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

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 Laghouat Province, Algeria 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 Djelfa, 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, Algiers, 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. 23 Sci Parasitol 19(1-2):23-33, December 2018 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE 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). 24 Sci Parasitol 19(1-2):23-33, December 2018 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE 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.

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