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Blastocystis is the most common parasite in humans and animals. Molecular studies based on the small subunit ribosomal RNA gene (SSU-rRNA) have shown that Blastocystis isolates have 17 subtypes and nine of these subtypes have been reported in human. The objective of the present study was to identify the subtype of Blastocystis. isolated from human in Shiraz city, southern Iran.
Materials and methods
A cross-sectional study was conducted from March to August 2019. A total of 802 fecal samples from persons who were referred to health centers of Shiraz University of medical sciences were collected and tested by wet mount method to find Blastocystis. Genomic DNA was extracted from the fecal samples to be positive for Blastocystis. SSU-rRNA gene was amplified by the polymerase chain reaction. The amplicons were sequenced and compared with published sequences in GenBank using BLAST system. Phylogenetic analysis was performed using the maximum likelihood method in the MEGA 5.0 software.
The microscopy method showed that 39 out of the 802 fecal samples were positive to Blastocystis. A 500 bp fragment of SSU-rRNA was amplified from isolates after the PCR. Sequence analysis identified four subtypes (STs) of Blastocystis consisted of ST1 (32.43%), ST2 (24.32%), ST3 (35.14%) and ST7 (8.11%).
Four subtypes (STs) of Blastocystis in human was identified in our study and subtype 3 was the most prevalent subtype. The common subtype (ST3) in this study was identical to the reports from other regions of Iran. For identification of the more subtypes of Blastocystis, comprehensive molecular studies with a large number of Blastocystis isolates are suggested.
Blastocystis is one of the most common protozoan parasite with worldwide distribution which inhabits in the intestine of humans and numerous animals such as farm animals, fishes, amphibians, birds, reptiles, rodents and cockroaches.
Different morphological forms of Blastocystis have been identified and four commonly forms are granular, vacuolar, amoeboid and cystic forms. The vacuolar form is the most common form and often observed under microscopic examination.
therefore, identification of the subtypes of Blastocystis is necessary for epidemiological studies. The current study aimed to estimate the prevalence rate of Blastocystis isolated from human in Shiraz city, southern Iran and identify its subtypes by PCR-sequencing method.
2. Material and methods
2.1 Study area
Shiraz city, capital of Fars province which is located in the south of Iran (29°37′N 52°32′E), is the fifth-most-populous city of the country. It has a moderate climate, and around 300 mm of rain falls each year in the city, almost entirely in the winter months.
2.2 Samples collection
A cross-sectional study was conducted from March to August 2019. A total of 802 fecal samples were collected from persons who were referred to health centers of Shiraz University of medical sciences, Shiraz city, Southern Iran. The stool specimens were placed in a clean plastic container and transferred to the parasitology laboratory of Shiraz University of medical sciences for microscopic examination. A structured questionnaire was filled out for each sample. The design of the study, including ethical aspects, was reviewed and approved by the ethics committee at the Shiraz University of Medical Sciences (code: IR.SUMS.MED.REC.1398.012).
2.3 Microscopy method
All the stool specimens were tested by wet mount (normal saline or Lugol's iodine) method to find Blastocystis. The samples contained Blastocystis were stored at −20 °C until DNA extraction.
2.4 Molecular method
2.4.1 DNA extraction and PCR
The genomic DNA from positive stool samples of Blastocystis was extracted using Stool DNA Isolation mini kit, with Proteinase (Yekta Tajhiz Azma kit, Cat. No. YT9032) according to the manufacturer's instructions. The small subunit ribosomal RNA (SSU-rRNA) gene was amplified using the PCR reaction. The forward (Blast 505–532: 5′-GGAGGTAGTGACAATAAATC-3′) and reverse (Blast 998–1017: 5′-TGCTTTCGCACTTGTTCATC -3′) primers were used for amplification of the SSU-rRNA gene.
The PCR reaction was performed in a final volume of 25 μl. Each reaction contained 12.5 μl of the PCR master mix (2x Master Mix RED (Ampliqon, Denmark), (1.25 U Taq DNA polymerase, 200 μM of dNTPs and 1.5 mM of MgCl2), 0.5 μl of each primer (12.5 pmol), 5 μl of template DNA and 6.5 μl of double-distilled water. The temperature profile was one cycle of 95 °C for 4 min to denature the double stranded DNA, followed by 35 cycles of 95 °C for 30 s (denaturation), 54 °C for 30 s (annealing), 72 °C for 30 s (extension), and a final extension of 72 °C for 5 min. Double-distilled water (DDW) instead of template DNA was included in each set of the PCR reaction as negative control and DNA extracted from Blastocystis as positive control. Amplicons were visualized by electrophoresis on a 1.5% agarose gel and stained with Gel Red (GelRed™ Nucleic Acid Gel Stain, 10,000X in Water, cat. No. 41003).
2.4.2 Sequencing and subtype identification
The PCR products were sequenced in two directions using the same forward and reverse primers used in the PCR by Sanger sequencing method. The SSU-rRNA sequence results were edited by the Geneious software (www.geneious.com) and the consensus sequences were compared with the reference sequences available in GenBank using BLAST (Basic Local Alignment Search Tool) system (http://www.ncbi.nlm.nih.gov/) for subtype identification. A phylogenetic tree was constructed with sequences obtained in the present study along with the reference sequences deposited in GenBank using the maximum likelihood method in the Molecular evolutionary genetic analysis version 5 (MEGA 5.0) software.
Bootstrap analyses (using 1,000 replicates) were carried out to determine the robustness of the finding.
A total of 39 (4.86%) out of 802 stool samples were positive to Blastocystis by microscopy method. The individuals infected with Blastocystis consisted of 17 males (43.6%) and 22 females (56.4%) with the mean age of 45.54 ± 17.29 years old (Table 1).
Table 1Demographic characterizations, subtype and accession number of the positive samples.
The positive stool samples were examined by the PCR method and a 500 bp fragment of SSU-rRNA was amplified from 39 isolates and the positive control, and no amplification was observed in the negative control (Fig. 1). Sequence analysis was performed for 37 PCR products to identify the subtypes of Blastocystis. The consensus sequences determined in this study were deposited in GenBank with accession numbers MN396272 to MN396308. BLAST analysis confirmed the presence of four subtypes (STs) of Blastocystis: ST1 (n = 12), ST2 (n = 9), ST3 (n = 13) and ST7 (n = 3). ST3 was the most prevalent subtype in this study with a prevalence of 35.14%, followed by ST1 (32.43%), ST2 (24.32%) and ST7 (8.11%). The consensus phylogenetic tree indicated that 13 ST3 of Blastocystis obtained in the current study based on the SSU-rRNA sequences were divided into 6 haplotypes, 12 ST1 of Blastocystis into 9 haplotypes, 9 ST2 of Blastocystis into 4 haplotypes and 3 ST7 of Blastocystis into 2 haplotypes (Fig. 2).
The results of the current study reported a prevalence of 4.86% for Blastocystis isolated from humans in Shiraz city, southern Iran. Neghab et al. (2006) reported that the prevalence rate of Blastocystis was 25.4% among 39 catering staff of a university canteen in the city of Shiraz, which was higher than the rate in our study.
This may be due to the type of the diagnostic method. We used the wet mount (normal saline or Lugol's iodine) method to find Blastocystis while they used formalin-ether concentration technique for three stool samples collected from each person on three consecutive days. Study conducted on randomly 4200 stool samples obtained from patients with gastroenteritis reported a lower prevalence rate (0.5%) than that in our study.
Prevalence analysis of Blastocystis in Iran by Badparva et al. demonstrated that the total prevalence of Blastocystis sp. in Iran was 3% between 2003 and 2015, and the results showed that prevalence of Blastocystosis had a decreasing trend in Iran.
The survived population, sample size, diagnostic method and personal and community hygiene could affect the prevalence of Blastocystis.
In this study, the subtypes of Blastocystis were identified using the PCR-sequencing, while Motazedian et al. reported the genomic diversity of Blastocystis from patients in Shiraz by PCR-RFLP, which is not a suitable method for the genotype identification of Blastocystis.
Four subtypes of Blastocystis (ST1, ST2, ST3 and ST7) were identified in our study and ST3 was the most prevalent subtype. Molecular studies on Blastocystis previously conducted in Iran showed ST1, ST2, ST3, ST4, ST5, ST6 and ST7 in humans, and ST3 was reported in all studies performed in Iran.
The subtype 3 was the most prevalent subtype of Blastocystis in our study, which is similar to the several studies performed in Iran about the subtype distribution of Blastocystis sp. isolated from humans,
In the present study, 13 ST3 of Blastocystis obtained based on the SSU-rRNA sequences were divided into 6 haplotypes, and 6 isolates (H2, H3, H7, H10, H11 and L9) were identical and exhibiting 100% homology with an isolate from human in Colombia (accession no. KC294189) and Lebanon (accession no. KC294196), also one ST3 sequence (L19) showed 100% identity with Blastocystis isolated from human in Turkey (accession no. AM779044). Twelve ST1 of Blastocystis were divided into 9 haplotypes, isolate L3 showed 100% homology to an isolate from Turkey (accession no. AM779059), another two ST1 sequences (L1 and L31) were identical to those from Libya (accession nos. KF306282). Nine ST2 of Blastocystis were divided into 4 haplotypes and two isolates (H14 and L12) did not differ from an isolate collected from human in Turkey (accession no. AM779052). Two sequences identified as ST7 (L2 and L17) had the similarity with those from human in France (accession no. AY591109).
Blastocystis sp. is the most common intestinal parasite in stool sample of both symptomatic and asymptomatic individuals worldwide. The ST1, ST2, ST3 and ST7 of Blastocystis can be found both in humans and animals. In this study, ST3 was the most common subtype, followed by ST1, ST2 and ST7. However, these results cannot be generalized to the all Shiraz population. Further studies are required to determine the distribution of subtypes of Blastocystis in the more population. Because of the zoonosis of Blastocystis, the prevention and control programs in human populations are necessary to help identify the sources of Blastocystis transmission to humans. Further studies will be needed to identify the subtypes of Blastocystis in different hosts and areas.
This work was supported by the office of the Vice Chancellor for Research at Shiraz University of Medical Sciences [grant number: 97-01-01-17058 and 97-01-01-17877 ].
Declaration of competing interest
The authors declare that there is no conflict of interests.
Blastocystis in humans and animals: new insights using modern methodologies.