Chronic kidney disease (CKD) affects over 10% of the global population, posing significant challenges due to its asymptomatic nature in early stages and progressive deterioration of renal function. The identification of reliable biomarkers for early diagnosis and monitoring of disease progression is crucial for improving prognosis and treatment. Neutrophil gelatinase-associated lipocalin (NGAL) is a promising candidate, already validated as a marker for acute kidney injury. Purpose: This study aimed to evaluate the potential of serum NGAL levels as a biomarker for diagnosis and monitoring the progression of CKD. Material/Methods: Seventy-one adult Caucasian patients at CKD stages G1–G4 according to the KDIGO criteria were divided into three groups: Group 1 (eGFR ≥90 mL/min/1.73m²), Group 2 (eGFR 45–89 mL/min/1.73m²), and Group 3 (eGFR 15–44 mL/min/1.73m²). Blood samples were collected and analyzed via standard biochemical and immunological techniques. Serum NGAL levels were measured using ELISA. Results: NGAL levels increased significantly with disease progression and strongly correlated with declining eGFR. Higher NGAL levels were linked to elevated serum creatinine and inflammatory markers, indicating progressive damage to the kidney tissue against the backdrop of persistent systemic inflammation. Conclusions: As a biomarker, NGAL has significant potential for identifying and monitoring kidney damage. Its application in clinical settings could complement and improve existing methods for diagnosing and observing CKD.

Vitamin K2 plays a key role in calcium homeostasis through activation of VKDP, including matrix Gla protein, a potent inhibitor of vascular calcification. Impaired vitamin K2 status results in increased circulating levels of inactive dephosphorylated uncarboxylated MGP (dpucMGP), which serves as a marker for vitamin K2 deficiency. CKD patients are at particularly high risk of vitamin K2 deficiency and its related complications. Purpose: To assess vitamin K2 status indirectly by measuring plasma dp-ucMGP levels in patients with predialysis CKD. Material/Methods: This cross-sectional study included 71 adult Caucasian patients with pre-dialysis CKD stages G1-G4, classified according to KDIGO criteria. Participants were divided into three groups based on estimated glomerular filtration rate (eGFR). Serum creatinine, calcium, inorganic phosphate, and plasma dp-ucMGP levels were measured. Statistical analyses were performed using appropriate parametric and non-parametric tests. Results: Significant differences were observed among the groups in age, renal function parameters, serum calcium, and phosphate levels (p < 0.05). Plasma dpucMGP concentrations did not differ significantly between CKD stages. However, when compared with recalculated reference values reported in the literature, dp-ucMGP levels in all groups were elevated, suggesting functional vitamin K2 deficiency. Sex-specific analysis revealed significantly higher dp-ucMGP levels in females with preserved to moderately reduced renal function, while no sex-related differences were observed in advanced CKD. Conclusion: Patients with pre-dialysis CKD demonstrated poor vitamin K2 status, as reflected by elevated dp-ucMGP levels. These findings support growing evidence that vitamin K2 deficiency is common and clinically relevant in the early tages of CKD.

The tick species Ixodes ricinus is the most widely distributed throughout Europe and serves as the primary vector for the spirochete Borrelia burgdorferi sensu lato (s.l.), which is responsible for Lyme borreliosis. The present study evaluated the prevalence of B. burgdorferi (s.l.) in I. ricinus ticks using nPCR amplification. Ticks were collected from three geographical regions in Bulgaria: the Black Sea Coast in the east, the Pleven region in the north, and the Stara Zagora region in the central south. This study focused on urban and peri-urban areas, including heavily traveled trails in parks. The results indicated statistically significant differences in infection rates, with the highest percentage of infected ticks found in Pleven (52.34%), followed by Stara Zagora (35.46%) and the lowest on the Black Sea Coast (23.08%). Furthermore, we conducted genospecies molecular identification of B. afzelii and B. garinii by PCR amplification of genospecies-specific nucleotide sequences in the 16S rRNA gene. The lowest prevalence of B. afzelii was recorded in Stara Zagora at 10.64%, followed by the Black Sea region at 12.5%, while the statistically highest frequency was observed in Pleven at 21.03%. The prevalence of B. garinii among the total number of I. ricinus ticks was greatest in Pleven at 15.89%, followed by the Black Sea region at 8.65%, and the lowest in the Stara Zagora region at 4.96%. Statistically significant differences were found only between Pleven and Stara Zagora (p = 0.002), but no significant differences in infection rates were observed between adults and nymphs in the regions examined. Overall, the prevalence of B. afzelii in each region exceeds that of B. garinii, and when considered collectively for Bulgaria, the frequency of B. afzelii (15.9%) is higher than that of B. garinii (10.89%) in infected ticks. In a controlled epidemiological context, managing the population of I. ricinus infected with B. burgdorferi s.l. can mitigate the health burden of Lyme disease, although this preventive strategy may not guarantee complete protection.

The uterine microbiota has been the subject of increasing study, but its interaction with the local immune system remains unclear. Successful embryo implantation relies on endometrial receptivity, which is pivotal for immunological tolerance to fetal antigens and precise regulation of inflammatory mediators. Emerging data suggest a dynamic interplay between endometrial microflora and the immune system, making dysbiosis a potential determinant of pregnancy outcomes. Imbalances in the regulation of immune cells in the endometrium and decidua have been associated with infertility, miscarriage, and obstetric complications. A thorough comprehension of the immune system in the female reproductive tract shows potential for improving women’s health and pregnancy outcomes. The objective of this study was to evaluate the patterns of endometrial microbiota in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) and to explore their implications for endometrial immune cells and chronic endometritis (CE). Immune cells in biopsies from 107 RIF and 93 RPL patients were examined using flow cytometry. The endometrial microbial composition was analyzed using real-time polymerase chain reaction (RT-PCR). The research uncovered disrupted endometrial microbiota in most women with RIF and RPL, which was often associated with significant effects on lymphocytes, T cells, and uNK cells.

Purpose: To describe age- and sex-related differences in COVID-19 symptoms in patients admitted to emergency departments during the four waves of the pandemic in Central Northern Bulgaria Materials and methods: Retrospective analysis of data collected from 5382 outpatients with positive result for SARS-CoV-2 by RT-PCR, admitted to 11 emergency departments in Central Northern Bulgaria, was carried out. Results: The median age of patients was 64 years, and 60.6% of them were old adults (≥ 60 years). Overall, more patients were males (51.8%) than females (48.2%). The most frequent symptoms were fever (84.8%), dyspnea (72.3%), and cough (69.9%) without significant difference in both sexes. Less common symptoms, such as sore throat, headache, loss of taste/smell, and fatigue, were found in 1.3% to 4.0% of patients and were more prevalent in women (p≤0.01). Fever remained constant, leading symptom in all ages. The incidence of cough and dyspnoea was less in young patients and showed an increase in middle-aged and old adults. Loss of taste and smell, muscle pain and fatigue displayed the opposite trend. For the leading symptoms, the male/female ratio was >1 in all age groups, whereas for all less common symptoms, this ratio was <1. Conclusions: Older age, male sex and cardiovascular disease were major risk factors for COVID-19 infection. Fever, dyspnoea and cough were the core symptoms and were more prevalent in men and in adult patients. The less common symptoms were more frequent in young people and women.

Background and Aims: Matrix metalloproteinase-12 (MMP-12) is an enzyme capable of degrading major extracellular matrix (ECM) proteins. Studies have suggested that MMP-12 deficiency is related with increased vascular ECM degradation and development of atherosclerotic plaques. Relatively little is known whether MMP-12 interacts with atherogenic lipoproteins in heart failure (HF). The aim of our study was to investigate a possible association between MMP-12 and atherogenic indices in chronic heart failure. Methods: 56 patients with chronic HF with midrange ejection fraction (HFmrEF) were examined, mean age 65.62±9.69 years, and 22 age and sex-matched healthy subjects, mean age 56.4±5.53 years. ELISA was used for measuring MMP-12 levels. The lipid profile and atherogenic indices (log TG/HDL, LDL/HDL, TC/HDL and TG/HDL) were also studied. Results: Serum MMP-12 levels were statistically significantly lower in patients than in controls: 0.0033 (0.0022-0.0071) vs. 0.0075 (0.0068-0.016) (KW=7.37; p=0.006). There was not statistically significant difference between the lipid indices in patients compared to healthy controls (p>0.05). MMP-12 showed correlation with LDL/HDL (r=0.39; p=0.03). Conclusions: Our data show an association between LDL/HDL ratio and MMP-12 in HF. A possible relationship is suggested between abnormal ECM degradation and serum lipoproteins in HF. Further studies are needed to clarify the role of MMP-12 in development of atherosclerotic plaques in HF.

Objective: Early diagnosis of nosocomial infections in newborns is a great challenge, because in the initial phase of systemic infection, clinical symptoms are often non-specific, and routinely used hematological markers are not sufficiently informative. The aim of this study was to determine the potential of early inflammatory markers to diagnose late-onset neonatal sepsis—procalcitonin (PCT), interleukin 6 (IL-6), interleukin 8 (IL-8) and endocan (ESM-1). Material and methods: A prospective clinical–epidemiological study was conducted in a third-level NICU in Pleven, Bulgaria. Patients with suspected late-onset sepsis and healthy controls were tested. A sandwich ELISA method was used to measure the serum concentrations of biomarkers. Results: Sixty newborns were included, of which 35% symptomatic and infected, 33.3% symptomatic but uninfected and 31.7% asymptomatic controls. The mean values of PCT, IL-6, I/T index and PLT differ significantly in the three groups. For ESM-1, IL-8 and CRP, the difference was statistically insignificant. The best sensitivity (78%) and negative predictive value (84%) was found for IL-6. The combinations of PCT + IL-6 and PCT + IL-6+ I/T+ PLT showed very good diagnostic potential. Conclusion: The introduction into the routine practice of indicators such as PCT and IL-6 may provide an opportunity to promptly optimize the diagnostic and therapeutic approach to LOS.

Summary The aim of this study was to determine the potential of early inflammatory markers to diagnose late-onset neonatal sepsis – interleukin 6 (IL-6), interleukin 8 (IL-8) and endocan (ESM-1), and to compare them with routinely used markers like C-reactive protein (CRP) and procalcitonin (PCT). A prospective (January, 2022 – January, 2023) clinical-epidemiological study was conducted in a third level NICU in Pleven, Bulgaria. Patients with suspected nosocomial infection and healthy controls were tested. A sandwich ELISA method was used to measure the serum concentrations. Sixty newborns with an average gestational age of 29.75±3.61 gestational weeks were included, of which 35% were symptomatic and infected, 33.3% were symptomatic but uninfected, and 31.7% were asymptomatic controls. The mean values of PCT and IL-6 differ significantly in the three groups. For ESM-1, IL-8 and CRP, the difference was statistically insignificant. The best sensitivity (78%) and negative predictive value (84%) was found for IL-6. The introduction into routine practice of indicators such as PCT and IL-6 may provide an opportunity to promptly optimize the diagnostic and therapeutic approach to LOS.

The aim of this study was to determine the potential of early inflammatory markers to diagnose late-onset neonatal sepsis - procalcitonin (PCT), interleukin 6 (IL-6), interleukin 8 (IL-8) and endocan (ESM-1), and to compare them with routinely used markers. A prospective (January, 2022 – January, 2023) clinical-epidemiological study was conducted in a third level NICU in Pleven, Bulgaria. Patients with suspected nosocomial infection and healthy controls were tested. A sandwich ELISA method was used to measure the serum concentrations. Sixty newborns with an average gestational age of 29.75 ± 3.61 gestational weeks were included, of which 35 % were symptomatic and infected, 33.3 % were symptomatic but uninfected, and 31.7 % were asymptomatic controls. The mean values of PCT, IL-6, I:T index and PLT differ significantly in the three groups. For ESM-1, IL-8 and CRP, the difference was statistically insignificant. The best sensitivity (78%) and negative predictive value (84%) was found for IL-6. The combination of PCT + IL-6 + I:T+ PLT demonstrated high precision (78%) and best sensitivity (94%) and negative predictive value (95%). The introduction into routine practice of indicators such as PCT and IL-6 may provide an opportunity to promptly optimize the diagnostic and therapeutic approach to LOS.

Background and Aims: Atherogenic indices are used to predict coronary artery disease (CAD) risk. Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) is a natural inhibitor of matrix metalloproteinases and also a protein with inhibitory effects on angiogenesis. The aim of our study was to investigate a possible association between atherogenic indices and TIMP-3 in chronic heart failure (CHF). Methods: 56 patients with chronic HF with midrange ejection fraction (HFmrEF) were examined, mean age 65.62±9.69 years, and 22 age and sex-matched healthy subjects, mean age 56.4±5.53 years. ELISA was used for measuring TIMP-3 levels. The lipid profile and atherogenic indices (log TG/HDL, LDL/HDL, TC/HDL and TG/HDL) were also studied. Results: Serum levels of TIMP-3 were significantly lower in patients compared to controls: 6.460 (1.007-12.520) vs. 5.051 (2.062-10.463); (p<0.05). There was not statistically significant difference between the lipid indices in patients compared to healthy controls (p>0.05). TIMP-3 showed correlation with LDL/HDL (r=0.33; p=0.02) and TC/HDL (r=0.30; p=0.04). Conclusions: Our data show an association between atherogenic indexes LDL/HDL, TC/HDL and TIMP-3 in CHF. A possible lipid interaction with tissue inhibitor of matrix metalloproteinase-3, favoring up-regulation of different pathogenic pathways of HF is suggested. Further studies are needed to clarify the underlying mechanisms of cross-talk between TIMP-3 and lipoproteins in heart failure.

The aim of the study was to determine prevalence of Anaplasmataceae-infected ticks in the Black Sea Coast and the Pleven regions of Bulgaria. A total of 350 ticks from different tick species were collected. Two hundred fifty-five ticks were removed from dogs in the Black Sea Coast region, and 95 Ixodes ricinus ticks were collected by flagging vegetation with a white flannel cloth in two areas in the region of Pleven. After the DNA isolation of the ticks, a genus-specific polymerase chain reaction (PCR) was performed to identify Anaplasmataceae. Second PCRs were performed with species-specific primers to identify Ehrlichia canis (E. canis) and Anaplasma phagocytophilum (A. phagocytophilum). The results showed that 26.9% of the Ixodes ricinus ticks were infected with Anaplasmataceae in the Black Sea Coast region and 36.8% in the Pleven region. The infection with E. canis was detected in 35.7% and A. phagocytophilum in 25.0% of positive ticks from the Black Sea Coast region. In the Pleven region, 22.9% of ticks were positive for E. canis, while 42.9% were positive for A. phagocytophilum. The molecular identification of E. canis in ticks collected from Bulgaria was performed for the first time. In conclusion, the present study revealed a higher prevalence of ticks infected with Anaplasmataceae, particularly A. phagocytophilum, in the Pleven region than in the Black Sea Coast region.

Purpose: To describe clinical characteristics of patients positive simultaneously for RdRp, N and E genes of SARS-CoV-2 and to analyse dynamics of symptoms and Ct values in the interval from day 0 to day 10 after symptoms onset. Materials and methods: Retrospective analysis of data obtained on the day of specimen collection from 1044 individuals was carried out. Detection of RdRp, E and N genes of SARS-CoV-2 was conducted by RT-PCR. Only patients positive for all three genes were included. Results: Out of all patients, 47.8% were female, and 52.2% were male with a median age of 60 years, and the elder (>50 years) population constituted 71.5%. The most common comorbidities were cardiovascular disease (44.5%), diabetes mellitus (11.0%), and chronic pulmonary disease (8.6%). The prevalent symptoms at the time of specimen collection were fever (74.7%), cough (67.1%), and dyspnea (39.6%). Fever and cough were leading symptoms in the time interval between zero and the tenth day since the onset of COVID-19. A linear trend for dry cough and dyspnea, with a daily increase of 2.2% and 2.3%, respectively, was observed. Dry cough was the primary discriminator in distinguishing mild and moderate infection. Gradual increase of Ct values of all genes was observed from day 0 to day 10, and day 3 was essential for separating two time intervals. Conclusions: Flu-like symptoms were leading over the study period. Male sex, older age, cardiovascular disease, diabetes mellitus, and chronic pulmonary disease were risk factors for infection. Cough, age over 65 years and male sex in the group 33-63 years were predictors of more severe disease.

We aimed to determine the presence and distribution of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks collected from urbanized and wild areas in Kaylaka Park (Bulgaria). A total of 546 ticks were collected over three years (2017–2019). The presence of Borrelia in 334 of the collected I. ricinus was detected by dark-field microscopy (DFM) and two nested PCRs (nPCR) targeting the borrelial 5S-23S rRNA intergenic spacer and Flagellin B (FlaB) gene. DFM was performed on a total of 215 ticks, of which 86 (40%) were positive. PCR was performed on 153 of the ticks. In total, 42.5% of the 5S-23S rRNA intergenic spacer and 49% of FlaB were positive. Considering as positive any single tick in which Borrelia sp. was detected regardless of the used method, the infection rate reached 37% (10/27) in the nymphs and 48.5% (149/307) in the adults (48.7% (77/158) females, 48.3% (72/149) males). The incidence of B. burgdorferi infection in I. ricinus did not differ statistically significantly between female, male, and nymph. This study provides evidence that Lyme disease spirochetes are present in various regions of Kaylaka Park with extremely high prevalence in their vectors.

Estradiol is an estrogen steroid hormone and is produced basically within the follicles of the ovaries. The decrease in serum estrogens concentration at menopause disrupts the metabolic balance, changes the lipid profile leading to visceral obesity, which caused an increase in serum estradiol levels, through aromatase activity. Estrogen deficiency also is a reason for the development of osteoporosis.We investigated the serum estradiol levels and changes in bone alpha estrogen receptor expression in ovariectomized rats. For this purpose, we used 20 female Wistar rats at reproductive age - 2 months divided into 2 groups: group 1 (G1)-10 animals were ovariectomized and group 2 (G2)-10 of which were sham-operated. All animals of G1 showed weight gain compared to group G2. The results showed that the values of serum 17β-estradiol in rats of G1 statistically increased compared to G2 (p <0.05). Immunohistochemical analysis revealed no difference in estrogen receptor expression between the both groups. Histomorphological analysis of femur from G1 showed the presence of pronounced osteoporosis. Ovariectomy led to the development of obesity, which caused an increase in serum estradiol levels, through aromatase activity, but this process did not prevent bone tissue from developing osteoporosis.

Hypertension (HTN) is a leading risk factor for cardiovascular (CV) disease. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are thought to be actively involved in the remodeling of the CV extracellular matrix (ECM) during hypertensive damage. Therefore, in this study, we aimed to investigate serum levels of MMP-1 and TIMP-1 in patients with essential HTN and compare them with those of normotensive individuals. We measured serum concentrations of MMP-1 and TIMP-1 in 60 patients with HTN and 20 healthy controls using an ELISA. The obtained results showed that in patients with HTN, the mean levels of MMP-1 (1.82 ± 0.9 ng/mL) were significantly higher (p = 0.03) than the mean levels in the control group (1.19 ± 0.7 ng/mL). The levels of TIMP-1 in patients with essential HTN (0.44 ± 0.1 ng/mL) were also significantly higher (p = 0.005) than those in the control group (0.33 ± 0.1 ng/mL). In HTN, elevated serum MMP-1 levels may be associated with increased collagen degradation in the CV ECM, whereas elevated TIMP-1 levels may favor its accumulation and the development of pathological remodeling and fibrosis of the heart and arterial vessels.

Introduction. The decrease in serum estrogens concentration at menopause disrupts the metabolic balance and leads to visceral obesity, which causes an increase in serum estradiol levels, through aromatase activity. Also, estrogen deficiency is a reason for the development of osteoporosis. The objective of the study was to investigate the serum estradiol levels and changes in bone alpha estrogen receptor expression in an experimental model of osteoporosis. Materials and methods. The study included 20 female Wistar rats at a reproductive age of two months, divided into two groups: group 1 (G1) – 10 ovariectomized rats, and group 2 (G2) -10 rats sham-operated. SPSS 20 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis of the results. For the immunohistochemical study, the nonparametric test – Mann Whitney was used, by comparing the median values. Data were presented as mean ± standard deviation, with p < 0.05 as the limit for statistical significance. Results. All animals of G1 had weight gain compared to group G2. The results showed that the values of serum 17β-estradiol in rats of G1 statistically significant increased compared to G2 (p<0.05). The immunohistochemical analysis revealed no difference in estrogen receptor expression between the groups. The histological analysis of femur in rats from G1 showed pronounced osteoporosis. Conclusions. Ovariectomy led to the development of obesity, which caused an increase in serum estradiol levels, through aromatase activity. However, this increase of serum estradiol did not prevent osteoporosis.

The increased glycation of elastin is an important factor in vascular changes in diabetes. Using the ELISA method, we determined serum levels of IgM and IgG autoantibodies to advanced glycation end products of vascular elastin (anti-AGE EL IgM and anti-AGE EL IgG) in 59 hypertensive patients with type 2 diabetes (T2D) and 20 healthy controls. Serum levels of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and the C-reactive protein (CRP) were also determined. The levels of anti-AGE EL IgM antibodies in the T2D group were similar to those in the control group, while those of anti-AGE EL IgG antibodies were significantly higher (p = 0.017). Significant positive correlations were found between the levels of anti-AGE EL IgM antibodies and MMP-2 (r = 0.322; p = 0.013) and between the levels of anti-AGE EL IgG antibodies and CRP (r = 0.265; p = 0.042). Our study showed that elevated anti-AGE EL IgG antibody levels may be an indicator of the enhanced AGE-modification and inflammatory-mediated destruction of vascular elastin in hypertensive patients with T2D. Anti-AGE EL IgM antibodies may reflect changes in vascular MMP-2 activity, and their elevated levels may be a sign of early vascular damage.

Asparuh Nikolov1 , Nicola Popovski2, Alexander Blazhev3 1Cardiovascular Diseases Investigations, Institute for Scientific Research, Medical University of Pleven 2Clinic of Obstetrics and Gynecology, University Hospital Pleven, Medical University of Pleven 3Division of Biology, Medical University of Pleven, Bulgaria Collagen type III to type I propeptides ratioan early onset preeclampsia indicator Collagen type III to type I propeptides ratio is а better indicator of early-onset preeclampsia than a single propeptide