Molecular identification and genotyping of Blastocystis spp. in children with clinical symptoms in southeast Iran using PCR-sequencing method

1. Introduction

Blastocystis spp. is a protozoan parasite that is both anaerobic and zoonotic. It has been found in the large intestine of humans and many other vertebrates. This parasite is isolated to a high extent from stool samples in parasitology laboratories. In the year 2000, using the SSU rRNA gene, Blastocystis spp. was included in the phylum Sarcomastigophora ( 1 , 2 ). Despite its discovery in human feces approximately a century ago, it was long considered a non-pathogenic parasite. However, in recent years, the significance of Blastocystis spp., particularly in individuals with certain diseases, has come to the fore ( 3 , 4 ). The pathogenic cysts of Blastocystis spp. have been shown to survive in water and to be transmitted directly between different hosts through contaminated water or food ( 5 ). The prevalence of Blastocystis spp. has been documented to range from 0.8% to 61.8% on a global scale ( 6 ). A preponderance of reports indicates that Blastocystis spp. is prevalent in tropical and subtropical regions. In developing countries, immigrants and refugees exhibit a higher rate of infection compared to the native population ( 7 , 8 ). The literature has demonstrated the pathogenicity of Blastocystis spp., with associated symptoms including acute and chronic diarrhea, abdominal pain and bloating, constipation, nausea, irritable bowel syndrome, and intestinal inflammation. The validity of these indicators and pathological changes has been substantiated in laboratory animals and cell cultures. However, the debate surrounding the pathogenicity of Blastocystis spp. persists, particularly in the context of asymptomatic carriers, as further discussed below. According to the established morphological criteria, the Blastocystis spp. isolated from both animals and humans are morphologically indistinguishable. According to molecular studies, 17 subspecies of this parasite have been identified ( 6 , 10 ), with subtypes 1 to 9 and ST12 isolated from humans and mammals, and subtypes 10 to 17 found in animals. According to the findings of these studies, subtypes 1 to 4 are predominantly associated with humans, and types 3 and 4 are the most prevalent subtypes isolated from humans and are associated with gastrointestinal symptoms. Subtypes ST3 and ST4 have been observed to exhibit the highest and lowest levels of diversity, respectively, among the subtypes ( 11 , 12 ). Blastocystis spp. is a mysterious and unique parasite with multiple evolutionary processes and various reproduction methods, including binary division, budding, endodyogeny, sporogony, schizogony, and various morphological forms (i.e., vacuolated, granulated, ameboid, and cystic forms with dimensions of 2-200 nm). This characteristic has led to a decline in the sensitivity and specificity of microscopy-based diagnostic methods ( 13 ). In the study of Funda Dogruman-Al, et al. in Turkey, the sensitivity of the Lugol's stain method, trichrome staining and IFA stain was of 36.7%, 50%, and 83%, respectively in contrast to the culture method (gold standard). PCR method has higher specificity and sensitivity compared to other methods ( 14 ). Identifying the diversity of parasite species in different geographical areas and identifying zoonotic strains can be useful in providing prevention strategies. Blastocystis spp. has a high genetic diversity and there is a possibility of transmission of animal species of the parasite to humans. So far, no study has been conducted in Zahedan city (southeast Iran) to determine the prevalence and genotypes of Blastocystis spp. in patients. Therefore, this study aimed at determining the prevalence of Blastocystis species in children with diarrheal stool under 10 years old in Zahedan using PCR-sequencing method.

2. Materials and Methods

2.1. The Target Community

This descriptive-cross-sectional study was conducted between 2019 and 2021. The inclusion criteria for the study comprised children under 10 years of age and children exhibiting gastrointestinal clinical symptoms, with a particular focus on diarrhea. Exclusion criteria included children without gastrointestinal clinical symptoms, especially diarrhea, and those who lacked consent to participate in the study.

2.2. Ethical Point of View

Prior to the conduction of any laboratory analysis or physical examination, written informed consent was obtained from the guardian (parents/guardians/LAR's= Legally Authorized Representative) of all participants. A unique code was assigned to each patient, and the data was kept confidential. The study protocol was approved by the Ethics Committee of Zahedan University of Medical Sciences (reference number: IR.ZAUMS.REC.1398.322).

2.3. Ethical Considerations

Ethical clearance was obtained from the ethical review committee of Zahedan University of Medical Sciences. Prior to the interview and stool examination, written consent was obtained from the children's parents/guardians/LARs.

2.4. Sampling

The samples were selected from children who met the inclusion criteria for the study (convenience sampling). An informed consent form regarding the collection of stool samples was prepared with the signature of the parents of children who met the inclusion criteria for the study. A total of 60 diarrheal stool samples were collected from children referred to Ali Asghar Children's Hospitals in Zahedan and the Reference Laboratory of Sistan and Baluchistan Province, Iran. The characteristics of each sample were recorded, and all stool samples were referred to these two centers. Therefore, the samples had a geographical distribution and were collected from all parts of the Sistan and Baluchistan Province, Iran. In the parasitology laboratory of Zahedan Medical School, Iran, daily collections were examined microscopically (i.e., direct examination of stool samples) for the presence of Blastocystis spp. Those samples that were microscopically positive were then entered into the molecular diagnosis stage.

2.5. DNA Extraction and PCR

DNA extracted using an extraction kit (AccuPrep® stool DNA extraction kit Bioneer, Daejeon, South Korea) according to the manufacturer's instructions and genotyping was performed at the locations of Blastocystis spp. specific sequences (SSU rRNA gene). Before using the extraction kit, the freezing ( -196˚с Liquid Nitrogen for 3 minutes (and thawing (95˚с) process was performed alternately (10 times) on the samples to break the parasite wall. The extracted DNA was kept at -20 ˚с until use. Polymerase chain reaction was performed using specific primers. The DNA extraction process was carried out in accordance with the manufacturer's instructions for the AccuPrep® stool DNA extraction kit (Bioneer, Daejeon, South Korea). Subsequently, genotyping was performed at the locations of Blastocystis spp. specific sequences (SSU rRNA gene). Prior to utilizing the extraction kit, a freezing (liquid nitrogen, -196˚C, 3 minutes) and thawing (95˚C) process was performed on the samples. This process was repeated ten times to disrupt the parasite wall. The extracted DNA was stored at -20˚C until use. Polymerase chain reaction (PCR) was subsequently performed using primers designed to target a specific region of the parasite's DNA.Blast 505-532 (5′ GGA GGT AGT GAC AAT AAATC 3) forward, Blast 998-1017 (5′ TGC TTT CGC ACT TGT TCATC 3′; reverse ( 15 ). The PCR reaction mixture comprised the following components: 250 µM deoxynucleoside triphosphates (dNTPs), 1.5 mM MgCl₂, 1 U of Taq polymerase, 1 µM of a forward Blast primer, 1 µM of a reverse Blast primer, and 2 µL of DNA, yielding a final volume of 15 µL. The reaction mixtures were then subjected to incubation in a thermocycler (Flexcycler2, Germany) according to the following thermal cycling protocol: an initial denaturation step at 95°C for a period of 4 minutes, followed by 35 cycles of 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds. The final extension step was conducted at 72°C for a duration of 5 minutes. Subsequent to the completion of the thermocycling protocol, the PCR products were subjected to electrophoresis in a 1.5% agarose gel. The positive control (Accession Number: MZ734401.1) was utilized. The expected PCR product size was determined to be between 479 and 500 base pairs (bp).

2.6. Sequencing and Phylogenic Analysis

Initially, the five samples of Blastocystis spp. sequences obtained from the new isolates were multi-aligned with ClustalW software against those of humans, which had been previously deposited in GenBank. Subsequent to this, the nucleotide sequences were examined using Chromas biotechnology software version 2.4 and CLC genomic work bench software 11. Thereafter, the sequences were aligned using BLAST database, and finally, they were compared with Blastocystis spp. reference genotypes in gene bank. The genotyping of the sequences was conducted using CLC genomic work bench software 11, and a phylogenic tree was constructed using MEGA7 software with the Neighbor-Joining statistical method, which applied the Kimura 2-parameter method based on nucleic acid. The ABI 310 automated fluorescent sequencing system was utilized to sequence high-quality PCR products, employing the indicated primers. The similarity between sequences of other Blastocystis spp. was performed by the Basic Local Alignment Search Tool (BLAST) software provided by the National Center for Biotechnology Information (NCBI) in GenBank. The sequences were then analyzed by CLC Genomics Workbench 12 software and manually edited. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software version X, with trees generated using the neighbor-joining method and the Kimura 2-parameter model. Bootstrap values were estimated using 100 replicate data sets at each node. Furthermore, a comparison was made between our samples and similar isolates from Iran and other regions of the world.

3. Results

3.1. Microscopic Examination and Molecular Diagnosis

A total of 60 stool samples from children were examined, and five samples were identified as positive for Blastocystis spp. This diagnosis was made based on the observation of morphological characteristics and the analysis of DNA. The presence of Blastocystis spp. was detected using the SSU rRNA gene. The PCR amplification product exhibited a fragment of 500 (479) base pairs.

3.2. Genotyping and Phylogenic Analysis

A subsequent analysis of the obtained sequences revealed the presence of four distinct subtypes, namely subtypes 1, 2, 3, and 5, among the existing isolates. The accession numbers of the five Blastocystis spp. isolates utilized in this study are documented in Gen Bank as follows: ON271191, ON271192, ON271193, ON271194, ON271195. The similarity percentage of the isolates was obtained by comparing the isolates available in the gene bank (Table 1). A comparison of the nucleotide changes in the sequences obtained from the studied isolates reveals the presence of significant nucleotide differences within the range of sequence 140 to 240, where deletions have occurred in the nucleotides of subtypes 1 and 2 compared to subtypes 3 and 5 within this range (Figure 1). Phylogenic analysis of the nucleotide sequence of the obtained isolates revealed a genetic affinity between subtypes 1 and 2. The same genetic affinity is also observed in the plotted tree between subtypes 3 and 5 (Figure 2).

Figure 1. Alignment of 5 Blastocystis spp. sequenced isolates from Zahedan and a schematic representation of nucleotide changes in a small subunit rRNA gene fragment.

Figure 2. The phylogenetic position of Blastocystis spp., ST1,2,3 and 5 isolated from children’s patients. Phylogenic tree was drawn using Neighbor-Joining statistical method applying Kimura 2-parameter method for different subtype Blastocystis spp.

4. Discussion

Small subunit ribosomal ribonucleic acid (SSU rRNA) constitutes the minor component of the two major RNA components of the ribosome. The SSU rRNA constitutes the small subunit of the ribosome and is an rRNA-encoding gene ( 16 ). The SSU-rDNA gene has a wide range of applications in fields such as phylogeny, taxonomy, and the analysis of evolutionary relationships among organisms. This is due to the fact that it is of ancient origin and is found in all known life forms ( 17 ). Due to the similarity of Blastocystis spp. with yeast, fat globules, and cyclospora species, the prevalence of this parasite is usually reported higher using microscopic methods ( 15 ). The prevalence of Blastocystis spp. in different regions of the world varies significantly, ranging from 0.8% to 61%, with a predominance observed in tropical and subtropical regions, including Argentina, Egypt, the Philippines, Malaysia, China, Japan, Iran, Singapore, England, Spain, Italy, and Turkey ( 18 - 21 ). Despite the decline in the number of microorganisms employing the same transmission method, Blastocystis spp. has been identified as one of the most prevalent parasites in 38.9% of studies, suggesting an escalating prevalence ( 22 , 23 ). Blastocystis spp. has been identified as an emerging parasite ( 24 ). Recent studies have documented a pooled prevalence of Blastocystis hominis of 3% in Iran ( 25 ). In the present study, 8.33% of the 60 samples examined were found to be infected, with the number of positive cases falling short of the reported cases. This discrepancy may be attributable to various factors, including weather conditions, low parasite concentrations, improper sample storage, inadequate sampling techniques, or the presence of fecal inhibitors. Notably, there was a 100% concordance between the microscopic and molecular results, underscoring the reliability of the diagnostic methods employed. A total of 17 subtypes have been identified in diverse mammalian, avian, and amphibian species based on the sequence of different genes and genetic analysis. Subtypes 1-9 have been isolated from humans, while subtypes 10-17 have been isolated from animals. It is noteworthy that subtypes 1-9 have been identified in other hosts in addition to humans. For instance, ST4 has been identified in rodents, ST6 in birds, and ST7-8 in birds. Additionally, ST1, ST2, ST5, ST8 have been detected in primates, pigs, and cattle, ST10 and ST15 in artiodactyls and non-human primates, ST11 in proboscideans, ST12 in artiodactyls and marsupials, ST13 in non-human primates and marsupials, ST14 in artiodactyls, and ST16 in marsupials. Finally, ST17 has been identified in rodents ( 26 ). As indicated by the findings of studies conducted on human subjects, the presence of ST1-9 and ST12 has been detected in certain cases ( 11 , 12 ). Subtype 5 is zoonotic and has been reported in Australia and China, which is one of the most common subtypes in animals ( 21 , 27 - 28 ). In the present study, subtypes 1, 2, 3, and 5 were identified in a total of five sequences examined from a sample of children exhibiting gastrointestinal clinical symptoms. This finding aligns with the prevalence of subtypes 1-4 reported in previous studies conducted in Iran and globally ( 29 ). The presence of subtypes 5 and 3 among the samples of children with diarrhea can be attributed to differences in lifestyle and reservoirs. Subtype 3 was isolated from 60% of Iranian cattle samples, and subtype 5 was found in pigs and cattle worldwide ( 30 - 32 ). The religious prohibition of pork in the Muslim population of Iran suggests the potential for zoonotic transmission of subtypes 3 and 5, highlighting the importance of public health measures to prevent the spread of these pathogens. A multitude of studies have reported subtype 1 as a pathogenic subtype in patients with gastroenteritis symptoms ( 33 ), and some studies have reported a high degree of infectivity in subtype 2. Subtype 3 has been identified as the most prevalent subtype in Iran and has been associated with AIDS and cancer patients in several studies ( 34 ). The presence of subtypes 1, 2, 3, and 5 in this study, as indicated by stool samples from children with diarrhea symptoms, aligns with the findings of previous studies ( 25 ). A previous study in this region reported the prevalence of intestinal protozoa in people with gastrointestinal discomfort as 2.2%, the average prevalence of this parasite based on common detection methods such as wet smear as 12.5%, and the low sensitivity of this method reducing the rate of parasite detection ( 35 ). The use of molecular methods directly on the stool sample can show the true prevalence of the parasite. The enigmatic nature of Blastocystis spp. may be a contributing factor to the observed increase in prevalence across most regions of the world. This increase could be attributed to either hitherto unknown modes of transmission or a lack of consensus among medical professionals regarding the pathogenesis and treatment of the condition. Conversely, it is imperative to acknowledge the rarity of this condition in clinical laboratories, where its presence must be systematically documented and reported. The presence of 7-8 Blastocystis spp. in each microscopic field indicates a need for treatment ( 36 ). The most prevalent subspecies in humans and animals are ST3 and ST5, respectively. These two subspecies have been documented in cattle in Iran, suggesting the potential for zoonotic transmission of these subspecies. Given the transmission of the parasite cyst via the fecal-oral route, further research is necessary to elucidate the precise mechanisms of transmission among humans, animals, and different hosts. This investigation should prioritize regions within Iran to obtain a comprehensive understanding of the pathogenicity and transmission dynamics of these subspecies.

Acknowledgment

We would like to express our profound gratitude to all the family members for their participation and cooperation in this study.

Authors' Contribution

The conceptualization of the study was initiated by HM and MRB, who also played a pivotal role in its design. MRB and HY were responsible for the collection of samples and the execution of tests. MZ and YSH contributed to the analysis of data and the interpretation of results. ED and SE were responsible for the drafting of the manuscript and the review of the literature. All authors have read and approved the final manuscript.

Ethics

Prior to the administration of any laboratory analysis or physical examination, written informed consent was obtained from the legal guardian of each participant. A unique identifier was assigned to each patient, and all data were kept confidential. The study protocol was approved by the Ethics Committee of Zahedan University of Medical Sciences (reference number: IR.ZAUMS.REC.1398.322).

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

The research leading to these results has received funding from the project titled "Molecular Diagnosis and Genotype Determination of Blastocystis hominis in Children by Sequencing-PCR in Zahedan, South-East Iran." This research was supported by Zahedan University of Medical Sciences (Grant agreement number "9219"). The research supervisors of Mansour Rahmati-Balaghaleh were entrusted with the management and oversight of the study.

Consent for Publication

We obtained fully informed written consent from parents of minor subjects for the publication of their clinical data.

Consent to Participate

Prior to the administration of any laboratory analysis or physical examination, written informed consent was obtained from the subject or their legal guardian. A unique identifier was assigned to each patient, and all data were kept confidential.

Data Availability

The authors affirm that the data substantiating the study's findings are accessible within the article itself and its supplementary materials.

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