nature publishing group
ORIGINAL CONTRIBUTIONS
1
Consecutive Monitoring of Fecal Calprotectin and Lactoferrin for the Early Diagnosis and Prediction of Pouchitis after Restorative Proctocolectomy for Ulcerative Colitis Takayuki Yamamoto, MD, PhD, FACG1, Takahiro Shimoyama, MD1, Takuya Bamba, MD1 and Koichi Matsumoto, MD, PhD1 OBJECTIVES:
This prospective study was conducted to evaluate the significance of consecutive monitoring of fecal calprotectin and lactoferrin for the early diagnosis and prediction of pouchitis after restorative proctocolectomy for ulcerative colitis (UC).
METHODS:
Sixty patients who had ileostomy closure following total proctocolectomy and ileal pouch-anal anastomosis for UC were included. Stool samples were collected for the measurement of calprotectin and lactoferrin every 2 months up to 12 months after the ileostomy closure. When patients had symptoms suggestive of pouchitis, endoscopic examination was immediately undertaken. All asymptomatic patients underwent endoscopy at 12 months. Pouchitis was defined as a pouchitis disease activity index score of ≥7.
RESULTS:
During the 12 months, 10 patients (17%) developed pouchitis. In patients with pouchitis, fecal calprotectin and lactoferrin levels were elevated already 2 months before the diagnosis of pouchitis. In contrast, these fecal biomarkers remained at low levels, and they did not change significantly in patients without pouchitis. A cutoff value of 56 μ g/g for calprotectin had a sensitivity of 100% and a specificity of 84% to predict pouchitis, whereas a cutoff value of 50 μ g/g for lactoferrin had a sensitivity of 90% and a specificity of 86%. At the time of endoscopy, the median calprotectin and lactoferrin levels were significantly higher in patients with pouchitis than those without pouchitis.
CONCLUSIONS: Elevated fecal calprotectin and lactoferrin levels appeared to be significant predictors of pouchitis
after restorative proctocolectomy for UC. Consecutive monitoring of these fecal biomarkers is useful for the early diagnosis of pouchitis. Am J Gastroenterol advance online publication, 28 April 2015; doi:10.1038/ajg.2015.129
INTRODUCTION Ileal pouch-anal anastomosis is currently accepted as the standard method to restore continence after total proctocolectomy for medically refractory ulcerative colitis (UC) and familial adenomatous polyposis (1). Pouchitis is the most common complication of total proctocolectomy and ileal pouch-anal anastomosis in UC (2). The incidence of a first episode of pouchitis at 1, 5, and 10 years postoperatively is 15, 33, and 45%, respectively (2,3). A variety of pathophysiological mechanisms have been proposed for pouchitis, but the precise pathogenesis remains unknown. The use of noninvasive biomarkers for the diagnosis and management of inflammatory bowel disease (IBD) has been increasing. Calprotectin
is a neutrophil cytosolic protein with antimicrobial properties, which is present at an increased concentration in stool during bowel inflammation (4). The stability of the protein to degradation keeps it stable in feces for up to 7 days at room temperature, making it an ideal analyte (5). Lactoferrin is an iron-binding glycoprotein and a major component of the secondary granules of polymorphonuclear neutrophils (6). During intestinal inflammation, leukocytes invade the mucosa, which may lead to an increase in the excretion of lactoferrin into the stool. Lactoferrin can be measured by using simple and inexpensive techniques, as it is stable in the stool for up to 5 days. Fecal calprotectin and lactoferrin are highly sensitive and specific biomarkers for detecting intestinal inflammation (7). These fecal
1
Inflammatory Bowel Disease Center, Yokkaichi Hazu Medical Center, Yokkaichi, Mie, Japan. Correspondence: Takayuki Yamamoto, MD, PhD, FACG, Inflammatory Bowel Disease Center, Yokkaichi Hazu Medical Center, 10-8 Hazuyamacho, Yokkaichi, Mie 510-0016, Japan. E-mail: [email protected] Received 22 December 2014; accepted 3 March 2015
© 2015 by the American College of Gastroenterology
The American Journal of GASTROENTEROLOGY
INFLAMMATORY BOWEL DISEASE
see related editorial on page x
INFLAMMATORY BOWEL DISEASE
2
Yamamoto et al.
biomarkers showed a close correlation with endoscopic inflammation in patients with IBD (8,9). Further, elevated fecal calprotectin and lactoferrin levels were associated with subsequent clinical relapse (10). Fecal calprotectin and lactoferrin determination may be useful in predicting clinical relapse in patients with IBD (11,12). A few studies (13–16) have been carried out to evaluate the value of fecal calprotectin or lactoferrin measurement in patients with pouchitis after restorative proctocolectomy for UC. In these studies, fecal calprotectin or lactoferrin level was determined by only one test using a single stool sample (13–16). So far, no studies have consecutively measured the levels of fecal calprotectin and lactoferrin in patients after ileal pouch surgery. Therefore, the value of consecutive fecal calprotectin and lactoferrin measurements after restorative proctocolectomy remains unknown. This prospective study was designed to evaluate the significance of consecutive monitoring of fecal calprotectin and lactoferrin for the early diagnosis and prediction of pouchitis after restorative proctocolectomy for UC.
Table 1. Baseline characteristics of 60 patients Median (range) age at entry
34 (21–64) years
Male:female (n)
37:23
Median (range) duration of UC before proctocolectomy
51 (13–112) months
Preoperative medications for UC (n) 5-aminosalicylic acid
57 (95%)
Corticosteroids (prednisolone 20–60 mg/day)
56 (93%)
Azathioprine or 6-mercaptopurine
32 (53%)
Tacrolimus
10 (17%)
Infliximab
7 (12%)
Extent of UC (n) Left-sided colitis
24 (40%)
a
36 (60%)
Extensive colitis
Extraintestinal manifestations (n)
METHODS Study design
This was a prospective, single-center study undertaken at the Yokkaichi Hazu Medical Center, a referral center treating a large number of patients with IBD. The study was conducted in accordance with good clinical practice and the Declaration of Helsinki principles. Our study protocol was reviewed and approved by our Institutional Review Board. Patients
Inclusion criteria were as follows: (i) patients who were between 20 and 70 years of age; (ii) patients who were diagnosed with histologically confirmed UC; and (iii) patients who had ileostomy closure following total proctocolectomy and ileal pouch-anal anastomosis for medically refractory UC. Exclusion criteria were as follows: (i) patients who developed pouch-related complications (anastomotic leak, pelvic abscess, pouch fistula, anastomotic stricture); and (ii) patients who were taking antibiotics, nonsteroidal anti-inflammatory agents, 5-aminosalicylic acids, corticosteroids, or immunosuppressive medications within 1 month of the entry to the study. A total of 60 patients who met the inclusion criteria were included in this study. Baseline characteristics of the 60 eligible patients are shown in Table 1. All patients underwent a total proctocolectomy and ileal pouch-anal anastomosis with a covering ileostomy. Postoperatively, all patients were in good health, were off steroid medication, and regained their normal body weight. At 3 months after restorative proctocolectomy, ileostomy closure was performed.
Arthritis
7 (12%)
Pyoderma gangrenosum
2 (3%)
UC, ulcerative colitis. a Involvement extends proximal to the splenic flexure.
acids, corticosteroids, or immunosuppressive medications before the diagnosis of pouchitis. In our clinic, peripheral blood samples were collected for the measurement of white blood cell (WBC) count, hemoglobin, platelet count, C-reactive protein (CRP), total protein, albumin, creatinine, urea, sodium, potassium, chloride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactic dehydrogenase, total bilirubin, and cholesterol. Endoscopic examination
When patients had clinical symptoms suggestive of pouchitis (increased stool frequency, urgency, tenesmus, incontinence, nocturnal seepage, abdominal cramping, and pelvic discomfort) at the clinic visits, endoscopic examination was immediately undertaken. In contrast, all asymptomatic patients underwent endoscopy at the end of this study (12 months after the ileostomy closure). During the endoscopy, the biopsies were obtained from the ileal pouch in each patient. The endoscopic and histologic severities of the mucosal inflammation were assessed according to the components of “endoscopic findings” and “histologic findings” in the PDAI, respectively. Pouchitis was defined as a PDAI score (maximum score: 18) of ≥7. Endoscopists were blinded to the results of fecal biomarkers.
Clinical assessment
All patients were reviewed in our clinic every 2 months up to 12 months after the ileostomy closure. At the clinic visits, clinical assessment was performed according to the component of “symptom” in a Pouchitis Disease Activity Index (PDAI) (17); stool frequency, rectal bleeding, fecal urgency, abdominal cramps, and fever. During the study period, patients were not allowed to take antibiotics, nonsteroidal anti-inflammatory agents, 5-aminosalicylic The American Journal of GASTROENTEROLOGY
Measurement of fecal calprotectin and lactoferrin
Patients provided a stool sample for the measurement of calprotectin and lactoferrin at the clinic visits, every 2 months after the ileostomy closure. Fecal calprotectin was measured by a quantitative enzyme immunoassay (Human Calprotectin enzyme-linked immunosorbent assay Kit, Cell Sciences, Canton, MA). Lactoferrin was measured by a colloidal gold agglutination reagent (Auto www.amjgastro.com
Lf-Plus, Alfresa Pharma, Osaka, Japan) by using a high-throughput discrete clinical chemistry analyzer (Hemo Techt NS-Plus C, Alfresa Pharma). Laboratory personnel were blinded to the clinical data.
diagnosed with pouchitis (PDAI score: median 3, range 0–6). No patient went on to develop Crohn’s disease in the ileal pouch during this study.
Statistical analysis
Fecal biomarkers before the diagnosis of pouchitis
Comparisons of frequencies were analyzed using the χ2 test with Yates’ correction. Differences between median values were compared using the Mann–Whitney U-test or the Kruskal– Wallis test if more than two groups were compared. The change in median values with time was evaluated by Wilcoxon signed rank test. Correlations were calculated by using the Spearman’s r-test. P0.05), and remained at low levels. However, these levels were significantly elevated 2 months before the diagnosis of pouchitis (calprotectin: P=0.005 and lactoferrin: P=0.005 vs. 4 months before the diagnosis of pouchitis), although those patients were asymptomatic. In contrast, in 50 patients without pouchitis, the median fecal calprotectin and lactoferrin levels remained at low levels, and did not change significantly during the entire study (all comparisons: P>0.05; Figure 2).
RESULTS Pouchitis
During the study period, 13 patients (22%) developed clinical symptoms suggestive of pouchitis. Endoscopic examination was undertaken in the 13 patients, of whom 10 (17%) were diagnosed with pouchitis (PDAI score: median 11, range 8–14). The duration from the ileostomy closure to the diagnosis of pouchitis was 6 months in 1 patient, 8 months in 3 patients, 10 months in 4 patients, and 12 months in 2 patients (median: 10 months). In the remaining 3 symptomatic patients, there was no apparent inflammation in the ileal pouch, and the PDAI score was
ORIGINAL CONTRIBUTIONS
1
Consecutive Monitoring of Fecal Calprotectin and Lactoferrin for the Early Diagnosis and Prediction of Pouchitis after Restorative Proctocolectomy for Ulcerative Colitis Takayuki Yamamoto, MD, PhD, FACG1, Takahiro Shimoyama, MD1, Takuya Bamba, MD1 and Koichi Matsumoto, MD, PhD1 OBJECTIVES:
This prospective study was conducted to evaluate the significance of consecutive monitoring of fecal calprotectin and lactoferrin for the early diagnosis and prediction of pouchitis after restorative proctocolectomy for ulcerative colitis (UC).
METHODS:
Sixty patients who had ileostomy closure following total proctocolectomy and ileal pouch-anal anastomosis for UC were included. Stool samples were collected for the measurement of calprotectin and lactoferrin every 2 months up to 12 months after the ileostomy closure. When patients had symptoms suggestive of pouchitis, endoscopic examination was immediately undertaken. All asymptomatic patients underwent endoscopy at 12 months. Pouchitis was defined as a pouchitis disease activity index score of ≥7.
RESULTS:
During the 12 months, 10 patients (17%) developed pouchitis. In patients with pouchitis, fecal calprotectin and lactoferrin levels were elevated already 2 months before the diagnosis of pouchitis. In contrast, these fecal biomarkers remained at low levels, and they did not change significantly in patients without pouchitis. A cutoff value of 56 μ g/g for calprotectin had a sensitivity of 100% and a specificity of 84% to predict pouchitis, whereas a cutoff value of 50 μ g/g for lactoferrin had a sensitivity of 90% and a specificity of 86%. At the time of endoscopy, the median calprotectin and lactoferrin levels were significantly higher in patients with pouchitis than those without pouchitis.
CONCLUSIONS: Elevated fecal calprotectin and lactoferrin levels appeared to be significant predictors of pouchitis
after restorative proctocolectomy for UC. Consecutive monitoring of these fecal biomarkers is useful for the early diagnosis of pouchitis. Am J Gastroenterol advance online publication, 28 April 2015; doi:10.1038/ajg.2015.129
INTRODUCTION Ileal pouch-anal anastomosis is currently accepted as the standard method to restore continence after total proctocolectomy for medically refractory ulcerative colitis (UC) and familial adenomatous polyposis (1). Pouchitis is the most common complication of total proctocolectomy and ileal pouch-anal anastomosis in UC (2). The incidence of a first episode of pouchitis at 1, 5, and 10 years postoperatively is 15, 33, and 45%, respectively (2,3). A variety of pathophysiological mechanisms have been proposed for pouchitis, but the precise pathogenesis remains unknown. The use of noninvasive biomarkers for the diagnosis and management of inflammatory bowel disease (IBD) has been increasing. Calprotectin
is a neutrophil cytosolic protein with antimicrobial properties, which is present at an increased concentration in stool during bowel inflammation (4). The stability of the protein to degradation keeps it stable in feces for up to 7 days at room temperature, making it an ideal analyte (5). Lactoferrin is an iron-binding glycoprotein and a major component of the secondary granules of polymorphonuclear neutrophils (6). During intestinal inflammation, leukocytes invade the mucosa, which may lead to an increase in the excretion of lactoferrin into the stool. Lactoferrin can be measured by using simple and inexpensive techniques, as it is stable in the stool for up to 5 days. Fecal calprotectin and lactoferrin are highly sensitive and specific biomarkers for detecting intestinal inflammation (7). These fecal
1
Inflammatory Bowel Disease Center, Yokkaichi Hazu Medical Center, Yokkaichi, Mie, Japan. Correspondence: Takayuki Yamamoto, MD, PhD, FACG, Inflammatory Bowel Disease Center, Yokkaichi Hazu Medical Center, 10-8 Hazuyamacho, Yokkaichi, Mie 510-0016, Japan. E-mail: [email protected] Received 22 December 2014; accepted 3 March 2015
© 2015 by the American College of Gastroenterology
The American Journal of GASTROENTEROLOGY
INFLAMMATORY BOWEL DISEASE
see related editorial on page x
INFLAMMATORY BOWEL DISEASE
2
Yamamoto et al.
biomarkers showed a close correlation with endoscopic inflammation in patients with IBD (8,9). Further, elevated fecal calprotectin and lactoferrin levels were associated with subsequent clinical relapse (10). Fecal calprotectin and lactoferrin determination may be useful in predicting clinical relapse in patients with IBD (11,12). A few studies (13–16) have been carried out to evaluate the value of fecal calprotectin or lactoferrin measurement in patients with pouchitis after restorative proctocolectomy for UC. In these studies, fecal calprotectin or lactoferrin level was determined by only one test using a single stool sample (13–16). So far, no studies have consecutively measured the levels of fecal calprotectin and lactoferrin in patients after ileal pouch surgery. Therefore, the value of consecutive fecal calprotectin and lactoferrin measurements after restorative proctocolectomy remains unknown. This prospective study was designed to evaluate the significance of consecutive monitoring of fecal calprotectin and lactoferrin for the early diagnosis and prediction of pouchitis after restorative proctocolectomy for UC.
Table 1. Baseline characteristics of 60 patients Median (range) age at entry
34 (21–64) years
Male:female (n)
37:23
Median (range) duration of UC before proctocolectomy
51 (13–112) months
Preoperative medications for UC (n) 5-aminosalicylic acid
57 (95%)
Corticosteroids (prednisolone 20–60 mg/day)
56 (93%)
Azathioprine or 6-mercaptopurine
32 (53%)
Tacrolimus
10 (17%)
Infliximab
7 (12%)
Extent of UC (n) Left-sided colitis
24 (40%)
a
36 (60%)
Extensive colitis
Extraintestinal manifestations (n)
METHODS Study design
This was a prospective, single-center study undertaken at the Yokkaichi Hazu Medical Center, a referral center treating a large number of patients with IBD. The study was conducted in accordance with good clinical practice and the Declaration of Helsinki principles. Our study protocol was reviewed and approved by our Institutional Review Board. Patients
Inclusion criteria were as follows: (i) patients who were between 20 and 70 years of age; (ii) patients who were diagnosed with histologically confirmed UC; and (iii) patients who had ileostomy closure following total proctocolectomy and ileal pouch-anal anastomosis for medically refractory UC. Exclusion criteria were as follows: (i) patients who developed pouch-related complications (anastomotic leak, pelvic abscess, pouch fistula, anastomotic stricture); and (ii) patients who were taking antibiotics, nonsteroidal anti-inflammatory agents, 5-aminosalicylic acids, corticosteroids, or immunosuppressive medications within 1 month of the entry to the study. A total of 60 patients who met the inclusion criteria were included in this study. Baseline characteristics of the 60 eligible patients are shown in Table 1. All patients underwent a total proctocolectomy and ileal pouch-anal anastomosis with a covering ileostomy. Postoperatively, all patients were in good health, were off steroid medication, and regained their normal body weight. At 3 months after restorative proctocolectomy, ileostomy closure was performed.
Arthritis
7 (12%)
Pyoderma gangrenosum
2 (3%)
UC, ulcerative colitis. a Involvement extends proximal to the splenic flexure.
acids, corticosteroids, or immunosuppressive medications before the diagnosis of pouchitis. In our clinic, peripheral blood samples were collected for the measurement of white blood cell (WBC) count, hemoglobin, platelet count, C-reactive protein (CRP), total protein, albumin, creatinine, urea, sodium, potassium, chloride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactic dehydrogenase, total bilirubin, and cholesterol. Endoscopic examination
When patients had clinical symptoms suggestive of pouchitis (increased stool frequency, urgency, tenesmus, incontinence, nocturnal seepage, abdominal cramping, and pelvic discomfort) at the clinic visits, endoscopic examination was immediately undertaken. In contrast, all asymptomatic patients underwent endoscopy at the end of this study (12 months after the ileostomy closure). During the endoscopy, the biopsies were obtained from the ileal pouch in each patient. The endoscopic and histologic severities of the mucosal inflammation were assessed according to the components of “endoscopic findings” and “histologic findings” in the PDAI, respectively. Pouchitis was defined as a PDAI score (maximum score: 18) of ≥7. Endoscopists were blinded to the results of fecal biomarkers.
Clinical assessment
All patients were reviewed in our clinic every 2 months up to 12 months after the ileostomy closure. At the clinic visits, clinical assessment was performed according to the component of “symptom” in a Pouchitis Disease Activity Index (PDAI) (17); stool frequency, rectal bleeding, fecal urgency, abdominal cramps, and fever. During the study period, patients were not allowed to take antibiotics, nonsteroidal anti-inflammatory agents, 5-aminosalicylic The American Journal of GASTROENTEROLOGY
Measurement of fecal calprotectin and lactoferrin
Patients provided a stool sample for the measurement of calprotectin and lactoferrin at the clinic visits, every 2 months after the ileostomy closure. Fecal calprotectin was measured by a quantitative enzyme immunoassay (Human Calprotectin enzyme-linked immunosorbent assay Kit, Cell Sciences, Canton, MA). Lactoferrin was measured by a colloidal gold agglutination reagent (Auto www.amjgastro.com
Lf-Plus, Alfresa Pharma, Osaka, Japan) by using a high-throughput discrete clinical chemistry analyzer (Hemo Techt NS-Plus C, Alfresa Pharma). Laboratory personnel were blinded to the clinical data.
diagnosed with pouchitis (PDAI score: median 3, range 0–6). No patient went on to develop Crohn’s disease in the ileal pouch during this study.
Statistical analysis
Fecal biomarkers before the diagnosis of pouchitis
Comparisons of frequencies were analyzed using the χ2 test with Yates’ correction. Differences between median values were compared using the Mann–Whitney U-test or the Kruskal– Wallis test if more than two groups were compared. The change in median values with time was evaluated by Wilcoxon signed rank test. Correlations were calculated by using the Spearman’s r-test. P0.05), and remained at low levels. However, these levels were significantly elevated 2 months before the diagnosis of pouchitis (calprotectin: P=0.005 and lactoferrin: P=0.005 vs. 4 months before the diagnosis of pouchitis), although those patients were asymptomatic. In contrast, in 50 patients without pouchitis, the median fecal calprotectin and lactoferrin levels remained at low levels, and did not change significantly during the entire study (all comparisons: P>0.05; Figure 2).
RESULTS Pouchitis
During the study period, 13 patients (22%) developed clinical symptoms suggestive of pouchitis. Endoscopic examination was undertaken in the 13 patients, of whom 10 (17%) were diagnosed with pouchitis (PDAI score: median 11, range 8–14). The duration from the ileostomy closure to the diagnosis of pouchitis was 6 months in 1 patient, 8 months in 3 patients, 10 months in 4 patients, and 12 months in 2 patients (median: 10 months). In the remaining 3 symptomatic patients, there was no apparent inflammation in the ileal pouch, and the PDAI score was
