Detection and molecular identification of Blastocystis isolates from humans and cattle in northern Egypt

Abstract

Blastocystis is one of the less well-understood waterborne protozoa of medical and veterinary importance. The present study aimed to detect, identify, and subtype Blastocystis in cattle and humans in northern Egypt and to investigate the potential for zoonotic transmission. Fecal samples were collected from 136 humans (68 in contact with animals and 68 no animal contact) and 190 cattle from a rural area in Kafr El-Sheikh Province, Egypt. All samples were microscopically examined by direct wet mount technique and cultured in Jones’ medium. Genomic DNA was extracted from positive cultures. A region of the Blastocystis small subunit ribosomal ribonucleic acid (SSU rRNA) gene was amplified using PCR. Blastocystis subtypes were identified by sequencing of the amplified SSU rRNA gene. The relatedness of sequence types obtained in this study and others submitted in GenBank was compared according to their genetic relatedness. Out of 136 human and 190 cattle samples, Blastocystis was detected in 53 (38.9%) and 37 (19.4%), respectively using either culture or direct wet mount. Blastocystis SSU rRNA gene was amplified in 55 samples (32 humans and 23 animals). Six subtypes were identified (STs 1–3 in humans and ST4, ST10 and ST14 in cattle) and were distinguishable with inter (up to 0.075) and intra (up to 0.024) subtype variations. A close phylogenetic relationship between human and animal isolated STs 1–4 was demonstrated. To the best of our knowledge, this is the first study to investigate the the potentiality for zoonotic transmission of certain Blastocystis subtypes in northern Egypt.

Introduction

Blastocystis is an intestinal parasite infecting humans and animals (Cian et al. 2017; Stensvold and Clark 2016). It is classified as a member of Stramenopiles (Silberman et al. 1996). The host specificity, genetic diversity, and ability of Blastocystis to cause disease remain not fully explained (Clark et al. 2013; Tan 2008). Worldwide, Blastocystis was detected in different animal hosts including domestic, pet, and wild animals (Cian et al. 2017; Ramirez et al. 2014). In Egypt, rates as low as 19.1% and as high as 67.4% were reported in humans (Eassa et al. 2016; El-Badry et al. 2018), while the prevalence in cattle was 72.2% (Mokhtar and Youssef 2018). In a previous study conducted in Senegal, Blastocystis spp. had the highest prevalence (100%) ever recovered worldwide in symptomatic individuals presenting with various gastrointestinal disorders or asymptomatic children (El Safadi et al. 2014).

Human to human transmission via the fecal-oral route is the principal route of acquiring the infection. However, the probability of zoonotic transmission of Blastocystis is supported by its high prevalence among animal handlers indicating that animals may represent a significant source for human infection (Salim et al. 1999) although this route of transmission should be investigated further (Paulos et al. 2018).

Blastocystis isolated from animals and humans were morphologically similar by light microscopic examination (Abe et al. 2002). In vitro culture is a simple sensitive technique used for the diagnosis of Blastocystis (Stensvold and Clark 2016). Furthermore, the culture isolated organisms can yield a large quantity of DNA that is suitable for molecular studies to differentiate between Blastocystis subtypes (STs) (Clark et al. 2013). Identification of Blastoctstis STs is performed either by using diagnostic subtype-specific sequence-tagged-site (STS) primers or by sequencing of small-subunit rRNA (SSU-rRNA) gene region. However, the former method targets only STs 1–7 with some false-negative results due to genetic variation in the STS loci. For SSU-rRNA sequencing, the barcode region is a valid representation for the whole gene and, additionally, the GenBank and the Blastocystis Subtype (18S) and Sequence Typing (MLST) Database contain numerous sequences for it (Stensvold 2012). The genus Blastocystis involves 26 STs with some overlap in STs isolated from humans and animals (Maloney et al. 2019). The first four Blastocystis STs are mainly recovered from humans and occasionally identified in animals (Stensvold and Clark 2016). ST5, ST6, ST7, and ST8 are principally recovered from animals and rarely identified in humans, ST9 is restricted to humans while STs with higher numbers are mainly identified in non-human hosts (Cian et al. 2017; Clark et al. 2013; Stensvold and Clark 2016). Recently, several subtypes (ST-3, ST-4, ST-5, ST-10, ST-14, ST-17, ST-21, ST-23 to ST-26) were identified in calves (Maloney et al. 2019). The role of cattle in the transmission of Blastocystis to humans remains unclear. In China, the possibility of transmission of Blastocystis subtypes ST4 and ST5 from cattle to humans was demonstrated (Zhu et al. 2017). However, in Lebanon, it was reported that cattle have a minor role in the transmission of Blastocystis to humans (Greige et al. 2019).

This study aimed to detect the prevalence and characterize Blastocystis STs in humans and cattle in Nile Delta, Egypt to investigate the potential risk of zoonotic transmission.

Materials and methods

Study population

The cross-sectional study from February 2018 to March 2019 was carried out on 136 participants (65 males and 71 females) and 190 cattle in Kafr El-Sheikh province in Nile Delta, Egypt. The demographic data (age and gender) and the presence of gastrointestinal symptoms among the study individuals were recorded. The age of the studied individuals ranged from 3 to 70 years with a mean of 33.05 years. The participants were classified into 68 in close contact with animals and 68 without contact. Out of 190 examined cattle, 120 were females and 70 were males. All were above 24 months of age. One hundred and twelve were raised in farm pastures while 78 were raised in a courtyard adjacent to the main building inhabited by the farmers. None of them exhibited clinical signs.

Isolation and identification of Blastocystis

Participants were asked to submit fresh stool specimens free from water and urine. Fresh fecal samples were collected from cattle. Part of each sample (about 2 mg) was immediately processed by a direct wet mount technique. Another part (about 50 mg) was directly inoculated into tubes containing 5 ml Jones’ medium enriched with 10% horse serum (Jones 1946) (kindly supplied by the Laboratory of Microbiology, Medical Research Institute, Alexandria, Egypt) and incubated at 37 °C for 72 h. The samples were examined daily using light microscopy for the detection of Blastocystis. Approximately 50 µL were removed from the sediment of positive cultures for additional subculture in fresh Jones’ medium to remove fecal debris (Zman and Khan 1994).

Seven ml Phosphate-buffered saline (PBS) was added to the subculture suspension and vortexed thoroughly then centrifuged for 1 min at 12.000 × g (Uobeed et al. 2015; Yoshikawa et al. 2004). The pellet was re-suspended in 1 ml of PBS solution and preserved at − 20 °C until processed.

Extraction of genomic DNA and PCR assay

DNA was extracted from culture-positive samples using commercial DNA MiniPrep™ kit (Zymo Research Corp., USA) following the manufacturer’s instructions. The eluted DNA was preserved at − 20 °C for further investigation. A fragment of 550–585 bp (barcode region) of the Blastocystis SSU rRNA gene was amplified using eukaryote-specific forward primer, RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) and Blastocystis-specific reverse primer, BhRDr (5′-GAG CTT TTT AAC TGC AAC AAC G-3′) (Scicluna et al. 2006). The PCR amplification reaction was performed using a thermocycler (Eppendorf, Germany) as described previously (Scicluna et al. 2006). PCR was performed using 2 µl DNA template, 5 µl 10 x Taq buffer (Genaxxon bioscience GmbH, Biberach, Germany), 0.2 µl Taq DNA polymerase (Genaxxon bioscience GmbH), 2 µl dNTP mix (2 mM each; Carl Roth GmbH, Karlsruhe, Germany) and 1 µl of both primers (10 mM; Metabion, Planegg, Germany) in a total volume of 50 µl. PCR products were analyzed by gel electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized under UV light.

Sequencing of PCR products

The resulting 550–585 bp amplicon was excised and DNA was purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. DNA sequencing of PCR products with adequate DNA concentration and quality was carried out using BhRDr and RD5 primers by cycle sequencing procedure with the BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Darmstadt, Germany). The Genetic Analyzer ABI Prism 310 was used to determine the sequences of the nucleotides.

After determining the sequence of Blastocystis rRNA gene fragments, the organism subtypes were identified using BLAST. The genetic divergence between the sequences of Blastocystis isolated in this study was analyzed using the bioinformatics tools of Geneious V10.2.3 analysis.

Statistical analysis

For data analysis IBM SPSS software suite version 20.0. (Armonk, NY: IBM Corp) was used (Kirkpatrick and Feeney 2013). The Chi-square test also was used. The significance level was set at 5% level.

Results

Identification of Blastocystis

Overall prevalence rates of Blastocystis in 136 human samples and 190 cattle samples were 53 (39%) and 37(19.4%), respectively using both culture and direct wet mount techniques. Blastocystis was demonstrated using both culture and direct wet mount technique in 23 human samples (16.9%) and 20 cattle samples (10.6%), by culture only in 17 humans (12.5%) and 11 cattle (5.8%) and by direct wet mount only in 13 humans (9.5%) and 6 cattle (3.1%) (Table 1).

Table 1.

The detection rate of Blastocystis using direct wet mount and culture techniques in human and cattle samples

Detection method	Human samples (n = 136)	Cattle samples (n = 190)
Direct wet mount and culture	23 (16.9%)	20 (10.6%)
Culture only	17 (12.5%)	11 (5.78%)
Direct wet mount only	13 (9.5%)	6 (3.1%)
Total	53 (39%)	37 (19.4%)

Analysis of potential risk factors for Blastocystis infection

Age, gender, and animal contact as potential risk factors had no significant association with Blastocystis infection (Table 2). Infection was more common in asymptomatic compared to symptomatic participants (41.5% versus 35.5%) but the difference was not significant. Blastocystis infection was significantly higher in backyard cattle (27%) than in farm cattle (14.2%) with a statistically significant difference (P-value 0.03) (Table 3).

Table 2.

Demographic data of the study population

Criteria	No. examined	Blastocystis positive by culture or direct microscopy	P-value
Age in years
3–<15	36	14 (38.8%)	0.8
15–30	31	10 (32.2%)
31–77	69	29 (42%)
Gender
Male	65	26 (40%)	0.6
Female	71	27 (38%)
Animal contact
Present	68	23 (33.8%)	0.8
Absent	68	30 (44.1%)
Total	136	53 (39%)

*Statistically significant at P < 0.05.

Table 3.

Prevalence of Blastocystis in cattle according to the origin

Animal origin	No. examined	Blastocystis positive	P-value*
Farm cattle	112	16 (14.2%)	0.03
Backyard cattle	78	21 (27%)
Total	190	37 (19.4%)

*Statistically significant at P < 0.05

Molecular subtyping

Out of 71 positive culture samples (40 human and 31 cattle), Blastocystis DNA was amplified in 55 positive culture samples. According to the quality and quantity of PCR amplicon, only 13 PCR products produced good quality sequences and produce an accurate length of the targeted gene. Sequencing detected thirteen different sequence types (six from human samples and seven from cattle samples) that were assigned to six different STs (ST1, ST2, and ST3 in humans and ST4, ST10, and ST14 in cattle). All the 13 sequences detected in the present study have been submitted in the GenBank database with the accession numbers (MH332370 to MH332382) (Table 4).

Table 4.

Blastocystis STs detected in humans and cattle in Kafr El-Sheikh province with GenBank accession numbers

ID	Subtype	Host	Animal contact/origin	Gender	Age /year	Clinical manifestation	Accession No.
H.KFS1	ST 2	Human	No animal contact	Male	40	Abdominal pain	MH332370
H.KFS2	ST 3	Human	No animal contact	Male	54	Diarrhea	MH332371
H.KFS3	ST 1	Human	contacted	Male	33	Abdominal pain	MH332372
H.KFS4	ST 2	Human	No animal contact	Female	8	Asymptomatic	MH332373
H.KFS5	ST 2	Human	Contacted	Male	47	Asymptomatic	MH332374
H.KFS6	ST 1	Human	Contacted	Female	53	Asymptomatic	MH332375
C.KFS1	ST 14	Cattle	Backyard	Female	2.5	Not applied	MH332376
C.KFS2	ST 14	Cattle	Backyard	Female	2	Not applied	MH332377
C.KFS3	ST 4	Cattle	Farm	Female	2	Not applied	MH332378
C.KFS4	ST 10	Cattle	Farm	Female	2.5	Not applied	MH332379
C.KFS5	ST 14	Cattle	Backyard	Female	2	Not applied	MH332380
C.KFS6	ST 14	Cattle	Backyard	Female	2	Not applied	MH332381
C.KFS7	ST 14	Cattle	Backyard	Female	2	Not applied	MH332382

Three subtypes were assigned for Blastocystis isolated from human samples (ST1, ST2, and ST3). ST1 was identified in two samples, one from an asymptomatic female and the other belonged to a male complaining of abdominal pain. ST2 was detected in three participants, two of them were asymptomatic and one male had abdominal pain. ST3 was found in a male patient complaining of diarrhea (Table 4).

Among cattle, ST14 was highly predominant as it was detected in five out of seven samples and all belonged to backyard cattle. In contrast, ST4 and ST10 were detected only in farm cattle (Table 4).

Genetic diversity analysis

The thirteen sequences obtained in this study were compared with each other and with Blastocystis sequence types obtained from GenBank to show the amount of divergence (Table 5).

Table 5.

The divergence between the sequences of Blastocystis isolated in the present study

	C.KFS1 (ST14)	C.KFS2 (ST14)	C.KFS3 (ST4)	C.KFS4 (ST10)	C.KFS5 (ST14)	C.KFS6 (ST14)	C.KFS7 (ST14)	H.KFS1 (ST2)	H.KFS2 (ST3)	H.KFS3 (ST1)	H.KFS4 (ST2)	H.KFS5 (ST2)	H.KFS6 (ST1)
C.KFS1 (ST14)		0.010	0.043	0.067	0.010	0.010	0.006	0.058	0.058	0.059	0.061	0.059	0.058
C.KFS2 (ST14)	0.010		0.045	0.063	0.012	0	0.009	0.050	0.056	0.057	0.049	0.048	0.056
C.KFS3 (ST4)	0.043	0.045		0.049	0.047	0.046	0.032	0.060	0.053	0.065	0.056	0.059	0.063
C.KFS4 (ST10)	0.067	0.063	0.049		0.067	0.061	0.059	0.068	0.058	0.072	0.062	0.070	0.075
C.KFS5 (ST14)	0.010	0.012	0.047	0.067		0.012	0.024	0.059	0.063	0.064	0.062	0.060	0.058
C.KFS6 (ST14)	0.010	0	0.046	0.061	0.012		0.008	0.049	0.052	0.054	0.048	0.047	0.055
C.KFS7 (ST14)	0.006	0.009	0.032	0.059	0.024	0.008		0.071	0.061	0.055	0.075	0.064	0.048
H.KFS1 (ST2)	0.058	0.050	0.060	0.068	0.059	0.049	0.071		0.066	0.025	0.002	0.004	0.023
H.KFS2 (ST3)	0.058	0.056	0.053	0.058	0.063	0.052	0.061	0.066		0.070	0.062	0.065	0.068
H.KFS3 (ST1)	0.059	0.057	0.065	0.072	0.064	0.054	0.055	0.025	0.070		0.024	0.020	0.005
H.KFS4 (ST2)	0.061	0.049	0.056	0.062	0.062	0.048	0.075	0.002	0.062	0.024		0.004	0.029
H.KFS5 (ST2)	0.059	0.048	0.059	0.070	0.060	0.047	0.064	0.004	0.065	0.020	0.004		0.022
H.KFS6 (ST1)	0.058	0.056	0.063	0.075	0.058	0.055	0.048	0.023	0.068	0.005	0.029	0.022

Analysis of 13 sequences obtained in this study revealed that the inter-subtype divergence was higher, up to 0.075 than intra-subtype divergence, up to 0.024 (Table 5). The divergence between the ST3 sequence and the other human sequences of the present study was higher than the divergence between this ST3 sequence and animal STs.

The two ST1 detected in the present study were genetically related to the sequences from humans and animals ST1 from different countries. Similarly, the three ST2 detected in this study were related to human and animal ST2s from different regions. One human ST3 from Czech Rep. (MT0427891) with two human ST3 from the Philippines (KY610153.1 and KY610145.1) and animal ST3 (AB107963.1) from Japan share the same clade with ST3 detected in the present study. The ST4 (C.KFS3) isolated from cattle in the present study was in the same clade as a human ST4 isolated from Thailand (MH197686) and animal STs4 from Indonesia (MH127488.1, MH127483.1and MH127487.1).

Discussion

Blastocystis is the most frequently reported intestinal parasite with a high prevalence worldwide (Eassa et al. 2016; Rebolla et al. 2016). The probability of zoonotic transmission and the public health significance of Blastocystis is currently the focus of intense research (Yoshikawa et al. 2016). The different clinical presentations of Blastocystis infection are may be related to genetic diversity at the ST level (Stensvold et al. 2009a).

In the present study, the prevalence of Blastocystis in human samples was 39% using a combination of culture and direct wet mount techniques. This result is in agreement with a previous study conducted in Ismailia, Egypt showing a rate of 35.7% (Mokhtar and Youssef 2018). High prevalence rates of Blastocystis were reported among children in Alexandria (67.4%) and Gharbeya (53%) governorates in Egypt (Eassa et al. 2016; EL-Marhoumy et al. 2015), while a lower prevalence rate (19.1%) was reported in patients attending Beni-Suef University Hospital, Egypt (El-Badry et al. 2018).

In the present study, the prevalence rate of Blastocystis in cattle was 19.4% which was lower than those previously reported in Egypt (72.2%) (Mokhtar and Youssef 2018) and Lebanon (63.4%) (Greige et al. 2019) but higher than that reported in China (10.3%) (Zhu et al. 2017).

The in vitro culture technique in this study was able to reveal more cases than the direct wet mount technique, which in agreement with previous reports in both humans (El-Badry et al. 2018; Elghareeb et al. 2015) and animal samples (Abe et al. 2002). However, stool cultures may miss some positive cases (Stensvold et al. 2009a) due to the degeneration of Blastocystis in culture (Tan 2008) and this may explain the cases missed in the present study (13 human and 6 animal cases).

In the present study, age and gender had no significant association with Blastocystis infection in humans. In contrast, a previous study observed higher Blastocystis infection among adults than children (Abdulsalam et al. 2013). While another previous study reported that children were more frequently infected than adults possibly due to the bad hygienic practices in the juvenile age (Rebolla et al. 2016). Another study found that males had a significantly higher rate of infection than females (Abdulsalam et al. 2013). Age and gender differences in Blastocystis infection rates may be related to the associated exposure risk and environmental conditions rather than host physiological properties (Li et al. 2007). The ability of Blastocystis to cause gastrointestinal symptoms remains unclear. In the present study, there was no significant difference between Blastocystis detection rates in symptomatic and asymptomatic participants. The possibility of asymptomatic carriage of Blastocystis was confirmed previously (Salvador et al. 2016). However, in another study, 70.2% of Blastocystis infected patients were symptomatic (Beyhan et al. 2015).

A higher rate of Blastocystis detection was observed in the present study among backyard cattle compared to farm cattle with a statistical significance difference (P-0.03). This might be attributed to different rearing conditions. In the present study, six different STs of Blastocystis were detected in humans and cattle. Three Blastocystis STs (STs 1–3) were identified in human samples. In Egypt, STs1-3 were previously detected with ST3 being the most common STs (El-Badry et al. 2018; Souppart et al. 2010).

The association between Blastocystis STs and abdominal symptoms is still controversial. Two previous studies in Egypt reported different results; one of them reported that ST1 was the most pathogenic subtype (Fouad et al. 2011). While the other one proved no definite correlation between genetically distinct genotypes and pathogenicity (Elwakil and Talaat 2009).

The divergence decreases when comparing the human ST3 sequence with animal STs sequences while the divergence increases when comparing the human ST3 sequence with the other human STs sequences.

This finding of this study showed the possible potential for zoonotic transmission of Blastocystis subtypes isolated from a rural area in Kafr El-Sheikh Province, Egypt. In the current study, a non-significant difference in Blastocystis infection rate was observed among contacts and non-contacts of animals. Minvielle et al. (2004) reported that Blastocystis prevalence was similar in both urban and rural areas and this may exclude zoonotic transmission. In contrast, Salim et al. (1999) observed that Blastocystis infection was significantly higher among animal handlers. The zoonotic transmission of Blastocystis may vary from one area to another depending on the prevailing environmental conditions, hygienic practice, and the level of exposure to other major sources of infection such as contaminated food and water. The occurrence of human-infectious subtypes in cattle suggests the potential zoonotic role of cattle (Yoshikawa 2004). Globally, different Blastocystis STs have been reported in cattle; ST1, ST3 and ST6 in Japan (Yoshikawa et al. 2004), ST5 and ST10 in the UK (Stensvold et al. 2009b), ST1, ST2, ST3, ST5, ST7, ST10 and ST14 in Lebanon (Greige et al. 2019). Further large scale studies should be conducted in Egypt to identify the role of cattle as zoonotic reservoirs of Blastocystis.

By analyzing the genetic relatedness, the six detected STs (ST1, ST2, ST3, ST4, ST10, and ST14) were distinguishable. Despite the previous report that ST2 exhibited more intra-genetic variation than ST1 (Alinaghizade et al. 2017), in the present study the divergence between the three isolated human ST2 (H.KFS1, H.KFS4, and H.KFS5) were 0.004 and 0.002 while the two ST1 sequences showed a divergence of 0.005.

The ST1 (H.KFS6) sequences detected from humans in this study were closely related to ST1 isolated from a human from Czech Rep. (MT039571.1), animals from Malaysia (MG831418.1), and Waste Water from the Philippines (MF737393.1). Additionally, they were genetically close to ST2 isolated from an animal in the UK (MN526898.1) and to a human isolated ST2 (MK100361.1) from Russia. In agreement with these results, Yoshikawa et al. (2009) found ST2 sequences shared between human and monkey isolates indicating that ST2 is not host-specific.

In the present study, the human ST3 (H.KFS2) shared the main clade with animals ST4 and ST10. This observation is in agreement with previous reports which confirmed the phylogenetic relationship between ST3, 4, and 10 as they were grouped within one cluster and shared a common ancestor (Alfellani et al. 2013; Betts et al. 2018; Stensvold et al. 2009b). Moreover, previous studies proved that ST3 was not restricted to humans but was reported from non-human primates closely contacted with humans (Abe et al. 2003; Stensvold et al. 2009b; Yoshikawa 2004).

A previous study has reported the detection of ST4 in cattle (Zhu et al. 2017). In the present study, the cattle ST4 (C.KFS3) was clustered with three Indonesian ST4 isolated from animals (MH127488.1, MH127483.1, and MH127487.1) together with human ST4 from Thailand (MH197686.1). Genetic similarities between animal and human-driven ST4 isolates were reported previously suggesting the potential for zoonotic transmission of this subtype (Stensvold et al. 2009b, 2012; Zhu et al. 2017).

ST10 and ST14 detected in the present study were not closely related to any of the human STs. The previous report confirmed that ST10 and ST14 were restricted to animals and excluded their zoonotic transmission (Alfellani et al. 2013).

Conclusion

Blastocystis is a common organism identified in humans and cattle in Kafr El-Sheikh province in northern Egypt. The presence of ST4 in cattle and the close genetic relation between human and animal isolates of STs1-3 support the potential for cross-transmission of Blastocystis of ST1, ST2, ST3, and ST4.

Authors’ contributions

SMA, HE, HFF, AE-b, HE-t, and HE-A planned and synchronized the study, performed the laboratory work, data analysis, writing of the manuscript, and helped to draft and review the manuscript. SMA and HAE were involved in the collection and examination of samples analysis and writing of the manuscript. HE participated in the study design, molecular investigation, and data interpretation, revised the paper, and approved the final version. All authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The data supporting the findings of this study are contained within the manuscript. The raw data are available by the corresponding author when requested.

Code availability

Not applicable.

Compliance with ethical standards

Conflict of interest

None.

Ethical approval

The study was approved by the Research Ethics Committee of the Medical Research Institute (MRI), Alexandria University (IORG 0008812). All procedures performed in studies involving human participants were under the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the study involving humans and animals were following the ethical standards of the Medical Research Institute (MRI), Alexandria University.

Consent to participate

Verbal consent was obtained from all participants.

Consent for publication

Verbal consent was obtained from all participants. Written informed consent was taken from all the study participants and children’s guardians after an explanation of the study's purpose.