Multifactorial analysis of clinical laboratory signs, the levels of IL-17A, IL-23, IL-33, IL-35, and specific antibodies in the serum of patients with Lyme borreliosis without erythema migrans
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Lyme disease, also known as Lyme borreliosis, is a natural focal vector-borne disease caused by gram-negative bacteria (Borrelia burgdorferi sensu lato) which are spread through the bite of infected Ixodes ticks. Lyme disease without erythema migrans manifests as an influenza-like syndrome and presents diagnostic challenges since the hallmark symptom, erythema migrans at the site of the tick bite, is lacking [1–10]. It was demonstrated the clinical signs of Lyme disease are determined by the host immune response [1–14]. Many current studies (including those on borreliosis) assess the immunological characteristics and criteria of unfavorable disease course as well as general parameters [12–15]. Multivariate analysis (principal component analysis) allows for identifying the effects of individual factors on the changes in an ensemble of parameters and the association between study parameters .
To determine the association between clinical signs, laboratory parameters, and the production of specific antibodies and cytokines (IL-17A, IL-23, IL-33, and IL-35) in Lyme borreliosis without erythema migrans by multifactorial analysis.
Patients and methods
Thirty patients with Lyme disease without erythema migrans aged 15–75 years (mean age 45.5 ± 15.73 years) who were admitted to the Kirov Infectious Clinical Hospital were examined in 2012–2014. The study was approved by the local ethics committee of the The Kirov State Medical University. The diagnosis of Lyme disease was based on medical history, clinical examinations, laboratory tests, and specific diagnostic tests. The diagnosis of Lyme disease without erythema was made using the E. Asbring classification (1988) and the N.N. Vorob′eva classification (2013) [1–4]. Serological tests for Lyme disease detect specific IgM and IgG against Borrelia afzelii and Borrelia garinii antigens using ImmunoChip. The levels of serum IL-17A, IL-23, IL-33, and IL-35 were measured during the acute period and convalescence. The control group included 30 gender- and age-matched individuals (mean age 45.6 ± 12.27 years, 15–75). Serum levels of cytokines were measured using Bender MedSystems GmbH (Austria) and Wuhan USCN Business Co. Ltd. (China) reagents.
Statistical analysis was performed using the StatSoft Statistica v 10.0 software (StatSoft Inc., USA). Qualitative data are represented as %. The distribution of quantitative data was evaluated using a histogram-based analysis or the Shapiro-Wilk test. If the data were normally distributed, the arithmetic mean and standard deviation (М ± σ) were calculated and compared using Student t-test. If the data were non-normally distributed, the median and the upper and lower quartiles (Me (Q25; Q75)) were calculated. To determine the significance of differences, Mann–Whitney U test was used when comparing independent samples and the Wilcoxon signed-rank test was used when comparing dependent samples. Spearman correlation coefficient was used to determine the correlation between two data series. Testing for the significance of differences was performed using a significance level of 95% (р < 0.05).
In 2000–2019, the incidence of Lyme disease in the Kirov region was 2.6–8.2 times higher than the average Russian incidence . Most (80%) patients with Lyme borreliosis without erythema migrans were people of working age (р < 0.001). 30% of patients denied any tick bites but were in the natural areas within 1–35 days before disease onset. Patients were infected with both B. garinii and B. afzelii, (mixed infection, 76.7%), B. garinii (20.0%), or B. afzelii (3.3%). In 21 patients, the incubation period was 2–25 days (on average, 12.7 ± 1.49 days). In 9 patients, the incubation period was not determined. Patients were admitted 4.1 ± 0.66 days after the onset of influenza-like syndrome with fever and fatigue (30 patients). Patients complained of headache (83.3%), dizziness (46.7%), chills (43.3%) myalgia (46.7%), arthralgia (16.7%), nausea (36.7%), sluggishness (43.3%). The duration of hectic period was 8.3 ± 1.27 days. Most (90%) patients were diagnosed with moderate disease. Severe disease with meningism (vomiting, severe headache, eye pain, neck stiffness) was diagnosed in 10% of patients with mixed infection. The central nervous system (CNS) dysfunctions (anxiety, insomnia, psychomotor agitation) were revealed in 30% of patients (mixed infection was diagnosed in 26.6% and mono-infection with В. garinii in 3.3%). Vago-insular crises were detected in 5 patients with mixed infection (16.7%). Sensory neuropathy of the upper and lower limbs (pain, cramps, stinging, numbness) was identified in 26.7%. Among these patients, mixed infection was diagnosed in 16.7%, mono-infection with B. garinii in 6.7%, and mono-infection with B. afzelii in 3.3%.
Arrhythmias and conduction disorders were reported in 40% of patients. Sinus bradycardia (54.2 ± 2.03 rpm) was diagnosed in 16.7% and sinus tachycardia (98 rpm) was diagnosed in 3.3%. Abnormal cardiac repolarization was diagnosed in 10%, intraventricular conduction delay in 6.7%, AV conduction disorders in 3.3%, and single AV extrasystoles in 3.3%. Chest pain and beat deafness were reported in 6.7%. Patients with cardiac disorders were diagnosed in mixed infection (36.7%) and mono-infection with B. garinii (3.3%).
Hepatitis without jaundice, as demonstrated by 1.1-2.6-fold elevation of ALAT (on average, 66.5 ± 7.51 U/l) was identified in 26.7% of patients (who were diagnosed with mixed infection).
Ceftriaxone was administered in 80%, cefotaxime in 16.7%, and penicillin G in 3.3%.
The disease course with severe influenza-like syndrome was associated with complete blood count (CBC) abnormalities and changes in the levels of specific IgM and IgG against B. garinii and B. afzelii (see Table 1). During treatment, a significant reduction in band and segmented neutrophil counts, a significant increase in platelet count, and a tendency to increase in specific antibodies were revealed compared to these parameters at admission.
Therefore, in the Kirov region, Lyme disease without erythema commonly occurs in people of working age (80%). In most patients (76.7%), mixed infection (B. garinii plus B. afzelii) associated with ECG abnormalities (40%) and cytolysis (26.7%) was diagnosed. In Lyme disease without erythema, blood smear abnormalities illustrate the severity of influenza-like syndrome during the acute period, improvement of hemostasis parameters, and increased activity of humoral immunity during convalescence.
The level of pro-inflammatory IL-23, a specific marker of cellular immune response, was also evaluated. Serum concentrations of IL-23 during the acute period (Me 1267.95 (Q25 399.18; Q75 1518.27) pg/ml) and convalescence (Me 1137.88 (Q25 553.42; Q75 1508.26) pg/ml) were significantly higher compared to healthy controls (Me 24.89 (Q25 17.56; Q75 40.20)) pg/ml; р1< 0.001; р2< 0.001). Pro-inflammatory IL-33, a specific marker of humoral immune response, stimulates the production of interleukins by T helper 2 (Th2) cells . Serum concentrations of IL-33 during the acute period (Me 84.68 (Q25 72.55; Q75 121.00) pg/ml) and convalescence (Me 76.17 (Q25 57.97; Q75 99.22) pg/ml) compared with healthy controls (Me 6.79 (Q25 4.35; Q75 11.67) pg/ml; р1< 0.001; р2< 0.001). Hence, in Lyme disease without erythema, elevated IL-23 and IL-33 concentrations illustrated the activity of inflammatory reactions required for the elimination of Borrelia. However, no correlations between IL-23 and IL-33 were revealed, thereby describing individual implementations of Th1- and Th2-mediated immunity.
The concentration of pro-inflammatory IL-17A, which is responsible for the level of autoimmune inflammatory reactions in the connective and nervous tissue was measured as well . The IL-17A levels during the acute period (Me 0.47 (Q25 0.26; Q75 0.63) pg/ml) and convalescence (Me 0.29 (Q25 0.17; Q75 0.63) pg/ml) were significantly higher than in healthy controls (Me 0.10 (Q25 0.00; Q75 0.64) pg/ml; р1 < 0.01; р < 0.05) thus demonstrating the level of autoimmune inflammation. IL-35 produced by T suppressors inhibits IL-17A synthesis . Serum levels of IL-35 during the acute period (Me 12.07 (Q25 2.03; Q75 19.92) pg/ml) and convalescence (Me 11.26 (Q25 2.03; Q75 20.65) pg/ml) were lower than in healthy controls (Me 21.24 (Q25 13.68; Q75 29.44) pg/ml; р1 < 0.01; р2 < 0.01), thus illustrating the deficiency of pro-inflammatory IL-35. No correlations between IL-17A and IL-35 production were revealed, thereby illustrating imbalance between pro-inflammatory and anti-inflammatory reactions.
Multivariate analysis was additionally performed to identify the most informative factors of the immune pathogenesis of Lyme disease without erythema (see Fig. 1). The interaction between clinical signs, laboratory tests, and levels of IL-17A, IL-23, IL-33, and IL-35 was evaluated. Two major components, “type” and “treatment” (which determine the variability of a set of parameters), were selected. Multivariate analysis has demonstrated that the most informative immunopathological factors are fever, ECG abnormalities, liver disorders, the levels of IL-23, IL-33, and IL-35, and low serum levels of specific antibodies. Elevated pro-inflammatory cytokines in early Lyme disease without erythema illustrate potential organ lesions and delayed antibody production.
The Kirov region is among the top regions with the highest incidence rate of Lyme disease in Russia; the highest incidence rate was 55.9 per 100,000 (15.03–16.88 per 100,000 in recent years). This is accounted for by climate and geographic features, i.e., the Kirov region resides in the sub-zones of the middle and south taiga (mild continental climate). A high incidence of Lyme disease is also reported in the Vologda region (22.85–23.04 per 100,000), the Kostroma region (11.33–17.39 per 100,000), and the Tyva Republic (23.51–32.17 per 100,000) . Many patients are people of working age, as demonstrated by other authors (56% in the Moscow region and 79.6% in the Primorsky region) [18, 19]. Most (86.7%) patients were diagnosed with mixed infection (B. garinii plus B. afzelii). Domestic and foreign authors also report on the high prevalence of mixed Borrelia infection [13, 18, 20, 22].
Our findings demonstrate that the incubation period of Lyme disease without erythema is, on average, 12.7 days. Other domestic authors report that the incubation period ranges from 11.6 to 11.9 days [18, 21]. Severe influenza-like syndrome (100%), ECG abnormalities (40%), and hepatitis without jaundice (26.7%) were detected. Domestic authors also report on fever (51.9%–100%), headache (50.0%–85.2%), fatigue (52.9%–68.7%), arthralgia (16.7%–50.0%), cardiac disorders (16.4%–84.4%), and hepatitis (15.0%–31.1%) in Lyme disease without erythema [18, 21, 23, 25].
We detected the abnormalities of CBC (which are accounted for by intoxication and inflammation) and hemostasis (resulting from a generalized infection). Similar abnormalities were revealed in patients with Lyme disease without erythema in the Perm region [3, 4]. The moderate elevation of specific IgM and IgG illustrates a limited tendency to the enhancement of humoral immunity. The antibody production in Lyme disease occurs 2–3 weeks after onset. Delayed and mild spirochetemia and antibiotic use in early disease account for this phenomenon [9, 24, 26]. The changes in cytokine profile and the imbalance between pro-inflammatory and anti-inflammatory interleukins are an important component of the immune pathogenesis of borreliosis [9, 10, 23–25, 27]. The successful outcome of Lyme disease and the eradication of Borrelia require the balance between early significant Th1 response and late Th2 response in the presence on anti-inflammatory reactions [24, 28]. We established a significant combined Th1/Th2 immune response (as demonstrated by elevated serum concentrations of pro-inflammatory IL-23 and IL-33) required for effective Borrelia eradication. Meanwhile, scarce immunosuppressive reactions (the reduced production of anti-inflammatory IL-35) and high IL-17A production (which mediates autoimmune inflammation) illustrate the potential long-term persistence of immunopathological processes.
1. The perennial incidence of Lyme disease in the Kirov region is 2.6–8.2 times higher than the average Russian incidence. Most (80%) patients with Lyme disease without erythema are people of working age. In most (76.7%) patients, mixed infection (B. garinii plus B. afzelii) is diagnosed.
2. Alterations in cytokine profile (overproduction of pro-inflammatory IL-17A, IL-23, and IL-33 and deficient production of immunosuppressive IL-35) are seen in the acute period and convalescence of Lyme disease without erythema.
3. Multifactorial analysis has demonstrated that the severity of immunological abnormalities in Lyme borreliosis without erythema migrans is correlated with fever, cardiac and liver disorders, high IL-23 and IL-33 levels, and lack of IL-35 and specific IgM and IgG.
About the authors:
Vera V. Sapozhnikova — Cand. of Sci. (Med.), assistant of the Department of Infectious Diseases, Kirov State Medical University, 112, Karl Marx str., Kirov, 610027, Russian Federation; ORCID iD 0000-0002-3930-0460.
Alla L. Bondarenko — Doct. of Sci. (Med.), Professor, Head of the Department of Infectious Diseases, Kirov State Medical University, 112, Karl Marx str., Kirov, 610027, Russian Federation; ORCID iD 0000-0002-9151-604X.
Contact information: Vera V. Sapozhnikova, e-mail: firstname.lastname@example.org. Financial Disclosure: no authors have a financial or property interest in any material or method mentioned. There is no conflict of interests. Received 15.09.2019, revised 03.10.2019, accepted 18.01.2020.
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