Molecular Identification of Blastocystis Subtypes

Total genomic DNA was extracted from approximately 250 mg of stool samples using the QIAamp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s recommended procedures then eluted in 200 μL of elution buffer (Qiagen GmbH, Hilden, Germany). All DNA samples were stored at −20 °C then transported to the Institut Pasteur of Lille (Lille, France). To detect and subtype Blastocystis sp., 2 µL of extracted DNA from each sample was subjected to highly sensitive real-time PCR (qPCR) assay using the Blastocystis-specific primers BL18SPPF1 (5′-AGTAGTCATACGCTCGTCTCAAA-3′) and BL18SR2PP (5′-TCTTCGTTACCCGTTACTGC-3′) targeting the small subunit (SSU) rDNA gene as previously described [41 (link)]. The corresponding amplified gene domain of about 300 bp has been shown to contain sufficient sequence information for accurate subtyping of Blastocystis sp. isolates. Positive (DNA from Blastocystis sp. ST7 axenic culture) and negative (DNA replaced by water) qPCR controls were included with each batch of samples analyzed. The qPCR product from each positive sample was purified and directly sequenced on both strands (Genoscreen, Lille, France). For a proportion of samples, sequence chromatograms analysis revealed the presence of double traces, suggesting mixed infections by at least two different Blastocystis STs that were not determined. The SSU rDNA sequences obtained in this study from samples presenting single infection were deposited in GenBank under accession numbers MW168447 to MW168610. Obtained sequences were compared with all Blastocystis sp. homologous sequences of known STs available from the National Centre for Biotechnology Information (NCBI) using the nucleotide Basic Local Alignment Search Tool (BLAST) program. STs were identified by determining the exact match or closest similarity against all known Blastocystis sp. STs [14 (link),26 (link)]. Moreover, the sequences of Blastocystis sp. isolates belonging to the same ST (ST1, ST2 or ST3) were aligned with each other using the BioEdit v7.0.1 package (Date of release 06/10/2019; http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to determine intra-ST diversity and identify so-called genotypes referring to genetically distinct strains within the same ST as described in recent surveys [9 (link),16 (link)]. Subsequently, ST1, ST2 and ST3 sequences from isolates previously identified in the local population of North Lebanon [4 (link),16 (link),35 (link),36 (link)] were extracted from databases and compared to genotypes reported herein from the cohort of Syrian refugees.