Investigation of intestinal parasites by conventional and molecular methods in children with gastrointestinal system complaints

Intestinal parasitic infections are a global health problem that causes morbidity and mortality, especially in children living in rural areas. In this study, stool samples of pediatric patients with gastrointestinal complaints were examined by conventional and molecular methods to determine the prevalence of intestinal parasites. A total of 100 pediatric patients with gastrointestinal complaints and 50 healthy children were included in the study. Stool samples were collected from each child and examined by direct microscopic examination (native-Lugol method), formol-ethyl acetate concentration technique, Kinyoun’s acid-fast staining, and Wheatley trichrome staining methods. Real-time PCR was used for the detection of Blastocystis spp. and D. fragilis in the stool samples. Sanger sequencing was used to identify Blastocystis spp. subtypes. One or more intestinal parasites were found in 12% (n = 100) of the patient group and 1% (n = 50) of the control group using conventional techniques. By using real-time PCR, Blastocystis spp. was discovered in 14% (14/100) of the patient group and 8% (4/50) of the control group. There was no significant difference in the frequency of Blastocystis spp. between the two groups. The most prevalent Blastocystis subtype was ST1 and the most frequent allele was a2 among the samples successfully amplified and sequenced. D. fragilis was detected in 17% (17/100) of the patient group and 8% (4/50) of the control group by real-time PCR. The prevalence of D. fragilis was not significantly different between the patient and control groups, as well. Blastocystis spp. and D. fragilis were found in high prevalence in pediatric patients with gastrointestinal complaints in this study. Although the role of these protists as a pathogen in humans is still controversial, it is supposed to the presence of the parasites are associated with gastrointestinal disorders such as diarrhea, abdominal pain, nausea, and vomiting. More case-control studies are needed to understand the pathogenic or commensal role of these parasites on the intestinal microbiota, especially in both patients with gastrointestinal disorders and healthy individuals.


Introduction
Acute gastrointestinal infections, which are an important cause of morbidity and mortality, especially in the pediatric age group, constitute a global public health problem, especially in developing and underdeveloped countries. Infectious microorganisms such as bacterial, viral, and parasitic agents can cause gastrointestinal disorders such as gastroenteritis (Riddle et al. 2018). Acute gastroenteritis leads to millions of deaths each year, particularly in children under 5 years, worldwide. It is also a common reason for admission to emergency departments in hospitals. Viral agents (Rotavirus, Norovirus, enteric Adenovirus, etc.) are the leading causes of acute gastroenteritis. Bacterial pathogens (Campylobacter jejuni, Salmonella spp., Clostridium difficile, Enterotoxigenic Escherichia coli, etc.), protozoa (Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, etc.) and helminths (Strongyloides stercoralis, etc.) can also be the cause of acute gastrointestinal infections (Elliott 2007;Florez et al. 2020). Rapid and accurate detection of the causative agent of gastrointestinal disorders has great importance in terms of initiating appropriate treatment immediately, preventing unnecessary drug use, Section Editor: Nawal Hijjawi * Filiz Demirel dr.filiz.demirel@gmail.com Extended author information available on the last page of the article 1 3 shortening hospital stays, and reducing morbidity and mortality (Tavares et al. 2011).
Intestinal parasitic infections (IPI) continue to be a common health problem in the pediatric population, especially in developing countries. Intestinal parasites may present with gastrointestinal symptoms such as abdominal pain, flatulence, nausea and vomiting, loss of appetite, weight loss, and diarrhea, as well as asymptomatic infections. Diagnosis and treatment of IPI are of great importance because these infections, when undiagnosed for a prolonged time, may lead to malnutrition, impairment in physical and neurological development, and loss of ability to attend school in children (Sandoval et al. 2015;Demirel and Dinç 2022;Harhay et al. 2010).
Blastocystis spp., G. intestinalis, and Cryptosporidium spp. are common intestinal parasites affecting pediatric patients. In addition to gastrointestinal clinical manifestations, subclinical infections of these parasites are also common during childhood (Hernández-Castro et al. 2023). Although the prevalence of Blastocystis spp. is reported more than 1 billion worldwide, the pathogenic significance of the parasite remains controversial (Matovelle et al. 2022). Currently, based on phylogenetic analysis of the small subunit (SSU) rRNA genes, there are at least 28 subtypes (STs) of Blastocystis isolated from mammals and birds. While ST1-10, 12, 14, 16, and 26 can be found in humans, the most common Blastocystis subtypes are ST1-4 (Stensvold 2013a, b;Clark et al. 2013;Stensvold et al. 2020;Zhou et al. 2022).
Dientamoeba fragilis is a neglected intestinal protist commonly found in humans worldwide. Although many studies show that D. fragilis is associated with gastrointestinal disorders such as abdominal pain and diarrhea, the pathogenicity and the life cycle of the parasite are still controversial. It is reported that D. fragilis tends to be more common in pediatric patients (Garcia 2016). In recent years, the detection of the parasite by molecular methods has replaced microscopic examinations, especially in developed countries. According to the reports, D. fragilis prevalence ranges from 0-62% depending on region, population, and the diagnostic method of the studies (Garcia 2016;Wong et al. 2018;van Gestel et al. 2019).
In this study, it was aimed to determine the frequency of intestinal parasites in paediatric patients with gastrointestinal complaints, to detect Blastocystis spp. and D. fragilis by molecular methods and to identify subtypes of Blastocystis spp. using DNA sequencing.

Ethical approval
The protocol of the study was reviewed and approved by Ankara Training and Research Hospital Non-Interventional Ethics Board/Committee (decision number: 87/2019). Written informed consent was obtained from both children and their parents.

Study population
A total of 100 paediatric patients who admitted to the Ankara Training and Research Hospital Paediatric Emergency Outpatient Clinic, Türkiye, because of gastrointestinal complaints such as nausea-vomiting, abdominal pain, diarrhea, constipation, abdominal flatulence, etc., between December 2019 and March 2020, were included in the study. As the control group, 50 children who applied to the hospital for general control or vaccination and did not have any gastrointestinal disorders were included in the study.
Inclusion criteria for the patient group were being younger than 18 years of age, having one or more gastrointestinal symptoms, not using any medication at least one month before sampling, not being immunosuppressive, and consenting to participate in the study. Exclusion criteria for the patient group were being older than 18 years of age, using any medication in the last month, being immunosuppressive, and refusing to participate in the study.
Inclusion criteria for the control group were being younger than 18 years of age, not having any gastrointestinal symptoms, not using any medication at least one month before sampling, not being immunosuppressive, and consenting to participate in the study. Exclusion criteria for the control group were being older than 18 years of age, having gastrointestinal symptoms, using any medication in the last month, being immunosuppressive, and refusing to participate in the study.

Collection of samples and microscopic examinations
Fresh stool samples were collected from each child and transferred to the laboratory immediately. All stool samples were first examined macroscopically for consistency, presence of blood, mucus, and visible adult parasitic forms. After macroscopic examination, stool samples were examined by direct microscopic examination (saline/iodine, wet mount preparation) using 100 × and 400 × magnifications of a light microscope (Mengist et al. 2018). The formalinethyl acetate concentration method was performed by using a commercial concentrator tube (Parasep® Fecal Parasite Concentrators, Apacor, USA) as previously described and the sediments of each centrifuged sample were examined microscopically by saline/iodine method for the detection of helminth eggs and protozoa cysts (Zeeshan et al. 2011). Additionally, all stool samples were stained using Wheatley's trichrome and Kinyoun's acid-fast staining techniques and then examined with an 100 × immersion objective, as previously described (Mokobi 2022;Mathison 2022). For molecular investigations, each faecal sample was stored frozen at -20 °C until DNA extraction.

Detection of adenovirus and rotavirus
To assess the presence of viral agents, a commercial immunochromatographic card test (Rotavirus and Adenovirus Combo Rapid Test Cassette, ExacTest, General Diagnostica, USA) that detects rotavirus and adenovirus was used.

Bacterial stool cultures
All stool samples were inoculated on MacConkey agar and Salmonella-Shigella (SS) agar and incubated overnight at 37 °C for the identification of Salmonella spp. and Shigella spp. Suspected colonies were identified by conventional techniques, as previously described (Humphries and Linscott 2015).

DNA extraction
A commercial DNA extraction kit (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) was used by the manufacturer's instructions to isolate genomic DNA from frozen stool samples. DNA extraction was performed every two weeks. Extracted and purified DNA samples were stored at − 20 °C until further molecular analysis.

Molecular detection of Blastocystis spp.
For the molecular detection of Blastocystis spp., real-time PCR method targeting a 118 bp fragment of the small subunit ribosomal RNA (ssu rRNA) gene of Blastocystis spp. was performed. The list of primers and probes used for Blastocystis spp. real-time PCR is given in Table 1. The amplification reaction was established in a Rotor-Gene 6000 real-time cycler (Rotor-Gene Q, Qiagen, Germany). All PCR reactions were carried out as previously indicated (Stensvold and Clark 2016).

Molecular characterization of Blastocystis spp.
Analysis of Blastocystis subtypes in samples with positive Blastocystis-qPCR results was achieved by barcoding PCR targeting a 600 bp segment of the Blastocystis ssu-rDNA as previously described (Scicluna et al. 2006;Stensvold 2013a, b) and Sanger sequencing method. Amplification reactions (25 μl) included 5 μl of template DNA and 0.5 μM of the pan-Blastocystis, barcode primer set RD5/BhRDr. Subtype confirmation and allele identification were performed on Blastocystis sequences submitted to the Blastocystis 18S database (http:// pubml st. org/ blast ocyst is/) (Sarzhanov et al. 2021). Barcoding primers used in the study are listed in Table 1.

Molecular detection of Dientamoeba fragilis
For the molecular detection of D. fragilis in stool samples, real-time PCR method targeting a 78-bp fragment of small subunit ribosomal RNA (ssu rRNA) was achieved (Stark et al. 2006). The list of primers and probes used for D. fragilis real-time PCR is given in Table 1.

Statistical analysis
The Chi-square test was used to compare differences in the frequency of protists among infected individuals according to sociodemographic variables, presence/absence of clinical manifestations, and parasite´s assemblages. A p value < 0.05 was considered evidence of statistical significance. Odds  (2006) ratios (OR) and 95% confidence intervals (CI) were calculated to assess the potential association between the occurrence of symptomatic infection and the protists causing the infection. Statistical analyses were carried out using the OpenEpi free software (https:// www. opene pi. com).

Results
The distribution of children included in the study by age and gender was given in Table 2. There was no statistically significant difference between the groups in terms of age and gender (p > 0.05). According to the macroscopic examination, 67% of the stool samples from the patient group were watery-amorphous, while all of the stools from the control group were soft consistency. Intestinal parasites detected by conventional methods were Blastocystis spp. (8%, n = 8), D. fragilis (2%, n = 2), Giardia intestinalis (2%, n = 2), Cryptosporidium spp. (1%, n = 1) and Hymenolepis nana (1%, n = 1). Using the immunochromatographic rotavirus and adenovirus quick diagnostic test, three children in the patient group tested positive for rotavirus, two for adenovirus, and one for both, while the control group did not show any positivity. Additionally, Salmonella spp. was identified in the stool culture of only one child in the patient group.
Blastocystis spp. was identified in 14% (14/100) of the patient group and 8% (4/50) of the control group by realtime PCR. Only three of the patients with positive Blastocystis real-time PCR were also positive for Blastocystis using conventional methods. Distribution of the patients whose stool samples revealed Blastocystis spp. using both conventional and molecular methods in terms of age, gender, and symptoms is given in Table 3. Age, gender, and symptoms did not statistically differ between the patient and control groups ( Table 3). The frequency of Blastocystis spp. between the two groups was not significantly different, as well.
Out of 14 samples in the patient group that tested positive for Blastocystis spp. using real-time PCR, 57.1% (8/14) were successfully amplified and sequenced at the ssu rRNA (barcode region). Out of four samples in the control group that tested positive for Blastocystis spp. using real-time PCR, 75% (3/4) were successfully amplified and sequenced, as well. ST1 was identified in 42.9% (6/14) of the patients, and 25% (1/4) of the control group. ST3 was identified in 14.2% (2/14) of the patients, and 50% (2/4) of the control group. According to the allelic discrimination, the most frequent allele was a2 (4/14), followed by a34 (2/14), a4 (1/14), and a88 (1/14) in the patient group. In the control group, a4 (1/4), a34 (1/4), and a36 (1/4) were identified in the subtypes mentioned. The distribution of the subtype and allele did not statistically differ between the patient and control groups (p > 0.05). Blastocystis subtypes and alleles distribution among the patients and healthy children are given in Table 4. Dientamoeba fragilis was detected in 17% (17/100) of the patient group and 8% (4/50) of the control group by real-time PCR. Two of the patients with positive D. fragilis real-time PCR were positive for D. fragilis by conventional methods. Gender and symptoms did not statistically differ between the patient and control groups ( Table 3). The frequency of D. fragilis between the patient and control groups was not significantly different, as well (p > 0.05). In terms of age groups, D. fragilis positivity in control patients was significantly higher only in the 13-18 age group (χ2 = 7.9, p = 0.02).
The diagnostic performance of conventional methods used in the diagnosis of Blastocystis spp. and D. fragilis were compared with real-time PCR (Table 5). Both Blastocystis spp. and D. fragilis were detected more frequently by real-time PCR than by saline/iodine, formalin-ethyl acetate concentration technique (FECT), and trichrome staining methods. When conventional methods were compared to Blastocystis sp. qPCR, the kappa value was found to be very low (k = 0.32, fair agreement).

Discussion
In the current study, intestinal parasites were investigated in children with and without gastrointestinal complaints in a territory hospital in Ankara, the capital city of Türkiye, using conventional and molecular methods. Prevalence of intestinal parasites among pediatric patients with gastrointestinal complaints was 12% by conventional methods. In Türkiye, the prevalence of intestinal parasites varies depending on the methods used, the population included in the study, and the region where the study was conducted. In a six-year retrospective study, İnal et al. reported an overall prevalence of IPs of 7.5%, in Ankara (İnal et al. 2022). In another retrospective study, IPs were detected at 3.1% and 10.2% in native and refugee patients, respectively (Demirel and Dinç 2022). Paediatric patients with chronic spontaneous urticaria (22.4%) and immunocompromised children with chronic diarrhea (20%) had higher IP prevalence rates in the same area, while immunocompromised adults had a prevalence rate (12%) similar to the general population (Vezir et al. 2019;Maçin et al. 2016;Kaya et al. 2021).
The majority of the children included in the current study had diarrhea and 67% of the stool samples were wateryamorphous. Acute infectious gastroenteritis (AGE) is one of the leading causes of death in children worldwide, especially those under the age of five. Viruses, bacteria, and parasites are infectious agents in children that cause AGE (Hasan et al. 2021). In our study, adenovirus and rotavirus infections were detected at lower rates (4% and 3%, respectively) than expected, and Salmonella spp. was identified  only in one patient, while IP was detected at a higher rate than viruses and bacteria. Bacterial and protozoal gastroenteritis can mimic viral gastroenteritis symptoms, but they often require a different treatment approach and may have a higher morbidity potential.The differential diagnosis of gastroenteritis, particularly in children, is critical for giving proper treatment (Stuempfig and Seroy 2022).
In this study, the most common IP detected by conventional methods in children was Blastocystis spp. (8%). By real-time PCR, the overall prevalence of Blastocystis spp. was found to be 14% (14/100) of the patient group and 8% (4/50) of the control group. In a previous epidemiological study that assessed the occurrence of enteric parasites in patients admitted to a hospital in Ankara, Blastocystis spp. was detected in 11.1% by conventional methods and in 16.4% by real-time PCR (Sarzhanov et al. 2021). In a meta-analysis study that examined the prevalence of IPI among children in Europe between 2015 and 2021, the overall IP prevalence rate was 5.9% and Blastocystis spp. was the most commonly detected parasite with a prevalence rate of 10.7% in children residing in European countries (Kantzanou et al. 2021). These rates are consistent with the data obtained in our study. Although the pathogenic role of Blastocystis spp. is still controversial, several recent studies have identified Blastocystis as an emerging pathogen. Certain populations, such as pediatric patients suffering from gastrointestinal diseases, are reported to be vulnerable to Blastocystis-related disorders (Kumarasamy et al. 2018). However, in the current study, the frequency of Blastocystis spp. between pediatric patients with gastrointestinal symptoms and healthy children was not significantly different. In a study conducted in Spain, Blastocystis spp. was detected in 4.8% of stool samples from children by microscopy and 28.1% by PCR. There was no difference in Blastocystis prevalence between the control and patient groups (Mormeneo Bayo et al. 2022). In contrast, some studies reported a positive correlation between Blastocystis and chronic abdominal pain (Légeret et al. 2020) and diarrhea (Perea et al. 2020) in children. It is obvious that more studies are needed to understand the relationship between Blastocystis with clinical symptoms in children.
According to earlier investigations of the molecular characterization of Blastocystis, the most common Blastocystis STs circulating in different Turkish human groups were ST1-4 (Dogan et al. 2017;Sankur et al. 2017;Malatyalı et al. 2019;Sarzhanov et al. 2021). In the current study, ST1 was noticeably higher in the children, although the distribution of the subtype did not statistically differ between the patient and control groups. In a study evaluating Blastocystis subtypes in children with functional abdominal pain and celiac disease in Türkiye, there was no difference in Blastocystis prevalence between study groups. ST2, ST3, and ST1 were the most common subtypes, respectively (Güreser et al. 2022). In a study that determined the prevalence and subtypes distribution of Blastocystis among school-aged children in Thailand, the most common subtypes were ST3, followed by ST1 and ST2 (Abu et al. 2022). In Spain, Blastocystis spp. was also reported as common in toddlers attending day-care centers. In addition, contrary to our research, ST2 and ST4 were described as the most prevalent subtypes in these children (Hernández-Castro et al. 2023).
Dientamoeba fragilis is another protozoan parasite with controversial pathogenicity that is frequently found in the human intestine. According to reports, the prevalence of D. fragilis varies with region, population, and the type of detection method utilized, from 0 to 62%. PCR has been reported as the best diagnostic test due to its high sensitivity and specificity (van Gestel et al. 2019). In the current study, D. fragilis was detected in 17% of the patient group and 8% of the control group using real-time PCR, while it was detected in only 2% of the patients using conventional methods. Although D. fragilis was detected more frequently in children with gastrointestinal complaints, the difference was not statistically significant. In a recent study in Türkiye, the overall prevalence of D. fragilis was 11.9% in patients with diarrhea (Sarzhanov et al. 2021). In another study held in Türkiye, D. fragilis was detected in 12.04% of the patients by real-time PCR, and diarrhea was found statistically significant in patients with D. fragilis (Aykur et al. 2019). In some other reports, there was no association found between abdominal pain and D. fragilis in children (Brands et al. 2019;de Jong et al. 2014). In another study, D. fragilis prevalence was detected in 10.3% of asymptomatic children attending day care centers, and higher frequencies were encountered among children aged four to six years. In our study, D. fragilis positivity was significantly higher in the 13-18 age group in control patients (Oliveira-Arbex et al. 2021).
The study's limitations include its limited sample size, its inability to raise the number of healthy controls, and the fact that it only took place in one center. The study's strength is that it examined the prevalence of D.fragilis in children, did analyses of Blastocystis spp. prevalence, subtype, and alleles, and compared the results to those of healthy kids.
In conclusion, our study contributes important knowledge on the prevalence of Blastocystis spp. and D. fragilis in children for molecular epidemiology. Data reported here show that real-time PCR-based approaches have higher diagnostic sensitivity than conventional microscopy, especially for the detection of less common intestinal parasites. Further multicenter studies with a larger sample size are required to better understand the pathogenic or commensal roles of these parasites based on intestinal microbiome in patients with gastrointestinal disorders and healthy individuals.