Fecal Calprotectin in patients with Infantile Colic: A case-control study

The precise etiology of infantile colic has not been elucidated after many years of research. In this study, we aimed to determine the association of fecal calprotectin with infantile colic. This case control study was performed on colicky infants referred to pediatrics clinics in Zanjan over a 6-month period. Infantile colic was clinically dened based on wessel criteria. The control group consisted of healthy infants matched for age, sex, weight, and type of feeding with the case group. Fecal calprotectin levels were measured in both groups by the ELISA method. statistically There was calprotectin regarding and nutrition in both colicky infants and controls

. Therefore; it may be hypothesized that the gastrointestinal in ammation may increase fecal in ammatory proteins such as Calprotectin in colicky infants.
Calprotectin is a protein of calgranulin family, secreted mainly from neutrophils with a regulatory function on in ammatory processes [9]. It may be used for diagnosis of In ammatory Bowel Disease, Necrotizing Enterocolitis and intestinal Cystic Fibrosis [10,11]. Few studies have investigated the amount of fecal Calprotectin in colicky infants. The aim of this study was to evaluate the fecal Calprotectin levels in infants with colic in comparison with normal infants.

Methods:
This case control study was performed on infants aged between 3 weeks to 4 months with diagnosis of colic, referred to the clinics of Moussavi hospital in Zanjan, Iran ,for a period of 6 months in 2019. This study has been approved by the ethics committee of Zanjan University of Medical Sciences (IR.ZUMS. REC.1398.191 ) Infantile colic was clinically de ned based on the Wessel criteria, which includes crying for more than 3 hours a day for more than 3 days a week and for more than three weeks. The case group was randomly selected from colicky infants aged 3 weeks to 4 months whose parents agreed to participate in the study.
Patients with diagnosis of in ammatory bowel disease, infectious enterocolitis such as Shigella and Salmonella, and parental dissatisfaction were excluded from the study. The control group was selected from healthy infants without colic who were referred to the clinics for control and were willing to be tested. They were matched with the cases in terms of age, sex, weight and type of nutrition. Stool samples were taken in both groups to measure the level of calprotectin in the specimen. Fecal calprotectin measurement was performed by Calprest-Dynex Elisa laboratory kit at Ayatollah Mousavi Hospital. The cutoff point of ≥ 120 (µg/g) was considered positive.
Finally, the Calprotectin levels along with the demographic information were entered into the SPSS V22 software and analyzed. Normal data distribution was investigated using the Kolmogorov-Smirnov test. The independent T test was used to compare quantitative variables. Qualitative data were compared using chi square or Exact Fisher's test. P < 0.05 was considered statistically signi cant.

Results:
Forty infants (75.5% boys and 42.5% girls) were equally divided into two groups. In the colic group, 11 boys (55%) and 9 girls (45%) had a mean age of 63 days and weighed between 3700 and 6500 g. In the control group there were 12 boys (60%) and 8 girls (40%) with mean age of 48 days and their weight ranged from 3400 to 7200 g. The mean head circumference of colicky patients and controls were 38.32 ± 1.28 and 37.62 ± 1.31 respectively and the difference between the two groups was not statistically signi cant (p = 0.095). There were no signi cant difference regarding sex (p = 0.749), age (p = 0.514), weight (p = 0.603), and type of nutrition (p = 0.887) between the two groups.
Fecal calprotectin was positive in 80% of the colic group and 30% of the control group. The Chi-Square (Ch2) test showed that fecal calprotectin was signi cantly higher in infants with colic compared to the control group (p 0.001). Table 1 shows the relation between fecal Calprotectin and sex, age, weight and type of nutrition in both groups. Discussion: This study was designed to evaluate the fecal Calprotectin levels in infants with colic compared to normal infants. Our results showed that fecal Calprotectin was signi cantly higher in colicky infants compared to controls but there was no signi cant difference in fecal calprotectin regarding sex, age, weight and type of nutrition in both groups.
Although the exact etiology of colic is not yet known [12], studies suggest that gut in ammation may play a major role in this issue [13]. Rhoads et al. exploring the pathophysiologic mechanism of colic, studied intestinal ora changes in 19 infants with colic versus 17 healthy infants and showed that the levels of fecal calprotectin in infants with colic were 2 times higher than in healthy infants [14]. A study conducted by Shahramian et al. in 2018 examined the level of fecal calprotectin in 100 infants and concluded that Calprotectin was signi cantly higher in the stool of colicky infants [15]. Rhoads et al. studied the factors in uencing infant bowel in ammation in the United States in 2018. A total of 65 infants were included in the study, 37 of whom had colic and the rest were healthy. The results of this study showed that fecal calprotectin levels in colicky infants were signi cantly higher than healthy infants [13]. The concept of alterations in the gut micro ora emerged several dietary interventions [12] A study by Savino et al. in 2015 evaluated the effect of treatment with Lactobacillus ruteri on the levels of calprotectin and the crying time in infants with colic. Forty-three infants, including 25 receiving probiotics and 18 receiving placebos were studied. The results of this study showed that fecal calprotectin levels were signi cantly higher in the colic group compared to controls and fecal Calprotectin as well as crying time decreased signi cantly after treatment with L Reuteri [16].
The results of these studies are consistent with our study and suggest that fecal calprotectin is a good diagnostic and predictive agent for infantile colic. Calprotectin is secreted from the cytoplasm of neutrophils into the intestinal lumen during in ammatory and infectious conditions. As a result, fecal calprotectin is an indicator of in ammation in the gastrointestinal tract [17,18]. Calprotectin lasts for a week in the stool which makes it a good marker for detecting in ammation in the gut [17,19]. However, other studies with different results have been reported. In the study of Olafsdottir, there was not any signi cant difference in fecal Calprotectin of colicky infants compared to healthy babies and the level of Calprotectin decreased signi cantly with age [20].
The results of our study showed that both in the infants with colic and in controls, fecal calprotectin levels did not differ with sex. Similar results are found in other studies [9,15,20].
Our ndings showed that the increase in age did not make a difference in the level of fecal calprotectin in the two groups. Shahramian and Asgarshirazi found the same results [9,15], but Olafsdottir concluded that calprotectin levels is high in infancy and decreases with age [20]. In a study by Dorosko et al. in the United States, infants were assessed for the level of fecal calprotectin depending on their diet. In this study, 77 fecal samples were collected from 32 infants 3 days to 6 months of age. Fifteen infants were exclusively breastfed and 21 infants had a mixed diet. The results of this study showed that increasing age signi cantly reduced the level of calprotectin [21]. This study is not consistent with our study.
We concluded that infant's weight had no effect on fecal calprotectin levels. Some studies have shown the same result [15]. In the study of Karabayir et al. on 70 infants, fecal Calprotectin was signi cantly higher in coliky infants compared to controls and similar to our study they showed that infant's birth weight had no effect on fecal calprotectin [22].
In our study there was no signi cant difference between calprotectin levels regarding dietary patterns. This nding is consistent with some studies [13,17,22].However, the results of Asgarshirazi's study showed that breastfed infants had signi cantly higher levels of calprotectin in their feces (9). Drosko studied 15 infants with exclusively breastfeeding and 21 infants with a combination feeding. The results of this study also showed that exclusive breastfeeding signi cantly increased fecal calprotectin levels [21].
Although the exact role of calprotectin is not yet known, its antibacterial and antifungal role has been proven in some studies [17,23]. On the other hand, the secretion of calprotectin increases in cases of in ammation and intestinal infection. More researches are needed to determine the exact role of calprotectin.

Conclusion:
In conclusion, the present study demonstrated that fecal Calprotectin of colicky infants is signi cantly higher and therefore an in ammatory process should be considered in the etiology of this disease. By conducting well designed clinical trials to eliminate in ammatory agents, we may achieve brilliant results in treating the disease. written informed parental consent was obtained to participate in the study.

Consent for publication
Not applicable Availability of data and materials : The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.