Blastocystis is a single-celled intestinal monoxenus parasite with fecal-oral transmission. To date, based on the SSU rDNA and MLST loci analysis, it was discovered to comprise 17 subtypes (STs), likely representing distinct species. They colonize humans, other mammals, birds, reptiles and amphibians. Some STs from animals are zoonotic to humans. Despite being among the most common intestinal parasites infecting humans, the pathogenicity of STs still remains uncertain. Recent findings indicate that some STs can be considered as "markers" of gastrointestinal health rather than of disease; so far, little is known about whether and how STs alter the faecal bacterial microbiota.
The Project aims to: 1) carry out a molecular epidemiological analysis of Blastocystis STs on: a) human groups from developing countries, HIV-positive and immunocompetent patients; b) domestic and sylvatic animals; 2) compare the faecal microbiota in human groups infected with STs; 3) detect protease in distinct STs and their gene expression level which could be implicated in the pathogenicity; 4) improve the rapid and specific molecular detection of STs in humans by RT-PCR DNA primers/probes systems; 5) evaluate the human immune response (IgA and IgE) to STs' antigens.
Results that will be obtained will add knowledge on the association between STs, faecal microbiota and intestinal disorders in different patients groups. Studying the genetic variation of STs, as well as gene expression analysis of molecules/proteins of STs having a pathogenic role, could further clarify the pathological aspects, which they could cause to humans. The improvement of qRT-PCR-DNA assay will represent a rapid, sensitive and specific tool in the diagnosis of STs in humans. The RT-RNA analysis of target proteins could be also used as "marker" of pathogeny of STs in humans. Finally, exploring the occurrence of STs in animals could add knowledge about possible sources of blastocystosis to humans, in Italy.
The exposure to Blastocystis infected animals alone is not sufficient to explain the result in an infection in humans. It has been suggested that variables, other than just body temperature, are determining the ability of Blastocystis STs to colonize the human gut. For example, the human gut flora could have an impact. Thus, the present Project, aiming to investigate whether the carriage of Blastocystis is associated with differences in the faecal microbiota, as a proxy of gut microbiota, and what might be the impact of this differences on gut homeostasis. Studies on the gut microbiota in humans with and without Blastocystis, are likely to provide valuable information to help in determine the role of Blastocystsis in human health and disease. Different populations will be studied among immunocompromised and immunocompetent patients from different settings in order to shed light on possible association of different STs with faecal microbiota in subjects with HIV infection and their possible implications to gut homeostasis.
Furthermore, the general epidemiological and clinical patterns of parasitic infection of HIV positive subjects in Italy will be explored, for the first time. Additionally, the faecal microbiota and STs in people from two different epidemiological scenarios will be compared, to our knowledge, for the first time in Italy. The possibility to study a human population of immigrants, it would also allow to detect if any variance exists between faecal microbiota and STs distribution in people residents at different latitudes and having a completely different diet.
The molecular detection and genetic characterization of STs occurring in stools of humans and animals will allow to clarify for the first time in Italy, the potential transmission of those zoonotic subtypes (STs) to humans. Indeed, despite the fact that this pathogen is highly occurring in humans in Italy, no epidemiological survey has been so far carried out on domestic and sylvatic animals. This will contribute to the knowledge about the risk of transmission to humans.
This will also represent the first study, in Italy, concerning the detection of STs by using a sensitive and specific detection method based on a qRT-PCR using STs-specific primers and probes. Conventional PCR-DNA analysis sometimes in not sensitive in STs detection; in addition, the sequences analysis needs of time, is money costing, and sometimes no good sequences are obtained. On the contrary, the application of Taqman RT-PCR DNA primers-probes system has the advantage to be rapid, specific, sensible, and, at least, less time and money consuming. This aspect will represent also a strong advantage to the screening tools used routinely in human diagnostic parasitology at Policlinico Umberto I.
The genetic characterization of the subtypes from stool samples from both humans and animals will allow to implement the epidemiological data concerning the occurrence of subtypes in animals (domestic and sylvatic), having also a zoonotic role to humans. In addition, the estimates of genetic polymorphism at the intra-subtype level, as preliminarly observed in some STs (Mattiucci et al., 2016), will be able to detect a possible association, in a multivariate analysis, between distinct haplotypes and degree of intestinal diseases in infected subjects (humans).
Finally, because the pathogenesis of STs, remains as a controversial issue, as it cannot be clearly assigned to certain ST, the gene expression analysis (by RNA seq and RT-RNA) of STs, which will be detected in the present study, will allow to evidence a possible differential pathogenic role to humans. The study some proteases activity in the detected STs, as well as the comparative analysis of gene expression of some antigenic proteins from the different STs, will increase the knowledge concerning aspects of the parasite life cycle, and parasite-host interactions. The RT-RNA analysis of some target proteins could be also used as a "biomarkers" of distinct ST affecting humans.