International Journal of Rheumatic Diseases 2015; 18: 46–51
ORIGINAL ARTICLE
Diagnostic value of the anti-Sa antibody compared with the anti-cyclic citrullinated peptide antibody in rheumatoid arthritis Cezary IWASZKIEWICZ, Mariusz PUSZCZEWICZ and Gra_zyna BIAŁKOWSKA-PUSZCZEWICZ Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Poznan´, Poland
Abstract Aim: To evaluate the prevalence and diagnostic significance of the autoantibody against citrullinated vimentin (anti-Sa) compared with the widely used anti-cyclic citrullinated peptide autoantibody (anti-CCP) in patients with rheumatoid arthritis (RA). Method: One hundred and sixty-nine patients hospitalized at the Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Pozna n, Poland, were enrolled in a cross-sectional study and divided into two groups. The RA group comprised 41 patients diagnosed as having RA. The non-RA control group included 128 individuals with a variety of rheumatic disorders. Serum anti-Sa and anti-CCP measurements were performed by enzyme-linked immunosorbent assay. Results: The sensitivity and specificity of anti-Sa for the diagnosis of RA was 36.6% and 96.9%, respectively. For the anti-CCP test, the sensitivity was 65.9% and the specificity was 95.3%. Concomitant presence of anti-Sa and anti-CCP was determined in 36.6% of the patients with RA, whereas isolated positivity of anti-Sa was not observed. Anti-Sa positive RA patients had significantly higher anti-CCP levels compared to anti-Sa negative subjects (P < 0.05). Conclusion: With regard to the relatively low diagnostic sensitivity and the lack of cases identified by anti-Sa alone, we were unable to demonstrate any additional diagnostic value of the anti-Sa autoantibody in comparison to the anti-CCP autoantibody. To the authors’ best knowledge, this is the first study among Polish patients verifying the clinical utility of anti-Sa in the diagnosis of RA. Key words: anti-CCP, anti-MCV, anti-Sa, citrullinated vimentin, rheumatoid arthritis.
INTRODUCTION Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown etiology. The disease acts on the connective tissue of the whole organism and, what is more, contributes to a reduced life expectancy. IrreCorrespondence: Mariusz Puszczewicz, Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, No. 135/147, 28 Czerwca 1956 St., 61–545 Pozna n, Poland. Email: [email protected]
versible joint damage begins from the first stages of the disease. Consequently, RA leads to severe disability and deterioration in the patient’s quality of life.1,2 Therefore, current treatment standards focus on preventing, or at least, limiting irreversible joint destruction. The optimum treatment strategy, which should be employed as early as possible and tailored to each patient’s expected severity, requires access to reliable diagnostic and predictive tools.3,4 In addition to rheumatoid factor (RF), anti-citrullinated protein/peptide autoantibodies (ACPA) are the second
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Anti-Sa compared with anti-CCP in RA
serological marker included in the 2010 American College of Rheumatology – European League Against Rheumatism (ACR–EULAR) RA Classification Criteria.3 ACPA represent a family of autoantibodies detected in the majority of patients with RA and characterized by high specificity for diagnosis of the disease.5 The antibody against citrullinated vimentin can be measured by enzyme-linked immunosorbent assay (ELISA) tests using citrullinated vimentin (the so-called Sa antigen) or mutated citrullinated vimentin (MCV) as an antigen.6,7 Therefore, this serological marker, which is a particular member of the ACPA family, is called anti-Sa or anti-MCV, depending on the substrate used in the assay. It should be noted that results obtained using different tests can vary to some extent.8 The autoantibody against citrullinated vimentin (anti-Sa) has been suggested to be a promising marker of RA;9 however, it has not yet been introduced as a routine diagnostic marker. To the best of our knowledge, there has been no research published verifying the clinical utility of anti-Sa in the diagnosis of RA among Polish patients. Furthermore, data from all around the world vary substantially. Given the above, we decided to evaluate the prevalence and diagnostic significance of anti-Sa in comparison to the widely used anti-cyclic citrullinated peptide antibody (anti-CCP) in patients suffering from RA and other rheumatic diseases.
MATERIALS AND METHODS One hundred and sixty-nine patients hospitalized at the Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Pozna n, Poland, were enrolled in a cross-sectional study and divided into two groups. The RA group comprised 41 patients, fulfilling the 2010 ACR–EULAR RA Classification Criteria.3 Thirty-five (85.4%) of these patients were positive for immunoglobulin M (IgM)-RF (seropositive RA) and six (14.6%) were negative for IgM-RF (seronegative RA). The non-RA control group included 128 individuals with a variety of rheumatic disorders: systemic lupus erythematosus (SLE) (n = 21); primary Sj€ ogren’s syndrome (pSS) (n = 12); undifferentiated connective tissue disease (UCTD) (n = 12); osteoarthritis (n = 11); psoriatic arthritis (n = 8); diffuse cutaneous systemic sclerosis (dcSSc) (n = 8); systemic vasculitis (n = 7); ankylosing spondylitis (n = 7); granulomatosis with polyangiitis (n = 6); reactive arthritis (n = 5); polymyositis (PM) (n = 3); limited cutaneous systemic sclerosis (n = 3); undifferentiated seronegative
International Journal of Rheumatic Diseases 2015; 18: 46–51
spondyloarthropathy (n = 3); juvenile idiopathic arthritis (n = 3); primary antiphospholipid syndrome (n = 2); calcium pyrophosphate dihydrate disease (n = 2); cutaneous lupus erythematosus (n = 2); limited systemic sclerosis (n = 2); radiculopathy (n = 1); fibromyalgia (n = 1); erythema nodosum (n = 1); adult Still’s disease (n = 1); mixed connective tissue disease (n = 1); Behcßet’s disease (n = 1); gout (n = 1); sarcoidosis (n = 1); polymyalgia rheumatica (n = 1); overlap syndrome: dcSSc and PM (n = 1) and overlap syndrome: SLE and dcSSc (n = 1). All patients were Caucasian of Polish origin. Selected demographic and clinical characteristics of the RA group and the non-RA group are compared in Table 1. The radiographic changes among the seropositive RA patients were evaluated according to the Steinbrocker staging method on a four-level scale, ranging from stage I to stage IV.10 Clinical diagnosis, IgM-RF status, C-reactive protein concentration (CRP), erythrocyte sedimentation rate (ESR), as well as demographic, radiographic and treatment data were taken from the patients’ charts. All laboratory analyses were performed on samples remaining after routine diagnostic tests. The study complied with the guidelines of the Bioethical Committee at Poznan University of Medical Sciences. The blood samples were obtained after written informed consent during patient’s hospital examinations and patient anonymity was preserved. Blood samples for the study were obtained from the patients during their hospitalization. The sera were separated by centrifugation and stored at 20°C until assayed. Serological analyses were performed by ELISA using commercially available kits and according to the manufacturer’s instructions: anti-Sa IgG (Euroimmun, L€ ubeck, Germany; cut-off value for positivity was 20 relative units/ml [RU/ml]) and anti-CCP IgG (secondgeneration anti-CCP assay; Euroimmun, L€ ubeck, Germany; values > 5 RU/mL were considered positive). Statistical analyses were performed using Statistica 9 PL Trial software (StatSoft, Krakow, Poland). The sensitivities and specificities of the anti-Sa and anti-CCP were calculated. All of the study variables were tested for normality with the Shapiro–Wilk test. For all the tests, the level of significance was set at P < 0.05. Spearman’s rank correlation coefficient was used to assess the relationship between anti-Sa levels and disease duration, Steinbrocker radiographic stage, as well as CRP, ESR and anti-CCP levels. The Mann–Whitney U-test was used to determine the statistical differences in disease duration, Steinbrocker radiographic stage, the number of current disease-modifying antirheumatic
47
C. Iwaszkiewicz et al.
Table 1 Demographic and clinical data of the RA group (n = 41) and the non-RA disease group (n = 128)
Age (years), mean SD (range) Sex, M:F ratio IgM-RF positive, n (%) IgM-RF negative, n (%) Disease duration (years) Median (range) Mean SD Steinbrocker stage (I–IV) Median (range) Mode CRP (mg/L) Median (range) Mean SD ESR (mm/1st h) Median (range) Mean SD Current antirheumatic therapy Patients on synthetic DMARDs (%) Patients on biological DMARDs (%) No. of DMARDs, mean SD (range) Patients on corticosteroids (%)
RA group
Non-RA group
51.7 11.8 (19–72) 1 : 3.6 35 (85.4%) 6 (14.6%)
46.1 15.2 (18–76) 1 : 3.3
9.0 (0.2–41.0) 10.7 9.4
5.5 (0.1–43.0) 9.53 9.3
III (I–IV) Multiple 5.8 (0.3–160.0) 17.1 30.4 19.0 (1.0–94.0) 27.4 24.3 94.7 15.8 1.2 0.5 (0–3) 84.2
CRP, C-reactive protein; DMARDs, disease-modifying antirheumatic drugs; ESR, erythrocyte sedimentation rate; IgM-RF, immunoglobulin M rheumatoid factor; RA, rheumatoid arthritis; SD, standard deviation.
drugs (DMARDs), as well as CRP and anti-CCP levels between anti-Sa positive and anti-Sa negative RA patients. Welch’s test was used to determine the statistical difference in ESR between anti-Sa positive and antiSa negative patients with RA. Homogeneity of variances was assessed using Levene’s test. Fisher’s exact test was used to assess the statistical differences in the proportion of patients taking corticosteroids and DMARDs between anti-Sa positive and anti-Sa negative RA patients.
RESULTS The sensitivity and specificity of anti-Sa for the diagnosis of RA was 36.6% and 96.9%, respectively. For antiCCP, the sensitivity was 65.9% and the specificity was 95.3%. These values are illustrated in Table 2. There were no patients with seronegative RA found to be positive for anti-Sa or anti-CCP. Among the non-RA group, anti-Sa positivity was observed in two (9.5%) patients with SLE, one (8.3%) patient with pSS and one (8.3%) patient with UCTD. The frequencies of positive results for anti-Sa and anti-CCP obtained among different diagnosis groups are demonstrated in Table 3. Concomitant presence of anti-Sa and anti-CCP was
48
determined in 15 (36.6%) patients with RA. Twelve (29.3%) individuals were anti-CCP positive only, whereas isolated positivity of anti-Sa was not observed. Fourteen (34.1%) RA patients were negative for both anti-Sa and anti-CCP (Table 4). The laboratory and clinical characteristics of anti-Sa positive and anti-Sa negative RA patients are compared in Table 5. There was no significant correlation between the levels of anti-Sa and disease duration, Steinbrocker radiographic stage and levels of CRP, ESR or anti-CCP. There was no significant difference between anti-Sa positive and anti-Sa negative RA patients with respect to disease duration, Steinbrocker radiographic stage, ESR, CRP level, the number of current DMARDs, as well as the proportion of patients taking corticosteroids and conventional or biological DMARDs (P > 0.05).
Table 2 Anti-Sa and anti-CCP in the diagnosis of RA
Sensitivity Specificity
Anti-Sa %
Anti-CCP %
36.6 96.9
65.9 95.3
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody.
International Journal of Rheumatic Diseases 2015; 18: 46–51
Anti-Sa compared with anti-CCP in RA
Table 3 The frequencies of positivity for anti-Sa and anti-CCP in different diagnosis groups
RA total, (n = 41) RA with IgM-RF, (n = 35) RA without IgM-RF, (n = 6) Non-RA total, (n = 128) SLE, (n = 21) pSS, (n = 12) UCTD, (n = 12) Osteoarthritis, (n = 11) Ankylosing spondylitis, (n = 7) JIA, (n = 3) Polymyalgia rheumatica, (n = 1)
Anti-Sa positive n (%)
Anti-CCP positive n (%)
15 (36.6) 15 (42.9) 0 4 (3.1) 2 (9.5) 1 (8.3) 1 (8.3) 0 0 0 0
27 (65.9) 27 (77.1) 0 6 (4.7) 0 1 (8.3) 1 (8.3) 1 (9.1) 1 (14.3) 1 (33.3) 1 (100.0)
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody; JIA, juvenile idiopathic arthritis; pSS, primary Sj€ ogren’s syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; UCTD, undifferentiated connective tissue disease.
However, anti-Sa positive RA patients had significantly higher anti-CCP levels (median 138.9 RU/mL) compared to anti-Sa negative subjects (median 61.4 RU/ mL) (P < 0.05).
DISCUSSION In our study, the specificity of the anti-CCP test versus non-RA disease controls was 95.3%, consistent with the results of van Venrooij et al.11 In their extensive review in 2008, which summarizes the results from 144 independent studies, the specificity of anti-CCP versus nonRA disease controls was demonstrated to be 94.2%. The sensitivity of this marker for established RA was 75.2%. In the present study, the sensitivity of anti-CCP was lower (65.9%). However, the results of anti-CCP sensitivity reported by different authors vary largely, depending on the diagnostic test used and on the study population.12 The diagnostic sensitivity of anti-Sa for RA is reported as between 31% and 44% and the specificity as 92–98%.13–16 In the present study, anti-Sa demon-
strated a sensitivity of 36.6%. It is important to note that the RA group almost entirely included individuals with established disease. In our study, the diagnostic specificity of anti-Sa was 96.9%. The non-RA control group comprised patients with a variety of rheumatic conditions, including nearly 30 disorders. The specificity of the marker evaluated in such a group has the highest clinical relevance, especially in terms of differential diagnosis. In the non-RA group, only 4/128 patients (3.1%) were found to be anti-Sa positive. Among these four patients: two had SLE, one had UCTD with clinical features more suggestive of SLE and one had pSS. Interestingly, three of the four non-RA anti-Sa positive individuals were negative for anti-CCP. This observation is in agreement with the results of Bang et al.7 and Wagner et al.,17 who have reported some cases of SLE and pSS that were anti-MCV positive and anti-CCP negative. Thus, more studies are needed to assess the prevalence of anti-Sa and anti-MCV in non-RA rheumatic diseases with particular focus on SLE and pSS. In the present study, neither anti-Sa nor anti-CCP autoantibodies were found in patients with IgM-RF negative RA, but a subgroup including only six individuals is too small from which to draw a valid conclusion. Among the RA group, isolated positivity of anti-Sa was not observed. A systematic review conducted by Luime et al.18 showed that the percentage of RA patients who are positive for anti-MCV alone oscillates between 1% and 20%. We observed that anti-Sa positive patients with RA had significantly higher anti-CCP levels compared to anti-Sa negative subjects, consistent with the results of Hou et al.16 In other words, anti-Sa positivity may be expected particularly in patients with high levels of serum anti-CCP. We did not observe any correlation between anti-Sa levels and disease duration or Steinbrocker radiographic stage. There were no significant differences in disease duration and Steinbrocker radiographic stage between anti-Sa positive and anti-Sa negative patients with RA. This may be explained by the reported association of the marker with disease activity at the time of obtaining the patient’s blood sample.16,19 Surprisingly, there was no
Table 4 Frequency of isolated and combined presence of anti-Sa and anti-CCP in rheumatoid arthritis (RA) patients (n = 41)
n (%)
Anti-Sa positive Anti-CCP positive
Anti-Sa positive Anti-CCP negative
Anti-Sa negative Anti-CCP positive
Anti-Sa negative Anti-CCP negative
15 (36.6)
0
12 (29.3)
14 (34.1)
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody.
International Journal of Rheumatic Diseases 2015; 18: 46–51
49
C. Iwaszkiewicz et al.
Table 5 Comparison of laboratory and clinical parameters between anti-Sa positive (n = 15) and anti-Sa negative (n = 26) RA patients; anti-Sa positive RA patients had significantly higher anti-CCP levels compared to anti-Sa negative subjects* Parameter Anti-CCP (RU/mL), median (range) Median (range) Mean SD Disease duration (years) Median (range) Mean SD Steinbrocker stage (I–IV), Median (range) Mode CRP (mg/L) Median (range) Mean SD ESR (mm/1st h) Median (range) Mean SD Current antirheumatic therapy Patients on synthetic DMARDs (%) Patients on biological DMARDs (%) No. of DMARDs, mean SD (range) Patients on corticosteroids (%)
Anti-Sa positive
Anti-Sa negative
138.9 (18.7–200.0) 124.0 74.9
61.4 (1.0–200.0) 77.0 77.0
13.0 (0.2–26.0) 11.7 8.3
8.5 (1.0–41.0) 10.4 9.7
III (II–IV) IV
III (I–IV) Multiple
6.5 (0.9–160.0) 26.5 47.4
4.5 (0.3–45.0) 10.7 13.5
18.0 (2.0–94.0) 34.9 30.5
22.0 (1.0–62.0) 22.8 15.6
92.3 15.4 0.8 100.0
96.0 16.0 1.3 76.0
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; DMARDs, disease-modifying antirheumatic drugs; RU, relative units; SD, standard deviation; *P < 0.05.
correlation between anti-Sa and CRP or ESR in our study. We also did not demonstrate a significant difference in CRP or ESR between anti-Sa positive and antiSa negative RA patients. It should be noted that a single determination of the autoantibody level is not the most adequate method for assessing its relationship with disease activity. To assess such a relationship, long-term prospective studies with serial anti-Sa measurement in patients during remission and exacerbation are needed. In the present study, there were no significant differences in the proportion of patients taking corticosteroids or DMARDs, as well as in the number of DMARDs being taken between anti-Sa positive and anti-Sa negative RA patients. It has been well documented that treatment with DMARDs can significantly reduce the levels of serum IgM-RF in patients with RA. Levels of ACPA have been considered to be much less affected by therapy, although the results reported by different authors are quite variable. The observed differences in the effect of antirheumatic treatment on RF and ACPA have emphasized the independence of these autoantibody systems.20,21 The main limitation of the present study was virtual lack of early-onset RA patients and a small number of IgM-RF negative subjects.
50
CONCLUSION With regard to the relatively low diagnostic sensitivity and the lack of cases identified by anti-Sa alone, we were unable to demonstrate any additional diagnostic value of the anti-Sa autoantibody in comparison to the anti-CCP autoantibody. Nevertheless, the prognostic capacity and correlation of anti-Sa with disease activity still remain an open question. The research on the utility of anti-Sa and anti-MCV in the diagnosis and monitoring of RA, including long-term prospective studies, is of high importance, and should be conducted in various ethnic groups. To the authors’ best knowledge, this is the first study among Polish patients verifying the clinical utility of anti-Sa in the diagnosis of RA.
REFERENCES 1 Abdul MA, Mohd Shahrir MS, Sazliyana S, Oteh M, Shamsul AS, Hussein H (2011) A cross-sectional study of diastolic dysfunction in rheumatoid arthritis and its association with disease activity. Int J Rheum Dis 14, 18–30. 2 Ho RC, Fu EH, Chua AN, Cheak AA, Mak A (2011) Clinical and psychosocial factors associated with depression
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and anxiety in Singaporean patients with rheumatoid arthritis. Int J Rheum Dis 14, 37–47. Aletaha D, Neogi T, Silman AJ et al. (2010) Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 62, 2569–81. Davatchi F, Abari IS, Soroosh S, Soroosh M, Sadeghi Abdollahi B (2012) Performance of the 2010 rheumatoid arthritis classification criteria. Int J Rheum Dis 15, 455–61. Puszczewicz M, Iwaszkiewicz C (2011) Role of anti-citrullinated protein antibodies in diagnosis and prognosis of rheumatoid arthritis. Arch Med Sci 7, 189–94. Vossenaar ER, Despres N, Lapointe E et al. (2004) Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin. Arthritis Res Ther 6, R142–50. Bang H, Egerer K, Gauliard A et al. (2007) Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum 56, 2503–11. Farid SSh, Azizi G, Mirshafiey A (2013) Anti-citrullinated protein antibodies and their clinical utility in rheumatoid arthritis. Int J Rheum Dis 16, 379–86. Innala L, Kokkonen H, Eriksson C, Jidell E, Berglin E, Dahlqvst SR (2008) Antibodies against mutated citrullinated vimentin are a better predictor of disease activity at 24 months in early rheumatoid arthritis than antibodies against cyclic citrullinated peptides. J Rheumatol 35, 1002– 8. Ravindran V, Rachapalli S (2011) An overview of commonly used radiographic scoring methods in rheumatoid arthritis clinical trials. Clin Rheumatol 30, 1–6. van Venrooij WJ, van Beers JJ, Pruijn GJ (2008) Anti-CCP Antibody, a Marker for the Early Detection of Rheumatoid Arthritis. Ann N Y Acad Sci 1143, 268–85. Zhu T, Feng L (2013) Comparison of anti-mutated citrullinated vimentin, anti-cyclic citrullinated peptides, anti-glucose-6-phosphate isomerase and anti-keratin antibodies and rheumatoid factor in the diagnosis of
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rheumatoid arthritis in Chinese patients. Int J Rheum Dis 16, 157–61. Hayem G, Chazerain P, Combe B et al. (1999) Anti-Sa antibody is an accurate diagnostic and prognostic marker in adult rheumatoid arthritis. J Rheumatol 26, 7–13. Hueber W, Hassfeld W, Smolen JS, Steiner G (1999) Sensitivity and specificity of anti-Sa autoantibodies for rheumatoid arthritis. Rheumatology 38, 155–9. L opez-Longo FJ, Rodrıguez-Mahou M, Sanchez-Ram on S et al. (2006) Anti-cyclic citrullinated peptide versus anti-Sa antibodies in diagnosis of rheumatoid arthritis in an outpatient clinic for connective tissue disease and spondyloarthritis. J Rheumatol 33, 1476–81. Hou YF, Sun GZ, Sun HS, Pan WP, Liu WB, Zhang CQ (2012) Diagnostic value of anti-Sa and anticitrullinated protein antibodies in rheumatoid arthritis. J Rheumatol 39, 1506–8. Wagner E, Skoumal M, Bayer PM, Klaushofer K (2009) Antibody against mutated citrullinated vimentin: a new sensitive marker in the diagnosis of rheumatoid arthritis. Rheumatol Int 29, 1315–21. Luime JJ, Colin EM, Hazes JM, Lubberts E (2010) Does anti-mutated citrullinated vimentin have additional value as a serological marker in the diagnostic and prognostic investigation of patients with rheumatoid arthritis? A systematic review. Ann Rheum Dis 69, 337–44. Zahran WE, Mahmoud MI, Shalaby KA, Abbas MH (2013) Unique correlation between mutated citrullinated vimentine IgG autoantibodies and markers of systemic inflammation in rheumatoid arthritis patients. Indian J Clin Biochem 28, 272–6. B€ ohler C, Radner H, Smolen JS, Aletaha D (2013) Serological changes in the course of traditional and biological disease modifying therapy of rheumatoid arthritis. Ann Rheum Dis 72, 241–4. Bobbio-Pallavicini F, Caporali R, Bugatti S, Montecucco C (2008) What can we learn from treatment-induced changes in rheumatoid factor and anti-citrullinated Peptide antibodies? J Rheumatol 35, 1903–5.
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ORIGINAL ARTICLE
Diagnostic value of the anti-Sa antibody compared with the anti-cyclic citrullinated peptide antibody in rheumatoid arthritis Cezary IWASZKIEWICZ, Mariusz PUSZCZEWICZ and Gra_zyna BIAŁKOWSKA-PUSZCZEWICZ Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Poznan´, Poland
Abstract Aim: To evaluate the prevalence and diagnostic significance of the autoantibody against citrullinated vimentin (anti-Sa) compared with the widely used anti-cyclic citrullinated peptide autoantibody (anti-CCP) in patients with rheumatoid arthritis (RA). Method: One hundred and sixty-nine patients hospitalized at the Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Pozna n, Poland, were enrolled in a cross-sectional study and divided into two groups. The RA group comprised 41 patients diagnosed as having RA. The non-RA control group included 128 individuals with a variety of rheumatic disorders. Serum anti-Sa and anti-CCP measurements were performed by enzyme-linked immunosorbent assay. Results: The sensitivity and specificity of anti-Sa for the diagnosis of RA was 36.6% and 96.9%, respectively. For the anti-CCP test, the sensitivity was 65.9% and the specificity was 95.3%. Concomitant presence of anti-Sa and anti-CCP was determined in 36.6% of the patients with RA, whereas isolated positivity of anti-Sa was not observed. Anti-Sa positive RA patients had significantly higher anti-CCP levels compared to anti-Sa negative subjects (P < 0.05). Conclusion: With regard to the relatively low diagnostic sensitivity and the lack of cases identified by anti-Sa alone, we were unable to demonstrate any additional diagnostic value of the anti-Sa autoantibody in comparison to the anti-CCP autoantibody. To the authors’ best knowledge, this is the first study among Polish patients verifying the clinical utility of anti-Sa in the diagnosis of RA. Key words: anti-CCP, anti-MCV, anti-Sa, citrullinated vimentin, rheumatoid arthritis.
INTRODUCTION Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown etiology. The disease acts on the connective tissue of the whole organism and, what is more, contributes to a reduced life expectancy. IrreCorrespondence: Mariusz Puszczewicz, Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, No. 135/147, 28 Czerwca 1956 St., 61–545 Pozna n, Poland. Email: [email protected]
versible joint damage begins from the first stages of the disease. Consequently, RA leads to severe disability and deterioration in the patient’s quality of life.1,2 Therefore, current treatment standards focus on preventing, or at least, limiting irreversible joint destruction. The optimum treatment strategy, which should be employed as early as possible and tailored to each patient’s expected severity, requires access to reliable diagnostic and predictive tools.3,4 In addition to rheumatoid factor (RF), anti-citrullinated protein/peptide autoantibodies (ACPA) are the second
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Anti-Sa compared with anti-CCP in RA
serological marker included in the 2010 American College of Rheumatology – European League Against Rheumatism (ACR–EULAR) RA Classification Criteria.3 ACPA represent a family of autoantibodies detected in the majority of patients with RA and characterized by high specificity for diagnosis of the disease.5 The antibody against citrullinated vimentin can be measured by enzyme-linked immunosorbent assay (ELISA) tests using citrullinated vimentin (the so-called Sa antigen) or mutated citrullinated vimentin (MCV) as an antigen.6,7 Therefore, this serological marker, which is a particular member of the ACPA family, is called anti-Sa or anti-MCV, depending on the substrate used in the assay. It should be noted that results obtained using different tests can vary to some extent.8 The autoantibody against citrullinated vimentin (anti-Sa) has been suggested to be a promising marker of RA;9 however, it has not yet been introduced as a routine diagnostic marker. To the best of our knowledge, there has been no research published verifying the clinical utility of anti-Sa in the diagnosis of RA among Polish patients. Furthermore, data from all around the world vary substantially. Given the above, we decided to evaluate the prevalence and diagnostic significance of anti-Sa in comparison to the widely used anti-cyclic citrullinated peptide antibody (anti-CCP) in patients suffering from RA and other rheumatic diseases.
MATERIALS AND METHODS One hundred and sixty-nine patients hospitalized at the Department of Rheumatology and Internal Medicine, Poznan University of Medical Sciences, Pozna n, Poland, were enrolled in a cross-sectional study and divided into two groups. The RA group comprised 41 patients, fulfilling the 2010 ACR–EULAR RA Classification Criteria.3 Thirty-five (85.4%) of these patients were positive for immunoglobulin M (IgM)-RF (seropositive RA) and six (14.6%) were negative for IgM-RF (seronegative RA). The non-RA control group included 128 individuals with a variety of rheumatic disorders: systemic lupus erythematosus (SLE) (n = 21); primary Sj€ ogren’s syndrome (pSS) (n = 12); undifferentiated connective tissue disease (UCTD) (n = 12); osteoarthritis (n = 11); psoriatic arthritis (n = 8); diffuse cutaneous systemic sclerosis (dcSSc) (n = 8); systemic vasculitis (n = 7); ankylosing spondylitis (n = 7); granulomatosis with polyangiitis (n = 6); reactive arthritis (n = 5); polymyositis (PM) (n = 3); limited cutaneous systemic sclerosis (n = 3); undifferentiated seronegative
International Journal of Rheumatic Diseases 2015; 18: 46–51
spondyloarthropathy (n = 3); juvenile idiopathic arthritis (n = 3); primary antiphospholipid syndrome (n = 2); calcium pyrophosphate dihydrate disease (n = 2); cutaneous lupus erythematosus (n = 2); limited systemic sclerosis (n = 2); radiculopathy (n = 1); fibromyalgia (n = 1); erythema nodosum (n = 1); adult Still’s disease (n = 1); mixed connective tissue disease (n = 1); Behcßet’s disease (n = 1); gout (n = 1); sarcoidosis (n = 1); polymyalgia rheumatica (n = 1); overlap syndrome: dcSSc and PM (n = 1) and overlap syndrome: SLE and dcSSc (n = 1). All patients were Caucasian of Polish origin. Selected demographic and clinical characteristics of the RA group and the non-RA group are compared in Table 1. The radiographic changes among the seropositive RA patients were evaluated according to the Steinbrocker staging method on a four-level scale, ranging from stage I to stage IV.10 Clinical diagnosis, IgM-RF status, C-reactive protein concentration (CRP), erythrocyte sedimentation rate (ESR), as well as demographic, radiographic and treatment data were taken from the patients’ charts. All laboratory analyses were performed on samples remaining after routine diagnostic tests. The study complied with the guidelines of the Bioethical Committee at Poznan University of Medical Sciences. The blood samples were obtained after written informed consent during patient’s hospital examinations and patient anonymity was preserved. Blood samples for the study were obtained from the patients during their hospitalization. The sera were separated by centrifugation and stored at 20°C until assayed. Serological analyses were performed by ELISA using commercially available kits and according to the manufacturer’s instructions: anti-Sa IgG (Euroimmun, L€ ubeck, Germany; cut-off value for positivity was 20 relative units/ml [RU/ml]) and anti-CCP IgG (secondgeneration anti-CCP assay; Euroimmun, L€ ubeck, Germany; values > 5 RU/mL were considered positive). Statistical analyses were performed using Statistica 9 PL Trial software (StatSoft, Krakow, Poland). The sensitivities and specificities of the anti-Sa and anti-CCP were calculated. All of the study variables were tested for normality with the Shapiro–Wilk test. For all the tests, the level of significance was set at P < 0.05. Spearman’s rank correlation coefficient was used to assess the relationship between anti-Sa levels and disease duration, Steinbrocker radiographic stage, as well as CRP, ESR and anti-CCP levels. The Mann–Whitney U-test was used to determine the statistical differences in disease duration, Steinbrocker radiographic stage, the number of current disease-modifying antirheumatic
47
C. Iwaszkiewicz et al.
Table 1 Demographic and clinical data of the RA group (n = 41) and the non-RA disease group (n = 128)
Age (years), mean SD (range) Sex, M:F ratio IgM-RF positive, n (%) IgM-RF negative, n (%) Disease duration (years) Median (range) Mean SD Steinbrocker stage (I–IV) Median (range) Mode CRP (mg/L) Median (range) Mean SD ESR (mm/1st h) Median (range) Mean SD Current antirheumatic therapy Patients on synthetic DMARDs (%) Patients on biological DMARDs (%) No. of DMARDs, mean SD (range) Patients on corticosteroids (%)
RA group
Non-RA group
51.7 11.8 (19–72) 1 : 3.6 35 (85.4%) 6 (14.6%)
46.1 15.2 (18–76) 1 : 3.3
9.0 (0.2–41.0) 10.7 9.4
5.5 (0.1–43.0) 9.53 9.3
III (I–IV) Multiple 5.8 (0.3–160.0) 17.1 30.4 19.0 (1.0–94.0) 27.4 24.3 94.7 15.8 1.2 0.5 (0–3) 84.2
CRP, C-reactive protein; DMARDs, disease-modifying antirheumatic drugs; ESR, erythrocyte sedimentation rate; IgM-RF, immunoglobulin M rheumatoid factor; RA, rheumatoid arthritis; SD, standard deviation.
drugs (DMARDs), as well as CRP and anti-CCP levels between anti-Sa positive and anti-Sa negative RA patients. Welch’s test was used to determine the statistical difference in ESR between anti-Sa positive and antiSa negative patients with RA. Homogeneity of variances was assessed using Levene’s test. Fisher’s exact test was used to assess the statistical differences in the proportion of patients taking corticosteroids and DMARDs between anti-Sa positive and anti-Sa negative RA patients.
RESULTS The sensitivity and specificity of anti-Sa for the diagnosis of RA was 36.6% and 96.9%, respectively. For antiCCP, the sensitivity was 65.9% and the specificity was 95.3%. These values are illustrated in Table 2. There were no patients with seronegative RA found to be positive for anti-Sa or anti-CCP. Among the non-RA group, anti-Sa positivity was observed in two (9.5%) patients with SLE, one (8.3%) patient with pSS and one (8.3%) patient with UCTD. The frequencies of positive results for anti-Sa and anti-CCP obtained among different diagnosis groups are demonstrated in Table 3. Concomitant presence of anti-Sa and anti-CCP was
48
determined in 15 (36.6%) patients with RA. Twelve (29.3%) individuals were anti-CCP positive only, whereas isolated positivity of anti-Sa was not observed. Fourteen (34.1%) RA patients were negative for both anti-Sa and anti-CCP (Table 4). The laboratory and clinical characteristics of anti-Sa positive and anti-Sa negative RA patients are compared in Table 5. There was no significant correlation between the levels of anti-Sa and disease duration, Steinbrocker radiographic stage and levels of CRP, ESR or anti-CCP. There was no significant difference between anti-Sa positive and anti-Sa negative RA patients with respect to disease duration, Steinbrocker radiographic stage, ESR, CRP level, the number of current DMARDs, as well as the proportion of patients taking corticosteroids and conventional or biological DMARDs (P > 0.05).
Table 2 Anti-Sa and anti-CCP in the diagnosis of RA
Sensitivity Specificity
Anti-Sa %
Anti-CCP %
36.6 96.9
65.9 95.3
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody.
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Anti-Sa compared with anti-CCP in RA
Table 3 The frequencies of positivity for anti-Sa and anti-CCP in different diagnosis groups
RA total, (n = 41) RA with IgM-RF, (n = 35) RA without IgM-RF, (n = 6) Non-RA total, (n = 128) SLE, (n = 21) pSS, (n = 12) UCTD, (n = 12) Osteoarthritis, (n = 11) Ankylosing spondylitis, (n = 7) JIA, (n = 3) Polymyalgia rheumatica, (n = 1)
Anti-Sa positive n (%)
Anti-CCP positive n (%)
15 (36.6) 15 (42.9) 0 4 (3.1) 2 (9.5) 1 (8.3) 1 (8.3) 0 0 0 0
27 (65.9) 27 (77.1) 0 6 (4.7) 0 1 (8.3) 1 (8.3) 1 (9.1) 1 (14.3) 1 (33.3) 1 (100.0)
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody; JIA, juvenile idiopathic arthritis; pSS, primary Sj€ ogren’s syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; UCTD, undifferentiated connective tissue disease.
However, anti-Sa positive RA patients had significantly higher anti-CCP levels (median 138.9 RU/mL) compared to anti-Sa negative subjects (median 61.4 RU/ mL) (P < 0.05).
DISCUSSION In our study, the specificity of the anti-CCP test versus non-RA disease controls was 95.3%, consistent with the results of van Venrooij et al.11 In their extensive review in 2008, which summarizes the results from 144 independent studies, the specificity of anti-CCP versus nonRA disease controls was demonstrated to be 94.2%. The sensitivity of this marker for established RA was 75.2%. In the present study, the sensitivity of anti-CCP was lower (65.9%). However, the results of anti-CCP sensitivity reported by different authors vary largely, depending on the diagnostic test used and on the study population.12 The diagnostic sensitivity of anti-Sa for RA is reported as between 31% and 44% and the specificity as 92–98%.13–16 In the present study, anti-Sa demon-
strated a sensitivity of 36.6%. It is important to note that the RA group almost entirely included individuals with established disease. In our study, the diagnostic specificity of anti-Sa was 96.9%. The non-RA control group comprised patients with a variety of rheumatic conditions, including nearly 30 disorders. The specificity of the marker evaluated in such a group has the highest clinical relevance, especially in terms of differential diagnosis. In the non-RA group, only 4/128 patients (3.1%) were found to be anti-Sa positive. Among these four patients: two had SLE, one had UCTD with clinical features more suggestive of SLE and one had pSS. Interestingly, three of the four non-RA anti-Sa positive individuals were negative for anti-CCP. This observation is in agreement with the results of Bang et al.7 and Wagner et al.,17 who have reported some cases of SLE and pSS that were anti-MCV positive and anti-CCP negative. Thus, more studies are needed to assess the prevalence of anti-Sa and anti-MCV in non-RA rheumatic diseases with particular focus on SLE and pSS. In the present study, neither anti-Sa nor anti-CCP autoantibodies were found in patients with IgM-RF negative RA, but a subgroup including only six individuals is too small from which to draw a valid conclusion. Among the RA group, isolated positivity of anti-Sa was not observed. A systematic review conducted by Luime et al.18 showed that the percentage of RA patients who are positive for anti-MCV alone oscillates between 1% and 20%. We observed that anti-Sa positive patients with RA had significantly higher anti-CCP levels compared to anti-Sa negative subjects, consistent with the results of Hou et al.16 In other words, anti-Sa positivity may be expected particularly in patients with high levels of serum anti-CCP. We did not observe any correlation between anti-Sa levels and disease duration or Steinbrocker radiographic stage. There were no significant differences in disease duration and Steinbrocker radiographic stage between anti-Sa positive and anti-Sa negative patients with RA. This may be explained by the reported association of the marker with disease activity at the time of obtaining the patient’s blood sample.16,19 Surprisingly, there was no
Table 4 Frequency of isolated and combined presence of anti-Sa and anti-CCP in rheumatoid arthritis (RA) patients (n = 41)
n (%)
Anti-Sa positive Anti-CCP positive
Anti-Sa positive Anti-CCP negative
Anti-Sa negative Anti-CCP positive
Anti-Sa negative Anti-CCP negative
15 (36.6)
0
12 (29.3)
14 (34.1)
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody.
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C. Iwaszkiewicz et al.
Table 5 Comparison of laboratory and clinical parameters between anti-Sa positive (n = 15) and anti-Sa negative (n = 26) RA patients; anti-Sa positive RA patients had significantly higher anti-CCP levels compared to anti-Sa negative subjects* Parameter Anti-CCP (RU/mL), median (range) Median (range) Mean SD Disease duration (years) Median (range) Mean SD Steinbrocker stage (I–IV), Median (range) Mode CRP (mg/L) Median (range) Mean SD ESR (mm/1st h) Median (range) Mean SD Current antirheumatic therapy Patients on synthetic DMARDs (%) Patients on biological DMARDs (%) No. of DMARDs, mean SD (range) Patients on corticosteroids (%)
Anti-Sa positive
Anti-Sa negative
138.9 (18.7–200.0) 124.0 74.9
61.4 (1.0–200.0) 77.0 77.0
13.0 (0.2–26.0) 11.7 8.3
8.5 (1.0–41.0) 10.4 9.7
III (II–IV) IV
III (I–IV) Multiple
6.5 (0.9–160.0) 26.5 47.4
4.5 (0.3–45.0) 10.7 13.5
18.0 (2.0–94.0) 34.9 30.5
22.0 (1.0–62.0) 22.8 15.6
92.3 15.4 0.8 100.0
96.0 16.0 1.3 76.0
Anti-CCP, anti-cyclic citrullinated peptide antibody; anti-Sa, anti-citrullinated vimentin antibody; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; DMARDs, disease-modifying antirheumatic drugs; RU, relative units; SD, standard deviation; *P < 0.05.
correlation between anti-Sa and CRP or ESR in our study. We also did not demonstrate a significant difference in CRP or ESR between anti-Sa positive and antiSa negative RA patients. It should be noted that a single determination of the autoantibody level is not the most adequate method for assessing its relationship with disease activity. To assess such a relationship, long-term prospective studies with serial anti-Sa measurement in patients during remission and exacerbation are needed. In the present study, there were no significant differences in the proportion of patients taking corticosteroids or DMARDs, as well as in the number of DMARDs being taken between anti-Sa positive and anti-Sa negative RA patients. It has been well documented that treatment with DMARDs can significantly reduce the levels of serum IgM-RF in patients with RA. Levels of ACPA have been considered to be much less affected by therapy, although the results reported by different authors are quite variable. The observed differences in the effect of antirheumatic treatment on RF and ACPA have emphasized the independence of these autoantibody systems.20,21 The main limitation of the present study was virtual lack of early-onset RA patients and a small number of IgM-RF negative subjects.
50
CONCLUSION With regard to the relatively low diagnostic sensitivity and the lack of cases identified by anti-Sa alone, we were unable to demonstrate any additional diagnostic value of the anti-Sa autoantibody in comparison to the anti-CCP autoantibody. Nevertheless, the prognostic capacity and correlation of anti-Sa with disease activity still remain an open question. The research on the utility of anti-Sa and anti-MCV in the diagnosis and monitoring of RA, including long-term prospective studies, is of high importance, and should be conducted in various ethnic groups. To the authors’ best knowledge, this is the first study among Polish patients verifying the clinical utility of anti-Sa in the diagnosis of RA.
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