Inflammatory Bowel Disease: Serologic Markers and Pharmacogenomic and Metabolic Assessment of Thiopurine Therapy

Number: 0249

  1. Aetna considers TPMT gene mutation or TPMT phenotypic assays (e.g., Prometheus TPMT Genetics, Prometheus TPMT Enzyme) medically necessary prior to initiation of 6-mercaptopurine or azathioprine therapy.  Only 1 genotypic or phenotypic assay of TPMT activity is necessary per member per lifetime.  TPMT gene mutation assays and TPMT phenotypic assays are considered experimental and investigational for all other indications because their effectiveness for indications other than the one listed above has not been established.

  2. Aetna considers testing for anti-chitobioside carbohydrate antibodies (ACCA), anti-laminaribioside carbohydrate antibodies (ALCA), anti-mannobioside carbohydrate antibodies (AMCA), anti-chitin IgA (Anti-C), and anti-laminarin IgA (Anti-L), anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisiae antibodies (ASCA), anti-outer membrane porin C (OmpC) antibodies, anti-CBir1 flagellin (anti-CBir1) antibodies, and I2 antibodies experimental and investigational to diagnose inflammatory bowel disease, to distinguish ulcerative colitis from Crohn's disease, and for all other indications because their effectiveness has not been established. 

  3. Aetna considers anti-smooth muscle antibodies (ASMA) experimental and investigational to diagnose inflammatory bowel disease or to distinguish ulcerative colitis from Crohn's disease because its effectiveness for these indications has not been established.  (Note: ASMA may be medically necessary to diagnose autoimmune hepatitis).

  4. Aetna considers measurements of 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN) (e.g., Prometheus Thiopurine Metabolites) medically necessary to monitor compliance in those not responding to 6-mercaptopurine and azathioprine therapy and to assess suspected toxicity.

  5. Aetna considers fecal measurement of calprotectin experimental and investigational for the management of inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis) and other indications because its clinical value has not been established.

  6. Aetna considers NOD2/CARD15 genotyping experimental and investigational because its clinical value has not been established.

  7. Aetna considers CD14 gene C-260T polymorphism testing, interleukin-10-1082A/G polymorphism testing, Prometheus Crohn's Prognostic, Prometheus IBD sgi diagnostic panel and the Prometheus IBS diagnostic panel (including BDNF [brain-derived neurotrophic factor], GRO-α [growth-regulated oncogene-alpha], IL-1β [interleukin -1 beta], NGAL [neutrophil gelatinase-associated lipocalin], TIMP-1 [tissue inhibitor of metalloproteinase-1], tTG [anti-human tissue transglutaminase IgA), and TWEAK [TNF-related weak inducer of apoptosis]) experimental and investigational for the diagnosis and management of inflammatory bowel diseases because its clinical value has not been established.

  8. Aetna considers the following tests experimental and investigational because their clinical value has not been established.

See also CPB 0341 - Remicade (Infliximab) (for measurements of serum levels of infliximab and antibodies to infliximab), and CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing


Serologic Markers of Inflammatory Bowel Disease:

A serology panel including anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisiae IgG and IgA antibodies (ASCA), and anti-OmpC antibodies (outer membrane porin from E. coli) are marketed by Prometheus Laboratories (San Diego, CA) as the IBD First Step. This panel has not been shown to have levels of specificity sufficient to distinguish ulcerative colitis (UC) from Crohn's disease (CD) in indeterminate cases.

Research into the pathogenesis of inflammatory bowel disease in the areas of mucosal immunology, genetics, the role of bacterial products, and mediators of tissue damage has identified new sets of “subclinical” serological markers known as anti-neutrophil cytoplasmic antibodies (ANCA).  ANCA have also been found to be associated with Wegener's granulomatosis and other forms of systemic vasculitides, and more recently with sclerosing cholangitis and other autoimmune liver diseases.

“Atypical” ANCA yielding a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils is primarily found in patients with UC; pANCA has been found to be detectable in 50 to 80 % of patients with UC, and 10 to 40 % of patients with CD.  Anti-Saccharomyces cerevisiae antibody (ASCA) is primarily detected in patients with CD; ASCA has been found to be detectable in 46 to 70 % of patients with CD and 6 to 12 % of patients with UC.

These tests, however, have insufficient sensitivity to diagnose UC or CD.  In a paper for the North American Society for Pediatric Gastroenterology and Nutrition, Griffiths concluded that “the relatively low sensitivities of serology for [Crohn's disease] and [ulcerative colitis] as documented in all studies argue against there being any greater value of ASCA/ANCA as routine or first-line screening tests for [inflammatory bowel disease] in comparison to clinical acumen and the equally sensitive (albeit less specific) measurement of acute phase reactants.  Moreover, the need for performance of definitive radiologic and endoscopic studies to guide therapy by defining the extent and nature of inflammatory bowel diseases (IBD) will not be averted by positive serologic tests.”  Gupta et al (2004) examined the concordance of serologic testing for inflammatory bowel disease with clinical diagnosis established by traditional testing in children. The investigators found that the sensitivity of serologic testing is insufficient to replace traditional studies when evaluating children for inflammatory bowel disease. The investigators evaluated the results of ANCA and ASCA testing in 107 children who had serologic testing for inflammatory bowel disease (IBD) at their center, and compared these results with their clinical diagnosis.  The investigators calculated that the sensitivity, specificity, positive and negative predictive values of serologic testing for ulcerative colitis were 69.2, 95.1, 90.0 and 87.1 %, respectively, and for CD were 54.1, 96.8, 90.9 and 80.8 % respectively.  The investigators concluded that “[t]he low sensitivity, especially for Crohn's disease, precludes the possibility that the IBD Diagnostic System can replace traditional studies when evaluating for inflammatory bowel disease.”

Some investigators have proposed using these serologic tests to differentiate CD from UC (e.g., Kornbluth and Sachar, 2004).  Differentiation of CD from UC is clinically problematic only when inflammation is confined to the colon.  A number of studies have reported that IgA and IgG ASCA titers are significantly greater and highly specific for CD, and that and that pANCA positivity is highly specific for UC.  However, there is much less published information concerning the subgroup of IBD patients with colitis only, where differentiation of UC from CD is clinically problematic.  One investigator reported ASCA positivity in only 47 % of 17 patients with Crohn's colitis.  Another investigator found only 32 % of 37 patients with Crohn's colitis were ASCA positive and pANCA negative.  Conversely, studies have found that the majority of Crohn's patients positive for pANCA have a UC-like presentations (Bentley et al, 2001; Ruemmele, 1998; Vasiliauskas et al, 1996).  Griffiths explained, “hence, the usefulness of serology is less (where it is needed most), given the higher prevalence of pANCA positivity and the lower prevalence of ASCA positivity in CD confined to the colon".

Similarly, whether or not ASCA/ANCA measurement may be helpful in classifying otherwise 'indeterminate' colitis can not as yet be ascertained.  Only a few patients have been studied, and follow-up is too limited.  In the only prospective study of serologic testing in indeterminate colitis, Joosens et al (2002) examined the results of serologic testing for ASCA or ANCA and final diagnosis of CD or UC after 6-year follow-up of 97 patients with indeterminate colitis.  The largest group (47) of subjects were negative for both ANCA and ASCA; 3 of these had a final diagnosis of CD, 4 had a final diagnosis of UC, and 40 had a final diagnosis of indeterminate colitis.  Of 26 subjects who were ASCA-positive and ANCA-negative, 8 had a final diagnosis of CD after 6 years follow- up, 2 had a final diagnosis of UC, and 16 remained with a diagnosis of indeterminate colitis.  Of 20 subjects who were ASCA-negative and ASCA-positive, 4 had a final diagnosis of CD, 7 had a final diagnosis of UC, and 9 had a final diagnosis of indeterminate colitis.  Only 4 of the 97 subjects were positive for both ANCA and ASCA; 2 of these had a final diagnosis of CD, 1 had a final diagnosis of UC, and 1 remained with a final diagnosis of indeterminate colitis.  Thus, about one-third (31 %) of subjects who were ASCA-positive and ANCA-negative progressed to CD during the 6-year follow-up period, and about one-third (35 %) of subjects who were ASCA-negative and ANCA-positive progressed to UC during the 6-year follow-up period.  Joosens et al (2002) calculated that, thus far, the sensitivity of ASCA+/ANCA- for CD was 66.7 % and the specificity is 77.8 %, and the sensitivity of ASCA-/ASCA+ for UC is 77.8 % and the specificity is 66.7 %.  Noting that these calculations exclude subjects who remained with the diagnosis of indeterminate colitis, a technology assessment of serologic testing for IBD by the Institute for Clinical Systems Improvement noted that only a “small number” (21) of subjects were included in this analysis of sensitivity and specificity. Based on their analysis of this prospective study and other published studies of serologic testing in indeterminate colitis, the ICSI concluded that the clinical utility of serologic testing in indeterminate colitis has not been established.  It has also been noted that this study does not provide direct evidence of improvement in clinical outcomes by basing the management of persons with indeterminate colitis on serologic testing.

ANCA and ASCA testing has not been proven to be useful in selecting therapeutic interventions.  “Although this would be desirable, there is no evidence as yet that serological test results can be used to predict the likelihood of therapeutic response to specific interventions,” Griffiths explained.  In a prospective clinical study of CD patients, Esters et al (2002) found no significant relationship between these serologic markers and response to anti-tumor necrosis factor (TNF) therapy.

In addition, studies have not demonstrated correlation of ANCA or ASCA with disease activity, duration of illness, extent of disease, extra-intestinal complications, or surgical or medical treatment in patients with IBD.  The Institute for Clinical Systems Improvement (ICSI) (2002) technology assessment of serologic testing for IBD concluded “[t]he clinical utility of serological testing is not yet established for the diagnosis of inflammatory bowel disease in patients presenting with symptoms suggestive of IBD” and “[t]he clinical utility of serological testing is not yet established for differentiating between UC and CD in patients with inflammatory bowel disease”.

Additional assays have been developed to use in conjunction with ANCA and ASCA in an effort to improve the diagnostic capabilities of serologic testing.  OmpC IgA is an autoantibody to outer membrane porin to E. coli included in the Prometheus serology panel to enhance detection of CD (Landers et al, 2002).  I2 is an IgA antibody that has been detected in patients with CD.  The I2 serologic response recognizes a novel homologue of the bacterial transcription-factor families from a Pseudomonas fluorescens associated sequence (Sandborn, 2004).  However, there are no studies of the clinical utility of anti-OmpC and I2 IgA antibodies in distinguishing CD from UC in persons with IBD whose diagnosis cannot be established by standard methods.

Landers et al (2002) reported on serologic test results of a referral center population of 151 patients with CD.  This study found that immune responses to specific antigens (ASCA, pANCA, OmpC, and I2) are not uniform among CD patients.  ASCA was detected in 56 % of patients, OmpC in 55 % of patients, I2 in 50 % of patients, and ANCA in 23 % of patients.  The investigators reported that 85 % of patients responded to at least 1 antigen and that only 4 % responded to all 4.  This study did not demonstrate, however, how these serologic test results relate to clinical behavior and response to therapy.  The authors stated that “[t]he relationship of these different patterns of immune responses to clinical behavior is not yet clear.”  The authors concluded “[d]efining how these antibody reactivities relate to clinical behavior and response to therapeutic modalities will require larger numbers of phenotypically well-characterized patients.”

Mow et al (2004) reported on the results of an hypothesis generating study, the aim of which was to determine whether Crohn's patients with predominant serum antibody reactivity toward bacterial antigens OmpC and/or I2 were more likely to achieve remission with antibiotics.  Study subjects were patients with moderately active right-sided colonic and/or small bowel CD who were participating in an 8-week randomized clinical trial comparing the steroid budesonide with or without the antibiotics metronidazole and ciprofloxacin.  Subject's serum was analyzed for ASCA, pANCA, anti-OmpC, and anti-I2 antibodies, and subjects were put into 1 of 4 “profile groups" (ASCA, pANCA, anti-OmpC/I2, and no or little antibody) depending upon the subjects' levels of antibody response.  Twenty-five of 121 subjects were excluded from the analysis because their level of antibody response did not fit the 4 predominant profile groups.  Only 2 subjects had an ANCA predominant profile, and these subjects were excluded from the analysis.  In the steroid plus antibiotic group, 5 of 11 subjects (45.5 %) with predominant OmpC and I2 antibodies achieved remission, 5 of 16 subjects with predominantly ASCA antibody (31.3 %) achieved remission (similar to the overall remission rate), and 5 of 21 subjects (23.8 %) with little or no antibodies achieved remission.  In the steroid only group, 7 of 16 subjects (43.8 %) with little or no antibodies achieved remission, 8 of 20 (40 %) with predominantly ASCA antibody achieved remission, and 3 of 10 (30 %) subjects with predominant OmpC and I2 antibodies achieved remission.  Although there was a trend toward greater responsiveness to therapy that includes antibiotics in subjects with predominant OmpC and I2 antibodies, and a trend toward less responsiveness in subjects with little or no antibodies, these trends did not achieve statistical significance.  The authors concluded that this hypothesis-generating study provides preliminary evidence to suggest that serologic information about CD patients may be helpful in defining patients who would best respond to therapy.  The authors noted, however, that, "[a]lthough these trends are provocative, they lack statistical significance.”  The authors concluded that '[p]rospective randomized placebo controlled trials that do not limit patient selection by disease location and do not have concomitant therapy are warranted to test this hypothesis."

Mow et al (2004) evaluated the sera of 303 patients with CD to determine whether expression of certain antibodies is associated with phenotypic manifestations.  The investigators found that patients expressing I2 were significantly more likely to have fibrostenosing CD (64.4 % versus 40.7 %), and to require small bowel surgery (62.2 % versus 37.4 %).  Patients with anti-OmpC were more likely to have internal perforating disease (50 % versus 30.7 %) and to require small bowel surgery (61.4 % versus 44.2 %).  The investigators stated that these findings suggest an association between these immune responses and CD complications.  The investigators concluded that "[i]n the future, knowledge of serological response may help the clinician determine the risk for more severe disease characteristics and predict disease behaviors.  As a result, it may be possible to tailor therapy more effectively on the basis of specific serological responses.  However, these findings must be confirmed by prospective studies that evaluate the presence of these antibody responses and the development of complicated small bowel disease phenotypes."  An editorial accompanying this study explained that this study is limited by its retrospective nature (Vermeire and Rutgeerts, 2004): "Therefore, it is important that these findings first be confirmed in independent series and more importantly, that prospective studies with these markers be conducted to assess the risk of microbial responses on the development of strictures and perforations and subsequent need for surgery."

The flagellin-like antigen, CBir1, is a associated with the presence of IBD.  In particular, serum response to anti-CBir1 identifies the presence of CD and is associated with a subset of patients with this form of IBD.  CBir1 is found in the C3H/HeJBir mouse model.  It is believed that the Cdcs1 locus of the C3H/HeJBir mouse confers severe colitis associated with a decrease in innate immune function and an increase in adaptive T-cell responses to commensal bacterial products. No evidence-based clinical guidelines from leading medical professional organizations recommend the use of this marker.

Targan et al (2005) assessed serum response to CBir1 flagellin in CD patients and compared this response to responses defined previously to ASCA, I2, OmpC, and pANCA, and determined anti-CBir1-associated phenotypes.  A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were evaluated for clinical phenotype associations.  The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently.  Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ UC patients.  Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features.  Levels of anti-CBir1 are increased in 50 to 55 % of patients with CD.  The authors concluded that serum responses to CBir1 independently identify a unique subset of patients with complicated CD.

Dubinsky et al (2006) examined the association of immune responses to microbial antigens with disease behavior and prospectively determined the influence of immune reactivity on disease progression in pediatric CD patients.  Sera were collected from 196 pediatric CD cases and tested for immune responses: anti-I2, anti-OmpC, anti-CBir1, and ASCA using enzyme-linked immunosorbent assay (ELISA).  Associations between immune responses and clinical phenotype were evaluated.  A total of 58 patients (28 %) developed internal penetrating and/or stricturing (IP/S) disease after a median follow-up of 18 months.  Both anti-OmpC (p < 0.0006) and anti-I2 (p < 0.003) were associated with IP/S disease.  The frequency of IP/S disease increased with increasing number of immune responses (p trend = 0.002).  The odds of developing IP/S disease were highest in patients positive for all 4 immune responses (odds ratio [OR] (95 % confidence interval [CI]): 11 (1.5 to  80.4); p = 0.03).  Pediatric CD patients positive for greater than or equal to 1 immune response progressed to IP/S disease sooner after diagnosis as compared to those negative for all immune responses (p < 0.03).  The authors concluded that the presence and magnitude of immune responses to microbial antigens are significantly associated with more aggressive disease phenotypes among children with CD.  This was the first study to prospectively demonstrate that the time to develop a disease complication in children is significantly faster in the presence of immune reactivity, thus predicting disease progression to more aggressive disease phenotypes among pediatric CD patients.

In a review on serological markers in IBD, Bossuyt (2006) stated that several antibodies have been associated with IBD, the 2 most comprehensively studied being autoantibodies to neutrophils (atypical pANCA) and ASCA.  New microbial target antigens such as OmpC, I2, and the flagellin CBir1 have been described in patients with CD.  There is evidence that the number and magnitude of immune responses to different microbial antigens are associated with the severity of the disease course.  However, this should be confirmed by additional studies.

Benor et al (2010) compared the predictive values of the Prometheus Inflammatory Bowel Disease (IBD) Serology 7 (IBD7) panel (Prometheus Laboratories, San Diego, CA) with the predictive values of routine blood tests in a population of children referred for initial evaluation of suspected IBD.  Medical records of pediatric patients referred for evaluation of IBD for whom IBD7 testing was performed at Prometheus Laboratories between January 2006 and November 2008 were reviewed.  Patients underwent diagnosis by pediatric gastroenterologists on the basis of clinical, radiologic, endoscopic, and pathologic evaluations.  A total of 394 records were identified.  These investigators excluded 90 records on the basis of age of greater than 21 years, previous diagnosis of IBD, or unclear diagnosis.  The prevalence of IBD in this cohort was 38 %.  The sensitivity, specificity, positive predictive value, negative predictive value, and kappa value for the serological panel were 67 %, 76 %, 63 %, 79 %, and 42 %, respectively, compared with values for a combination of 3 abnormal routine laboratory test results of 72 %, 94 %, 85 %, 79 %, and 47 %.  The anti-flagellin antibody assay, the newest assay added to the panel, had sensitivity of 50 % and specificity of 53 %.  Repeat serological testing failed to produce consistent results for 4 of 10 patients.  The authors concluded that despite its recent inclusion of the anti-flagellin assay, the IBD7 panel has lower predictive values than routine laboratory tests in pediatric screening for IBD.

In summary, the testing for ANCA, ASCA, OmpC antibodies, I2 antibodies, and anti-CBir1antibodies appears to be a promising approach to diagnose IBD, and to distinguish UCfrom CD.

The anti-smooth muscle antibody (ASMA) test detects autoantibodies directed against smooth muscle.  While this test has been employed in the management of patients with autoimmune hepatitis and cirrhosis, its use in the diagnosis of IBD has not been established.

The North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the Crohn’s and Colitis Foundation of America's consensus conference report on differentiating UC from CD in children and young adults (Bousvaros et al, 2007) stated that the clinical value of serology in patients with indeterminate colitis (IC) remains a topic of research, and further investigation should ascertain, among other areas, the role of surrogate laboratory markers (e.g., genetics, microbiology, and serology) in distinguishing these entities.  A proposed algorithm to aid clinicians in differentiating UC from CD does not include serological testing.

Mokrowiecka et al (2009) stated that the role of pANCA and ASCA assessment in IBD diagnosis and differentiating is still imprecise and controversial.  These researchers determined the accuracy of pANCA and ASCA in patients with IBD subgroups.  The study was performed in 125 patients: 71 patients with UC, 31 with CD and 23 with IC.  Control group consists of 45 patients with functional intestinal disorders; pANCA and ASCA (IgA and IgG) were measured with ELISA, using commercial antibody panel.  In UC patients the prevalence of pANCA was 68 %, which was significantly higher than in CD -- 29 %.  ASCA were found significantly more often in CD -- 80.6 % than in UC patients -- 26.8 %.  Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of pANCA for UC diagnosis was 68 %, 84 %, 75 % and 78 %; and ASCA for CD: 81 %, 78 %, 45,5 % and 95 %, respectively.  The combined use of these 2 markers gave changes in diagnostic accuracy: pANCA+/ASCA- in UC: 42 %, 100 %, 100 % and 43 %, and for pANCA-/ASCA+ in CD: 52 %, 98.6 % 94 % and 82 %, respectively.  The authors concluded that the specificity of these combined markers tends to be higher than sensitivity, what made them more useful in the differentiation of the IBD subtypes rather than population screening.  The characteristic IC serotype pANCA(-)ASCA(-) leads to further controversies about origin of this IBD subtype.

The American College of Gastroenterology practice guidelines for management of CD in adults (Lichtenstein et al, 2009) stated that serological studies evaluating ASCA, ANCA, anti-CBir1, anti-OmpC are evolving to provide adjunctive support for the diagnosis of CD; however, they are not sensitive or specific enough to be recommended for use as a screening tools.

Lembo and colleagues (2009) stated that currently, no single serum biomarker can reliably differentiate IBS from other functional gastrointestinal disorders or organic diseases of the gastrointestinal tract.  These researchers attempted to develop and validate a diagnostic test using serum biomarkers to detect IBS.  A total of 10 serum biomarkers (ANCA, ASCA-IgA, BDNF [brain-derived neurotrophic factor], CBir1, GRO-α [growth-regulated oncogene-alpha], IL-1β (interleukin -1 beta], NGAL [neutrophil gelatinase-associated lipocalin], TIMP-1 [tissue inhibitor of metalloproteinase-1], tTG [anti-human tissue transglutaminase IgA], and TWEAK [TNF-related weak inducer of apoptosis]) were selected from a potential panel of 140 for their ability to differentiate IBS from non-IBS disease in blood samples from patients with IBS, other gastrointestinal disorders and healthy volunteers.  A predictive modelling tool was developed to assess patterns and relationships among the 10 serum biomarkers that best differentiated IBS patients from healthy controls and patients with non-IBS gastrointestinal disease.  This model was tested in a different cohort of patients and healthy controls (n = 516) to determine the predictive accuracy of differentiating IBS from non-IBS.  The sensitivity and specificity of the 10-biomarker algorithm for differentiating IBS from non-IBS was 50 % and 88 %, respectively.  The positive predictive value was 81 %, and the negative predictive value was 64 % at 50 % IBS prevalence in the validation cohort.  Overall accuracy was 70 %.  The value of this panel of biomarkers in the management of patients with IBS needs to be validated.

In a review on the importance of early diagnosis in patients with IBS, Halpert (2010) stated that the diagnosis of IBS is challenging because symptoms can vary between patients and overlap with those of other disorders.  This investigator examined the current diagnostic approach in IBS and discussed new tools that may improve diagnostic confidence earlier in the process.  The author noted that accumulating evidence suggests that fecal and/or serum biomarkers may be helpful in differentiating IBS from non-IBS disorders.  These tools may help minimize unnecessary testing and diagnostic delays.  As biomarkers are further studied and developed, they are likely to become an integral part of the diagnosis of IBS and reduce the potential for incorrect diagnosis and treatment delays.

Dotan (2010) stated that IBD are chronic intestinal disorders where, in genetically susceptible hosts, an intestinal microorganism triggers an over-reactive immune response.  Antibodies against luminal antigens are specifically associated with CD.  In addition to the previously described ANCA, ASCA, OmpC, I2 and CBir1 Flagellin, new anti-glycan antibodies were recently added to the armamentarium of serologic markers in IBD.  The anti-glycan antibodies are directed against laminaribioside, chitobioside, mannobioside and mannan residues and are designated anti-laminaribioside carbohydrate antibodies (ALCA), anti-chitobioside carbohydrate antibodies (ACCA), anti-mannobioside carbohydrate antibodies (AMCA) and gASCA, respectively.  Anti-laminarin IgA (Anti-L), and anti-chitin IgA (Anti-C) are new members of this family.  Laminarin and chitobioside are capable of stimulating the innate immune system, thus the finding of antibodies against these glycans suggests a connection between the adaptive and innate arms of the immune response in CD patients.  The contribution of serologic markers, specifically the anti-glycan antibodies, to IBD diagnosis may be in differentiating IBD from other gastrointestinal diseases, and between CD and UC, in better classifying undetermined colitis and for decision-making prior to proctocolectmy in UC patients.  The anti-glycan antibodies are specifically important in ASCA-negative CD patients.  Correlation between serologic markers and genetic variations may contribute to re-classifying IBD into new and more homogeneous subclasses.  Their significance in diagnosing populations at risk, such as unaffected relatives of IBD patients and CD patients prior to diagnosis, is under current investigation.

Silberer et al (2005) compared 5 different leukocyte proteins in feces of patients with chronic IBD, irritable bowel syndrome (IBS) and healthy persons who underwent prophylactic colonoscopy.  The leukocyte proteins calprotectin, lactoferrin, lysozyme, myeloperoxidase (MPO), and PMN-elastase were determined with immunoassays in fecal samples of 3 consecutive feces (e.g., 3 days) in 40 healthy persons, 39 patients with chronic IBD (of these 21 with Crohn's disease and 18 with ulcerative colitis), and 40 patients with IBS.  Receiver operating characteristics (ROC) curves calculated for healthy persons and patients with IBD yielded the following areas under the curves (AUCs): PMN-elastase 0.916, calprotectin 0.872, MPO 0.750, lysozyme 0.726, and lactoferrin 0.693.  The AUCs of PMN-elastase and calprotectin were not significantly different (p = 0.327), whereas PMN-elastase or calprotectin versus the other proteins were significantly different (p < 0.001).  PMN-elastase and calprotectin correlated with the endoscopically classified severity of inflammation.  All fecal leukocyte markers in IBS were found in the range of the healthy persons.  Data on storage stability of leukocyte proteins in fecal supernatants are given.  The authors concluded that fecal PMN-elastase and calprotectin support the differentiation of chronic IBD from IBS and correlate with the severity of inflammation.

Lettesjo et al (2006) examined if inflammatory markers could be detected in feces from patients with IBS and collagenous colitis (CC), and elucidated if such analyses could be used as non-invasive tools to distinguish between these disorders.  Stool samples were obtained from 18 patients with CC, 46 patients with IBS and 20 healthy controls (HC).  Eosinophil protein X (EPX), MPO, tryptase, interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) were measured in supernatants from processed feces using immunoassays.  EPX levels were enhanced in feces from CC patients (median 3.8 microg/g (0.47 to 16.2)) compared to patients with IBS (0.44 microg/g (0.25 to 1.8)), p < 0.001, and HC (0.46 microg/g (0.21 to 1.3)), p < 0.001.  In addition, MPO was increased in CC patients (11.7 microg/g (2.0-124)) compared to IBS patients (1.7 microg/g (0.81 to 5.2)), p < 0.01, and HC (2.5 microg/g (1.1 to 6.3)), p < 0.05.  Tryptase was found in 9/18 patients with CC, 6/46 with IBS and 1/19 HC.  IL-1beta was only enhanced in 2/11 CC patients and TNFalpha was not detected in any sample.  The authors concluded that increased levels of EPX, MPO and tryptase were observed in stools from CC patients, whereas the levels in IBS patients did not differ from healthy controls.  These findings suggested that fecal markers could be used as part of the clinical work-up to determine which patients should be biopsied and evaluated for CC.

A recent review by Navaneethan et al (2009) on laboratory tests for ilieal pouch-anal anastomosis noted the need for further studies validating the role of anti-CBIR1 as a laboratory marker to predict pouchitis, and concluding that serologic tests are primarily for research purposes.

Booth et al (2011) noted that the evidence for testing TPMT enzymatic activity or genotype before starting therapy with thiopurine-based drugs is unclear.  These investigators examined the sensitivity and specificity of TPMT genotyping for TPMT enzymatic activity, reducing harm from thiopurine by pre-testing, and the association of thiopurine toxicity with TPMT status in adults and children with chronic inflammatory diseases.  MEDLINE, EMBASE, the Cochrane Library, and Ovid HealthSTAR (from inception to December 2010) and BIOSIS and Genetics Abstracts (to May 2009) were used for this review.  Two reviewers screened records and identified relevant studies in English.  Data on patient characteristics, outcomes, and risk for bias were extracted by one reviewer and independently identified by another.  A total of 54 observational studies and 1 randomized, controlled trial were included.  Insufficient evidence addressed the effectiveness of pre-testing.  Genotyping sensitivity to identify patients with low and intermediate TPMT enzymatic activity ranged from 70.33 % to 86.15 % (lower-bound 95 % CI: 54.52 % to 70.88 %; upper-bound CI: 78.50 % to 96.33 %).  Sparse data precluded estimation of genotype sensitivity to identify patients with low to absent enzymatic activity.  Genotyping specificity approached 100 %.  Compared with non-carriers, heterozygous and homozygous genotypes were both associated with leukopenia (OR, 4.29 [CI: 2.67 to 6.89] and 20.84 [CI: 3.42 to 126.89], respectively).  Compared with intermediate or normal activity, low TPMT enzymatic activity was significantly associated with myelotoxicity and leukopenia.  The authors concluded that insufficient evidence addresses the effectiveness of TPMT pre-testing in patients with chronic inflammatory diseases -- insufficient evidence demonstrating that this strategy is effective in reducing harm or is superior to the established clinical standard of hematological monitoring.

The new PROMETHEUS IBD sgi Diagnostic is the 4th-generation irritable bowel disease (IBD) diagnostic test and the first and only test to combine serologic, genetic, and inflammation markers in the proprietary Smart Diagnostic Algorithm for added diagnostic clarity.  This test aids healthcare providers in differentiating IBD versus non-IBD as well as CD versus UC in 1 comprehensive blood test.  This assay includes 9 serological markers including the proprietary Anti-Fla-X, Anti-A4-Fla2, anti-CBir1, anti-OmpC, and DNAse-sensitive pANCA that helps identify patients with IBD and utilizes Smart Diagnostic Algorithm Technology to improve the predictive accuracy.  Genetic susceptibility influences immune responses and this assay includes evaluation of ATG16L1, STAT3, NKX2-3, and ECM1.  Inflammatory markers include VEGF, ICAM, VCAM, CRP, SAA.  While most other labs only offer assay values, PROMETHEUS IBD sgi Diagnostic provides added clarity in diagnosing IBD, UC, and CD.

One of the references cited in the Prometheus website was a study on genetics of the ulcerative colitis by Thompson and Lees (2010).  The authors concluded that "[t]hese advances will not in short term provide additional diagnostic or prognostic value.  There is also no reason necessarily why polymorphisms that contribute to disease susceptibility should predict disease course, and almost certainly have no bearing on treatment response and intolerance.  Prospective studies are required to address the genetic contributions to these clinically important areas, and will become a major focus in the next few years".

Adler et al (2011) noted that CD is often purely inflammatory at presentation, but most patients develop strictures and fistulae over time (complicated disease).  Many studies have suggested that nucleotide-binding oligomerization domain 2 (NOD2) mutations are associated with a varying but increased risk of complicated disease.  An accurate and sufficiently powerful predictor of complicated disease could justify the early use of biological therapy in high-risk individuals.  These researchers performed a systematic review and meta-analysis to obtain accurate estimates of the predictive power of the identified mutations (such as p.R702W, P.G908R, and p.Leu1007fsX1008) in NOD2 for the risk of complicated disease.  An electronic search of MEDLINE, Embase, and Web of Science identified 917 relevant papers.  Inclusion required specification of genetic mutations at the individual level and disease phenotypes by Vienna classification (inflammatory (B1), stricturing (B2), and fistulizing (B3)).  A total of 49 studies met these criteria, which included 8,893 subjects, 2,897 of whom had NOD2 mutations.  Studies were weighted by median disease duration.  Studies not providing duration data were weighted at the level of the study with the shortest disease duration (3.9 years).  The relative risk (RR) of the presence of any NOD2 mutant allele for complicated disease (B2 or B3) was 1.17 (95 % confidence interval (95 % CI): 1.10 to 1.24; p < 0.001).  P.G908R was associated with an RR of complicated disease of 1.33 (95 % CI: 1.11 to 1.60; p = 0.002).  NOD2 did not predict peri-anal disease (p = 0.4).  The RR of surgery was 1.58 (95 % CI: 1.38 to 1.80; p < 0.001).  There was substantial heterogeneity across all studies (I(2) = 66.7 %).  On the basis of logistic regression of these data, the sensitivity of any mutation in predicting complicated disease was 36 % and specificity was 73 %, with the area under the receiver operating characteristic curve 0.56.  The authors concluded that the presence of a single NOD2 mutation predicted an 8 % increase in the risk for complicated disease (B2 or B3), and a 41 % increase with 2 mutations.  Surgery risk is increased by 58 % with any NOD2 mutation, whereas peri-anal disease was unchanged.  The predictive power associated with a single NOD2 mutation is weak.  The RR of any NOD2 mutations for complicated disease was only 17 % across 36 studies.  However, the presence of two NOD2 mutations had 98 % specificity for complicated disease.  These data provide insufficient evidence to support top-down therapy based solely on single NOD2 mutations, but suggest that targeted early-intensive therapy for high-risk patients with two NOD2 mutations might be beneficial, if prospective trials can demonstrate changes in the natural history in this subset of patients.

Bi et al (2011) stated that UC and CD are 2 distinct forms of IBD that can overlap radiologically, endoscopically, and pathologically.  This difficulty complicates surgical options.  The development of new technologies providing accurate diagnosis of IBD is needed.  Raman spectroscopy is a non-invasive method that uses the intrinsic properties of tissue and that tissue's vibrational energy in reaction to light. These investigators hypothesized that Raman spectroscopy can detect the structural and compositional changes that occur in the tissue during the development of IBD, and thus may offer increased diagnostic certainty in the differentiation between CD and UC.  Fresh frozen colon tissue biopsies from patients with UC (n = 12) and with CD (n = 9) were measured in-vitro using a custom-designed Raman fiber-optic probe.  For spectra collection, the probe was placed in gentle contact with the mucosa surface for 3 seconds, with excitation power at 150 mW.  Five spectra were acquired from each biopsy to increase the signal-to-noise ratio and to ensure repeatability of data collection.  Mean spectra were analyzed for peak difference and molecular origin.  Significant difference was observed between the spectra from each disease in the spectral regions assigned to nucleic acid, phenylalanine, and lipids.  Tissue samples from patients with UC demonstrated higher content of lipid and lower amount of phenylalanine and nucleic acid.  These characteristic Raman features could serve as spectral markers that can potentially be applied to distinguish UC and CD.  The authors concluded that this study presented the only application of Raman spectroscopy in the diagnosis of IBD.  The feasibility of this technique in differentially detecting molecular alterations in UC and CD has been demonstrated, indicating the potential to improve diagnostic accuracy of IBD.

Wang et al (2012) noted that the gene encoding CD14 has been proposed as an IBD-susceptibility gene with its polymorphism C-260T being widely evaluated, yet with conflicting results.  These researchers investigated the association between this polymorphism and IBD by conducting a meta-analysis.  A total of 17 articles met the inclusion criteria, which included a total of 18 case-control studies, including 1,900 UC cases, 2,535 CD cases, and 4,004 controls.  Data were analyzed using STATA software.  Overall, association between C-260T polymorphism and increased UC risk was significant in allelic comparison (OR = 1.21, 95 % CI: 1.02 to 1.43; p = 0.027), homozygote model (OR = 1.44, 95 % CI: 1.03 to 2.01; p = 0.033), as well as dominant model (OR = 1.36, 95 % CI: 1.06 to 1.75; p = 0.016).  However, there was negative association between this polymorphism and CD risk across all genetic models.  Subgroup analyses by ethnicity suggested the risk-conferring profiles of -260T allele and -260 TT genotype with UC in Asians, but not in Caucasians.  There was a low probability of publication bias.  The authors concluded that expanding previous results of individual studies, these findings demonstrated that CD14 gene C-260T polymorphism might be a promising candidate marker in susceptibility to UC, especially in Asians.

Zhu et al (2013) stated that a large number of studies have shown that the interleukin-10 (IL-10)-1082A/G polymorphism is implicated in susceptibility to IBD.  However, the results are inconsistent.  These investigators performed a meta-analysis to estimate the association between -1082A/G polymorphism in the IL-10 gene and IBD susceptibility.  A total number of 18 case-control studies including 17,585 subjects were identified.  No association was found between -1082A/G polymorphism and UC susceptibility.  However, increased risk of CD was associated with -1082A/G polymorphism in the dominant genetic model (GG+GA versus AA: OR = 1.22, 95 % CI: 1.02 to 1.46, p = 0.028) and the heterozygote comparison (GA versus AA: OR = 1.28, 95 % CI: 1.05 to 1.55, p = 0.015).  The authors concluded that the results of this meta-analysis provided evidence for the association between IL-10-1082A/G polymorphism and susceptibility of CD.  However, they noted that due to several limitations in the present study, well-designed epidemiological studies with large sample size among different ethnicities should be performed in the future.

Kovacs and associates (2013) stated that mannose-binding lectin (MBL) is a pattern-recognition molecule of the innate immune system and may be involved in the pathogenesis of IBD.  These researchers evaluated the prevalence of MBL deficiency in a cohort of patients with pediatric-onset IBD and examined if it is associated with the clinical manifestations, serum antibody formation, or genetic factors.  This prospective study included 159 pediatric patients (mean age of 14.0 years) with IBD (107 patients with CD and 52 patients with UC).  Furthermore, 95 controls were investigated.  Serum samples were determined for MBL by ELISA and for serologic markers (ASCA and pANCA) by indirect immunofluorescent assay.  NOD2/CARD15 variants were tested by PCR/restriction fragment length polymorphism.  The MBL serum concentration was significantly lower in IBD patients (both with CD and UC) compared to controls (IBD, p = 0.007; CD, p = 0.04; UC, p = 0.004).  Prevalence of low MBL level (less than 500 ng/ml) was significantly higher in both CD and UC groups compared to controls (p = 0.002 and p = 0.006).  Furthermore, low MBL level was associated with isolated ileal involvement (p = 0.01) and MBL deficiency (less than 100 ng/ml) with male gender (p = 0.004) in patients with CD.  These investigators failed to confirm any correlation between MBL deficiency and serum autoantibodies or NOD2/CARD15 variants.  The authors concluded that these findings suggested that low MBL associated with pediatric-onset IBD and ileal CD may be considered an additional marker of the IBD pathogenesis.

Moreover, an UpToDate review on “Clinical manifestations, diagnosis and prognosis of Crohn's disease in adults” (Peppercorn, 2013) does not mention measurement of mannose-binding lectin as a management tool.

Mitsuyama and colleagues (2014) stated that various non-invasive tests have been studied to screen for patients with CD, and were found to have limited accuracy and sensitivity, particularly in Asian populations.  These investigators explored the possible diagnostic utility of antibodies to the CD peptide (ACP) in patients with CD.  In a multi-center study using ELISA, serum ACP levels were determined in 196 patients with CD, 210 with UC, 98 with other intestinal diseases, 132 with other inflammatory diseases, and 183 healthy controls; and then examined for correlation to clinical variables.  The diagnostic utility of ACP was evaluated by ROC analysis and compared with ASCA.  Levels of ACP were significantly elevated in the CD patients, but not in the other groups that included UC, other intestinal diseases, other inflammatory diseases and the healthy controls.  Among these other groups, ACP levels were not significantly different.  In the CD patients, ACP had a higher sensitivity and specificity (63.3 % and 91.0 %, respectively) than ASCA (47.4 % and 90.4 %).  Levels of ACP were negatively associated with disease duration, but not with Crohn's Disease Activity Index (CDAI), disease location, or medical treatment.  The authors concluded that ACP, a newly proposed serologic marker, was significantly associated with CD and was highly diagnostic.  Moreover, they stated that further investigation is needed across multiple populations of patients and ethnic groups, and more importantly, in prospective studies to ascertain the clinical value of ACP.

Furthermore, an UpToDate review on “Clinical manifestations, diagnosis and prognosis of Crohn's disease in adults” (Peppercorn, 2013) does not mention measurement of antibodies to the CD peptide as a management tool.

Fecal Markers of Inflammatory Bowel Disease

The fundamental pathological process behind IBD is intestinal inflammation.  As the precise cause of IBD is not yet completely understood, current treatment strategies are aimed at reducing or eliminating the inflammation.  Endoscopic examination and histological analysis of biopsy specimens remain the “gold standard' methods for detecting and quantifying bowel inflammation; however, these techniques are costly, invasive, and repeated examinations are unpopular with patients.  Disease activity questionnaires and laboratory `inflammatory markers', although widely used, show an unreliable correlation with endoscopy and histology.  New markers are needed for detecting and quantifying bowel inflammation.

Calprotectin is a calcium- and zinc-binding protein of the S100 family derived mainly from neutrophils and monocytes.  It is excreted in excess in stools during IBD.  Fecal calprotectin level has been reported to parallel intestinal inflammation and can predict relapse of UC (Hanai et al, 2004).

Fecal measurement of calprotectin is emerging as a tool for the differential diagnosis of inflammatory (e.g., CD, UC) from non-inflammatory gastrointestinal disease (e.g., IBS), for monitoring patients' response to therapy and for predicting recurrence of IBD.

The available literature reports high fecal calprotectin levels in patients with active inflammation of the bowel. As such, it differs from erythrocyte sedimentation rates and other tests that are general markers of inflammation and not specific to bowel inflammation. The fecal calprotectin test, however, cannot distinguish among different causes of bowel inflammation. Several diseases other than inflammatory bowel disease, including colorectal neoplasia and gastrointestinal infection, can also increase fecal calprotectin. Because fecal calprotectin is a nonspecific marker of bowel inflammation, endoscopic workup remains crucial to determine the underlying cause of colitis.

In March 2006, the PhiCal (Genova Diagnostics) quantitative ELISA test for measuring concentrations of fecal calprotectin in fecal stool was cleared for marketing by the U.S. Food and Drug Administration (FDA) through the 510(k) process. Thus, the manufacturer was not required to provide the evidence of efficacy necessary to support a premarket approval application (PMA).

Studies have compared fecal calprotectin levels in patients with confirmed inflammatory bowel disease versus normal controls (Tibble, et al., 2000; Schoepfer, et al., 2010) or persons with irritable bowel syndrome (Schoepfer, et al., 2007). Joishy, et al., (2008) also had a control group of children with gastrointestinal symptoms, although the causes were not specified in detail in that report. Almost all of the studies published so far have been performed in prediagnosed groups of patients or in patients in whom the major clinical problem was to distinguish between Crohn’s disease and irritable bowel syndrome. These studies did not consider the wide spectrum of other organic causes of chronic diarrhea that must be distinguished from inflammatory bowel disease.

Studies have reported correlations of fecal calprotectin levels with endoscopic, histologic, clinical and serum markers of inflammation in patients with confirmed diagnoses of inflammatory bowel disease (Tibble, et al., 2000; Bunn, et al., 2001; Gaya, et al., 2005; Sipponen, et al., 2008; Vieira, et al., 2009; Quail, et al., 2009; Fagerberg, et al., 2007). Sipponen, et al., 2010 examined correlations between fecal calprotectin and endoscopic response to therapy. Other studies (Kallel, et al. 2010; Gisbert, et al., 2009) examined correlations between fecal calprotectin levels and relapse. However, the usefulness of these findings in the management of patients with inflammatory bowel disease has not been shown. There is a lack of clinical outcome studies of the use of fecal calprotectin in lieu of a standard workup for inflammatory bowel disease. Although some authors claim that fecal calprotectin can be used to avoid invasive testing, there are no studies demonstrating an actual reductions in number of invasive tests through use of fecal calprotectin measurement.

Berni et al (2004) examined fecal calprotectin values in different pediatric gastrointestinal diseases (n = 281; age ranging from 13 to 216 months) comparing them with those obtained in healthy children (n = 76; age ranging from 13 to 209 months).  These investigators concluded that fecal calprotectin is a sensitive, but not disease specific, marker to easily detect inflammation throughout the whole gastrointestinal tract.  It may help in identifying an organic disease characterized by intestinal mucosa inflammation and in the differential diagnosis of functional bowel disorders.  Wassell et al (2004) posited that a single measurement of fecal calprotectin may help gastroenterologists in the differential diagnosis of CD and IBS.

Tibble et al (2000) examined if measurement of intestinal permeability and inflammation could predict relapse of IBD.  Forty-three patients with CD and 37 with UC in clinical remission provided a stool sample to be assayed for calprotectin and patients with CD additionally underwent a small intestinal permeability test.  Relapse was defined using clinical disease activity indices.  Twenty-five (58 %) patients with CD and 19 (51 %) with UC had a relapse over the 12-month period.  Median calprotectin levels in the relapse groups (122 mg/L for CD, 123 mg/L for UC; normal less than 10 mg/L) differed significantly (p < 0.0001) from those of the non-relapse groups (41.5 mg/L for CD, 29.0 mg/L for UC).  At 50 mg/L, the sensitivity and specificity of calprotectin for predicting relapse in all patients with IBD were 90 % and 83 %, respectively.  Permeability in the CD patients who relapsed (median, 0.075; normal less than 0.04) differed significantly (p = 0. 004) from that in the non-relapse group (median, 0.038).  At the level of 0.05, the sensitivity and specificity of permeability in predicting relapse were 84 % and 61 %, respectively.  The authors concluded that fecal calprotectin may be useful in predicting clinical relapse of disease activity in patients with CD and UC, whereas small intestinal permeability may be a useful predictor of relapse in patients with small intestinal CD.

Although the study by Tibble et al (2000) suggested that high fecal calprotectin levels may identify IBD patients in remission who are at risk for early relapse, there are reports that there may be differences in relapse prediction in patients with CD compared to those with UC.

Costa et al (2005) examined if the predictive value of fecal calprotectin is different in CD and UC.  Seventy-nine consecutive patients with a diagnosis of clinically quiescent IBD (38 CD and 41 UC) were followed for 12 months, undergoing regular clinical evaluations and blood tests.  A single stool sample was collected at the beginning of the study from each patient and the calprotectin concentration was measured by a commercially available enzyme linked immunoassay.  In CD, median calprotectin values were 220.1 microg/g of stool in those patients who relapsed during follow-up, and 220.5 microg/g in non-relapsing patients (p = 0.395).  In UC, median calprotectin values were 220.6 microg/g and 67 microg/g in relapsing and non-relapsing patients, respectively (p < 0.0001).  The multi-variate Cox regression model, after adjustment for possible confounding variables, showed a 2- and 14-fold increase in the relapse risk, respectively, in those patients with CD and UC in clinical remission who had a fecal calprotectin concentration higher than 150 microg/g.  The authors concluded that fecal calprotectin proved to be an even stronger predictor of clinical relapse in UC than in CD, which makes the test a promising non-invasive tool for monitoring and optimizing therapy.

In a commentary on the role of biomarkers for predicting relapse in patients with IBD, Pardi and Sandborn (2005) stated that based on the studies of Tibble et al (2000) as well as Costa et al (2005), fecal calprotectin appears to be a relatively sensitive and specific marker of the risk of relapse for UC.  It also appears to be a sensitive marker of relapse risk in CD but the data on specificity are conflicting at this juncture.  However, these data need to be interpreted carefully since the number of studies is small, and in both studies using calprotectin to predict relapse risk, most patients were on medical therapy.  Calprotectin may behave differently in patients who are not on therapy.  Thus, before fecal calprotectin or any other biomarker of inflammatory activity in the gastrointestinal tract can be incorporated into routine clinical practice, other studies in larger and diverse groups of patients will be needed to further clarify its role.

In the guidelines for chronic diarrhea compiled at the request of the British Society of Gastroenterology, Thomas et al (2003) stated that stool markers of gastrointestinal inflammation such calprotectin are of considerable research interest.  However, these tests have not been introduced into clinical practice.  Moreover, in a review on the diagnostic and therapeutic strategies in the IBD, Cremonini and Talley (2004) stated that the usefulness of fecal tests such as calprotectin to exclude organic bowel disease is not adequately established.  Furthermore, the use of calprotectin is not mentioned in the practice parameters on the management of CD in adults by the American College of Gastroenterology (Hanauer et al, 2001) and the practice guideline on the management of UC by the Society of Surgery of the Alimentary Tract (2001).

D'Inca et al (2008) evaluated the role of calprotectin tests in predicting clinical relapse in IBD patients.  A total of 97 patients with UC and 65 with CD in clinical remission were prospectively included in the study.  A 10-g stool sample was collected for calprotectin assay.  The cut-off level was set at 130 mg/kg of feces.  Patients were followed-up for 1 year after the test or until relapse.  The cumulative proportion of relapses was estimated by the Kaplan-Meier analysis.  Statistics for equality of survival distribution were tested using the log-rank test.  The calprotectin test was positive in 44 UC patients and 26 of them relapsed within 1 year, while 11 of 53 UC patients with a negative calprotectin test relapsed within the same time frame.  Thirty CD patients had a positive calprotectin test and 13 of them relapsed within 1 year, as did 7 of the 35 with a negative test result.  A significant correlation emerged between a positive calprotectin test and the probability of relapse in UC patients (p = 0.000).  In CD patients, only cases of colonic CD showed a significant correlation between a positive calprotectin test and the probability of relapse, i.e., 6 colonic CD patients were positive for the calprotectin test and 4 relapsed (p = 0.02).  The authors concluded that measuring calprotectin may help to identify UC and colonic CD patients at higher risk of clinical relapse.  The author also noted that while the marker has an impressive profile in discriminating organic versus functional intestinal diseases, its role in monitoring IBD remains promising but still unclear.  These investigators are currently evaluating the usefulness of calprotectin levels for predicting relapses in a more careful manner by measuring calprotectin every 3 months, and are also planning to verify the usefulness of this marker in monitoring response to treatments.

Gisbert and McNicholl (2009) noted that fecal calprotectin has been proposed as a non-invasive surrogate marker of intestinal inflammation in IBD.  Close correlation between fecal calprotectin concentration and fecal leukocyte excretion quantified with (111)indium has been described.  Fecal calprotectin has a good diagnostic precision for separating organic and functional intestinal diseases.  However, the specificity for the diagnosis of IBD is lower than desirable, as several diseases other than IBD -- specially colorectal neoplasia and gastrointestinal infection -- can also increase fecal calprotectin.  High concentration of calprotectin in feces is a strong argument to carry out a colonoscopy in order to rule out the presence of IBD or other organic pathologies.  Parallelism between fecal calprotectin levels and IBD activity has been confirmed, although this fecal marker appears to better reflect the disease activity in UC than in CD.  Fecal calprotectin's capacity to predict IBD relapse is promising.  It has been suggested that, in IBD patients receiving treatment, a normalization or decrease in fecal calprotectin concentrations is an accurate indicator of endoscopic healing.  Greater fecal calprotectin concentration has been shown in asymptomatic first-degree relatives of patients with IBD, suggesting that there is a high prevalence of sub-clinical intestinal inflammation in them.

One of the proposed uses of this test is to evaluate patients presenting with gastrointestinal symptoms, where irritable bowel syndrome (a noninflammatory condition) and inflammatory bowel disease (Crohn's disease or ulcerative colitis) are in the differential. A metaanalysis from the Netherlands (van Rheenen, et al., 2010) examined studies of the potential use of fecal calprotectin for inflammatory bowel disease screening. The pooled analysis showed that a normal fecal calprotectin test result reduced the probability of inflammatory bowel disease in adults to 3 percent; however the confidence intervals were wide, ranging from 1 percent to 11 percent. The test did not perform as well in children, where a normal fecal calprotectin test reduced the probability of inflammatory bowel disease to 15 percent, with confidence intervals ranging from 7 percent to 28 percent. The investigators calculated false negative results in 6 percent of adults and 8 percent of children. Based upon this analysis, it has been suggested that adults with a normal fecal calprotectin can forgo endoscopy, and be treated presumptively as having irritable bowel syndrome. Based upon that assumption, the authors presenting a modeling scenario where they estimated that screening by measuring fecal calprotectin levels would result in a 67% reduction in the number of adults requiring endoscopy. This is a modeling estimate, based upon an assmption that all adults with a normal fecal calprotectin test would forgo endoscopy. Based on this analysis, there has been some support in Europe for the use of fecal calprotectin in this way. A guideline on irritable bowel syndrome from the World Gastroenterology Organisation that lists markers of inflammation ("e.g., faecal calprotectin") in the workup of persons suspected of having irritable bowel syndrome.

This metaanalysis by Van Rheem, et al. (2010) of fecal calprotectin presents a number of limitations. One has to do with the wide confidence intervals of the estimates. For adults, the upper end of the confidence interval is 11 percent, and for children, the upper end of the confidence interval is 28 percent. Thus, for adults, the actual proportion of adults with a normal fecal calprotectin may be more than 10 percent. The wide confidence intervals are a reflection of the heterogeneity of the results of the studies included in the review. The substantial differences in the estimates of the accuracy of fecal calprotectin testing among studies means that there is substantial uncertainty on the true diagnostic performance of this test. A critique of this metaanalysis by the Centre for Reviews and Dissemination (2010) stated that the authors of the metaanalysis, while recognizinng the heterogeneity of study results, failed to do a statistical analysis of the heterogenetity, and that the substantial heterogeneity of study results suggests that pooling of the results into a single metaanlysis may have been inappropriate. In reviewing the conclusions of this study, one commentator (Rex, et al., 2010) questioned whether, in the United States, given the current medical legal environment, a gastroentrologist would be willing to forgo endoscopy knowing that there may be an 11 percent chance that they will misdiagnose a person with inflammatory bowel disease as having irritable bowel syndrome. Rex (2010) stated that, in the United States, given the medical legal burden to exclude inflammatory bowel disease, gastroenterologists would be unlikely to forgo endoscopy because of this imprecision.  In addition, endoscopy detects more than just inflammatory bowel disease, and can detect other conditions, such as diverticulosis, that may be in the differential or contribute to the patient's symptoms.

The authors of this metaanalysis (Van Rheem, et al., 2010) stated that the estimates of test performance for this indication have been in referral populations and not in primary care. The absence of information on the performance of the fecal calprotectin test in the primary care population was also the conclusion of a recent Cochrane review. The test performance may be very different in primary care, given the differences in disease spectrum in the primary care population compared to the population of patients that are seen in secondary or tertiary care settings. For example, the prevalence of inflammatory bowel disease would be expected to be much lower in the primary care population, and one would expect to have a higher prevalence of acute inflammatory bowel disorders that may lead to falsely positive results. Thus, the results of this metaanalysis cannot be extrapolated for use in primary care.

There is an absence of evidence to support the use of fecal calprotectin testing to guide the management of patients with inflammatory bowel disease. There are no current guidelines from leading medical professional organizations that recommend the use of fecal calprotectin for this purpose. The FDA-approved labeling of products used to treat inflammatory bowel disease do not discuss the use of fecal calprotectin for this purpose. There is a lack of reliable evidence from clinical studies where fecal calprotectin is used to guide management. There is a need for clinical studies of the use of fecal calprotectin demonstrating that, compared to standard of care management without fecal calprotetin, the use of fecal calprotectin results in better clinical outcomes, fewer endoscopies, or some other important clinical outcome. Current evidence for fecal calprotectin to guide management describes the test performance in guaging inflammation in persons with inflammatory bowel disease; this type of evidence is hypothesis generating, and suggests that fecal calprotectin has promising potential for this use. 

Henderson and colleagues (2014) stated that fecal calprotectin (FC) is increasingly used during the diagnosis of inflammatory bowel disease (IBD), out-performing blood markers during investigation in children.  Tests that reduce endoscopy rates in children with suspected gut inflammation would be beneficial.  These investigators determined the usefulness of FC in children undergoing their primary investigation for suspected IBD by systematic review and meta-analysis.  An electronic search was performed with keywords relating to IBD and calprotectin in multiple electronic resources from 1946 to May 2012; a hand search was also performed.  Inclusion criteria were studies that reported FC levels before the endoscopic investigation of IBD in patients less than 18 years old.  Studies were evaluated using the Quality Assessment of Diagnostic Accuracy Studies tool, and a meta-analysis was performed using a hierarchical summary receiver operating curve model.  A total of 8 papers met the inclusion criteria (6 prospective and 2 retrospective case-control studies); methodological quality was determined in detail for each study.  The 8 studies presented FC levels at presentation in 715 patients, 394 pediatric IBD patients, and 321 non-IBD controls.  Pooled sensitivity and specificity for the diagnostic utility of FC during the investigation of suspected pediatric IBD were 0.978 (95 % confidence interval (CI): 0.947 to 0.996) and 0.682 (95 % CI: 0.502 to 0.863), respectively; the positive and negative likelihood ratios were 3.07 and 0.03, respectively.  The authors concluded that FC has a high sensitivity and a modest specificity during the diagnosis of suspected pediatric IBD.  Moreover, they stated that further work is required to determine the effect of FC levels on endoscopy rates and its role during the re-evaluation of those with confirmed disease.

Also, an UpToDate review on “Clinical manifestations, diagnosis and prognosis of Crohn's disease in adults” (Peppercorn, 2013) states that “Testing for fecal calprotectin may help identify patients with intestinal inflammation, though it is not routinely done in clinical practice”.

Lasson et al (2014) examined if the concentration of FC reflects the endoscopic findings 1 year after ileo-caecal resection and evaluated the variation of FC in individual patients during 6 months prior to the ileo-colonoscopy.  A total of 30 patients with CD and ileo-caecal resection performed within 1 year were included.  Stool samples were delivered monthly until an ileo-colonoscopy was performed 1 year post-operatively.  One year after surgery the median values of FC were not significantly different between the patients in endoscopic remission (n = 17) and the patients with an endoscopic recurrence (189 (75 to 364) versus 227 (120 to 1,066) μg/g; p = 0.25).  However, most patients with low values were in remission and all patients with high (greater than 600 μg/g) calprotectin values had recurrent disease.  The variability of the FC concentration was most pronounced in patients with diarrhea.  The authors concluded that they found no statistical difference in the concentrations of calprotectin between patients in endoscopic remission and patients with a recurrent disease 1 year after ileo-caecal resection for CD.  However, among the minority of patients with low or high values, FC indicated remission and recurrence, respectively.  There was significant variation of the FC concentrations over time, which affects the utility of calprotectin in clinical practice.

Pharmacogenomic and Metabolic Assessment of Thiopurine Therapy

The PRO-Predict series of tests were developed by Prometheus, Inc. with the intent of providing guidance in determining therapeutic direction and predicting therapeutic response in individual patients.  PRO-Predict 6MP is for 6-MP/Azathioprine remission and toxicity monitoring, PRO-Predict TPMT is for 6-MP/azathioprine response stratification, and PRO-Predict TNF is for anti-TNF response stratification.  However, there is insufficient scientific evidence in the current medical literature to support the routine use of these tests in clinical practice.

Although there is a potential to regulate azathioprine and mercaptopurine according to measurement of metabolites, the optimal mode of therapeutic monitoring remains to be established through prospective clinical trials demonstrating that clinical outcomes are improved through metabolite monitoring. Current evidence is largely limited to studies exploring correlations between metabolite levels and azathioprine and mercaptopurine toxicity and response. These studies have shown conflicting results in the strength of the relationship between metabolite levels, toxicity, and response. There are no well-designed prospective clinical studies demonstrating that azathioprine or mercaptopurine therapy based upon results of metabolite testing leads to improved clinical outcomes compared to therapy based upon clinical assessment and standard testing including routine blood counts and liver enzymes.

A significant association has been found between erythrocyte 6-thioguanine levels and the likelihood of clinical remission with azathioprine and 6-mercaptopurine (Osterman et al, 2006); however, studies of the relationship between 6-thioguanine levels and the likelihood of clinical remission have not shown consistent results (Cuffari et al. 1996; Gupta et al, 2001; Lowry et al, 2001; Belaiche et al, 2001; Gilissen et al, 2004).  A similar association with clinical remission has not been found with levels of 6-methylmercaptopurine (Cuffari et al, 1996; Dubinsky et al, 2000; Gilissen et al, 2004).  Despite an overall correlation with clinical response rates, clinical studies have shown a wide range of rates of response and toxicity across a range of 6-TG levels (e.g., Cuffari et al, 1996; Dubinsky et al, 2000).  In addition, studies have shown that the predictive value of metabolite levels in individual patients is low (e.g., Cuffari et al, 2006; Goldenberg et al, 2004).  Another limitation to using metabolites levels to guide drug dosing is that there may be substantial variation in levels between measurements in individual patients (e.g., Wright et al, 2004).

Bone marrow toxicity due to azathiorpine or 6-mercaptopurine has been found to correlate with elevated levels of 6-thioguanine, and liver toxicity has been found to correlate with levels of 6-methylmercaptopurine (Ciffari et al, 1996; Dubinsky et al, 2000; Ooi et al, 2007).  However, clinical studies have shown that 6-methylmercaptopurine levels are not an accurate predictor of liver toxicity in individual patients (e.g., Goldenberg et al, 2004; Shaye et al, 2007; Heneghan et al, 2006; Reinshagen et al, 2007).  In addition, other adverse reactions, such as pancreatitis or leukopenia, can occur unrelated to serum metabolite levels (Cuffari et al, 1996; Ooi et al, 2007).  Thus, measurement of serum metabolite levels can not replace regular monitoring for toxicity with blood counts and liver enzymes.

In addition, measurement of serum metabolite levels must be compared with commonly available standard laboratory methods.  Waljee et al (2010) reported that algorithms that use age and laboratory values (CBC and chemistries) can differentiate clinical response, nonadherence, and shunting of thiopurine metabolism among patients who take thiopurines, and that this approach was more accurate than 6-TGN metabolite measurements in predicting clinical response.

A Cochrane Collaboration meta-analysis of literature on azathioprine and 6-mercaptopurine in Crohn's disease (Sandborn et al, 2002) concluded: “Another potential area of investigation is individualization of therapy.  6-Mercaptopurine is inactivated by methylation catalyzed by thiopurine methyltransferase (TPMT) whereas the active metabolites are the 6-thioguanine nucleotides (6-TGN).  The toxicity and effectiveness of 6-mercaptopurine therapy in childhood lymphocytic leukemia is inversely correlated with the activity of TPMT and directly correlated with the erythrocyte concentrations of 6TGN.  Measurement of both TPMT enzyme activity and erythrocyte 6TGN (therapeutic drug monitoring) in patients with Crohn's disease may allow individualized dosing to produce an optimal therapeutic effect while minimizing the potential for leukopenia.  Colanna and Korelitz (1994) have advocated using the presence of mild leukopenia as an indicator of the appropriate dose of 6-mercaptopurine.  In their recent retrospective analysis of 6-mercaptopurine therapy in 98 patients with refractory CD, they demonstrated a correlation between leukopenia and the achievement and maintenance of remission.  However, another retrospective study reported that leukopenia did not predict response in patients with UC treated with 6-mercaptopurine.  Prospective studies are needed to determine whether any of these attempts to optimize therapy with azathioprine or 6-mercaptopurine will be of use in routine clinical practice.”

Guidelines from the American College of Gastroenterology (2001) on management of CD state that, although there is a potential to regulate azathioprine and mercaptopurine according to measurement of metabolites, the optimal mode of therapeutic monitoring remains to be established through clinical trials.  “It remains to be determined how to optimize dose and whether induction of leukopenia or therapeutic monitoring of 6-thioguanine metabolites offer improved means of assuring a long-term response.”  American College of Gastroenterology guidelines on UC (2004) reached similar conclusions about the utility of metabolite monitoring.  “The utility of thiopurine metabolite testing requires prospective controlled evaluation before routine use can be recommended.”

A position statement from the American Gastroenterological Association (Lichtenstein et al, 2006) provides a Grade C recommendation for the use of thiopurine metabolite monitoring in the treatment of patients with 6-MP or AZA when attempting to determine medical noncompliance.  The guidelines note that thiopurine metabolite monitoring “may be helpful” for optimizing dose and monitoring for toxicity.  The guidelines, however, do not provide specific recommendations for use of thiopurine monitoring for these indications.  “Given the conflicting data, the retrospective nature of these studies, and the limited positive and negative predictive values for these particular uses, the utility of these tests needs prospective controlled evaluation before their routine use can be recommended.”

More recently, an American College of Gastroenterology (ACG) (Lichtenstein et al, 2009) guideline on CD in adults stated that, although retrospective analyses indicate that determination of 6-TGN and 6 MMP levels can be helpful to assess lack of response, patient adherence, leukopenia, or elevations in liver enzymes, "at the present time, there are inadequate data to recommend routine measurement of these metabolites...." The discussion of metabolite monitoring in the supporting ACG technical review cites a European evidence based consensus on the diagnosis and management of Crohn's disease (2006), where none of the consensus used 6-thioguanine concentrations to adjust the dose. The discussion in the AGA technical review also cites an American Gastroenterological Association Technical Review (Lichtenstein, et al., 2006), which stated that “[t]hiopurine metabolite monitoring in the treatment of patients with 6-MP or AZA is useful when attempting to determine medical noncompliance …”. It should be noted that this is a grade C recommendation based upon low quality evidence (defined as “evidence based on clinical experience, descriptive studies, or reports of expert committees”). Although thiopurine metabolite monitoring has been advocated for assessing compliance, there are no rigorous study data on this use. The guidelines also state that thiopurine metabolite monitoring “may be helpful” (grade C recommendation) for optimizing dose and monitoring for toxicity. The guidelines, however, do not provide specific recommendations for use of thiopurine monitoring for these indications. The technical review reports that “[s]ome, but not all, recent retrospective studies have suggested that measurement of AZA and 6-MP metabolites may be useful in dose adjustments…. Given the conflicting data, the retrospective nature of these studies, and the limited positive and negative predictive values for these particular uses, the utility of these tests needs prospective controlled evaluation before their routine use can be recommended. The utility of these metabolite markers, however, can assist an individual in determining whether a patient is noncompliant with their immunomodulator therapy.” The technical review also states that “Future prospective studies with measurement of dose optimization based on clinical efficacy and remission compared with measurements of TPMT, 6-TGN, and 6-MMP levels are still needed to better define appropriate treatment algorithms and doses in patients with UC. These particular studies can also assess the level (if any is present) of incremental benefit to the traditional routine of monitoring complete blood count, liver-associated laboratory chemistries, and clinical response.” It also should be noted that the FDA-approved labeling for Imuran (azathioprine) and mercaptopurine include recommendations for TPMT genotyping and phenotyping, but include no recommendation for use of thiopurine metabolites in dosing and administration.

The role of 6-TGN for assessing compliance, and in combination with 6-MMP to assess drug-failure attributable to high methylator status has been advocated but has not been substantiated by rigorous study data.

Prospective clinical studies are needed to determine whether outcomes are improved by tailoring individual drug regimens using azathioprine metabolite testing.  A prospective, randomized controlled clinical trial found that 6-TGN-adapted azathioprine therapy did not lead to higher remission rates than standard therapy in patients with chronic active CD.  Reinshagen et al (2007) reported on a prospective randomized trial that examined whether 6-TGN concentration-adapted azathioprine therapy is clinically superior to a standard dose of 2.5 mg/kg/day azathioprine in patients with CD.  After 2 weeks of standard therapy, patients (n = 71) were randomized into standard (n = 32) or adapted-dose (n = 25) groups; 14 patients dropped out before randomization.  In the adapted group, the azathioprine dose was adjusted to maintain 6-TGN concentrations between 250 and 400 pmol/8 x 10(8) erythrocytes (Ery).  Response criteria were the number of patients in remission after 16 weeks without steroids (primary) and remission after 24 weeks, frequency of side effects, and quality of life (secondary).  After 16 weeks, 14 of 32 (43.8 %) patients in the standard group versus 11 of 25 (44 %) in the adapted group were in remission without steroids (intent-to-treat analysis).  After 24 weeks, 43.8 % versus 40 % were in remission.  The researchers found no significant differences concerning quality of life, disease activity, 6-TGN concentrations, azathioprine dose, or dropouts due to side effects.  Sixty-six patients had a wild-type TPMT genotype, with TPMT activities of 8 to 20 nmol/(mL Ery x h).  Five patients (dropouts after randomization) were heterozygous, with TPMT activities less than 8 nmol/(mL Ery x h). 6-MMP) concentrations greater than 5,700 pmol/8 x 10(8) Ery were not associated with hepatotoxicity.  The investigators concluded that standard and adapted dosing with the provided dosing scheme led to identical 6-TGN concentrations and remission rates.  The investigators reported that adapted dosing had no apparent clinical benefit for patients with TPMT activity between 8 and 20 nmol/(mL Ery x h).  The investigators also found that 6-MMP monitoring had no predictive value for hepatotoxicity.  Additional prospective controlled clinical studies of thiopurine metabolite monitoring have been sponsored by the National Institutes of Health (NIDDK, 2010).

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of azathioprine, 6-mercaptopurine and thioguanine.  Several studies have reported correlations between mutations in the TPMT gene with susceptibility to leukopenia from azathioprine and mercaptopurine therapy, and investigators have proposed using the results of TPMT gene mutation testing to select candidates for azathioprine therapy.  However, almost all studies that have been published to date have only tested patients exhibiting toxicity for TPMT gene mutations and not a broader group of patients; thus, the sensitivity and specificity of TPMT gene mutation testing to toxicity susceptibility is unknown (Evans et al, 2001; Colombel et al, 2000).  In those studies where all patients were tested for TPMT gene mutations prior to therapy, TPMT mutations were detected in only a minority of patients exhibiting drug-induced leukopenia (Naughton et al, 1999; Dubinsky et al, 2000; Relling et a., 1999).  This has led investigators to conclude that myelosuppression may be due to other factors in addition to variable TPMT therapy, and that monitoring of blood counts throughout azathioprine or mercaptopurine therapy is essential in all patients, regardless of the presence or absence of TPMT gene mutations.  In addition, there are no prospective studies demonstrating improvement in outcomes by guiding azathioprine therapy or mercaptopurine therapy based on the results of TPMT gene mutation testing.

Prometheus Laboratories, manufacturer of the ProPredict Series of Tests, has obtained United States distribution rights for Imuran (azathioprine).  The FDA has agreed to include information about TPMT genotyping and phenotyping in the Imuran product labeling.  It should be noted that the product labeling emphasizes that TPMT testing cannot substitute for complete blood count monitoring in patients receiving Imuran.

A position statement from the American Gastroenterological Association (Lichtenstein et al, 2006) notes that information about TPMT testing is included in azathioprine product labeling.  The position statement explains that “[c]urrent Food and Drug Administration (FDA) recommendations suggest that individuals should have thiopurine methyltransferase (TPMT) genotype or phenotype assessed before initiation of therapy with AZA [azathioprine] or 6-MP [6-mercaptopurine] in an effort to detect individuals who have low enzyme activity (or who are homozygous deficient in TPMT) in an effort to avert AZA or 6-MP therapy and thus avoid potential adverse events.”  The position statement notes, however, that individuals who have intermediate or normal TPMT activity (wild type or heterozygotes) need measurement of frequent complete blood counts (as above) in addition to TPMT assessment because these individuals may still develop myelosuppression subsequent to use of azathioprine or 6-mercaptopurine.

Guidelines from the British Society of Gastroenterology (2004) stated that TPMT testing is not necessary.  The guidelines state that research has shown that the majority (77 %) of inflammatory bowel disease patients with azathioprine (AZA)-induced bone marrow suppression did not carry a TPMT mutation.  The guidelines noted that evidence that TPMT activity predicts other side effects or outcome is limited.  The guidelines noted that TPMT testing “cannot yet be recommended as a prerequisite to therapy, because decades of experience has shown clinical AZA to be safe in UC [ulcerative colitis] or CD [Crohn's Disease].”

Guidelines from the American College of Gastroenterology (Kornbluth and Sachar, 2004) are in agreement.  “6-MP and its prodrug azathioprine are both metabolized by thiopurine methyltransferase (TPMT), and enzyme that exhibits variation as a result of a genetic polymorphism of its alleles and this enzyme can now be measured by commercial laboratories.  Approximately 0.3 % of the general population have low to absent enzyme activity, 11 % have intermediate, and 89 % have normal to high levels of activity.  However, only about a quarter of cases of leukopenia in practice are associated with one of these genetic polymorphisms.  Therefore, prospective studies of dose-optimization based on measurements of TPMT, 6-TG, or 6-MP levels to monitor clinical response are still needed before the routine use of these assays can be recommended as providing much incremental benefit to the traditional routine of monitoring the CBC, liver associated laboratory chemistry abnormalities, and clinical response.”

Teml et al (2007) summarized clinical pharmacological aspects of thiopurines in the treatment of chronic IBD.  These investigators noted that on the basis of an excellent phenotype-genotype correlation for TPMT, genotyping has become a safe and reliable tool for determination of a patient's individual phenotype.  Based on several cost-benefit analyses, assessment of TPMT activity is recommended prior to thiopurine therapy in patients with IBD.  They also stated that although the therapeutic response appears to be related to 6-TGN concentrations above a threshold of 230 to 260 pmol/8 x 10(8) red blood cells, currently therapeutic drug monitoring of 6-TGN can be recommended only to estimate patients' compliance.

In a cross-sectional study, Firooz et al (2008) examined the role of TPMT activity in the safety and effectiveness of azathioprine in the treatment of pemphigus vulgaris.  A total of 139 patients were included in this study.  The TPMT activity in red blood cells was measured using high-performance liquid chromatography.  Severe adverse effects were defined as those judged serious enough that azathioprine therapy be discontinued.  These investigators also evaluated the relationship of clinical response and TPMT concentration in 52 patients who had been treated with a combination of prednisolone and azathioprine for at least 1 year, and the clinical response was considered favorable if there was no recurrence of pemphigus vulgaris in the first year of treatment.  The median activity of TPMT was 44.7 ng/ml/h (interquartile range of 28.7 ng/ml/h).  Eleven patients (7.9 %) had low TPMT activity (TPMT-HL), 127 patients (91.4 %) had normal TPMT activity (TPMT-HH), and 1 patient (0.7 %) had supra-normal enzyme activity; TPMT activity was noted in all patients.  Serious adverse effects occurred in 14 patients (10.1 %).  There was no relationship between development of adverse effects and TPMT activity (p = 0.29).  Eleven patients with low TMPT activity had been treated with azathioprine for a mean (SD) of 10.2 (4.1) months.  Only 1 patient exhibited serious adverse effects.  The TPMT enzyme activity was not different in 28 patients with unfavorable clinical response compared with 24 patients with favorable clinical response (p = 0.09).  The authors concluded that larger prospective studies are needed to determine the clinical relevance of TPMT activity and to determine accurate azathioprine dosing guidelines based on TPMT activity.

A study by Heneghan et al (2006) of patients with autoimmune hepatitis receiving azathioprine found that advanced fibrosis, rather than TPMT genotype or phenotype, predicted drug toxicity.  In addition, the investigators found that measurement of 6-TG and 6-MMP levels was not useful.  The investigators retrospectively evaluated data on 6-TG and 6-MMP levels and on TPMT genotyping and enzyme activity in patients with autoimmune hepatitis who received azathioprine therapy with or without prednisone.  From 1995 through 2001, 86 patients began courses of azathioprine; to maintain remission, 26 received azathioprine alone, 19 received prednisone alone, 26 received azathioprine plus prednisone, 9 received alternative therapies (mycophenolate mofetil, sirolimus, or cyclosporine), and the rest received no therapy.  TPMT genotyping was performed and 6-TG and 6-MMP levels were measured in all patients.  Twenty-two patients had at least 1 episode of azathioprine-related toxicity.  Neither TPMT genotype nor enzyme activity predicted such episodes, although cirrhosis was more common among patients who developed azathioprine toxicity than among those who did not (p = 0.043).  No threshold levels of 6-TG or 6-MMP predicted remission.

Cao et al (2009) identified the TPMT gene polymorphisms in Chinese IBD patients and studied the relationship between TPMT status and thiopurine-related toxicity in these patients.  A total of 189 IBD patients, 87 with CD and 102 with UC, and 273 healthy controls were enrolled.  All subjects were from the Han Chinese ethnic group.  Polymorphisms in TPMT*2, *3A, *3B and *3C were analyzed using allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism.  Direct sequencing was used to confirm the mutation results.  Exons of the TPMT gene from patients who suffered from azathioprine-induced toxicity were amplified and sequenced to detect TPMT mutations.  No TPMT*2, *3A or *3B mutant alleles were detected.  The allele frequency of TPMT*3C in the IBD group was 1.59 %, which was similar to that of the healthy control group (1.59 % versus 1.47 %, p = 1.000).  Forty-three patients were treated with azathioprine therapy, 4 experienced myelotoxicity, and 1 experienced hepatotoxicity, so the incidence of drug toxicity was 11.7 % (5/43).  No TPMT*2, *3A, *3B or *3C polymorphisms were detected in these 5 patients.  After directly sequencing the exons of the TPMT gene in these 5 patients, a synonymous single-nucleotide polymorphism (TPMT*1S), which does not alter the encoded amino acid, was found in 3 patients.  The authors concluded that TPMT*3C seemed to be a unique variant allele in this Han Chinese population.  The overall frequencies of variant TPMT alleles in this population were lower than those in Caucasians, but thiopurine toxicity in Han Chinese IBD patients is not low.  The authors reported that factors other than TPMT polymorphisms may be responsible for the development of toxicity.  Similarly, Takatsu et al (2009) found that adverse reactions to azathioprine can not be predicted by thiopurine S-methyltransferase genotype in Japanese patients with inflammatory bowel disease.

There is no indication for repeat pharmacogenomic testing for TPMT, as a person’s TPMT genotypic or phenotypic status remains unchanged over one’s lifetime.

NOD2/CARD15 Genotyping for Crohn's Disease

Nucleotide-binding oligomerization domain (NOD) proteins are cytosolic proteins that include principal regulators of apoptosis such as the apoptotic protease activating factor 1.  NOD proteins have also been described as intra-cellular activators of the caspase and nuclear factor-kappaB (NF-kappaB) signaling pathways.  In particular, NOD1, NOD2, cryopyrin, and ICE protease-activating factor (ICE refers to interleukin-1b converting enzyme that is also known as caspase-1) have been implicated in protective immune responses against pathogens.  Moreover, a large number of NOD proteins contain leucine-rich repeats (LRR), hence referred to as NOD-LRR proteins, which include human NOD2, cryopyrin, and MHC class II trans-activator (CIITA), as well as mouse neuronal apoptosis inhibitory protein 5.  NOD2 participates in the signaling events triggered by host recognition of specific motifs in bacterial peptidoglycan and, upon activation, induces the production of pro-inflammatory mediators.  Neuronal apoptosis inhibitory protein 5 is needed in macrophages to restrict intra-cellular growth of Legionella pneumophila, whereas CIITA plays a critical role in antigen presentation and development of antigen-specific T lymphocytes.  Thus, NOD-LRR proteins appear to be involved in a diverse array of processes needed for host immune reactions against pathogens (Inohara and Nunez, 2003; Inohara et al, 2005; Eckmann and Karin, 2005).

Epidemiological data, notably concordance rates in twin pairs as well as sibling pairs, have provided support for the importance of the genetic contribution to inflammatory bowel diseases, especially in CD.  Studies have found that genetic variations in several NOD-LRR proteins resulting in the abolishment of NF-kappaB signal transduction of pattern recognition receptors such as NOD/caspase recruitment domain (NOD/CARD) receptors is associated with inflammatory disease or increased susceptibility to microbial infections.  The identification of the IBD1 gene on chromosome 16 as NOD2 (also known as CARD15) is an important finding.  Specifically, mutations in the gene encoding NOD2 have been reported to be associated with the predisposition to CD and Blau syndrome.  It is estimated that at least 5 additional genes are also involved in susceptibility to CD.  These findings provided the impetus for laboratory-based studies of the molecular genetics of IBD, CD and UC.  Although many issues regarding gene function and expression remain to be resolved, there is much enthusiasm that useful clinical applications may follow (Satsangi et al, 2003; Girardin et al, 2003; Carneiro et al, 2004; Philpott and Viala, 2004).

Mutations in the gene that encodes NOD2 occur in a small portion of patients with CD.  Hampe,et al.(2001) noted that background genetic predisposition to IBD has been shown by epidemiological and linkage studies.  These researchers sequenced the coding region of the NOD2 gene and genotyped an insertion polymorphism affecting the leucine-rich region of the protein product in 512 individuals with IBD from 309 German or British families, 369 German trios (namely, German patients with sporadic IBD and their unaffected parents), and 272 normal controls.  They then tested for association with CD and UC.  Family-based association analyses were consistently positive in 95 British and 99 German affected sibling pairs with CD (combined p < 0.0001); the association was confirmed in the 304 German trios with CD.  No association was seen in the 115 sibling pairs and 65 trios with UC.  The genotype-specific disease risks conferred by heterozygous and homozygous mutant genotypes were 2.6 (95 % CI: 1.5 to 4.5) and 42.1 (4.3 to infinity), respectively.  These investigators stated that the insertion mutation in the NOD2 gene confers a substantially increased susceptibility to CD, but not to UC.  The lack of effect of the described mutation on UC might be due to the use of different pathways in this disorder.  Also, the NF-kappaB activation is stronger in CD than UC.  Moreover, the authors noted that this NOD2 frame-shift mutation is rather rare -- approximately 6.5 % of CD patients are homozygous for it.  They estimated that about 18 % of the genetic risk in the population can be attributed to this mutation; and did not find any homozygote in the control group.

Ahmad and co-workers (2002) stated that mutations in the NOD2 gene have been associated with CD, but are found in only 25 % of patients.  No data regarding their contribution to specific disease subtypes exist.  The authors reported a detailed genotype-phenotype analysis of accurately characterized patients.  A total of 244 white patients with CD recruited from a single center in the United Kingdom were studied.  All patients were phenotyped and followed-up for a median time of 16 years.  By using linkage disequilibrium mapping, these researchers studied 340 polymorphisms in 24 HLA genes and 3 NOD2 polymorphisms.  They demonstrated that NOD2 mutations determine ileal disease only, and confirmed that alleles on specific long-range HLA haplotypes determine overall susceptibility to CD.  These investigators concluded that clinical pattern of CD may be defined by specific genotypes.

There is also ethnic variation in the relationship of NOD2 genotype and clinical phenotype.  Guo et al (2004) noted that an insertion mutation at nucleotide 3020 (3020insC) in NOD2 is associated with CD.  The C-insertion mutation at nucleotide 3020 (3020insC) in the LRR region results in a frame-shift in the 10th LRR followed by a premature stop codon, which is responsible for the inability to activate NF-kappaB in response to bacterial lipopolysaccharide.  These researchers aimed to genotype NOD2 gene 3020insC frame-shift mutation in Chinese patients with IBD.  They genotyped an insertion polymorphism affecting the leucine-rich region of the protein product by the allele specific PCR in 74 unrelated patients with ulcerative colitis of Han nationality in Hubei Province of China, 15 patients with CD and 172 healthy individuals.  No significant differences were found in the genotype and allele frequencies of the C-insertion mutation of NOD2 gene among patients with CD and UC and healthy controls.  These investigators concluded that NOD2 gene 3020insC frame-shift mutation is not a major contributor to the susceptibility to both CD and UC in Chinese Han patients.  Moreover, Tosa et al (2006) noted that Japanese patients with CD do not have any of the common NOD2/CARD15 variants that are associated with CD in Caucasians.

Economou et al (2004) stated that 3 variants of the NOD2 gene -- SNP8, SNP12, and SNP13 -- have been associated with CD.  These investigators assessed the impact of NOD2 variants on the CD risk across diverse populations and examined possible associations with disease phenotype.  In a meta-analysis, a total of 42 eligible studies contributed data on 206 comparisons.  No variants were detected in Asians.  In non-Jewish descent Caucasians, carriage of SNP8, SNP12, or SNP13 had an odds ratio (OR) for CD of 2.20 (95 % CI: 1.84 to 2.62), 2.99 (95 % CI: 2.38 to 3.74), and 4.09 (95 % CI: 3.23 to 5.18), respectively.  For Jewish descent patients, the corresponding ORs were 1.74, 1.93, and 2.45, respectively.  The OR in carriers of at least 2 alleles was 17.1 (95 % CI: 10.7 to 27.2).  Large studies tended to yield more conservative estimates than smaller studies, so publication or other bias can not be excluded.  Among patients with CD, carrying at least 1 high-risk variant increased slightly the risk for familial disease (OR = 1.49, (95 % CI: 1.18 to 1.87)), modestly the risk of stenosing CD (OR = 1.94, (95 % CI: 1.61 to 2.34)), and more prominently the risk of small bowel involvement (OR = 2.53, (95 % CI: 2.01 to 3.16)).  The authors concluded that SNP8, SNP12, and SNP13 have differential effects on CD risk, with SNP13 having the strongest genetic effect.  The authors found that these NOD2 variants may also be significant risk factors for CD phenotype, especially ileal location.

Uyar and co-workers (2006) stated that 3 common genetic variations -- R702W, G908R, and 1007fs -- on CARD15 have been shown to increase the risk for CD in Caucasian populations.  The authors ascertained the frequencies of these CARD15 variants by genotyping in 56 patients with CD and 100 healthy ethnically matched controls from Turkey.  Overall frequency of all 3 variants was 10.7 % in patients with CD, compared with 1.5 % in controls (OR: 7.9).  Among them, the frequency of the G908R variant allele was 8 % in CD cases, compared with 0 % in controls (OR: 36.8).  The allele frequencies of 3 CD-related CARD15 variants were considerably lower in the control group compared to the reported Caucasian populations.  Among the described CARD15 variants, G908R confers an increased susceptibility to CD, whereas the more frequently reported associations in Europeans with R702W and 1007fs are not confirmed in this Turkish population.

In addition to studies on the role of NOD2 genetic variations in the development of intestinal strictures, questions also arise regarding whether the response to treatment or need for surgery can be predicted by genotype.  Preliminary reports analyzing the association between these variants and the need for surgeries have produced inconsistent results.  Alvarez-Lobos and associates (2005) examined the predictive value of NOD2 gene variants along with disease phenotypic characteristics for requirement of initial surgery and for surgical recurrence in CD. A  total of 170 CD patients were included prospectively in the study and followed-up regularly for a mean of 7.4 +/- 6.1 years.  Clinical characteristics of CD, time and indication for surgery, and recurrence were noted.  NOD2 gene variants were determined by DNA sequencing analysis.  It was reported that surgery for stricturing disease was significantly more frequent in patients with NOD2 variants in the uni-variate analysis (OR, 3.63; 95 % CI: 1.42  to 9.27), and it was required at an earlier time p = 0.004).  Only NOD2 variants (OR, 3.58; 95 % CI: 1.21 to 10.5) and stricturing phenotype at diagnosis of CD (OR, 9.34; 95 % CI: 2.56 to 33.3) were independent predictive factors of initial surgery for stricturing lesions in the multivariate analysis.  Among 70 patients that required surgery, post-operative recurrence was also more frequent in patients with NOD2 variants in the uni-variate and multi-variate analysis (OR, 3.29; 95 % CI: 1.13 to 9.56), and re-operation was needed at an earlier time (p = 0.03).  The authors concluded that NOD2 genotyping may have a useful clinical application as a major marker of evolution of CD, especially an early need of initial surgery due to stricturing disease and need of re-operation.  They noted that although determination of the NOD2 genotype presently cannot be used to guide indications for surgery, it might define a subgroup of patients with a severe course of the disease, who may require a more aggressive therapeutic approach to prevent the appearance of complications.  Moreover, these investigators stated that confirmation of these results in future studies is needed before interventional studies based on NOD2 genotyping are designed.

Fries and associates (2005) noted that a defect of gastrointestinal barrier function is considered to represent an important step in the pathogenesis of CD; but the mechanisms leading to an increased intestinal permeability (IP) are poorly understood.  Since IP is influenced by pro-inflammatory mediators, it seems likely that a genetically determined abnormal immune response may lead to a loss of barrier function.  In a geographic area in Southern Italy with high incidence of CD, these researchers examined IP (by means of lactulose/mannitol testing) together with the 3 main mutations of the NOD2/CARD15 and the D299G polymorphism of the toll-like receptor (TLR)-4 gene in 23 families of CD patients (patients and 1st-degree relatives).  A total of 48 % of CD patients and 40 % of their healthy relatives were found to have an abnormal IP compared to 5 % of an appropriate control population (p < 0.0001).  However, IP was not associated with the L1007finsC mutation of the NOD2/CARD15 or the D299G variant of the TLR-4 gene.  Allele frequency of the only L1007finsC mutation of CARD15 was significantly increased in patients (8.7 %, p < 0.003) and in relatives (8.3 %, p < 0.024) compared with controls (2.4 %), whereas the D299G variant of the TLR-4 gene was found to be increased only in relatives (8.3 %, p < 0.022), but not in patients (4.3 %) compared with the control population (1.7 %).  These investigators concluded that there was no association between IP and genetic markers.  These findings showed a very high proportion of healthy 1st-degree relatives to bare alterations suggested to constitute determinants of CD.  Mutations of NOD2/CARD15 or TLR-4, however, do not lead to permeability defects emphasizing the importance of additional environmental and/or genetic factors for pathogenesis.

While research has been focused on trying to explain the biological role of NOD2 and how mutations can contribute to the development of CD, it is important to note that the association between NOD2 gene variants and CD is not absolute (i.e., not everyone who has one of the variants will get the disease, and not everyone with CD has an alteration in NOD2).  Mascheretti and Schreiber (2005) noted that genetic testing for the NOD2/CARD15 variants has only a modest relevance in clinical practice.  This is in agreement with the observations of Vermeire (2004) who stated that the relevance of NOD2/CARD15 genotyping for clinical practice is modest.  The current data show that NOD2/CARD15 mutations in CD are associated with small-bowel involvement.  More studies are needed to ascertain if NOD2/CARD15 mutations are also associated with a fibro-stenotic behavior of the disease.  If CARD15 variants would predict a more aggressive disease course, then a more aggressive treatment may be justified in these patients after NOD2/CARD15 genetic testing.  It is not clear whether NOD2/CARD15 genotyping is helpful in differentiating indeterminate colitis patients.  Although CARD15 variants do not predict response to the tumor necrosis factor-alpha monoclonal antibodies, the role of the gene in response to other drugs is not known.  Finally, screening unaffected relatives of CD patients is not recommended until preventive strategies are available.  It is also interesting to note that although there is evidence to suggest that NOD2/CARD15 may influence individuals' susceptibility to colorectal cancer, there is no evidence to indicate that mutations of this gene predict the clinicopathological characteristics of this disease (Roberts et al, 2006).  Chamaillard and colleagues (2006) stated that the current etiologic model for IBD emphasizes an interaction between susceptibility and modifier genes along with environmental factors.  Together, these lead to disease progression.  However, further work should clarify the pathophysiological mechanisms leading to IBD and how innate immune signaling confers susceptibility to intestinal inflammation.

There is currently insufficient scientific evidence to support the clinical use of NOD2/CARD15 genotyping of CD.  More research is needed to define more precisely the relationship between NOD2/CARD15 genotype, clinical phenotype, and the effect of ethnic variation on this relationship.  Further investigation is also needed to better understand the mechanisms involved in gene-environment interactions in the gastrointestinal tract, and to examine the role of genetic variations in the NOD2/CARD15 gene on the natural cause of the disease, response to therapies and need for surgery.

CPT Codes / HCPCS Codes / ICD-10 Codes
Information in the [brackets] below has been added for clarification purposes.   Codes requiring a 7th character are represented by "+":
ICD-10 codes will become effective as of October 1, 2015 :
TPMT gene mutation assays or TPMT phenotypic assays (eg, Prometheus TPMT Genetics, Prometheus TPMT Enzyme):
CPT codes covered if selection criteria are met:
81401 Molecular pathology procedure, Level 2 (eg, 2-10 SNPs, 1 methylated variant, or 1 somatic variant [typically using nonsequencing target variant analysis], or detection of a dynamic mutation disorder/triplet repeat) (TPMT thiopurine S-methyltransferase) (eg, drug metabolism), common variants (eg, *2, *3) [Prometheus TPMT Genetics, Prometheus TPMT Enzyme]
82491 Chromatography, quantitative, column (e.g., gas liquid or HPLC); single analyte not elsewhere specified, single stationary and mobile phase
82657 Enzyme activity in blood cells, cultured cells, or tissue, not elsewhere specified; nonradioactive substrate, each specimen
CPT codes not covered for indications listed in the CPB:
82492 Chromatography, quantitative, column (eg, gas liquid or HPLC); multiple analytes, single stationary and mobile phase
83788 Mass spectrometry and tandem mass spectrometry (MS, MS/MS), analyte not elsewhere specified; qualitative, each specimen
83789     quantitative, each specimen
86256 Fluorescent noninfectious agent antibody; titer, each antibody
Anti-chitobioside carbohydrate antibodies (ACCA), Anti-laminaribioside carbohydrate antibodies (ALCA), Anti-mannobioside carbohydrate antibodies (AMCA) anti-chitin IgA (Anti-C), anti-laminarin IgA (Anti-L):
Anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisae antibodies (ASCA), anti-outer membrane porin C (OmpC) antibodies, anti-CBir1 flagellin (anti-CBir1) antibodies, 12 antibodies, and Anti-smooth muscle antibodies (ASMA):
CPT codes not covered for indications listed in the CPB:
82397 Chemiluminescent assay
83516 Immunoassay for analyte other than infectious agent antibody or infectious agent antigen, qualitative or semi-quantitative; multiple step method
83518 Immunoassay for analyte other than infectious agent antibody or infectious agent antigen; qualitative or semiquantitative, single step method (eg, reagent strip)
83519     quantitative, by radioimmunoassay (eg, RIA)
83520 Immunoassay, analyte, quantitative; not otherwise specified
86021 Antibody identification; leukocyte antibodies [ANCA antibodies]
86255 Fluorescent noninfectious agent antibody; screen, each antibody
86671 Antibody; fungus, not elsewhere specified
88347 Immunofluorescent study, each antibody; indirect method
6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN) (Prometheus Thiopurine Metabolites):
CPT codes covered if selection criteria are met:
82491 Chromatography, quantitative, column (e.g., gas liquid or HPLC); single analyte not elsewhere specified, single stationary and mobile phase
CPT codes not covered for indications listed in the CPB:
8299 Quantitation of therapeutic drug, not elsewhere specified
Other HCPC codes related to the CPB:
J7500 Azathioprine, oral, 50 mg
J7501 Azathioprine, parenteral, 100 mg
CPT codes not covered for indications listed in the CPB:
83520 Immunoassay, analyte, quantitative; not otherwise specified
83993 Calprotectin, fecal
Other HCPCS codes related to the CPB:
J7500 Azathioprine, oral, 50 mg
J7501 Azathioprine, parenteral, 100 mg
S0108 Mercaptopurine, oral 50 mg
NOD2/CARD15 genotyping:
No specific code
The Prometheus IBD sgi diagnostic panel and Prometheus IBS diagnostic panel (see next entry) :
(including BDNF [brain-derived neurotrophic factor], GRO-a [growth-regulated oncogen-alpha], IL1B [interleukin-1beta], NGAL [neutrophil gelatinase-associated lipocalin], CD14 gene C-260T polymorphism testing, interleukin-10-1082A/G polymorphism testing, Prometheus Crohn's Prognostic
No specific code
TIMP-1 [tissue inhibitor of metalloproteinase-1], tTG [anti-human tissue transglutaminase IgA) and TWEAK [TNF-related weak inducer of apoptosis):
No specific code
CPT codes not covered for indications listed in the CPB:
82397 Chemiluminescent assay
83520 Immunoassay, analyte, quantitative; not otherwise specified
86140 C-reactive protein
88347 Immunofluorescent study, each antibody; indirect method
Crohn’s disease peptide antibody testing, eXalBD test (a quantitative PCR test for 6 genes [BLCAP, UBE2G1, RAP1A, CALM3 AND NONO]), Measurement of serum mannose-binding lectin :
No specific code
Myeloperoxidase antibody testing:
CPT codes not covered for indications listing in the CPB:
83876 Myeloperoxidase (MPO)
Proteinase-3 antibody testing:
CPT codes not covered for indications listed in the CPB:
83516 Immunoassay for analyte other than infectious agent antibody or infectious agent antigen, qualitative or semi-quantitative; multiple step method
83520 Immunoassay, analyte, quantitative; not otherwise specified
86021 Antibody identification; leukocyte antibodies
Raman spectroscopy:
No specific code
ICD-10 codes covered if selecion criteria are met:
K50.0 - K51.919 Regional enteritis and ulcerative colitis
ICD-10 codes not covered if selection criteria are met:
K58.0 - K58.9 Irritable bowel syndrome

The above policy is based on the following references:

    Serologic Testing in Inflammatory Bowel Disease

    1. Griffiths AM. Serological diagnosis of IBD. Excerpts from the 1999 NASPGN Course Syllabus. North American Society for Pediatric Gastroenterology and Nutrition Position Papers. Flourtown, PA: NASPGN; 1999. Available at: Accessed October 20, 2000.
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    26. Deusch K, Oberstadt K, Schaedel W, et al. p-ANCA as a diagnostic marker in ulcerative colitis. Adv Exp Med Biol. 1993;336:527-531.
    27. Cambridge G, Rampton DS, Stevens TR, et al. Antineutrophil antibodies in inflammatory bowel disease: Prevalence and diagnostic role. Gut. 1992;33:668-674.
    28. Ruemmele FM, Targan SR, Levy G, et al. Diagnostic accuracy of serological assays in pediatric inflammatory bowel disease. Gastroenterology. 1998;115:822-829.
    29. Vasiliauskas EA, Plevy SE, Landers CJ, et al. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology. 1996;110:1810-1819.
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    39. Vasiliauskas EA, Kam LY, Karp LC, et al. Marker antibody expression stratifies Crohn's disease into immunologically homogenous subgroups with distinct clinical characteristics. Gut. 2000;47:487-496.
    40. Abreu MT, Vasiliauskas EA, Kam LY, Dubinsky MC. Use of serologic tests in Crohn's disease. Clin Gastroenterol Hepatol. 2001;4(3):158-164.
    41. Fleshner PR, vasiliauskas EA, Kam LY, et al. High level perinuclear antineutrophil cytoplasmic antibody (pANCA) in ulcerative colities patients before colectomy predicts the development of chronic pouchitis after ileal pouch-anal anastomosis. Gut. 2001;49:671-677.
    42. Dubinsky MC, Johanson JF, Seidman EG, Ofman JJ. Suspected inflammatory bowel disease - The clinical and economic impact of competing diagnostic strategies. Am J Gastroenterol. 2002;97(9):2333-2342.
    43. Ooi CJ, Lim BL, Cheong WK, et al. Antineutrophil cytoplasmic antibodies (ANCAs) in patients with inflammatory bowel disease show no correlation with proteinase 3, lactoferrin, myeloperoxidase, elastase, cathepsin G and lysozyme: A Singapore study. Ann Acad Med Singapore. 2000;29(6):704-707.
    44. Halme L, Turunen U, Helio T, et al. Familial and sporadic inflammatory bowel disease: comparison of clinical features and serological markers in a genetically homogeneous population. Scand J Gastroenterol. 2002;37(6):692-698.
    45. Linskens RK, Mallant-Hent RC, Groothuismink ZM, et al. Evaluation of serological markers to differentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobic coccoid rods. Eur J Gastroenterol Hepatol. 2002;14(9):1013-1018.
    46. Khan K, Schwarzenberg SJ, Sharp H, et al. Role of serology and routine laboratory tests in childhood inflammatory bowel disease. Inflamm Bowel Dis. 2002;8(5):325-329.
    47. Institute for Clinical Systems Improvement (ICSI). Serum antibodies for the diagnosis of inflammatory bowel disease (IBD): pANCA for ulcerative colitis (UC) and ASCA for Crohn's disease (CD). Technology Assessment Report No. 65. Bloomington, MN: ICSI; November 2002. 
    48. American Gastroenterological Association Clinical Practice Committee. American Gastroenterological Association medical position statement: Perianal Crohn's disease. Gastroenterology. 2003;125(5):1503-1507.
    49. Nakamura RM, Matsutani M, Barry M. Advances in clinical laboratory tests for inflammatory bowel disease. Clin Chim Acta 2003;335:9-20.
    50. Teml A, Kratzer V, Schneider B, et al. Anti-Saccharomyces cerevisiae antibodies: A stable marker for Crohn's disease during steroid and 5-aminosalicylic acid treatment. Am J Gastroenterol. 2003;98(10):2226-2231.
    51. Vasiliauskas E. Recent advances in the diagnosis and classification of inflammatory bowel disease. Curr Gastroenterol Rep. 2003;5;493-500.
    52. Weinshilboum R. Inheritance and drug response. N Engl J Med. 2003;348(6):529-537.
    53. Weinshilboum R. Thiopurine pharmacogenetics: Clinical and molecular studies of thiopurine methyltransferase. Drug Metab Disp. 2001;29(4 Pt 2):601-605.
    54. Landers CJ, Cohavy O, Misra R, et al. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto-and microbial antigens. Gastroenterology. 2002;123(3):689-699.
    55. Vernier G, Sendid B, Poulain D, Colombel JF. Relevance of serologic studies in inflammatory bowel disease. Curr Gastroenterol Rep. 2004;6(6):482-487.
    56. Saibeni S, Folli C, de Franchis R, et al. Diagnostic role and clinical correlates of anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (p-ANCA) in Italian patients with inflammatory bowel diseases. Dig Liver Dis. 2003; 35(12):862-868.
    57. Nakamura RM, Matsutani M, Barry M. Advances in clinical laboratory tests for inflammatory bowel disease. Clin Chim Acta. 2003;335(1-2):9-20.
    58. Forcione DG, Rosen MJ, Kisiel JB, Sands BE. Anti-Saccharomyces cerevisiae antibody (ASCA) positivity is associated with increased risk for early surgery in Crohn's disease. Gut. 2004;53(8):1117-1122.
    59. Gupta SK, Fitzgerald JF, Croffie JM, et al. Comparison of serological markers of inflammatory bowel disease with clinical diagnosis in children. Inflamm Bowel Dis. 2004; 10(3):240-244.
    60. Sandborn WJ. Serologic markers in inflammatory bowel disease: State of the art. Rev Gastroenterol Disord. 2004;4(4):167-174.
    61. Sutton CL, Kim J, Yamane A, et al. Identification of a novel bacterial sequence associated with Crohn's disease. Gastroenterology. 2000;119:23-31.
    62. Wei B, Huang T, Dalwadi H, et al. Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen. Infect Immun. 2002;70:6567-6575.
    63. Dalwadi H, Wei B, Kronenberg M, et al. The Crohn's disease-associated bacterial protein I2 is a novel enteric T cell superantigen. Immunity. 2001;15:149-158.
    64. Kornbluth A, Sachar DB. Ulcerative colitis practice guidelines in adults (update): American College of Gastroenterology, Practice Parameters Committee. Am J Gastroenterol. 2004;99(7):1371-1385.
    65. Mow WS, Landers CL, Steinhart AH, et al. High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn's disease: A pilot study. Digest Dis Sci. 2004;49(718):1280-1286.
    66. Mow WS, Vasiliauskas EA, Ying-Chao L, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterol. 2004;126(2):414-424.
    67. Vermeire S, Rutgeerts P. Antibody responses in Crohn's disease. Gastroenterol. 2004;126(2):601-604.
    68. Elitsur Y, Lawrence Z, Tolaymat N. The diagnostic accuracy of serologic markers in children with IBD: The West Virginia experience. J Clin Gastroenterol. 2005;39(8):670-673.
    69. Targan SR, Landers CJ, Yang H, et al. Antibodies to CBir1 flagellin define a unique response that is associated independently with complicated Crohn's disease. Gastroenterology. 2005;128(7):2020-2028.
    70. Dubinsky MC, Lin YC, Dutridge D, et al; Western Regional Pediatric IBD Research Alliance. Serum immune responses predict rapid disease progression among children with Crohn's disease: Immune responses predict disease progression. Am J Gastroenterol. 2006;101(2):360-367.
    71. Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem. 2006;52(2):171-181.
    72. North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition; Colitis Foundation of America, Bousvaros A, Antonioli DA, Colletti RB, et al. Differentiating ulcerative colitis from Crohn disease in children and young adults: Report of a working group of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the Crohn's and Colitis Foundation of America. J Pediatr Gastroenterol Nutr. 2007;44(5):653-674.
    73. Mokrowiecka A, Daniel P, Słomka M, et al. Clinical utility of serological markers in inflammatory bowel disease. Hepatogastroenterology. 2009;56(89):162-166.
    74. Lichtenstein GR, Hanauer SB, Sandborn WJ, and the Practice Parameters Committee of the American College of Gastroenterology. Management of Crohn’s disease in adults. Am J Gastroenterol. 2009;104(2):465-483. 
    75. Lembo AJ, Neri B, Tolley J, et al. Use of serum biomarkers in a diagnostic test for irritable bowel syndrome. Aliment Pharmacol Ther. 2009;29(8):834-842.
    76. Halpert AD. Importance of early diagnosis in patients with irritable bowel syndrome. Postgrad Med. 2010;122(2):102-111.
    77. Dotan I. New serologic markers for inflammatory bowel disease diagnosis. Dig Dis. 2010;28(3):418-423.
    78. Benor S, Russell GH, Silver M, et al. Shortcomings of the inflammatory bowel disease Serology 7 panel. Pediatrics. 2010;125(6):1230-1236.
    79. Silberer H, Küppers B, Mickisch O, et al. Fecal leukocyte proteins in inflammatory bowel disease and irritable bowel syndrome. Clin Lab. 2005;51(3-4):117-126.
    80. Lettesjo H, Hansson T, Peterson C, et al. Detection of inflammatory markers in stools from patients with irritable bowel syndrome and collagenous colitis. Scand J Gastroenterol. 2006;41(1):54-59.
    81. Navaneethan U, Shen B. Laboratory tests for patients with ileal pouch-anal anastomosis: Clinical utility in predicting, diagnosing, and monitoring pouch disorders. Am J Gastroenterol. 2009;104(10):2606-2615.
    82. Booth RA, Ansari MT, Loit E, et al. Assessment of thiopurine S-methyltransferase activity in patients prescribed thiopurines: A systematic review. Ann Intern Med. 2011;154(12):814-823.
    83. Thompson AI, Lees CW. Genetics of ulcerative colitis. Inflammatory.Bowel Diseases. 2011;17(3):831-848.
    84. Adler J, Rangwalla SC, Dwamena BA, Higgins PD. The prognostic power of the NOD2 genotype for complicated Crohn's disease: A meta-analysis. Am J Gastroenterol. 2011;106(4):699-712.
    85. Bi X, Walsh A, Mahadevan-Jansen A, Herline A. Development of spectral markers for the discrimination of ulcerative colitis and Crohn's disease using Raman spectroscopy. Dis Colon Rectum. 2011;54(1):48-53. 
    86. Wang Z, Hu J, Fan R, Zhou J, Zhong J. Association between CD14 gene C-260T polymorphism and inflammatory bowel disease: A meta-analysis. PLoS One. 2012;7(9):e45144.
    87. Zhu H, Lei X, Liu Q, Wang Y. Interleukin-10-1082A/G polymorphism and inflammatory bowel disease susceptibility: A meta-analysis based on 17,585 subjects. Cytokine. 2013;61(1):146-153.
    88. Kovacs M, Papp M, Lakatos PL, et al. Low mannose-binding lectin (MBL) is associated with paediatric inflammatory bowel diseases and ileal involvement in patients with Crohn disease. J Crohns Colitis. 2013;7(2):134-141.
    89. Peppercorn MA. Clinical manifestations, diagnosis and prognosis of Crohn's disease in adults. UpToDate [online serial]. Waltham, MA: UpToDate; reviewed December 2013.
    90. Mitsuyama K, Niwa M, Masuda J, et al; the Kyushu ACP Study Group. Possible diagnostic role of antibodies to Crohn's disease peptide (ACP): Results of a multicenter study in a Japanese cohort. J Gastroenterol. 2014;49(4):683-691.

    Fecal Markers of Inflammatory Bowel Disease

    1. Tibble JA, Sigthorsson G, Bridger S, et al. Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease. Gastroenterology. 2000;119(1):15-22.
    2. Hanauer SB, Sandborn W; Practice Parameters Committee of the American College of Gastroenterology. Management of Crohn's disease in adults. Am J Gastroenterol. 2001;96(3):635-643.
    3. Society of Surgery of the Alimentary Tract (SSAT). Management of ulcerative colitis. SSAT Patient Care Guidelines. Beverly, MA: SSAT; 2001. Available at: Accessed June 15, 2005.
    4. Kane SV, Sandborn WJ, Rufo PA, et al. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol. 2003;98(6):1309-1314.
    5. Thomas PD, Forbes A, Green J, et al. Guidelines for the investigation of chronic diarrhoea, 2nd edition. Gut. 2003;52 Suppl 5:v1-v15.
    6. Cremonini F, Talley NJ. Diagnostic and therapeutic strategies in the irritable bowel syndrome. Minerva Med. 2004;95(5):427-441.
    7. Hanai H, Takeuchi K, Iida T, et al. Relationship between fecal calprotectin, intestinal inflammation, and peripheral blood neutrophils in patients with active ulcerative colitis. Dig Dis Sci. 2004;49(9):1438-1443.
    8. Berni Canani R, Rapacciuolo L, Romano MT, et al. Diagnostic value of faecal calprotectin in paediatric gastroenterology clinical practice. Dig Liver Dis. 2004;36(7):467-470.
    9. Wassell J, Dolwani S, Metzner M, et al. Faecal calprotectin: A new marker for Crohn's disease? Ann Clin Biochem. 2004;41(Pt 3):230-232.
    10. Costa F, Mumolo MG, Ceccarelli L, et al. Calprotectin is a stronger predictive marker of relapse in ulcerative colitis than in Crohn's disease. Gut. 2005;54(3):364-368.
    11. Pardi DS, Sandborn WJ. Predicting relapse in patients with inflammatory bowel disease: What is the role of biomarkers? Gut. 2005;54(3):321-322.
    12. Kolho KL, Raivio T, Lindahl H, Savilahti E. Fecal calprotectin remains high during glucocorticoid therapy in children with inflammatory bowel disease. Scand J Gastroenterol. 2006;41(6):720-725.
    13. Denis MA, Reenaers C, Fontaine F, et al. Assessment of endoscopic activity index and biological inflammatory markers in clinically active Crohn's disease with normal C-reactive protein serum level. Inflamm Bowel Dis. 2007;13(9):1100-1105.
    14. D'Incà R, Dal Pont E, Di Leo V, et al. Can calprotectin predict relapse risk in inflammatory bowel disease? Am J Gastroenterol. 2008;103(8):2007-2014.
    15. Gisbert JP, McNicholl AG. Questions and answers on the role of faecal calprotectin as a biological marker in inflammatory bowel disease. Dig Liver Dis. 2009;41(1):56-66.
    16. Turner D, Leach ST, Mack D, et al. Faecal calprotectin, lactoferrin, M2-pyruvate kinase and S100A12 in severe ulcerative colitis: A prospective multicentre comparison of predicting outcomes and monitoring response. Gut. 2010;59(9):1207-1212.
    17. Bunn SK, Bisset WM, Main MJ, Golden BE. Fecal calprotectin as a measure of disease activity in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2001;32(2):171-177.
    18. Fagerberg UL, Lööf L, Lindholm J, et al. Fecal calprotectin: A quantitative marker of colonic inflammation in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2007;45(4):414-420.
    19. Gaya DR, Lyon TD, Duncan A, et al. Faecal calprotectin in the assessment of Crohn's disease activity. QJM. 2005;98(6):435-441.
    20. Gisbert JP, Bermejo F, Pérez-Calle JL, et al. Fecal calprotectin and lactoferrin for the prediction of inflammatory bowel disease relapse. Inflamm Bowel Dis. 2009;15(8):1190-1198.
    21. Joishy M, Davies I, Ahmed M, et al. Fecal calprotectin and lactoferrin as noninvasive markers of pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2009;48(1):48-54.
    22. Kallel L, Ayadi I, Matri S, et al. Fecal calprotectin is a predictive marker of relapse in Crohn's disease involving the colon: A prospective study. Eur J Gastroenterol Hepatol. 2010;22(3):340-345.
    23. Quail MA, Russell RK, Van Limbergen JE, et al. Fecal calprotectin complements routine laboratory investigations in diagnosing childhood inflammatory bowel disease. Inflamm Bowel Dis. 2009;15(5):756-759.
    24. Schoepfer AM, Beglinger C, Straumann A, et al. Fecal calprotectin correlates more closely with the Simple Endoscopic Score for Crohn's disease (SES-CD) than CRP, blood leukocytes, and the CDAI. Am J Gastroenterol. 2010;105(1):162-169.
    25. Schoepfer AM, Trummler M, Seeholzer P, et al. Accuracy of four fecal assays in the diagnosis of colitis. Dis Colon Rectum. 2007;50(10):1697-1706.
    26. Sipponen T, Björkesten CG, Färkkilä M, et al. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn's disease treatment. Scand J Gastroenterol. 2010;45(3):325-331.
    27. Sipponen T, Savilahti E, Kolho KL, et al. Crohn's disease activity assessed by fecal calprotectin and lactoferrin: Correlation with Crohn's disease activity index and endoscopic findings. Inflamm Bowel Dis. 2008;14(1):40-46.
    28. Tibble J, Teahon K, Thjodleifsson B, et al. A simple method for assessing intestinal inflammation in Crohn's disease. Gut. 2000;47(4):506-513.
    29. van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of patients with suspected inflammatory bowel disease: Diagnostic meta-analysis. BMJ. 2010;341:c3369.
    30. Vieira A, Fang CB, Rolim EG, et al. Inflammatory bowel disease activity assessed by fecal calprotectin and lactoferrin: Correlation with laboratory parameters, clinical, endoscopic and histological indexes. BMC Res Notes. 2009;2:221.
    31. World Gastroenterology Organisation Global Guidelines. Inflammatory bowel disease: A global perspective. WGO Global Guideline. June 2009.
    32. Peppercorn MA. Clinical manifestations, diagnosis and prognosis of Crohn's disease in adults. UpToDate [online serial]. Waltham, MA: UpToDate; reviewed December 2013.
    33. Henderson P, Anderson NH, Wilson DC. The diagnostic accuracy of fecal calprotectin during the investigation of suspected pediatric inflammatory bowel disease: A systematic review and meta-analysis. Am J Gastroenterol. 2014;109(5):637-645.
    34. Lasson A, Strid H, Ohman L, et al. Fecal calprotectin one year after ileocaecal resection for Crohn's disease -- a comparison with findings at ileocolonoscopy. J Crohns Colitis. 2014;8(8):789-795.

    Pharmacogenomic and Metabolic Assessment of Thiopurine Therapy

    1. Sandborn W, Sutherland L, Pearson D, et al. Azathioprine or 6-mercaptopurine for induction of remission in Crohn's disease (Cochrane Review). In: The Cochrane Library, Issue 2, 2002. Oxford, UK: Update Software.
    2. Cuffari C, Theoret Y, Latour S, Seidman G. 6-Mercaptopurine metabolism in Crohn's disease: Correlation with efficacy and toxicity. Gut. 1996;39(3):401-406.  
    3. Hanauer SB, Sandborn W; Practice Parameters Committee of the American College of Gastroenterology. Management of Crohn's disease in adults. Am J Gastroenterol. 2001;96(3):635-643.
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    NOD2/CARD15 Genotyping

    1. Hampe J, Cuthbert A, Croucher PJ, et al. Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet. 2001;357(9272):1925-1928.
    2. Ahmad T, Armuzzi A, Bunce M, et al. The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology. 2002;122(4):854-866.
    3. Inohara N, Nunez G. NODs: Intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003;3(5):371-382.
    4. Girardin SE, Hugot JP, Sansonetti PJ. Lessons from Nod2 studies: Towards a link between Crohn's disease and bacterial sensing. Trends Immunol. 2003;24(12):652-658.
    5. Satsangi J, Morecroft J, Shah NB, Nimmo E. Genetics of inflammatory bowel disease: Scientific and clinical implications. Best Pract Res Clin Gastroenterol. 2003;17(1):3-18.
    6. Carneiro LA, Travassos LH, Philpott DJ. Innate immune recognition of microbes through Nod1 and Nod2: Implications for disease. Microbes Infect. 2004;6(6):609-616.
    7. Economou M, Trikalinos TA, Loizou KT, et al. Differential effects of NOD2 variants on Crohn's disease risk and phenotype in diverse populations: A metaanalysis. Am J Gastroenterol. 2004;99(12):2393-2404
    8. Guo QS, Xia B, Jiang Y, et al. NOD2 3020insC frameshift mutation is not associated with inflammatory bowel disease in Chinese patients of Han nationality. World J Gastroenterol. 2004;10(7):1069-1071.
    9. Philpott DJ, Viala J. Towards an understanding of the role of NOD2/CARD15 in the pathogenesis of Crohn's disease. Best Pract Res Clin Gastroenterol. 2004;18(3):555-568.
    10. Vermeire S. NOD2/CARD15: Relevance in clinical practice. Best Pract Res Clin Gastroenterol. 2004;18(3):569-575.
    11. Inohara N, Chamaillard M, McDonald C, Nunez G. NOD-LRR proteins: Role in host-microbial interactions and inflammatory disease. Annu Rev Biochem. 2005;74:355-383.
    12. Eckmann L, Karin M. NOD2 and Crohn's disease: Loss or gain of function? Immunity. 2005;22(6):661-667.
    13. Alvarez-Lobos M, Arostegui JI, Sans M, et al. Crohn's disease patients carrying Nod2/CARD15 gene variants have an increased and early need for first surgery due to stricturing disease and higher rate of surgical recurrence. Ann Surg. 2005;242(5):693-700.
    14. Fries W, Renda MC, Lo Presti MA, et al. Intestinal permeability and genetic determinants in patients, first-degree relatives, and controls in a high-incidence area of Crohn's disease in Southern Italy. Am J Gastroenterol. 2005;100(12):2730-2736.
    15. Mascheretti S, Schreiber S. Genetic testing in Crohn disease: Utility in individualizing patient management. Am J Pharmacogenomics. 2005;5(4):213-222.
    16. Roberts RL, Gearry RB, Allington MD, et al. Caspase recruitment domain-containing protein 15 mutations in patients with colorectal cancer. Cancer Res. 2006;66(5):2532-2535.
    17. Chamaillard M, Iacob R, Desreumaux P, Colombel JF. Advances and perspectives in the genetics of inflammatory bowel diseases. Clin Gastroenterol Hepatol. 2006;4(2):143-151.
    18. Tosa M, Negoro K, Kinouchi Y, et al. Lack of association between IBD5 and Crohn's disease in Japanese patients demonstrates population-specific differences in inflammatory bowel disease. Scand J Gastroenterol. 2006;41(1):48-53.
    19. Uyar FA, Over-Hamzaoglu H, Ture F, et al. Distribution of common CARD15 variants in patients with sporadic Crohn's disease: Cases from Turkey. Dig Dis Sci. 2006;51(4):706-710.
    20. Figueroa C, Peralta A, Herrera L, et al. NOD2/CARD15 and Toll-like 4 receptor gene polymorphism in Chilean patients with inflammatory bowel disease. Eur Cytokine Netw. 2006;17(2):125-130. 
    21. Bene J, Magyari L, Talian G, et al. Prevalence of SLC22A4, SLC22A5 and CARD15 gene mutations in Hungarian pediatric patients with Crohn's disease. World J Gastroenterol. 2006;12(34):5550-5553.
    22. Alvarez-Lobos M, Arostegui JI, Sans M, et al. Combined type-1 plasminogen activator inhibitor and NOD2/CARD15 genotyping predicts complicated Crohn's disease behaviour. Aliment Pharmacol Ther. 2007;25(4):429-440.
    23. Morgan AR, Han DY, Lam WJ, et al. Association analysis of 3p21 with Crohn's disease in a New Zealand population. Hum Immunol. 2010;71(6):602-609.
    24. Stein JM, Lammert F, Zimmer V, et al. Clinical periodontal and microbiologic parameters in patients with Crohn's disease with consideration of the CARD15 genotype. J Periodontol. 2010;81(4):535-545.
    25. Lin Z, Hegarty JP, John G, et al. NOD2 mutations affect muramyl dipeptide stimulation of human B lymphocytes and interact with other IBD-associated genes. Dig Dis Sci. 2013;58(9):2599-2607.

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