Fungal translocation measured by serum 1,3‐ß‐D‐glucan correlates with severity and outcome of liver cirrhosis—A pilot study

Abstract Background & Aims On a global scale, liver cirrhosis is attributable to ~1 million deaths per year. This systemic disease comes along with diverse sequelae, including microbiota alterations, increased gut permeability and translocation of microbial components into the systemic circulation. Alongside the extensively studied influence of bacterial translocation and its host–pathogen interactions, far less is known about the role and impact of fungal components once having crossed the intestinal barrier. Methods Including 70 patients with different aetiologies of liver cirrhosis, we investigated the relationship between fungal translocation, measured by 1,3‐β‐D‐glucan (BDG), and biomarkers of gut integrity, inflammation and severity/outcome of liver disease. Results Patients with cirrhosis Child–Pugh class (CPC)‐B were more likely to have positive serum BDG (aOR 5.4, 95% CI 1.2–25.2) compared to patients with cirrhosis CPC‐A. BDG showed a moderate positive correlation with several markers of inflammation (sCD206, sCD163, Interleukin 8, interferon‐gamma‐induced protein). Mortality differed significantly between patients with positive versus negative BDG (log‐rank test, p = 0.015). The multivariable Cox regression model yielded an aHR of 6.8 (95% CI 1.8–26.3). Discussion We observed trends for increased fungal translocation depending on the severity of liver cirrhosis, an association of BDG with an inflammatory environment and the adverse effects of BDG on disease outcome. In order to gain more in‐depth knowledge about (fungal‐)dysbiosis and its detrimental consequences in the setting of liver cirrhosis, these trends need to be studied in more detail including prospective sequential testing in larger cohorts together with mycobiome analyses. This will further elucidate complex host–pathogen interactions and potentially introduce points of application for therapeutic interventions.


| INTRODUC TI ON
Liver cirrhosis is a severe systemic disease widely prevalent in low-, middle-, and high-income countries and attributable to ~1 million worldwide deaths annually. 1 Despite major efforts in cirrhosisrelated research having led to an improved understanding of pathogenesis, diagnosis and treatment, various aspects of disease-related complications remain to be understood in detail. Of particular interest are the relationship between the gut microbiota and microbial translocation acting as a potential driver of persistent inflammation involving adverse effects on clinical disease course and outcome.
Fungi are commensals of the human skin, respiratory tract and gut. The fungal part of the human gut microbiota is referred to as the mycobiota. Alongside bacteria, fungi account for the second most abundant population of the gut microbiota. 2 Predominant human intestine fungal species comprise ascomycetes like Candida albicans and other yeasts as well as moulds like Aspergillus spp. and Fusarium spp. 3 One common characteristic feature shared by the above-mentioned fungi is the production of the polysaccharide 1,3-ß-D-glucan (BDG), a fungal cell wall component. To date, BDG is mainly serving as a pan-fungal serum biomarker for the diagnosis of invasive infection, however, the presence of BDG in blood has also been correlated with gut dysbiosis, immune activation, persistent inflammation, microbial translocation and prediction of non-AIDS events in patients with HIV infection. [4][5][6][7][8][9] While research in liver cirrhosis has mainly focused on bacterial-host interactions, the fungal part and potential cross-kingdom microbial interactions remain enigmatic. It has been acknowledged that liver cirrhosis and its multifaceted sequelae, including portal hypertension leading to intestinal hypoperfusion and gut microbiome dysbiosis, are associated with increased gut permeability. 10 Alterations of the gut barrier result in increased translocation of bacterial as well as fungal components, which is associated with an increased risk of infections. [10][11][12][13] Further, the presence of microbial elements in the bloodstream induced a hyperinflammatory state and an increased rate of complications in cirrhosis patients. 14 Narrowing down on the fungal part, fungal translocation measured by serum BDG levels had various effects on immunologic pathways as well as the microbiome composition of mice chronically exposed to ethanol administration, and a small cohort of patients with alcoholic cirrhosis. 15   Discussion: We observed trends for increased fungal translocation depending on the severity of liver cirrhosis, an association of BDG with an inflammatory environment and the adverse effects of BDG on disease outcome. In order to gain more in-depth knowledge about (fungal-)dysbiosis and its detrimental consequences in the setting of liver cirrhosis, these trends need to be studied in more detail including prospective sequential testing in larger cohorts together with mycobiome analyses. This will further elucidate complex host-pathogen interactions and potentially introduce points of application for therapeutic interventions.  15 Larger scale evaluations that correlate fungal translocation with the severity of liver cirrhosis, extent of the "leaky gut", and disease outcome in humans are lacking. For this aim, we prospectively measured levels of serum BDG and investigated associations with additional biomarkers for gut integrity, microbial translocation, inflammation and severity/outcome of patients with liver cirrhosis.

| Study design
Biosamples were collected as part of the study "Influence of

| Measurements
BDG was tested according to previously described methods using reagents from the Fungitell® Assay (Associates of Cape Cod, Falmouth, MA). 16  an in-house ELISA assay as previously described. 19 For endotoxin measurement, HEK-Blue LPS Detection Kit 2 (Invivogen, Toulouse, France) was used as previously described. 20 The differential sugar absorption test was performed with lactulose (10 g) and mannitol (5 g) and analysed by nuclear magnetic resonance spectroscopy as previously described. 20 Interleukins (IL) 1β, 6, 8, 10, tumour necrosis factor (TNF) α, interferons (IFN) α, β, γ, interferon-gamma induced protein (IP) and monokine induced by gamma-interferon (MIG) were assessed in sodium citrate plasma using ProcartaPlex® Multiplex Immunoassay (Affimetrix, Vienna, Austria) according to manufacturer's instruction and measured with a Bio-Plex 200 (Biorad, Hercules, USA). For BDG positivity, we used the cut-off of ≥80 pg/mL, which is recommended by the manufacturer for diagnosing invasive fungal infections. Importantly, this is not the detection limit for the assay, which is 15.4 pg/mL 16 ; the test is linear between about 15 pg/mL and about 250 pg/mL, with modest deviations from linearity below 15 pg/mL (higher slope) and above about 300 pg/mL (slightly lower slope). To date, definition of an appropriate cut-off for fungal translocation is lacking. 4 Thus, relying upon a high cut-off value, such as 80 pg/mL, provides a more conservative estimate of the utility of the BDG titre where a significant relationship with outcome, symptom severity or another biomarker was also observed in various other non-fungal infection disease states [e.g., CKD, inflammatory bowel disease, COPD, Lupus 21-24 ].

| Statistical analysis
Continuous variables are reported as median plus interquartile range (IQR). Correlation between various biomarkers was calculated using Spearman's rho correlation analysis due to the non-normal distributions. Correlation matrixes were calculated and graphically depicted as heat diagrams. Logistic regression was used to assess predictors

| DISCUSS ION
We found an association between serum BDG levels with liver dis- shown to be higher compared to a healthy cohort. 29 Lastly, we found a significant difference in mortality between patients with positive versus negative BDG. In the multivariable Cox model, we found an aHR of 6.8 (95% CI 1.8-26.3) for death in patients with positive BDG ≥80 pg/mL. Another pilot study showed that BDG blood levels were not influenced by plant-origin BDG-rich nutrition in people with advanced liver cirrhosis, and HIV, indicating that the interpretation of serum BDG levels may not need to be adjusted for nutrition and daytime. 13 While animal and human studies have demonstrated the role of hepatic clearance in the elimination of circulating BDG 32,33 and also that a minor component of circulating BDG is cleared renally, 34 indicating that less secretion or less metabolism may contribute to sustained elevated BDG levels. It has to be emphasized, however, that BDG has to enter the bloodstream via translocation in the first place before the above-mentioned affects might come into play.
BDG has been studied as a marker of fungal translocation in various clinical settings (e.g., sepsis, haemodialysis, abdominal surgery). 26,28,35 All these conditions lead to increased gut permeability through different pathophysiological pathways, resulting in microbial translocation, including fungal translocation that can be measured by serum BDG. Besides, detailed investigations between non-AIDS events and serum BDG levels were conducted and found that elevated serum BDG levels were able to predict and correlated with higher clinical event rates. [7][8][9] Interestingly, we could not find clear relations between serum BDG and other biomarkers of increased gut permeability. Similar findings were obtained in a very recent study where BDG levels in HIV patients decreased after 1 year of anti-retroviral therapy. 36 The absence of correlation with other permeability markers might also be partially explained by another mechanism acting a part, which is a decrease in BDG clearance by the hepatic reticuloendothelial system with increasing hepatic inflammation and hence higher levels in peripheral blood. 32 In a mouse model, Yang et al. observed that a decrease in intestinal fungal overgrowth by non-absorbable Amphotericin B did not alter the gut barrier function and permeability remained increased. 15 These findings may indicate that fungal dysbiosis might not be alone driving the disruption of gut integrity and that other factors such as hypoperfusion of the gut occurring in patients with liver cirrhosis, as well as persistent low-level inflammation caused by microbial products and BDG entering the bloodstream, and underlying diseases might play important roles as well. Nevertheless, non-absorbable Amphotericin B led to a decrease in serum BDG levels and had a positive effect on disease severity. 15 Limitations of our study are the retrospective cohort and the In conclusion, we showed that BDG levels were higher in patients with CPC-B cirrhosis compared to CPC-A cirrhosis patients, and that higher BDG leveles were associated with mortality. Further, BDG levels significantly correlated with markers of inflammation.
In order to gain more in-depth knowledge about (fungal-)dysbiosis and its detrimental consequences in the setting of liver cirrhosis, these trends need to be studied in more detail including prospective sequential testing in larger cohorts and micro-mycobiome studies.
This will further elucidate complex host-pathogen interactions and potentially introduce points of application for timely therapeutic interventions.

FU N D I N G I N FO R M ATI O N
BDG test kits were provided by the Associates of Cape Cod. The clinical cohort was built up as part of the Austrian Science Fund project P 24362.

F I G U R E 3
Kaplan-Maier survival estimation.

CO N FLI C T O F I NTER E S T S TATEM ENT
Malcolm Finkelman was an employee of Associates of Cape Cod, Inc. the manufacturer of the Fungitell® kit. Other authors declare no conflict of interest for this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S A PPROVA L S TATEM ENT
The study was approved by the research ethics committee of the Medical University of Graz (23-096 ex 10/11).