Original Article

Anti CCP antibody assay: a diagnostic dilemma in diagnosis of tubercular Synovitis.

^1,Saurabh Singh ²Usha Singh, ¹AbhijeetKunwar ²Neha Chaurasia

• ¹Department of Orthopedics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
• ²Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

• Submitted:Wednesday, April 2, 2014
• Accepted:Saturday, April 26, 2014
• Published:Sunday, April 27, 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Abstract

Objective

Anti-CCP antibodies assays have been reported to be a specific diagnostic marker than rheumatoid factor in the diagnosis of rheumatoid synovitis. There may be diagnostic dilemma if anti CCP antibody assays are also positive in cases of tubercular synovitis. In this study we look for prevalence of anti CCP in cases of active tubercular synovitis of knee and compare it with controls.

Methods

Serum levels of anti CCP antibodies were measured in 31 patients of tubercular arthritis of knee and were compared with equal number of age matched controls.

Results

Discussion

Anti CCP assay for diagnosis of rheumatoid synovitis may give false positive results in patients of tubercul ararthritis

Conclusion

Test for anti CCP antibody is positive in many patients of tuberculous synovitis.

Keywords

Tuberculosis, rheumatoid arthritis, false positive

Introduction

The global incidence of tuberculosis (TB) is around 8.8 million and prevalence 14 million as per World Health Organization reports and of this 1%–3% is skeletal disease [1]. TB arthritis is characteristically monoarticular [2] and most often affects the spine and weight-bearing joints such as the knee and hip. In spite of monoarthritis being the most common presentation in weight bearing joints oligo or polyarthitis is not a rare presentation resembling the clinical picture of spondylo arthropathies [3] or rheumatoid arthritis (RA) [4]. Patients of tuberculosis may have rheumatoid factor in their serum, in 40% cases [5].

In a recent study, anti-CCP antibody positivity has been reported in 32% of patients with active pulmonary TB [6]. With increasing prevalence of TB in developed nations and their continued presence in developing nations this may create diagnostic dilemma in establishing a case of mono or pauciarticular arthritis as that of tuberculosis or of rheumatoid arthritis. Anti-CCP antibodies have been reported to be a novel and more specific diagnostic marker than rheumatoid factor (RF) in the diagnosis of RA. Antibodies that are formed against CCP primarily belong to the immunoglobulin G class and are 97% specific for RA [7,8]. With emerging evidence linking anti CCP positivity with pulmonary tuberculosis there is need to further investigate the association of anti CCP positivity with various forms of tuberculosis. In this study we intend to study prevalence of anti CCP positivity in cases of active TB arthritis of knee and compare it with the prevalence in healthy controls.

Patients and Method

A total of 31 patients (20 males and 11 females) of newly diagnosed active TB arthritis of knee (29 unilateral and 2 bilateral knee diseases) were included in the study. Provisional diagnosis of TB arthritis was made on the basis of history of fever, chronic knee pain, clinical examination and plain radiography findings. All patients were subjected to open synovial biopsy and only patients who proved positive for tuberculosis on histopathological examination were included in the study.

Serum sample for anti CCP was taken upon histopathological confirmation of TB arthritis. Only newly diagnosed patients of tubercular arthritis of knee were included and patients on prior anti tubercular drug therapy were excluded from the study.The kit used for the test was Genesis Diagnostics’ (Cambridgeshire, UK, www.elisa.co.uk) EDRA Genesis CPA kit. This is an Enzyme linked immune sorbent assay (ELISA) test and the steps of handling of serum sample and of the test were followed as per the instructions of the manufacturer of the kit. In this test a value of more than 6.25 is considered to be positive. A questionnaire was used to determine the presence of rheumatological symptoms like pain in other joints, myalgia, rash, mucocutaneous symptoms, family history of autoimmune diseases. Along with this, ACR/EULAR classification criteria (2010) [9] was used to rule out the possibility of RA in the cases and control groups. According to the ACR/EULAR 2010 criteria a score of more than or equal to 6 out of 10 is needed for the patients to be termed as of rheumatoid arthritis [9].

Statistical analysis

Statistical analysis was performed by the SPSS 17 software, using Student’s t test and a Chi square test to compare antibody titres or positivity rate, respectively, between patients with TB and controls. Pearson correlation coefficients were used to study the relationship between clinical measures and the levels of anti-CCP. A value of p<0.05 was considered significant.

Results

Table 1 summarizes the results of the clinical characteristics of patients with TB and healthy controls.

The patients had a mean duration of fever of 3.4 months, 74.19% had fever. Only a small minority had symptoms such as arthralgia [3.2%], myalgias [6.4%]. None of the patients had signs typical of rheumatoid arthritis such as arthritis, morning stiffness or rheumatoid nodules or could get a 6 or higher score on ACR/EULAR2010 classification criteria [9].

Serum levels of anti-CCP

The mean (SD) levels of anti-CCP were significantly increased in patients with TB arthritis in comparison with controls: [9.95[10.81] V 4.09[0.83]) (p value<0.005). Serum levels above the upper normal limits (6.25 IU) were found in 12/31 (38.7%) patients in comparison with 1/47 (2.1%). Gupta et al determined the sensitivity and specificity of use of anti CCP antibody assay in 63 Indian patients and found fifty-four of 63 RA patients (85.71%) were positive for anti-CCP, while 9 patients tested negative for anti-CCP antibodies. Their study found a sensitivity of 85% and a specificity of 90.19% in regards to the use of anti-CCP antibodies assay in patients with synovitis and joint pain to correctly identify rheumatoid arthritis [10].

Discussion

Antibodies recognising cyclic citrullinated peptides are highly specific for RA [11]. The specificity for RA has been shown to be up to 98% in comparison with 0–1% of healthy controls and 2–5% of disease controls. Anti-cyclic citrullinated proteins (anti-CCP) are present early in the disease process and may even pre-date the onset of RA by many years [12].

In 1964, Nienhuis and Mandema described an autoantibody they called antiperinuclear factor. Detected by indirect immunofluorescence test on human buccal mucosa cells, antiperinuclear factor recognized antigens present in keratohyalin granules surrounding the nucleus [13]. Antiperinuclear factor was present in up to 90% of established RA patients, with 73–99% specificity [14]. Young and colleagues later detected antikeratin antibodies using indirect immunofluorescence on cryosections of rat esophagus [15]. The reported sensitivity of the antikeratin assay in RA patients was 36–59% and the specificity was 88–99% [14]. Despite the high specificity for RA, these tests were not widely used because of difficulty in standardization of natural substrates and arbitrary interpretation of the indirect immunofluorescence pattern. In 1995, Sebbag and colleagues demonstrated that both of these antibodies belonged to a family of autoantibodies directed against citrullinated filaggrin, an epithelial cell protein [16]. Citrullination is a posttranslational modification of arginine to citrulline by the enzyme peptidyl arginine deiminase. This process occurs naturally during inflammation, apoptosis, and keratinization [17]. Thus it can be postulated that anti CCP antibody may be present in other inflammatory disorders. Although filaggrin is not present in the synovium [18], several citrullinated proteins, including fibrinogen and fibronectin, are present in RA synovium, and other citrullinated epitopes have been identified as targets of highly RA-specific autoantibodies [19,21]. The first commercially available ACPA assay (first-generation CCP [CCP-1]) was developed by Euro-Diagnostica (Arnhem, The Netherlands) and was used in early studies (2000 to 2001). This ELISA-based test employed a single CCP derived from filaggrin. The assay detected autoantibodies in 53% of established RA patients, with 96% specificity [22]. Peptide libraries were then screened for better epitopes. Since 2000, second-generation CCP (CCP-2) and third-generation CCP (CCP-3) assays have been developed. The compositions of many new CCP-3 peptides are not yet publicly available because patents are pending [23]. We in this study have used anti CCP 2assay.

Anti CCP antibody assays are able to predict the development of RA in healthy subjects [24], and are prognostic markers of erosive disease progression [25]. It has also been shown that this marker system can help in discriminating between RA and other forms of erosive arthritis that can simulate rheumatoid arthritis [26]. Shankar et al [27] in their study of 211 patients with established RA concluded that anti CCP-2 antibodies were the most important factor inpredicting erosive disease.They were of the opinion that these antibodies were strong predictors of erosive disease in both seropositive and seronegative RA and had an additional role in predicting cumulative radiological damage over and above that predicted by rheumatoid factor. Anti CCP antibodies are rarely detectable in other diseases, and in these cases usually with low titers [28]. There are many differential diagnosis that have to be kept in mind while labeling a patient as of rheumatoid arthritis especially when the disease in monoarticular or pauciarticular involving the large joints. Amongst them one is early stage of TB arthritis.TB arthritis is commonly is monoarticular [2] and most often affects the spine and weight-bearing joints such as the knee and hip. The mode of transmission is hematogenous from visceral foci such as the lung or kidneys [1]. Articular disease often starts as a synovitis progressing to arthritis, with demineralization, marginal erosions, and ultimate joint destruction [29]. While the time period from synovitis to arthritis and erosions may be long, progression to joint destruction can be rapid, particularly in weight-bearing joints.Thus there is a need for early diagnosis of the disease and institution of appropriate treatment [30].

Patients with TB can exhibit various forms of arthropathy, which in some cases may mimic RA [31]. Some of these patients' diseases may mimic early rheumatoid arthritis and may be difficult to diagnose. Thus, anti-CCP–positive test results in these patients can be misleading [31].

In a recent study, the prevalence of anti-CCP antibodies was shown to be 37% in Japanese patients with active pulmonary TB[32]. Elkayam et al [6] compared anti-CCP antibody positivity and Immunoglobulin M (IgM) RF status in 47 patients with active pulmonary TB infection and 39 healthy controls. Anti-CCP titers were above normal in 32% of the patients with active TB infection, while the rate was only 2.6% in controls. Mori et al [33] in their study, we found positivity rate of anti-CCP2in the cases of active lung tuberculosis to be 6.7% (six of 89 patients) and 3.4% of these patients were strongly positive. They found the prevalence of anti-CCP2 inhealthy individuals to be 0.4%.

In our study, the prevalence of anti CCP2 antibody positivity in cases of active tubercular arthritis was found to be 38.7% and that in healthy controls to be 2.1%. Anti CCP antibody assay has also been found to be positive in other disease also.It has also been shown that prevalence of anti-CCP3 in patients with leprosy, another disease caused by mycobacterialinfection is 3.1% [34]. It has also been reported that anti CCP antibodies (using a CCP2 test) can be detected in 9% of patients with autoimmune hepatitis 1, in absence of recognizable rheumatoid arthritis overlap, and in some cases with high titers, comparable to those observed in rheumatoid arthritis [35].

The mechanism of production of anti CCP antibody in patients of TB is still not very evident. The mechanism cited in literature is as follows. Citrulline, a non-standard amino acid, is not incorporated into proteins during translation; citrullinated proteins are generated by post translational conversion of protein-contained arginine residues to citrulline by peptidyl arginine deiminase enzyme. Citrullinated proteins are present in a wide range of inflammatory tissues, with little to no expression in non inflammatory tissues, suggesting that the citrullination of cellular proteins is not specific to RA but is rather a common phenomenon in various inflammatory conditions [36,38]. Citrullination may occur at sites of inflammation in patients with active TB, even without arthritis, and its occurrence may be related to the degree of inflammation. It is possible, therefore, that anti-citrullinated antibodies maybe generated in response to citrullination in some TB patients without arthritis [33]. Kakumanu et al [32] showed that many anti-CCP-positive sera obtained from patients of pulmonary tuberculosis alsoreact with a corresponding non-citrullinated argininecontainingpeptide (CAP) and that High anti-CCP : anti-CAP ratios (>2.0) were seen far more commonly in anti-CCP–positive RA patients than in anti-CCP–positive TB patients (94% versus 22%). They also revealed that the anti-CCP reactivity, in sera from the RA patients is nearly completely inhibited bypreincubation with CCP but this does not happen in serafrom tuberculosis patients [32]. These data suggest that anti-CCP in sera from RA patients is specific to CCP, whereas theanti-CCP reactivity seen in the sera of tuberculosis patients may be citrulline-independent [33].

Thus in the light of these new revelations the validity of anti CCP antibody assay in diagnosis of RA has come into question. With increasing prevelance of TB in developed nations and continued presence of the disease in the developing nations, the use of anti CCP antibody in diagnosis of RA must also take into account other clinical and laboratory features of rheumatoid arthritis. Early onset TB arthritis should be kept as a differential and must be ruled out systematically in patients presenting with large joint mono or pauciarticular synovitis with positive anti CCP antibody assay.

Conclusion

Anti CCP antibody assay may be false positive in many patients of tubercular synovitis. Patients presenting with mono or pauciarticular pain of large joints with positive anti CCP antibody assay have to be investigated to rule out the possibility of early stage of tubercular arthritis before labeling them as of rheumatoid arthritis. Low threshold for suspicion and thorough investigative workup is essential for early diagnosis, appropriate treatment and good outcome in tubercular arthritis.

Authors Contributions

SS : Designed the study and clinical diagnosis

US: Laboratory investigations done under her supervision, writing of laboratory part

 AK: Clinical diagnosis and assisted in manuscript compilation

 NC : Assisted in laboratory investigations and drafting of manuscript

Conflict of interest

The authors declare that there is no conflict of interests

Acknowledgements

None

Ethical considerations

The study was approved by the Institute Ethics Committee, written informed consent was obtained from all the subjects participating in the study.

References

[1]Tuli SM. General principles of osteoarticular tuberculosis. ClinOrthop 2002; 398:11-9[pubmed]

[2]Sequeira W, Co H, Block JA. Osteoarticular tuberculosis: current diagnosis and treatment. Am J Ther 2000; 7:393-8[pubmed]

[3]Hammoudeh M, Khanjar I. Skeletal tuberculosis mimicking seronegativespondyloarthropathy. Rheumatol Int. 2004; 24: 50–2, Epub 2003 May 29.. [pubmed]

[4]Bush DC, Schneider LH. Tuberculosis of the hand and wrist. J Hand Surg [Am] 1984; 9:391–8[pubmed]

[5]Isenberg DA, Maddison P, Swana G, et al. Profile of autoantibodies in the serum of patients with tuberculosis, klebsiella and other gram-negative infections. ClinExpImmunol 1987;76:516–23[pubmed]

[6]Elkayam O, Segal R, Lidgi M, et al.Positive anti-cyclic citrullinated proteins and rheumatoid factor during active lung tuberculosis. Ann Rheum Dis 2006; 65: 1110-2[pubmed]

[7]Van Boekel MA, Vossenaar ER, van den Hoogen FH,etal.Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res 2002; 4: 87-93[pubmed]

[8]Schellekens GA,Visser H, de Jong BAW, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing anti-cyclic citrullinated peptide. Arthritis Rheum 2000; 43: 155-63. [pubmed]

[9]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. 2010;62:2569-81[pubmed]

[10]Gupta R, Thabah MM, Aneja R, et al. Usefulness of anti -CCP antibodies in rheumatic diseases in Indian patients. Indian J Med Sci 2009; 63: 92-100[[pubmed]

[11]Van Venrooj WJ, Hazes JMW, Visser H. Anti-citrullinated protein/peptideantibody and its role in the diagnosis and prognosis of early rheumatoidarthritis. Neth J Med 2002;60:383–8.

[12]van Gaalen FA, Linn-Rasker SP, van Venrooij WJ, et al. Autoantibodies to cyclic citrullinated peptides predictprogression to rheumatoid arthritis in patients with undifferentiated arthritis: aprospective cohort study. Arthritis Rheum 2004;50:709–15[pubmed]

[13]Nienhuis RL, Mandema E. A new serum factor in patients with rheumatoid arthritis: the antiperinuclear factor. Ann Rheum Dis1964; 23: 302–5[pubmed]

[14]Hoet RM, Boerbooms AM, Arends M, et al. Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin. Ann Rheum Dis1991; 50: 611–8[pubmed]

[15]Young BJ, Mallya RK, Leslie RD, et al. Anti-keratin antibodies in rheumatoid arthritis. Br Med J1979; 2: 97–9[pubmed]

[16]Sebbag M, Simon M, Vincent C,et al. The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J Clin Invest1995; 95: 2672–9[pubmed]

[17]Lee DM, Schur PH. Clinical utility of the anti-CCP assay in patients with rheumatic diseases. Ann Rheum Dis2003; 62: 870–4[pubmed]

[18]]Baeten D, Peene I, Union A,et al. Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies. Arthritis Rheum2001; 44: 2255–62.[pubmed]

[19]Schellekens GA, de Jong BA, van den Hoogen FH, et al. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest1998; 101: 273–81[pubmed]

[20]Nogueira L, Sebbag M, Vincent C,et al. Performance of two ELISAs for antifilaggrin autoantibodies, using either affinity purified or deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis.Ann Rheum Dis2001; 60: 882–7[pubmed]

[21]Vincent C, Nogueira L, Sebbag M,et al. Detection of antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent assay: a highly effective test for the diagnosis of rheumatoid arthritis. Arthritis Rheum2002; 46: 2051–8[pubmed]

[22]Schellekens GA, Visser H, de Jong BA, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000; 43: 155–63[pubmed]

[23]Aggarwal R, Liao K, Nair R, et al. Anti–citrullinated peptide antibody assays and their role in the diagnosis of rheumatoid arthritis. Arthritis Care & Research 2009; 61: 1472–83[pubmed]

[24]Rantapää‐Dahlqvist S, de Jong B A, Berglin E, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48: 2741–9[pubmed]

[25]Vencovský J, Machacek S, Sedova Let al. Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis. Ann Rheum Dis 2003;62: 427–30[pubmed]

[26]Mediwake R, Isenberg D A, Schellekens G A, et al. Use of anti‐citrullinated peptide and anti‐RA33 antibodies in distinguishing erosive arthritis in patients with systemic lupus erythematosus and rheumatoid arthritis. Ann Rheum Dis 2001;60: 67–8[pubmed]

[27]Shankar S, Grover R, Handa R. Role of anti cycliccitrullinated peptide antibodies in erosive disease in patients with rheumatoid arthritis. Indian J Med Res 2006; 124: 689-96[pubmed]

[28]Bizzaro N, Mazzanti G, Tonutti E, et al. Diagnostic accuracy of the anti‐citrulline antibody assay for rheumatoid arthritis. ClinChem 2001; 47: 1089–93[pubmed]

[29]]Furia JP, Box GG, Litner DM. Tubercular arthritis of the knee presenting as a meniscal tear. Am J Orthop 1996;25:138-42[pubmed]

[30]Al-Shaikh R, Goodman SB. Delayed onset mycobacterium tuberculosis infection with staphylococcal super-infection after total knee replacement. Am J Orthop 2003;32:302-5.[pubmed]

[31]Franco-Paredes C, Diaz-Borjon A, Senger MA, et al. The ever-expanding association between rheumatologic diseases and tuberculosis.Am J Med 2006; 119: 470–7.[pubmed]

[32Kakumanu P, Yamagata H, Sobel ES, et al. Patients with pulmonary tuberculosis are frequently positive for anti-cyclic citrullinated peptide antibodies, but their sera also react with unmodified arginine-containing peptide. Arthritis Rheum 2008 Jun; 58: 1576-81[pubmed]

[33Mori S, Naito H, Ohtani S, et al. Diagnostic utility of anti-cyclic citrullinated peptide antibodies for rheumatoid arthritis in patients with active lung tuberculosis. ClinRheumatol 2008; 28: 277-83. [pubmed]

[34Guedes-Barbosa LS, Mangueira C, Scheinberg M.Anticitrullinepeptide antibodies (CCP3) in leprosy sera: a negativeassociation. ClinRheumatol 2008; 27:515–6[pubmed]

[35].Fusconi M, Vannini A, Dall'aglio A C, et al.Anti‐cyclic citrullinated peptide antibodies in type 1 autoimmune hepatitis. Aliment PharmacolTher 2005. 22951–5.[pubmed]

[36]. Makrygiannakis D, Klint E, Lundberg IE, et al. Citrullination is an inflammation-dependent process. Ann RheumDis 2006; 65:1219–22[pubmed]

[37Vossenaar ER, Nijenhuis S, Helsen MM et al.Citrullinationof synovial proteins in murine models of rheumatoid arthritis.Arthritis Rheum 2003; 48:2489–500[pubmed]

[38Vossenaar ER, Smeets TJ, Kraan MC, et al.The presence of citrullinated proteins is notspecific for rheumatoid synovial tissue. Arthritis Rheum 2004; 50:3485–94[pubmed]