The diagnostic value of soluble urokinase-type plasminogen activator receptor (suPAR) for the discrimination of vertebral osteomyelitis and degenerative diseases of the spine

Vertebral osteomyelitis is a primary infection of the end-plates of the vertebral bodies with secondary infection of the adjacent intervertebral discs [1]. Concomitant abscesses are detected in about a third of the patients, potentially leading to neurological deficits at a rate of approximately 20% [2, 3]. The overall incidence rate of vertebral osteomyelitis increased from 0.5 cases per 100,000 person years 1978–1982 to 2.2 in 1995 and 5.8 in 2008. It is most common among older persons with a higher incidence among men [3‐5]. Clinical symptoms, especially in the early stages, are unspecific. Patients suffer from back pain, and fever occurs only in 50% of all cases [6]. Given that current markers including leucocyte count, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are also unspecific, several weeks may elapse between the first symptoms and the final diagnosis of vertebral osteomyelitis [3, 7].

Vertebral osteomyelitis is primarily caused by hematogenous seeding leading to monomicrobial infections. Staphylococcus aureus is most frequently isolated followed by streptococci species and Escherichia coli [2, 8] while coagulase-negative staphylococci are more often found after spinal surgery [9]. Nevertheless, worldwide vertebral osteomyelitis is mostly caused by Mycobacterium tuberculosis, and brucellosis is more frequently found than pyogenic infection in the Mediterranean and Middle East countries [10].

To identify the pathogens for an effective therapy tailored to the causative agent, blood cultures, computed tomography (CT)-guided fine-needle aspiration or open biopsies [11, 12] may be needed. Nevertheless, also due to previous antibiotic treatment the pathogen can only be identified in approximately two thirds of the patients [5, 13]. Magnetic resonance imaging (MRI) is the gold standard of imaging to detect vertebral osteomyelitis [14].

Treatment of an advanced vertebral osteomyelitis consists of removal of the necrotic tissue, stabilization of the affected vertebral bodies and concomitant antibiotic therapy [15]. Currently, there are different recommendations for the duration of antibiotic treatments but 6 weeks were shown to be suitable [16]. For evaluating the therapy response, clinical improvement and the CRP value are used. Nevertheless, due to the low specificity of CRP, new biomarkers are needed for improvement of diagnosis and treatment monitoring to prevent long periods with symptoms and destructive changes of the spine.

The urokinase plasminogen activator (uPA) is a proteolytic enzyme, which converts the proenzyme plasminogen to the active serine protease plasmin [17]. The urokinase-type plasminogen activator receptor (uPAR) is a glycoprotein, which is expressed on various immunologically active cells, and is released during inflammation and infection. uPAR is cleaved from the cell surface by proteolysis to produce the soluble urokinase-type plasminogen activator receptor (suPAR), which can be found in urine, blood, and cerebrospinal fluid [18]. The suPAR levels are low in healthy patients [17, 19] while levels are significantly increased during immune activation [20, 21]. A recent report showed that suPAR correlated highly with the C-reactive protein (CRP) in patients with prosthetic joint infection [21].

Our goal was to establish a non-invasive method, which allows discrimination of vertebral osteomyelitis and degenerative diseases of the spine. The potential of such a diagnostic method lies in the reduction of morbidity and mortality due to vertebral osteomyelitis and reducing medical costs. To this end, blood samples from patients with vertebral osteomyelitis or erosive osteochondrosis (a non-infectious, degenerative disease of the spine with similar surgical treatment as vertebral osteomyelitis) were collected and analyzed for suPAR levels.

To determine the suitability of suPAR for vertebral osteomyelitis diagnosis, the suPAR concentrations in serum from vertebral osteomyelitis patients (n = 16) and from a control group with erosive osteochondrosis (n = 20) were measured pre-OP and post-OP (3–5 days, 6–11 days, 40–56 days, and 63–142 days). The suPAR and CRP concentrations were compared at each interval within each group.

Due to variations of more than 20% between the duplicate measurements for suPAR, 6 values were excluded 3-5 days post-OP from control patients 29, 30, 34, 37, 38, and 39 and because of the lack of a valid corresponding CRP value, the 6–11 days post-OP value of control patient 32 was excluded. Mean values of suPAR concentrations ranged from 3.61 ± 0.33 (3–5 days post-OP) to 4.78 ± 0.54 ng/mL (40–56 days post-OP) in vertebral osteomyelitis patients while these values were 2.65 ± 0.22 (pre-OP) to 3.79 ± 0.28 ng/mL (40–56 days post-OP) in controls (Fig. 1a). Generally, within the same interval, suPAR values from vertebral osteomyelitis patients were higher than those from controls (pre-OP, p = 0.0041; 3–5 days post-OP, p = 0.0402; 6–11 days post-OP, p = 0.0060; 40–56 days post-OP, p = 0.1192; 63–142 days post-OP, p = 0.0744) and were, therefore, significantly different from each other pre-OP, 3–5 days post-OP, and 6–11 days post-OP. Over all post-OP intervals, differences between the vertebral osteomyelitis group and the controls were significant (p = 0.0167).

The CRP values for both patient groups are shown in Fig. 1b. In the vertebral osteomyelitis patients, the CRP concentration was 75.75 ± 24.44 mg/L pre-OP and increased to 102.73 ± 10.84 mg/L 3–5 days post-OP, decreasing continuously until the end of the study to 9.29 ± 3.08 mg/L. A similar pattern was observed for the controls. Concentrations increased from 3.49 ± 0.90 mg/L pre-OP to 112.99 ± 13.06 mg/L 3–5 days post-OP and decreased to 3.45 ± 0.82 mg/L 40–56 days post-OP and 3.8 ± 0.91 mg/L 63–142 days post-OP. Significantly higher CRP values were observed in the vertebral osteomyelitis group than in the controls pre-OP (p = 0.0098) and 6–11 days post-OP (p = 0.048). Over all post-OP intervals, differences between the vertebral osteomyelitis group and the controls were significant (p = 0.0490).

Measurements for suPAR and CRP were positively correlated in the vertebral osteomyelitis group in the pre-OP period, r = 0.55 (95% CI: 0.07–0.82), p = 0.023, and in all patients in the pre-OP period, r = 0.63 (95% CI: 0.37–0.79), p < 0.001 and 6–11 days post-OP, r = 0.45 (95% CI: 0.13–0.68), p = 0.0059. However, for the overall post-OP period, the suPAR and CRP correlation was positive but not significant; r = 0.39 (95% CI: − 0.04 to 0.68), p = 0.0688. In the controls pre-OP as well as overall post-OP, the suPAR and CRP correlations were not significant, pre-OP: r = − 0.06 (95% CI: − 0.49–0.40), p = 0.8082 and post-OP: r = 0.14 (95% CI: − 0.48 to 0.66), p = 0.6706.

Figure 2 summarizes the main findings of the logistic regression analyses for suPAR and CRP stratified by interval and overall post-OP for all intervals post-OP. Figure 2a and Fig. 2b show the odds ratios together with their 95% CI by interval and overall post-OP for suPAR and CRP, respectively. Logistic regression of patient status with respect to suPAR as well as CRP and adjusted for sex reveals a significant predictive potential of these parameters for diagnosis of vertebral osteomyelitis for both pre-OP as well as for post-OP overall. For example, the odds ratio in the univariate logistic regression for suPAR pre-OP is 2.46 (95% CI: 1.27–4.76), p = 0.0078. This means that the odds of developing vertebral osteomyelitis increases by the factor 2.46 per change in the suPAR measurement by 1 ng/mL. Adjusting for the sex of the patients increases the odds ratio per ng/mL to 2.92 (95% CI: 1.34–6.38), p = 0.0071. Likewise, the odds ratio in the univariate logistic regression for CRP pre-OP is 1.22 (95% CI: 1.02–1.47), p = 0.0278. This means that the odds of developing vertebral osteomyelitis increases by the factor 1.22 (i.e., 22% increase) per change in the CRP measurement by 1 mg/L. Adjusting for the sex of the patients increased the odds ratio slightly to 1.24 (95% CI: 1.02–1.5), p = 0.0281.

The accuracy of a diagnostic test depends on how well the test separates the group being tested into those with and without the disease or condition in question. Receiver operating characteristics (ROC) curve analysis revealed that the values for the AUC based on logistic regression of patient status with respect to suPAR and CRP measurements and adjusted for sex were 0.88 (95% CI: 0.76–1.00) and 0.93 (95% CI: 0.85–1.00) for pre-OP, and 0.84 (95% CI: 0.71–0.97) and 0.77 (95% CI: 0.62–0.93) for the overall model post-OP, respectively, as shown in Fig. 3 and Table 5. The AUC based on logistic regression for the combination of suPAR and CRP and likewise adjusted for sex showed higher results both in the pre-OP, 0.98 (95% CI: 0.96–1.00), as well as in the overall post-OP period, 0.91 (95% CI: 0.82–1.00). The cut-off levels, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for suPAR and CRP for diagnosis of vertebral osteomyelitis are shown in Table 5.

Current diagnostic methods for vertebral osteomyelitis are based on structural changes in the spine, delaying early diagnosis and treatment. To the best of our knowledge, this is the first study to explore the potential of suPAR for differentiating between vertebral osteomyelitis and degenerative diseases of the spine. Microbiological analyses are necessary to identify the causative pathogen. Notably, the current results show that suPAR is a suitable adjunct biomarker to CRP for diagnosing vertebral osteomyelitis. Furthermore, the potential for diagnosing vertebral osteomyelitis before surgery was higher with CRP than with suPAR, the latter showing a higher specificity. The diagnostic potential of the combination of both biomarkers was superior to the use of the single biomarkers prior to surgery as well as in the post-OP period.

To date, there is only one report about suPAR concentrations and diseases of the spine [22]. Toldi et al. found plasma suPAR levels of 2.57 to 3.80 ng/mL in patients suffering from ankylosing spondylitis and 2.06 to 3.42 ng/mL in healthy patients, therefore showing no significant differences between both groups. In contrast, in the present study, suPAR values were significantly higher in vertebral osteomyelitis patients ranging from 3.61 to 4.78 ng/mL compared to 2.65 to 3.79 ng/mL in controls. The differences in the results obtained by Toldi et al. and our results for vertebral osteomyelitis could be because ankylosing spondylitis is an immune-mediated rheumatoid disease resulting in chronic inflammation in the vertebrae with systemic manifestations at a later stage of this mild disease [22].

In the present study, a significant positive correlation between suPAR and CRP was found only prior to surgery in the vertebral osteomyelitis group. A positive correlation between suPAR and CRP was also reported for critically ill intensive care patients with or without sepsis [23] and prosthetic joint infection [21]. However, none was found in patients with rheumatic diseases [24], pneumococcal bacteraemia [25], and severe sepsis [26]. Therefore, the mostly weak and insignificant correlations between suPAR and CRP in the post-OP intervals in the vertebral osteomyelitis group in the current study are consistent with the latter reports.

In the plasma of healthy humans, suPAR is found in low constant concentrations [17, 19]. Increased suPAR levels were found in several bacterial diseases including bacteraemia [25, 27‐30], sepsis [26, 31], tuberculosis [31], purulent meningitis [32], and prosthetic joint infections [21]. Previous reports show that suPAR levels approximated 1.0 to 20.0 ng/mL in patients with different infections [21, 22, 33, 34]. The suPAR levels were summarized by Eugen-Olsen [20] to be < 4 ng/mL in healthy, > 4 < 10 ng/mL for low-grade inflammation, and > 10 ng/mL for critical illness. The suPAR levels determined in the present study averaged 3.61 to 4.78 ng/mL in the vertebral osteomyelitis patients while concentrations in the control group were 2.65 to 3.79 ng/mL. According to the classification of Eugen-Olsen for suPAR, vertebral osteomyelitis in our patient cohort can be considered a low-grade infection.

Cut-off levels for suPAR may be used for diagnosis but this approach would depend on the patient cohort and disease of concern. Cut-off levels, sensitivities, and specificities, respectively, were reported to be 10.0 ng/mL, 0.38, and 0.95 for diagnosis of Streptococcus pneumoniae bacteraemia [25], 2.7 ng/mL, 0.35, and 0.67 for diagnosis of bacterial infection in SIRS patients [35], and 2.96 ng/mL, 0.69 and 1.00 in the present study. Therefore, suPAR measurement may be useful in monitoring the therapy response in patients. After 8 months of treatment for tuberculosis in patients with and without HIV, suPAR levels decreased significantly by 0.56 to 2.07 ng/mL among sputum-positive patients, levels being comparable to those of tuberculosis-negative patients [31]. Ostrowski et al. also reported a decrease in plasma suPAR 1 year after the induction of therapy in patients suffering from HIV who had a high baseline suPAR level [36]. Significant decreases in suPAR levels were also observed subsequent to a 4-to-7 day antimicrobial therapy for SIRS in children [37]. In the present study, the vertebral osteomyelitis patients received antibiotics perioperatively and post-OP. In contrast to the CRP levels, which decreased with time, no significant decrease was observed in the suPAR levels in both groups throughout the study. To the authors’ knowledge, there are no reports concerning the mechanism responsible for this observation. Since two of the abovementioned studies also revealed decreasing suPAR values within longer periods of follow-up, notably 8 months to 1 year [31, 36], it is possible that the duration of the present study of up to 5 months was insufficient to observe decreasing suPAR concentrations. Therefore, the present data show that monitoring of the therapy success can be performed using CRP but not suPAR.

Specific inflammation parameters are needed in the diagnostic work-up and evaluation of treatment success of vertebral osteomyelitis, especially in cases with low-virulent causative agents where CRP values are normal or low. A greater challenge is posed because the CRP value alone is not always helpful to distinguish between vertebral osteomyelitis and degenerative diseases of the spine. As shown in the present study, suPAR is only elevated in vertebral osteomyelitis patients and therefore is a specific biomarker for differentiating between vertebral osteomyelitis and degenerative diseases of the spine pre-operatively. Therefore, in difficult cases, additional specific parameters such as suPAR are needed to determine the pre- and intra-operative diagnostic pathways. Notably, the significantly different concentrations of suPAR in the patients with vertebral osteomyelitis compared to the control patients shortly after surgery could reveal a potential use of suPAR in diagnosing the infection since CRP is of limited use for this purpose also due to the strong influence of surgery on the non-specific CRP concentration [38, 39].

There are many strengths of the present study. We were able to do a 5-month follow-up with 5 intervals in patients, thus increasing the impact of the study. The suPAR assay employed in the present study is a double monoclonal antibody sandwich assay, which measures all circulating suPAR including full-length and cleaved forms of the receptor. Furthermore, suPAR is highly stable in serum and plasma for 24 h at room temperature [40, 41] or 72 h at 4 °C [40] and is not affected by circadian rhythm [42], repeated freeze-thaw cycles [40, 41] nor surgery [43, 44]. The latter results were also confirmed in the present study for suPAR, in contrast to CRP, where CRP values were comparable for both groups 3-5 days post-OP.

However, there are some limitations of this study. Due to the fact that suPAR levels are also elevated due to co-morbidities, some of which have been mentioned above, it is considered a non-specific biomarker. Since suPAR concentrations may remain stable for a long period after treatment, as mentioned above, it may not be a suitable marker for monitoring the therapy success. Even though the patients’ co-morbidities were reported consistently, the influence of possible undetected diseases on suPAR levels cannot be excluded which may have an impact on the mean values because of the relatively small number of patients included in this single-center study. Furthermore, there is a certain form of erosive osteochondrosis (MODIC Type 1), which has an immunological active character [45, 46]. As it is not investigated yet, the effect of this form on the suPAR concentrations remains unclear and should be part of further studies. Therefore, control patients should be examined by imaging as was done with the vertebral osteomyelitis group.

Our results show that suPAR is more specific than CRP whereas CRP is more sensitive than suPAR for discrimination of vertebral osteomyelitis and degenerative diseases of the spine. Furthermore, improvement in the diagnostic potential can be achieved by a combination of both suPAR and CRP. Also, the present study reveals a potential use of suPAR as a biomarker for detection of post-operative infections and therefore, opportunities for further research.