AS A BIOCHEMICAL CRITERION OF A GENERAL INFLAMMATORY SYNDROME IN LYME DISEASE IN CHILDREN

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INTRODUCTION
Lyme borreliosis (LB) is a multisystem infectious disease caused by Borrelia burgdorferi sensu lato and transmitted to humans by a tick bite. The reported estimated global Borrelia burgdorferi seropositivity is relatively high with the top three regions: Central Europe, Western Europe and Eastern Asia. Using the western blotting (WB) to confirm Borrelia burgdorferi serological results could significantly improve the accuracy [1]. More studies are needed to improve the accuracy of global Lyme borreliosis burden estimates. Lyme borreliosis has become more common among children. Improving the methods of non-specific biochemical markers of Lyme disease is especially important [1]. C-reactive protein (CRP) is an acute inflammatory protein that increases up to 1,000-fold at sites of infection or inflammation [2]. C-reactive protein is produced as a homopentameric protein, termed native CRP (nCRP), which can irreversibly dissociate at sites of inflammation and infection into five separate monomers, termed monomeric CRP (mCRP) [2].

MATERIALS AND METHODS
There were 42 children with a tick bite in study group and 20 children in the control group -residents of Ternopil region, who applied to Ternopil Regional Children Hospital, 2019-2021, whose aged 8-17 years.
The group of investigated children had clinical symptoms of Lyme borreliosis and antibodies to specific proteins of B.burgdorferi antigen. The diagnosis of ring-shaped EM was established clinically and based on characteristic manifestations and epidemiological history. Lyme borreliosis diagnoses were confirmed according to the classification by Y. V. Lobzyn, 2000 [3], Diagnostic criteria Serologic Diagnostic Criteria recommended by the Second National Conference on Serologic Diagnosis of Lyme Disease (1994) [4].
The data was interpreted according to the WHO guidelines: Guidance Lyme disease: diagnosis and treatment [5].
All patients were divided into the following groups. The first group: EM+arthritis (n=16), Second group -EM (n=26).
Eight children were investigated during two phases. During the investigation it was defined the CRP, level of leucocytes taking into consideration the general blood analysis with the next definition of the correlation between them. The definition of CRP level in children with Lyme disease after tick's bite and after the treatment was investigated in the first group. The correlation comparison of CRP level with the level of leucocytes in the general blood analysis was made.
Blood samples were collected twice: before antibiotic therapy (sample 1) and after the treatment (sample 2) in about 14 days. Standard two-stage tests based on indirect fluorescent antibody (IFA) assay and immunoblotting currently serve as the primary supports for the laboratory diagnosis of LB and assessment of the disease development. The objective of this study was to identify blood proteins with altered abundances in patients with early stage Lyme disease as compared to healthy controls. Such proteins are candidate biomarkers requiring further validation that may Педіатрія ultimately lead to the development of improved diagnostic tests for early Lyme disease especially in patients with a negative antibody-based serologic test.
Treatment of patients with an erythematous form of LB was performed according to the following schemes: patients received Doxycycline, tablets 100 mg, 10 pcs. 2 times/day after meals during 14 days at a dose 4 mg/kg.
The effectiveness of thе treatment was assessed by the dynamics of clinical manifestations of the disease and the level of the concentrations of acute-phase response proteins including C-reactive protein (CRP) in children with Lyme disease. CRP was determined by turbidimetric method with latex reinforcement.
Less than 1 (mg/l) is a low result, from 1 to 3 (mg/l) is average result or risk, more than 3 (mg/l) is a high result.
Statistical analysis of the results was performed using the methods of parametric and nonparametric statistics "Microsoft Office Excel" and "STATISTICA". The study was conducted within the framework of the scientific research "Research on epidemiology, pathogenesis, clinics and prevention of Borreliosis", which is a part of a joint Ukrainian-Polish project under the auspices of the European Union.

RESULTS AND DISCUSSION
Inclusion criteria: clinical, laboratory and instrumental signs of Lyme disease.There were no significant age and sex differences between the groups in this study.
The definition of CRP and leucocytes was made twice before the treatment and after the treatment.
The mean interval between tick suction and clinical symptoms was 12-14 days.
EM was observed in 14 patients within 24 hours, it was 31.9 % of all patients. After 24-48 hours, the appearance of EM was observed in 13 patients (29.5 %); on the 3 rd day it was observed in 2 children (4.5 %); after a few months -in 1 patient (2.3 %).
Patients in the first group had arthritis syndrome, which occurred in one month after a tick bite with manifestations of EM. The children complained for persistent pain, oligoarthritis and myalgia. The main symptoms of 12 children of the second group were: subfebrile body temperature, redness of the skin and general intoxication. Nonspecific intoxication syndrome of fatigue and myalgia was observed in 10% of patients without erythematous forms of the disease.
In the first group of the investigation it was revealed a statistic true increase of the level of CRP (Table 1).
Children with Lyme disease who had not yet received antibacterial therapy did not have normal CRP level at admission. In children of the first group, whose disease was accompanied by arthritis and EM (migration erythema), the level of CRP was higher than the CRP indicator in which Lyme disease proceeded with EM and was (6.8±0.6) mg/l and (5.2±0.5) mg/l respectively, which did not make a significant difference of 0.05. In 5 children (11.9 %) from the first group and 3 children (7.2 %), the CRP level showed high indicators.
In children with Lyme disease, which did not have antibiotic therapy, there were no normal CRP levels. In children from the first group with Lyme disease and arthritis and ME, the level of CRP was higher than the level of CRP and in those children, which had Lyme disease and EM it was (6.8±0.6) mg/l and (5.2±0.5) mg/l correspondingly that showed no significant difference, it was 0.05. In 5 children (11.9 %) of the first group and 3 children (7.25 %) the CRP level was of a high indication.
In the 1 st group of children, there were 6 examined (14.3 %), the CRP level remained at high levels after the treatment. In 10 examinees (23.8 %), the average level of CRP was (2.2±0.4) mg/l, which corresponded to the average level of the protein index. In the 2 nd group of the examined children, whose disease occurred only with erythema migrans, the average CRP level was (3.0±0.3), which was not significantly different from the indicator in the first group of the sick children (EM+Arthritis) more than 0.05.
High levels of CRP after the treatment were noticed in 10 children of the second group (23.8%) and were (3.8±0.4). In the rest children, 16 (38.1 %) were kept at the level of medium risk and increased to (2.2±0.2).
In both groups of children, after the treatment, normalization of CRP was noticed in almost the same number of children from the first group, 4 (9.5 %) and from the second group, 3 (7.1 %). In 8 children, 5 (11.9 %) from the first group and 3 (7.1 %) from the second group, CRP ranged from 0.5 to 0.9 mg/l, which corresponded to a low level of the indicator.

Normal index
Notes*p<0.5

Педіатрія
In both groups of children after the treatment, the normalization of CRP was admitted almost in equal quantity of children from the first group 4 children (9.5 %), from the second group 3 children (7.1 %). Among 8 children 5 of them (11.9 %) from the first group and 3 (7.1 %) from the second group, the CRP level of which was from 0.5 to 0.9, what was correspondent to a low level of its indication.
The number of leukocytes in patients of both groups before the treatment and after the treatment was normal. The comparative analysis between the children of both groups did not show a significant difference.
Therefore, in all patients with Lyme disease, an increase in CRP was observed before antibacterial treatment, the high level of which was noticed in almost 5 percent of children (19.1 %). After the treatment, the maximum results were maintained in those patients who had elevated results at the time of admission and were ((11.2±6.4) mg/l) and normalization of the results was not observed.
2. Second item; clinical manifestations of leukocytosis are increased fatigue and malaise for no apparent reason, increased body temperature more than 37.5 0 C, which occurred in the examined group of patients.
Clinical manifestations of leukocytosis include increased fatigue and malaise for no apparent reason , increased body temperature in 38.0 %, which occurred in the examined group of patients .
The level of leucocytes was increased in 5 % of the examined children. In 95 % of children the level of peripheral leucocytes was within a referent norm.
It is known that Lyme disease is a polysystemic disease with a polymorphic clinical picture, we tried in the first phase of the research to establish criteria for assessing the disease [2,3]. Inclusion criteria were as follows: -epidemiological (living in an endemic area), clinical complaints of patients (erythematous skin lesions, Lyme arthritis), infectious diagnosis confirmation: 2-stage research scheme, laboratory serological detection methods with the following standard laboratory tests for identifying Bb including conventional serological methods such as ELISA, IFA test, protein biochemical test, line blot and western blotting (WB) [4, 5]. Acute-phase proteins that are part of the innate immune system and can respond within hours of bacteria entering the skin include CRP, complement factors [6].
Nowadays, approximately CRP is a highly sensitive marker of infection and inflammation that binds to various ligands present on the surface of pathogens or exposed to stress or autologous cell death by acting through opsonin deposition and complement pathway activation, in addition to direct interaction with phagocytic cells [6].
CRP as a marker of inflammation shows high serum levels in invasive bacterial infections [7]. There are scientific studies of the potential for measuring CRP in different phases of acute bacterial meningitis in children to predict the results [8]. Thus, Peltola and Heikki [9] in their study determined the initial values of CRP in 669 children. There were obtained useful prognostic information, CRP measurements on the 3rd or 4th day carried out in 275 children with pathology of the nervous system. Hearing impairment although not total deafness was found to be 3-7 times more likely if CRP was above the median level shortly after hospitalization.
So Elsammak, Mohamed [12] studied serum procalcitonin (РСТ) and C-reactive protein (CRP) in children with streptococcal tonsillopharyngitis or non-streptococcal tonsillopharyngitis which was similar to healthy children. PCТ had greater specificity than CRP for the detection of bacterial tonsillopharyngitis. Differentiating between bacterial and non-bacterial conditions is very difficult. On the example of differential diagnosis of viral pneumonia and bacterial community-acquired pneumonia in children [10] in the works of Robert G. Badik, 2008, evaluated serum concentrations of C-reactive protein (CRP) as a predictor of bacterial pneumonia [13].
It should be noted that Yong Zhou, Shizhen Qin (2019) [14] in a longitudinal cohort of 40 Lyme disease patients and 20 healthy controls, identified 10 proteins with significantly altered serum levels in patients at the time of diagnosis. In an independent cohort of patients with erythema migrans, six of these proteins, APOA4, C9, CRP, CST6, PGLYRP2 and S100A9, were confirmed to show significantly altered serum levels in patients at time of presentation. According to (Elrod, Julia, 2022) [15], the most important predictors of a long-term increased frequency of infectious complications are an increased preoperative level of C-reactive protein, a high intraoperative assessment of severity, and insufficient primary antibiotic treatment [16].
We examined the relationship between baseline peripheral white blood cell count and C-reactive protein (CRP) values with outcomes in 42 children. A highly sensitive method of determining CRP established that the initial level of peripheral leukocytes corresponded to normal limits, while the level of CRP ranged from the normal reference values to (7.2 mg/l).
The same data were obtained by researchers Derek and Williams, 2015, [10] who studied the relationship between baseline peripheral leukocyte and C-reactive protein (CRP) values with outcomes among 153 children hospitalized with pneumonia.
To our opinion the CRP concentration is thus a very useful nonspecific biochemical marker of inflammation, measurement of which contributes importantly to: a) screening for organic disease; b) monitoring of the response to treatment of inflammation and infection; c) detection of intercurrent infection in immunocompromised individuals, and in the few specific diseases characterized by modest or absent acute-phase responses.
CRP is a biochemical criterion of a general inflammatory syndrome in Lyme disease in children.

CONCLUSIONS.
1. Nowadays, the markers of inflammation in Lyme disease in children have not been sufficiently investigated.
2. Taking into consideration that the level of C-reactive protein fluctuates within (up to 3.0), it shows that this is a marker of high bacterial load in Lyme disease in children.