Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2155-2
ARTICLE
Serum annexin A2 levels in acute brucellosis and brucellar spondylodiscitis N. Aktug Demir & S. Kolgelier & S. Sumer & A. C. Inkaya & S. Ozcimen & L. S. Demir & O. Ural & A. Arpaci
Received: 2 April 2014 / Accepted: 5 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Brucellosis is a chronic granulomatous infection and may present with various clinical manifestations. Brucellar spondylodiscitis symptoms are initially subtle and nonspecific. Annexin A2 (ANXA2) is involved in various biological functions, including osteoclast formation, bone resorption, and cell growth regulation. In this study, we aimed to determine the clinical significance of serum ANXA2 levels in acute brucellosis and brucellar spondylodiscitis. This prospective study included 96 acute brucellosis patients and 51 healthy controls. Acute brucellosis was diagnosed by a 1/160 or higher titer in a standard tube agglutination (STA) test or a four-fold increase in titers between two STA tests performed two weeks apart in the presence of clinical N. Aktug Demir : S. Sumer (*) : O. Ural Department of Infectious Diseases and Clinical Microbiology, Selcuk University, Faculty of Medicine, Konya, Turkey e-mail: [email protected] S. Sumer e-mail: [email protected] S. Kolgelier Department of Infectious Diseases and Clinical Microbiology, Adiyaman University, Faculty of Medicine, Adiyaman, Turkey A. C. Inkaya Department of Infectious Diseases and Clinical Microbiology, Hacettepe University, Faculty of Medicine, Ankara, Turkey S. Ozcimen Department of Infectious Diseases and Clinical Microbiology, Konya State Hospital, Konya, Turkey L. S. Demir Department of Public Health, Necmettin Erbakan University, Faculty of Medicine, Konya, Turkey A. Arpaci Department of Biochemistry, Adiyaman University, Faculty of Medicine, Adiyaman, Turkey
symptoms within the last eight weeks and/or growth of Brucella spp. in appropriately prepared culture media. ANXA2 levels were determined with an enzyme-linked immunosorbent assay (ELISA). Forty (41.7 %) of 96 acute brucellosis patients were male and 56 (58.3 %) were female. Serum ANXA2 levels were elevated in patients compared to healthy controls (p=0.001). Eighteen of 96 (18.7 %) acute brucellosis patients had brucellar spondylodiscitis. The serum ANXA2 levels of patients with brucellar spondylodiscitis were higher than those of patients with acute disease without brucellar spondylodiscitis (p=0.001). ANXA2, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) values were elevated in the brucellar spondylodiscitis group compared to patients without brucellar spondylodiscitis. Serum ANXA2 measurement together with ESR and CRP is thought to be indicative in the diagnosis of brucellar spondylodiscitis, a common complication of brucellosis.
Introduction Brucellosis is a chronic granulomatous infection caused by Brucella spp. and may lead to morbidity and loss of productivity. The prevalence of brucellosis is high in particular geographic areas in the world, like Mediterranean countries. Acute brucellosis may present with various clinical manifestations. Brucella spp. may persist for a long time and cause chronic complications [1, 2]. Brucellar spondylodiscitis is a common complication of brucellosis, affecting 10–21.4 % of patients. After the transmission of Brucella spp. was ingested by host cells, one-third of the invading organisms survive in phagolysosomes, which creates a unique site for bacterial growth and immune escape [1]. Annexins are a family of Ca2+/lipid-binding proteins that contain Ca2+-binding proteins. Twelve annexin subfamilies have been identified in vertebrates. Annexin A2 (ANXA2),
Eur J Clin Microbiol Infect Dis
also called annexin 2, is one of the best characterized in the group of annexins. ANXA2 is involved in various biological functions, including exocytosis, endocytosis, cell–cell adhesion, cell proliferation, cell surface fibrinolysis, osteoclast formation, bone resorption, cell growth regulation, and apoptosis [3]. Therefore, ANXA2 levels have been investigated in different disease groups, such as neoplasms and infections [4–18]. The aim of this study is to evaluate the serum ANXA2 levels in acute brucellosis and brucellar spondylodiscitis that may have a course with different clinical findings and complications.
Materials and methods Patients This study was carried out at the Infectious Disease and Clinical Microbiology Clinics of Adiyaman University, Faculty of Medicine, between March 2012 and February 2013. Ninety-six patients who were diagnosed with acute brucellosis and 51 healthy controls were included in this study. Inclusion criteria were age 18 years old or above at the time of the study initiation, not having a diagnosis of brucellosis previously or having treatment for the disease, not having immunosuppression, and not being pregnant. Ethics This work was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Approval was obtained from the ethical committee of Adiyaman University (2012/03-1.2). Informed consent was received from all patients involved in this study. Diagnosis of acute brucellosis Acute brucellosis was diagnosed by a 1/160 or higher titer in a standard tube agglutination (STA) test or a four-fold increase in titers between two STA tests performed two weeks apart in the presence of clinical symptoms (a compatible clinical picture, such as arthralgia, fever, sweating, chills, headache, and malaise) within the last eight weeks and/or growth of Brucella spp. in appropriately prepared culture media. The control group consisted of healthy individuals who had normal leukocyte counts, normal C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values, and a negative Brucella STA test. Contrast-enhanced lumbar magnetic resonance (MR) images were taken from the patients who were diagnosed with acute brucellosis and who had a complaint of lower lumbar
pain. The MR images were evaluated using the format defined by Pourbagher et al. [19]. Determination of ANXA2 All blood samples were drawn before the treatment was initiated. These blood samples were centrifuged for 5,000 cycles for 3 min to separate the serum. The serum samples were stored at −80 °C until the assays were performed. Serum ANXA2 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Uscn® E91944HU, Wuhan, Hubei, China). A total of 100 μL of sample or standard was placed in each tube, and the tubes were incubated for 2 h at 37 °C. Following the incubation, 100 μL of reagent A were added, and the tubes were incubated for another hour at 37 °C. They were washed three times, followed by the addition of 100 μL of reagent B and a third incubation for 30 min at 37 °C. Then, they were washed five times. Following another incubation at 37 °C with the addition of 90 μL of substrate, 50 μL of stop solution were added, and readings were taken immediately at 450 nm. Statistical analysis SPSS version 18.0 (Statistical Package for the Social Sciences, Chicago, IL) was used to analyze the data. The percentage distribution and mean standard deviation values were used as descriptive statistics. The Mann–Whitney U-test was used for analyzing the data. p-Values less than 0.05 were accepted as significant.
Results Forty (41.7 %) of the 96 acute brucellosis patients were male and 56 (58.3 %) were female. There were 25 male (49.0 %) and 26 (51.0 %) female volunteers in the control group. The mean age of the patient group was 40.9±15.4 years versus 33.8±9.3 years in the control group. Serum ANXA2 levels were elevated in the patients compared to the healthy controls (p=0.001) (Table 1, Fig. 1). Serum ANXA2 levels were not different between genders. There was no association between the patients’ complaints and ANXA2 levels (Table 2). Table 1 Serum annexin A2 levels in patients and control groups
Annexin A2 (ng/dl)*
Acute brucellosis patients (n=96)
Healthy controls (n=51)
p-Value
7.24±10.4
2.1±4.7
0.001
*Mean±standard deviation
Eur J Clin Microbiol Infect Dis
Fig. 1 Serum annexin A2 levels in the patient and control groups
Thirty-one (32 %) of the 96 patients with acute brucellosis had a complaint of lower lumbar pain (Table 2). Brucellar spondylodiscitis was found in 18 of the 31 patients with lower lumbar pain. Eighteen of 96 (18.7 %) acute brucellosis patients had brucellar spondylodiscitis. The serum ANXA2 levels of the patients with brucellar spondylodiscitis were higher than those patients with acute disease but without brucellar spondylodiscitis (p=0.001). ANXA2, ESR, and CRP values were elevated in the brucellar spondylodiscitis group compared to patients without brucellar spondylodiscitis (Table 3).
Discussion ANXA2 expression has been found to be up-regulated in several types of bacterial and viral infections [12–18]. However, this change in ANXA2 levels is known to be important Table 2 Serum annexin A2 levels with respect to the gender and symptoms of patients n (%)
Gender Fatigue Fever over 38 °C Night sweats Lumbar pain Arthralgia
Male Female Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative
40 (41 56 (59 67 (70 29 (30 55 (57 41 (43 66 (69 30 (31 31 (32 65 (68 78 (81 18 (19
%) %) %) %) %) %) %) %) %) %) %) %)
Annexin A2 (ng/ml)
p-Value
7.07±9.8 7.3±10.3 7.9±10.4 5.7±9.1 7.5±10.4 6.8±9.5 6.0±9.4 9.8±11.0 6.0±9.4 7.8±10.3 6.5±9.7 10.4±10.9
0.890 0.307 0.727 0.109 0.419 0.131
in the diagnosis and monitoring of infectious diseases. Our literature survey did not reveal any study that evaluated ANXA2 levels in brucellosis. The present study examines the change in ANXA2 levels in brucellosis and the relationship of this change to spondylodiscitis. In our study, ANXA2 levels in patients with acute brucellosis were found to be higher than those of the control group, and this difference was statistically significant (Table 1, Fig. 1). This result was consistent with the results of other studies that demonstrated elevated ANXA2 levels in infectious diseases [18, 20, 21]. This increase in the patient group was associated with multiple cellular functions of ANXA2. When the relationship between ANXA2 levels and the distribution of patients according to gender and symptoms was evaluated, no statistically significant difference was found (Table 2). Brucellosis causes diagnostic and therapeutic challenges due to its similarities to other diseases regarding clinical presentation. Among the focal manifestations, osteoarticular involvement constitutes the majority of complications. Brucellar spondylodiscitis is a challenging diagnosis, particularly in low endemic areas; delayed diagnosis and inappropriate treatment may lead to prolonged morbidity [22]. In the studies conducted by Taşova et al. [23], Hashemi et al. [22], and Aydin et al. [24], the rate of brucellar spondylitis was found to be 13.8 %, 21.4 %, and 10 %, respectively, in cases with brucellosis. In our study, brucellar spondylodiscitis was detected in 18 (18.7 %) of 96 cases with acute brucellosis. ANXA2 levels of acute brucellosis cases with spondylodiscitis were higher than those without spondylodiscitis (p=0.001) (Table 3). Significantly higher ANXA2 levels, especially in patients with spondylodiscitis compared to other patients, were associated with the functions of this molecule in osteoclast formation, bone resorption, cell growth regulation, and apoptosis. In addition, ESR and CRP levels were also found to be higher in patients with brucellar spondylodiscitis compared to those without spondylitis (Table 3). Colmenero et al. [25] reported that ESR values were elevated in brucellosis patients with osteoarticular involvement. These studies in the literature, however, do not report any relationship between brucellar spondylodiscitis and CRP values [22, 25, 26]. Table 3 Serum CRP, ESR, and annexin A2 levels in patients with and without spondylitis Patients with Patients without p-Value spondylitis (n=18) spondylitis (n=78) ESR (mm/h) 41.2±19.9 CRP (mg/dl) 31.9±24.0 Annexin A2 (ng/dl) 14.3±10.3
26.2±19.4 19.5±18.7 5.6±9.2
ESR: erythrocyte sedimentation rate, CRP: C-reactive protein
0.004 0.018 0.001
Eur J Clin Microbiol Infect Dis
Based on our literature survey, our study is the first that has assessed ANXA2 levels in acute brucellosis. The limitations of our study include the following: the follow-up and end-oftreatment ANXA2 levels of acute brucellosis patients could not be evaluated; a cut-off level could not be determined for ANXA2; ANXA2 levels were not assessed on the molecular level but instead in serum; and blood culture could not be obtained from outpatients. In conclusion, it is believed that, together with ESR and CRP levels, ANXA2 levels that are shown to be elevated in infectious diseases may be indicative in the diagnosis of brucellar spondylodiscitis, a common complication in regions where brucellosis is an endemic disease. Acknowledgments The authors would like to express their gratitude to the Adiyaman University Biochemistry Laboratory staff. Authorship Nazlim Aktug Demir and Servet Kolgelier participated in obtaining the kits used in this study, collecting the data, and writing the paper. Sua Sumer, Ahmet Cagkan Inkaya, Serap Ozcimen, and Onur Ural collected the serum samples, stored the samples, interpreted the findings, and obtained the serum samples of the controls. Abdullah Arpaci investigated the biochemical parameters of the serum of the patients and the control group. Lutfi Saltuk Demir conducted the statistical evaluations. Funding support There is no financial or material support for this research and work. Financial disclosure The authors have no financial interests related to the material in the manuscript.
References 1. Pappas G, Akritidis N, Bosilkovski M, Tsianos E (2005) Brucellosis. N Engl J Med 352:2325–2336. doi:10.1056/NEJMra050570 2. Colmenero JD, Ruiz-Mesa JD, Plata A, Bermúdez P, Martín-Rico P, Queipo-Ortuño MI, Reguera JM (2008) Clinical findings, therapeutic approach, and outcome of brucellar vertebral osteomyelitis. Clin Infect Dis 46:426–433. doi:10.1086/525266 3. Zhao Y, Ben H, Qu S, Zhou X, Yan L, Xu B, Zhou S, Lou Q, Ye R, Zhou T, Yang P, Qu D (2010) Proteomic analysis of primary duck hepatocytes infected with duck hepatitis B virus. Proteome Sci 8:28. doi:10.1186/1477-5956-8-28 4. Mohammad HS, Kurokohchi K, Yoneyama H, Tokuda M, Morishita A, Jian G, Shi L, Murota M, Tani J, Kato K, Miyoshi H, Deguchi A, Himoto T, Usuki H, Wakabayashi H, Izuishi K, Suzuki Y, Iwama H, Deguchi K, Uchida N, Sabet EA, Arafa UA, Hassan AT, El-Sayed AA, Masaki T (2008) Annexin A2 expression and phosphorylation are upregulated in hepatocellular carcinoma. Int J Oncol 33:1157– 1163. doi:10.3892/ijo_00000105 5. Paciucci R, Torà M, Díaz VM, Real FX (1998) The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro. Oncogene 16:625–633 6. Emoto K, Sawada H, Yamada Y, Fujimoto H, Takahama Y, Ueno M, Takayama T, Uchida H, Kamada K, Naito A, Hirao S, Nakajima Y (2001) Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma. Anticancer Res 21:1339–1345
7. Guzmán-Aránguez A, Olmo N, Turnay J, Lecona E, Pérez-Ramos P, López de Silanes I, Lizarbe MA (2005) Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5. J Cell Biochem 94:178– 193. doi:10.1002/jcb.20293 8. Reeves SA, Chavez-Kappel C, Davis R, Rosenblum M, Israel MA (1992) Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma. Cancer Res 52: 6871–6876 9. Liu JW, Shen JJ, Tanzillo-Swarts A, Bhatia B, Maldonado CM, Person MD, Lau SS, Tang DG (2003) Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration. Oncogene 22:1475–1485. doi:10. 1038/sj.onc.1206196 10. Rodrigo Tapia JP, Pena Alonso E, García-Pedrero JM, Florentino Fresno M, Suárez Nieto C, Owen Morgan R, Fernández MP (2007) Annexin A2 expression in head and neck squamous cell carcinoma. Acta Otorrinolaringol Esp 58:257–262 11. Pena-Alonso E, Rodrigo JP, Parra IC, Pedrero JM, Meana MV, Nieto CS, Fresno MF, Morgan RO, Fernandez MP (2008) Annexin A2 localizes to the basal epithelial layer and is down-regulated in dysplasia and head and neck squamous cell carcinoma. Cancer Lett 263: 89–98. doi:10.1016/j.canlet.2007.12.029 12. Tobe T (2010) Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: role of EspL2 in adherence and an alternative pathway for modulating cytoskeleton through Annexin A2 function. FEBS J 277:2403–2408. doi:10.1111/j.17424658.2010.07654.x 13. Raynor CM, Wright JF, Waisman DM, Pryzdial EL (1999) Annexin II enhances cytomegalovirus binding and fusion to phospholipid membranes. Biochemistry 38:5089–5095. doi:10. 1021/bi982095b 14. Wright JF, Kurosky A, Pryzdial EL, Wasi S (1995) Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts. J Virol 69:4784–4791 15. Malhotra R, Ward M, Bright H, Priest R, Foster MR, Hurle M, Blair E, Bird M (2003) Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells. Microbes Infect 5: 123–133. doi:10.1016/S1286-4579(02)00079-5 16. Kirschnek S, Adams C, Gulbins E (2005) Annexin II is a novel receptor for Pseudomonas aeruginosa. Biochem Biophys Res Commun 327:900–906. doi:10.1016/j.bbrc.2004.12.089 17. Ma G, Greenwell-Wild T, Lei K, Jin W, Swisher J, Hardegen N, Wild CT, Wahl SM (2004) Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection. J Exp Med 200:1337–1346. doi:10.1084/jem.20041115 18. Swisher JF, Khatri U, Feldman GM (2007) Annexin A2 is a soluble mediator of macrophage activation. J Leukoc Biol 82:1174–1184. doi:10.1189/jlb.0307154 19. Pourbagher A, Pourbagher MA, Savas L, Turunc T, Demiroglu YZ, Erol I, Yalcintas D (2006) Epidemiologic, clinical, and imaging findings in brucellosis patients with osteoarticular involvement. AJR Am J Roentgenol 187:873– 880. doi:10.2214/AJR.05.1088 20. Römisch J, Schüler E, Bastian B, Bürger T, Dunkel FG, Schwinn A, Hartmann AA, Pâques EP (1992) Annexins I to VI: quantitative determination in different human cell types and in plasma after myocardial infarction. Blood Coagul Fibrinolysis 3:11–17 21. Ulvestad E, Kristoffersen EK, Jensen TS, Matre R (1994) Identification of a soluble Fc gamma-binding molecule (annexin II) in human serum using a competitive ELISA. APMIS 102:667–673 22. Hashemi SH, Keramat F, Ranjbar M, Mamani M, Farzam A, JamalOmidi S (2007) Osteoarticular complications of brucellosis in Hamedan, an endemic area in the west of Iran. Int J Infect Dis 11: 496–500. doi:10.1016/j.ijid.2007.01.008
Eur J Clin Microbiol Infect Dis 23. Ta şova Y, S alto ğ lu N, Sa hin G, Ak su HS ( 19 99 ) Osteoarthricular involvement of brucellosis in Turkey. Clin Rheumatol 18:214–219 24. Aydin M, Fuat Yapar A, Savas L, Reyhan M, Pourbagher A, Turunc TY, Ziya Demiroglu Y, Yologlu NA, Aktas A (2005) Scintigraphic findings in osteoarticular brucellosis. Nucl Med Commun 26:639–647
25. Colmenero JD, Reguera JM, Fernández-Nebro A, Cabrera-Franquelo F (1991) Osteoarticular complications of brucellosis. Ann Rheum Dis 50:23–26 26. Turan H, Serefhanoglu K, Karadeli E, Togan T, Arslan H (2011) Osteoarticular involvement among 202 brucellosis cases identified in Central Anatolia region of Turkey. Intern Med 50:421–428. doi:10. 2169/internalmedicine.50.4700
ARTICLE
Serum annexin A2 levels in acute brucellosis and brucellar spondylodiscitis N. Aktug Demir & S. Kolgelier & S. Sumer & A. C. Inkaya & S. Ozcimen & L. S. Demir & O. Ural & A. Arpaci
Received: 2 April 2014 / Accepted: 5 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Brucellosis is a chronic granulomatous infection and may present with various clinical manifestations. Brucellar spondylodiscitis symptoms are initially subtle and nonspecific. Annexin A2 (ANXA2) is involved in various biological functions, including osteoclast formation, bone resorption, and cell growth regulation. In this study, we aimed to determine the clinical significance of serum ANXA2 levels in acute brucellosis and brucellar spondylodiscitis. This prospective study included 96 acute brucellosis patients and 51 healthy controls. Acute brucellosis was diagnosed by a 1/160 or higher titer in a standard tube agglutination (STA) test or a four-fold increase in titers between two STA tests performed two weeks apart in the presence of clinical N. Aktug Demir : S. Sumer (*) : O. Ural Department of Infectious Diseases and Clinical Microbiology, Selcuk University, Faculty of Medicine, Konya, Turkey e-mail: [email protected] S. Sumer e-mail: [email protected] S. Kolgelier Department of Infectious Diseases and Clinical Microbiology, Adiyaman University, Faculty of Medicine, Adiyaman, Turkey A. C. Inkaya Department of Infectious Diseases and Clinical Microbiology, Hacettepe University, Faculty of Medicine, Ankara, Turkey S. Ozcimen Department of Infectious Diseases and Clinical Microbiology, Konya State Hospital, Konya, Turkey L. S. Demir Department of Public Health, Necmettin Erbakan University, Faculty of Medicine, Konya, Turkey A. Arpaci Department of Biochemistry, Adiyaman University, Faculty of Medicine, Adiyaman, Turkey
symptoms within the last eight weeks and/or growth of Brucella spp. in appropriately prepared culture media. ANXA2 levels were determined with an enzyme-linked immunosorbent assay (ELISA). Forty (41.7 %) of 96 acute brucellosis patients were male and 56 (58.3 %) were female. Serum ANXA2 levels were elevated in patients compared to healthy controls (p=0.001). Eighteen of 96 (18.7 %) acute brucellosis patients had brucellar spondylodiscitis. The serum ANXA2 levels of patients with brucellar spondylodiscitis were higher than those of patients with acute disease without brucellar spondylodiscitis (p=0.001). ANXA2, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) values were elevated in the brucellar spondylodiscitis group compared to patients without brucellar spondylodiscitis. Serum ANXA2 measurement together with ESR and CRP is thought to be indicative in the diagnosis of brucellar spondylodiscitis, a common complication of brucellosis.
Introduction Brucellosis is a chronic granulomatous infection caused by Brucella spp. and may lead to morbidity and loss of productivity. The prevalence of brucellosis is high in particular geographic areas in the world, like Mediterranean countries. Acute brucellosis may present with various clinical manifestations. Brucella spp. may persist for a long time and cause chronic complications [1, 2]. Brucellar spondylodiscitis is a common complication of brucellosis, affecting 10–21.4 % of patients. After the transmission of Brucella spp. was ingested by host cells, one-third of the invading organisms survive in phagolysosomes, which creates a unique site for bacterial growth and immune escape [1]. Annexins are a family of Ca2+/lipid-binding proteins that contain Ca2+-binding proteins. Twelve annexin subfamilies have been identified in vertebrates. Annexin A2 (ANXA2),
Eur J Clin Microbiol Infect Dis
also called annexin 2, is one of the best characterized in the group of annexins. ANXA2 is involved in various biological functions, including exocytosis, endocytosis, cell–cell adhesion, cell proliferation, cell surface fibrinolysis, osteoclast formation, bone resorption, cell growth regulation, and apoptosis [3]. Therefore, ANXA2 levels have been investigated in different disease groups, such as neoplasms and infections [4–18]. The aim of this study is to evaluate the serum ANXA2 levels in acute brucellosis and brucellar spondylodiscitis that may have a course with different clinical findings and complications.
Materials and methods Patients This study was carried out at the Infectious Disease and Clinical Microbiology Clinics of Adiyaman University, Faculty of Medicine, between March 2012 and February 2013. Ninety-six patients who were diagnosed with acute brucellosis and 51 healthy controls were included in this study. Inclusion criteria were age 18 years old or above at the time of the study initiation, not having a diagnosis of brucellosis previously or having treatment for the disease, not having immunosuppression, and not being pregnant. Ethics This work was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Approval was obtained from the ethical committee of Adiyaman University (2012/03-1.2). Informed consent was received from all patients involved in this study. Diagnosis of acute brucellosis Acute brucellosis was diagnosed by a 1/160 or higher titer in a standard tube agglutination (STA) test or a four-fold increase in titers between two STA tests performed two weeks apart in the presence of clinical symptoms (a compatible clinical picture, such as arthralgia, fever, sweating, chills, headache, and malaise) within the last eight weeks and/or growth of Brucella spp. in appropriately prepared culture media. The control group consisted of healthy individuals who had normal leukocyte counts, normal C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values, and a negative Brucella STA test. Contrast-enhanced lumbar magnetic resonance (MR) images were taken from the patients who were diagnosed with acute brucellosis and who had a complaint of lower lumbar
pain. The MR images were evaluated using the format defined by Pourbagher et al. [19]. Determination of ANXA2 All blood samples were drawn before the treatment was initiated. These blood samples were centrifuged for 5,000 cycles for 3 min to separate the serum. The serum samples were stored at −80 °C until the assays were performed. Serum ANXA2 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Uscn® E91944HU, Wuhan, Hubei, China). A total of 100 μL of sample or standard was placed in each tube, and the tubes were incubated for 2 h at 37 °C. Following the incubation, 100 μL of reagent A were added, and the tubes were incubated for another hour at 37 °C. They were washed three times, followed by the addition of 100 μL of reagent B and a third incubation for 30 min at 37 °C. Then, they were washed five times. Following another incubation at 37 °C with the addition of 90 μL of substrate, 50 μL of stop solution were added, and readings were taken immediately at 450 nm. Statistical analysis SPSS version 18.0 (Statistical Package for the Social Sciences, Chicago, IL) was used to analyze the data. The percentage distribution and mean standard deviation values were used as descriptive statistics. The Mann–Whitney U-test was used for analyzing the data. p-Values less than 0.05 were accepted as significant.
Results Forty (41.7 %) of the 96 acute brucellosis patients were male and 56 (58.3 %) were female. There were 25 male (49.0 %) and 26 (51.0 %) female volunteers in the control group. The mean age of the patient group was 40.9±15.4 years versus 33.8±9.3 years in the control group. Serum ANXA2 levels were elevated in the patients compared to the healthy controls (p=0.001) (Table 1, Fig. 1). Serum ANXA2 levels were not different between genders. There was no association between the patients’ complaints and ANXA2 levels (Table 2). Table 1 Serum annexin A2 levels in patients and control groups
Annexin A2 (ng/dl)*
Acute brucellosis patients (n=96)
Healthy controls (n=51)
p-Value
7.24±10.4
2.1±4.7
0.001
*Mean±standard deviation
Eur J Clin Microbiol Infect Dis
Fig. 1 Serum annexin A2 levels in the patient and control groups
Thirty-one (32 %) of the 96 patients with acute brucellosis had a complaint of lower lumbar pain (Table 2). Brucellar spondylodiscitis was found in 18 of the 31 patients with lower lumbar pain. Eighteen of 96 (18.7 %) acute brucellosis patients had brucellar spondylodiscitis. The serum ANXA2 levels of the patients with brucellar spondylodiscitis were higher than those patients with acute disease but without brucellar spondylodiscitis (p=0.001). ANXA2, ESR, and CRP values were elevated in the brucellar spondylodiscitis group compared to patients without brucellar spondylodiscitis (Table 3).
Discussion ANXA2 expression has been found to be up-regulated in several types of bacterial and viral infections [12–18]. However, this change in ANXA2 levels is known to be important Table 2 Serum annexin A2 levels with respect to the gender and symptoms of patients n (%)
Gender Fatigue Fever over 38 °C Night sweats Lumbar pain Arthralgia
Male Female Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative
40 (41 56 (59 67 (70 29 (30 55 (57 41 (43 66 (69 30 (31 31 (32 65 (68 78 (81 18 (19
%) %) %) %) %) %) %) %) %) %) %) %)
Annexin A2 (ng/ml)
p-Value
7.07±9.8 7.3±10.3 7.9±10.4 5.7±9.1 7.5±10.4 6.8±9.5 6.0±9.4 9.8±11.0 6.0±9.4 7.8±10.3 6.5±9.7 10.4±10.9
0.890 0.307 0.727 0.109 0.419 0.131
in the diagnosis and monitoring of infectious diseases. Our literature survey did not reveal any study that evaluated ANXA2 levels in brucellosis. The present study examines the change in ANXA2 levels in brucellosis and the relationship of this change to spondylodiscitis. In our study, ANXA2 levels in patients with acute brucellosis were found to be higher than those of the control group, and this difference was statistically significant (Table 1, Fig. 1). This result was consistent with the results of other studies that demonstrated elevated ANXA2 levels in infectious diseases [18, 20, 21]. This increase in the patient group was associated with multiple cellular functions of ANXA2. When the relationship between ANXA2 levels and the distribution of patients according to gender and symptoms was evaluated, no statistically significant difference was found (Table 2). Brucellosis causes diagnostic and therapeutic challenges due to its similarities to other diseases regarding clinical presentation. Among the focal manifestations, osteoarticular involvement constitutes the majority of complications. Brucellar spondylodiscitis is a challenging diagnosis, particularly in low endemic areas; delayed diagnosis and inappropriate treatment may lead to prolonged morbidity [22]. In the studies conducted by Taşova et al. [23], Hashemi et al. [22], and Aydin et al. [24], the rate of brucellar spondylitis was found to be 13.8 %, 21.4 %, and 10 %, respectively, in cases with brucellosis. In our study, brucellar spondylodiscitis was detected in 18 (18.7 %) of 96 cases with acute brucellosis. ANXA2 levels of acute brucellosis cases with spondylodiscitis were higher than those without spondylodiscitis (p=0.001) (Table 3). Significantly higher ANXA2 levels, especially in patients with spondylodiscitis compared to other patients, were associated with the functions of this molecule in osteoclast formation, bone resorption, cell growth regulation, and apoptosis. In addition, ESR and CRP levels were also found to be higher in patients with brucellar spondylodiscitis compared to those without spondylitis (Table 3). Colmenero et al. [25] reported that ESR values were elevated in brucellosis patients with osteoarticular involvement. These studies in the literature, however, do not report any relationship between brucellar spondylodiscitis and CRP values [22, 25, 26]. Table 3 Serum CRP, ESR, and annexin A2 levels in patients with and without spondylitis Patients with Patients without p-Value spondylitis (n=18) spondylitis (n=78) ESR (mm/h) 41.2±19.9 CRP (mg/dl) 31.9±24.0 Annexin A2 (ng/dl) 14.3±10.3
26.2±19.4 19.5±18.7 5.6±9.2
ESR: erythrocyte sedimentation rate, CRP: C-reactive protein
0.004 0.018 0.001
Eur J Clin Microbiol Infect Dis
Based on our literature survey, our study is the first that has assessed ANXA2 levels in acute brucellosis. The limitations of our study include the following: the follow-up and end-oftreatment ANXA2 levels of acute brucellosis patients could not be evaluated; a cut-off level could not be determined for ANXA2; ANXA2 levels were not assessed on the molecular level but instead in serum; and blood culture could not be obtained from outpatients. In conclusion, it is believed that, together with ESR and CRP levels, ANXA2 levels that are shown to be elevated in infectious diseases may be indicative in the diagnosis of brucellar spondylodiscitis, a common complication in regions where brucellosis is an endemic disease. Acknowledgments The authors would like to express their gratitude to the Adiyaman University Biochemistry Laboratory staff. Authorship Nazlim Aktug Demir and Servet Kolgelier participated in obtaining the kits used in this study, collecting the data, and writing the paper. Sua Sumer, Ahmet Cagkan Inkaya, Serap Ozcimen, and Onur Ural collected the serum samples, stored the samples, interpreted the findings, and obtained the serum samples of the controls. Abdullah Arpaci investigated the biochemical parameters of the serum of the patients and the control group. Lutfi Saltuk Demir conducted the statistical evaluations. Funding support There is no financial or material support for this research and work. Financial disclosure The authors have no financial interests related to the material in the manuscript.
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