Identification and Genetic Characterization of Blastocystis Species in Patients from Makkah, Saudi Arabia

Figures & data

Table 1 Gender and Age Group of Blastocystis sp.-Infected and Uninfected Patients

Age Group (N)	Females (N=35)		Males (N=105)
	Infected	Uninfected	Infected	Uninfected
<20 yrs. (4)	0 (0%)	2 (5.7%)	1 (0.95%)	1 (0.95%)
21–40 yrs. (94)	16 (45.7%)	6 (17.1%)	54 (51.4%)	18 (17.1%)
41–60 yrs. (37)	2 (5.7%)	8 (22.9%)	21 (20%)	6 (5.7%)
>60 yrs. (5)	1 (2.9%)	0 (0%)	1 (0.95%)	3 (2.9%)
Total (140)	19 (54.3%)	16 (45.7%)	77 (73.3%)	28 (26.7%)

Notes: Stool samples from patients were examined microscopically for the presence of Blastocystis sp. The numbers (N) of infected and uninfected patients are presented according to gender and age group.

Figure 1 Median joining network sequence analysis of SSU rRNA gene-PCR products demonstrated different haplotypes (H1-15) within Blastocystis sp. subtypes. Maximum parsimony (MP) analysis was used as the optional post-processing calculation using Network 4.6.1.3 software. Each haplotype represents a distinct subtype. The haplotypes are numbered from H1 to H15.

Table 2 Inter- and Intra-Subtype Genetic Variations Characterize Blastocystis sp. Subtypes/Haplotypes

	ST2	ST3	ST3	ST3	ST3	ST3	ST3	ST3	ST3	ST1	ST1	ST1	ST1	ST1	ST1
ST2	0.000
ST3	0.1950	0.000
ST3	0.2133	0.0236	0.000
ST3	0.2041	0.0212	0.0284	0.000
ST3	0.1950	0.0164	0.0212	0.0236	0.000
ST3	0.2133	0.0188	0.0260	0.0260	0.0332	0.000
ST3	0.2163	0.0308	0.0356	0.0332	0.0332	0.0236	0.000
ST3	0.2071	0.0117	0.0236	0.0308	0.0188	0.0260	0.0332	0.000
ST3	0.2102	0.0260	0.0284	0.0308	0.0332	0.0117	0.0236	0.0308	0.000
ST1	0.1315	0.2010	0.1920	0.2010	0.2041	0.2102	0.2257	0.2102	0.2071	0.000
ST1	0.0991	0.1830	0.1950	0.1950	0.1890	0.1980	0.2102	0.1920	0.2010	0.0529	0.000
ST1	0.1343	0.2163	0.2133	0.2194	0.2226	0.2288	0.2352	0.2226	0.2320	0.0405	0.0705	0.000
ST1	0.1371	0.1950	0.2041	0.2102	0.2133	0.2102	0.2194	0.2071	0.2163	0.0554	0.0680	0.0284	0.000
ST1	0.1483	0.2163	0.2288	0.2194	0.2352	0.2288	0.2480	0.2226	0.2352	0.0731	0.0834	0.0504	0.0332	0.000
ST1	0.1151	0.1950	0.2071	0.2071	0.2071	0.2102	0.2163	0.2071	0.2133	0.0260	0.0454	0.0454	0.0529	0.0756	0.000

Notes: The JC model with Mega X software program was used for the analysis of sequences of PCR products. Sequence analysis identified inter-subtype genetic variations with a minimum JC=0.0991, between ST1 and ST2 subtypes, to a maximum JC=0.248 between ST1 and ST3. High intra-subtype genetic variations were also detected between ST1 sequences (JC=0.0834) followed by ST3 (JC=0.0356).

Table 3 Genetic Variability of Blastocystis sp. Haplotypes

Parameters	ST3	ST1
Number of mutations	28	67
Parsimony informative sites	15	26
Nh (haplotype number)	8	6
Hd (haplotype diversity)	1.000	1.000
π (nucleotide diversity)	0.02243	0.05981
K (average number of nucleotide differences)	10.071	26.733
S (number of polymorphic sites)	21	54
Tajima’s test	−0.35616	−0.57236
Fu’s Fs	−2.160	−0.19732

Notes: Several parameters were used to identify genetic variations in ST1 and ST3 haplotypes. ST2 could not be analyzed as it was obtained in only one sample.

Table 4 GenBank References for the Blastocystis sp. Subtypes Used to Construct the Phylogenetic Tree

No.	Accession no.	Host	Subtype	Reference
1.	U51151	Human	1	[54]
2.	AB070989	Human	1	[55]
3.	AB091241	Chicken	1	[55]
4.	EU445492	Human	1	[56]
5.	JQ665860	Human	1	[57]
6.	JQ665863	Human	1	[57]
7.	JQ665846	Human	1	[57]
8.	JQ665867	Human	1	[57]
9.	EF200010	Human	2	[58]
10.	AB070987	Human	2	[55]
11.	AB070988	Human	3	[55]
12.	AB107965	Cattle	3	[59]
13.	EU445494	Human	3	[56]
14.	AB071000	Rat	4	[55]
15.	AY244620	Human	4	[28]
16.	AB107966	Cattle	5	[59]
17.	EU445485	Chicken	6	[56]
18.	JQ665850	Human	6	[57]
19.	AB070991	Human	7	[55]
20.	AY135412	Duck	7	[60]
21.	AF408425	Human	9	[28]
22.	AF408426	Human	9	[28]
23.	KC148207	Camel	10	[12]
24.	KC148205	Cattle	14	[12]
25.	KC148210	Camel	15	[12]
26.	KU147329	Human	1	[61]
27.	AM275364	Human	7	[62]
28.	KF242029.1	Human	1	[63]
29.	MN876884.1	Cattle	10	[64]
30.	MN014064.1	Human	2	[64]

Figure 2 Phylogenetic relationship of Blastocystis sp. haplotypes identified in the current study to other Blastocystis sp. isolates, based on SSU rRNA gene sequences. The reference GenBank sequence accession numbers of all isolates are shown, including our haplotypes accession numbers in bold. Proteromonas lacertae served as the outgroup sequence. *Indicates the haplotypes identified in the current study.

Pintong A, Sunyanusin S, Prasertbun R, et al. Blastocystis subtype 5: predominant subtype on pig farms, Thailand. Parasitol Int. 2018;67(6):824–828. doi:10.1016/j.parint.2018.08.009

 Web of Science ®Google Scholar

Arisue N, Hashimoto T, Yoshikawa H. Sequence heterogeneity of the small subunit ribosomal RNA genes among blastocystis isolates. Parasitology. 2003;126(Pt 1):1–9. doi:10.1017/s0031182002002640

 Web of Science ®Google Scholar

Rivera WL. Phylogenetic analysis of Blastocystis isolates from animal and human hosts in the Philippines. Vet Parasitol. 2008;156(3):178–182. doi:10.1016/j.vetpar.2008.06.001

 Web of Science ®Google Scholar

Thathaisong U, Siripattanapipong S, Mungthin M, et al. Identification of Blastocystis subtype 1 variants in the home for girls, Bangkok, Thailand. Am J Trop Med Hyg. 2013;88(2):352–358. doi:10.4269/ajtmh.2012.12-0237

 Web of Science ®Google Scholar

Leelayoova S, Siripattanapipong S, Thathaisong U, et al. Drinking water: a possible source of blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand. Am J Trop Med Hyg. 2008;79(3):401–406. doi:10.4269/ajtmh.2008.79.401

 Web of Science ®Google Scholar

Abe N. Molecular and phylogenetic analysis of Blastocystis isolates from various hosts. Vet Parasitol. 2004;120(3):235–242. doi:10.1016/j.vetpar.2004.01.003

 Web of Science ®Google Scholar

Yoshikawa H, Wu Z, Kimata I, et al. Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitol Res. 2004;92(1):22–29. doi:10.1007/s00436-003-0995-2

 PubMed Web of Science ®Google Scholar

Noël C, Peyronnet C, Gerbod D, et al. Phylogenetic analysis of Blastocystis isolates from different hosts based on the comparison of small-subunit rRNA gene sequences. Mol Biochem Parasitol. 2003;126(1):119–123. doi:10.1016/S0166-6851(02)00246-3

 Web of Science ®Google Scholar

Alfellani MA, Taner-Mulla D, Jacob AS, et al. Genetic diversity of Blastocystis in livestock and zoo animals. Protist. 2013;164(4):497–509. doi:10.1016/j.protis.2013.05.003

 Web of Science ®Google Scholar

Villegas-Gómez I, Martínez-Hernández F, Urrea-Quezada A, et al. Comparison of the genetic variability of Blastocystis subtypes between human carriers from two contrasting climatic regions of México. Infect Genet Evol. 2016;44:334–340. doi:10.1016/j.meegid.2016.07.036

 Web of Science ®Google Scholar

Stensvold CR, Traub RJ, von Samson-himmelstjerna G, Jespersgaard C, Nielsen HV, Thompson RCA. Blastocystis: subtyping isolates using pyrosequencing technology. Exp Parasitol. 2007;116(2):111–119. doi:10.1016/j.exppara.2006.12.002

 Web of Science ®Google Scholar

Bart A, Wentink-Bonnema EMS, Gilis H, et al. Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands. BMC Infect Dis. 2013;13(1):2–7. doi:10.1186/1471-2334-13-389

 Web of Science ®Google Scholar

Sharifi Y, Abbasi F, Shahabi S, Zaraei A, Mikaeili F, Sarkari B. Comparative genotyping of Blastocystis infecting cattle and human in the south of Iran. Comp Immunol Microbiol Infect Dis. 2020;72:101529. doi:10.1016/j.cimid.2020.101529

 Web of Science ®Google Scholar