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Serum levels of anti-CCP antibodies were determined by enzyme-linked immunosorbent assay,and levels of RF were determined by turbidimetry on a latex-enhanced agglutination assay in 266 patients with RA and 134 patients with non-RA rheumatic diseases.
Results: Among the 266 patients with RA, 188 patients (70.7%) tested positive for anti-CCP antibodies, and 123 patients (46.2%) tested positive for RF. The sensitivity, specificity, positive predictive value and negative predictive value of anti-CCP antibodies for diagnosing RA were 70.76%, 85.07%, 90%, and 59% respectively. Those for RF were 46.26%, 90.29%, 90%, and 45% respectively.
Conclusion:The anti-CCP antibodies assay is is a useful test for diagnosing RA. According to its high PPV and NPV, it is an important diagnostic test in accurate diagnosis of disease.The use of anti-CCP antibodies and RF in combination further increases the diagnostic value for RA.
Key words: rheumatoid arthritis; Citrullinated Peptide; Rheumatoid factor; Comparison
Rheumatoid Arthritis (RA) is the most frequent autoimmune rheumatic disease, affecting; 1-2% of the population in Western countries (1). It is characterized by chronic synovitis leading to severe disability and premature mortality. Thus early diagnosis and starting effective disease-suppressive therapy to prevent or minimize joint destruction and systemic sequel is necessary. Disease severity in RA may vary from mild to severe which has a bearing on the intensity of the treatment offered to the patient. Several factors predict disease severity in RA including female sex, early development of erosions, genotype, acute-phase reactants, extra-articular manifestations and duration of disease ( 2). However, there is a need to identify markers that predict aggressive RA even before the appearance of erosions. Conventionally, the serology test routinely used in RA is the determination of serum rheumatoid factor (RF). The more specific auto antibodies for the diagnosis of RA, anti-cyclic citrullinated peptide (anti-CCP) antibodies, were discovered in 1964 (3). Accumulating evidence shows that anti-CCP antibodies are very useful in the diagnosis of RA (4). They may be present very early in the disease course (5) and are also considered as a prognostic factor for joint destruction (6). Several studies have shown that anti-CCP antibodies are moderately sensitive but highly specific for the diagnosis of RA, and their specificity is higher than RF (7) therefore, we conducted a cross-sectional study to identify the diagnostic value of anti-CCP antibodies and RF in patients with RA. We have also studied the presence of anti-CCP antibodies in RF negative patients with RA.
Materials and Methods:
The study was approved by the university ethics committee, and all the participants were informed about the research, and informed consents were obtained.
In this cross-sectional study we studied 400 serum samples: 266 from definite RA patients according on American College of Rheumatology (ACR) criteria(8) ( (228 women and 38 men; mean age 45.5 ± 13.8 years; range, 8-74 years) and consecutively recruited from the Rheumatology out-patient clinic of Shahid Sadoughi hospital of Yazd,Iran. 198 (61%) of these patients were classified as having early RA because the diagnosis was made <1 year before this study and radiological examinations revealed no lytic lesions at the wrists, hands, and feet. To provide data on assay specificity, 134 controls (matched for age and sex including 22 males and 112 females with non-RA rheumatic diseases) selected on the basis of their clinical diagnosis, were also studied.
Anti-CCP antibodies were tested by first-generation ELISA (AESKULISA).The anti-CCP was considered positive at values greater.The RF was measured by turbidimetry on a latex-enhanced agglutination assay (Roche Integra, Penzberg, Germany). The RF was considered positive at values greater than 10 U/mL. Each of these tests was performed and evaluated by operators who were blinded to other serological results and unaware of the patients' clinical data. The sensitivity and specificity for each assay was determined with respect to the clinical diagnosis. The statistical analysis was performed using SPSS, version 16 (SPSS Inc., Chicago, IL, USA) using the Chi square and Student t test. Pearson correlation coefficients were determined between diagnostic groups. A P value <0.05 was considered to be significant.
The demographic data is summarized in Tables 1&2.
Based on the cut-off value suggested by the manufacturer, in the RA group, 188 sera were positive for anti-CCP at>18unit/ml (table-3). The sensitivity was 70.67% .In the non-RA group, only 20 sera (9.61%) were positive. The Specificity was 85.07% .
Based on the cut-off value suggested by the manufacturer in the RA group, 123 sera were positive for RF at>10 IU/mL. The sensitivity was 46.24% .In the non-RA group, 13 sera were positive for RF at>10 IU/mL. The specificity was 90.29 % .
The anti-CCP antibodies had PPV, and NPV for diagnosis of RA of 90.38%, and 59.37% respectively. Those for RF were 90.44%, and 45.83% respectively. Receiver operating characteristic (ROC) curve was generated by the method of Metz8 for anti-CCP, and its accuracy was measured by the area under the ROC curve. The area under the curve for anti-CCP was 0.779.The accuracy was therefore considered as good(fig-1).
Regarding to anti-CCP titer , 96% of cases with titer higher than 201(201-1500) had definite RA whereas , 55.4% of patients with titer less than 101(0.2-101)had definite RA. Ninety eight (72.1%)serum samples were positive for both RF and anti-CCP and 154(58.3%) serum samples were negative for both of tests. Calculated concordance percent was 63%. Among patients with early RA disease 138 (69.7%) cases were positive for anti-CCPand 84(42.4%) cases were positive for RF. Among patients with symptoms lasting more than one year, 47cases (78.3%) had positive anti-CCP results and 35(58.3%) patients were positive for RF test.
Recent papers have highlighted the importance of early treatment of Rheumatoid Arthritis with Disease Modifying Anti Rheumatic Drugs (DMARDs). Consequently, these findings have shifted the medical problem from ''how to treat'' to ''how to make an early diagnosis of rheumatoid arthritis''. Some studies have demonstrated the value of biological markers by using diagnostic models of erosive arthritis (9). Markers such as rheumatoid factor (RF) and anti-cyclic citrullinated (filaggrin) peptide antibodies (anti-CCP antibodies) were selected from among other markers. Rheumatoid factor (RF) has been widely used as a screening test for patients with arthritis. Although RF is prognostic ally useful, as it correlates with functional (7) and radiographic (10) outcomes in both RA and early inflammatory polyarthritis (11) and also constitute one of the classification criteria proposed by the American College of Rheumatology (ACR), as a diagnostic test it performs poorly, with low sensitivity and moderate specificity (12).RF is present in patients with other autoimmune and infectious diseases, and even in a noticeable proportion of normal healthy subjects, particularly in old individuals( 13). More recently anti-CCP (anti-cyclic citrullinated peptide) has been described for RA. About 35-40% of RF-negative patients are anti-CCP antibody-positive. Anti-CCP antibodies have also demonstrated prognostic utility with regard to radiographic outcomes (14).The present study compared diagnostic performances of these two tests in RA patients in comparison to those in non-RA rheumatic diseases.In our study the sensitivity and specificity of RF were 46.2 and 90.29%, respectively. These values are similar to those reported by Aflaki using latex fixation test (15) but was inferior to some previous studies (8). Possible explanation for the discrepancy could arise from racial and geographical differences. In our study 57.6% of patients with early RA had negative RF results. Although negative RF results are consistent with conditions other than RA but, because of low sensitivity, do not rule out RA. Because RF-negative patients may seroconvert, follow up testing during the first year of disease may be useful .In the present study the anti-CCP antibody test was positive in 110 cases (41.7 %)of seronegative RA patients. Although some studies, using latex fixation or nephelometry, reported that RF had similar specificities and sensitivities (16) therefore, the use of latex agglutination test for RF probably did not contribute to lower sensitivity in the present study, mean while other studies showed that specificity and sensitivity of RF have been improved by the development of enzyme-linked immunosorbent assays (ELISA). They have found out that RF can be detected in up to 70-80% of RA patients by ELISA method (17).
In the current study, we found that RF was positive in only 58.3% of the advanced RA cases. This might be due to treatment with DMARDs. Several studies documented that the level of the RF decrease with the administration of DMARDs.(18).
Current study showed that RF was also positive in 13 patients with non-RA rheumatic diseases . These findings are similar to those of a previous study which showed RF was positive in many other rheumatic and non-rheumatic diseases (19). Thus it seems that two major disadvantages of RF are low specificity and possible absence in the first year of the disease .Our study showed that anti- CCP had a sensitivity of 70.67% and specificity of 85.07%. The sensitivity was similar to that reported by Hussin (20) but more than that reported by Sharif (21). The specificity of anti-CCP in the present study was somehow lower than that reported by Hussin (20)but similar to that of Sharif et al(21). To explain the differences between reported sensitivity and specificity of anti-CCP in different studies it must be considered that1- anti-CCP antibodies are a heterogeneous group of antibodies directed against different epitopes on the citrulline molecule, that each patient's serum contains different subsets of antibodies, and that the synthetic peptide used in this assay represents a relatively small set of antigenic determinants that do not entirely encompasses the antigenic determinants present on the as yet unknown antigenic molecule in the joint (22 ). 2-In addition in laboratory assessment, we used the first generation of anti-CCP-1 kit. However, the first-generation anti-CCP assay has low analytical sensitivity (ranging from 48 to 68%) (23).3-In one study it was showed that the specificity and sensitivity of anti-CCP antibodies may depend on patient's race (24). 4- Another explanation for the discrepancy is that the differences in the patient populations (especially disease duration) among these studies might have influenced the results. For example in the study of Kamali et al. (25) the sensitivity of the anti-CCP antibody for RA is higher than ours. Their patients had at least 1 year of disease duration, in contrast to the symptom duration of less than one year in nearly 70% of our patients. Nevertheless in this study it was interesting to evaluate anti-CCP behavior in RA patients in relation to the duration of disease. In patients with early arthritis the correlation with anti-CCP was significant, thus indicating that this assay may be used even in the early phases of disease. 5-Specificity in each diagnostic test is negatively related to frequency of false positive results. So differences in specificity of anti-CCP might be due to frequency of positive anti-CCP results in non-RA patients.
The high specificity of anti-CCP assays in some studies did not exclude some false positive results, some patients with various non-RA diseases demonstrated high anti-CCP titers.
In our study the positive predictive value of anti-CCP was 90.38% and ppv of RF was 90.44%. The negative predictive value of anti-CCP was 59% and negative PV of RF was 45%. It should be noted that the value of any diagnostic test has been related to its disease prediction ability. Positive predictive value in diagnostic studies is related to disease prevalence and thus in population with lower prevalence we suspected to have low PPV. In one study it was noted that anti-CCP positivity was significantly higher in RA patients with severe than those with minimal joint destruction (26). Although in the present study we didn't evaluate this parameter but we found out that in higher titers of anti-CCP the diagnosis of RA was more accurate.
This study showed that anti-CCP indeed is a good serological marker for RA. Anti-CCP is well suited as a front line diagnostic test for RA and especially early RA. In RF seronegative patients anti-CCP can be helpful in confirming the diagnosis of RA.A combination of RF and anti-CCP has a higher diagnostic and prognostic potential than either of these serological markers alone, it seems logical to add anti-CCP as an eighth criterion.
We thank shahid sadoughi university of medical sciences for their support.
Conflict of interest: not declared.
Funding support: This work was supported by Shahid Sadoughi University of medical sciences and health services, Yazd,Iran
Table-1:Shows demographic information according to gender.
Tables-2: shows demographic information according to age.
Age groups RA
Table-3: shows relative distribution of anti-CCP results in studied patients according to RA diagnosis
Figure 1 Receiver operating curve for anti-cyclic citrullinated
peptide antibody in rheumatoid arthritis.