Serum changes in pyridinoline, type II collagen cleavage neoepitope and osteocalcin in early stage male brucellosis patients

Musculoskeletal changes are the most common clinical manifestation of brucellosis. The main objective of this study was to provide a better understanding of this disease, while also attempting to identify potential markers that can identify the early stage musculoskeletal changes associated with human brucellosis. In this case–control study, 41 male early-stage brucellosis patients (within 6 months of diagnosis) who had not received drug therapy and 44 matched controls were examined. Venous blood samples were collected and serum pyridinoline (PYD), type II collagen cleavage neoepitope (C2C) and osteocalcin (OC) levels were quantified using an enzyme-linked immunosorbent assay (ELISA). In the brucellosis group, the median serum levels of PYD (278.53 µg/L), C2C (82.23 µg/L) and OC (8.41 µg/L) were significantly elevated relative to the control group (Z = 5.686, 3.997, 3.579; P = 0.000). Serum PYD, C2C, and OC levels were increased in early-stage male brucellosis patients, and these factors appear to have promise as potential indicator biomarkers that can reflect the osteoarticular changes that occur in the early stage of human brucellosis.

www.nature.com/scientificreports/ may serve as potential biomarkers able to identify early-stage brucellosis-induced musculoskeletal changes. It is hoped that these findings will contribute to a better understanding of this disease.

Materials and methods
patients and sample processing. In this case-control study, serum samples were collected at the Qinghai Institute for Endemic Disease Prevention and Control (Qinghai, China) from 2017 and 2018. Each patient was examined serologically and by X-ray imaging, and any subjects with a joint disease, such as rheumatic fever, osteoarthritis, rheumatic arthritis, paratyphoid fever, or tuberculosis, were excluded as these joint diseases have similar clinical manifestations to human brucellosis. Patients were determined brucellosis positive following clinical examination, serological examination, and epidemiological examination, and 41 male patients who were within 6 months of receiving a diagnosis and had not received drug therapy were selected. For some of these patients, a definite time of infection was unclear, so acute and subacute patients were also defined as at the early period of brucellosis. Clinically, of some of the brucellosis patients presented with fever, weakness, and headache, whereas others had no clear symptoms, but were examined due to their increased chance of occupational exposure or living environment, both. Additionally, 44 matched male control samples were also obtained and had similar occupational risks or living environments. Serum samples from fasting blood were collected and stored at − 80 °C until assayed. Serum levels of PYD, C2C, and OC were detected using enzyme-linked immunosorbent assay (ELISA) kits (Beijing Air-hx biological technology Co. Ltd., Beijing, China) according to the manufacturer's protocols(the detecting ranges were 1.0-200 nmol/L, 1.0-80 μg/L, 0.1-8 μg/L, respectively, and intra-and inter-assay CVs were less than 9% and 15%). This study was performed in compliance with the ethical principles in the Declaration of Helsinki and was approved by the ethics committee of the Qinghai Institute for Endemic Disease Prevention and Control. All subjects provided informed consent.
Brucellosis diagnostic criteria. Patients were diagnosed based on Chinese brucellosis diagnosis criteria (WS 269-2007). The brucellosis patients were divided into three groups, including acute (< 3 months postinfection, high serum titer (≥ 1:100), and high fever), subacute (3-6 months post-infection, positive serum titer (≥ 1:100), and low fever) and chronic (> 6 months post-infection, positive serum titer, and normal body temperature). The predominant clinical manifestations were fever, weakness, headache, hyperhidrosis, and joint and muscle aches. Serological tests were also utilized to establish disease, including the rose bengal plate test (RBPT, a primary screen test) and a serum agglutination test (SAT, titer ≥ 1:100), meanwhile, SAT is a confirmation test with high sensitivity and high specificity The matched controls tested negative for these serological tests.
Exclude criteria. At first, all the study population were excluded normal joint diseases, such as rheumatic fever, osteoarthritis, rheumatic arthritis, paratyphoid fever, and tuberculosis which these joint diseases were similar to human brucellosis at the clinical manifestation .Then, we asked the disease history for each subjects to exclude thyroid dysfuctions, chronic liver and/or kidney diseases, Ehlers-Danlos Syndrome and malignancies with bone metastasis.
Statistical analysis. Data were analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). The data was analyzed using a t-test and data are displayed as a mean ± standard, with P < 0.05 deemed significant. The data for PYD, C2C, and OC expression levels were nonparametric and thus analyzed using a Wilcoxon rank-sum test. consent for publication. All of the authors have read the manuscript and have agreed to submit it in its current form for consideration for publication.

Results
According to epidemiological data, clinical expression and serological test (RBPT positive and SAT titer ≥ 1:100), all the patients were diagnosed as early patients of brucellosis. The average age of the brucellosis patients was 39.69 ± 9.98 years and for the control group was 42.07 ± 13.70 years, with no significant difference in age between the groups (t = 0.912, P = 0.364). When examining serum PYD, C2C, and OC levels, the median levels in the brucellosis group were 278.53 nmol/L, 82.23 µg/L, and 8.41 µg/L, respectively, whereas in the control group, the median levels were 210.54 nmol/L, 72.74 µg/L, and 7.43 µg/L, respectively. When comparing the levels in both groups using a Wilcoxon rank-sum test, the PYD (Z = 5.686), C2C (Z = 3.997), and OC (Z = 3.579) levels were all significantly different between the two groups (P = 0.000, Table 1).

Discussion
Human brucellosis can cause serious complications and obtaining an early diagnosis is very difficult and is often met with delays or even misdiagnoses 7,13 . Furthermore, while radiography is a useful method for diagnosing brucellosis-induced musculoskeletal changes, it is only able to provide a low level of sensitivity during the early stages; thus, relying on radiography can often delay a diagnosis 14 . The brucellosis-induced musculoskeletal changes are frequently destructive and associated with osteopenia and cartilage damage 15 . Components that have been identified as associated with brucellosis include metalloproteinases, which may be promising indicators for determining osteoarticular changes 16 , and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), which are involved in brucellosis pathophysiology and inflammatory activation 17 . However, these biomarkers are not specific and cannot provide a definitive clinical diagnosis. So, it is necessary to find biomarkers that reliably reflect the musculoskeletal changes associated with human brucellosis. PYD is derived from collagen breakdown and has been shown to be an indicator of bone resorption in OA patients 8 . Furthermore, urinary PYD was found to be significantly correlated with radiographic OA grades and associated with the synthesis of osteophytes, subchondral bone sclerosis and synovial degeneration, as well as cartilage degeneration in the joints 18 . C2C is a type II collagen degradation product (3/4 fragment) that appears to reflect the extent of cartilage degradation in OA patients 9 . C2C has also been shown to correlate with knee degeneration in patients with symptomatic knee osteoarthritis 19 . Moreover, OC is one of the most abundant non-collagen bone proteins in the bone matrix and has been used as a biomarker for monitoring bone formation and bone turnover 20 , with its synthesis significantly increased during the remodeling processes 21 . In this study, the PYD, C2C, and OC serum levels were all statistically elevated in brucellosis individuals relative to the healthy controls, and their elevation indicates abnormal changes in the collagen metabolism in cartilage, bone formation, and bone turnover.
Brucellosis more commonly affects males rather than females 22 , with a ratio of 5:2-5:3 in endemic areas 13 . It is possible that the increased prevalence in male patients reflects the exposure pattern of human brucellosis. Furthermore, male patients predominately experience spinal brucellosis, which is the foremost cause of the debilitating and disabling complications of brucellosis 23 . Thus, PYD, C2C, and OC serum changes may be useful in providing an early brucellosis diagnosis in males, but to make these factors useful markers in females, the effects of osteoporosis due to menopause and age would have to be ruled out.
Meanwhile, there were a few limitations in study. Firstly , there are many types of osteoarticular changes among brucellosis patients, and, some of brucellosis patients at an early stage do not have imaging changes of osteoarticular changes, so, this study only have laboratory investigations without radiological evidence. Secondly, these biomarkers can reflect not each type of osteoarticular changes. Thus, future research should focus on comparing PYD, C2C, and OC changes in individuals with brucellosis, osteoarthritis, and osteoarthritis/ brucellosis to determine the true specificity of these markers. Furthermore, the study set should be expanded in future studies as abnormal changes could happen at early stages and knowing how these proteins change over time would be insightful. Also, a larger study set can help confirm the findings presented here.

conclusion
In this study, PYD, C2C, and OC serum levels were increased in male brucellosis patients at an early stage. These factors are associated with abnormal changes in the collagen metabolism in cartilage, bone formation, and bone turnover. These findings suggest that these factors may potentially serve as indicator biomarkers for osteoarticular changes that occur during the early stage of human brucellosis. Future research should focus on examining the relationship between these biomarkers and early-stage brucellosis patients with/without osteoarticular clinical symptoms.

Data availability
All the data is contained within the manuscript.