 days of the disease and before the study were stored in 70 ° ethyl alcohol in the freezer at minus 20°C..
Nucleic acids were extracted individually from each sample of whole blood and serum, as well as from biopsy samples, using the AmplePrime RIBO-prep kit (FBSI CRIE) in accordance with the manufacturer's recommendations [12].
The detection of the TE virus, Borellia burg-dorferi sl, Anaplasma phagocytophilum, Ehrlichia chaffeensis / Ehrlichia muris was carried out after the preliminary extraction of nucleic acids by poly-merase chain reaction (PCR) in real time with the help of the set of reagents AmpliSens TBEV, B.burgdorferi sl, A. phagocytophilum, E.chaffeen-sis / E.muris-FL» (FBSI CRIE) according to the protocol recommended by the manufacturer [13].
The detection of rickettsia of the TSF group was carried out by real-time PCR, proposed by J. Stenos et al. [14] in the modification of the FBSI CRIE. Subsequent typing of rickettsia in positive samples was performed by confirming PCR and sequencing of two fragments of gltA citrate synthase genes and OmpA outer membrane protein. Sequencing of the resulting fragments was carried out using an automatic capillary sequencing device "ABI-Prism 3500 XL device" (Applied Biosystems,
USA). The resulting sequences were compared with the reference sequences of R. spp., presented in GenBank NCBI and based on their maximum homology, their species identity was determined.
The material for serological testing was paired sera obtained from the venous blood of the patient. The first serum was taken on the day of admission to hospital (4.7 + 0.2 day of illness), the second - after 7-10 days. Detection of specific IgM and IgG antibodies to the agents of HME and HGA by the enzyme immunoassay (EIA) was carried out by commercial immunoenzyme test systems "HME-EIA-IgM", "HME-EIA -IgG" and "HGA-EIA-IgM", "HGA-EIA -IgG" produced by LLC "Omniks", (St. Petersburg).
Detection of specific antibodies of IgM and IgG class to the causative agent of TE was carried out by commercial immunoenzyme test systems "TBEvirus (FSME) IgM EIA" and "TBEvirus (FSME) IgG EIA" (Humburg, Germany). In connection with the presence of a cross-serological reaction between the rickettsia of the TSF group, specific IgM and IgG antibodies to the TR pathogen were determined in the "Rickettsia conorii EIA IgM / IgG" test system (Vircell, Spain) according to the manufacturer's instructions.
The sera were tested for the presence of antibodies to tick-borne borreliosis pathogens with the help of a set of "ImmunoChip Borreliosis" reagents produced by the FBSI CRIE of Rospotrebnadzor. This set of reagents allows for differentiation in a single treatment to differentiate a large spectrum of IgG and IgM antibodies against antigens of Borrelia afzelii and Borrelia garinii, the main pathogenic genes widely distributed in Russia.
In 28 (20.1%) cases TR was diagnosed on the basis of clinical and epidemiological data (sucking and/or crawl of a tick, presence of feverish intoxication syndrome, characteristic exanthema and the primary affect and regional lymphadenitis). In other cases (79.9%), TR diagnosis was established on the basis of the DNA typing of rick-ettsia in various biological materials from patients (skin biopsy, whole blood clot, blood serum) and/ or the detection of antibodies of IgM class by EIA in paired sera increase of seropositivity coefficient, or only in the second serum with negative results in the first serum. In this case, all patients with TR had no markers of other tick-borne infections.
Diagnosis of ITB in 10 (21,7%) people was established on the basis of epidemiological history (sucking or crawling of the tick), and the presence of erythema 3-5 or more centimeters in diameter on the body. In the remaining 36 (78.3%), the diagnosis was confirmed serologically by the determination of antibodies of the IgM class on immuno-capses to various antigens of borrelia in paired sera or only in the second serum with negative results in the first. At the same time, all patients with ITB lacked markers of other tick-borne infections.
The diagnosis of TE (4 people), HGA (1 person) is based on the detection of antibodies of IgM class by EIA in the second serum from patients, provided that there are no markers of other tick-borne infections.
The diagnosis of tick-borne mixed infections (23 people) is based on the detection of antibodies of IgM class by the method of EIA (immunochips for ITB) to the target pathogens and/or typing of DNA pathogens in various biological materials from patients (for TR).
Various methods of statistical processing are used in the work, depending on the type of random variables and the research task.
To compare the frequencies of qualitative characteristics in independent samples, the criterion x2 was used. In the presence of low frequencies (less than 10), the Yates correction for continuity was used for this criterion. At frequencies less than 5, Fisher's four-field conjugacy tables were used. The relationship between the characteristics was determined using the Spearman rank correlation method.
The level of statistical significance in verifying the null hypothesis was taken as the corresponding p <0.05. The processing and graphical representation of the data was carried out using computer programs Statistica 10.0 (Russified version), Excel 2007.
Results
Diagnosis of hospitalized patients: 139 (65.3%) patients were diagnosed with tick-borne rick-ettsiosis, including in one case, TE caused by R. heilongjiangensis, 46 (21.6%) - diagnosed with ixodic tick-borne borreliosis, 4 (1.8%) - tick-borne encephalitis, and granulocytic anaplasmosis was diagnosed in one patient. In 23 (10,8%) patients, a mixed infection caused by pathogens of these diseases in various combinations, was detected. By studying the diagnostic significance of PCR and the serological method (EIA), data of the results of the examination of patients who had a laboratory verified diagnosis of TE (110 people) were used. A total of these patients were withdrawn and examined by real-time PCR, proposed by J. Stenos et al. (2005), in the modification of the FBSI CRIE with subsequent typing sequencing of the obtained amplicons of 27 biopsies from the place of primary affect (crusts), 110 whole blood samples (clots) and 110 paired sera collected with an interval of 7-10 days.
In "PCR-positive" patients, Rickettsia sibirica DNA sensu stricto was detected in various biological materials: a biopsy from the place of primary affect, serum of blood, a blood clot.
The study of samples of skin biopsy specimens collected on 8.0 + 0.3 day of the disease (against antibiotics) revealed 22 positive and 5 negative results. The diagnostic significance of PCR in de-
tecting rickettsia DNA in skin biopsy samples was 81.5%.
By the study of serum samples, 13 positive and 97 negative results were obtained, by the study of the whole blood clot - 6 and 104 results, respectively. The effectiveness of the method for detecting rickettsia DNA in serum is 11.8%, in whole blood - 5.5%. Simultaneous study of whole blood and serum samples in TR patients made it possible to increase the detectability of rickettsia DNA only up to 17.3%. It should be noted, that the DNA of rickettsia was found only in the first serum collected on 4.7 + 0.2 day of the disease before the initiation of etiotropic therapy. The presence of significant differences in the frequency of positive results in the PCR biopsy specimen from the place of primary affect and the first blood serum (p <0.05) testifies to the high diagnostic significance of the PCR method when examining biopsies from the site of primary affect. An undoubted advantage of PCR is the possibility of subsequent typing sequencing of the obtained amplicons of the pathogen for the purpose of its species differentiation, which is relevant for regions in which different rickettsia from the group of TSF pathogens are simultaneously encountered.
By the study of serum samples by EIA, antibodies of class Ig M to rickettsia of the TSF group were determined in 99 samples, and 11 samples gave negative results. Thus, the effectiveness of the EIA method for detecting antibodies to rickettsia in the TSF group in patients with CR was 90.0%. In 44 patients (44.4%), Ig M antibodies to rickettsia of the TSF group were detected in the first serum. The study of paired sera collected at an interval of 7-10 days, made it possible to increase the detectability of Ig M class antibodies by 55.6%.
Comparative analysis of the results of EIA and PCR of blood serum in patients with verified diagnosis of TR showed that the coincidence of positive results occurred only in 10 (9.1%) cases. In this case, there are significant differences in the frequency of positive results when using EIA in comparison with PCR and there is no correlation between them (r = 0,1).
The obtained data testify that the EIA method in the diagnosis of CR is sufficiently effective under the condition of the study of paired sera taken with an interval of 7-10 days.
Features of laboratory diagnostics of tick-borne borreliosis.
In the period from 2013 to 2016, 46 patients with ixodic tick-borne borreliosis were under observation, including 10 people with an arterial form (21.7%).
Detection of Borrelia DNA in blood serum of patients was carried out by real-time PCR method using the set of reagents "AmpliSens TBEV, B.burg-dorferi sl, A. phagocytophilum, E. chaffeensis /
E.muris-FL" (FBSI CRIE). Studies of sera in all patients gave negative results, which does not contradict the literature data on the low sensitivity of PCR in the study of this biological material [15; 16].
Taking into account the absence of non-ery-thematous forms of the disease, as well as the low sensitivity of PCR, the establishment of a diagnosis of ITB only on the basis of clinical and epidemi-ological data presents certain difficulties and requires the examination of patients by serological methods.
The serological examination of the patients included the study of paired sera collected on the first day of patients' admission to the hospital and later on in dynamics - after 7-10 days, by means of the immunochip method using the "ImmunoChip Bor-relios" diagnostic system produced by the Central Research Institute of Epidemiology of Rospotreb-nadzor. This method allows detecting antibodies of Ig M and Ig G class to various antigens of Borrelia.
In cases of diagnosis on the basis of clinical and epidemiological data (21.7%), the first blood sera were collected on the day of admission to hospital, which corresponded to 2.0 + 0.3 day of the disease and in dynamics - on the 7-10th day of inpatient treatment (9.6 + 0.5 day sickness).
In 36 patients (78.3%), the diagnosis was confirmed serologically - the Ig M class antibodies to various Borrelia antigens: in paired blood sera - in 25 people (64.4%), and in 11 people (30.6%) -only in the second serum. In patients in whom Ig M antibodies were detected already in the first serum, it was withdrawn on 8.1 + 1.1 days of illness (i.e., in the second week of the disease). In the absence of antibodies of Ig M class in the first serum, but their presence in the second serum, the first serum was withdrawn in the first week of the disease (2.6 + 0.3 day of the disease) (p <0.05). The second serum of blood in patients with serological confirmation of the diagnosis was withdrawn on 21.4 + 1.4 days of illness (i.e. at the end of the third week of the disease), i.e. later than in patients without se-rological confirmation of the diagnosis (9.6 + 0.5 day of the disease), (p <0.05). This is due to the length of stay of patients in the hospital.
Thus, the serological diagnosis of ITB with the determination of antibodies to various antigens of Borrelia by the method of immunochips is effective in the collection of biological material from the second week of the disease. If this condition is met, the method of paired sera makes it possible to increase the detectability of Ig M by 30.6%. The highest frequency of positive results is observed at the end of the third week of the disease (21.4 + 1.4 days of illness).
Discussion
An integrated approach to conducting a survey of patients with tick-borne infections with
the simultaneous use of molecular genetic and se-rological methods of investigation made it possible to verify the diagnosis of tick infections in all 213 patients. Due to a comprehensive examination of patients for the full range of tick-borne infections, it was found that the simultaneous infection of individuals with a tick attack by several pathogens is 10.8%.
A comparative study of the informativity of the two modern laboratory diagnostic methods, EIA and PCR, in the diagnosis of CR showed that the serological test (EIA) confirmed the clinical diagnosis with the greatest frequency (90.0%). At the same time, the study of paired sera collected at an interval of 7-10 days made it possible to increase the detectability of Ig M class antibodies by 55.6%.
The low efficiency of the PCR method in detecting rickettsia DNA in blood serum (11.8%) and in whole blood (5.5%), the possibility of detection of the pathogen only in the first serum collected before the etiotropic therapy does not allow to recommend this method as routine diagnosis of TR.
The diagnostic significance of PCR in detecting rickettsia DNA in skin biopsy samples was 81.5%. The presence of significant differences in the frequency of positive results when studying the PCR biopsy from the place of primary affect and the first blood serum (p <0.05) testifies to the high diagnostic significance of the PCR method in the study of biopsy specimens even when etiotropic therapy is performed at 8.0 ± 0.3 day of illness.
The diagnosis was confirmed serological-ly in 78.3% of all patients with ITB. The study of paired sera made it possible to increase the de-tectability of Ig M by 30.6%. In patients with Ig M class antibodies already detected in the first serum, it was significantly relieved later (by 8.1 + 1.1 days of the disease) than in patients without serolog-ical confirmation of the diagnosis (2.0 + 0, 3 day of illness). In all cases of ITB, in serum collected at the end of the third week of the disease (21.4 + 1.4 days of the disease), specific Ig M antibodies to Borrelia were detected.
None of the patients with a serologically confirmed diagnosis of ITB by PCR in real time in the serum did not detect Borrelia DNA. Thus, the method of immunochips can be recommended as the main method of confirming the diagnosis of ITB. This method shows its effectiveness in the study of sera from the second week of the disease. The maximum diagnostic value of serological examination is observed in the third week of the disease. The study by the PCR method of sera and whole blood from patients can not be recommended as a routine method of diagnosing ITB.
Conclusions
1. Laboratory diagnosis of tick infections requires the obligatory use of several methods of research at the same time.
2. For laboratory diagnosis of TR, the leading method is EIA, which allows to confirm the diagnosis in 90.0% of patients.
3. The PCR method for TR diagnosis shows the greatest diagnostic efficiency in the study of skin biopsies from the place of primary affect (81.5%).
4. The method of immunochips is an effective laboratory method for the diagnosis of ITB, provided it is used from the second week of the disease.
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Contacts
Corresponding author: Beskhlebova Olga Vasily-evna, teaching assistant of the Department of infectious diseases and phthisiology of ASMU, Barnaul. Tel: 9-961-995-04-80. E-mail: olg.deriglazova@yandex.ru
