INTRODUCTION
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn disease (CD), represent a spectrum of diseases characterized by an idiopathic and chronic inflammation affecting the gastrointestinal (GI) tract. Pediatric and adult patients with IBD may present with a variety of clinical symptoms (including abdominal pain and diarrhea) that can be nonspecific. The pathogenesis of IBD remains unknown, and it is generally thought to be caused by a combination of genetic, environmental, and host immune factors. Recent studies suggest that the incidence of these disorders in children may be as high as 7/100,000 per year (1)
Tissue samples from patients with active IBD demonstrate a chronic inflammatory process characterized by an accumulation of activated polymorphonuclear neutrophils (PMNs) in the lamina propria and intestinal lumen. These activated cells are capable of mounting a potent host innate immune response through the release of proteins with proinflammatory properties. Among these is lactoferrin, an 80-kDa member of the transferrin family (2–8) (2,3,9) (10–17)
Clinicians rely on several biochemical studies to help either support a diagnosis of IBD or gauge a patient's disease activity during a particular office visit. These studies include complete blood counts and measurement of acute-phase reactants such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (18–20)
The goal of this study was to evaluate the degree to which quantitative FLA levels reflect disease activity in pediatric patients with known or suspected IBD. Previous studies have demonstrated that FLA levels differ significantly in patients with inflammatory and noninflammatory diarrheal disease (10) (10,13,16,21)
PATIENTS AND METHODS
Consecutive pediatric and young adult patients (≤21 years of age) with known or suspected IBD or irritable bowel syndrome (IBS) were recruited from the inpatient and ambulatory services at Children's Hospital Boston from March 1998 to September 2003. Accrual of study participants was delayed by research resources, and there was no systematic exclusion to bias the selection. Patients characterized as having clinically active or inactive disease were asked to participate. Classification of IBD was based on endoscopic and histological criteria (22)
This study protocol was approved by the Committee on Clinical Investigation at Children's Hospital. Written informed consent was obtained from all parents or legal guardians. Patient assent was also obtained when appropriate. Disease activity was determined prospectively by study personnel at the time of enrollment, using relevant disease activity indices and available laboratory data that were extracted from the medical record.
Stool samples were collected from inpatients in the GI service at Children's Hospital Boston. Ambulatory patients who were unable to provide a fecal specimen at the time of enrollment were permitted to collect samples at home and return them (refrigerated) using a commercial overnight delivery service in accordance with IPAA regulations. For study participants mailing fecal samples by overnight courier, concurrent disease activity was assessed by written questions (included in the sample mailing kit and/or collected by telephone).
Radiological data extracted from upper GI contrast studies were scored (presence or absence of disease) within anatomic regions of the esophagus, stomach, and small intestine. Histological data were similarly coded for each section of the GI tract when available. Radiological and endoscopic data were not typically obtained coincident with study enrollment and did not necessarily reflect the location, degree, or extent of mucosal inflammation at the time of FLA sample collection. In patients with multiple previous studies, data were coded from the radiological or endoscopic study occurring most closely to the time when the fecal sample was collected.
Data Collection
Pediatric Crohn Disease Activity Index (PCDAI) and Harvey Bradshaw Activity Index (HBAI) scores were calculated for patients with CD. Not all patients had these data available for subsequent analysis. The PCDAI categorizes patients into 1 of 3 categories: inactive (<10), mildly active (10–30), and moderate to severe (>30) (23) (10,24) (25) (26–30) (31–38)
Lactoferrin Assay
Collected fecal specimens were labeled, stored at –80°C, and batched for subsequent analysis. Fecal lactoferrin was quantified by use of a commercially available polyclonal-based enzyme-linked immunosorbent assay (IBD-SCAN ; TECHLAB, Inc) and reported as microgram per milliliter of feces, as previously described (10) (10,21) (39)
Statistical Analysis
Data were collected in an SPSS database (SPSS, Chicago), and subsequent analysis was performed by use of SAS (SAS Institute, Cary, NC). Fecal lactoferrin levels were not normally distributed in our study population. Accordingly, we used the Mann-Whitney test in statistical assessments. We present raw FLA measurements when reporting means, distribution, and sensitivity and specificity values. Log transformation permitted the use of parametric testing in studies evaluating the correlation between FLA levels and disease activity indices and biochemical parameters. Fecal lactoferrin was compared across diagnoses (IBD, CD, and UC) and level of disease activity. The relationship between FLA levels and other biochemical markers of inflammation was assessed by Spearman rank correlation. Receiver operating characteristic (ROC) curves were generated to determine the sensitivity and specificity of FLA in discriminating patients with IBD from healthy control individuals and the ability of FLA to discriminate the presence or absence of active disease in patients with IBD. Area under the curve (AUC) with 95% confidence interval was calculated for each ROC curve, and AUCs were compared between different markers of inflammation by use of the statistical package Medcalc. Mean values are reported together with standard deviations, unless otherwise specified. P < 0.05 were considered significant.
RESULTS
FLA Levels Discriminated Pediatric Patients With IBD From Healthy Control Participants
A total of 170 (101 male, 69 female) individuals with a mean age of 13.4 years (range 2–21 years) provided single fecal specimens for use in this study (Table 1 ). Of the 170 study participants, 107 had IBD (56 CD, 51 UC), and blood work was completed within 1 week of specimen collection. These data were used in analyses correlating FLA levels with existing biochemical parameters of intestinal inflammation. Levels of FLA were significantly greater (Fig. 1 ) in patients with UC (1880 ± 565 μg/mL) (mean ± SE) or CD (1701 ± 382 μg/mL) than in healthy control individuals. The upper limit of normal for healthy control individuals was 7.25 μg/mL, corresponding to a log FLA 0.086. Mean FLA levels measured in healthy control individuals (1.17 ± 0.47 μg/mL) fell below the 7.25-μg/mL cutoff and were statistically indistinguishable from those measured in the 7 patients with IBS (2.08 ± 0.90 μg/mL).
TABLE 1: Demographic characteristics of the study groups
FIG. 1: FLA levels in patients with IBS, CD, and UC and in healthy control individuals. FLA levels were significantly greater (P < 0.001) in all patients with UC (1880 ± 565 μg/mL) (mean ± SE) or CD (1701 ± 382 μg/mL) than in healthy control individuals (1.17 ± 2.20 μg/mL) or in patients with IBS (2.08 ± 0.90 μg/mL). No significant differences in mean FLA levels were observed in patients with UC vs CD, or in patients with IBS vs healthy control individuals. Data presented as box plots, with median values indicated by horizontal lines.
Similarly, we did not detect a significant difference in FLA levels between patients with UC and those with CD (P = 0.603). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in distinguishing individuals with IBD from those without IBD (IBS and healthy control individuals) were 83.5%, 96.6%, 99%, and 55%, respectively. All 7 patients with IBS, 21 of 22 healthy control individuals (1 individual had FLA = 8.09), and 23 of 141 patients with IBD had normal (<7.25 μg/mL feces) FLA levels. Only 2 of 23 patients with IBD and normal FLA levels were scored as active according to either the HBAI (HBAI ≥4) or the PGA.
Receiver operating characteristic curves comparison demonstrated that FLA levels displayed greater sensitivity and specificity (AUC 0.932 ± 0.026, P < 0.001) for identifying patients with IBD than did ESR (AUC 0.750 ± 0.085, P = 0.04) (Fig. 2 ). Including all patients without IBD (IBS and healthy control individuals), the AUC was 0.952 ± 0.015, P < 0.001. Despite the small number of patients with IBS, we next evaluated FLA measurements as a screening tool for discriminating 87 patients with active GI symptoms (HBAI score >2) as having IBD or IBS. In this symptomatic population we found that the sensitivity of elevated FLA was 97% (91%–99%, 95% CI) and the specificity was 100% (57%–100%). The corresponding AUC of the ROC in this subset of patients was 0.983 ± 0.013 (P < 0.001, data not shown).
FIG. 2: ROC curve analysis of FLA and ESR levels in patients with IBD and healthy control individuals. ROC analysis calculated from FLA (n = 146) and ESR (n = 113) values collected from individuals with and without IBD. AUC was 0.932 ± 0.026 for FLA, and AUC was 0.750 ± 0.085 for ESR.
FLA Levels Correlated With Disease Activity in Patients With IBD
We next examined the correlation between FLA and clinical disease severity in subsets of patients with IBD, CD, or UC (Table 2 ). Disease activity was assessed according to several previously validated disease activity indices. PGA and HBAI and scores were available for 130 and 135 of 141 patients with IBD, respectively. PGA was assigned retrospectively (and blinded to FLA levels) based on the assessment of a patient's treating gastroenterologist and characterized into 1 of 3 categories: active, inactive, and steroid weaning. By use of the PGA 84 patients were classified as having active disease and 55 as having inactive disease. Fecal lactoferrin levels were higher in patients with active disease (2782 ± 509 μg/mL) than in those with inactive disease (250 ± 94 μg/mL, P < 0.001) when stratified by PGA. Patients with active IBD (defined as an HBAI score ≥ 4) had significantly higher FLA (2803 ± 571 μg/mL) than did those with inactive disease (695 ± 276 μg/mL, P < 0.001). Fecal lactoferrin levels correlated well with continuous HBAI scores (Spearman correlation coefficient of 0.575, P < 0.001).
TABLE 2: FLA levels in patients with active and inactive IBD
We next examined FLA levels in patients with CD stratified by PCDAI. We first compared levels in those with inactive disease (PCDAI <10) and those with mildly active disease (PCDAI 10–30). There was no difference in FLA levels in these 2 populations. Therefore, for further analysis, these values were combined, and they were termed inactive.
When we stratified patients with CD by PGA, we found that FLA levels measured in patients with active disease were significantly higher (2472 ± 574 μg/mL) than in patients with inactive disease (398 ± 172 μg/mL, P < 0.001). Similarly, we found that patients with moderate to severe CD (PCDAI >30) displayed significantly greater FLA levels (6177 ± 1793 μg/mL) than did those with inactive disease (658 ± 260 μg/mL, P = 0.004). FLA levels were also higher in patients with active CD (2465 ± 691 μg/mL) than in those with inactive disease (1055 ± 429 μg/mL) when assessed by HBAI (P = 0.002). In an additional analysis we compared FLA levels with continuous PCDAI scores and found a highly significant Spearman correlation (0.598) between disease activity as assessed by PCDAI and FLA measurements (P < 0.001).
FLA levels were equally sensitive predictors of disease activity in patients with UC. Physician's Global Assessment, modified HBAI, and Kozarek scores were available for 61, 60, and 39 of a total of 62 patients with UC, respectively (Table 2 ). FLA levels differed substantially in patients with active UC (3215 ± 926 μg/mL) in comparison with levels observed in patients with inactive UC (84 ± 33 μg/mL), as assessed by PGA (P < 0.001). Similarly, FLA levels were significantly higher (3121 ± 904 μg/mL) in patients with active UC than in patients with inactive disease (79 ± 34 μg/mL, P < 0.001) when categorized by HBAI. Spearman analysis revealed FLA levels to be highly correlated (0.701) with HBAI scoring (P < 0.001). Finally, FLA levels (93 ± 58 μg/mL) distinguished patients with clinically inactive disease (Kozarek score 0–3) from those with moderately active disease (Kozarek score 4–6) (2940 ± 1728 μg/mL, P = 0.006) and those with severe disease (Kozarek score >6) (3035 ± 979 μg/mL, P < 0.001). FLA levels in patients with moderately active (Kozarek score 4–6) and severely active (>6) disease did not differ significantly and were grouped together in comparison with patients with inactive disease (<4).
As previously noted, the PGA used in this study categorized patients receiving steroid therapy as having active disease irrespective of the presence or absence of clinical symptoms. We therefore completed a subgroup analysis that compared FLA levels in 15 patients with active disease and completing steroid tapers, 69 patients with active disease but not receiving steroid therapy, and 55 individuals with clinically inactive disease at the time of study enrollment. Interestingly, we found that patients with IBD and who were being weaned from steroid therapy displayed log-transformed FLA levels (2.22 ± 0.15 μg/mL) that were significantly higher than those measured in individuals with clinically inactive disease (1.32 ± 0.15 μg/mL, P = 0.003) but less than those measured in patients with clinically active disease but not receiving steroids (3.04 ± 0.091, P < 0.001). To compare data from patients with UC and CD, we examined log-transformed FLA measurements in each category described above. As shown in Figure 3 , and as expected, FLA levels differed significantly in patients with active disease, those with inactive disease, and those who were being weaned from steroids. However, they did not discriminate patients with UC from those with CD (Fig. 3 ).
FIG. 3: FLA levels and disease activity as measured by PGA. Log transformation of FLA produced normalized data for analysis. Log FLA levels measured in patients with active disease and completing steroid therapy were significantly lower (2.22 ± 0.15 μg/mL) than in patients with active disease who were not being weaned from steroids (3.04 ± 0.091 μg/mL) but remained significantly elevated relative to FLA levels in patients with clinically inactive disease (1.32 ± 0.15 μg/mL).
Existing data suggest that FLA levels should rise with increasing levels of intestinal inflammation (10,40)
FLA May Predict Recurrence in Patients With IBD Who Are in Clinical Remission
In our study population 141 patients carried a diagnosis of IBD and 55 were in remission (assessed by PGA) at the time of enrollment. We retrospectively examined the clinical course of these patients (blinded to FLA) during the subsequent 2-month period. Of the patients who were in remission at the time of enrollment, 10% (5/55) went on to experience a clinical flare in their disease. Subgroup analysis revealed that FLA levels in the 5 patients (4 CD, 1 UC) in whom active disease subsequently developed (845 ± 452 μg/mL) were significantly higher than those observed in the 50 patients who remained in clinical remission (190 ± 90 μg/mL, P < 0.01). Concomitant ESR values that were available in 4 of these 5 patients at the time of sample collection were 14, 19, 23, and 79 (normal <20 mm/h). Although our sample size was small, these data suggest that elevated FLA levels may identify patients at risk for disease recurrence, or at least those who may merit closer follow-up. Larger prospective studies are necessary to further address this intriguing hypothesis.
FLA Levels Correlated With Biochemical Markers of Inflammation
Clinicians typically define disease activity in their pediatric and adult patients with IBD using a combination of clinical and laboratory dimensions incorporating the use of ESR, hematocrit (Hct), serum albumin (Alb), and platelet count (Plt). We wanted to determine how well a patient's quantitative FLA levels compared with commonly acquired laboratory indices. To that end, we used a Spearman linear regression to measure the correlation between FLA levels and biochemical studies that were completed within 1 week of fecal sample collection (Table 3 ). Analyzing all of the study participants, we found significant correlations between FLA levels and ESR, Hct, Alb, and Plt. Correlations between FLA levels and ESR, Alb, Hct, and Plt were significant (P < 0.05) in patients with active or inactive disease as defined by PGA. These correlations were present when similar subgroup analyses were performed in patients with either UC or CD. Receiver operating characteristic curves comparing the relative abilities of FLA and ESR to discern disease activity in patients with IBD revealed that both markers are sensitive and specific indices of disease activity (defined by PGA, Fig. 4 ). Fecal lactoferrin levels displayed comparable sensitivity and specificity profiles in distinguishing patients with active IBD from those with inactive IBD (AUC 0.9 ± 0.4 when compared with serum ESR, AUC 0.8 ± 0.05). However, the difference between these surrogate markers of intestinal inflammation reached statistical significance only in patients with UC (P < 0.05).
TABLE 3: FLA levels and biochemical markers of inflammation in patients with IBD
FIG. 4: FLA levels measured in patients with active and inactive IBD. ROC curve comparing FLA with ESR in distinguishing active from inactive IBD as defined by PGA. AUC for FLA (0.90 ± 0.04) was greater (P < 0.001) than that observed for ESR (0.80 ± 0.05).
DISCUSSION
The data collected in this large pediatric population demonstrate that FLA levels correlate with disease activity (assessed prospectively) determined by previously validated disease activity indices. Our data also suggest that FLA levels may be elevated in patients who are clinically well but subsequently experience a flare in disease activity. The data are consistent with findings reported in previous adult series (10,13,16,21)
The acute and chronic active inflammation observed in patients with IBD is characterized by an influx of activated neutrophils into and across the mucosal lining, resulting in the formation of crypt abscesses in the intestinal lumen. Clinicians have used the identification of fecal leukocytes as a way to differentiate patients with inflammatory (ie, invasive bacterial infection, IBD) from those with noninflammatory (ie, viral, toxigenic) diarrheal illness. Fecal leukocyte analysis, however, is limited by its lack of sensitivity, its dependence on a fresh stool sample, and the skill of the laboratory technician (41–44) (43) (10,13,16,21)
Several factors support the inclusion of FLA levels in the diagnosis and interval assessment of IBS. First, because lactoferrin can be measured in feces, it is likely to be a more direct measure of intestinal inflammation than are serum markers such as ESR (as demonstrated in the present study) that reflect systemic inflammation. Second, because FLA measurements are collected noninvasively, they are especially attractive for use in pediatric patients, in whom phlebotomy is both anxiety provoking and technically difficult. Third, the relative ease of sample collection and the prospect of monitoring FLA in samples collected and then sent through the mail (similarly to samples for ova and parasites examination) or collected at a patient's bedside, makes FLA measurements especially well suited for serial assay in the same patient over time. Finally, quantitative FLA levels are easily and reliably measured in the stool by use of a commercially available diagnostic test that has been cleared by the US Food and Drug Administration (10,17,42)
It should be noted that there are limitations to the use of FLA measurements and other stool-based assays that clinicians must keep in mind when applying surrogate markers in the diagnosis and treatment of their patients with IBD. It may be more difficult for parents to collect samples from less cooperative pediatric patients. In addition, elevated FLA levels indicate intestinal inflammation that may be caused by infections and by clinical flares in IBD activity. Therefore, clinicians need to consider obtaining stool cultures from patients presenting with rectal bleeding or symptoms of colitis.
Most fecal markers studied to date, including α-1-antitrypsin, elastase, lysozyme, esterase, and myeloperoxidase, have not been fully validated for clinical use, and it remains unclear how closely their levels reflect the presence or extent of intestinal inflammation (14,45) (10,45,46) (47) (40,46,48–53) (49,54,55)
We further evaluated the potential utility of FLA measurements in predicting patients who are at risk for disease flares by comparing the short-term (2-month) clinical outcome in patients with inactive disease at the time of specimen collection. We found that FLA levels were significantly increased in patients who then experienced flares within 2 months of sample collection in comparison with patients who remained in remission. Although our sample size was relatively small, the results were significantly different, and they raise the possibility that elevations in FLA may presage clinical flares. This question is being addressed in a multicenter study evaluating the utility of serial lactoferrin measurements in pediatric and adult patients with active and inactive IBD. Our data further demonstrate that quantitative FLA measurements are highly correlated with commonly used biochemical metrics of inflammation, including ESR, hematocrit, Alb, and Plt count.
An inherent weakness of our study, and perhaps all studies evaluating the use of surrogate markers in the assessment of patients with IBD, is the lack of an accepted gold standard of disease activity against which to measure their validity. Although endoscopic and histological assessments are generally accepted as definitive benchmarks, their application is limited by their cost, invasive nature, and potential interrater reliability. As such, we were unable to correlate FLA levels with disease location and severity in this study. Similarly, we were not able to correlate FLA measurements, clinical activity, and disease location in subgroups of patients with primarily small or large bowel IBD. In our study population 5 of 62 (8%) patients with UC had disease limited to the left colon, and no patients had isolated proctitis, which was consistent with previous reports of disease distribution in pediatric patients (1)
The development of valid, noninvasive, and readily obtainable surrogate markers of intestinal disease activity will contribute significantly to patient care. In addition, the application of surrogate markers, such as FLA, could play an important role in pharmaceutical trials by reducing both the cost and the length of time necessary to determine the effects of novel anti-inflammatory therapies and by limiting the need for more invasive diagnostic studies. Further studies are needed to define how to optimize the inclusion of FLA measurements into the prospective management of IBD in children and adults.
Acknowledgments
The authors thank the Children's Hospital Clinical Research Program and General Clinical Research Center (MO1 RR-02172) for assistance with this study, Dr. Richard J. Grand for his time and effort in revising this manuscript, and the Children's Hospital Center for Inflammatory Bowel Disease, the Wolpow Family Fund, and the community of patients and providers for their support in completing this clinical study.
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