Infectious diarrhea causes gastrointestinal disease and increases the economic burden of the health care system, and the pediatric population is especially vulnerable to these gastrointestinal infections. According to a World Health Organization report in 2003, the median incidence of diarrhea for all of the children younger than 5 years was 3.2 episodes per child-year, and this number has not changed significantly since the 1980s (1). Viruses are the most significant causes of diarrhea and cause approximately 40% to 60% of cases (2,3). Rotavirus is a well-known pathogen (prevalence 13.2%–35.4%) that causes diarrhea in children, but norovirus (5%–29.3%), adenovirus (0.5%–5.4%), astrovirus (0%–2.7%), and sapovirus (0%–2.8%) are also important pathogens (2–7). The prevalence of bacterial gastroenteritis, such as Salmonella (2.2%–19.5%) and Campylobacter (2%–15.7%) infections, increased in many developing and developed countries; other pathogens, such as Shigella (0%–2.8%), Yersinia (0%–2.5%), and pathogenic Escherichia coli (0%–11.2%), were also important (2–6). In Taiwan, the enteric pathogen–associated mortality is low, but the social burden and economic costs are substantial because of the high incidence. Although acute infectious diarrhea is a common clinical disease in children, few reliable and noninvasive diagnostic tools have been used as biological markers in patients with acute infectious diarrhea or persistent digestive symptoms.
Calprotectin is a heterodimer of 2 calcium-binding proteins, S100A8 and S100A9. Both subunits are found at increased levels in the colon. Calprotectin is a 36-kDa calcium- and zinc-binding protein and constitutes approximately 60% of soluble cytosolic proteins in neutrophilic granulocytes. Therefore, calprotectin is a marker of neutrophil influx and is elevated in a number of inflammatory conditions (8,9). Elevated calprotectin levels are a good predictor of inflammation as well as associated with standard inflammatory markers (eg, serum C-reactive protein [CRP]). Calprotectin probably has a high diagnostic value; if the fecal calprotectin level is low, then there is a high probability that no organic intestinal disease exists. Naughton et al (10) also found increased levels of calprotectin in Salmonella-infected animals. Fecal calprotectin is emerging as a useful marker to quantify mucosal inflammation, not in the least because calprotectin appears to be stable in feces, which can be obtained by noninvasive means (11). In the literature, there are few data comparing acute infectious diarrhea in children (viral or bacterial) and calprotectin values as a function of the various pathogens and severity of the acute illness course.
Few scales are available to evaluate gastroenteritis disease severity. One of the most commonly used scoring scales is the Vesikari 20-point scale (12) in which an episode of gastroenteritis with a score of ≥11 is considered moderate and a score of ≥16 is considered severe. Our present, prospective study was conducted to explore the role of fecal calprotectin in gastrointestinal infection to include the following factors: to predict bacterial or viral infection, to evaluate the extent to which values may be associated with the severity of gastroenteritis based on the Vesikari 20-point scale, and to monitor patients during the course of gastrointestinal infection, which may be informative for evaluation and follow-up.
The present prospective study enrolled and analyzed children who were being treated in Chang Gung Children's Hospital located in Northern Taiwan. After all of the subjects had provided written informed consent, 3 fecal samples were collected from each.
Enrollment was performed between September 2008 and May 2010. Diarrhea was defined as 3 or more outputs of loose or liquid stools per day. The inclusion criteria were as follows: between 3 months and 10 years of age and being hospitalized with acute infectious diarrhea for <5 days. The exclusion criteria were immunodeficiency, allergy, noninfectious causes for the symptoms, history of inflammatory bowel disease (IBD), history of gastrointestinal tract surgery, and the administration of antibiotics or anti-inflammatory medications. The study protocol was approved by the institutional review board of Chang Gung Memorial Hospital. Informed consent was obtained from the parents of all eligible children. The study was performed in accordance with the Declaration of Helsinki.
Upon enrolling in the study, the hospitalized patients received treatments that consisted of intravenous fluid and oral rice water or half-strength formula. The severity of diarrhea was evaluated according to the following parameters: volume of stools, fecal consistency, and frequency. Other clinical symptoms or signs, including fever, vomiting, abdominal pain, daily intake, and appetite, were also assessed. All of the participants underwent first-step hematology and biochemistry tests (including blood cell counts, serum CRP, and electrolytes) as well as fecal pus cell and mucus analysis. The disease severity was recorded with the severity scoring method termed the Vesikari 20-point scale (supplementary Table 1, http://links.lww.com/MPG/A121). The fecal samples of several patients were collected at 3 different time points, which included the initial stage of infectious diarrhea, 3 to 5 days later and 7 to 10 days later. Series follow-ups of fecal calprotectin level were measured by enzyme-linked immunosorbent assay (ELISA). The control group comprised 30 children (mean age 3.7 years; range 1–10 years) without diarrhea. We compared and analyzed the levels of fecal calprotectin collected from the different patients at the same time interval.
To assess the etiology of infectious diarrhea, the fecal specimens were collected for the detection of Salmonella, Shigella, or Campylobacter colonies on specifically prepared agar plates. The fecal specimens were also sent for evaluating rotavirus or adenovirus antigen levels by ELISA as well as norovirus RNA by real-time polymerase chain reaction.
The stool samples were prepared and analyzed for calprotectin levels according to the manufacturer's instructions (PhiCal Calprotectin ELISA Kit; Immundiagnostik, Bensheim, Germany). The stool samples were collected in plastic containers and sent to the laboratory by mail either the same day or the following day. The weight of each sample (100–500 mg) was measured, and an extraction buffer containing citrate was added in a weight/volume ratio of 1:50. The samples were mixed by a vortex mixer for 30 seconds and homogenized for 25 minutes. One milliliter of the homogenate was transferred to a tube and centrifuged for 20 minutes at 10,000g. Finally, the supernatant was collected and frozen at −20°C. The supernatants were thawed, and calprotectin levels were analyzed with the quantitative calprotectin ELISA and read at OD 450 nm. Calprotectin was expressed as micrograms per gram of feces (13).
Simple univariate correlation coefficients (Spearman rank correlation) were calculated using baseline data only. Independent associations between the variables of interest were investigated by generalized estimating equations (GEEs). The use of GEE is a regression technique that allows the investigation of longitudinal data while adjusting for within-patient correlation. GEE analysis requires a predefined working correlation structure for the dependent variable (calprotectin), and based on the first-level and follow-up data, an exchangeable correlation structure was chosen here. The GEE approach was developed to correct for repeated outcomes within the same subject (14). While using data from >2 time points, GEE analysis was used for longitudinal analysis (associations).
A univariate comparison between groups was performed with a t-test for repeated measures, and a chi-squared test and Fisher exact test were used with the categorical data. Analyses were performed on the intention-to-treat population. A P value <0.05 was considered significant, and the statistical tests were 2-tailed. The GraphPad Software Prism 3.03 (GraphPad Software, Inc, San Diego, CA) and SPSS Software, version 15.0 (SPSS Inc, Chicago, IL) were used for the statistical analysis.
Sample description. A total of 198 participants were screened between September 2008 and May 2010. From that cohort, 45 patients were excluded from further study because no confirmed pathogen was identified from the stool examination. Among these individuals (n = 153) included in the study, rotavirus infection was diagnosed in 52 patients, norovirus infection was diagnosed in 31 patients, and adenovirus infection was diagnosed in 8 patients. In addition, Salmonella infection was diagnosed in 40 patients, and Campylobacter infection in 22 patients. No Shigella or Yersinia was isolated. The demographic details are presented in Table 1.
The data include 451 evaluations for 153 individuals across 3 different time points. The pattern of assessment was as follows: 145 subjects (94.7%) had 3 assessments, and 8 subjects (5.3%) had 2 assessments. The mean age of the subjects was 3.46 years (standard deviation [SD] = 2.18; range 3 months–10 years), and of these subjects, 82 (53.6%) were boys.
The median and range of fecal calprotectin concentration was 765 (range 252–1246) μg/g in patients with Salmonella infection, 689 (307–1046) μg/g in patients with Campylobacter infection, 89 (11–426) μg/g in patients with rotavirus infection, 93 (25–405) μg/g in patients with norovirus infection, 95 (65–224) μg/g in patients with adenovirus infection, and 43 (10–145) μg/g in the control group (Fig. 1A). Collectively, the fecal calprotectin concentration was 89 (11–426) μg/g in virus-infected patients and 754 (252–1246) μg/g in bacteria-infected patients (Fig. 1B). Concentrations of each fecal marker for patients with either form of bacterial infection were significantly elevated compared with the concentrations of the virus-infected patients. The P values for calprotectin were <0.05. There were no significant differences observed in the fecal calprotectin concentrations between clinically confirmed Salmonella and Campylobacter infections. The P values for calprotectin were 0.65.
The median and range of fecal calprotectin concentrations were 843 (284–1246) μg/g in patients with severe disease activity (ie, Vesikari score ≥16), 402 (71–995) μg/g in patients with moderate disease activity (ie, Vesikari score ≥11), and 87 (11–438) μg/g in patients with mild disease activity (ie, Vesikari score <11, Fig. 2). The fecal calprotectin concentrations for patients with either form (ie, viral or bacterial) of severe or moderate disease activity were significantly elevated compared with the concentration associated with mild disease activity. The P values for calprotectin were <0.05.
Univariate Linear Regression Analysis
Certain parameters are most likely associated with calprotectin levels, including white blood cells (WBCs)/differential counts (DCs), CRP, fecal mucus, fecal pus cells, dehydration, duration of fever, vomiting, diarrhea before treatment or after treatment, and clinical severity (as indicated by Vesikari scores), which were recorded and analyzed. To determine the correlation between these parameters and fecal inflammatory markers, we performed a univariate linear regression analysis.
The univariate linear regression analysis revealed that Vesikari score, fecal pus cells, fecal mucus, and dehydration all correlated with the fecal calprotectin level. We found that concentrations of fecal calprotectin differed significantly among patients (n = 153) with bacterial and viral gastroenteritis. In patients with only bacterial gastroenteritis (n = 62), we perform a further analysis showing the correlation of calprotectin level and severity of bacterial gastroenteritis. Moreover, a similar finding was also demonstrated by the correlation of calprotectin level and severity of viral gastroenteritis (n = 91) (Table 2). The data suggested that fecal calprotectin may be a useful marker in evaluating the clinical severity of infectious diarrhea and the fecal inflammatory status of intestinal infection.
GEE Analysis Results
Table 3 reveals the results of a multivariate analysis of the predictive value of fecal calprotectin with time variations. Subjects with higher Vesikari severity scores had higher fecal calprotectin levels initially (time = 0), and the fecal calprotectin levels may have decreased when followed up at different times (time > 0).
(Calprotectin = 67.6863 + 915.5821 × Vesikari score + 55.321 × Time − 203.139 × Time × Vesikari score)
Calprotectin is a calcium- and zinc-binding protein of the S100 family, which is derived predominantly from neutrophils and monocytes. It is detectable in body fluids and tissue samples and is emerging as a valuable marker in the diagnosis, monitoring, and determination of the prognosis of commonly encountered gastroenterological conditions. Fecal calprotectin in particular has long been regarded as a promising marker of gastrointestinal pathology (15). Fecal calprotectin has been proposed as a marker of gastrointestinal inflammation (13). Our study demonstrates the usefulness of fecal calprotectin for detecting colonic inflammation in children with gastrointestinal symptoms, such as infectious diarrhea or enterocolitis.
Children usually experience pain while a blood sample is collected through venipuncture or when invasive endoscopy techniques are performed. Noninvasive tests, such as those with stool samples, are greatly recommended for children and can avoid their fear of pain; however, there are limitations of detecting and measuring calprotectin levels. A study reported significantly higher fecal calprotectin levels in babies between age 1 week and 10 months, although the study showed a slight, and not significant, negative correlation with age (16). Calprotectin is a useful marker of bowel inflammation because it is remarkably resistant to proteolytic degradation in the presence of calcium (17). Calprotectin (ie, S100a8 and S100a9) is also upregulated by Salmonella infection (10,18). According to our study, fecal calprotectin levels are higher in patients with bacterial gastrointestinal infections, such as Salmonella and Campylobacter infection, but lower in patients with rotavirus, adenovirus, or norovirus infection.
The detection of calprotectin may have advantages because it accounts for 60% of the cytosolic protein of neutrophils. A noticeable intestinal infection has intense infiltration of neutrophils, macrophages, lymphocytes, and other inflammatory cells in the epithelial lining and lamina propria of the colonic mucosa (19). Neutrophils have been shown to be involved in the maintenance of inflammation in the gut in acute infections that are caused by Salmonella(20) and Shigella(21). Promising results have shown that the fecal calprotectin level correlates well with the degree of colonic inflammation in IBD (22,23) and can be predictive of relapse into IBD (24). Target stability is a major concern in any assay of fecal molecules, and the protein molecules in stools may be undetectable because of degradation or denaturation of the target. Calprotectin is resistant to heat and proteolytic enzymes when bound to calcium. In this form, it is stable at room temperature, and the sample can be stored for 4 to 7 days (25), which permits the patient to collect the sample at home without preparations and send it to the laboratory by regular mail. The improved method of fecal calprotectin monitoring is now available as a commercial test, and a spot sample of 40 to 120 mg feces is sufficient (26).
Few studies have explored the role of fecal calprotectin in acute infectious diarrhea; 1 study (27) enrolled children whose age was <3 years, and another study (28) investigated adult bacteria diarrhea. Our study explored fecal calprotectin values in acute infectious diarrhea of children whose age was between 3 months and 10 years. Because traditional fecal bacteria culture usually required at least 2 to 3 days to acquire results and many species of bacteria are difficult to identify, fecal calprotectin is a quick method to evaluate the presence of a bacterial infection. Shastri et al (28) reported that the calprotectin test could generate results within hours to support a presumptive diagnosis of infectious diarrhea, which can determine the suitability of stool samples for culture. Furthermore, Schroder et al (29) reported that fecal calprotectin is also an accurate marker in several types of chronic diarrhea.
In our study, the data included 451 evaluations for 153 individuals across 3 different time points. The fecal calprotectin concentrations were significantly elevated in patients with severe or moderate disease activity compared with those with mild disease activity (P < 0.05 for each marker by disease activity level). A univariate linear regression analysis revealed that the Vesikari score, fecal pus cells, fecal mucus, and dehydration are correlated with the calprotectin level. The Vesikari score parameters include the number and duration of diarrhea, number and duration of vomiting, body temperature, and severity of dehydration. Bacterial pathogens, including Salmonella spp, Campylobacter spp, and Shigella spp, can cause invasive diarrhea. These pathogens have the capacity to invade the mucosa of the distal small intestine and colon, stimulate local and systemic inflammatory responses, and may cause hemorrhage and ulceration of the mucosa. In our study, the Vesikari score (ie, clinical severity) is usually higher in bacteria-infected patients compared with those with a viral infection. Fecal calprotectin levels may correlate with clinical severity that includes diarrhea, vomiting, fever, and dehydration. Fecal pus cells are usually positive in bacteria-infected patients, and higher fecal calprotectin levels may indicate a bacterial infection. Increased fecal mucus may be associated with intestinal mucosal inflammation that is caused by a pathogen. Collectively, fecal calprotectin may correlate with disease activity, which may include the number and duration of diarrhea or vomiting episodes, severity of fever or dehydration, as well as fecal pus cells and fecal mucus secretion.
There are several limitations in the present study, which include the age and inflammatory status of the patients. Normal values of fecal calprotectin may vary with age; higher cutoff values are observed during the first year of life (30,31). An age distribution of the etiology of diarrhea and clinical severity is shown in supplementary Table 2 (http://links.lww.com/MPG/A122). Seven patients were younger than 1 year in our study. One patient had a bacterial infection with a moderate severity based on the Vesikari score and a calprotectin level of 487.2 μg/g. The other 6 patients had viral infections with mild or moderate severity based on their Vesikari scores and calprotectin levels that were <305 μg/g. In 42 presumably healthy children ages between 6 months and 1 year, Rugtveit et al (32) reported that the fecal calprotectin median and upper 95% confidence interval were 67 to 79 μg/g and 291 μg/g, respectively; however, fecal calprotectin levels should be carefully evaluated in younger children, especially during the first year of life. Additionally, fecal cytokines as well as calprotectin are often elevated in IBD or other inflammatory disorders. Therefore, patients with a history of either IBD or gastrointestinal tract surgery or those receiving treatment with anti-inflammatory medications were excluded from our study.
GEE analysis is a regression technique that allows the investigation of longitudinal data and corrects the repeated outcomes within the same subject. The GEE approach requires a predefined working correlative structure for the dependent variable (ie, calprotectin) and is derived from the first-level and follow-up data. In our study, we found that fecal calprotectin detection on the first evaluation and on follow-up visit is greatly associated with Vesikari scores. The above results indicate that fecal calprotectin may be useful for correlation with the severity of infectious diarrhea during the course of the disease and may be informative for managing gastrointestinal infection. According to our study, the measurement of fecal calprotectin levels may be a useful, noninvasive test for evaluating intestinal infections or inflammation. For children with persistent diarrhea or recurrent digestive symptoms after 1 episode of gastrointestinal infection, fecal calprotectin monitoring could be a helpful tool to provide present information for physicians. Repeated blood collection is difficult and painful; therefore, it will be beneficial to use a noninvasive fecal sampling method that is easily available, with calprotectin levels measured quickly by ELISA. Fecal calprotectin is a useful quantitative marker to approach and monitor patients with persistent diarrhea or digestive symptoms.
In conclusion, the noninvasive marker fecal calprotectin can differentiate bacterial or viral infection, and the relative values may be associated with the severity of gastroenteritis, which corresponds to the Vesikari scores. Furthermore, fecal calprotectin levels may be useful for correlation with the severity and the course of gastrointestinal infection, which may be informative for patient evaluation, management, and follow-up.
The authors thank Shao-Yu Lin for assistance with the statistical analysis.
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