Aspects of Genetic Diversity, Host Specificity and Public Health Significance of Single-Celled Intestinal Parasites Commonly Observed in Humans and Mostly Referred to as 'Non-Pathogenic'.

Clinical microbiology involves the detection and differentiation of primarily bacteria, viruses, parasites and fungi in patients with infections. Billions of people may be colonised by one or more species of common luminal intestinal parasitic protists (CLIPPs) that are often detected in clinical microbiology laboratories; still, our knowledge on these organisms' impact on global health is very limited. The genera Blastocystis, Dientamoeba, Entamoeba, Endolimax and Iodamoeba comprise CLIPPs species, the life cycles of which, as opposed to single-celled pathogenic intestinal parasites (e.g., microsporidia and sporozoa), do probably not include gut-invasive stages that could result in pathological processes and thereby disease (except for Entamoeba histolytica). All five genera are parasites in the sense that they use a host to complete their life cycle; still, by many specialists, these are considered to be of limited clinical relevance and could possibly be referred to as 'eukaryotic endobionts' or even 'endosymbionts', in case they would have health-protective effects. The articles included in this thesis exemplify the work and the data that support the view that it might be more relevant to study these genera in a public health and gut ecology context than in a clinical microbiology context. Essential to investigating the impact of intestinal parasites on health and disease are accurate diagnostic tools, including DNA-based technology such as PCR and sequencing, plus accurate reference databases. Small subunit (SSU) ribosomal RNA (rRNA) genes consistently present in both pro- and eukaryotic organisms are today avidly used as taxonomic markers. DNA-based methods have been developed for genetic characterisation of microorganisms and provided data on species/subtypes/genotypes, etc. Metagenomics and metabarcoding (the use of low-specific PCR coupled with next-generation sequencing) can provide information on co-infection/co-colonisation with other organisms and enable screening for genetic diversity, even in complex matrices. By developing and implementing sensitive and specific DNA-based diagnostic tools and typing assays primarily based on the SSU rRNA gene, we have increased insight into the diversity, distribution and significance of CLIPPs. With these tools, we have shown that the genera Blastocystis and Dientamoeba are far more common than previously thought. Only 10-15 years ago, hypotheses on their distribution typically relied on data generated by traditional parasitological diagnostic methods, such as light microscopy. Hence, we have shown that most older children in Nigeria host Blastocystis, and that most children in day-care institutions in Denmark, if not all, get colonised by Dientamoeba at some point. Single-celled non-pathogenic intestinal parasites can be hosted by patients with diarrhoea and functional or inflammatory bowel diseases. However, emerging data appear to suggest that CLIPPs are generally more common in gut-healthy individuals than in patients with gastrointestinal symptoms. The research we have carried out on associations between CLIPPs and gut bacteria suggests that colonisation with these parasites is seen primarily in individuals with a healthy 'gut flora' (eubiosis). This observation should prompt future research projects focusing on the use of CLIPPs as biomarkers, and it should be investigated to which extent manipulation with CLIPPs could lead to changes in the gut flora and thereby be used as probiotics. In the event that it makes sense to speak of 'infection' by CLIPPs, we still lack tools to differentiate between colonisation and infection. We have known for decades that morphologically similar parasites can differ in terms of clinical impact and be genetically distinct, a feature that we refer to as 'cryptic genetic diversity'. One example is E. histolytica, which cannot be differentiated from Entamoeba dispar by cyst morphological features. However, whereas E. histolytica can be invasive and give rise to amoebic dysentery and amoebiasis, E. dispar is by most specialists considered non-invasive and generally non-pathogenic. This insight led us to investigate genetic diversity among other species of Entamoeba as well as other CLIPPs genera. If we could demonstrate similar-or higher-degrees of diversity within Blastocystis, Dientamoeba, Endolimax and Iodamoeba, these differences might be key to explaining differences in parasite phenotype and thereby differences in the ability of the parasites to cause symptoms. Despite the disclosure of striking genetic diversity among some CLIPPs, we have found little support for such theories; however, more studies are needed. As for Dientamoeba, we have observed a more or less clonal expansion of one of the two genotypes known to exist, and this genotype appears to have global predominance. In contrast, extensive genetic diversity is observed between and within subtypes of Blastocystis: to date, more than 30 species, the so-called subtypes, have been acknowledged. We, and many others, have sought to identify whether one or more of these subtypes could be linked to the development of intestinal symptoms, but there is little evidence to support this hypothesis. We know that Subtypes 1-4 reflect about 95% of Blastocystis colonisation in humans, and we have shown that individuals with zoonotic subtypes (e.g., ST6, ST7 and ST8) might typically experience symptoms. We have disclosed astonishing genetic variation among other CLIPPs, which has led to the recognition of Iodamoeba bütschlii, Endolimax nana, Entamoeba coli and Entamoeba hartmanni as species complexes, where each species should be regarded as a complex of species (referred to as 'subtypes' or 'ribosomal lineages') with overlapping morphology. And where E. histolytica and E. dispar differ by only 1%-2% diversity across the SSU rRNA gene, we have observed up to at least 10% and 30% genetic difference among ribosomal lineages within E. coli and I. bütschlii, respectively, challenging species concepts currently applied. Our research has resulted in the recognition of three ribosomal lineages within both E. coli and E. hartmanni, as well as two ribosomal lineages of E. nana and I. bütschlii. Moreover, we have discovered a new lineage of Entamoeba moshkovskii. Molecular characterisation of intestinal parasites collected from different host species (humans, non-human primates, other mammals, birds, etc.) can help identify opportunity for transmission between human and non-human hosts. We have shown that pigs can host a few species/lineages that can readily colonise humans, such as Entamoeba hartmanni and I. bütschlii. Many other species of larger mammals are common hosts of Blastocystis and Entamoeba. However, for the two latter genera, the species/genetic variants observed in non-human hosts are typically different from those observed in humans, which could indicate that many species of CLIPPs have adapted to their respective hosts over a long period, resulting in relatively high host specificity. For Blastocystis, we have shown that even though a given subtype may be found in more than one host species, it is possible to demonstrate cryptic host specificity at allele level. For instance, even though both human and non-human primates can be colonised by ST3, host species-specific strains of ST3 circulate within these two host populations. With regards to E. coli, it is possible that ST1 has adapted to human hosts, while E. coli ST2 has adapted to a broader host range, including non-human primates and rodents. It has become clear that CLIPPs are common colonisers of the human background population, and even though we cannot disprove the existence of infection by any of these, it should be reasonable to consider clinical and medical intervention redundant in most cases. Perhaps it might even be so that one should try not to eradicate these organisms from the gut when first established. However, more studies are warranted to elucidate the significance of the pronounced genetic diversity observed in some CLIPPs with regards to transmission patterns and clinical significance. Future research in CLIPPs should also include studies that can elucidate those factors that favour colonisation with CLIPPs and what role CLIPPs have in host-gut ecology, metabolism and overall health condition. Finally, as human and non-human hosts share these parasitic genera, and as some protozoa possibly contribute to overall gut function in ruminants, it would be interesting to study these in domesticated and wild animals to learn more about the role of these parasites in health and disease, including investigations into whether some CLIPPs might be endosymbionts.