- Email: [email protected]
Fecal calprotectin as a factor that supports the pathogenicity of Dientamoeba fragilis
Microbial Pathogenesis 139 (2020) 103868
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Fecal calprotectin as a factor that supports the pathogenicity of Dientamoeba fragilis
T
Mehmet Aykura,b,∗,1, Guliz Armaganc, Rukiye Vardard, Hande Dagcia a
Department of Parasitology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey Department of Parasitology, Gaziosmanpaşa University, Faculty of Medicine, Tokat, Turkey c Department of Biochemistry, Ege University, Faculty of Pharmacy, Bornova, Izmir, Turkey d Department of Gastroenterology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dientamoeba fragilis Fecal calprotectin Pathogenicity Gastrointestinal symptoms
Calprotectin is a protein that is mostly released from neutrophils, monocytes, macrophages and submucosal epithelial cells. Fecal calprotectin (f-CP) is a marker of intestinal inflammation. There are some discussions about the pathogenicity of D. fragilis in the gastrointestinal tract. In this study, we investigated whether f-CP level is a factor supporting the pathogenicity of D. fragilis. The f-CP levels were evaluated in patients with only D. fragilis positive in comparison with healthy controls. Moreover, the levels of f-CP were investigated in fecal samples of D. fragilis negative patients with gastrointestinal complaints. The fecal samples were collected from three groups. Three groups of fecal samples were examined directly microscopy, trichrome staining, cultivation, enzyme immunoassay (EIA) and real-time PCR assay. In the first group (Group 1, n = 34), patient stool samples with gastrointestinal symptoms (without other pathogens) found only with D. fragilis were included. In the second group (Group 2, n = 31), there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative (but there may be other pathogenic agents). In the control group (Group 3, n = 23), we used fecal samples collected from healthy volunteers without any infection or gastrointestinal complaints. The collected fecal samples were stored at −20 °C until analysis. Levels of f-CP were determined by using human calprotectin ELISA kits. Total of 88 patients were enrolled in three different groups. We obtained f-CP levels as follows: 33.40 ng/mg protein in the group 1, 15.99 ng/mg protein in the group 2 and 1.54 ng/mg protein in the group 3. Statistically significant difference in f-CP levels of the group 1 and the group 2 were obtained when compared with healthy controls (p < 0.0001). However, the f-CP levels of the group 1 were not significantly different from the group 2 (p > 0.99). In conclusion, increased levels of f-CP are shown as a marker of an inflammatory disease of the lower gastrointestinal tract in infected humans. There is continues controversy about the pathogenicity of D. fragilis in symptomatic and asymptomatic patients. The findings of this study contribute to the ongoing debate about the pathogenicity of D. fragilis. In our study, the potential pathogenicity of D. fragilis is associated with increased f-CP concentrations with parasite detection in the fecal samples and therefore we assume that the parasite is not only a harmless commensal. In summary, higher levels of f-CP found in D. fragilis positive patients suggest the importance of researches that support the pathogenicity of indicated parasite.
1. Introduction Dientamoeba fragilis is a common flagellate protozoan of the human gastrointestinal tract [1]. The reported prevalence of D. fragilis varies from 0.2 to 82% in different regions of the worldwide [2–4]. Nevertheless, the parasite is a neglected parasite of the gastrointestinal tract
[5]. The first commensal parasite was descripted by Dobell and Jepps in 1918 [6]. However, the presence of D. fragilis in patients without other pathogens causing gastrointestinal symptoms should be considered as a pathogen that causes symptoms [5]. The patients infected with D. fragilis have common gastrointestinal symptoms such as diarrhea or abdominal pain. The symptoms such as anorexia, weight loss, nausea,
∗
Corresponding author., Department of Parasitology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey. E-mail address: [email protected] (M. Aykur). 1 Current address: Department of Parasitology, Ege University, Faculty of Medicine, Bornova/Izmir, Turkey. https://doi.org/10.1016/j.micpath.2019.103868 Received 5 July 2019; Received in revised form 21 October 2019; Accepted 11 November 2019 Available online 12 November 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
vomiting or fatigue are also reported [7,8]. Although there are findings supporting the possible pathogenicity of D. fragilis, there are still some doubts about its expression as a pathogen [9]. Some of studies were stated that D. fragilis is not associated with gastrointestinal symptoms and may even be regarded as commensal [3,10–12]. On the other hand, it is associated with a wide range of symptoms in patients suffering from gastrointestinal symptoms [7]. Treatment of patients infected with D. fragilis together with the disappearance of symptoms may be an indicative of the evidence that this parasite is a pathogen [1]. Some studies have suggested that the most common symptoms in patients with D. fragilis were diarrhea and abdominal pain and that these symptoms disappear after treatment with metronidazole and secnidazole or ornidazole [13,14]. In recent years experimental animal studies have performed an animal model by infecting mice with D. fragilis and these studies provided significant information regarding to its pathogenic role [15]. The animals infected with D. fragilis were histopathological demonstrated an inflammation in the large intestine mucosa compared to the control group [15]. Moreover, D. fragilis has been shown to infect mucosal crypts between the caecum and the rectum [16]. Therefore, it shows the pathogenicity of this parasite. Fecal calprotectin (f-CP) is a calcium- and zinc-binding protein found between 30 and 60% of neutrophil cytosolic protein [17]. Concentration of f-CP have been measured in various bacterial, viral, parasitic infection, colorectal cancer and inflammatory bowel diseases (IBD) in gastrointestinal tract [18]. Measuring the level of f-CP in the feces is accepted as a good indicator of inflammation in the gastrointestinal tract [18,19]. In the case of pathogens in the gastrointestinal tract, neutrophils migrate into intestinal lumen, lead to the release of the calprotectin protein and cause damage to the mucosal epithelial cells (Fig. 1) [20]. In this case, diarrhea is observed in patients with gastrointestinal symptoms, increasing intestinal permeability and chlorine ion balance [18]. Recently, in an experimental mice study infected with D. fragilis, fCP levels in feces have been shown to be significantly higher when compared to the control group [15]. It has been reported that f-CP level changes are similar in feces of patients suffering from parasitic infections such as giardiasis and intestinal schistosomiasis [21,22]. In this study, we investigated whether fecal calprotectin is a factor supporting the pathogenicity of D. fragilis. For this purpose, f-CP levels were evaluated in patients with only D. fragilis positive in comparison with healthy controls. Moreover, the levels of f-CP were investigated in
D. fragilis negative patients with gastrointestinal complaints. It is important in terms of being a study that supports pathogenicity by forming a group for patients infected with the only D. fragilis. 2. Materials and methods 2.1. Ethics statement The present study was approved by Dokuz Eylül University Clinical Research Ethics Committee (Protocol No: 2013/16-02). Written informed consent was obtained from all adult subjects provided informed consent, and a parent or guardian of any child participant provided informed consent on the child's behalf. 2.2. Study groups In this study, stool samples of patients which we had investigated and published before for the prevalence values of D. fragilis were used [23]. These used specimens belonged to the patients with gastrointestinal symptoms such as diarrhea with during 3–5 weeks, abdominal pain or etc … and patients in outpatient clinic in department of gastroenterology. These samples were divided into two groups. In the first group (Group 1), patient stool samples with gastrointestinal symptoms (without other pathogens) found only with D. fragilis were included. In the second group (Group 2), there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative (but there may be other pathogenic agents). In the control group (Group 3), we used fecal samples collected from healthy volunteers without any infection or gastrointestinal complaints. A questionnaire was conducted to collect data about clinical symptoms of all patients and healthy volunteers. 2.3. Fecal samples collection and preparation The fecal samples were collected as fresh and preserved in a screw top container. The containers were delivered immediately to the Parasitology Laboratory of Ege University Faculty of Medicine. Three groups of fecal samples were examined for intestinal parasites as directly microscopy and as using trichrome staining. After that, we performed Robinson culture medium, enzyme immunoassay (EIA) and real-time PCR assay [23]. Moreover, group 1 (n = 34) was investigated in terms of the presence for other viral (Rotavirus and Adenovirus) and bacterial pathogens (Salmonella sp. and Shigella sp. and E. coli O157:H7) causing gastrointestinal disorders. The fecal samples in group 2 (n = 31) and group 3 (n = 23) were examined for the presence of intestinal parasites only using the methods mentioned above. However, these were not tested the presence for other viral and bacterial pathogens causing gastrointestinal disorders. The fecal samples were stored at – 20 °C until further analysis. The preparation of fecal samples was performed according to the instructions of the commercial manufacturer (Mybiosource. com, San Diego, CA, USA). Briefly, fecal samples were taken up to approximately 100 mg on dry ice before melting and diluted with PBS buffer (1:10). After that, homogenates were centrifuged for approximately 20 min at 5000 rpm and collected carefully the supernatants and stored – 20 °C until using. 2.4. Measurement of BCA protein and fecal calprotectin The amount of protein in all fecal samples was determined by using Pierce BCA protein assay kit. Briefly, this method was added 25 μL of supernatants from each sample to the well of 96-well plates with two wells of each sample on dry ice. Then it was added 200 μL of working reagent to each well and mixed the plate well on shaker for 30 s. The microplates were covered with aluminum foil and incubated at 37 °C for 30 min. It was measured the absorbance at 562 nm in the plate reader.
Fig. 1. Schematic mechanism of fecal calprotectin releasing. It shows the formation of a mucosal inflammation in the presence of D. fragilis in the gastrointestinal tract and also calprotectin release from neutrophils passing into the intestinal lumen. This figure was created using templates from Servier Medical Art website (https://smart.servier.com/). 2
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
The obtained value was expressed as μg protein/ml. Thus, the amount of protein of all samples has been equalized before starting the test of fecal calprotectin. Fecal calprotectin levels were measured with quantitative enzymelinked immune assay (Mybiosource. com, San Diego, CA, USA) according to the instructions of the commercial manufacturer's protocol [24]. 50 μl of protein equalized samples were added to each standard well in duplicate to the plate. After added 100 μl of HRP-conjugate reagent to each well, the plate was covered with a Closure Plate Membrane and incubated for 60 min at 37 °C. After incubation, the plates were washed four times with the wash solution (1x). After that, 50 μl Chromogen Solution A and 50 μl Chromogen Solution B were to each well successively. Then it was incubated for 15 min at 37 °C in dark. It was added 50 μl Stop Solution to each well. After the color in the wells changed from blue to yellow, it was read the optical density at 450 nm using an ELISA reader within 15 min after adding Stop Solution. The fecal calprotectin levels were calculated from the standard curve obtained with the kit standards.
Fig. 2. The relationship of fecal calprotectin values between groups. Fecal calprotectin values in healthy control volunteers (Group 3) and in patients affected by gastrointestinal symptoms compared with detected D. fragilis positive (Group 1) and D. fragilis negative (Group 2). Statistically as significantly was defined as ****p < 0.0001 and #p > 0.05.
2.5. Statistical analysis
obtained to be 15.99 ng/mg protein in the group 2 and was found to be significant when compared with the group 3 f-CP level (p < 0.0001) (Fig. 2). However, there was no statistically significant difference between the group 1 and the group 2 (p > 0.05) (Fig. 2). When we grouped according to the age of the participants, it was divided into three groups: < 20 years old, 20–40 years old and > 40 years old. Among the three different groups included in the study, it was not statistically significant when compared to three different age groups in the same group (p > 0.05). However, the f-CP levels were found to be significant between the group 1 and the group 3 in all age groups (p < 0.01) (Fig. 3). Although there was no significance between f-CP level of the group 1 and the group 2 in < 20 years old, it was found to be significant when compared with 20–40 years old and > 40 years old groups (p < 0.05) (Fig. 3). The f-CP levels of patients’ fecal samples with gastrointestinal symptoms reported in the group 1 and the group 2 were shown in Table 2. The same patient had presented with at least one or more gastrointestinal symptoms. The most common gastrointestinal symptoms in these two groups were diarrhea and abdominal pain. When f-CP values in fecal samples of the patients with gastrointestinal symptoms were compared between these two groups, the gastrointestinal symptoms such as diarrhea, abdominal pain and weight loss were found statistically significant, respectively (p = 0.007, p = 0.030, p = 0.016) (Table 2). Receiver operating characteristic (ROC) curve analysis was used to detect sensitivity, specificity and the best of cut-off values of f-CP. We, also, used ROC curve analysis, to determine cut-off f-CP values for predicting mucosal inflammation. The area under the curve of the ROC was 1.00 (95% CI 0.8972 to 1.000) compute for identifying f-CP value in the group 1 (D. fragilis positive) and the group 3 (healthy control) (Fig. 4). The area under the curve revealed sensitivity and specificity of f-CP value, respectively (100%, 95% CI, 89.72–100.0%; 95.65%, 95% CI, 78.05–99.89%) (Fig. 4). ROC curve was calculated that f-CP values of 5.045 μg/ml was the best cut-off in order to distinguish between the group 3 and the group 1 (Fig. 4).
The statically analysis was performed using the Graph Pad Prism 6 software package for macOS high sierra. Fecal calprotectin levels were expressed as the mean and 95% confidence interval. Fecal calprotectin value in among different groups was compared to by Mann–Whitney U test and by the Kruskal–Wallis. The receiver operating characteristic (ROC) curve for f-CP values was obtained with Graph Pad Prism 6 software package program. We considered P values of < 0.05 as statistically significant. 3. Results A total of 88 patients were enrolled in three the different groups in this study and their demographic information was present in Table 1. Group 1, the patient stool samples with gastrointestinal symptoms (without other pathogens) found with only D. fragilis positive were used. The others pathogens that would cause gastrointestinal complaints for these other pathogens were excluded by microscopy, culture method, EIA and real-time PCR assay [23]. Of the 34 patients included in group 1, 13 (38.2%) were male and 21 (61.7%) were female, with ages ranging from 4 to 80 years. Group 2, there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative. Since this group was not excluded bacterial and viral pathogens in group 1 that cause gastrointestinal symptoms, there may be these pathogenic agents [23]. Thirty-one patients were included in group 2, 13 (41.9%) were males and 18 (58.1%) were females and their ages ranged from 1 to 80 years. Group 3 consisted of 23 stool samples collected from healthy volunteers. In this group, no any a pathogen was detected when the stool samples were investigated by microscopic, culture and realtime PCR assay [23]. Four of the healthy volunteers (17.3%) were male and 19 (82.6%) were female and their ages ranged from 18 to 59 years. After equalizing the total protein content of each sample to 1 μg protein/ml, we found that f-CP levels were 33.40 ng/mg protein in the group 1. The f-CP level of the group 3 was 1.54 ng/mg protein. When fCP level of the group 3 and the group 1 were compared, it was found statistically significant (p < 0.0001) (Fig. 2). The level of f-CP was
4. Discussion Table 1 The demographic characteristics of participants in the study group. Groups
Group 1 Group 2 Group 3
Patients No.
34 31 23
Gender
Age
Male/Female n (%)
Means
Range
13 (38.2%)/21 (61.7%) 13 (41.9%)/18 (58.1%) 4 (17.3%)/19(82.6%)
31.7 38.9 34.6
4–80 1–80 18–59
D. fragilis was actually described by Jeeps and Dobell a hundred years ago as a non-pathogenic protozoan organism [6]. Although many publications have supported the pathogenic potential of D. fragilis for years, it has been reported to be the commensal an organism in some studies [3,10,25,26]. In most studies, it is reported that D. fragilis is associated with gastrointestinal symptoms and the symptoms disappear after treatment, and consequently D. fragilis could be pathogenic [1,26,27]. However, there is still continues controversy about the life 3
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
Fig. 3. The evaluation of fecal calprotectin levels by age groups. Age-related distribution of fecal calprotectin (ng/mg protein) in patient which in include three different groups (D. fragilis positive, D. fragilis negative and healthy control). Statistically of significance was defined as *p < 0.05, **p < 0.01.
cycle, mode of transmission and pathogenicity of this protozoan [7,8]. In our study, the most common gastrointestinal symptoms were found as diarrhea, abdominal pain and weight loss in the group 1, respectively (80.0%, 52.9% and 23.5%). Moreover, the absence of any pathogens other than D. fragilis in the group 1 to cause gastrointestinal symptoms has supported the pathogenicity of D. fragilis. In another study similar to our study, the most common gastrointestinal symptoms seen in most patients infected with D. fragilis were diarrhea (83.3%), abdominal pain (77.7%), and loose stools (72.2%), respectively [1]. Calprotectin is a 36-kDa calcium-and zinc-binding protein which is found from neutrophils, monocytes, and macrophages. This protein is composed of about 60% as dissolved in the cytosol of neutrophils. Calprotectin can be measured in various body fluids, such as plasma, urine, and cerebrospinal fluid, and in feces and tissue samples. The changes in levels of f-CP are clinically useful for gastroenterologists by measuring directly as a marker of inflammation of the mucous membrane [18,28]. The use of f-CP as a non-invasive stool marker to monitor inflammation have been discussed in several studies [29–31]. Currently, it is well-known that f-CP is the best available surrogate marker for mucosal inflammatory activity in IBD [32,33]. The resistance to degradation and stability in the stool for up to four days in room temperature are major advantages of calprotectin while evaluating the biological changes in diseases [34]. In our study, it was observed that f-CP concentration was approximately six times higher in the group 1 (33.40 ng/mg protein) compared to the cut-off value
Fig. 4. Receiver operating characteristic curve graph. Receiver Operating Characteristic (ROC) curve defining an optimal f-CP cut-off value in order to distinguish between the healthy control group and D. fragilis positive group. The area under the curve, sensitivity and specificity values are also shown, respectively (1.00 (95% confidence interval [CI], 0.8972 to 1.000; 100%, 95% CI, 89.72–100.0%; 95.65%, 95% CI, 78.05–99.89%).
Table 2 Gastrointestinal symptoms and calprotectin values of patients enrolled in the study. Symptomsa
Diarrhea Abdominal pain Weight loss Nausea Itching Fever Lack of appetite Vomiting
Group 1
Group 2 b
n: 34 (%)
f-CP
28 (80.0%) 18 (52.9%) 8 (23.5%) 7 (20.5%) 5 (14.7%) 5 (14.7%) 4 (11.7%) 3 (8.8%)
33.05 33.60 45.59 29.82 32.46 49.14 46.13 51.66
± ± ± ± ± ± ± ±
5.53 6.81 8.88 10.48 15.16 10.98 15.24 18.69
Abbreviations: f-CP = fecal calprotectin. * Statistically significant difference was defined as p < 0.05. a The same patient had presented with at least one or more gastrointestinal symptoms. b Fecal calprotectin values given as mean ± SE of the mean. 4
P value* b
n: 31 (%)
f-CP
21 (67.7%) 11 (35.4%) 11 (35.4%) 7 (22.5%) 7 (22.5%) 4 (12.9%) 11 (35.4%) 6 (19.3%)
15.61 16.26 16.56 16.47 18.13 17.71 16.43 17.60
± ± ± ± ± ± ± ±
1.08 1.53 1.48 2.31 1.96 1.58 1.56 2.14
0.007* 0.030* 0.016* 0.271 0.435 0.127 0.212 0.187
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
(5.045 μg/ml protein). Therefore, this group of patients showed an inflammation of the mucosa of the gastrointestinal tract. The f-CP value in feces of D. fragilis-infected experimental animals (69 ng/ml) was approximately two-fold higher than the control group (33 ng/ml) and indicative of the presence of intestinal inflammation in gastrointestinal mucosa [15]. In the first study of f-CP in humans, it was reported that there was no significant difference between the f-CP values of D. fragilis positive (40–55 μg/g) fecal samples and D. fragilis negative fecal samples (40–75 μg/g) [35]. In this study, it was the first study found to be statistically significant when the f-CP values of the group 1 (D. fragilis positive patient samples (33.40 ng/mg protein)) and the group 3 (the control group samples (1.54 ng/mg protein)) were compared. In this case, the f-CP value supports the pathogenicity of D. fragilis. There are a number of studies in recent advances in the relationship between bacterial, viral gastroenteritis and f-CP [36,37]. Although there were limited studies the association with between the intestinal symptoms caused by parasites and f-CP, the first time a significant relationship between the symptoms induced by D. fragilis and f-CP was investigated in the only animal model [15,16]. Our study measuring fCP levels in a group of patients with gastrointestinal symptoms, caused only by D. fragilis was the first study and in terms of supporting the pathogenicity of D. fragilis. When f-CP levels of gastrointestinal symptoms of patients with the group 1 and the group 2 were compared, it was found statistically significant in patients with diarrhea (33.05 ± 5.53, 15.61 ± 1.08; p = 0.007), abdominal pain (33.60 ± 6.81, 16.26 ± 1.53; p = 0.030) and weight loss (45.59 ± 8.88, 16.56 ± 1.48; p = 0.016), respectively. In the group 2 patients, f-CP levels were statistically significant when compared with the healthy control group (the group 3) because other etiologic agents causing gastrointestinal symptoms were not excluded. Some the reviews had reported that f-CP levels of patients with gastrointestinal disorders were higher than healthy controls [38]. With the migration of neutrophils into the lumen in the gut, it is thought that calprotectin is released and triggers inflammation by induction of proinflammatory chemokines [20]. Therefore, the intestinal mucosal epithelial cells are damaged and the protein is secreted and thus causes diarrhea together with increased intestinal permeability and chlorine ion [16,34]. The group 1 (only D. fragilis positive patients) has been found to have gastrointestinal symptoms and high f-CP concentration, and in this case has supported a potential pathogenicity of D. fragilis. There was no significant difference in comparing f-CP levels in the group 1 and the group 2 without age groups. However, when divided into three groups according to age, f-CP levels in between the group 1 and the group 2 were found to be significant in 20–40 years old and > 40 years old. However, some studies support the idea that f-CP should not be used as a marker of inflammation because of its high expression up to the age of 4 years [39,40]. The reason for this may be raised early in life to compensate for an immature and developing immune system in newborns [40]. Increased intestinal permeability, bacterial microbiota formation and the giving of diet may also affect fCP levels in healthy newborns [41]. In this study, f-CP levels of the group 1 were distinguished from cutoff values according to ROC analysis. However, it cannot be distinguished from the group 2. This is given to the exclusion of other pathogens that will cause gastrointestinal symptoms. In the future, its performance could be further improved by increasing the number of patients with D. fragilis positive, negative and healthy control groups. Although the f-CP level is not a fully indicative indicator of the presence of D. fragilis, it shows that it is a potential pathogen with real-time PCR, which is the gold standard. The lesions that D. fragilis had previously formed in the large intestine were defined as colitis [15,16]. These pathogenic changes may be because of the secretion of calprotectin (a neutrophil protein) and its existence in the stool. This is determinative of neutrophilic infiltration into the gut linked inflammatory processes [16]. D. fragilis is among the possible etiological agents for patients suffering from intestinal
disorders including Inflammatory Bowel Syndrome (IBS) [1]. In addition to its antimicrobial activity, calprotectin has been shown to regulate innate immune response by activating toll-like receptor 4 [16,42]. High levels of calprotectin have also been shown in some parasitic infections such as giardiasis and intestinal schistosomiasis [21,22]. In our study, significant increases in calprotectin levels were observed in patients with only D. fragilis positive as an indicative of intestinal inflammation. The debates about the pathogenicity of D. fragilis are still ongoing. Several potential pathogenicity has been described in D. fragilis as amoebapore-like proteins and putative immunomodulatory proteins [43]. The plentiful detected virulence factors are members of the cathepsin L-like cysteine protease family. Plenty cysteine proteases have been identified in D. fragilis [5,8,43]. On the other hand, f-CP is a stimulates immunomodulation and antibacterial effects of neutrophil [17]. It has also important roles in the trans epithelial migration and accumulation at mucosal surfaces of neutrophil. The presence of calprotectin levels in feces is a good predictor of inflammation. In experimental studies, D. fragilis related colon inflammation was observed [15,16]. In our study; higher levels of f-CP found in D. fragilis positive patients are of great importance to be a research that supports the pathogenicity of the specified parasite and to be searched for the first time. In conclusion, the findings of this study contribute to the ongoing debate about the pathogenicity of D. fragilis. The f-CP level can be used as a good indicator of gastrointestinal systems inflammation. In our study, the potential pathogenicity of D. fragilis is associated with increased f-CP concentrations with parasite detection in the fecal samples and therefore we assume that the parasite is not only a harmless commensal. This is the first study to support the pathogenicity of D. fragilis by looking at the f-CP concentration in human fecal samples of D. fragilis positive. Further studies are needed to determine the pathogenicity of D. fragilis by looking at the f-CP level by forming a larger sample group than the D. fragilis positive patient group. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This study was partly supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant number: 13-TIP-092). References [1] D. Stark, J. Barratt, T. Roberts, D. Marriott, J. Harkness, J. Ellis, A review of the clinical presentation of dientamoebiasis, Am. J. Trop. Med. Hyg. 82 (2010) 614–619. [2] J. Ogren, S. Van Nguyen, M.K. Nguyen, J. Dimberg, A. Matussek, Prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp in Da Nang, Vietnam, detected by a multiplex real-time PCR, APMIS 124 (2016) 529–533. [3] G.A. Holtman, J.J. Kranenberg, M.H. Blanker, A. Ott, Y. Lisman-van Leeuwen, M.Y. Berger, Dientamoeba fragilis colonization is not associated with gastrointestinal symptoms in children at primary care level, Fam. Pract. 34 (2017) 25–29. [4] S.M. Caccio, Molecular epidemiology of Dientamoeba fragilis, Acta Trop. 184 (2017) 73–77, https://doi.org/10.1016/j.actatropica.2017.06.029. [5] J.L. Barratt, J. Harkness, D. Marriott, J.T. Ellis, D. Stark, A review of Dientamoeba fragilis carriage in humans: several reasons why this organism should be considered in the diagnosis of gastrointestinal illness, Gut Microb. 2 (2011) 3–12. [6] M.W. Jepps, C. Dobell, Dientamoeba fragilis n. g., n. sp., a new Intestinal Amoeba from Man, Parasitology 10 (1918) 352–367. [7] L.S. Garcia, Dientamoeba fragilis, one of the neglected intestinal Protozoa, J. Clin. Microbiol. 54 (2016) 2243–2250. [8] D. Stark, J. Barratt, D. Chan, J.T. Ellis, Dientamoeba fragilis, the neglected trichomonad of the human bowel, Clin. Microbiol. Rev. 29 (2016) 553–580. [9] J.L. Barratt, J. Harkness, D. Marriott, J.T. Ellis, D. Stark, The ambiguous life of Dientamoeba fragilis: the need to investigate current hypotheses on transmission, Parasitology 138 (2011) 557–572.
5
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
819–823. [27] H.Z. Rayan, O.A. Ismail, E.K. El Gayar, Prevalence and clinical features of Dientamoeba fragilis infections in patients suspected to have intestinal parasitic infection, J. Egypt. Soc. Parasitol. 37 (2007) 599–608. [28] A. Poullis, R. Foster, M.A. Mendall, M.K. Fagerhol, Emerging role of calprotectin in gastroenterology, J. Gastroenterol. Hepatol. 18 (2003) 756–762. [29] T. Sipponen, E. Savilahti, K.L. Kolho, H. Nuutinen, U. Turunen, M. Farkkila, Crohn's disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn's disease activity index and endoscopic findings, Inflamm. Bowel Dis. 14 (2008) 40–46. [30] P. Rutgeerts, G. Van Assche, W.J. Sandborn, D.C. Wolf, K. Geboes, J.F. Colombel, et al., Adalimumab induces and maintains mucosal healing in patients with crohn's disease: data from the EXTEND trial, Gastroenterology 142 (2012) 1102. [31] C. Caimmi, E. Bertoldo, A. Venturini, P. Caramaschi, L. Frulloni, R. Ciccocioppo, et al., Relationship between increased fecal calprotectin levels and interstitial lung disease in systemic sclerosis, J. Rheumatol. (2018) 171445 jrheum. [32] J.D. Lewis, The utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease, Gastroenterology 140 (2011) 1817-U160. [33] S.C. Wei, Could fecal calprotectin enter mainstream use for diagnosing and monitoring inflammatory bowel disease? Intest. Res. 14 (2016) 293–294. [34] A. Voganatsi, A. Panyutich, K.T. Miyasaki, R.K. Murthy, Mechanism of extracellular release of human neutrophil calprotectin complex, J. Leukoc. Biol. 70 (2001) 130–134. [35] M.R. Brands, E. Van de Vijver, S.M. Haisma, A. Heida, P.F. van Rheenen, No association between abdominal pain and Dientamoeba in Dutch and Belgian children, Arch. Dis. Child. 104 (7) (2019) 686–689, https://doi.org/10.1136/archdischild2018-316383. [36] C.C. Chen, J.L. Huang, C.J. Chang, M.S. Kong, Fecal calprotectin as a correlative marker in clinical severity of infectious diarrhea and usefulness in evaluating bacterial or viral pathogens in children, J. Pediatr. Gastroenterol. Nutr. 55 (2012) 541–547. [37] M. Duman, P. Gencpinar, M. Bicmen, N. Arslan, O. Ozden, O. Uzum, et al., Fecal calprotectin: can be used to distinguish between bacterial and viral gastroenteritis in children? Am. J. Emerg. Med. 33 (2015) 1436–1439. [38] I.D. Kostakis, K.G. Cholidou, A.G. Vaiopoulos, I.S. Vlachos, D. Perrea, G. Vaos, Fecal calprotectin in pediatric inflammatory bowel disease: a systematic review, Dig. Dis. Sci. 58 (2013) 309–319. [39] M. Garg, S.T. Leach, M.J. Coffey, T. Katz, R. Strachan, T. Pang, et al., Age-dependent variation of fecal calprotectin in cystic fibrosis and healthy children, J. Cyst. Fibros. 16 (2017) 631–636. [40] M. Garg, S.T. Leach, A.S. Day, C.Y. Ooi, Fecal calprotectin concentrations in young children with cystic fibrosis: authors response, J. Cyst. Fibros. 17 (2018) e10–e11. [41] E. Olafsdottir, L. Aksnes, G. Fluge, A. Berstad, Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children, Acta Paediatr. 91 (2002) 45–50. [42] T. Vogl, K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M.A. van Zoelen, et al., Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock, Nat. Med. 13 (2007) 1042–1049. [43] J.L.N. Barratt, M. Cao, D.J. Stark, J.T. Ellis, The transcriptome sequence of Dientamoeba fragilis offers new biological insights on its metabolism, kinome, degradome and potential mechanisms of pathogenicity, Protist 166 (2015) 389–408.
[10] P. Jokelainen, B. Hebbelstrup Jensen, B.U. Andreassen, A.M. Petersen, D. Roser, K.A. Krogfelt, et al., Dientamoeba fragilis, a commensal in children in Danish day care centers, J. Clin. Microbiol. 55 (2017) 1707–1713. [11] R. Enserink, R. Scholts, P. Bruijning-Verhagen, E. Duizer, H. Vennema, R. de Boer, et al., High detection rates of enteropathogens in asymptomatic children attending day care, PLoS One 9 (2014) e89496. [12] M.J. De Jong, J.J. Korterink, M.A. Benninga, M. Hilbink, J. Widdershoven, J.M. Deckers-Kocken, Dientamoeba fragilis and chronic abdominal pain in children: a case–control study, Arch. Dis. Child. 99 (12) (2014) 1109–1113, https://doi.org/ 10.1136/archdischild-2014-305942 25053737. [13] O. Kurt, N. Girginkardesler, I.C. Balcioglu, A. Ozbilgin, U.Z. Ok, A comparison of metronidazole and single-dose ornidazole for the treatment of dientamoebiasis, Clin. Microbiol. Infect. 14 (2008) 601–604. [14] N. Girginkardesler, S. Coskun, I. Cuneyt Balcioglu, P. Ertan, U.Z. Ok, Dientamoeba fragilis, a neglected cause of diarrhea, successfully treated with secnidazole, Clin. Microbiol. Infect. 9 (2003) 110–113. [15] V.S. Munasinghe, N.G. Vella, J.T. Ellis, P.A. Windsor, D. Stark, Cyst formation and faecal-oral transmission of Dientamoeba fragilis–the missing link in the life cycle of an emerging pathogen, Int. J. Parasitol. 43 (2013) 879–883. [16] E.K. El-Gayar, A.B. Mokhtar, W.A. Hassan, Study of the pathogenic potential of Dientamoeba fragilis in experimentally infected mice, Parasite Epidemiol. Control 1 (2016) 136–143. [17] F. Costa, M. Mumolo, Bellini Ma, M. Romano, L. Ceccarelli, P. Arpe, et al., Role of faecal calprotectin as non-invasive marker of intestinal inflammation, Dig. Liver Dis. 35 (2003) 642–647. [18] C.W. McMahon, R. Chhabra, The role of fecal calprotectin in investigating digestive disorders, J. Lab. Precis. Med. 3 (2018). [19] H. Manceau, V. Chicha-Cattoir, H. Puy, K. Peoc'h, Fecal calprotectin in inflammatory bowel diseases: update and perspectives, Clin. Chem. Lab. Med. 55 (2017) 474–483. [20] Y.A. Lam, S.M. Warouw, A.M. Wahani, J.I. Manoppo, P.M. Salendu, Correlation between gut pathogens and fecal calprotectin levels in young children with acute diarrhea, Paediatr. Indones. 54 (2014) 193–197. [21] K. Hanevik, T. Hausken, M.H. Morken, E.A. Strand, K. Morch, P. Coll, et al., Persisting symptoms and duodenal inflammation related to Giardia duodenalis infection, J. Infect. 55 (2007) 524–530. [22] A.L. Bustinduy, J.C. Sousa-Figueiredo, M. Adriko, M. Betson, A. Fenwick, N. Kabatereine, et al., Fecal occult blood and fecal calprotectin as point-of-care markers of intestinal morbidity in Ugandan children with Schistosoma mansoni infection, PLoS Neglected Trop. Dis. 7 (2013). [23] M. Aykur, C. Caliskan Kurt, D. Dirim Erdogan, C. Biray Avci, R. Vardar, S. Aydemir, et al., Investigation of Dientamoeba fragilis Prevalence and Evaluation of Sociodemographic and Clinical Features in Patients with Gastrointestinal Symptoms, Acta Parasitol 64 (2019) 162–170. [24] K.M. Goodrich, G. Fundaro, L.E. Griffin, M.W. Hulver, M.A. Ponder, A.P. Neilson, Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occludin and reduces fecal calprotectin: a secondary analysis of healthy Wistar Furth rats, Nutr. Res. (N.Y.) 32 (2012) 787–794. [25] M.J. Spencer, M.R. Chapin, L.S. Garcia, Dientamoeba fragilis: a gastrointestinal protozoan infection in adults, Am. J. Gastroenterol. 77 (1982). [26] G. Banik, J. Barratt, D. Marriott, J. Harkness, J. Ellis, D. Stark, A case-controlled study of Dientamoeba fragilis infections in children, Parasitology 138 (2011)
6
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Fecal calprotectin as a factor that supports the pathogenicity of Dientamoeba fragilis
T
Mehmet Aykura,b,∗,1, Guliz Armaganc, Rukiye Vardard, Hande Dagcia a
Department of Parasitology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey Department of Parasitology, Gaziosmanpaşa University, Faculty of Medicine, Tokat, Turkey c Department of Biochemistry, Ege University, Faculty of Pharmacy, Bornova, Izmir, Turkey d Department of Gastroenterology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dientamoeba fragilis Fecal calprotectin Pathogenicity Gastrointestinal symptoms
Calprotectin is a protein that is mostly released from neutrophils, monocytes, macrophages and submucosal epithelial cells. Fecal calprotectin (f-CP) is a marker of intestinal inflammation. There are some discussions about the pathogenicity of D. fragilis in the gastrointestinal tract. In this study, we investigated whether f-CP level is a factor supporting the pathogenicity of D. fragilis. The f-CP levels were evaluated in patients with only D. fragilis positive in comparison with healthy controls. Moreover, the levels of f-CP were investigated in fecal samples of D. fragilis negative patients with gastrointestinal complaints. The fecal samples were collected from three groups. Three groups of fecal samples were examined directly microscopy, trichrome staining, cultivation, enzyme immunoassay (EIA) and real-time PCR assay. In the first group (Group 1, n = 34), patient stool samples with gastrointestinal symptoms (without other pathogens) found only with D. fragilis were included. In the second group (Group 2, n = 31), there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative (but there may be other pathogenic agents). In the control group (Group 3, n = 23), we used fecal samples collected from healthy volunteers without any infection or gastrointestinal complaints. The collected fecal samples were stored at −20 °C until analysis. Levels of f-CP were determined by using human calprotectin ELISA kits. Total of 88 patients were enrolled in three different groups. We obtained f-CP levels as follows: 33.40 ng/mg protein in the group 1, 15.99 ng/mg protein in the group 2 and 1.54 ng/mg protein in the group 3. Statistically significant difference in f-CP levels of the group 1 and the group 2 were obtained when compared with healthy controls (p < 0.0001). However, the f-CP levels of the group 1 were not significantly different from the group 2 (p > 0.99). In conclusion, increased levels of f-CP are shown as a marker of an inflammatory disease of the lower gastrointestinal tract in infected humans. There is continues controversy about the pathogenicity of D. fragilis in symptomatic and asymptomatic patients. The findings of this study contribute to the ongoing debate about the pathogenicity of D. fragilis. In our study, the potential pathogenicity of D. fragilis is associated with increased f-CP concentrations with parasite detection in the fecal samples and therefore we assume that the parasite is not only a harmless commensal. In summary, higher levels of f-CP found in D. fragilis positive patients suggest the importance of researches that support the pathogenicity of indicated parasite.
1. Introduction Dientamoeba fragilis is a common flagellate protozoan of the human gastrointestinal tract [1]. The reported prevalence of D. fragilis varies from 0.2 to 82% in different regions of the worldwide [2–4]. Nevertheless, the parasite is a neglected parasite of the gastrointestinal tract
[5]. The first commensal parasite was descripted by Dobell and Jepps in 1918 [6]. However, the presence of D. fragilis in patients without other pathogens causing gastrointestinal symptoms should be considered as a pathogen that causes symptoms [5]. The patients infected with D. fragilis have common gastrointestinal symptoms such as diarrhea or abdominal pain. The symptoms such as anorexia, weight loss, nausea,
∗
Corresponding author., Department of Parasitology, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey. E-mail address: [email protected] (M. Aykur). 1 Current address: Department of Parasitology, Ege University, Faculty of Medicine, Bornova/Izmir, Turkey. https://doi.org/10.1016/j.micpath.2019.103868 Received 5 July 2019; Received in revised form 21 October 2019; Accepted 11 November 2019 Available online 12 November 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
vomiting or fatigue are also reported [7,8]. Although there are findings supporting the possible pathogenicity of D. fragilis, there are still some doubts about its expression as a pathogen [9]. Some of studies were stated that D. fragilis is not associated with gastrointestinal symptoms and may even be regarded as commensal [3,10–12]. On the other hand, it is associated with a wide range of symptoms in patients suffering from gastrointestinal symptoms [7]. Treatment of patients infected with D. fragilis together with the disappearance of symptoms may be an indicative of the evidence that this parasite is a pathogen [1]. Some studies have suggested that the most common symptoms in patients with D. fragilis were diarrhea and abdominal pain and that these symptoms disappear after treatment with metronidazole and secnidazole or ornidazole [13,14]. In recent years experimental animal studies have performed an animal model by infecting mice with D. fragilis and these studies provided significant information regarding to its pathogenic role [15]. The animals infected with D. fragilis were histopathological demonstrated an inflammation in the large intestine mucosa compared to the control group [15]. Moreover, D. fragilis has been shown to infect mucosal crypts between the caecum and the rectum [16]. Therefore, it shows the pathogenicity of this parasite. Fecal calprotectin (f-CP) is a calcium- and zinc-binding protein found between 30 and 60% of neutrophil cytosolic protein [17]. Concentration of f-CP have been measured in various bacterial, viral, parasitic infection, colorectal cancer and inflammatory bowel diseases (IBD) in gastrointestinal tract [18]. Measuring the level of f-CP in the feces is accepted as a good indicator of inflammation in the gastrointestinal tract [18,19]. In the case of pathogens in the gastrointestinal tract, neutrophils migrate into intestinal lumen, lead to the release of the calprotectin protein and cause damage to the mucosal epithelial cells (Fig. 1) [20]. In this case, diarrhea is observed in patients with gastrointestinal symptoms, increasing intestinal permeability and chlorine ion balance [18]. Recently, in an experimental mice study infected with D. fragilis, fCP levels in feces have been shown to be significantly higher when compared to the control group [15]. It has been reported that f-CP level changes are similar in feces of patients suffering from parasitic infections such as giardiasis and intestinal schistosomiasis [21,22]. In this study, we investigated whether fecal calprotectin is a factor supporting the pathogenicity of D. fragilis. For this purpose, f-CP levels were evaluated in patients with only D. fragilis positive in comparison with healthy controls. Moreover, the levels of f-CP were investigated in
D. fragilis negative patients with gastrointestinal complaints. It is important in terms of being a study that supports pathogenicity by forming a group for patients infected with the only D. fragilis. 2. Materials and methods 2.1. Ethics statement The present study was approved by Dokuz Eylül University Clinical Research Ethics Committee (Protocol No: 2013/16-02). Written informed consent was obtained from all adult subjects provided informed consent, and a parent or guardian of any child participant provided informed consent on the child's behalf. 2.2. Study groups In this study, stool samples of patients which we had investigated and published before for the prevalence values of D. fragilis were used [23]. These used specimens belonged to the patients with gastrointestinal symptoms such as diarrhea with during 3–5 weeks, abdominal pain or etc … and patients in outpatient clinic in department of gastroenterology. These samples were divided into two groups. In the first group (Group 1), patient stool samples with gastrointestinal symptoms (without other pathogens) found only with D. fragilis were included. In the second group (Group 2), there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative (but there may be other pathogenic agents). In the control group (Group 3), we used fecal samples collected from healthy volunteers without any infection or gastrointestinal complaints. A questionnaire was conducted to collect data about clinical symptoms of all patients and healthy volunteers. 2.3. Fecal samples collection and preparation The fecal samples were collected as fresh and preserved in a screw top container. The containers were delivered immediately to the Parasitology Laboratory of Ege University Faculty of Medicine. Three groups of fecal samples were examined for intestinal parasites as directly microscopy and as using trichrome staining. After that, we performed Robinson culture medium, enzyme immunoassay (EIA) and real-time PCR assay [23]. Moreover, group 1 (n = 34) was investigated in terms of the presence for other viral (Rotavirus and Adenovirus) and bacterial pathogens (Salmonella sp. and Shigella sp. and E. coli O157:H7) causing gastrointestinal disorders. The fecal samples in group 2 (n = 31) and group 3 (n = 23) were examined for the presence of intestinal parasites only using the methods mentioned above. However, these were not tested the presence for other viral and bacterial pathogens causing gastrointestinal disorders. The fecal samples were stored at – 20 °C until further analysis. The preparation of fecal samples was performed according to the instructions of the commercial manufacturer (Mybiosource. com, San Diego, CA, USA). Briefly, fecal samples were taken up to approximately 100 mg on dry ice before melting and diluted with PBS buffer (1:10). After that, homogenates were centrifuged for approximately 20 min at 5000 rpm and collected carefully the supernatants and stored – 20 °C until using. 2.4. Measurement of BCA protein and fecal calprotectin The amount of protein in all fecal samples was determined by using Pierce BCA protein assay kit. Briefly, this method was added 25 μL of supernatants from each sample to the well of 96-well plates with two wells of each sample on dry ice. Then it was added 200 μL of working reagent to each well and mixed the plate well on shaker for 30 s. The microplates were covered with aluminum foil and incubated at 37 °C for 30 min. It was measured the absorbance at 562 nm in the plate reader.
Fig. 1. Schematic mechanism of fecal calprotectin releasing. It shows the formation of a mucosal inflammation in the presence of D. fragilis in the gastrointestinal tract and also calprotectin release from neutrophils passing into the intestinal lumen. This figure was created using templates from Servier Medical Art website (https://smart.servier.com/). 2
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
The obtained value was expressed as μg protein/ml. Thus, the amount of protein of all samples has been equalized before starting the test of fecal calprotectin. Fecal calprotectin levels were measured with quantitative enzymelinked immune assay (Mybiosource. com, San Diego, CA, USA) according to the instructions of the commercial manufacturer's protocol [24]. 50 μl of protein equalized samples were added to each standard well in duplicate to the plate. After added 100 μl of HRP-conjugate reagent to each well, the plate was covered with a Closure Plate Membrane and incubated for 60 min at 37 °C. After incubation, the plates were washed four times with the wash solution (1x). After that, 50 μl Chromogen Solution A and 50 μl Chromogen Solution B were to each well successively. Then it was incubated for 15 min at 37 °C in dark. It was added 50 μl Stop Solution to each well. After the color in the wells changed from blue to yellow, it was read the optical density at 450 nm using an ELISA reader within 15 min after adding Stop Solution. The fecal calprotectin levels were calculated from the standard curve obtained with the kit standards.
Fig. 2. The relationship of fecal calprotectin values between groups. Fecal calprotectin values in healthy control volunteers (Group 3) and in patients affected by gastrointestinal symptoms compared with detected D. fragilis positive (Group 1) and D. fragilis negative (Group 2). Statistically as significantly was defined as ****p < 0.0001 and #p > 0.05.
2.5. Statistical analysis
obtained to be 15.99 ng/mg protein in the group 2 and was found to be significant when compared with the group 3 f-CP level (p < 0.0001) (Fig. 2). However, there was no statistically significant difference between the group 1 and the group 2 (p > 0.05) (Fig. 2). When we grouped according to the age of the participants, it was divided into three groups: < 20 years old, 20–40 years old and > 40 years old. Among the three different groups included in the study, it was not statistically significant when compared to three different age groups in the same group (p > 0.05). However, the f-CP levels were found to be significant between the group 1 and the group 3 in all age groups (p < 0.01) (Fig. 3). Although there was no significance between f-CP level of the group 1 and the group 2 in < 20 years old, it was found to be significant when compared with 20–40 years old and > 40 years old groups (p < 0.05) (Fig. 3). The f-CP levels of patients’ fecal samples with gastrointestinal symptoms reported in the group 1 and the group 2 were shown in Table 2. The same patient had presented with at least one or more gastrointestinal symptoms. The most common gastrointestinal symptoms in these two groups were diarrhea and abdominal pain. When f-CP values in fecal samples of the patients with gastrointestinal symptoms were compared between these two groups, the gastrointestinal symptoms such as diarrhea, abdominal pain and weight loss were found statistically significant, respectively (p = 0.007, p = 0.030, p = 0.016) (Table 2). Receiver operating characteristic (ROC) curve analysis was used to detect sensitivity, specificity and the best of cut-off values of f-CP. We, also, used ROC curve analysis, to determine cut-off f-CP values for predicting mucosal inflammation. The area under the curve of the ROC was 1.00 (95% CI 0.8972 to 1.000) compute for identifying f-CP value in the group 1 (D. fragilis positive) and the group 3 (healthy control) (Fig. 4). The area under the curve revealed sensitivity and specificity of f-CP value, respectively (100%, 95% CI, 89.72–100.0%; 95.65%, 95% CI, 78.05–99.89%) (Fig. 4). ROC curve was calculated that f-CP values of 5.045 μg/ml was the best cut-off in order to distinguish between the group 3 and the group 1 (Fig. 4).
The statically analysis was performed using the Graph Pad Prism 6 software package for macOS high sierra. Fecal calprotectin levels were expressed as the mean and 95% confidence interval. Fecal calprotectin value in among different groups was compared to by Mann–Whitney U test and by the Kruskal–Wallis. The receiver operating characteristic (ROC) curve for f-CP values was obtained with Graph Pad Prism 6 software package program. We considered P values of < 0.05 as statistically significant. 3. Results A total of 88 patients were enrolled in three the different groups in this study and their demographic information was present in Table 1. Group 1, the patient stool samples with gastrointestinal symptoms (without other pathogens) found with only D. fragilis positive were used. The others pathogens that would cause gastrointestinal complaints for these other pathogens were excluded by microscopy, culture method, EIA and real-time PCR assay [23]. Of the 34 patients included in group 1, 13 (38.2%) were male and 21 (61.7%) were female, with ages ranging from 4 to 80 years. Group 2, there were patients’ stool samples with gastrointestinal symptoms that D. fragilis was negative. Since this group was not excluded bacterial and viral pathogens in group 1 that cause gastrointestinal symptoms, there may be these pathogenic agents [23]. Thirty-one patients were included in group 2, 13 (41.9%) were males and 18 (58.1%) were females and their ages ranged from 1 to 80 years. Group 3 consisted of 23 stool samples collected from healthy volunteers. In this group, no any a pathogen was detected when the stool samples were investigated by microscopic, culture and realtime PCR assay [23]. Four of the healthy volunteers (17.3%) were male and 19 (82.6%) were female and their ages ranged from 18 to 59 years. After equalizing the total protein content of each sample to 1 μg protein/ml, we found that f-CP levels were 33.40 ng/mg protein in the group 1. The f-CP level of the group 3 was 1.54 ng/mg protein. When fCP level of the group 3 and the group 1 were compared, it was found statistically significant (p < 0.0001) (Fig. 2). The level of f-CP was
4. Discussion Table 1 The demographic characteristics of participants in the study group. Groups
Group 1 Group 2 Group 3
Patients No.
34 31 23
Gender
Age
Male/Female n (%)
Means
Range
13 (38.2%)/21 (61.7%) 13 (41.9%)/18 (58.1%) 4 (17.3%)/19(82.6%)
31.7 38.9 34.6
4–80 1–80 18–59
D. fragilis was actually described by Jeeps and Dobell a hundred years ago as a non-pathogenic protozoan organism [6]. Although many publications have supported the pathogenic potential of D. fragilis for years, it has been reported to be the commensal an organism in some studies [3,10,25,26]. In most studies, it is reported that D. fragilis is associated with gastrointestinal symptoms and the symptoms disappear after treatment, and consequently D. fragilis could be pathogenic [1,26,27]. However, there is still continues controversy about the life 3
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
Fig. 3. The evaluation of fecal calprotectin levels by age groups. Age-related distribution of fecal calprotectin (ng/mg protein) in patient which in include three different groups (D. fragilis positive, D. fragilis negative and healthy control). Statistically of significance was defined as *p < 0.05, **p < 0.01.
cycle, mode of transmission and pathogenicity of this protozoan [7,8]. In our study, the most common gastrointestinal symptoms were found as diarrhea, abdominal pain and weight loss in the group 1, respectively (80.0%, 52.9% and 23.5%). Moreover, the absence of any pathogens other than D. fragilis in the group 1 to cause gastrointestinal symptoms has supported the pathogenicity of D. fragilis. In another study similar to our study, the most common gastrointestinal symptoms seen in most patients infected with D. fragilis were diarrhea (83.3%), abdominal pain (77.7%), and loose stools (72.2%), respectively [1]. Calprotectin is a 36-kDa calcium-and zinc-binding protein which is found from neutrophils, monocytes, and macrophages. This protein is composed of about 60% as dissolved in the cytosol of neutrophils. Calprotectin can be measured in various body fluids, such as plasma, urine, and cerebrospinal fluid, and in feces and tissue samples. The changes in levels of f-CP are clinically useful for gastroenterologists by measuring directly as a marker of inflammation of the mucous membrane [18,28]. The use of f-CP as a non-invasive stool marker to monitor inflammation have been discussed in several studies [29–31]. Currently, it is well-known that f-CP is the best available surrogate marker for mucosal inflammatory activity in IBD [32,33]. The resistance to degradation and stability in the stool for up to four days in room temperature are major advantages of calprotectin while evaluating the biological changes in diseases [34]. In our study, it was observed that f-CP concentration was approximately six times higher in the group 1 (33.40 ng/mg protein) compared to the cut-off value
Fig. 4. Receiver operating characteristic curve graph. Receiver Operating Characteristic (ROC) curve defining an optimal f-CP cut-off value in order to distinguish between the healthy control group and D. fragilis positive group. The area under the curve, sensitivity and specificity values are also shown, respectively (1.00 (95% confidence interval [CI], 0.8972 to 1.000; 100%, 95% CI, 89.72–100.0%; 95.65%, 95% CI, 78.05–99.89%).
Table 2 Gastrointestinal symptoms and calprotectin values of patients enrolled in the study. Symptomsa
Diarrhea Abdominal pain Weight loss Nausea Itching Fever Lack of appetite Vomiting
Group 1
Group 2 b
n: 34 (%)
f-CP
28 (80.0%) 18 (52.9%) 8 (23.5%) 7 (20.5%) 5 (14.7%) 5 (14.7%) 4 (11.7%) 3 (8.8%)
33.05 33.60 45.59 29.82 32.46 49.14 46.13 51.66
± ± ± ± ± ± ± ±
5.53 6.81 8.88 10.48 15.16 10.98 15.24 18.69
Abbreviations: f-CP = fecal calprotectin. * Statistically significant difference was defined as p < 0.05. a The same patient had presented with at least one or more gastrointestinal symptoms. b Fecal calprotectin values given as mean ± SE of the mean. 4
P value* b
n: 31 (%)
f-CP
21 (67.7%) 11 (35.4%) 11 (35.4%) 7 (22.5%) 7 (22.5%) 4 (12.9%) 11 (35.4%) 6 (19.3%)
15.61 16.26 16.56 16.47 18.13 17.71 16.43 17.60
± ± ± ± ± ± ± ±
1.08 1.53 1.48 2.31 1.96 1.58 1.56 2.14
0.007* 0.030* 0.016* 0.271 0.435 0.127 0.212 0.187
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
(5.045 μg/ml protein). Therefore, this group of patients showed an inflammation of the mucosa of the gastrointestinal tract. The f-CP value in feces of D. fragilis-infected experimental animals (69 ng/ml) was approximately two-fold higher than the control group (33 ng/ml) and indicative of the presence of intestinal inflammation in gastrointestinal mucosa [15]. In the first study of f-CP in humans, it was reported that there was no significant difference between the f-CP values of D. fragilis positive (40–55 μg/g) fecal samples and D. fragilis negative fecal samples (40–75 μg/g) [35]. In this study, it was the first study found to be statistically significant when the f-CP values of the group 1 (D. fragilis positive patient samples (33.40 ng/mg protein)) and the group 3 (the control group samples (1.54 ng/mg protein)) were compared. In this case, the f-CP value supports the pathogenicity of D. fragilis. There are a number of studies in recent advances in the relationship between bacterial, viral gastroenteritis and f-CP [36,37]. Although there were limited studies the association with between the intestinal symptoms caused by parasites and f-CP, the first time a significant relationship between the symptoms induced by D. fragilis and f-CP was investigated in the only animal model [15,16]. Our study measuring fCP levels in a group of patients with gastrointestinal symptoms, caused only by D. fragilis was the first study and in terms of supporting the pathogenicity of D. fragilis. When f-CP levels of gastrointestinal symptoms of patients with the group 1 and the group 2 were compared, it was found statistically significant in patients with diarrhea (33.05 ± 5.53, 15.61 ± 1.08; p = 0.007), abdominal pain (33.60 ± 6.81, 16.26 ± 1.53; p = 0.030) and weight loss (45.59 ± 8.88, 16.56 ± 1.48; p = 0.016), respectively. In the group 2 patients, f-CP levels were statistically significant when compared with the healthy control group (the group 3) because other etiologic agents causing gastrointestinal symptoms were not excluded. Some the reviews had reported that f-CP levels of patients with gastrointestinal disorders were higher than healthy controls [38]. With the migration of neutrophils into the lumen in the gut, it is thought that calprotectin is released and triggers inflammation by induction of proinflammatory chemokines [20]. Therefore, the intestinal mucosal epithelial cells are damaged and the protein is secreted and thus causes diarrhea together with increased intestinal permeability and chlorine ion [16,34]. The group 1 (only D. fragilis positive patients) has been found to have gastrointestinal symptoms and high f-CP concentration, and in this case has supported a potential pathogenicity of D. fragilis. There was no significant difference in comparing f-CP levels in the group 1 and the group 2 without age groups. However, when divided into three groups according to age, f-CP levels in between the group 1 and the group 2 were found to be significant in 20–40 years old and > 40 years old. However, some studies support the idea that f-CP should not be used as a marker of inflammation because of its high expression up to the age of 4 years [39,40]. The reason for this may be raised early in life to compensate for an immature and developing immune system in newborns [40]. Increased intestinal permeability, bacterial microbiota formation and the giving of diet may also affect fCP levels in healthy newborns [41]. In this study, f-CP levels of the group 1 were distinguished from cutoff values according to ROC analysis. However, it cannot be distinguished from the group 2. This is given to the exclusion of other pathogens that will cause gastrointestinal symptoms. In the future, its performance could be further improved by increasing the number of patients with D. fragilis positive, negative and healthy control groups. Although the f-CP level is not a fully indicative indicator of the presence of D. fragilis, it shows that it is a potential pathogen with real-time PCR, which is the gold standard. The lesions that D. fragilis had previously formed in the large intestine were defined as colitis [15,16]. These pathogenic changes may be because of the secretion of calprotectin (a neutrophil protein) and its existence in the stool. This is determinative of neutrophilic infiltration into the gut linked inflammatory processes [16]. D. fragilis is among the possible etiological agents for patients suffering from intestinal
disorders including Inflammatory Bowel Syndrome (IBS) [1]. In addition to its antimicrobial activity, calprotectin has been shown to regulate innate immune response by activating toll-like receptor 4 [16,42]. High levels of calprotectin have also been shown in some parasitic infections such as giardiasis and intestinal schistosomiasis [21,22]. In our study, significant increases in calprotectin levels were observed in patients with only D. fragilis positive as an indicative of intestinal inflammation. The debates about the pathogenicity of D. fragilis are still ongoing. Several potential pathogenicity has been described in D. fragilis as amoebapore-like proteins and putative immunomodulatory proteins [43]. The plentiful detected virulence factors are members of the cathepsin L-like cysteine protease family. Plenty cysteine proteases have been identified in D. fragilis [5,8,43]. On the other hand, f-CP is a stimulates immunomodulation and antibacterial effects of neutrophil [17]. It has also important roles in the trans epithelial migration and accumulation at mucosal surfaces of neutrophil. The presence of calprotectin levels in feces is a good predictor of inflammation. In experimental studies, D. fragilis related colon inflammation was observed [15,16]. In our study; higher levels of f-CP found in D. fragilis positive patients are of great importance to be a research that supports the pathogenicity of the specified parasite and to be searched for the first time. In conclusion, the findings of this study contribute to the ongoing debate about the pathogenicity of D. fragilis. The f-CP level can be used as a good indicator of gastrointestinal systems inflammation. In our study, the potential pathogenicity of D. fragilis is associated with increased f-CP concentrations with parasite detection in the fecal samples and therefore we assume that the parasite is not only a harmless commensal. This is the first study to support the pathogenicity of D. fragilis by looking at the f-CP concentration in human fecal samples of D. fragilis positive. Further studies are needed to determine the pathogenicity of D. fragilis by looking at the f-CP level by forming a larger sample group than the D. fragilis positive patient group. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This study was partly supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant number: 13-TIP-092). References [1] D. Stark, J. Barratt, T. Roberts, D. Marriott, J. Harkness, J. Ellis, A review of the clinical presentation of dientamoebiasis, Am. J. Trop. Med. Hyg. 82 (2010) 614–619. [2] J. Ogren, S. Van Nguyen, M.K. Nguyen, J. Dimberg, A. Matussek, Prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp in Da Nang, Vietnam, detected by a multiplex real-time PCR, APMIS 124 (2016) 529–533. [3] G.A. Holtman, J.J. Kranenberg, M.H. Blanker, A. Ott, Y. Lisman-van Leeuwen, M.Y. Berger, Dientamoeba fragilis colonization is not associated with gastrointestinal symptoms in children at primary care level, Fam. Pract. 34 (2017) 25–29. [4] S.M. Caccio, Molecular epidemiology of Dientamoeba fragilis, Acta Trop. 184 (2017) 73–77, https://doi.org/10.1016/j.actatropica.2017.06.029. [5] J.L. Barratt, J. Harkness, D. Marriott, J.T. Ellis, D. Stark, A review of Dientamoeba fragilis carriage in humans: several reasons why this organism should be considered in the diagnosis of gastrointestinal illness, Gut Microb. 2 (2011) 3–12. [6] M.W. Jepps, C. Dobell, Dientamoeba fragilis n. g., n. sp., a new Intestinal Amoeba from Man, Parasitology 10 (1918) 352–367. [7] L.S. Garcia, Dientamoeba fragilis, one of the neglected intestinal Protozoa, J. Clin. Microbiol. 54 (2016) 2243–2250. [8] D. Stark, J. Barratt, D. Chan, J.T. Ellis, Dientamoeba fragilis, the neglected trichomonad of the human bowel, Clin. Microbiol. Rev. 29 (2016) 553–580. [9] J.L. Barratt, J. Harkness, D. Marriott, J.T. Ellis, D. Stark, The ambiguous life of Dientamoeba fragilis: the need to investigate current hypotheses on transmission, Parasitology 138 (2011) 557–572.
5
Microbial Pathogenesis 139 (2020) 103868
M. Aykur, et al.
819–823. [27] H.Z. Rayan, O.A. Ismail, E.K. El Gayar, Prevalence and clinical features of Dientamoeba fragilis infections in patients suspected to have intestinal parasitic infection, J. Egypt. Soc. Parasitol. 37 (2007) 599–608. [28] A. Poullis, R. Foster, M.A. Mendall, M.K. Fagerhol, Emerging role of calprotectin in gastroenterology, J. Gastroenterol. Hepatol. 18 (2003) 756–762. [29] T. Sipponen, E. Savilahti, K.L. Kolho, H. Nuutinen, U. Turunen, M. Farkkila, Crohn's disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn's disease activity index and endoscopic findings, Inflamm. Bowel Dis. 14 (2008) 40–46. [30] P. Rutgeerts, G. Van Assche, W.J. Sandborn, D.C. Wolf, K. Geboes, J.F. Colombel, et al., Adalimumab induces and maintains mucosal healing in patients with crohn's disease: data from the EXTEND trial, Gastroenterology 142 (2012) 1102. [31] C. Caimmi, E. Bertoldo, A. Venturini, P. Caramaschi, L. Frulloni, R. Ciccocioppo, et al., Relationship between increased fecal calprotectin levels and interstitial lung disease in systemic sclerosis, J. Rheumatol. (2018) 171445 jrheum. [32] J.D. Lewis, The utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease, Gastroenterology 140 (2011) 1817-U160. [33] S.C. Wei, Could fecal calprotectin enter mainstream use for diagnosing and monitoring inflammatory bowel disease? Intest. Res. 14 (2016) 293–294. [34] A. Voganatsi, A. Panyutich, K.T. Miyasaki, R.K. Murthy, Mechanism of extracellular release of human neutrophil calprotectin complex, J. Leukoc. Biol. 70 (2001) 130–134. [35] M.R. Brands, E. Van de Vijver, S.M. Haisma, A. Heida, P.F. van Rheenen, No association between abdominal pain and Dientamoeba in Dutch and Belgian children, Arch. Dis. Child. 104 (7) (2019) 686–689, https://doi.org/10.1136/archdischild2018-316383. [36] C.C. Chen, J.L. Huang, C.J. Chang, M.S. Kong, Fecal calprotectin as a correlative marker in clinical severity of infectious diarrhea and usefulness in evaluating bacterial or viral pathogens in children, J. Pediatr. Gastroenterol. Nutr. 55 (2012) 541–547. [37] M. Duman, P. Gencpinar, M. Bicmen, N. Arslan, O. Ozden, O. Uzum, et al., Fecal calprotectin: can be used to distinguish between bacterial and viral gastroenteritis in children? Am. J. Emerg. Med. 33 (2015) 1436–1439. [38] I.D. Kostakis, K.G. Cholidou, A.G. Vaiopoulos, I.S. Vlachos, D. Perrea, G. Vaos, Fecal calprotectin in pediatric inflammatory bowel disease: a systematic review, Dig. Dis. Sci. 58 (2013) 309–319. [39] M. Garg, S.T. Leach, M.J. Coffey, T. Katz, R. Strachan, T. Pang, et al., Age-dependent variation of fecal calprotectin in cystic fibrosis and healthy children, J. Cyst. Fibros. 16 (2017) 631–636. [40] M. Garg, S.T. Leach, A.S. Day, C.Y. Ooi, Fecal calprotectin concentrations in young children with cystic fibrosis: authors response, J. Cyst. Fibros. 17 (2018) e10–e11. [41] E. Olafsdottir, L. Aksnes, G. Fluge, A. Berstad, Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children, Acta Paediatr. 91 (2002) 45–50. [42] T. Vogl, K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M.A. van Zoelen, et al., Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock, Nat. Med. 13 (2007) 1042–1049. [43] J.L.N. Barratt, M. Cao, D.J. Stark, J.T. Ellis, The transcriptome sequence of Dientamoeba fragilis offers new biological insights on its metabolism, kinome, degradome and potential mechanisms of pathogenicity, Protist 166 (2015) 389–408.
[10] P. Jokelainen, B. Hebbelstrup Jensen, B.U. Andreassen, A.M. Petersen, D. Roser, K.A. Krogfelt, et al., Dientamoeba fragilis, a commensal in children in Danish day care centers, J. Clin. Microbiol. 55 (2017) 1707–1713. [11] R. Enserink, R. Scholts, P. Bruijning-Verhagen, E. Duizer, H. Vennema, R. de Boer, et al., High detection rates of enteropathogens in asymptomatic children attending day care, PLoS One 9 (2014) e89496. [12] M.J. De Jong, J.J. Korterink, M.A. Benninga, M. Hilbink, J. Widdershoven, J.M. Deckers-Kocken, Dientamoeba fragilis and chronic abdominal pain in children: a case–control study, Arch. Dis. Child. 99 (12) (2014) 1109–1113, https://doi.org/ 10.1136/archdischild-2014-305942 25053737. [13] O. Kurt, N. Girginkardesler, I.C. Balcioglu, A. Ozbilgin, U.Z. Ok, A comparison of metronidazole and single-dose ornidazole for the treatment of dientamoebiasis, Clin. Microbiol. Infect. 14 (2008) 601–604. [14] N. Girginkardesler, S. Coskun, I. Cuneyt Balcioglu, P. Ertan, U.Z. Ok, Dientamoeba fragilis, a neglected cause of diarrhea, successfully treated with secnidazole, Clin. Microbiol. Infect. 9 (2003) 110–113. [15] V.S. Munasinghe, N.G. Vella, J.T. Ellis, P.A. Windsor, D. Stark, Cyst formation and faecal-oral transmission of Dientamoeba fragilis–the missing link in the life cycle of an emerging pathogen, Int. J. Parasitol. 43 (2013) 879–883. [16] E.K. El-Gayar, A.B. Mokhtar, W.A. Hassan, Study of the pathogenic potential of Dientamoeba fragilis in experimentally infected mice, Parasite Epidemiol. Control 1 (2016) 136–143. [17] F. Costa, M. Mumolo, Bellini Ma, M. Romano, L. Ceccarelli, P. Arpe, et al., Role of faecal calprotectin as non-invasive marker of intestinal inflammation, Dig. Liver Dis. 35 (2003) 642–647. [18] C.W. McMahon, R. Chhabra, The role of fecal calprotectin in investigating digestive disorders, J. Lab. Precis. Med. 3 (2018). [19] H. Manceau, V. Chicha-Cattoir, H. Puy, K. Peoc'h, Fecal calprotectin in inflammatory bowel diseases: update and perspectives, Clin. Chem. Lab. Med. 55 (2017) 474–483. [20] Y.A. Lam, S.M. Warouw, A.M. Wahani, J.I. Manoppo, P.M. Salendu, Correlation between gut pathogens and fecal calprotectin levels in young children with acute diarrhea, Paediatr. Indones. 54 (2014) 193–197. [21] K. Hanevik, T. Hausken, M.H. Morken, E.A. Strand, K. Morch, P. Coll, et al., Persisting symptoms and duodenal inflammation related to Giardia duodenalis infection, J. Infect. 55 (2007) 524–530. [22] A.L. Bustinduy, J.C. Sousa-Figueiredo, M. Adriko, M. Betson, A. Fenwick, N. Kabatereine, et al., Fecal occult blood and fecal calprotectin as point-of-care markers of intestinal morbidity in Ugandan children with Schistosoma mansoni infection, PLoS Neglected Trop. Dis. 7 (2013). [23] M. Aykur, C. Caliskan Kurt, D. Dirim Erdogan, C. Biray Avci, R. Vardar, S. Aydemir, et al., Investigation of Dientamoeba fragilis Prevalence and Evaluation of Sociodemographic and Clinical Features in Patients with Gastrointestinal Symptoms, Acta Parasitol 64 (2019) 162–170. [24] K.M. Goodrich, G. Fundaro, L.E. Griffin, M.W. Hulver, M.A. Ponder, A.P. Neilson, Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occludin and reduces fecal calprotectin: a secondary analysis of healthy Wistar Furth rats, Nutr. Res. (N.Y.) 32 (2012) 787–794. [25] M.J. Spencer, M.R. Chapin, L.S. Garcia, Dientamoeba fragilis: a gastrointestinal protozoan infection in adults, Am. J. Gastroenterol. 77 (1982). [26] G. Banik, J. Barratt, D. Marriott, J. Harkness, J. Ellis, D. Stark, A case-controlled study of Dientamoeba fragilis infections in children, Parasitology 138 (2011)
6