1.       Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive damage of synovial-lined joints and variable extra-articular manifestations [1]. The therapeutic approach provides an increasingly early and aggressive treatment as it is evident that an early intervention can reduce or stop the progression of the disease [2]. There are numerous drugs available for the treatment of RA and include conventional synthetic and biologic Disease Modifying Anti-Rheumatic Drugs (csDMARDs and bDMARDs). Early treatment, the availability of biologic therapies and treat-to-target strategies have made the remission a realistic goal in RA [3-7]. However, most patients are still on treatment with only csDMARDs, both because they have obtained a good clinical response to this therapy so that a biological therapy is not considered necessary or because they have contraindications for biological agents (i.e. infectious diseases, cancer) [8].

The sDMARDs continue to be the first-line treatment for both the favorable risk-benefit profile and the pharmacoeconomic aspect and in many patients, are sufficient to achieve remission [9]. Accurate assessment of disease activity and joint damage in RA is important for monitoring treatment efficacy and for prediction of the outcome of the disease [10,11]. Different composite clinical indices are now available to monitor the disease activity and treatment efficacy [12]. But are clinical and laboratory parameters sufficient to establish whether the inflammatory process is stopped and thus exclude unfavorable progression over time [13]? It is essential to answer this question to properly guide therapy in individual patients during follow-up in order to prevent relapse and ensure the best possible long-term outcome. In fact, as shown by various studies, some patients considered to be in remission on the basis of DAS28 score (DAS 28<2.6), the most commonly used criteria in clinical practice, show frequent reactivations of the disease and/or a progression of structural joint damage [14]. The last is one of the most important consequences of active disease and it can lead to joint deformities and functional limitation [15].

The Ultrasound Examination (US) responds to the need to deepen the clinical and laboratory evaluation, helping to define the remission more stringently, so that the concept of "Deep Remission" has recently been introduced to indicate the ultrasound remission. Today we have ultrasound instruments with an increasing sensitivity in detecting morph-structural joint alterations in RA, such as thickening of the synovial membrane, cartilage damage, bone damage and tendon commitment. In particular, alongside the Power-Doppler ultrasound technique (PD), it is possible to distinguish the joints with active synovitis from those in which inflammation has been stopped [16-19].

The primary objectives of this study are to:

·     evaluate the presence of subclinical disease activity, through US study, in patients in clinical remission according to DAS28 criteria, in order to obtain an early identification of subjects with a high probability of future evolution of structural damage;

·         identify the main clinical and laboratory predictors for failure to reach a state of deep remission (confirmed on ultrasound).

The secondary objectives are to:

·         assess whether other remission scores are actually more accurate (SDAI, CDAI, BOOLEAN) to exclude subclinical activity;

·         evaluate the “Janus” contribution of steroids in disease activity: they can be necessary but not sufficient for the clinical assessment of the disease, but, on the other hand, they can be a source of potential false negative in PD US evaluation.

2.       Materials and Methods

2.1.  Patients

We retrospectively reviewed all patients aged over 18 years affected by RA who were referred to the Rheumatology Department in Brescia (Italy) during the period from 01/01/2015 to 28/02/2015. Of these patients, we considered only those who met the 2010 ACR/EULAR classification criteria of RA and who were being treated with csDMARDs (methotrexate, MTX; sulfasalazine, SSZ; leflunomide, LEF; hydroxychloroquine, HCQ); concomitant treatment with bDMARDs was an exclusion criterion of the study. We finally considered only those who met DAS remission criteria (DAS28 <2.6) for at least 6 months. Based on the medical records of each patient included in the study, we considered different parameters, that we can subdivide into: clinical and demographic; laboratory and instrumental data.

2.2.  Clinical and Demographic Evaluation

About clinical data, we retrospectively collected data about the duration of the disease, the number of swollen and tender joints and the judgment of physician and patient about the degree of overall disease activity, expressed by analogue scale (VAS medical doctor and VAS patient, respectively). The latter data were evaluated at the onset along with the follow-up of the patient, up to 84 months after the first evaluation (every 6 months for the first year and every 12 months after that) and were used to calculate the composite index of disease activity, such as DAS28, SDAI and CDAI. Moreover, we evaluated the age, the biometric indexes (BMI), concomitant therapy with csDMARDs and steroids, with the respective dosages, smoking status, the presence of any comorbidities and concomitant therapies (focusing mainly on statins and ACE -inhibitors for the documented anti-inflammatory effect), the presence of extra-articular involvement and the level of functional disability by the Health Assessment Questionnaire (HAQ). According to literature we considered patients in remission if DAS28 <2.6; other remission criteria used during statistical evaluation was the following: SDAI <3.3, CDAI <2.3 and Boolean criteria.

2.3.  Laboratory and Instrumental Evaluation

Concerning the laboratory data, we examined whether patients were rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) seropositive and their title, stratifying between low level (<3x normal range) and high level (> 3x normal range), as well as the positivity of the other main autoantibodies: Anti–Nuclear Antibodies (ANA), anti-native DNA antibodies (dsDNA), antiextractable nuclear antigens (ENA) and antiphospholipid antibodies (anti-cardiolipin and antibeta2glicoprotein I, lupus anticoagulant). We also collected data about the main values of inflammatory markers, C-reactive protein (CRP) and Erythrocyte Sedimentation Rate (ESR), from the onset and subsequent assessments.

Finally, from the instrumental point of view, we evaluated the presence or absence of marginal erosions by traditional radiography and the possible progression of structural damage during follow-up. All clinical, laboratory and instrumental data were collected by rheumatologists who did not

perform the US evaluation.

2.4.  Ultrasound Evaluation

All patients were subjected to US, according to the Backhaus score, of the following joints: wrist, Metacarpophalangeal (MCP) II, MCP III, Proximal Interphalangeal (PIP) II, PIP III, Metatarsophalangeal (MTP) II, MTP V. For each joint, evaluated bilaterally, we evaluated the presence of joint effusion and synovial hypertrophy (grading from 1 to 3 according to the Scheele method), the degree of synovial vascularization with PD (grading from 1 to 3 according to the Szkudlarek method), the presence of marginal or subchondral erosivity (dichotomous rating). In addition, we evaluated the presence of tenosynovitis (dichotomous rating). For each joint area we performed both axial and coronal evaluations, studying the dorsal (all), volar (all) and lateral-medial (wrist, MCP II, PIP II, PIP III, MTP V) sides [20-22].

According to the OMERACT guidelines, the US study was performed by an independent expert rheumatologist in musculoskeletal ultrasound who was unaware of the clinical evaluation (blind). For all the feedback, we used the latest generation ultrasound scanner (Esaote MyLab 70 X vision), with multi-frequency linear probe (6-18 MHz). For the study in B-MODE we used this setting: frequency of 18 MHz; gain of 50%; the PD frequency was 10 MHz, the PRF (pulse repetition frequency) was 750 Hz. The study included a period of 15-20 minutes for each patient; all the US were performed in the morning. If a synovial hypertrophy PD+ with grading> 1 in at least 2 joints resulted, the patient was not considered to be in deep remission (subclinical disease activity).

2.5.  Statistical Evaluation

Data were analyzed using the following statistical tests:

·         χ2 test with Fisher's correction, used to compare qualitative parameters;

·         Mann-Whitney test, to compare the quantitative parameters.

We used these two non-parametric tests because the samples in the study were small in size (<30 patients each) and the distribution of the samples was not normal. A p value <0.05 was considered

as statistically significant.

3.       Results

3.1.  Demographic and Clinical Features

There were 575 patients over the age of 18 with RA who were evaluated in the Rheumatology Department in Brescia (Italy) in the January-February 2015 period. Of these, 388 were receiving bDMARDs and 187 csDMARDs; we considered only the latter in the study. Of the 187 patients receiving csDMARDs, 98 were found to be in clinical remission according to DAS criteria (DAS28 <2.6). Among these, 40 patients who met the criteria diagnostic ACR / EULAR 2010 at the time of diagnosis were randomized and summoned to undergo an ultrasound examination of their hands and feet.

Patients included in the study had an average age of 64 and were represented by 70% women (F/M:28/12). Most were FR (70%) and ACPA (73.68%) seropositive, although data relating to this last item was missing in 2 patients. In 33 patients (82.5%) the diagnosis of disease was made within two years from the clinical onset (early arthritis); the mean disease duration was 11.8 months. All patients were in DAS28 remission for at least 6 months (inclusion criteria) but 9 subjects (97.5%) for at least 1 year (mean 1.65). The mean values of the other two composite indices, SDAI and CDAI, were respectively 3.28 and 2.75. 50% of patients also respected the Boolean remission criteria.

The demographic and clinical features of the analyzed sample are reported in Table 1 and Table 2 respectively.

3.2.  Pharmacological Features

At the time of evaluation, 65% of patients were receiving MTX, 37.5% HCQ, 7.5% LEF, at a mean dosage of 10.67 mg/week, 186.66 mg/day and 13.81 mg/day respectively. No one was taking SSZ. 67.5% of patients were also taking steroids, at a mean dosage of 23.85 mg/week. Only 4 patients were treated with steroids at higher dosage (> 35mg/week).

The pharmacological features of studied patients are reported in Table 3.

3.3.  Sonographic Features

Ultrasound investigation carried out on 14 joints provided by the Backhaus score showed that 38 patients had B-MODE synovitis at least in one joint (with a range of joints involved ranging from 1 to 12), 32 at least an eroded joint (range: 1 -8) and 15 showed at least one PD+ synovitis (range 1-11). The mean number of involved joints for each patient was 4.13, considering B-MODE synovitis, 3.65 with regards to the erosions and 3.06 for PD +. Two patients with PD + synovitis had both hands and feet involvement (II MTF and / or V MTF); no patients had PD + synovitis instead synovitis exclusively in the feet.

There were 23 patients that had both DAS28 <2.6 and SDAI <3.3 (57.5%) and 11 (47%) of these showed positivity of the PD signal to at least one of the joints examined (range: 1 -11), with a mean value of 3.27 joints per patient; similarly, in patients with CDAI <2.3 the mean of PD positive joints for each patient was of 3.18. Of the 20 patients in Boolean remission, 8 (40%) showed joints with PD positive signal, between a minimum of 1 and a maximum of 5 joints involved (mean of 2.37 joints per patient). 15% of the patients also had tenosynovitis, but this was never found in the absence of joint involvement. In particular, the flexor tendons were involved in 5 patients (12.5% of the cases); the extensor tendons in 2 patients (5% of cases).

The US characteristics of the studied sample are summarized in (Table 4).

Considering the patients with PD positive signal associated with synovitis, the most involved joints were the right MCP II (72.7%), the left MCP II (54.5%) and the left wrist (45.4%), as indicated in Table 5.

Based on US criteria the patients were divided into two groups: subjects with PD + synovitis (PD+) and subjects with PD-synovitis or absence of synovitis (PD-). The first group was represented by those who had at least 2 joints with PD positive signal in the ultrasound examination and included 11 patients (27.5%), the second included the remaining 29 patients. We considered patients with one single joint involved as negative so as to be even more stringent in our evaluation, avoiding the possibility of any false positives. Thus, 27.5% of the patients have actually shown subclinical disease activity.

3.4.  Demographic, Clinical and Pharmacological Features Comparison

Each group was stratified for all demographic, clinical, pharmacological and US parameters. With regards to the demographic and clinical aspects there were no significant differences (p>0.05). Only for the age and the ACPA seropositivity (especially at high level) there was a tendency to statistical significance.

The two groups were homogeneous with regards to the ongoing drug treatment, both for csDMARDs and steroids. Significant differences between the two groups were found in US results, especially in the median number of synovitis and eroded joints for each patient (p <0.05), in both cases higher in the positive PD group (Table 6).

The data are expressed as median and interquartile range [0,25-0,75] if not specified.

4.       Discussion

Recent studies have shown a greater sensitivity of ultrasound (US) with respect only to the clinical evaluation in the detection of joints structural abnormalities and inflammatory activity. It has also a higher predictive value with regards to patient outcome in terms of erosivity and disease reactivation [23,24]. With our study we evaluated the status of "Deep Remission" in RA patients treated with csDMARDs, considered in remission on the basis of DAS criterion [25]. The Backhaus score (assessed on large series) was used for US evaluation and particular emphasis was given to the PD signal associated with synovitis, parameter by which we have obtained two samples of subjects, positive Power-Doppler (PD +) and negative Power-Doppler (PD-)patients. The first group included patients with two or more joints with synovitis associated with PD signal the second those with less than two joints involved (the latter represented the control population). Considering negative even those patients who showed only one articulation PD+ we wanted to be as stringent as possible, minimizing the false positive.

The results show a homogeneity of the two samples regarding demographic, clinical and pharmacological features. There were no significant differences regarding steroids therapy and its dosage. As to this aspect, it should be stressed that the majority of our patients were in the low dose steroids despite the state of clinical remission (<5mg / day) and the dosage of csDMARDs (MTX, LEF, HCQ) was in most cases submaximal. Also composite indexes with which we evaluate the disease activity (DAS28, SDAI, CDAI) in daily practice were not significantly different in the two groups of patients and therefore not able to identify patients PD +. This could be related to the fact that they consider the patient's judgment about the activity of the disease based on a visual analogue scale of pain difficult to confirm objectively. But we must highlight the fact that seropositivity to ACPA, especially at high level, is more frequent in PD + showing a trend to statistical significance, that was not reached probably because of the low number of the sample. Most joints affected by synovitis accompanied by PD signal were found to be the right MCP II and the left MCP II, in which the PD signal also had a greater intensity, and this can be associated with greater use of the same in everyday life.

The left wrist appears frequently involved and this aspect could be an interesting topic for discussion given the greater number of right-handers in the population. Our study, like many other studies, confirms a higher prevalence of smoking than expected. In light of the results obtained with our study, we can say that the only clinical and laboratory data, including composite indexes usually used, are not sufficient to confirm deep remission state. In particular, the greater ultrasound examination sensitivity compared to the simple clinical evaluation is confirmed. This suggests an important role of this examination in the monitoring of patients with RA. 27.4% of the patients in fact showed the presence of inflammatory activity despite the clinical evaluation having classified them as patients in remission. We had optimized the drug therapy and intensified the US follow-up in these patients.

The US evaluation is a non-invasive examination, very well tolerated by patients and relatively inexpensive. However, it is an operator-dependent examination and there is still no standardized score for his performance [26]. The use of the Backhaus US score in which the joints mainly affected by inflammatory activity are included reduces the time required for the ultrasound examination requiring an average of 15 minutes for each patient, but at the same time allowing an accurate evaluation [27]. Our pilot study had some limitations: the low number of samples; unavailability of some laboratory data (not available from the medical records of some patients); moreover, it was a retrospective study and not prospective. Despite these limitations, which will be subject to future revaluations the study leads us to conclude that the use of joint ultrasound examination is essential in the periodic monitoring of disease activity. In fact, this could early identify patients who might otherwise be mistakenly considered in remission, with consequent reduction of immunosuppressive therapy. In addition, it would allow us to optimize the therapeutic strategy in patients where subclinical disease activity is detected.

In the future, it would be useful to explore the predictive significance of ACPA positivity, especially at high level, recruiting a larger number of patients. This would allow to further optimize the monitoring of RA patients, both in terms of cost and time.

5.      Acknowledgements: None

Table 1: Demographic and laboratory features of the patients at the time of US evaluation.

Table 2: Clinical features of the patients in the study at the time of US evaluation.

Table 3: Pharmacological features of patients at the time of US evaluation.

Table 4: US features of studied patients.

Table 5: PD + signal and mean intensity of the signal in studied joints.

Table 6: Ultra sonographic features of PD + e PD – patients.

1. Grassi W, De Angelis R, Lamanna G, Cervini C (1998) The clinical features of rheumatoid arthritis, European Journal of radiology 27: S18-24.
2. Montecucco C (2004) Fattori predittivi dell’outcome nell’artrite reumatoide. Reumatismo 56: 21-27.
3. Goekoop-Ruiterman YP, de Vries-Bouwstra JK, Allaart CF, van Zeben D, Kerstens PJ, et al. (2005) Clinical and radiographic outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the Best study): a randomized, controlled trial. Arthritis Rheum 52: 3381-3390.
4. Mottonen T, Hannonen P, Leirisalo-Repo M, Nissilä M, Kautiainen H, et al. (1999) Comparison of combination therapy with single-drug therapy in early rheumatoid arthritis: a randomized trial. FIN-RACo trial group 353: 1568-1573.
5. Grigor C, Capell H, Stirling A, McMahon AD, Lock P, et al. (2004) Effect of a treatment strategy of tight control for rheumatoid arthritis (the TICORA study): a single-blind randomised controlled trial 364: 263-269.
6. Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pavelka K, et al. (2006) The PREMIER study: A multi-center, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not previous methotrexate treatment. Arthritis Rheum 54: 26-37.
7. Canete JD, Pablos JL (2013) Biologic Therapy in rheumatoid arthritis. Curr Top Med Chem 13: 752-759.
8. Jyoti RP, Durga PM, Anupam W, Vikas A (2015) Is non-biological treatment of rheumatoid arthritis as good as biologics? World J Orthop 6: 278-283.
9. Verstappen SM, Jacobs JW, van der Veen MJ, Heurkens AH, Schenk Y, et al. (2007) Intensive treatment with methotrexate in early rheumatoid arthritis: aiming for remission. Computer Assisted Management in Early Rheumatoid Arthritis (CAMERA, an open-label strategy trial). Ann Rheum Dis 66: 1443-1449.
10. Saag KG, Teng GG, Patkar NM, Anuntiyo J, Finney C, et al. (2008) American College of Rheumatology 2008 recommendations for the use of nonbiologic disease-modifyng antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum 59: 762-784.
11. Smolen JS, Landawè R, Breedveld FC, Dougados M, Emery P, et al. (2010) EULAR recommendations for the management of rheumatoid arthritis with syntetic and biological disease-modifying ant rheumatic drugs. Ann Rheum Dis 69: 964-975.
12. Ohrndorf S, Backhaus M (2013) Advances in sonographic scoring of rheumatoid arthritis. Ann Rheum Dis 72: ii69-75.
13. Saleem B, Brown AK, Keen H, Nizam S, Freeston J, et al. (2011) Should imaging be a component of rheumatoid arthritis remission criteria? A comparison between traditional and modified composite remission scores and imaging assessments. Ann Rheum Dis 70: 792-798.
14. Felson D (2012) Defining remission in rheumatoid arthritis. Ann Rheum Dis 71: i86-i88
15. Pincus T, Sokka T (2002) Partial control of Core Data Set measures and Disease Activity Score (DAS) measures of inflammation does not prevent long-term joint damage: evidence from longitudinal observations over 5-20 years. Clin Exp Rheumatol 20: S42-47.
16. Szkudlarek M, Court-Payen M, Strandberg C, Klarlund M, Klausen T, et al. (2001) Power Doppler ultrasonography for assessment of sinoviti in the metacarpophalangeal joints of patients with rheumatoid arthritis: a comparison with dynamic magnetic resonance imaging. Arthritis Rheum 44: 2018-2023.
17. Ramirez J, Celis R, Usategui A, Ruiz-Esquide V, Cuervo A, et al. (2016) Immunopathology characterization of ultrasound-defined synovitis in rheumatoid arthritis patients in clinical remission. Arthritis Res Ther 18:74.
18. Naredo E, Monteagudo I (2014) Doppler techniques. Clin Exp Rheum 32: S12-S19.
19. Wakefield RJ, Gibbon WW, Emery P (1999) The current status of ultrasonography in rheumatology: Rheumatology Oxford 38: 195-198.
20. Schell AK, Hermann KG, Kahler E, Pasewaldt D, Fritz J, et al. (2005) A novel ultra-sonographic sinoviti scoring system suitable for analyzing finger joint inflammation in rheumatoid arthritis. Arthritis Rheum 52: 733-743.
21. Szkudlarek M, Court- Payen M, Jacobsen S, Klarlund M, Thomsen HS, et al. (2003) Interobserver agreement in ultrasonography of the finger and toe joints in rheumatoid arthritis. Arthritis Rheum 48: 955-962.
22. Backhaus M, Ohrndorf S, Kellner H, Strunk J, Backhaus TM, et al. (2009) Evaluation of anovel 7-joint ultrasound score in daily rheumatologic practice: a pilot project. Arthritis Rheum 61: 1194-1201.
23. Brown A. K, Conaghan P.G, Karim Z, Quinn MA, Ikeda K, et al. (2008) An explanation for the apparent dissociation between clinical remission and continued structural deterioration in rheumatoid arthritis. Arthritis Rheum 58: 2958-2967.
24. Wakefield RJ, Green MJ, Marzo-Ortega H, Conaghan PG, Gibbon WW, et al. (2004) Should oligoarthritis be reclassified? Ultrasound reveals a high prevalence of subclinical desease. Ann Rheum Dis 63: 382-385.
25. Anderson JK, Zimmerman L, Caplan L, Michaud K (2011) Measures of Rheumatoid Arthritis Disease Activity. Arthritis Care & Research 63: S14-S36.
26. Wakefield RJ, Balint PV, Szkudlarek M, Filippucci E, Backhaus M, et al. (2005) Muscoloskeletal ultrasound including definitions for ultrasonographic pathology. J Rheumatol 32: 2485-2487.
27. Brown AK, Quinn MA, Karim Z, Conaghan PG, Peterfy CG, et al. (2006) Presence of significant sinoviti in rheumatoid arthritis patients with disease-modifying antirheumatic drug-induced clinical remission: evidence from an imaging study may explain structural progression. Arthritis Rheum 54: 3761-3773.