Sci Parasitol 19(1-2):23-33, December 2018 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE

Subtyping of Blastocystis Isolates from Cultivated Human Stool Samples in Province,

Soumia Sebaa1,5, Mehtap Demirkazık2, Djamel Baroudi3, Said Amer4, Mohamed Debagha5, Ahcene Hakem1, Ismail S. Koltas 2

1 – Laboratory of Exploration and Valorization of Steppic Ecosystems, Faculty SNV, University of Ziane Achour, 17000 , Algeria. 2 – Department of Parasitology, School of Medicine, University of Çukurova, Balcalı, 01330, Adana, Turkey. 3 – Ecole Nationale Supérieure Vétérinaire, Rue Issad Abbes, El-Alia, , Algeria. 4 – Department of Zoology, Faculty of Science, Kafr El Sheikh University, Kafr El Sheikh 33516, Egypt. 5 – Private Laboratory of Medical Analyzes, Dr. Debagha El Gharbia, Laghouat, Algeria. Correspondence: Tel. +905353059393, Fax +903223386572, E-mail [email protected]

Abstract. Blastocystis is an ubiquitous parasite, however, little is known on occurrence and subtypes (ST) of this protist in Algeria. This study was designed to determine the prevalence and subtypes of Blastocystis parasites in Laghouat province, Algeria. A total of 287 stool samples collected from symptomatic and asymptomatic patients were screened by microscopy and subtyped by PCR using sequenced-tagged site (STS) primers. Results indicated that the overall infection rate was 32.1% (92/287), with higher occurrence in symptomatic patients (80.4%; 74/92) compared to that in asymptomatic ones (19.6%; 18/92). Also, the infection rate was higher in patients from rural areas (66.3%; 61/92) compared to that in those from urban regions (33.7%; 31/92) and in patients used to consume tap water compared to those used to drink bottled water (77.2 vs 22.8%). Subtyping of 25 symptomatic and 5 asymptomatic samples indicated a higher prevalence of ST1 (63.3%), followed by ST4 (23.3%), ST2 (13,3%), ST7 (13.3%), ST3 (10%), and ST5 (6.7%). Mixed subtypes were identified in 30% (9/30) of analyzed samples. Findings of this study suggest that Blastocystis is a common parasite in humans in Algeria with anthroponotic and zoonotic transmission cycles were both likely to occure. Drinking of tap water and contact with animals were high risk factors in the infection with this parasite. To the best of our knowledge, this is the first report on subtyping of Blastocystis in Algeria.

Keywords: Blastocystis; Prevalence; PCR; Subtype; Algeria. Received 12.11.2018. Accepted 14.12.2018.

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Introduction determined by microscopy and subtyping was done on in vitro cultured fecal samples by PCR Blastocystis (Brumpt, 1912) is a cosmopolitan analysis using Sequenced-Tagged Site (STS) intestinal parasite of humans and a wide range primers. of animals (Stenzel and Boreham, 1996; Noel et al., 2005). The infection rate varies widely from Materials and methods one region to another, with occurrence in developing countries is commonly higher than Ethical approval that reported from developed ones (Tan, 2008; El Safadi et al., 2014). Although most infections The study was ethically approved by the running asymptomatic, the clinical signs Faculty of Science of Nature and Life, Djelfa include abdominal pain, watery diarrhoea, University, Algeria (Ref:AT04/E.V.E.S/2017) anorexia, perianal pruritus and urticaria (Al- and informed consent was obtained from each Fellani et al., 2007; Kaya et al., 2007; Beyhan et patient or guardian of the children patients al., 2015). Irritable bowel syndrome and participated in this study. inflammatory bowel disease may also be associated with the presence of Blastocystis Study area and sample collection (Yakoob et al., 2004; Dogruman-Al et al., 2009a). This cross-sectional study was carried out in the province of Laghouat situated in the centre Fresh microscopic examination of Blastocystis of the country at 400 km to the south of the is being low sensitive due to the pleomorphic capital Algiers, between latitude 33° 48’ north nature of the parasite including granular, and longitude 02° 53’ east. This province is vacuolar, cystic and amoebic shapes. Vacuolar characterized by an agro-pastoral activity and and granular forms are seen more commonly covers about 25052 km2 for a population in fecal samples and cultures while the estimated to 520188 inhabitants. amoeboid forms and the cyst stages are seen less frequently (Tan, 2008). Up to this end in Collection of samples vitro culture is reported to be more sensitive than microscopic examination in several A total of 287 stool samples were collected studies (Dogruman-Al et al., 2009b; Dagci et al., during the October 2017 to March 2018 from 2014). Recently, molecular biological analyses symptomatic and asymptomatic patients aged using stools directly or after culturing have 1-78 yrs (24.42±18.36), who attended to been described as a sensitive and clinical laboratories for copro-parasitological discriminatory tool (Snowden et al., 2000; examination. Each patient or his guardian Wong et al., 2008; Fouad et al., 2011; filled a datasheet that included patient Yoshikawa et al., 2011; Koltas and Eroglu, identification (sex and age), clinical 2016). Seventeen subtypes have been symptoms, geographical origin (urban or recognized based on PCR/sequence analysis of rural), and risk factors. the small subunit (SSU) rRNA (Yoshikawa et al., 2004; Skotarczak, 2018). Whereas subtypes 1- Stool examination 9 have been reported in humans, other subtypes have been described from other Direct-light microscopy (DLM) and Modified animal hosts (Hussein et al., 2008; Al-Fellani et Ziehl-Neelsen (MZN) stain al., 2013; El Safadi et al., 2014; Ben Abda et al., 2017). Individual fecal samples were emulsified in one drop of normal saline (0,9%) and one drop of Yet, little information on distribution and Lugol’s iodine solution and subjected for direct genetic profiling of Blastocystis are available microscopic investigation (DLM). Additional from Algeria. Therefore, the present study individual fecal smears were fixed with aimed to determine the distribution and methanol and stained by modified Ziehl- subtypes of Blastocystis in Laghouat Province, Neelsen (MZN) as previously described (El- Algeria. Up to this end, infection rate was Marhoumy et al., 2015; Farghaly et al., 2017).

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For the quantitative criteria, Blastocystis was staning. The fragments were separated by size reported as abundant when detected number ≥ using of a DNA ladder (100–1,000 bp DNA 5/high-power (X400 magnification) field markers, BioBasic). (Zierdt, 1991). Statistical analysis Xenic in vitro culture (XIVC) Obtained data were analyzed statistically Two xenic culture mediums were used in this using the XLStat (2017) software. Pearson’s study: Jones’ and Modified Boeck and Chi-square test was applied to determine Drbohlav Locke-egg (LE). Approximately 50 association between variables. Values were mg of stool sample was inoculated in 5 ml considered significant at P < 0,05. The Kappa screw caped tube with medium of Jones coefficient (K) was used to calculate the (Jones, 1946) and Locke-egg (LE) media agreement between the different methods containing 10% horse serum, and incubated at used in this study with K< 0 indicating poor 37C° (Saksirisampant et al., 2010) for 48-72 agreement, 0.0 to 0.20 slight agreement, 0.21 hours. Microscopically positive cultures were to 0.40 fair agreement, 0.41 to 0.60 moderate subsequently sub-cultured into fresh medium agreement, 0.61 to 0.80 substantial once every 5 days. A representative number agreement, and 0.81 to 1.00 perfect (30 samples, including 25 symptomatic agreement (Landis and Koch, 1977; Dogan et patients and 5 asymptomatic patients) of al., 2017). The 95% confidence intervals (CI) positive Blastocystis cultures were shipped to for sensitivity, specificity, accuracy, positive University of Çukurova, School of Medicine, predictive value (PPV), negative predictive Department of Parasitology, Adana, Turkey value (NPV) and prevalence of infection were for subtyping. calculated using Medcalc website: https://www.medcalc.org/calc/diagnostic_tes DNA extraction from culture and PCR t.php. amplification Results Genomic DNA was extracted from 30 Blastocystis positive culture samples using Obtained result indicated that the overall QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, infection rate of Blastocystis in Laghouat Germany) according to the manufacturer’s province, Algeria, was 32.1% (92/287; 95% instructions. The PCR was performed using CI: 26.7% -37.8%). The relationships of the subtype-specific sequence tagged site (STS) Blastocystis infected cases with demographic primers; SB83 (ST1), SB155 (ST2), SB227 factors in the study area are presented in (ST3), SB332 (ST4), SB340 (ST5), SB336 (ST6), table 2. Of the 92 patients harboring and SB337 (ST7) as described by Yoshikawa et Blastocystis, 53 (57.6%) were males and 39 al. (2004). Primer sequences of Blastocystis are (42.4%) were females, the differences of illustrated in table 1. PCR reaction mixtures frequency were not statistically significant (X2 (25 μl of total volume) consisted of PCR buffer = 7.01; p = 0.26). The median (min ± max) age 1X [10 mM Tris-HCl, pH 8.8, and 50 mM KCl], of patients infected with Blastocystis was 13.5 1.5 mM MgCl2, 2.5 U/μl of Taq DNA (2 ± 72years) and the highest infection rate polymerase (Fermantas, SB38), 1.25 μM of (37%) was found in the 0-9 age group that each dNTPs (Fermantas, RO191), 0.5 pmol of decreased gradually with the advance of age each primer, and 5 μl of the DNA sample. The (X2 = 216.5; p < 0.001). Additionally, the PCR conditions consisted of a denaturation prevalence of Blastocystis was significantly cycle at 94°C for 5 min, 40 cycles of higher among symptomatic patients denaturation at 94°C for 30s, annealing at 57°C compared to asymptomatic ones (80.4 vs for 30s and extention at 72°C for 60s. An 19.6%; X2 = 90.093; P = 0,007). Recorded additional elongation cycle at 72°C for 5 min clinical signs of symptomatic cases included was included. The PCR products were abdominal pain (75.7%), diarrhea (32.4%), electrophoresed on 2% agarose gel and vomiting (14.9%), nausea (4.1%) and perianal visualized by UV light after ethidium bromide pruritus (2.7%).

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Univariate analysis of the risk association for was a perfect agreement between Locke-egg Blastocystis in this study showed that those (LE) and Jones medium (K = 0.95; P < 0.0001) who were living in rural area (66.3%), who and moderate agreement between Locke-egg were consuming tap water (77.2%) and who (LE) and DLM (K = 0.42; P < 0.0001) and also were raising animal (37%) had a higher risk of moderate agreement between Locke-egg (LE) acquiring Blastocystis compared to and MZN stain (K = 0.46; P < 0.0001). For the counterparts who were living in urban areas, comparison between the three techniques used consuming bottled water and had no animal regarding the morphological details; the Iodine contact (table 2). staining technique identified the vacuolar, granular and cystic forms (figure 1A), MZN The number of Blastocystis organisms ≥ 5/field staining technique identified only vacuolar and was found in 19 (76%) of the 25 patients with cystic forms (figure 1B). While using in vitro gastrointestinal symptoms. In contrast 6 culture, nearly all forms of Blastocystis were symptomatic and 5 asymptomatic patients identified as vacuolar, granular, cystic and have intensity <5 organism/field (x400 amoeboid forms (figure 2). magnification) (table 3). Statistical analysis indicated that there is strong association Subtyping analysis revealed the occurrence of between intensity of infection and 6 subtypes including ST1 in 13/30 (43.3%), gastrointestinal complaints (X2 = 10.364; p = ST4 in 3/30 (10%), ST7 in 3/30 (10%) and ST3 0.001). in 2/30 (6.7%). Also, nine samples (30%) have two different subtypes including ST1/ST2 in Comparison of the sensitivity and specificity of 2/30 (6.7%), ST1/ST4 in 2/30 (6.7%), different diagnostic techniques in this study ST1/ST3 in 1/30 (3.3%), ST1/ST5 in 1/30 showed that in vitro culture is more sensitive (3.3%), ST2/ST4 in 1/30 (3.3%), ST2/ST7 in than DLM or MZN stain. Locke-egg (LE) 1/30 (3.3%), and ST4/ST5 in 1/30 (3.3%). medium identify all positive cases of infection with Blastocystis 32.1% (92/287), and Jones’ Analysis of the variable risk factors potentially medium detect 30% (86/287), while the involved in the distribution of Blastocystis detection rate with DLM was 11.1% (32/287) single and mixed subtypes was statistically and MZN stain was 12.2% (35/287). significant (p < 0.0001) (table 5). For the quantitative criteria the number of Blastocystis Using Locke-egg (LE) medium as the gold organism did not correlate with the standard the sensitivity of DLM, MZN and Jones distribution of subtypes (X2 = 8.026; p = 0.626). medium were 34.8%, 38% and 93.5% Figure 3 shows clustering of the detected respectively and specificity was 100% for all subtypes based on SSU rRNA phylogeny using (table 4). Statistical analysis showed that there agarose gel fraction size.

Table 1. Primer sequences of Blastocystis

STS primer sets Sequences of forward (F) Product length GenBank Subtypes and reverse (R) primers (bp) accession no. SB83 F GAAGGACTCTCTGACGATGA 351 AF166086 1 R GTCCAAATGAAAGGCAGC SB155 F ATCAGCCTACAATCTCCTC 650 AF166087 2 R ATCGCCACTTCTCCAAT SB227 F TAGGATTTGGTGTTTGGAGA 526 AF166088 3 R TTAGAAGTGAAGGAGATGGAAG SB332 F GCATCCAGACTACTATCAACATT 338 AF166091 4 R CCATTTTCAGACAACCACTTA SB340 F TGTTCTTGTGTCTTCTCAGCTC 704 AY048752 5 R TTCTTTCACACTCCCGTCAT SB336 F GTGGGTAGAGGAAGGAAAACA 317 AY048751 6 R AGAACAAGTCGATGAAGTGAGAT SB337 F GTCTTTCCCTGTCTATTCTGCA 487 AY048750 7 R AATTCGGTCTGCTTCTTCTG

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Table 2. The relationships of the Blastocystis infected cases with demographic factors in Laghouat province, Algeria

Infected Characteristic number=92 95% CI X2 P-value (frequency %) Gender Male 53 (57.6%) 47.5-67.7 7.201 0.126 NS Female 39 (42,4%) 32,3-52,5 0-9 34 (37%) 27.1-46.8 10-19 27 (29.4%) 20-38.7 20-29 11 (12%) 5.3-18.6 Age group 30-39 6 (6.5%) 1.5-11.6 216.501 p< 0.001 (year) 40-49 9 (9.8%) 3.7-15.9 50-59 4 (4.4%) 0.2-8.5 60 + 1 (1.1%) 0-3.2 Clinical Symptomatic 74 (80.4%) 72.3-88.5 90.093 p=0.007 manifestation Asymptomatic 18 (19.6%) 11.5-27.7 Area Urban 31 (33.7%) 24-43.4 4.054 0.399 NS Rural 61 (66.3%) 56.7-76 Drinking water Tap water 71 (77.2%) 68.6-85.7 4.411 0.353 NS resource Bottled water 21 (22.8%) 14.3-31.4 Raising animal Yes 34 (37%) 27.1-46.8 1.228 0.873 NS No 58 (63%) 53.2-72.9 NS: not significant.

A B

Figure 1. (A): Iodine stained fecal smear showing vacuolar form (arrow) and granular form (star) of Blastocystis (X 400). (B): MZN stained fecal smear showing multiple vacuolar forms (arrow) and cystic forms (star) of Blastocystis (X1000).

A B

Figure 2. (A): In vitro culture showing vacuolar forms (arrow) and cyst form (star) of Blastocystis (X400). (B): In vitro culture showing binary fission of Blastocystis (X400).

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M ST1 M ST2 M ST3

M ST4 M ST5 M ST7

Figure 3. Agarose gel electrophoresis of polymerase chain reaction products, according to STS primers. M; 100 bp DNA marker (Biobasic).

Discussion reported from African countries including Senegale, Côte d'Ivoire, Madagascar and In the present study, copro-parasitological Nigeria (El Safadi et al., 2014; Poulsen et al., investigation of samples from patients from 2016; D'Alfonso et al., 2017; Greigert et al., Laghouat Province, Algeria, revealed the 2018) as well as Asian United Arab Emirates presence of Blastocystis in 92/287 (32.1%). (44.4%) (AbuOdeh et al., 2016). Interestingly, These results are in more or less concordance results of the current study indicated higher with those reported from different infection (37%) in the age group 0-9 years, geographical regions. A similar Blastocystis contrasting those reported by M'bondoukwé et infection rate (35%) was reported from al. (2018) that adults had a higher prevalence northern Spain (Paulos et al., 2018), however, a of Blastocystis in Gabon. Infection with lower rate (23-28%) was reported from Blastocystis is a multifactorial issue and is more Columbia, Brazil and Thailand (Espinosa likely to be associated with sanitary Aranzales et al., 2018; Seguí et al., 2018; infrastructures, hygienic levels, occupational Yowang et al., 2018). On the other hand, a activities, age, and basic health conditions. higher rate of Blastocystis 58-100% was These factors may be individually or

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collectively implicated for the occurrence of (LE) culture medium showed that prevalence infection reported in the present study. of Blastocystis is 32.1% in Laghouat, Algeria by high sensitivity and specificity. Many previous In addition, the vacuolar form was the most study showed that culture is more sentive common to be detected by all techniques used. compared to fecal smears and concentration While the gold standard method used to methods (Leelayoova et al., 2002; Suresh and identify Blastocystis is accepted as Locke-egg Smith, 2004).

Table 3. Blastocystis subtype results of the 30 Algerian samples and demographic factors

No. Clinical Water Blastocystis/ Subtype Raising animal Area Code manifestation resource high-power field (x400) A1 ST1 Abdominal pain No Urban Bottled ≥5 water A2 ST2/ST7 Diarrhea+ Yes Rural Tap water ≥5 abdominal pain A3 ST1/ST2 Asymptomatic Yes Rural Tap water <5 A4 ST1/ST2 Diarrhea+ Yes Urban Tap water ≥5 abdominal pain A5 ST1/ST5 Constipation No Rural Tap water <5 A6 ST1/ST4 Diarrhea+ No Urban Tap water ≥5 abdominal pain A7 ST1 Abdominal pain No Rural Tap water ≥5 A8 ST1 Abdominal pain Yes Rural Bottled ≥5 water A9 ST7 Diarrhea+ Yes Urban Tap water ≥5 abdominal pain A10 ST4 Asymptomatic Yes Urban Tap water <5 A11 ST7 Abdominal pain No Rural Bottled <5 water A12 ST1 Asymptomatic Yes Urban Tap water <5

A13 ST1 Abdominal pain+ No Rural Tap water ≥5 Vomiting A14 ST1/ST4 Abdominal pain Yes Urban Tap water ≥5 A15 ST2/ST4 Abdominal pain No Rural Bottled ≥5 water A16 ST1 Abdominal pain Yes Urban Tap water ≥5 A17 ST4/ST5 Abdominal pain Yes Rural Bottled <5 water A18 ST1 Diarrhea No Rural Tap water ≥5 A19 ST1 Vomiting No Rural Tap water <5 A20 ST1/ST3 Flatulence Yes Urban Tap water <5 A21 ST1 Asymptomatic No Urban Tap water <5 A22 ST1 Diarrhea+ Yes Rural Tap water ≥5 abdominal pain A23 ST1 Diarrhea+ Yes Rural Tap water ≥5 abdominal pain A24 ST1 Asymptomatic Yes Rural Tap water <5 A25 ST3 Vomiting Yes Rural Tap water <5 A26 ST3 Abdominal pain Yes Rural Bottled ≥5 water A27 ST4 Abdominal pain Yes Rural Tap water ≥5 A28 ST7 Diarrhea No Rural Tap water ≥5 A29 ST1 Abdominal pain Yes Rural Tap water ≥5 A30 ST4 Diarrhea+ Yes Rural Tap water ≥5 abdominal pain

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Table 4. Sensitivity and specificity of using different methods and their agreement with Boeck and Drbohlav (LE) medium

DLM MZN Jones medium Sensitivity% (95% CI) 34.8 (25.2-45.4) 38 (28.1-48.8) 93.5 (86.3-97.6) Specificity% (95% CI) 100 (98.1- 100) 100 (98.1- 100) 100 (98.1-100) NPV% (95% CI) 76.5 (73.7-79.1) 77.4 (74.5-80) 97 (93.8-98.6) PPV% 100 100 100 Accuracy% (95% CI) 79.1 (73.9-83.7) 80,1 (75.1-84.6) 97.9 (95.5-99.2) K K= 0.42 K= 0.46 K= 0,95 NPV: negative predictive value, PPV: positive predictive value. 95% CI: 95% confidence intervals. K: Kappa coefficients.

Table 5. Analysis of the variable risk factors potentially involved in the distribution of Blastocystis single and mixed subtypes

Subtype Clinical manifestation Area Water resource Raising animal Symp Asymp Urban Rural Tap water Bottled Yes n(%) No n(%) n(%) n(%) n(%) n(%) n(%) water n(%) ST1 15(78.9) 4(21.1) 8(42.1) 11(57.9) 17(89.5) 2(10.5) 11(57.9) 8(42.1) ST2 3(75) 1(25) 1(25) 3(75) 3(75) 1(25) 3(75) 1(25) ST3 3(100) 0(0) 1(33.3) 2(66.7) 2(66.7) 1(33.3) 3(100) 0(0) ST4 6(85.7) 1(14.3) 1(14.3) 6(85.7) 5(71.4) 2(28.6) 5(71.4) 2(28.6) ST5 2(100) 0(0) 0(0) 2(100) 1(50) 1(50) 1(50) 1(50) ST7 4(100) 0(0) 1(25) 3(75) 3(75) 1(25) 2(50) 2(50) Mixed 8(88.9) 1(11.1) 4(44.4) 5(55.6) 7(77.8) 2 (22.2) 6(66.7) 3(33.3) Subtype P-value < 0.0001 < 0.0001 < 0.0001 < 0.0001 Subtypes involved in mixed subtype were added to the distribution (30 cases of subtype distribution + 9 cases of mixed subtype).

Our results indicated that ST1 was the most In the present study we found that ST1 was frequent Blastocystis subtype in 19/30 significantly higher among symptomatic (63.3%), followed by ST4 in 7/30 (23.3%), patients (78.9%) compared with asymptomatic ST2-ST7 in 4/30 (13.3%), ST3 in 3/30 (10%) ones (21.1%). In general agreement, Eroglu et and ST5 in 2/30 (6.7%). Mixed subtypes were al. (2009) and Tan (2008) reported ST1 identified in 30% (9/30) of analyzed samples exclusively from symptomatic patients versus (table 3). We add the subtypes involved in major fraction (75% and 100%, respectively) mixed infection in the distribution. Reportedly, of ST3 from asymptomatic ones. The ST4 ST1 is the most prevalent subtypes in Libya, subtype is the most obtained in rural areas and Tanzania, Nigeria, Côte d'Ivoire and Brazil ST1 is the most obtained subtype from urban followed by ST2 and ST3 (Al-Fellani et al., areas. It was found relation between the other 2013; Forsell et al., 2016; Poulsen et al., 2016; subtype and transmission from raising animal D'Alfonso et al., 2017; Seguí et al., 2018). Small (table 5, p < 0,0001). This subtype was first fractions of ST7 were reported in one case reported in in Angola (Dacal et al., 2018) from Nigeria (Poulsen et al., 2016) as well as and in this study. Because subtype 5 and 7 ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%) in were reported from both human and animals, Brazil (Seguí et al., 2018). On contrast, ST3 was their high frequency in rural areas may the most prevalent in United Arab Emirates indicate to potential zoonotic cycle (Lee et al., (AbuOdeh et al., 2016), Egypt (Fouad et al., 2012) versus the potential anthroponotic cycle 2011), Tunisia (Ben Abda et al., 2017), Senegal of ST1 in urban area. (El Safadi et al., 2014) and Angola (Dacal et al., 2018) followed varying percentages of other The risk factors of Blastocystis transmission in subtypes. Also, subtypes ST2 (62.3%), ST3 the present study included drinking of tap (17.0%), ST1 (13.2%) and ST4 (7.5%) were water and contact with animals. Tap water reported from Spain (Paulos et al., 2018). transmissions of Blastocystis were described by Ocurrence of ST7 in this study was similar to Eroglu and Koltas (2010). However it was that reported previously from Egypt (Fouad et found a close relation among subtype 5, al., 2011) and Nigeria (Poulsen et al., 2016). subtype 7 of Blastocytsis isolates and zoonotic

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potential. These subtypes are generally Brumpt E. 1912. Blastocystis hominis N. sp. et isolated from animals as well as from humans. formes voisines. Bull. Soc. Pathol. Exot. 5:725- The multivariate analysis indicate a clearly 730. association between occurrence of symptoms Dacal E., Saugar J.M., de Lucio A., Hernández-de- and increased number of the parasite (more Mingo M., Robinson E., Köster P.C., Aznar-Ruiz- de-Alegría M.L., Espasa M., Ninda A., Gandasegui than five parasites per field x 400 J., Sulleiro E., Moreno M., Salvador F., Molina I., magnification) (p = 0,001). Same result was Rodríguez E., Carmena D. 2018. Prevalence and found in study from Libya (Al-Fellani et al., molecular characterization of Strongyloides 2007). However the distribution of subtypes stercoralis, Giardia duodenalis, Cryptosporidium was not statically significant with the intensity spp., and Blastocystis spp. isolates in school of Blastocystis (p = 0.626). children in Cubal, Western Angola. Parasit. Vectors 11:67. Conclusion Dagci H., Kurt O., Demirel M., Mandiracioglu A., Aydemir S., Saz U., Baret A., Van Gool T. 2014. Results of the present study indicated that Epidemiological and diagnostic features of Blastocystis infection in symptomatic patients in Blastocysis parasites are common in Algeria. Izmir Province, Turkey. Iranian J. Parasitol. Contact with animals and drinking of tap water 9(4):519-529. are key risk factors in Blastocystis infection. D'Alfonso R., Santoro M., Essi D., Monsia A., Kaboré Gastrointestinal symptoms were more likely to Y., Glé C., Di Cave D., Sorge RP., Di Cristanziano be associated with intensity of Blastocystis. V., Berrilli F. 2017. Blastocystis in Côte d'Ivoire: More proper sanitation measures to be taken molecular identification and epidemiological for the water supply inhabited areas and for data. Eur. J. Clin. Microbiol. Infect. Dis. 36:2243- people working with animals. More studies are 2250. warranted to spot light on epidemiology of Dogan N., Aydin M., Tuzemen N.U., Dinleyici E.C., Blastocystosis in different geographic areas in Oguz I., Dogruman-Al F. 2017. Subtype distribution of Blastocystis spp. isolated from Algeria. children in Eskisehir, Turkey. Parasitol. Int. 66:948-951. Dogruman-Al F., Kustimur S., Yoshikawa H., Tuncer References C., Simsek Z., Tanyuksel M., Araz E., Boorom K. 2009a. Blastocystis subtypes in irritable bowel AbuOdeh R., Ezzedine S., Samie A., Stensvold C.R., syndrome and inflammatory bowel disease in ElBakri A. 2016. Prevalence and subtype Ankara, Turkey. Mem. Inst. Oswaldo Cruz distribution of Blastocystis in healthy 104(5):724-727. individuals in Sharjah, United Arab Emirates. Dogruman-Al F., Yoshikawa H., Kustimur S., Balaban Infect. Genet. Evol. 37:158-162. N. 2009b. PCR-based subtyping of Blastocystis Al-Fellani A.M., Khan A.H., Al-Gazoui, R.M., Zaid M.K., isolates from symptomatic and asymptomatic Al-Ferjani M.A. 2007. Prevalence and Clinical individuals in a major hospital in Ankara, Features of Blastocystis hominis Infection among Turkey. Parasitol. Res. 106:263-268. Patients in Sebha, Libya. Sultan Qaboos Univ. El Safadi D., Gayeeb L., Meloni D., Cian A., Poirier P., Med. J. 7:1. Wawrzyniak I., Delbac F., Dabboussi F., Delhae Al-Fellani A.M., Stensvold C.R., Vidal-Lapiedra A., L., Seck M., Hamze M., Riveau G., Viscogliosi E. Onuoha E.S.U., Fagbenro-Beyioku A.F., Clark C.G. 2014. Children of Senegal River Basin show the 2013. Variable geographic distribution of highest prevalence of Blastocystis sp. ever Blastocystis subtypes and its potential observed Worldwide. BMC Infect. Dis. 14:164. implications. Acta Trop. 126:11-18. El-Marhoumy S.M., Abd EL-Nouby K., Salah Shoheib Ben Abda I., Maatoug N., Ben Romdhane R., Z., MostafaSalama A. 2015. Prevalence and Bouhelmi N., Zallegua N., Aoun K., Viscogliosi E., diagnostic approach for a neglected protozoon Bouratbine A. 2017. Prevalence and Subtype Blastocystis hominis. Asian Pac. J. Trop. Dis. Identification of Blastocystis sp. in Healthy 5(1):51-59. Individuals in the Tunis Area, Tunisia. Am. J. Eroglu F., Genc A., Elgun G., Koltas I.S. 2009. Trop. Med. Hyg. 96(1): 202-204. Identification of Blastocystis hominis isolates Beyhan Y.E., Yilmaz H., Cengiz T.Z., Ekici A. 2015. from asymptomatic and symptomatic patients Clinical significance and prevalence of by PCR. Parasitol. Res. 105(6):1589-1592. Blastocystis hominis in Van, Turkey. Saudi Med. Eroglu F., Koltas I.S. 2010. Evaluation of the J. 36(9):1118-1121. transmission mode of B. hominis by using PCR method. Parasitol. Res. 107(4):841-845.

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