Molecular identification and subtypes analysis of Blastocystis sp. in South Khorasan province, Eastern Iran

doi:10.21203/rs.3.rs-4812511/v1

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Molecular identification and subtypes analysis of Blastocystis sp. in South Khorasan province, Eastern Iran

https://doi.org/10.21203/rs.3.rs-4812511/v1

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Background

Blastocystis sp. is a prevalent intestinal parasitic protest that infects both birds and animals. There are at least 17 subtypes (ST) of Blastocystis sp., with ST1-ST9 being found in humans. The correlation between specific subtypes and pathogenicity has not been definitively established. This study aimed to identify the genetic diversity within subtypes of Blastocystis sp. in stool samples collected from individuals who referred medical laboratories in the cities of Birjand, Darmian, Nehbandan, and Ferdows, Eastern Iran.

Methods

1800 stool specimens were randomly collected from referred individuals randomly selected medical laboratories of South Khorasan province. DNA was extracted and PCR, sequencing and subtyping was performed for characterization and phylogenetic analysis.

Results

A total of 118 (6.5%) cases of Blastocystis sp. through microscopic observations was confirmed. A ~620-bp segment of the SSU rRNA gene of Blastocystis sp. was amplified and were genetically evaluated using MEGA6 and DnaSP. 30 samples of Blastocystis sp-positive cases were sequenced. BLAST analyses identified 3 distinct subtypes including ST1 (11, 36.66 %), ST2 (5, 16.68 %), ST3 (14, 46.66) within our samples. Intra-subtype discrimination showed the similarity of 95.88%, 91.56% and 92.14% for ST1 and ST2, ST1 and ST3, and ST2 and ST3, respectively.Additionally, allele 4 for ST1; allele 12 for ST2; alleles 34, 36 for ST3 were detected.

Conclusions

As results, ST1 exhibited the highest nucleotide diversity (π: 0.00113), suggesting a high degree of genetic diversity within this subtype. Phylogenetic analysis showed that, subtype 3 was clustered to animal isolates from cattle and cats, indicated the potential zoonotic transmission of Blastocystissp.

Blastocystis sp

Subtyping

Phylogenetic analysis

South Khorasan province

Iran

Blastocystis sp. are single-celled parasites that are highly prevalent in humans and are often detected during routine stool examinations (1, 2). There are currently 17 identified subtypes of Blastocystis sp, designated as ST1 to ST17. These subtypes show genetic diversity and have been detected in various host species, such as humans and other animals (3, 4). The prevalence of Blastocystis sp. in Iran has been reported to range from 3–50% in various studies in addition to, higher prevalence rates have been reported in rural areas compared to urban areas (5, 6). The correlation between specific subtypes and pathogenicity has not been definitively established (7, 8). Blastocystis sp. subtypes exhibit remarkable genetic diversity, with at least 29 subtypes identified to date. While some subtypes have been associated with gastrointestinal symptoms and clinical manifestations, others have been found in asymptomatic individuals, leading to debates about their pathogenic potential (9). In humans, the majority of Blastocystis sp. infections are associated with ST1 to ST9 and ST12 (10). However, it is worth noting that infections with ST1 to ST4 are relatively more commonly seen in humans. Determining the prevalence and distribution of Blastocystis sp. subtypes is crucial for understanding their epidemiology, transmission dynamics, and clinical significance(11). Zoonotic subtypes of Blastocystis sp. are subtypes that can be transmitted between animals and humans. Some of the zoonotic subtypes of Blastocystis sp. include ST1, ST2, ST3, ST4, ST6, and ST8. These subtypes have been found in both animal and human hosts, raising concerns about potential cross-species transmission and zoonotic infections (12, 13).

Various diagnostic methods, including microscopy, culture, and molecular techniques, have been employed to detect and differentiate this parasite (14, 15). Importantly, molecular techniques like polymerase chain reaction (PCR) and sequencing have greatly enhanced our ability to identify specific species and subtypes within these parasite groups (16). The initial aim of this research was to identify different subtype’s Blastocystis sp. of the region of Khorasan. In this study, Blastocystis sp. subtypes were investigated in the individuals who are referred to medical centers' laboratories in the South Khorasan province. The presence of different Blastocystis sp. subtypes was identified and analyzed in relation to demographic information of the patient. The study aimed to understand the distribution of Blastocystis sp. subtypes with gastrointestinal issues in the patients. Each Blastocystis sp. subtype may display distinct features and potential links to health conditions, including different disorders. Additional research is necessary to investigate the impact of each subtype on human health and diseases. Moreover, Zoonotic subtypes of Blastocystis sp.are subtypes that can be transmitted between animals and humans. Which is essential for disease control strategies.

Geographical Location of the Study Area

South Khorasan Province is located in eastern Iran and borders Afghanistan. It has a total population of 898,000 people living in 11 cities (Fig. 1). Birjand city serves as the capital of the province, with about half of the population residing in the city and its suburbs (Fig. 1)

Sample collection

This cross-sectional study was performed from April 2020 to the end of March 2021 in South Khorasan Province. In this study, a total of 1800 stool specimens were collected randomly from individuals who visited 10 different medical laboratories in the cities of Birjand, Darmian, Nehbandan, and Ferdows. For every participant, we completed a demographic questionnaire that included the following information: age, gender, education, occupation, location, type of laboratory (hospital or health center), reason for referral (intestinal symptoms, asymptomatic, or regular check-up), and history of contact with livestock.

DNA Extraction

Approximately 200–250 µl of the positive Blastocystis sp. suspension were washed three times with phosphate-buffered saline (PBS) and centrifuged at 2,500 x g for 5 min, prior to extraction.

The DNA was extracted using the FAVORGEN Biotech DNA Stool Mini Kit (Taiwan) following the instructions provided by the manufacturer. The extracted DNA was then stored at -20°C until PCR analysis.

PCR amplification, Sequencing and subtyping

Molecular subtyping was conducted by utilizing the primers RD5 (5’-ATCTGGTTGATCCTGCCAGT-3’) and BhRDr (5’-GAGCTTTTTAACTGCAACAACG-3’) as described by Scicluna et al. (2006). These primers targeted a ~ 620-bp segment of the SSU rRNA gene of Blastocystis sp. PCR amplifications were carried out following standard protocols, which included an initial denaturation step at 95°C for 5 minutes, followed by 35 cycles. as follow: denaturing step at 94°C for 30 sec, annealing step of 59°C for 30 sec, and 30 sec of extension at 72°C. A final extension at 72°C was performed for 5 min. PCR products (5 µL of each) were analyzed on 1.5% agarose gel. After purification of amplicons, PCR products were sequenced with ABI 3130 sequencer

To amplify the barcoding region of the SSU rRNA gene of Blastocystis sp., PCR was conducted using specific primers as previously described. For the first reaction, outer forward (5’ GAGCTTTTTAACTGCAACAACG-3’) and reverse primers (5’ATCTGGTTGATCCTGCCAGTA-3’) were used at an annealing temperature of 58°C, with a product size of about 600 bp. The PCR was performed using the Taq DNA Polymerase Master Mix Red (Amplicon III, Denmark). The PCR products were observed by running them on 1.5% agarose gels using electrophoresis, and then the gels were stained with ethidium bromide. The secondary PCR products were sequenced using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) in a 3130xl-ABI Genetic Analyzer. All sequences were assembled and edited manually using the Chromas program version 1.0.0.1. The results were compared using Basic Local Alignment Search Tool (BLAST). Bootstrap analyses were carried out with 1000 replicates, and phylogenetic analyses were performed using the maximum likelihood method in MEGA 5.05.

Phylogenetic and Nucleotide diversity analysis

The phylogeny tree of this study was constructed by the MEGA version 11 software (17) using the MLM method and with the substitution model. In order to evaluate Neutrality and diversity indices of subtypes; haplotype (Sequence-type) diversity (Hd), Nucleotide diversity (π), number of segregating sites (S), average number of pairwise nucleotide differences (K), neutrality indices including Tajima’s D from obtained subtypes, DnaSP V5 (Rozas et al., 2003) software was used in this study.

Statistical Analysis

The collected data were analyzed using the chi-square (X2) test with the SPSS software (version 21, SPSS Inc., Chicago, IL). A p-value of less than 0.05 was considered statistically significant.

Ethical Statement

All the participants in the study completed a written informed consent before their specimens were collected. The study received approval from the ethics committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1398.741).

Demographic data

In this study, although there were more positive cases in men (63/7%) than in women 652 (36/3%), Fisher's exact test did not indicate a significant statistical correlation (P>0.05). The research revealed that individuals with lower literacy levels were at a higher risk of blastocyst infection, with a statistically significant relationship (P≤0.05). Furthermore, an association was found between age and the incidence of blastocyst infection, with a majority of positive cases occurring in individuals aged 31 to 50, which was statistically significant (P≤0.05). The table displays additional demographic variables from the study (Table 1).

Subtype and alleles analysis

Thirty samples of Blastocystis sp-positive cases were sequenced, and subsequent BLAST analysis revealed three different subtypes: ST1 (11, 36.66%), ST2 (5, 16.68%), and ST3 (14, 46.66%), as detailed in the Table. The website Pubmlst.org/ Blastocystis / was consulted to determine the alleles of these subtypes. All sequences were submitted in GenBank (Table 2). The allele analysis results showed allele 4 in ST1, allele 12 in ST2, and alleles 34 and 36 in ST3, as outlined in Table 2.

Statistical analysis indicated no significant correlation between the presence of Blastocystis sp. and season, animal contact, or location (P>0.05). Subsequent multiple sequence alignment revealed inter-subtype variations based on the sequenced SSU rRNA gene fragment. The similarity percentages were calculated as 95.88% for ST1 and ST2, 91.56% for ST1 and ST3, and 92.14% for ST2 and ST3. Notably, ST1 and ST2 isolates exhibited the highest similarity, whereas ST2 and ST3 showed the lowest similarity among the subtypes.

Phylogenetic analysis, genetic diversity and neutrality test

A phylogenetic analysis demonstrated clear differentiation among the three subtypes, with three distinct clusters identified. Maximum Likelihood (ML) analysis supported the robustness of these clusters, exhibiting highly consistent topological patterns (Fig2). Furthermore, the analysis confirmed the allelic distinctions, with ST3 showing the highest diversity of sequence types, while ST1 and ST2 displayed the least variation. Furthermore, the sequences obtained from other countries and deposited in GenBank showed similarities with Blastocystis sp. sequences from Iran in this regard, The isolates from our study are clustered with isolates from Switzerland, Malaysia, France, Egypt, Poland, and Turkey (Fig2).

Based on the DnaSP analysis results, the number of haplotypes (Hn) was 3, 2, and 2 for ST1, ST2, and ST3, respectively (Table 2). The highest haplotype diversity was observed in ST1 (Hd: 0.345), while the lowest was in ST2 (Hd: 0.400) and ST3 (Hd: 0.440). Moreover, ST1 exhibited the highest nucleotide diversity (π: 0.00113) and number of segregating sites (n=3), indicating significant genetic diversity within this subtype. Neutrality indices were calculated, showing negative Tajima’s D values in all three subtypes but were not statistically significant (P > 0.10) for ST1, ST2, and ST3. The negative values of Tajima's D suggest an excess of rare variation, which is consistent with population growth or positive selection (Table3).

Over the past twenty years, numerous studies have been reported on the prevalence of blastocysts in the worldwide, with findings indicating an increasing prevalence in both developing and developed countries (18-21). However, despite variations in distribution rates across regions, there is still no conclusive evidence on the global prevalence of Blastocystis sp. In the past two decades, multiple studies have conducted the prevalence of Blastocystis sp. worldwide, revealing an increasing trend in both developing and developed countries. Despite regional variations in distribution rates, the true global prevalence of Blastocystis sp. remains uncertain, with reported rates reaching up to 70%.(22). Studies on the biology of the parasite have revealed that Blastocystis sp.is a molecularly divergent microorganism. On the other hand, many studies have conducted, the widespread distribution of Blastocystis sp.in Iran (5, 23).

The prevalence rate of Blastocystis sp.infections in our study closely aligns with that reported in several other studies (24-26) ). Additionally, our subtype analysis indicated a frequent occurrence of subtype 3, consistent with findings in related studies (25, 27). In our research, we identified subtypes 1, 2, and 3, contrasting with studies conducted in Iran (28, 29) where subtypes like ST7 and ST6 were reported. Unlike the research by Nobilah Amelia Mohammad et al. (2017) (30), our study did not find a significant association between Blastocystis sp. prevalence and animal contact. . In our study, a notable proportion of individuals infected with Blastocystis sp.fell within the 31-50 age range, demonstrating a statistically significant correlation between this particular age group and the prevalence of Blastocystis sp. infection. This finding aligns with the study conducted by Mirjalali et al (2017) on individuals with Inflammatory Bowel Disease (IBD) patients, highlighting a correlation between the presence of Blastocystis sp. and the age range of 31-50 years (31). Our findings presented a higher prevalence of Blastocystis sp. cases in the autumn months, illustrating a significant association. In contrast, a study by Salehi et al. in (2017), highlighted a significant link between Blastocystis sp. prevalence and the spring season (32). In Algeria, a study conducted by Amina et al. in 2023(33) found a higher incidence of Blastocystis sp. cases during the fall season, with a significant correlation, aligning with the results of our study.

Studies by Alfellani et al. (2013) (34) and Ramirez et al. (2014) (35) have highlighted that allele discrimination can offer valuable insights into host specificity and geographical distribution. This is further supported by research from Ramirez et al. in 2016 and 2017 (2, 36). Additionally, the association between alleles and clinical manifestations has been proposed by Casero et al. (2015) (37). In the present study, the alleles of the identified subtypes were retrieved; revealing results including alleles 4, 12, 34, and 36 were reported from all subtypes. Our study found that the most common allele among ST1 is allele 4, This observation aligns with the results reported by Mehmet Aykar et al. in 2023. Another study, reported over 90% of ST1 cases in Iran corresponded to allele 4 (38) whereas Rahimi's research demonstrated allele 4 as the predominant variant in ST1 among domestic animals (39). In our investigation, ST2 was characterized by allele 12, in contrast, Rezaei et al. (2019) (40) indicated allele 11 as the primary allele for ST2 in Iran. This finding is in line with Ramirez et al.'s study in 2017 conducted in Colombia, where allele 12 was identified in ST2.(41), The total allele diversity among subtypes was lower compared to recent studies. It appears that allele 4 is commonly associated with ST1, while alleles 34 and 36 are frequently reported for ST3.

In our study, alleles 34 and 36 were identified as the predominant alleles for ST3 (36, 39) consistent with findings from Ramirez et al. (2017) (36, 42). Totally, allele diversity among subtypes was lower than recent studies (40). it seems that allele 4 and all 34,36 are common alleles reported from ST1 ST3 respectively (34, 43).

Interestingly, in the current study, DNAsp analysis indicated that ST1 exhibited the highest diversity, with three segregating sites compared to one segregating site in ST2 and ST3. Furthermore, the number of haplotypes (n=3) in ST1 exceeded that of the other subtypes (n=2) included in the study and exhibited the highest diversity.

In the phylogenetic tree generated using the maximum likelihood method, the subtypes isolated in our study are clustered alongside isolates from Switzerland (MW653091) and Malaysia (MH932091). Furthermore, subtype 3 from our study is positioned close to animal isolates from cattle (MW658899) and cats (MW295501). This placement suggests the potential zoonotic transmission of Blastocystis sp. Epidemiological studies indicate that ST1-ST8 and ST12 are zoonotic, ST9 is a subtype anthroponotic, while ST10, ST11, and ST13-ST26 have been exclusively found in animals (44, 45). Previous research has detected subtype 1 in various animals, and phylogenetic analysis has indicated the zoonotic potential of this subtype (46, 47). The haplotype diversity analysis revealed that subtype 1 exhibits greater diversity (48) Moreover, Tajima test results, showed a negative value for all subtypes. This indicates a selective pressure aimed at increasing the population of all subtypes. A study of parasitic infections among primary school children in a rural community in central Thailand found a high prevalence of Blastocystis sp. subtype 1. Additionally, Blastocystis sp. subtype 1 was detected in the drinking water (43). in this research, no association was discovered between the clinical manifestations of patients and the existence of Blastocystis sp., possibly because individuals referred to the medical care in that region, did not display any particular disorder. The primary aim of this investigation was to evaluate the presence of Blastocystis sp and the distribution of its subtypes in Khorasan Province. Examining a gastrointestinal disorder and its correlation with the presence of Blastocystis sp. may lead to encouraging findings.

Totally, the efforts to characterize subtype distributions and genetic variability in Blastocystis sp. among individuals in various parts of the world are important, a deeper understanding of the impact of Blastocystis sp. on human health and disease may be achieved by studying Blastocystis sp. genomes.

Genetic diversity analysis revealed that ST1 exhibited the highest number of haplotypes and the largest haplotype diversity followed by ST2 and ST3, which, suggesting a high degree of genetic diversity within this subtype. Phylogenetic analysis showed that, subtype 3 was clustered to animal isolates from cattle and cats, indicated the potential zoonotic transmission of Blastocystis sp. This study aimed to identify different subtypes of Blastocystis sp. in the region of the South Khorasan province, Iran.

Acknowledgments

The present article is financially supported by "Research Department of the School of Medicine Shahid Beheshti University of Medical Sciences" (Grant No 25680). We would like to express our special thanks for the cooperation and support of all the personnel of the Deputy of Research and Technology of Birjand University of Medical Sciences, South Khorasan province, Iran.

Author contributions

Conceived and designed: AH. Sample collection and questionnaire: MRB. Methodology and implementation: MRB, MM. Statistical and Phylogenetic analysis: EJ, SN. Reviewing and editing the manuscript: AH, MRB. All authors read and approved the final version of the manuscript.

Funding

This work was supported by Research Department of the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (Grant number 25680).

Data availability

All generated data from the current study are included in the article.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Informed consent

Informed consent was obtained from all subjects and/or their legal guardian(s). All authors declare that they have seen and approved the submitted version of this manuscript.

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Table 1 Demographic characteristics of the study participants, related to individuals referred to different medical laboratories in South Khorasan Province

Demographic variables		Number	Percentage (%)	Number of *Blastocystis* spp	Percent of *Blastocystis* spp (%)	P _value
Gender	male	1148	63/7	75	63/6	P>0.05
Gender	Female	652	36/3	43	36/4	P>0.05
Drinking water source	Purified	1798	99/8	118	100	P>0.05
Drinking water source	Untreated	2	0.2	0	0	P>0.05
Animal contact	YES	1690	93/8	110	93/2	P>0.05
Animal contact	NO	110	6/2	8	6/8	P>0.05
Location	City	1572	87/3	96	81/4	P>0.05
Location	Village	228	12/7	22	18/6	P>0.05
Level of Education	Illiterate to elementary	920	51/1	83	70/3	P≤0.05 ***
	High school	750	41/7	27	22/9
	University	130	7/2	8	6/8
Reason for referral	Check up	324	18	12	10/2	P≤0.05 ***
	Digestive	1032	57/3	82	69/5
	Non - digestive	444	24/7	24	20/3
Age	6>	65	3.6	1	0/8	P≤0.05 ***
	7-12	78	4.3	5	4/2
	13-18	157	8.7	11	9/3
	19-30	512	28.4	27	22/9
	31-50	691	38.5	43	36/5
	51<	297	16.5	31	26/3
Season	Spring	180	25	8	6/8	P≤0.05 ***
	Summer	180	25	23	19/5
	Autumn	180	25	49	41/5
	Winter	180	25	38	32/2

*** Statistically significant p<0.05

Table 2 GenBank accession numbers for the different Blastocystis sp. subtypes and frequency of Blastocystis sp. alleles among subtypes obtained in the current study.

No	Organisms	subtype	accession number	Allele
1	Blastocystis sp	ST1	(OL958706)	Allele 4
2	Blastocystis sp	ST1	(OL960191)	Allele 4
3	Blastocystis sp	ST1	(OL960201)	Allele 4
4	Blastocystis sp	ST1	(OL960584)	Allele 4
5	Blastocystis sp	ST1	(OL962696)	Allele 4
6	Blastocystis sp	ST1	(OL962725)	Allele 4
7	Blastocystis sp	ST1	(OL963594)	Allele 4
8	Blastocystis sp	ST1	(OL963607)	Allele 4
9	Blastocystis sp	ST1	(OL963687)	Allele 4
10	Blastocystis sp	ST1	(OL963691)	Allele 4
11	Blastocystis sp	ST1	(OL981378)	Allele 4
12	Blastocystis sp	ST2	(OL960197)	Allele 12
13	Blastocystis sp	ST2	(OL960547)	Allele 12
14	Blastocystis sp	ST2	(OL963525)	Allele 12
15	Blastocystis sp	ST2	(OL963561)	Allele 12
16	Blastocystis sp	ST2	(OL963690)	Allele 12
17	Blastocystis sp	ST3	(OL957050)	Allele 36
18	Blastocystis sp	ST3	(OL958702)	Allele 36
19	Blastocystis sp	ST3	(OL958709)	Allele 36
20	Blastocystis sp	ST3	(OL958711)	Allele 34
21	Blastocystis sp	ST3	(OL960194)	Allele 34
22	Blastocystis sp	ST3	(OL960199)	Allele 34
23	Blastocystis sp	ST3	(OL960555)	Allele 34
24	Blastocystis sp	ST3	(OL960563)	Allele 36
25	Blastocystis sp	ST3	(OL960566)	Allele 34
26	Blastocystis sp	ST3	(OL962697)	Allele 34
27	Blastocystis sp	ST3	(OL962722)	Allele 34
28	Blastocystis sp	ST3	(OL962723)	Allele 34
29	Blastocystis sp	ST3	(OL962724)	Allele 34
30	Blastocystis sp	ST3	(OL963606)	Allele 34

No competing interests reported.

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Molecular identification and subtypes analysis of Blastocystis sp. in South Khorasan province, Eastern Iran

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Abstract

Figures

Introduction

Materials and methods

Geographical Location of the Study Area

Sample collection

DNA Extraction

PCR amplification, Sequencing and subtyping

Phylogenetic and Nucleotide diversity analysis

Statistical Analysis

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1