Management in Health, Vol 16, No 3 (2012)

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UTILITY AND SIGNIFICANCE OF SEROLOGIC DETECTION OF IMMUNE MARKERS OF INFLAMMATION IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

UTILITY AND SIGNIFICANCE OF SEROLOGIC DETECTION OF IMMUNE MARKERS OF INFLAMMATION IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

 

Olga BRUSNIC1 - MD, PhD candidate

Professor Dr. Daniela DOBRU1- Chief Physician, Head of Doctoral School,

Danusia ONIŞOR1 - MD, PhD candidate,

Ofelia PASCARENCO1 - MD, PhD candidate,

Mircea STOIAN2- MD, PhD candidate,

 

1 Mureş County Clinical Hospital, Clinical Department of Gastroenterology

2 Mureş County Clinical Hospital, Department of Anesthesia and Intensive Therapy

 

 

 

INTRODUCTION: The recent advance in the area of diagnostic testing is focusing on serologic immune markers: atypical perinuclear anti-neutrophil citoplasmic antibody (pANCA) and anti-Saccharomyces cervisiae antibody (ASCA), and her utility in differentiating between ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis (IC). The aim of this study was to investigate the diagnostic value of pANCA and ASCA in inflammatory bowel diasease (IBD) diagnosis and for the differential diagnosis of UC from CD.

MATERIAL AND METHOD: A prospective study was conducted in 31 patients with new or established diagnoses of UC (n=15), CD (n=9) or IC (n=7) and also controls (n=7). Antibodies status has been measured with ELISA. A definitive diagnosis was reached using conventional techniques (colonoscopy or ileoscopy).

RESULTS: Sensitivity and specificity of pANCA for UC diagnosis was 66.67% and 77.78%, respectively; and ASCA for CD: 20% and 22.22%, respectively. The combined use of these two markers gave changes in diagnosis accuracy: pANCA+/ASCA- in UC and pANCA-/ASCA+ in CD: 75% and 72.73%, respectively. In phase II, for 23 of 38 patients a definitive diagnosis was reached using conventional techniques (colonoscopy and ileoscopy). In IC group, after 1-year follow-up, a definitive diagnosis was reached in 5 of the 7 patients.

CONCLUSION: The combined use of atypical pANCA and ASCA test results substantially affects pretest-posttest probability in distinguishing UC from CD in patients with IBD. This may be of help in patients in whom distinction between CD or UC is not obvious with the classic diagnostic tools.

 

Key words: Ulcerative colitis, Crohn's disease, Indeterminate colitis, perinuclear anti-neutrophil citoplasmic antibody , anti-Saccharomyces cervisiae antibody.

 

 

 

INTRODUCTION: Classically, three major entities of inflammatory bowel disease (IBD) have been defined based on symptoms of disease and standard clinical laboratory, endoscopic, radiologic and histologic parameters: Crohn's disease (CD), ulcerative colitis (UC) and indeterminate colitis (IC). The diagnosis of indeterminate colitis is usually a temporary diagnosis, and many patients with IC will be diagnosed with UC or CD over time [1].

The recent advance in the area of diagnostic testing is focusing on serologic immune markers. Numerous studies have investigated the utility of 2 serologic markers in differentiating between UC and CD: atypical perinuclear anti-neutrophil citoplasmic antibody (pANCA) and anti-Saccharomyces cervisiae antibody (ASCA). These determinations represent ideal non-invasive diagnostic test for IBD patients, especially in severe exacerbation, in cases where immediate endoscopy may be contraindicated or areas of the bowel are otherwise inaccessible [1]. Given the CD and UC-specific characteristics of ASCA and pANCA, these markers were initially introduced as markers that have increased and also improved test sensitivity, consideration has been given to these tests are adjunctive diagnostic tools and as possible prognostic indicators given their association with disease phenotype [2].

The aim of this study was to investigate the diagnostic value of pANCA and ASCA in IBD diagnosis and for the differential diagnosis of UC from CD.

 

MATERIAL AND METHOD: A prospective study was performed at the Gastroenterology Clinic, Targu Mures, in 2010. The reason for admission were workup of abdominal pain, altered bowel habit, and/or anorectal bleeding. In the first phase (phase I) a diagnosis of colonic disease was prospectively established based on clinical history and examination, laboratory findings (hemoglobin level, leukocyte count, platelet counts, electrolytes, serum albumin, ESR and CRP), abdominal ultrasonography and stool samples for exclusion of infectious diseases (fecal microbiology and fecal test for Clostridium difficile toxin). Out of the total of 164 IBD patients, in study were included 31 patients with new or established diagnoses of UC (n=15), CD (n=9) or IC (n=7) and also controls (n=7) - healthy subjects, with similar age and sex.

All serum samples were stored at -20C until analyzed. Determination of p-ANCA was performed by enzyme linked immunosorbent assay (ELISA) using myeloperoxidase IgG. According to the manufacturer's instructions, the cut-off value is 20 RU/ml; values higher than 20 RU/ml were interpreted as positive. The evaluation of ASCA was done using the ASCA IgA and IgG ELISA kits. Samples were interpreted as positive if ASCA was high than 20 RU/ml.

A definitive diagnosis was reached using conventional techniques (colonoscopy, ileoscopy or capsule endoscopy) for 23 of 38 patients (phase II). The extension of disease in UC group was determined by total colonoscopy, and categorized by Montreal classification into extensive UC (pancolitis), left-sided UC or ulcerative proctitis. The clinical activity was evaluated using the Mayo score and categorized into mild, moderate and severe.

The extension of disease in CD group were classified by Montreal classification into ileal, colonic, ileocolonic and isolated upper disease. The severity of disease was determined on clinical basis according to the Crohn's Disease Activity Index (CDAI) and categorized into mild-moderate, moderate-severe and severe-fulminant disease. CDAI higher than 150 was predicted as active disease in CD. The endoscopic Activity Score for CD was determined by SES-CD (Simplified endoscopic score for Crohn's disease).

Statistical analysis: Data were processed by statistical tools in Excel (Microsoft Excel 2003), and with statistical program GraphPad Prism 5. A p-value <0.05 was considered to be significant.

 

RESULTS: A total of 38 patients were participated in the study. All patients were including in three subgroups: 31 in IBD groups respectively: UC group (n=15), CD group (n=9) and IC group (n=7), and 7 healthy subjects - in the control group (CG). Demographic characteristics of the included patients are shown in Table 1.

There were 21 women and 17 men, with a mean age of 38.54 years. In the UC group, proctitis was present in 20%, left-sided colitis in 46.66% and extensive colitis in 33.33% (Figure 1).

Clinical activity were analyzed by UCDAI score: in the UC, no patient was in clinical remission; 2 patients had mild disease (4-5 points); 10 moderate disease (6-10 points); and 2 severe disease (11-12 points) (Fig.2).

In the CD group small bowel location was present in 11.11%, ileocolonic location in 33.33% and colonic location in the 55.55% (Fig. 3).

Clinical disease activity index in CD group were calculated by SES-CD and were in the range at 150 to 460, respectively mild-moderate disease in 22%, moderate-severe in 67% and severe in 11%.

pANCA was detected by ELISA in 10 of 15 (66.6%) samples from UC patients, in 2 of 9 (22.2%) samples from CD patients and in 3 of 7 (42.85%) samples of IC patients. pANCA was not detected in the CG (Table II). The difference between the prevalence of positive value pANCA in IBD and control groups was statistically significant (P value <0.0007). 5 (33%) patients with pancolitis and 7 (47%) patients with left-sided colitis were positive for pANCA. ASCA was detected by ELISA in 3 of 15 (20%) samples of UC patients, in 7 of 9 (77.7%) samples of CD group and in 2 of 7 (28,57%) samples of IC patients. Not found positive value for ASCA in CG. (Table 2).

The difference between the prevalence of positive value ASCA in IBD and control groups was statistically significant. The pANCA negative value in UC group were associated with mild disease cases of proctitis and left-sided colitis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of pANCA for UC diagnosis was 0.666, 0.7778, 0.8333 and 0.583; and ASCA for CD: 0.2000, 0.2222, 0.3000, 0.1429, respectively. The highest sensitivity for detecting IBD was achieved by using both pANCA and ASCA antibodies together. The combined use of these two markers gave changes in diagnosis accuracy: pANCA+/ASCA- in UC: 0.7500, 0.7273, 0.7500 and 0.7273, and for pANCA-/ASCA+ in CD: 07273, 0.7500, 0.7500 and 0.7273 respectively. pANCA-/ASCA- value were detected in 2 patients with IC.

 

In phase II, for 23 of 38 patients a definitive diagnosis was reached using conventional techniques (colonoscopy and ileoscopy). In IC group (n=7) endoscopic and histological diagnosis were UC (n=3), CD (n=2) and IC (n=2). After 1-year follow-up, a definite diagnosis was reached in 5 of the 7 patients.

 

DISCUSSIONS: Serologic testing using pANCA and ASCA has also been proposed as a method to reach a definitive diagnosis of Cron's diasease or ulcerative colitis in patients with indeterminate colitis [3-7]. pANCA has been shown repeatedly to be prevalent in the sera of approximately 60% and 20% of UC and CD patients, respectively [8-10]. In our study pANCA were detected in 10 of 15 patients (66.66%) with UC, in 2 of 9 cases (22.22%) with CD, and in 3 of 7 patients (42,85%) with IC. A distinct subset of patients manifesting left-sided Crohn's colitis may be positive for pANCA [11]. Koutrobakis et al. reported pANCA positivity as 30% in colonic involvement of CD patients [12]. We found 22.22% (2/9) in colonic involvement and these findings are in accordance with data from literature. The combination of atypical pANCA and ASCA may be useful in the differential diagnosis of UC and CD in patients with IBD. The UC associated pattern was pANCA +/ ASCA-, whereas the CD associated patern was ASCA+/pANCA-. The combined evaluation of pANCA and ASCA had a higher specificity (>90% in most studies and > 80% in all studies) to differentiate CD from UC than the separate use of either pANCA or ASCA. In patients with IBD, the PPV of the combination of a positive ASCA test with a negative pANCA test for UC has been reported to be 92,5% by Quinton et al. [9], 95% by Peeters et al. [13] and 77% by Koutrobakis et al.[12]. The PPV places test specificity in the context of disease prevalence and indicates what percentage of patients with positive test results actually have the disease. The our data for specificity and sensitivity is in accordance with literature.

Thus, the combined use of pANCA and ASCA results could be an addition to conventional technique (the patient's history, radiologic examination, endoscopy and biopsy) in the differential diagnosis between UC and CD.

 

CONCLUSIONS: In summary, the testing for pANCA, ASCA and other antibodies appears to be a promising approach to diagnose inflammatory bowel disease, and to distinguish UC from CD. Serologic evaluation of pANCA and ASCA could be of help in patients with indeterminate colitis. In this patients, early knowledge of the exact diagnosis could be of clinical importance with regard to therapeutic decisions and prognosis. The combination pANCA+/ ASCA- predicted UC in 57.1% of IC patients, whereas pANCA-/ASCA+ predicted CD in 42.8% of the patients. The 64.27% of patients did not have antibodies to either ASCA or pANCA and these seronegative patients remained indeterminate.

The combined use of atypical pANCA and ASCA test results substantially affects pretest-posttest probability in distinguishing UC from CD in patients with IBD. This may be of help in patients in whom distinction between CD or UC is not obvious with the classic diagnostic tools.

 

Table 1 - Demographic characteristics of the patients

 


 

UC

CD

IC

CG

Number of patients

15

9

7

7

Gender

 

 

 

 

Male

6

4

3

4

Female

9

5

4

3

Age (Mean value)

41.66

43.22

34.57

34.71

Location of disease (Montreal classification)

Ulcerative proctitis

3

-

-

-

Left-sided UC

7

-

-

-

Extensive UC

5

-

-

-

Small bowel

-

1

-

-

Ileocolonic

-

3

-

-

Colon

-

5

-

-

Clinical disease activity

 

 

 

 

CDAI

-

180.66 (150-460)

-

-

Mayo score (UCDAI)

8.66 (4-12)

-

-

-

Endoscopic activity index (EAI)

2.92 (1-4)

-

3 (2-4)

-

 

 

 

 

 

Simplified Endoscopic Activity Score for Crohn's Disease (SES-CD)

-

2.44 (1-4)

2.5 (2-3)

-

Treatment

 

 

 

 

Masalzine

10

9

-

-

Oral steroid

6

9

-

-

Ciprofloxacin

2

1

-

-

Azathioprin

3

-

-

-

Infliximab

2

-

-

-

Smoking status

7

4

4

5

 

 

Figure 1 - Disease extent in Ulcerative colitis

 

Figure 2 - Clinical activity analyzed by UCDAI score

 

 

Figure 3 - Location of disease in CD group

 

 

Table 2 - Prevalence of pANCA and ASCA in patients with UC, CD, IC and CG

 

 

 


CU

BC

IC

GC

 

(n=15)

(n=9)

(n=7)

(n=7)

pANCA+

10

2

3

0

pANCA-

5

7

4

7

ASCA+

3

7

2

0

ASCA-

12

2

5

7

pANCA+/ ASCA-

13

7

3

-

pANCA-/ ASCA+

6

11

2

-

 

 

 

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3.       LICHTENSTEIN, Gary-Clinical focus - Challenges in the Diagnosis of Ulcerative Colitis and Crohn's disease. Medscape education, 2006.

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8.       RUEMMELE, F.M., TARGAN, S.R., LEVY, G. et al.-Diagnostic accuracy of serological assays in pediatric, Inflammatory bowel disease; 115: 822-9, Gastroenterology, 1998,

9.       QUINTON, J.F., SENDID, B. REUMAUX, D. et al.-Anti-Saccharomyces cervisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease: prevalence and diagnostic role. 42: 788-791; Gut. 1998,

10.    HOFFENBERG, E.J., FIDANZA, S., SAUAIA, A.-Serologic testing for inflammatory bowel disease, J Pediat; 134: 447-52, 1999,

11.    VASILIAUSKAS, E.A., PLEVY, SE., LANDERS, C.J. et al.-Perinuclear antineutrophil cytoplasmic autoantibodies in patients with Crohn's disease define a clinical subgroup.; 110:1810-9, Gastroenterology, 1996,

12.    KOUTROBASKIS, I.E., PETINAKI, E., MOUZAS, I.A., et al.-Anti-Saccharomyces cervisiae mannan antibodies and antineutrophil cytoplasmic autoantibodies in Greek patients with inflammatory bowel disease; 96(2): 449-54., Am J Gastroenterol, 2001,

13.    PETERS, M., JOOSSENS, S., VERMEIRE, S., VLIETINCK, R., BOSSUYT, X., RUTGEERTS, P.-Diagnostic value of anti-Saccharomyces cervisiae and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease,; 96:730-4, Am J Gastroenterology, 2001.

 

 

 

 



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