Ultrasound Assessment of Entheses and Correlations Between Disease Activity and Ultrasound Scores in Ankylosing Spondylitis: A Cross Sectional Study


 Background: An increasing number of studies have applied ultrasound (US) to evaluate enthesitis in spondyloarthritis. However, there is no clear agreement on which sites should be evaluated for enthesitis. Furthermore, there are different opinions on whether US can monitor disease activity. The objectives of this study were to evaluate the common involvement of entheses and correlations between disease activity and US scores in ankylosing spondylitis (AS).Methods: A cross-sectional, monocentric, and controlled study was performed. US was used to scan 34 entheses per person and the Madrid sonography enthesitis index (MASEI) score was used. US scores were used to evaluate the elemental lesions of hypoechogenicity, thickness, erosion, calcification, bursitis, and Doppler signal. Disease activity was assessed by Disease Activity Score–C reactive protein (ASDAS-CRP) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI).Results: 104 patients with AS and 50 control subjects were included. A total of 5236 entheses were assessed. Compared with the control subjects, only three entheses – the Achilles tendon (AT), sternoclavicular joint (SCJ), and costochondral joint (CCJ) – showed significant differences in the AS group (p < 0.05). Patients with high disease activity (ASDAS-CRP ≥ 2.1) had higher scores than patients with low activity (ASDAS-CRP < 2.1) in erosion (p = 0.02). Patients who were categorized in very high disease activity (ASDAS-CRP ≥ 3.5) had a higher score in erosion (p = 0.02). The erosion score was moderately associated with ASDAS-CRP and BASDAI (r = 0.44, r = 0.21).Conclusions: The commonly involved entheses were the AT, SCJ, and CCJ in AS. Erosion occurred more often in patients with disease activity or high activity. Erosion would be more useful for monitoring disease activity in AS.


Background
Enthesitis is a characteristic sign of ankylosing spondylitis (AS). It has been validated that enthesitis is important for the diagnosis of AS and monitoring disease activity [1]. AS can cause physical damage and disability, declining the patient's quality of life and bringing about a nancial burden to both their families and society as a whole [2,3]. The prevalence of AS ranges from 0.1-0.9% [4]. Early diagnosis and treatment can decrease the probability of disability and improve the outcome. However, the diagnosis of AS was usually delayed by an average of 6-9 years after the onset of clinical symptoms [5,6].
X-rays are still the rst line of investigation for the evaluation of enthesitis, although the changes may occur in the late phase [7]. Computed tomography (CT) can demonstrate bone erosion, but it is limited by radiation exposure and low resolution for soft tissue. Magnetic resonance imaging (MRI) has been considered more valuable, as it can reveal bone edema and soft tissue swelling. Nonetheless, MRI is limited by its availability and high cost. Additionally, it cannot assess many entheses due to it being a time-consuming process. Ultrasound (US) has many advantages, such as being cheap, portable, nonradioactive, done in real-time, and can be easily compared to the opposite side. It is becoming increasingly accepted by rheumatologists as a suitable method. Also, some studies reported that US can evaluate enthesitis for disease diagnosis, even in the early stages of spondyloarthritis [8,9].
Recently, more studies have applied US to evaluate enthesitis in spondyloarthritis, where the main type of spondyloarthritis is AS [8][9][10][11]. Nowadays the standardized de nition of US enthesitis is in accordance with the de nition given by Outcome Measures in Rheumatology (OMERACT) [12]. However, there is no clear agreement on which sites should be evaluated for enthesitis. Most studies have focused on the lower limbs [11,13,14], while some others investigated the upper limbs [15,16]. However, few articles pay attention to the anterior chest wall (ACW) [17]. The ACW has been demonstrated as the second most commonly involved site in spondyloarthritis just behind the sacroiliac joint [18]. Furthermore, there are different opinions on whether US can monitor disease activity [19][20][21]. More studies are needed to assess whether disease activity is correlated with US elemental lesions.
The main objective of the present study was to evaluate the common involvement entheses in AS, including the upper limbs, lower limbs, trunk, and ACW. Second, the correlations between disease activity and US scores were assessed. Further study is needed to determine whether enthesis US can monitor disease activity in AS.

Methods
Patients and study design. This was a cross-sectional study with health control. The study was performed following the Declaration of Helsinki principles and local regulations. The approval was permitted by the ethical committee of Peking University Shenzhen Hospital. All patients and healthy individuals signed informed consent.
Between December 2017 and December 2019, the study nally enrolled 104 patients with AS who ful lled the modi ed New York criteria [22]. All the patients were consecutively enrolled from the Department of Rheumatology and Immunology at Peking University Shenzhen Hospital.
The exclusion criteria included the following: a history of joint or ACW surgery; corticoid injection at the enthesis within the last six weeks; peripheral neuropathy; age <18 years; diseases other than AS; and an unwillingness to sign for consent, or did not nish all the examinations or patient-reported outcome measures (PROMs). If patients met one of the above conditions, they were excluded.

Controls.
Fifty healthy persons were matched with AS patients in age, gender, and body mass index (BMI). They were selected among the hospital workers and healthy volunteers who did not have any symptoms at the entheses, without the diagnosis of spondyloarthritis or family history.

Data collection.
Data was collected by PROMs, laboratory data, and US assessments.
PROMs and laboratory data. All patients were examined by a quali ed rheumatologist. The rheumatologist used PROMs. For each patient, the laboratory tests consisted of C reactive protein (CRP) and human leukocyte antigen B27 (HLA-B27). Disease activity was assessed by Disease Activity Score-CRP (ASDAS-CRP) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). The interval between each examination was less than one week. The ASDAS-CRP value (V ASDAS-CRP ) indicated the disease activity of AS. When the value was <1.3, the disease was inactive. When the value was 1.3 ≤ V ASDAS-CRP < 2.1, the disease activity was low. When the index was 2.1 ≤ V ASDAS-CRP < 3.5, the disease activity was high. When the index was ≥3.5, the disease activity was very high. BASDAI was a selfassessment form to measure disease activity. It had six questions, and each question was scored from 0 to 10. When the overall score was ≥4, it indicated disease activity. Otherwise, it indicated disease inactivity. A higher score re ected a more active disease. US assessment. Ultrasonography was performed using Toshiba Aplio 400 equipment with a linear transducer with a frequency of 7-18 MHz by an experienced sonographer who had taken part in musculoskeletal US for more than ve years. All subjects were examined by grey scale and power Doppler ultrasonography. Doppler settings were standard with a pulse repetition frequency of 1000 Hz, low wall lter, and Doppler gain at a level of just below random noise. The sonographer was blinded to clinical information, and people were advised not to talk with the US examiner. US de nition. Elemental US lesions of enthesitis were as follows ( Figure 1): hypoechogenicity with lack of the normal homogeneous brillar pattern; increased thickness measured at the insertion of 2 mm near the bone cortex, and the data followed Glasgow Ultrasound Enthesitis Scoring System (GUESS) [11]; erosion with cortical breakage with contour defect in both longitudinal and transverse planes; calci cation: hyperechoic foci or bony prominence at the end of the bone contour, with or without acoustic shadow; bursitis: the locations of bursitis consisted of suprapatellar, infrapatellar, and retrocalcaneal bursas, with the normal anteroposterior diameter less than 5 mm, 2 mm, and 3 mm, respectively [23]; and Doppler signal at the insertion 2 mm near the bone surface with the tendon or ligament at a relaxation position. Enthesis US scores followed the MASEI: calci cation (0-3), erosion (0 or 3), Doppler signal (0 or 3), hypoechogenicity (0-1), increased thickness (0-1), and bursitis (0-1). The US images were read by two sonographers. If there was a difference in opinion, a third sonographer would help to make a decision. A de nition of enthesis US positive was at least one abnormal enthesis in one site. Acute echogenicity consisted of hypoechogenicity, increased thickness, bursitis, and Doppler signal. Chronic echogenicity included erosion and calci cation.
Statistical analysis.
The quantitative data were shown by mean ± standard deviation (SD), and the qualitative data were shown by percentages.

Demographic and clinical data
With a total of 247 potential AS patients, the study nally enrolled 104 patients with AS in the end ( Figure  2). In addition, 50 control subjects were included. There were 34 entheses by US assessment in each person ( Figure 3a). Thus, a total of 5236 entheses were evaluated, consisting of 3536 sites in patients with AS and 1700 sites in the controls. The AS and control groups matched in age, gender, and BMI. The demographic and clinical data were summarized in Table 1 (Table 2). However, compared with the control group, only the three entheses of AT, SCJ, and CCJ showed signi cant differences in the AS group (p < 0.05), as seen in Figure  3b. The AT and ACW were the common involvement sites in AS.

Comparing enthesis US scores in different groups assessed by ASDAS-CRP and BASDAI
In contrast to the control group, the enthesis US scores of MASEI, hypoechogenicity, thickness, erosion, Doppler signal, and acute and chronic echogenicity were higher, except for calci cation and bursitis in the AS group, as presented in Table 3.
The enthesis US scores did not show signi cant differences between patients with disease activity (ASDAS-CRP ≥ 1.3) and patients with disease inactivity (ASDAS-CRP < 1.3), as seen in Table S1 (the supplemental le). In addition, patients with high disease activity (ASDAS-CRP ≥ 2.1) had higher scores compared to patients with low activity (ASDAS-CRP < 2.1) in erosion and bursitis (p = 0.02, p = 0.04).
Patients who were categorized as having very high disease activity (ASDAS-CRP ≥ 3.5) had a higher score in erosion (p = 0.02).

Correlations between disease activity and US scores in AS
The disease activity was assessed by ASDAS-CRP and BASDAI, and the erosion score was associated with both of them ( Figure 3c). However, the calci cation score did not correlate with them. Moreover, all of the enthesis US scores except the thickness score had signi cant associations with the ASDAS-CRP. The range of correlation coe cient was from 0.22 to 0.44 (Figures S1 and S2 in the supplemental le).

Discussion
It has been validated that US is a useful tool for enthesitis in AS, especially within the last 20 years. An increasing number of rheumatologists prefer to assess peripheral enthesitis by US, which has the advantages of being readily available, inexpensive, and non-radioactive, while also exhibiting the bene ts of having high resolution and bedside convenience. To disseminate this technique in daily work, the standardization of enthesis US should be considered, which would be helpful for further multicenter studies. An international group of OMERACT US experts was formed in 2005. This group aimed to standardize the de nition of US elemental lesions for enthesitis [24]. However, there was no determined consensus about which sites should be examined.
The authors observed a total of 5236 entheses in the present study. So far, it was the largest number of sites. More entheseal areas were evaluated and more information was gathered. In the present study, the entheses included the upper limbs, lower limbs, trunk, and ACW, whereas most studies have only focused on the upper and lower limbs [13][14][15]. The ndings of the present study have shown that the three entheses of AT, SCJ, and CCJ were more common in the AS group compared to the control group. The SCJ and CCJ belonged to the ACW, suggesting that enthesis US should assess the sites of the ACW. Furthermore, several studies reported that the rate of ACW involvement was 30-50% in spondyloarthritis [25,26], and an article from the DESIR cohort revealed 44% in clinical involvement with the ACW in early spondyloarthritis [27]. Therefore, the ACW was also observed in addition to the upper and lower limbs. The authors' previous studies found that the ACW sites were the common entheses in AS by US assessment. Thus, the ACW should be considered to evaluate enthesitis in patients with AS.
Monitoring disease activity remains a challenge in AS, because each method is not fully evaluable. In clinical practice, there are a lot of methods for disease activity assessment, including physical examination, laboratory tests (CRP), composite index (BASDAI or ASDAS), or imaging examinations (Xrays, CT, MRI, or US). US has been proven to be a valuable tool for monitoring disease activity and therapeutic effect evaluation [12,20,28]. However, one study from the DESIR cohort found that US could not be helpful for monitoring disease activity in 402 patients with spondyloarthritis [21]. Therefore, further studies still need to be conducted to demonstrate the association between US lesions and disease activity. Patients with high disease activity (ASDAS-CRP ≥ 2.1) or very high disease activity (ASDAS-CRP ≥ 3.5) had a higher score in erosion. It represented that erosion could occur more often in patients with disease activity or high activity. In particular, erosion seems to be an indicator for enthesis US in disease Even so, there were still some limitations in the present study. First, the included patients were all inpatients, and most of them were male. As such, there were selection and gender biases. More female patients should have been recruited from the clinics. Second, the disease activity index did not include physical examination because of the low sensitivity and speci city. Third, US scanning was time consuming. Many sites throughout the entire body were investigated for enthesitis to nd which entheses were more involved. The next step simpli ed the entheses.

Conclusions
In conclusion, the common involvement entheses were the AT, SCJ, and CCJ in patients with AS, which suggested that the AT and ACW should be evaluated by enthesis US. Erosion occurred more often in patients with disease activity or high activity. More research is needed to determine if erosion can be implemented to monitor disease activity in AS.

Declarations
Ethics approval: The study was approved by the Ethics Committee of Peking University Shenzhen Hospital, with approval ID 2020030.
Patient consent: Not applicable.
Availability of data and materials: The datasets used and analyzed during the current study were available from the corresponding authors upon reasonable request.
Competing interests: The authors declare that they have no competing interests. Author contributions QW and LL conceived and designed the study. QD and YZ read the ultrasound images. HL did the ultrasound examination. GZ asked about patient-reported outcome measures and JL extracted the clinical data from the medical les in the information system. WZ and JZ extracted data from the laboratory system. HX and BW wrote the paper. HL and DS analyzed the data. All the authors have read and approved the manuscript.

Additional Material
Additional le Table S1: Comparing enthesis US scores in different groups assessed by ASDAS-CRP and BASDAI.
Additional le Figure