Subtypes and phylogenetic analysis of Blastocystis sp. isolates from West Ismailia, Egypt

In Egypt, Blastocystis sp. is not yet on the diagnostic list of parasitology reports, and information about its subtypes (STs) is scarce. This study investigated its prevalence and its STs/alleles, performed phylogenetic analysis, and considered the distribution of risk factors associated with Blastocystis sp. infections in West Ismailia, Ismailia governorate. Sociodemographic data, exposure factors, and previous parasitic infection status were recorded for symptomatic and asymptomatic individuals. Microscopy, polymerase chain reaction, sequencing, and phylogenetic analysis for Blastocystis sp. isolated from fecal samples were performed. Eighty Blastocystis sp.-infected individuals (15.3%) were examined. The age of the individuals ranged between 0.60 and 85.0 (mean 17.10 ± 15.70), the male/female ratio was 33/47, and the asymptomatic/symptomatic ratio was 55/25. The findings demonstrate clear evidence of direct contact with animals, poor water quality, and previous parasitic infections. Eleven samples yielded three Blastocystis STs (ST1: allele 4, ST2: alleles 9 and 12, and ST3: allele 34), with ST3 (45.5%) representing the most common subtype. Phylogenetic analysis with a robust bootstrap revealed three distinct clades for isolates of each subtype. This study updates the epidemiological knowledge of the distribution of Blastocystis sp. STs in Egypt and expands the current understanding of the prevalence, risk factor frequencies, and genetic diversity of this protist in the studied area.

Descriptive data concerning patient sociodemographics and the distribution of risk factors is illustrated for the 80 Blastocystis-positive samples (Tables 1 and 2). Microscopic and molecular examinations of Blastocystispositive samples are shown in detail ( Table 3, Fig. 2, and Table 4). Of the 15 samples sent for sequencing, 11 were successfully sequenced with high-quality yield (see "Methods" section for details). The genetic diversity and genotype distribution of Blastocystis sp. isolates are detailed in Table 5. Information regarding Blastocystis sp. STs in the current investigation, compared against studies of different Egyptian governorates, appears in Table 6. The current study's sequence isolates were submitted to phylogenetic analysis (Fig. 3).
Blastocystis sp. was identified in a total of 80/520 (15.3%) fecal samples, according to direct light microscopy of smears (saline and iodine examination). The 80 Blastocystis-positive subjects included those with no available data (NAD = 47) and those with complete historical data available (SAD = 33). For NAD samples, the only available data were age, sex, and symptomatology status, with no information on exposure factors or previous parasitic infections (PPIs). Both SAD and NAD subjects were classified as "symptomatic" (gastrointestinal symptoms) or "asymptomatic" (no gastrointestinal symptoms), based on responses to a standardized questionnaire ( Table 1).
The 80 Blastocystis-positive individuals ranged in age from 0.6 to 85 years. Most patients (55/80) had no gastrointestinal symptoms; those who did mostly complained of abdominal pain and diarrhea. Other nonspecific symptoms such as vomiting, dehydration, and fever were also described ( Table 1). Those aged between 5 and 18 (52/80) were most affected by Blastocystis sp. infection (Table 1). Approximately 60% (31) of this group were asymptomatic; the remainder were symptomatic. Females in this group represented 55.8% (29), and males represented 44.2% (23).
Most of the SAD subjects reported owning domestic animals (poultry, livestock, and pets). About 76.6% reported direct contact with their animals and that the floors of their animal farms were covered with sand. The water supplies at the studied subjects' residences appeared mostly clear. However, some participants mentioned that the water sometimes became yellow and turbid with a smell (Table 1).
Tests performed using BLAST software revealed that each of the SSU rRNA gene sequences obtained in this study demonstrated a high degree of homology with previously reported sequences from other Blastocystis sp. isolates. Although ST1 and ST2 sequences showed some single-nucleotide polymorphism mutations against the reference strains, ST3 demonstrated 100% homology with the reference strain. Furthermore, 10/11 sequenced    www.nature.com/scientificreports/  www.nature.com/scientificreports/ isolates belonged to asymptomatic subjects, with only one isolate (ST1) belonging to a symptomatic subject ( Table 5). The alignment of Blastocystis sequences showed three to five nucleotide differences. Three samples--IsKa 7 (ST2), IsKa 38 (ST2), and IsKa 75 (ST1)--demonstrated three nucleotide differences. Despite these differences, the BLAST search revealed that the first two of these isolates belong to the same Blastocystis ST. One sample, IsKa 29 (ST1), showed four nucleotide differences, while two samples, IsKa 13 (ST1) and IsKa 56 (ST2), showed five nucleotide differences. All of the sequence data for Blastocystis nucleotides obtained from the current study were deposited in GenBank with accession numbers OL845600-OL845610 as follows: ST3 represented by five samples (OL845600-OL845604), ST2 represented by three samples (OL845605-OL845607), and ST1 represented by three samples (OL845608-OL845610) ( Table 5). In terms of Blastocystis alleles, ST3 and ST1 produced completely homogeneous isolates with alleles 34 and allele 4, respectively. Meanwhile, ST2 exhibited low genetic variation in one isolate (IsKa 7), which corresponded to allele 12, with its remaining two isolates corresponding to allele 9.
A phylogenetic tree was constructed using eleven nucleotide sequences representative of the current study and GenBank database reference sequences, with Proteromonas lacertae (U37108) used as the outgroup. Three subtypes were distinguishable: ST1, ST2, and ST3 (Fig. 1).

Discussion
This study's findings revealed Blastocystis sp. to be prevalent in 15.3% of the West Ismailia population, which is consistent with the observations of El-Badry et al. (2018). Other studies have revealed variations in Blastocystis prevalence 27,30,31 . PCR in Cairo revealed a prevalence of 35.5% for Blastocystis in both patient and control groups 30 . Patients with irritable bowel syndrome in Beni-Suef were found to have a prevalence of Blastocystis of 16.5% by microscopy and 19.1% by culture 27 . In the same governorate, 53.6% of patients with acute diarrhoea had Blastocystis 31 . Such disparities can be attributed to various epidemiological parameters, including the target population, detection method, and the presence or absence of symptoms. www.nature.com/scientificreports/ In this study, Blastocystis colonization was more common among children aged 6 to 18 than among children under five years old and adults, which aligns with two previous Egyptian studies observing schoolchildren to be the most affected 32,33 . Infection with Blastocystis sp. has also been demonstrated by global surveys to be prevalent among schoolchildren 18,[34][35][36] , likely owing to this age group's lax hygiene standards. Blastocystis infection age disparities may be influenced by associated exposure risk factors, children's immunity, and environmental variables [34][35][36] .
This study classifies Blastocystis infections as silent, with most infected subjects not exhibiting symptoms. Three factors may influence the asymptomatic status of Blastocystis infections among Egyptians: (a) awareness, which is limited in the rural Egyptian population, of the need to seek medical attention for diarrhea; (b) the diagnostic ability of Egyptian laboratories to identify Blastocystis sp. in primary care settings (i.e., primary health care units), leaving Blastocystis outside their diagnostic scope 29 ; and (c) in the case of Blastocystis infection, the concept that it is a commensal protist, which encourages Egyptian physicians to disregard treating patients despite the presence of distressing symptoms 29 . These factors may enable Blastocystis sp. to colonize the host for an extended period without causing disease.
Most Egyptians, and particularly those living in rural areas, keep domestic animals and birds in and around their homes. This is especially evident in West Ismailia, where domestic animals, especially poultry, coexist with humans in traditional Egyptian residences without separate yards, exposing owners to high concentrations of Blastocystis infective stages for extended periods. This has led to the prediction that the risk of Blastocystis infection is tenfold greater in rural areas than in urban areas among the Egyptian population with irritable bowel syndrome 27 . Elsewhere, strong molecular evidence has confirmed zoonotic transmission between animals and their human caregivers 37,38 . Notably, humans in direct contact with animals were found to have the same Blastocystis STs in two studies conducted in Northern and Eastern Egypt (ST1, ST2, ST3, and ST4) 37,39 . Additionally, poultry can harbor the human-transmissible Blastocystis ST6 and ST7 40 , although this has only been reported in a recent Egyptian study of colorectal cancer patients 41 .
The Sweet Water Canal serves as the primary water source for municipal divisions in West Ismailia and is used directly for animal bathing, dishwashing, and laundry. Consequently, water used for residential or recreational purposes becomes contaminated, creating a risk of transmission of gastrointestinal diseases to humans and animals alike. Rural communities in West Ismailia have been particularly hard-hit, with some settlements in remote locations lacking access to safe drinking water, which increases the possibility of contamination during transportation and processing. Numerous protozoan contaminants, including Blastocystis sp., have been detected in Egypt's Dakahlia, Ismailia, and El-Minia governorates in potable water, water tanks, pumps, waterworks, and surface water (i.e., River Nile, ponds, and canals) 24,42,43 . Most SAD residents were observed to utilize tap water without a filter, with the water supplied appearing clear. However, because it is only used to remove rust, insects, and dust, the presence or absence of a filter would not substantially impact the purification of water from protozoa stages. Thus, even if the water appears clear, it may contain Blastocystis sp. Furthermore, in recent years, soil pollution has been identified as a source of Blastocystis sp. infection 20 ; as a major farming region, soil is extremely likely to be a source of infection for the West Ismailia population.
PPI are prevalent in rural Ismailia (author's observation, unpublished data); when questioned, this study's participants were fully aware of this. Over half of participants failed to take their parasitic infection medications as prescribed and did not complete the entire course of treatment (dose and duration). Consequently, infected individuals have acted as carriers facilitating anthroponotic protist transmission, while also experiencing infection maintenance, chronicity, and consequences.
This study's investigations also revealed polyparasitism in the West Ismailia population. Although most samples represented single Blastocystosis infections, the presence of pathogenic (G. duodenalis, E. histolytica, H. nana) and non-pathogenic (E. coli, C. mesnilli, non-pathogenic species of E. histolytica) parasites mixed with Blastocystis sp. infection suggests multiple sources of infection. Mixed parasitic infections are highly predicted in rural areas due to the presence of multiple risk factors, as has been previously documented [44][45][46][47] . Consumption of contaminated water and unwashed vegetables, lack of fingernail trimming and hand washing, children playing in the dirt, barefoot walking, low socioeconomic status, lack of sanitation, and large numbers of family members sharing a single room have all been observed among the residents of West Ismailia 27,31,48,49 . Furthermore, rats, cockroaches, fleas, ants, and flies were observed to spread in numerous locations, especially during hot weather, alongside sewage rash. Such behavioral, social, and sanitary factors are almost certainly implicated in developing mixed parasitic infections and perpetuating the life cycle of those infections. Thus, nearly all the transmission routes required for Blastocystis sp. appear open.
Three subtypes of Blastocystis sp. were characterized in the current investigation via the molecular analysis of isolates, namely, ST3, ST2, and ST1, with the latter two recording equal distribution. This study's findings corroborate those of Souppart et al. (2010), who discovered that ST3 had the highest prevalence (61.9%) and that ST1 and ST2 had equal prevalence (19.05%).
Several Egyptian studies have subtyped and sequenced Blastocystis sp. 39,41,50 , with five STs (ST1, ST2, ST3, ST4 and ST7) identified at varying frequencies in distinct Egyptian groups using PCR sequenced-tagged sites, PCR restriction fragment length polymorphisms, and PCR sequencing (barcoding) ( Table 6). The high prevalence of Blastocystis sp. ST1-ST4 in the Egyptian community suggests that most infections are transmitted from person to person.
The current investigation has revealed that ST3 is responsible for the vast majority of Blastocystis infections in West Ismailia, aligning with observations for other Egyptian governorates across 12 other studies (Table 6), which have revealed ST3 to be the most prevalent Blastocystis subtype in six distinct Egyptian locations; furthermore, it is the ST most closely related to various gastrointestinal symptoms ( www.nature.com/scientificreports/ of Egyptian studies 28,39 . Notably, STs and their relative frequencies appear to vary significantly within a single country (Table 6). Almost all the isolates sequenced in this investigation were asymptomatic, except for one patient who suffered diarrhea and abdominal pain and was subtyped as ST1, an observation consistent with the findings of other studies 10,[55][56][57] . Blastocystis is more frequent in healthy individuals, with its existence also linked to altered composition and increased richness of the bacterial gut microbiota 14,15 . It is unclear whether Blastocystis directly promotes a healthy gut and microbiome or whether it prefers to colonize and persist in a healthy gut environment. A study of Blastocystis sp. ST3 indicated that Blastocystis sp. may modify the gut ecosystem in a protective manner and facilitate faster recovery from disturbances 12 . The presence of Blastocystis among healthy individuals has also been linked to reduced levels of fecal calprotectin, a sign of intestinal inflammation according to a comparative investigation conducted in Mexico 56 . In contrast, some researchers have suggested that particular Blastocystis sp. isolates may produce an imbalance of the gut microbiota [58][59][60][61][62][63] . The context of the environment and hosts must be considered when discussing whether Blastocystis is a pathogen or a mutualist.
The present study's genetic analysis reveals that all 11 isolates detected in Blastocystis subtypes cross-corresponded to previously observed alleles. Intriguingly, ST3 isolates produced the highest frequency of isolates matching allele 34, the most common variant found in humans worldwide, with ST2 isolates exhibiting low levels of genetic diversity and multiple nucleotide substitutions corresponding to two different alleles (9 and 12). Although ST1 isolates demonstrated limited genetic diversity, all isolates corresponded to allele 4. In Egyptian isolates from Cairo, genetic diversity was detected in three subtypes in the same pattern, with ST1 and ST2 exhibiting nucleotide differences ranging from 1 to 11 and ST3 exhibiting reduced genetic variability of up to four nucleotide differences 50 . However, no further allelic analysis was conducted. On the contrary, there was no evidence of genetic diversity in the Blastocystis subtypes (ST3 and ST1) isolated from individuals with irritable bowel syndrome in the Beni-Suef governorate 27 .
A phylogenetic tree demonstrated that the 11 nucleotide sequences in this study clustered into the same subtype cluster, with high bootstrap support, and could be classified into three subtypes: ST1, ST2, and ST3. Each Blastocystis sp. ST formed a distinct clade, implying that the West Ismailian Blastocystis population can be divided into three subgroups. Another Egyptian study found the same phylogenetic distribution pattern for ST1 and ST3 subtype clusters in patients with irritable bowel syndrome 27 .
Among the drawbacks of the present investigation is the infeasibility of sequencing every isolate. Because samples were collected via a large-scale survey, there were no epidemiological data for some participants, which hindered presenting comprehensive information.
Blastocystis sp. infections are significantly under-and mis-diagnosed in Egypt, particularly in rural and remote areas such as West Ismailia. Additional research can illuminate the epidemiological situation of Blastocystis sp. in Egypt, enabling more effective control efforts against Blastocystis sp. infections and other parasitic disorders.

Conclusions
The current study updates the epidemiological situation and distribution of Blastocystis STs in Egypt. Phylogenetic analysis has revealed three distinct clades for isolates pertaining to each subtype, adding to our current understanding of Blastocystis's prevalence and genetic diversity. The widespread presence of ST3 in the West Ismailia population and throughout Egypt necessitates subtyping analysis, which has become indispensable for elucidating the relationship between Blastocystis subtypes and pathogenicity in the Egyptian population.
We highlight the need to invest in parasite education programs, specific to Blastocystis sp., that involve the general public along with doctors and laboratory technicians. Moreover, further studies are needed in the underrepresented areas of Egypt to verify the distribution of Blastocystis sp. throughout the country.

Methods
Study area and sample collection. The Ministry of Health and Population in West Ismailia's municipal divisions (villages around the Sweet Water Canal's geographical line) conducted a screening survey of 598 fecal samples for schistosomiasis eradication campaigns. Samples were collected randomly, without regard for age or gender, from the nearest sampling area.
Thus, this descriptive study used data from screens for gastrointestinal parasites of individuals in the municipal divisions of West Ismailia (El-Kassassin, El-Mahsama, El-Talelkbeer, and Abu-Suwayr). A total of 80 fecal samples positive for Blastocystis sp. were analyzed. The flowchart in Fig. 4 describes the current study's process.
Five hundred and twenty samples were selected after considering their suitability based on inclusion and exclusion criteria. The 520 fecal samples and their questionnaire forms were sent to Suez Canal University's Parasitology Laboratory for ovum and parasite examination, using wet mount and iodine microscopy to screen for gastrointestinal parasites. Blastocystis-positive samples were separated and selected for the current study analysis. The amount of the received fecal samples (a full tablespoon, i.e., 15-20 gr) was used as a guideline for rolling in or rolling out the received fecal samples.
To collect and transfer the fecal samples, participants were given a clean, labeled plastic container with an applicator stick. Patients were given verbal explanations in Arabic regarding the collection and transfer processes and the amount of stool sample required. Stool samples were excluded if contaminated with urine and water or if the amount was too small (less than a full tablespoon, i.e., 15-20 gr). Subsequently, the parasitology laboratory at El-Mahsama Family Practice Center divided the stool samples into two parts: one part for the eradication program campaign (primary health care unit at the parasitology laboratory) and one part for processing at the Suez Canal University's Medical Department's parasitology laboratory in Ismailia. Extraction of genomic DNA. Before extraction, the preserved 1 mL fecal samples were washed and centrifuged several times in phosphate-buffered saline until the supernatant became clear. Then, the supernatant was discarded, and 200 µL of the 1 mL sediment was exposed to the InhibitEX lysis buffer from the Qiagen DNA Stool Mini Kit (Qiagen, Germany, GmbH) according to the manufacturer's protocol. Using 100 µL of elution buffer, the protocol was slightly modified, and the extracted DNA was stored at -20 °C for further molecular investigation.
PCR amplification and sequencing. Financial restrictions meant that only 15 samples were sent to the Istituto Superiore di Sanità in Rome, Italy, for molecular characterization. For amplification of the SSU rRNA region in Blastocystis isolates, Blasto F (5′-TCT GGT TGA TCC TGC CAG T-3′) and Blasto R (5′-AGC TTT TTA ACT GCA ACA ACG-3′) primers were used according to the protocol described by Meloni et al. 65 . A proofreading enzyme (GoTaq Promega) was used to amplify the PCR. The Promega Wizard SV Gel and PCR Clean-Up System kit was used to purify the obtained 600 bp amplicon, and purified products were sent for MGW Sanger sequencing.
Phylogenetic analysis. Molecular and Evolution Genetic Analysis software (https:// www. megas oftwa re. net/) was used to produce a phylogenetic tree for nucleotide sequences using the maximum likelihood method. One thousand bootstrap replicates were used to test the phylogenetic tree's reliability and the statistical support for the topology. Evolutionary distances were calculated using the Tamura-3 parameter model. www.nature.com/scientificreports/ Statistical analysis. A descriptive analysis was used to report sociodemographic characteristics and the frequency and distribution of Blastocystis subtypes among the affected subjects. After uploading all questionnaire data, the IBM SPSS software package version 26.0 was used to analyze the data (IBM Corporation, Armonk, NY). Numbers and percentages were used to represent categorical data; mean and standard deviation were used to represent numerical data. The chi-squared test was used to examine the relationship between a PPI and its treatment as two qualitative variables. The p-value was statistically significant at 0.05.
Ethical considerations. Fecal sample collection and medical history questionnaires were reviewed and approved by the Scientific Research Committee and Bioethics Board of Suez Canal University, Faculty of Medicine, Egypt (approval no. 5089). All methods were performed in accordance with the relevant guidelines. Participants in the study were asked to sign a written informed consent form that clearly detailed in Arabic the study's objectives, sociodemographic questionnaire, symptomatology, exposure factors, and procedures. The collected data were kept private during and after the collection and analysis. The study's participants were informed that they could withdraw at any time. Patients whose fecal specimens were positive for gastrointestinal parasites were referred to physicians for further treatment.