Blastocystis is the intestinal protist most commonly identified during coproparasitological examination in clinical microbiology laboratories regardless of the presence/absence of symptoms in the affected patients (Stensvold et al 2011; Souppart et al 2009; Clark et al 2013). The epidemiology of Blastocystis in Morocco is largely unknown. Only a few microscopy-based studies have previously investigated the presence of Blastocystis in asymptomatic schoolchildren in Tiflet (22.3%) (Tligui et Agoumi 2006), Salé (33.4%) (Tagajdid et al 2012), and Tetouan (64.0%) (El Fatni et al 2014), and in patients of paediatric age in Kenitra (28.7%) (El Guamri et al 2011). Using the same detection method, a recent study conducted by our research group has reported a Blastocystis prevalence of 39.8% among food handlers in the region of Fez-Meknes (Boutahar et al 2023).
The culture-based prevalence of Blastocystis determined in the present study was much higher than that obtained by conventional microscopy (51.4% vs. 21.0%, respectively). These results are consistent with previous reports demonstrating the superior diagnostic performance of culturing over light microscopy (El-Badry et al 2018; Fathy 2011; Radwan et al 2017; Suresh et Smith 2004). The Blastocystis prevalence recorded in our study (51.4%) was higher than that observed in most previous studies conducted in Morocco (El Guamri et al 2011; Tagajdid et al 2012; Tligui et Agoumi 2006) and in neighbouring countries using optical microscopy as a diagnostic technique. An infection rate of 13.0% has been reported in apparently healthy individuals in Tunisia (Ben Abda et al 2017), 22.1% in outpatients in Libya (Abdulsalam et al 2013), and 32.1% in symptomatic and asymptomatic subjects in Algeria (Sebaa et al 2018). Higher prevalence rates ranging from 71–100% have been documented by PCR in patients with clinical manifestations in Syria (Darwish et al 2023), in paediatric populations in Nigeria (Poulsen et al 2016), and in apparently healthy individuals in Qatar (Abu-Madi et al 2015). Using the same method, prevalence rates ranging from 31–63% have been described different human populations in Lebanon (Osman et al 2016), Spain (Paulos et al 2018), and Italy (Gabrielli et al 2021), and from 11–18% in France (El Safadi et al 2016) and Saudi Arabia (Mohamed et al 2017). Variations in Blastocystis prevalence worldwide could be attributed to differences in drinking water quality and sanitary conditions, sample collection and diagnostic methods used, surveyed populations, and environmental factors.
Out of 54 Blastocystis-positive samples by culturing, 90.7% (49/54) were successfully subtyped by Sanger sequencing. Failure to amplify the remaining isolates could be potentially explained by several factors including insufficient DNA quality/quantity or inefficient removal of PCR inhibitors during the extraction and purification procedure.
In our study, Sanger sequence analysis identified three Blastocystis subtypes: ST1, ST2, and ST3. ST3 was most frequently observed (42%), followed by ST1 (34%) and ST2 (12%). These results corroborate those reported by several previous studies conducted in different human populations in countries of the Mediterranean basin (Ben Abda et al 2017; Ahmed et al 2022; Aykur et al 2023; Cakir et al 2019; Piubelli et al 2019; Rahimi et al 2022). We did not identify the presence of ST4 in our patient population. This result was somehow expected as ST4 has a marked geographical distribution and is mainly observed in European individuals (Alfellani et al 2013; Popruk et al 2021; Jiménez et al 2019). However, various investigations revealed that ST4 is much less frequently detected or absent in Africa, the Middle East, Asia, and North America (Stensvold et al 2011; Poirier et al 2011).
The distribution of STs in our study resembles that reported in Tunisia, where analysis carried out on healthy individuals registered that ST3 was the most abundant Blastocystis genetic variant (51.0%), followed by ST1 (30.0%) and ST2 (16.0%) (Ben Abda et al 2017). Similarly, Algerian research conducted among kitchen staff showed a comparable trend, with a predominance of ST3 (50.0%), followed by ST1 (33.3%) and ST2 (13.3%) (Bachi et al 2022). In contrast, in Libya, a study among symptomatic and asymptomatic people revealed a different scenario, in which ST1 was the most prevalent (51.1%), followed by ST2 (24.4%) and ST3 (17.8%) (Abdulsalam et al 2013). The high presence of ST1 and ST3 in our clinical population could be attributed to a primarily anthropic transmission. Studies conducted in Bangkok and Malaysia indicated that ST1 is transmitted from person to person (Noradilah et al 2017; Thathaisong et al 2013). Similarly, research in two healthcare facilities in Osaka confirmed that ST3 is also transmitted from person to person (Yoshikawa et al 2000).
The question of the pathogenesis associated with Blastocystis sp. remains controversial due to their frequent detection in both symptomatic and asymptomatic individuals (Kataki et al 2019; Sebaa et al 2021; Souppart et al 2009). This controversy arises from several complex elements. First, the high genetic diversity of Blastocystis complicates the establishment of direct links with the clinical manifestations observed in patients. Second, it turns out to be difficult to exclude the confounder effect caused by coinfections involving other intestinal pathogens of parasitic, bacterial or viral nature capable of causing similar digestive symptoms. Third, the different immune responses and the specific composition of the intestinal microbiota of each individual contribute might have an effect on the pathogenic potential of Blastocystis infections (Tan et al 2008; Guilavogui et al 2022; Stensvold et al 2020).
Our study did not reveal any statistically significant association between Blastocystis infections (regardless of the ST involved) and the gender or age group of the affected patients, which corroborates the findings of previous research (Lhotská et al 2020; Arbat et al 2021; Yersal et al 2016). Similarly, no clear links were demonstrated between clinical status and Blastocystis subtypes. Consistent with earlier studies, ST1 (41.2%) and ST3 (57.1%) were common in asymptomatic subjects, whereas ST2 (83.3%) was predominant in symptomatic subject (Tan et al 2008; Aydin et al 2019; Bachi et al 2022; Dagci et al 2014; Yowang et al 2018). It should be noted that the opposite trend has also been described, with ST1 and ST3 being associated with symptomatic individuals (Ahmed et al 2022; El Safadi et al 2014; Jones et al 2009; Stensvold et Clark 2020) and ST2 with asymptomatic individuals (El Safadi et al 2014; Dogruman et al 2008). Among all clinical manifestations considered in this survey, only flatulence was positively correlated with the presence of Blastocystis ST2 (P = 0.035). This very same result has been described in some previous studies (Fréalle et al 2015; Abdulsalam et al 2013), whereas in others flatulence was linked to ST3 (Tan et al 2008; Souppart et al 2010).
Other Blastocystis STs including ST1, ST4, and ST7 has been proposed as more pathogenic by some authors (Deng et al 2022; Ramirez et al 2014). ST1 has been frequently observed in patients suffering from diarrhoea (Aykur et al 2023), abdominal pain (Eroglu et al 2009), irritable bowel syndrome (IBS) (Fouad et al 2011), and colorectal cancer (Mohamed et al 2017). Additionally, experimental studies in murine models showed increased pathogenicity in rats infected with ST1 isolates of human origin (Hussein et al 2008). As for ST4, it has been associated with cases of acute and chronic diarrhoea in patients from Denmark (Stensvold et al 2023), Italy (Mattiucci et al 2016), and Spain (Domínguez-Márquez et al 2009). Regarding ST7, it has been described as a prevalent Blastocystis genetic variant in clinical cases of diarrhoea and irritable bowel syndrome (Ramirez et al 2014; Deng et al 2021). These claims have been supported by in vitro and in vivo studies demonstrating Blastocystis-derived detrimental effects on gut bacterial profiles altering the permeability of intestinal epithelial cells (Nourrisson et al 2016; Yason et al 2019).
To date, most molecular-based studies have favoured Sanger sequencing as the preferred method to identify Blastocystis STs and alleles (Lhotská et al 2020; Scanlan et al 2015). However, this technology does not allow the detection of mixed ST infections characterised by producing overlapped traces during chromatogram inspection (Stensvold et al 2020; Šloufová et al 2022). Indeed, our Sanger analysis revealed the presence of five samples with unreadable sequences that very likely correspond to such mixed infections. Recently, a new methodology based on next-generation amplicon sequencing has been proven useful to detect low abundance STs that would be missed by Sanger sequencing (Maloney et al 2023). The use of this method should be considered in epidemiological surveys conducted in Blastocystis endemic areas to accurately assess the true proportion of mixed infections involving two or more STs of the protist.
This study has some limitations that might have hampered the accuracy of the results obtained and the conclusions reached. First, sample size is limited and might have compromised the significance of the statistical analyses conducted. Second, results obtained might not be representative of the national scenario. Third, lack of detection of other parasitic, bacterial and viral intestinal pathogens might have biased the perception of symptomatic cases. And fourth, we were unable to identify potential mixed infections involving two or more STs.