Blastocystis spp. are the most common enteric parasites found in human stool and yet, the life cycle of the organism is unknown and the clinical relevance uncertain. Robust cysts transmit infection, and many animals carry the parasite. Infection in humans has been linked to Irritable bowel syndrome (IBS). Although Blastocystis carriage is much higher in IBS patients, studies have not been able to confirm Blastocystis spp. are the direct cause of symptoms. Moreover, eradication is often unsuccessful.
A number of approaches were utilised in order to investigate the clinical relevance of Blastocystis spp. in human IBS patients. Deconvolutional microscopy with time-lapse imaging and fluorescent spectroscopy of xenic cultures of Blastocystis spp. from IBS patients and healthy individuals was performed. Green autofluorescence (GAF), most prominently in the 557/576 emission spectra, was observed in the vacuolated, granular, amoebic and cystic Blastocystis forms. This first report of GAF in Blastocystis showed that a Blastocystis-specific fluorescein-conjugated antibody could be partially distinguished from GAF. Surface pores of 1μm in diameter were observed cyclically opening and closing over 24 hours and may have nutritional or motility functions. Vacuolated forms, extruded a viscous material slowly over 12 hours, a process likely involving osmoregulation. Tear-shaped granules were observed exiting from the surface of an amoebic form but their identity and function could not be elucidated.
Seventeen different subtypes of Blastocystis based on the 18S subunit of ribosomal DNA have been described. Using subtyping techniques, the human and animal household contacts of 11 symptomatic Blastocystis-positive patients were studied. All household contacts were found to be positive for Blastocystis spp. The specific subtypes within households had high similarity with the symptomatic patients (94% in human and 88% in animal contacts). Although 18S rDNA subtyping was not sufficient to discriminate genetically identical organisms, the ST findings suggest that intra-household transmission of Blastocystis is almost universal.
IBS is a common, heterogeneous condition with no objective biomarker. We studied eradication rates of Blastocystis following administration of different antimicrobial drug regimes. Currently recommended first line therapy for eradication, metronidazole 400 mg three times daily or sulfamethozazole/trimethoprim 160/80mg were administered to 11 symptomatic patients for 14 days. No patient was negative for Blastocystis carriage after therapy with either drug. In a later study 10 diarrhoea-predominant IBS Blastocystis-positive patients were given 14 days of triple antimicrobial therapy, namely diloxanide furoate 500mg and secnidazole 400 mg three times daily, and trimethoprim-sulfamethoxazole 160/80 mg twice daily, with 60% of the patients eradicating the organism.
It is not certain if particular Blastocystis organisms may have more pathogenic potential than others. Many different subtypes of Blastocystis were found in all symptomatic patients studied in this thesis and thus far there is no support for particular subtypes either being pathogenic or commonly transmitted from non-domestic animal contacts. Our study of household pets, combined with current knowledge in the literature, suggests that Blastocystis may well be a reverse zoonosis when it involves companion domestic pets such as dogs and cats.
If there is no correlation between the Blastocystis subtype and pathogenic potential it is possible that the host immunological response determines symptomatology. The serological responses to infection in 40 symptomatic diarrhoea-predominant IBS patients (26 positive, 14 negative for Blastocystis) and 40 healthy controls (24 positive, 16 negative for Blastocystis) were examined. Low serum immunoglobulin A (IgA) levels were found to be associated with carriage of Blastocystis but not symptoms (p<0.001). All IBS patients, but more commonly the Blastocystis-positive group, had significantly greater reactivity with Blastocystis proteins of 27kDa (likely a cysteine protease), 50 and 75-95 kDa molecular weight. Specific anti-protease antibodies were shown to react with Blastocystis proteins suggesting that metalloproteases, as well as cysteine proteases, may be important Blastocystis antigens.
The faecal microbiota (FM) is recognised to influence our health and is altered in IBS. We examined the FM in 40 patients with IBS (26 positive, 14 negative for Blastocystis) and 57 healthy controls (42 positive and 15 negative for Blastocystis). Characteristic changes in the FM profile of IBS patients at a phylum and genus level were observed. However, when the subgroups were analysed there were significant changes seen at different taxonomic levels between the Blastocystis positive and negative IBS groups. The lower serum IgA level present in Blastocystis carriers was hypothesised to result in changes in the FM that enable Blastocystis to flourish with increased epithelial cell contact. Other previously described host factors, such as cytokine polymorphisms in IBS patients that increase IL-8 production and decrease IL-10, may select subjects for immune activation and symptom development.
In summary, Blastocystis spp. remain enigmatic organisms that are difficult to clear from our gastrointestinal tracts. Subtyping has increased our epidemiological understanding and infection within households is almost universal. Host immune responses and FM are significantly different in Blastocystis-positive IBS patients suggesting that this organism may contribute to IBS in some subjects.