The potential significance of the faecal yeast Candida sp. in CoeD
The human mycobiome (fungi and their genome) is a relatively new advance in characterising the residents of healthy individual’s gastrointestinal tracts. One gastrointestinal mycobiome characterisation study identified 66 fungal genera and 184 fungal species, with
Candida as the dominant fungal genera [
21]. Fungi have been associated with a number of gastrointestinal diseases, with a dominant focus on the mycobiome of patients with IBD and graft-versus-host disease [
21].
The clinical significance of increased numbers of commensal yeasts is not fully understood in those other than individuals who are critically ill or immune compromised. To our knowledge this is the first study reporting statistically significant differences between the prevalence of faecal
Candida sp. counts of people with CoeD reporting persistent symptoms compared to people without CoeD. However, in vitro, animal and clinical studies have identified potential mechanisms of action as to the causal relationship between
candida and CoeD and these will be discussed briefly here. The role of candida in triggering aberrant immune response to dietary proteins has been supported by an animal study that demonstrated that gastrointestinal
Candida colonisation promotes sensitisation against food antigens, partly due to mast cell mediated hyperpermeability in the gastrointestinal mucosa [
22]. Dysregulation of the normal integrity of the small intestinal mucosa, i.e. small intestinal hyperpermeability is a feature of the pathophysiology of CoeD [
23,
24]. An association between an intestinal candida infection in those genetically predisposed CoeD has been proposed as a potential trigger of the disease process [
25,
26]. This association was first described by Nieuwenhuizen et al., who hypothesised that the virulence factor of
C. albicans-hyphal wall protein 1 (HWP1) contains amino acid sequences that are identical or highly homologous to known CoeD-related α-gliadin and γ-gliadin T cell epitopes [
26]. The HWP1 is used by
C. albicans to adhere to the intestinal epithelium. It is thought that tTg and endomysium components link to the yeast and act as an adjuvant that activates the immune system to fight the HWP1 and gluten, thereby forming autoimmune antibodies against tTg and endomysium, resulting in the characteristic villous atrophy of CoeD [
26]. More recently, Courage et al. proposed that the common denominator in the humoral cross-reactivity observed between HWP1 and gliadin in both CoeD and candida infection was transglutaminase further suggesting that candida infection could be a trigger of CoeD in the genetically predisposed [
25].
Other research groups have identified at least three species of
Candida sp. that produce proteases that can degrade immunoglobulin-A1 (Ig-A1), Ig-A2 and sIgA. Interestingly, of 2098 patients with CoeD in one study, 2.6% had an sIgA deficiency, representing a 10–16-fold increase over that of sIgA deficiency in the general population [
27]. Furthermore, it has been shown that sIgA deficiency may be a predisposing factor to autoimmune diseases [
28], and to recurrent infections [
29]. In this present study, there were no cases of sIgA deficiency in the non-CoeD control group. However, we did not have data on the levels of the sIgA from the CoeD group at point of diagnosis nor did we measure sIgA as the focus of this study was on the role of the microbiota as cause of persistent symptoms not as an aetiological factor. Nevertheless, the detection of measureable counts of
Candida sp. in 33% of the CoeD group compared to 0% in the control group (p = 0.000) raises the question; can
Candida sp. act as an immunosuppressant through an ability to reduce sIgA, thus allowing colonisation of the intestine and triggering autoimmune responses such as CoeD in the genetically predisposed? The findings from this study and those from the studies discussed above warrants further research that explores the role of
Candida sp. and the relationship between
Candida and sIgA and the onset of CoeD.
A possible link between
Candida albicans and the aetiology of the inflammatory bowel disease (IBD) Crohns disease has been reported [
30].
Candida taxa have been found in increased abundance in the faceal microbiota of children with IBD compared to children without IBD with the authors concluding that it is important to explore the fungal microbiota as playing a possible role in the pathogenesis of the disease [
31]. In a mouse model study,
Candida albicans colonisation was found to augment dextran sulphate sodium induced inflammation and conversely inflammation was strongly promoted
Candida albicans colonisation [
32]. A possible interpretation of this finding as it relates to CoeD could be that the chronic small intestinal inflammation characteristic of CoeD, is potentially induced by intestinal
Candida albicans colonisation thus contributing to the pathophysiology of the disease. In established CoeD, intestinal
Candida albicans colonisation may augment and promote inflammation resulting in only partial clinical and/or histological improvements.
While
Candida sp. may or may not be a fungal environmental trigger for CoeD, this study found that
Candida sp. was more prevalent in the faeces of people with mild to moderate persistent symptoms of CoeD compared to those with gastrointestinal symptoms from other causes. Antibiotics have a profound impact on the composition of the composition of the gastrointestinal microbiome and are considered the most common cause of opportunistic colonisation by
C. albicans. As part of the inclusion criteria for this study no participant in the CoeD group had taken antibiotics within 4 weeks of undertaking testing. However, they did report a history of recurrent antibiotic use as children, i.e. >once per year in the first 12 years of life. This is higher than estimated use by the general western population where it has been estimated that half of the paediatric population of most Western countries receive antibiotics at least once per year [
33]. Antibiotic use in childhood has been implicated as potential trigger for CoeD onset [
34]. In this present study we did not gather data regarding the specific proximity of antibiotic use and CoeD onset due to the difficulty in determining the duration of CoeD prior to a medical diagnosis. In line with the discussion regarding the opportunistic growth of
candida sp being secondary to antibiotic administration, is the important consideration that the increased prevalence of these yeasts could also be secondary to the altered architecture and function of the small intestine that is characteristic of CoeD. Therefore, it is theoretically possible that as the small intestine recovers to normal structure and function that the increased counts of yeasts could be self-limiting. However, should opportunistic yeast overgrowth of the small intestine be identified as secondary to the pathophysiology of CoeD in future studies, small intestinal yeast overgrowth in addition to small intestinal bacterial overgrowth can be considered in those failing to respond to dietary treatment.
The potential significance of the faecal yeasts Saccharomyces sp. in CoeD
To our knowledge, it has not been reported previously that people with CoeD have a significantly higher prevalence of
Saccharomyces sp. counts in their faeces compared to those without CoeD. A potential confounding factor in the interpretation of this finding could be related to dietary ingestion and transient numbers of
S. cerevisiae being detected in the faeces due to its prevalent use in the manufacturing of wine, beer and bread [
35]. Anti-
S. cerevisiae antibodies (ASCAs) have been found in 43% of patients with CoeD at diagnosis and these antibodies disappeared during treatment with a GFD [
36]. The disappearance of ASCAs after treatment was found to be more pronounced in children than in adults. It is suggested that ASCAs are more likely to persist in treated adult CoeD patients due to the more profound damage of the intestinal wall (as a consequence of their delayed diagnosis); therefore, resolution of intestinal permeability is slower [
36]. This present study’s findings would suggest that treated adult CoeD patients with persistent symptoms have higher indigenous faecal counts of
Saccharomyces sp. and the presence of ASCAs may not be secondary to intestinal permeability, but rather a suggestion that there is colonisation.
Saccharomyces boulardi is commonly prescribed as a probiotic supplement for individuals with gastrointestinal symptoms such as diarrhoea and has attracted research attention by many groups for its therapeutic potential [
37,
38]. None of the CoeD group reported were taking
s. boulardi prior to this baseline test. The potential clinical implications of taking supplemental probiotic formulations of
s. boulardi in these individuals is not known. CoeD patients who present to their health care professionals with persistent symptoms or partial symptom improvement despite adherence to a GFD are often faced with further invasive and non-invasive investigations and are subject to a dietary audit. If these investigations yield no explanation for their symptoms they are sent home with a range of pharmaceuticals that may provide symptomatic relief. This study indicates that gastrointestinal dysbiosis is prevalent in this population and faecal assessment may provide important clinical information in this sub-group of CoeD patients.
Study limitations
The addition of ASCAs as a biomarker would have strengthened the design of this study and provided greater insight as to the potential clinical relevance of the molecular detection of saccharomyces in faecal samples of indications with CoeD. Future studies, exploring this question further are encouraged to include ASCAs as a biomarker in evaluating this question.
There are a number of limitations for studies exploring components of the microbiome of specific populations including this study. Firstly, there are only general microbial markers of what constitutes a healthy intestinal microbiome and there is much ambiguity around the clinical significance of alterations in the measures of specific commensal microbial residents. In light of this, our findings are potentially limited by the fact we did not use a healthy control group. This present study employed control data obtained from a group of heterogeneous individuals who attended a doctor with symptoms consistent with irritable bowel syndrome and had excluded CoeD as part of their medical assessment. Conversely, while this may be deemed as a limitation, it may also be interpreted as strength in further differentiating groups of people troubled by gastrointestinal symptoms.
Secondly, sub-speciation and employing Sabroud culture methods of the yeasts detected may have provided more clinically significant information. Lastly, there is currently no gold standard for the molecular assessment of the intestinal microbiota with each technique having strengths and limitations that may result in discrepancies between findings and result bias. This study may have been strengthened through having the faecal specimens analysed by two laboratories to verify the findings.
Given the limitations of this study we cannot conclude there is a relationship of cause and effect but instead seek to report an important observation. We strongly encourage larger scale, rigorously designed clinical studies to further investigate these findings.