The clinical value of faecal calprotectin and lactoferrin measurement in postoperative Crohn’s disease

Abstract

Most patients with Crohn’s disease (CD) ultimately require one or more operations over their lifetime. Nevertheless, surgery is not a cure and postoperative CD recurrence is common. Ileocolonoscopy has been considered to be the gold standard in the diagnosis and monitoring of postoperative recurrence in patients with CD. However, endoscopy is a time-consuming and invasive procedure. Simple and non-invasive methods for the detection of postoperative recurrence are desirable. Faecal inflammatory biomarkers such as calprotectin and lactoferrin provide an accurate and non-invasive diagnostic and monitoring modality for inflammatory bowel disease. However, there have been limited data on the role of faecal biomarkers in the postoperative setting. Recently, several studies evaluated the value of faecal calprotectin and lactoferrin measurement after surgery for CD. This review was conducted to assess the role of faecal calprotectin and lactoferrin measurements in patients with postoperative CD.

Introduction

In patients with Crohn’s disease (CD), ileocolonoscopy has been considered to be the gold standard in the diagnosis and monitoring of postoperative recurrence.¹ The severity of the endoscopic lesions in the neo-terminal ileum during 6–12 months after ileocolonic resection appears to be a reliable predictor for future clinical recurrence.^1,2 However, patients do not undergo endoscopic examination, especially when their disease is inactive, because endoscopy is a time-consuming and invasive procedure. Simple and non-invasive procedures for the assessment of postoperative CD recurrence are desirable.

Faecal inflammatory biomarkers

Faecal inflammatory biomarkers such as calprotectin and lactoferrin are neutrophil proteins and are resistant to proteolysis and unaffected by multiple freeze-thaws, providing useful markers in faeces as indicators of inflammation.³ Faecal calprotectin and lactoferrin are stable in stool for as long as seven and five days, respectively.⁴ Calprotectin is often measured with an enzyme-linked immunosorbent assay (ELISA). Since ELISA is time-consuming and mostly suited for analysing samples in batch, faster and more user-friendly techniques have been developed.⁵ Recently, point-of-care or bedside faecal calprotectin tests have become available which may be advantageous in clinical circumstances when a more rapid result is required. These tests can be performed in less than 30 min.

Faecal biomarkers in inflammatory bowel disease

Both faecal calprotectin and lactoferrin are directly associated with the inflammatory process, are easy to measure, and therefore may have the potential to act as non-invasive markers for inflammatory bowel disease (IBD). Clinical implications of faecal biomarkers in IBD include differential diagnosis of IBD or non-IBD, monitoring endoscopic activity, and prediction of the clinical course. Recent systematic reviews confirmed that faecal calprotectin and lactoferrin provide an accurate and non-invasive diagnostic and monitoring modality for IBD.^6–8 Faecal biomarkers may avoid the need for invasive radiological or endoscopic investigation, and inform treatment decisions. Further, faecal calprotectin is useful in predicting disease relapse in quiescent IBD patients.⁹

Faecal biomarkers in postoperative Crohn’s disease

Clinical studies

There have been limited data on the role of faecal biomarkers in the postoperative setting. Recently, however, a number of studies^10–19 evaluated the value of faecal lactoferrin and calprotectin measurement after surgery for CD. Quantitative pooling of studies was not feasible due to the various types of designs in the available studies. A narrative account of the study characteristics and results was therefore undertaken. The main findings of each study are summarised in Table 1.

Table 1.

Summary of clinical studies for patients with postoperative Crohn’s disease

Authors	No. of patients	Faecal biomarker	Main findings and suggestions
Orlando et al.10	50	Calprotectin	Calprotectin measurement at three months after resection was useful to predict endoscopic recurrence at one year following surgery.
Scarpa et al.11	63	Calprotectin Lactoferrin	Lactoferrin significantly correlated with CRP, and was useful in predicting clinical recurrence.
Lamb et al.12	117	Calprotectin Lactoferrin	Both biomarkers were more accurate in predicting clinical disease activity than CRP or platelet count.
Yamamoto et al.13	20	Calprotectin Lactoferrin	Both biomarkers positively correlated with the endoscopic scores. The values of both biomarkers were significantly higher in patients with endoscopic recurrence than in those without endoscopic recurrence. The levels of both biomarkers were significantly higher in patients with future clinical recurrence than those in remission.
Lobatón et al.14	115	Calprotectin	Calprotectin correlated more closely with endoscopic severity than leucocytes, platelets or CRP.
Papamichael et al.15	59	Calprotectin	Calprotectin was a better surrogate marker of endoscopic activity than clinical activity, CRP or ESR.
Lasson et al.16	30	Calprotectin	There was no significant difference in the values of calprotectin between patients with and without endoscopic recurrence. However, among patients with low (<100 µg/g) or high (>600 µg/g) values, calprotectin indicated remission and recurrence, respectively.
Sorrentino et al.17	12	Calprotectin	Calprotectin was a better surrogate marker of endoscopic activity than CRP or ESR.
Sorrentino et al.18	25	Calprotectin	Calprotectin progressively decreased in patients responding to infliximab, while it remained high in non-responders.
Kamm et al.19	174	Calprotectin	Calprotectin but not CRP or CDAI significantly correlated with endoscopic disease activity.

CDAI: Crohn’s disease activity index; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate.

Orlando et al.¹⁰ consecutively studied 50 patients who had undergone resection for CD to prospectively evaluate the role of calprotectin as a predictive marker for endoscopic recurrence in comparison with ultrasound scan (US). Faecal calprotectin was measured, and US was performed at three months after resection. A colonoscopy was conducted at one year following surgery. Thirty-nine out of the 50 patients were evaluated by performing a colonoscopy; 19 patients had an endoscopic recurrence (Rutgeerts score ≥i2). The most reliable cut-off value of calprotectin for the prediction of endoscopic recurrence was 200 mg/l (Table 2). In contrast, the sensitivity and specificity of US for the prediction of endoscopic recurrence were 26% and 90%, respectively. US is more specific than calprotectin in predicting endoscopic recurrence, and faecal calprotectin (>200 mg/l) seems to have a better sensitivity than US. The authors suggest that a high value (>200 mg/l) of calprotectin can be an indication for colonoscopy in the group of patients with negative US in order to detect early endoscopic recurrence. Thus, faecal calprotectin measurement at three months after resection may be useful to predict endoscopic recurrence at one year following surgery.

Table 2.

Accuracy of faecal biomarker testing for the detection and prediction of recurrence

Authors	Detection or prediction of recurrence	Faecal biomarker (measuring method)	Cut-off level	Sensitivity (95% CI)	Specificity (95% CI)	Positive predictive value (95% CI)	Negative predictive value (95% CI)
Orlando et al.10	Prediction of endoscopic recurrence	Calprotectin (ELISA)	200 mg/l	63%	75%	–	–
Yamamoto et al.13	Detection of endoscopic recurrence	Calprotectin (ELISA)	140 µg/g	70% (42–98%)	70% (42–98%)	70% (42–98%)	70% (42–98%)
		Lactoferrin (colloidal gold agglutination assay)	125 µg/g	70% (42–98%)	60% (30–90%)	64% (35–92%)	67% (36–97%)
	Prediction of future clinical recurrence	Calprotectin (ELISA)	170 µg/g	83% (54–113%)	93% (79–106%)	83% (54–113%)	93% (79–106%)
		Lactoferrin (colloidal gold agglutination assay)	140 µg/g	67% (29–104%)	71% (48–95%)	50% (15–85%)	83% (62–104%)
Kamm et al.19	Detection of endoscopic recurrence	Calprotectin (not recorded)	100 µg/g	89%	–	–	91%

CI: confidence interval; ELISA: enzyme-linked immunosorbent assay.

Scarpa et al.¹¹ studied 63 patients who had undergone ileocolonic resection for CD with a median follow-up of 40.5 months. The mean ( ± standard error) lactoferrin level was 21 ± 3.9 µg/g, and the mean calprotectin level was 247 ± 22.7 ng/ml. High levels of lactoferrin and calprotectin were observed even in case of clinical remission. There was a strong correlation between the two faecal biomarkers. However, only lactoferrin significantly correlated with C-reactive protein (CRP) and showed a reliable threshold value for systemic inflammation and also seemed to predict the presence of episodes of clinical recurrence (CD activity index (CDAI) score >150 or its increase of ≥70 points). The lactoferrin threshold value for being predictive of systemic inflammation (CRP > 0.6 mg/dl) is 11 µg/g with a sensibility of 71% and a specificity of 90%. This study suggests that faecal lactoferrin level is a reliable indicator for intestinal inflammation influencing the systemic inflammatory status during the postoperative follow-up in patients with CD.

In the study by Lamb et al.,¹² a postoperative cohort of 13 patients was followed prospectively for one year with regular faecal calprotectin and lactoferrin measurements. A second postoperative cohort (median 24 months after resection) of 104 patients provided a single stool sample. Both faecal calprotectin and lactoferrin correlated significantly with the Harvey Bradshaw Index. Twenty-eight patients with severely clinically active disease had high mean (±standard error) levels of calprotectin (661.1 ± 119.1 µg/g) and lactoferrin (116.6 ± 32.2 µg/g); and 43 with clinically inactive disease had low levels of calprotectin (70.2 ± 27.1 µg/g) and lactoferrin (5.9 ± 2.4 µg/g). In patients with mild to moderately clinically active disease, faecal calprotectin and lactoferrin identified individuals with and without recurrent inflammatory disease. Faecal biomarkers were more accurate in predicting clinical disease activity than CRP, platelet count or endoscopic appearance. The authors confirmed that faecal calprotectin and lactoferrin measurement is valuable in identifying CD recurrence in symptomatic postoperative patients.

Yamamoto et al.¹³ conducted a prospective study to investigate the relationship between endoscopic activity, and faecal calprotectin and lactoferrin, and assess the predictive value of these biomarkers for future clinical recurrence. Twenty patients who remained in remission during 6–12 months after ileocolonic resection for CD were studied. A stool sample was collected for measurement of calprotectin and lactoferrin, and patients were then followed up for 12 months. The mean duration from surgery to the endoscopic examination was 7.2 months. There was a significant correlation between calprotectin and lactoferrin levels. Both calprotectin and lactoferrin positively correlated with the endoscopic (Rutgeerts) scores. The mean (±standard error) values of faecal calprotectin and lactoferrin were significantly higher in patients with endoscopic recurrence (≥i2) than in those without endoscopic recurrence (calprotectin 229.5 ± 34.8 vs 102.3 ± 19.0 µg/g, lactoferrin 161.4 ± 19.1 vs 83.7 ± 23.2 µg/g). The most reliable cut-off values for the detection of endoscopic recurrence were 140 µg/g for calprotectin and 125 µg/g for lactoferrin (Table 2). There was no significant correlation between the CDAI and the levels of faecal biomarkers. Laboratory measurements including leucocyte count, platelet count, albumin, and CRP at the time of endoscopy did not correlate with faecal biomarkers. During the 12-month follow-up, six patients developed clinical recurrence (CDAI > 150 with an increase of ≥70 points). Both calprotectin and lactoferrin levels were significantly higher in patients with clinical recurrence than those in remission. The most reliable cut-off values for the prediction of future clinical recurrence were 170 µg/g for calprotectin and 140 µg/g for lactoferrin (Table 2). These results suggest that both calprotectin and lactoferrin levels correlate well with endoscopic activity after ileocolonic resection for CD. Calprotectin and lactoferrin could be clinically relevant biomarkers for predicting postoperative recurrence. Calprotectin shows a higher sensitivity and specificity than lactoferrin.

In the study by Lobatón et al.,¹⁴ faecal calprotectin was determined simultaneously by an ELISA and a new quantitative point-of-care test (QPOCT) for the prediction of endoscopic remission and postoperative recurrence in CD patients. A total of 115 ileocolonoscopies were performed. Faecal calprotectin levels correlated more closely with the CD Endoscopic Activity Index of Severity (CDEIS) than leucocytes, platelets or CRP. The prediction of endoscopic remission (CDEIS < 3), using the QPOCT (cut-off 272 µg/g) and the ELISA (cut-off 274 µg/g) presented an area under the curve of 0.933 and 0.935, respectively. Median QPOCT levels discriminated endoscopic (Rutgeerts) score i0–i1 from i2–i4 (98 vs 234.5 µg/g). These results may suggest that faecal calprotectin determined by rapid quantitative test predicts endoscopic remission and endoscopic postoperative recurrence in CD patients.

Papamichael et al.¹⁵ studied 59 patients who had undergone ileocaecal resection for CD to assess the value of clinical, serological and faecal markers for the prediction of postoperative endoscopic recurrence. Faecal calprotectin was measured by a rapid hemi-quantitative point-of-care immuno-chromatographic test, with a cut-off value of 15 µg/g. Endoscopic recurrence (Rutgeerts score ≥i2) and clinical recurrence (CDAI > 150) rates were 6.8% and 1.7% at six months, 16.9% and 6.8% at one year, and 25.4% and 11.6% at two years after surgery, respectively. Among 15 patients who developed endoscopic recurrence at two years after resection, faecal calprotectin was persistently elevated (>60µg/g) in all patients (100%) compared vs CRP levels was elevated (>0.5 mg/dl) in 8 patients (53.3%; p = 0.017). In patients with endoscopic recurrence, CRP was moderately and erythrocyte sedimentation rate (ESR) was poorly correlated to the Rutgeerts score. CRP identified only patients with severe endoscopic recurrence (i3, i4). The authors suggest that a semi-quantitative faecal calprotectin test is a better surrogate marker of endoscopic activity and indicator of endoscopic recurrence than clinical activity or routine biomarkers.

Lasson et al.¹⁶ studied 30 patients with CD and ileocaecal resection performed within one year. Stool samples were delivered monthly until an ileocolonoscopy was performed one year postoperatively. The median levels of faecal calprotectin were not significantly different between patients in endoscopic remission (Rutgeerts score = i0, i1) and patients with endoscopic recurrence (≥i2) one year after resection; 189 (range, 75–364) vs 227 (range, 120–1066) µg/g. Further, there was no significant difference in faecal calprotectin levels when patients with a normal ileocolonoscopy (i0) were compared with patients with any endoscopic recurrence (≥i1); 150 (range, 64–287) vs 264 (94–717) µg/g. However, six (86%) of seven patients with a calprotectin value >600 µg/g had an endoscopic recurrence (≥i2). Moreover, six (75%) of eight patients with a calprotectin <100 µg/g, were in endoscopic remission (i0, i1). This study found no significant difference in the concentrations of calprotectin between patients in endoscopic remission and patients with endoscopic recurrence one year after ileocaecal resection for CD. However, among the minority of patients with low or high values, faecal calprotectin indicated remission and recurrence, respectively.

In other studies^17–19 which assessed the efficacy of biologic agents for postoperative CD, the value of faecal calprotectin for the detection and prediction of recurrence was additionally evaluated. Sorrentino et al.¹⁷ found that calprotectin was a much more reliable surrogate marker of endoscopic activity than CRP or ESR. In another study by Sorrentino et al.,¹⁸ 25 patients with endoscopic recurrence (Rutgeerts score ≥i2) were studied. Faecal calprotectin was measured approximately every two months. The levels of faecal calprotectin were elevated in all patients at entry. It progressively decreased in patients responding to infliximab, while it remained high throughout the duration of the study in non-responders. In a recent study by Kamm et al.,¹⁹ faecal calprotectin significantly correlated with endoscopic recurrence (Rutgeerts score ≥i2) and endoscopic (Rutgeerts) score; however, CRP and CDAI did not. Faecal calprotectin >100 µg/g indicated endoscopic recurrence with a sensitivity of 89% and a negative predictive value of 91%, potentially allowing avoidance of colonoscopy in 41% of patients.

Value of faecal biomarkers

Based on the results in the above-mentioned studies, faecal biomarkers are more highly correlated with endoscopic activity after resection for CD than routine inflammatory markers such as CRP, ESR, leucocyte count or platelet count. Two studies^13,19 reported high diagnostic accuracy of faecal biomarkers for the detection of endoscopic recurrence (Table 2). Calprotectin appeared to show a higher sensitivity and specificity than lactoferrin.¹³ Systemic markers of inflammation such as CRP and ESR are used for predicting IBD activity, but are not specific for intestinal inflammation and correlate poorly with endoscopic activity. Faecal calprotectin will be an additional or alternative biomarker to CRP in the monitoring of disease activity in patients with CD.

One study¹³ suggests that both calprotectin and lactoferrin are useful in predicting future clinical course and identifying patients at a high risk of clinical recurrence after ileocolonic resection for CD. The impact of clinical and systemic serological parameters on postoperative recurrence is weaker than these faecal biomarkers. It seems most likely that measurement of faecal biomarkers is picking up an ongoing escalating inflammatory process that gives clinical symptoms when sufficiently severe.

In the other study,¹⁸ faecal calprotectin levels decreased in patients who responded to biologic therapy, while they remained high in non-responders. Faecal calprotectin measurements may be useful in monitoring disease activity and evaluating the response to treatment (e.g. mucosal healing) during medical therapy. If measurement of faecal calprotectin is used to demonstrate an adequate response to biologic therapy, it may aid decisions regarding shortening of dosage interval, dose escalation, or switching to an alternative agent.

Limitations of the clinical studies

The previous studies have several limitations that should be addressed before faecal biomarkers become more widely used in clinical practice. First, the sample size is relatively small. Small studies can produce false-positive results, or they over-estimate the magnitude of an association. Clearly, large scale studies are necessary to evaluate the role of faecal biomarkers in patients with postoperative CD.

Recently, several different types of assay are available in the measurement of faecal biomarkers. Large quantitative differences were observed between the different calprotectin assays.⁵ This variation makes it impossible to use methods interchangeably and enforces the urgent need for standardisation in measurement procedures. Further, the optimal cut-off values for the detection and prediction of postoperative CD recurrence should be determined.

Because there is significant variation in the levels of a faecal biomarker, a single measurement seems to be of limited clinical utility.^16,20 A set of two to three samples at each measurement may be a possible solution for the variability. However, in a recent cohort of quiescent CD, day-to-day variability of faecal calprotectin was low.²¹ Further research on the variability of faecal biomarkers is needed.

Conclusions

Faecal calprotectin, and to a lesser degree lactoferrin, may be useful in monitoring disease activity, and in predicting future clinical course after surgery for CD. Although faecal biomarkers will never fully replace endoscopy and radiology, assays of faecal calprotectin and lactoferrin should serve as non-invasive, inexpensive and reproducible biomarkers. This should spare the patients from going through complicated endoscopy procedures. These biomarkers are likely to have a greater role in our future diagnostic and therapeutic pathways in postoperative CD. Nonetheless, further well-designed large scale studies are needed to evaluate the role of faecal biomarkers in patients after resection for CD.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The author declares that there is no conflict of interest.