76 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 DOI 10.11603/ijmmr.2413-6077.2021.2.12657 EVALUATION OF IMMUNOBLOT RESULTS FOR DETERMINATION OF ANTIBODIES TO LYME DISEASE PATHOGENS IN CHILDREN OF TERNOPIL REGION *S.O. Nykytyuk, S.I. Klymnyuk, I.M. Klishch, S.S. Levenets I. HORBACHEVSKY TERNOPIL NATIONAL MEDICAL UNIVERSITY, TERNOPIL, UKRAINE Background. Lyme disease (LD) is a multisystem disorder caused by Borrelia burgdorferi and other similar tick-borne Borrelia. Objective. The aim of the research was to compare the results of the serological examination of children with different forms of Lyme disease. Methods. We observed the group of children (n=178) aged 1 to 14 years who were bitten by ticks. The control group consisted of 30 healthy children. Ticks were identified using a stereomicroscopic SEO system which included a stereomicroscope, a colour digital camera and a photoadapter. B. burgdorferi sensu lato (sl) (B. burgdorferi sensu stricto, B. afzeliі and B. garinii), B. miyamotoi, A. phagocytophilum DNA in blood were determined by real- time PCR. Baseline investigations related to clinical and immunological studies, including ELISA and Immunoblot, were performed. Results. The survey covered 178 child parents bitten by ticks. Borrelia burgdorferi sensu lato (B. afzelii, B. burgdorferi sensu stricto and B. garinii), B. miyamotoi and A. phagocytophilum were identified. Serological results in children with different forms of Lyme disease were compared. Conclusions. It is established that B. burgdorferi sensu lato; B. miyamotoi; and A. phagocytophilum are pathogens that cause erythema migrans in children. The presence of specific IgG (only positive results) to B. burgdorferi s.l. by immunoblot was confirmed in 83.8% of individuals who had positive and intermediate results in the ELISA test. KEYWORDS: Lyme disease; borreliosis; ELISA; immunoblot; tick bite. *Corresponding author: Svitlana Nykytyuk, MD, PhD, Associate Professor, I. Horbachevsky Ternopil National Medical Univer- sity, Ternopil, 46001, Ukraine. E-mail: androx@tdmu.edu.ua International Journal of Medicine and Medical Research 2021, Volume 7, Issue 2, p. 76-82 copyright © 2021, TNMU, All Rights Reserved Introduction Lyme disease (LD) is a multisystem disorder caused by Borrelia burgdorferi and other si- milar tick-borne Borrelia. This acute systemic disease often occurs in children and is characterized by the presence of erythema migrans (EM), and in some untrea- ted patients of inflammatory arthritis, erythema migrans as well. Lyme disease, caused by Borrelia burgdorferi, is the most common vector-borne disease. In Western Europe it is caused by B. afzelii та B. garinii, [2] whereas in the United States – by B.burgdorferi [3]. The epidemics of Lyme disease is challenging in Poland and Ukraine. In western Ukraine, B. burgdorferi s.l were revealed in 14.2- 17.2% of adult Ixodes scapularis ticks [4]. In 2020-2021, the disease incidence in Ukraine and Ternopil region was 10.62 and 20.05 per 100 thou sand population, respectively (Public Health Center.org.ua). On the basis of MKH-10 allocate “Lyme disease” A69.2. Clinical manifestations of Lyme disease can be divided into three stages: the early localized stage characterized by erythema migrans at the site of the tick bite, multiple Erythema migrans, Borrelia lymphocytoma; early disseminated form with early symptoms confined to peripheral nervous system, carditis or arthritis, late disseminated form – late symp- toms confined to peripheral nervous and central nervous system, manifestations of late arth ri tis, cardiac complications. The patho- genesis, ecology, and epidemiology of Lyme disease are well described; the developed and suggested antimicrobial treatment is very effective [2, 5]. The study aimed to assess the incidence of clinical suspicion of LD among children in Ternopil region (Western Ukraine) by serological examination of children bitten by ticks. Methods The children’s blood tests were studied in the laboratory of the Centre for the Study of S.O. Nykytyuk et al. 77 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 S.O. Nykytyuk et al. Lyme Disease and Other Tick-Borne Infections of I. Horbachevsky Ternopil National Medical University. Ticks were identified using a stereo­ microscopic SEO-IMAGLAB system. Special defining tables were used for iden­ tification of ticks [6]. Databases regarding the incidence of LD in children of Ternopil region in 2017-2021 were used to evaluate ret ro- spective results. This study consists of two parts : the first part describes the data from the questionnaire and clinical examination of the patients, epi- sodes of tick bites, and the second part is serological examination of the blood by ELISA and immunoblot. The study involved 178 children aged 1 to 18 years, who visited to the centre for Study of Lyme Disease of Ternopil National Medical University after being bitten by ticks. The control group consisted of 30 healthy boys and girls living in Ternopil and Ternopil region. They were not bitten by ticks and did not suffer from LD previously. The age and sex distribution in the control group corresponded to that in the control group. Ticks were identified for transmissible in­ fections. DNA of B. burgdorferi sensu lato (sl) (B. burgdorferi sensu strictо, B. afzeliі and B. gari- nii), B. miyamotoi, A. phagocytophilum were determined by real-time PCR using Vector-Best production test system (Germany). The serological examination of the children with LD was made by two-stage diagnosis procedures, primarily using ELISA and immu- noblot for confirmation of the secondary re­ sults. Antibodies to antigens of the B. burgdorferi s.l. complex in blood serum were determined by ELISA using test systems by Euroimmun AG (Germany): class of IgM­test system Anti­Bor­ relia burgdorferi ELISA (IgM), IgG – Anti-Borrelia plus VLSE ELISA (IgG). The results were evaluated quantitatively. The indicator > 22U/ml was considered positive, 16-22 U/ml – intermediate, < 16 U / ml – nega- tive. To detect only IgM against Borrelia antigens, a specific line of the RN­AT system was used, which contained natural purified OspC Borrelia antigens of three species (B. af- zelii, B. burgdorferi ss, B.garinii) and antigens p 39, p 41 and VIѕE. To diagnose specific IgG a line of the RN­AT system was used, which contained VLSE anti- gens of Borrelia of three species (B. afzelii, B. burg dorferi s. s, and B. garinii) and other spe- cific antigens: p18, 19, p20, p21, p58, OspC (p25), p39, p83, Lipid Ba, Lipid Bb. Statistical processing of the results was performed using the methods of parametric and nonparametric statistics by computer programs Microsoft Office Excel and STATISTICA, estimating the absolute (n) and relative amount (%) of the indicators. The analysis of frequency was performed using Pearson’s test χ2 and two-sided Fisher’s exact test, the statistical significance of which was p<0.05. All the studies were performed according to the Conclusion of the Commission on Bioethics of I. Horbachevsky Ternopil Na- tional Medical University, dated September 1, 2021 (Minutes No. 65). Results Erythema migrans was observed in 113 (63.4%), and arthritis in 15 (8.4%) individuals. Nervous system disorders were present in 30 (16.8%) children; 18 (9.5%) children had an erythema-free form of the disease; 2 children (1.1%) complained about the cardiovascular disorders. All children bitten by ticks were divided into the following age groups (Table 1). The largest age group was children of 8-12 years old (42.1% of the surveyed). Among the surveyed, there were 83 (46.6%) boys and 95 (53.4%) girls. Only 143 (80.3%) children had a tick bite, while others did not remember the bite. The examination of patients with an erythematous form of LD took into account the presence of a tick bite in the anamnesis and the accompanying intoxication­inflammatory syndrome, the pre­ sence of lesions of various organs and systems. In the clinical diagnosis of an erythematous form of LD the prevalent symptoms were: primary skin lesion, which was manifested by the erythema migrans, and the epidemiological history. Subsequent examination revealed the presence of various pathogens in this category of patients. In children with erythema migrans, i.e. in 24.7% of 113 people, tick-borne infection with identified pathogens was confirmed (Fig. 1), and in children with the disseminated form of the disease only the effects of the bite were observed. Table 1. The age groups of children affected by ticks Age categories of children (years) Categories 1–3 4–7 8–12 13–18 Number of children 18 39 75 46 78 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 When using the western immunoblot during the first 4 weeks of the disease (an early form of LD), both immunoglobulin M (IgM) and immunoglobulin G (IgG) were determined. Since the probability of a false-positive test result for current infection is high, a positive IgM test result is not recommended when determining the active phase of the disease in people who are sick for longer than 1 month. Verification of the presence of specific IgM antibodies was performed in the sera of 179 patients, 71 of whom had positive (56 people) or intermediate (15) results when tested by the ELISA test. (Table 2). It was found that in 56 patients with positive results, using the method of immunoblot (EUROLINE Borrelia RN-AT) also found positive results in 17 (30.4%) persons, while intermediate was not detected. In children living in Ternopil region, a po- sitive ELISA test was confirmed in 56 children (31.3%), an intermediate test in 15 (8.4%), and a negative test in 108 (60.3%) individuals. The results of blood screening for the presence of IgG in ELISA were positive in 28 subjects (15.6%), intermediate – in 3 (1.7%), negative – in 148 (82, 7%) (Table 2). Subsequently, these results were confirmed by immunoblotting. Thus, from the examined group of patients (179 people) the immunoblot confirmed the total (IgM + IgG) absolute number of positive blood results in (20 + 26) 46 children (25.7%). To determine the etiological structure of LD, the presence of IgM antibodies to the immu- no genic external surface protein OspC (a mar- ker of the early immune response) of three species was determined: B. garinii, B. burgdorferi, B. afzelii separately in patients of both groups (Table 3). Antibodies of this class to OspC B. afzelii were found in the sera of 11 (55%) of the 20 subjects, to OspC B. garinii, respectively, in 11 (55%), to OspC B. burgdorferi s.s. – in 6 (30%) patients. IgM antibodies to antigens p41, p39, and VLsE were also determined in the sera of the examined patients. It was found that antibodies to antigens p41 were detected in 17 (85%) patients, to p39 antigen – in 4 (20%), respectively, to VLsE – not detected in any of the examined groups of children (Table 3). Simultaneously, the presence of IgG anti- bodies (only positive results) to VLsE (recom- binant highly immunogenic lipoprotein of the outer membrane, variable like sequence exp- ressed) of Borrelia of different genes in the sera of 31 patients with ME LD was determined with a positive result in all children. To determine the etiological structure of LD, the presence of IgG antibodies to the immunogenic external Fig. 1. Frequency of detection of infectious agents in children with erythema migrans. Bbs – B. burgdorferi sensu lato; Brm – B. miyamotoi; Aph – Anaplasma рhagocytophilum. Table 2. The content of IgM and IgG to B. burgdorfery s.l. determined by different methods in children living in Ternopil region IgM IgG Elisa EUROLINE Borrelia RN-AT Elisa EUROLINE Borrelia RN-AT Result Total (n=179) Result Total (n=71) Result Total (n=179) Result Total (n=31) Abso- lute value % Abso- lute value % Abso- lute value % Abso- lute value % Positive 56 31.3 Positive 17 30.4 Positive 28 15.6 Positive 25 89.3 Interme- diate 0 0 Interme- diate 0 0 Negative 39 69.6 Negative 3 10.7 Interme- diate 15 8.4 Positive 3 20.0 Interme- diate 3 1.7 Positive 1 33.3 Interme- diate 0 0 Interme- diate 0 0 Negative 12 80.0 Negative 2 66.7 Negative 108 60.3 Negative 148 82.7 9,70% 12,40% 0,80% 1,70% Bb Аph Brm Bb+А S.O. Nykytyuk et al. 79 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 S.O. Nykytyuk et al. surface protein VLsE (a marker of the early im- mune response) of three species was deter mi- ned: B. garinii, B. burgdorferi, B. afzelii separately in the patients of both groups (Table 4). OsC B. garinii antigens of IgM immuno- globulin predominated over OspC B. afzelii, OspC B. burgdorteri in Ternopil region. Vlse B.burgdorteri antigens of immuno- globulin IgG prevailed over Vlse B.afzelii, Vlse B. garinii in Ternopil region. Discussion Lyme disease (LD) is an endemic disease in many countries. In Europe, North America, and Asia, it is the most common vector disease [8,9]. It is caused by B. burgdorferi sensu lato and is transmitted to humans by ticks of the Ixodes ricinus mite complex; up to 20% of them are infected with this bacterium. Only 2-4% of bites are clinically manifested that is one of the diagnostic challenges [10, 11]. In the presence of erythema migrans, there was a significant difference in the ELISA results for immunoglobulin M, in particular a significant predominance of negative IgM values. At the same time, the erythematous form of ME was characterized by positive results of IgM. In a small amount of IgM to flagellin (41 kB) and membrane protein OspC Borrelia begin to appear in the first days of the disease. Their titres increase within 4-6 weeks, and a little longer in untreated patients. During the gene- ralization of the infectious process, IgG anti- bodies appear against several proteins, e.g. P39 and P58 [7]. The frequency of various pathogens that caused erythema migrans was established: the leading pathogen was Anaplasma phago- cytophilym in (12.4%) cases, B. burgdorferi sensu lato in 9.7% of cases. It is proved that if erythema migrans deve- lops in a patient after a tick bite in an endemic area [12], treatment tactics should be suggested immediately. However, if the diagnosis of Lyme disease is uncertain, it is recommended to first determine the sensitivity of the ELISA reaction. Only ELISA­positive cases should be confirmed with a more specific immunoblot results [7]. Table 4. Antigenic load to В. Burgdorferi IgG Antigens Absolutely value (IgG-31) Relative value (%) OspC B. afzelii 0 0 VlsE B. burgdorferi ss 16 51,6 VlsE B. afzelii, 13 41,9 VlsE B. garinii 12 38,7 p39 0 0 P41 21 67,7 Lipid Ba 4 12,9 Lipid Bb 2 6,4 P21 4 12,9 P18 3 9,6 P58 3 9,6 Fig. 2. Average rate of antigenic load to anti-B. burgdorferi IgM antibodies in Ternopil region. Table 3. Antigenic load to anti-B. burgdorferi IgM Antigens Absolute value (Ig M-20) Relative value (%) OspC Bg (B. garinii) 11 55 OspC Bb (B. burgdorferi) 6 30 OspC Ba (B. afzelii) 11 55 P39 4 20 P41 17 85 VLsE 0 0 80 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 It is established that the differentiation between septic arthritis and Lyme arthritis in endemic areas can be a difficult task, and therefore it causes serious consequences for the treatment of the patient [14]. In the acute and late stages, Lyme disease can be difficult to distinguish from other painful processes . To establish a prediction algorithm for the diffe- rentiation of septic arthritis from Lyme disease in children with knee pain and exhaustion [13], a two-stage diagnosis is recommended. The main surface antigens of OspA, OspB, OspC proteins, which determine the difference of individual strains [14], can vary significantly; thus determining the possibility of long-term (for many years) persistence of the pathogen in the human body [14, 15]. Many antigenic determinants of the outer shell of Borrelia of different species are similar to each other and even to some bacteria of other genera, which explains the possibility of cross-immune reactions [7, 16]. Serum samples from children with disse- minated or late stage LD almost always have a strong IgG response to Borrelia burgdorferi antigens [7, 8, 15]. In the evaluation and interpretation of serological test results, both the class of anti- bodies to specific B. burgdorferi antigenic pro- teins and the type of bacterial antigen, for which these antibodies are produced, are important [14]. External surface proteins (Osp) are important in the immune response to infection because they are highly immunogenic. The aantibodies to OspC are characteristic of recent infection. According to the manufacturer’s recommendations, the presence of specific IgM antibodies was considered positive, interme- diate, or negative, depending on the combi- nations of OspC antigens of three species of Borrelia (B. afzelii, B. burgdorferi ss, and B. garinii), p39, and VLsE Bb. At the same time, the presence of IgG was considered positive or negative depending on the combinations of VLsE antigens of three species of Borrelia (B. afzelii, B. burgdorferi ss, and B. garinii) and other specific antigens: p18, p19, p20, p21, p58, OspC (p25 ), p39, p83, Lipid Ba, Lipid Bb. In our immunological study, the genotype B. burgdor- feri sensu stricto was detected in children with erythema migrans, arthritis, and neurolaim, which have statistically significant results (Table 4). The diagnosis of Lyme disease should be established by a laboratory (serological tests (ELISA and Western blot) investigations, indi- cating the presence of specific anti­B. Burgdorferi IgM / IgG antibodies), which confirms clinical manifestations of the disease. This is very im- portant because physicians often seek serolo- gical evidence of B. burgdorfery infection in patients with undefined diffuse complaints [15,18]. According to the list of symptoms compatible with Lyme disease, the most common symptoms in children of Ternopil region were erythema migrans (84.9%). This corresponds to the results of other studies [19, 20]. Conclusions It was estanlished that B. burgdorferi sensu lato; B. miyamotoi; and A. Phagocytophilum are pathogens that cause erythema migrans in children of Ternopil region. The presence of specific IgG (only positive results) to B. Burg- dorferi s.l. was confirmed by immunoblotting in 83.8% of individuals who had positive and intermediate results in the ELISA test. 30,7% 36,1% 26,8% 0,0% 5,0% 10,0% 15,0% 20,0% 25,0% 30,0% 35,0% 40,0% VlsE Borrelia afzelii (VlsE-Ba) VlsE Borrelia burgdorferi (VlsE-Bb) VlsE Borrelia garinii (VlsE-Bg) VlsE Borrelia afzelii (VlsE-Ba) VlsE Borrelia burgdorferi (VlsE-Bb) VlsE Borrelia garinii (VlsE-Bg) Fig. 3. Average rate of antigenic load to anti-B. burgdorferi IgG antibodies in Ternopil. S.O. Nykytyuk et al. 81 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2021 Vol. 7 issue 2 S.O. Nykytyuk et al. Conflict of Interests Authors declare no conflict of interests. Acknowledgements Laboratory staff of the Centre for the Study of Lyme Disease and Other Tick-Borne Infections of I. Horbachevsky Ternopil National Medical University. Author’s Contributions Svitlana Oleksiivna Nykytyuk – formal analysis, writing – original draft, writing – reviewing and editing; Sergiy Ivanovych Klymnyuk – con- ceptualization, writing – original draft, writing – reviewing and editing; Ivan Mykolayovych Klishch – methodology, writing – reviewing and editing; Sofia Sergiivna Levenets – investigation, formal analysis. ОЦІНКА РЕЗУЛЬТАТІВ ІМУНОБЛОТУ ДЛЯ ВИЗНАЧЕННЯ АНТИТІЛ ДО ПАТОГЕНІВ ХВОРОБИ ЛАЙМА У ДІТЕЙ ТЕРНОПІЛЬСЬКОЇ ОБЛАСТІ *С.О. Никитюк, С.І. Климнюк, І.М. Кліщ, С.С. Левенець ТЕРНОПІЛЬСЬКИЙ НАЦІОНАЛЬНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І. Я. ГОРБАЧЕВСЬКОГО МОЗ УКРАЇНИ, ТЕРНОПІЛЬ, УКРАЇНА Вступ. Вступ. Лайм бореліоз (LD) є мультисистемним захворюванням, спричиненим Borrelia burgdorferi та іншими подібними кліщовими Borrelia. Мета. Визначити і порівняти серологічних результатів крові при різних формах хвороби Лайма у дітей. Методи. Під нашим спостереженням знаходилась група дітей (n=178) у віці від 1 до 14 років, укушених кліщами. Контрольна група становила 30 здорових дітей. Кліщів ідентифікували за допомогою стереомікроскопічної системи SEO та визначника. Фрагменти ДНК B. burgdorferi sensu lato (sl) (B. burgdorferi sensu strictо, B. afzeliі та B. garinii), B. miyamotoi, A. phagocytophilum визначали у крові методом ПЛР в реальному часі. Кліщів ідентифікували за допомогою стереомікроскопічної системи SEO. Були проведені базові дослідження, пов’язані з клінічними та імунологічними дослідженнями, включаючи дані Elisa та Immunoblot. Результати. Опитування охопило 178 батьків дітей, на яких напали кліщі. Виявлено Borrelia burgdorferi sensu lato (B. afzelii, B.burgdorferi sensu stricto та B. garinii), B. miyamotoi та A. phagocytophilum. Проведено порівняння серологічних результатів крові при різних формах хвороби Лайма у дітей. Висновки. Встановлено, що B. burgdorferi sensu lato; B. miyamotoi; та A. Phagocytophilum є збудниками, які викликають у дітей мігруючу еритему. Наявність специфічних антитіл IgG (тільки позитивні результати) до B. Burgdorferi s.l. імуноблотинг був підтверджений у 83,8% осіб, які мали позитивні та проміжні результати в тесті ІФА. КЛЮЧОВІ СЛОВА: хвороба Лайма; бореліоз ; ІФА; імуноблот; укус кліща. Information about the authors Svitlana Oleksiivna Nykytyuk – MD, PhD, Associate Professor, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine ORCID: 0000­0003­3146­9664, e­mail: androx@tdmu.edu.ua Sergiy Ivanovych Klymnyuk – MD, Professor, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID: 0000­0002­1308­3250, e­mail: klymnyuk@yahoo.com Ivan Mykolaiovych Klishch – Professor, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID: 0000­0001­6226­4296, e­mail: klishch@tdmu.edu.ua Sofia Sergiivna Levenets – PhD, Associate Professor, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. 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