Fungal signature differentiates alcohol-associated liver disease from nonalcoholic fatty liver disease

Figures & data

*post-hoc p values.

	Control vs ALD	Control vs NAFLD	ALD vs NAFLD
Age	0.004	<0.001	<0.001
BMI	0.002	<0.001	<0.001
AST	<0.001	<0.001	0.016
ALT	<0.001	<0.001	0.462
GGT	<0.001	<0.001	0.077
AP	<0.001	<0.001	0.474
Bilirubin	0.098	0.029	0.340
Albumin	0.057	0.638	0.005
CAP [dB/m]	<0.001	<0.001	0.210
Stiffness [kPa]	0.070	0.004	0.070

Table 1. Baseline demographic and laboratory data of the study population.

	n	Control (n = 34)	Alcohol-associated liver disease (n = 58)	Nonalcoholic fatty liver disease (n = 78)	p value
Gender [male], n [%]	170	20 (58.8%)	40 (69.0%)	38 (48.7%)	0.061
Age [years]	170	33.5 [30.6;44.9]	43.5 [36.2;53.0]	55.6 [43.3;63.4]	0.001
BMI [kg/m^Citation2	170	21.1 [20.0;24.1]	24.2 [22.0;27.5]	30.1 [27.5;33.5]	<0.001
AST [IU/L]	160	19.0 [16.0;25.0]	53.5 [29.0;101]	35.0 [28.0;52.0]	<0.001
ALT [IU/L]	160	13.0 [9.00;15.0]	46.5 [24.2;87.5]	48.0 [33.0;78.0]	<0.001
GGT [IU/L]	160	16.0 [11.0;20.4]	108 [42.0;289]	72.0 [46.0;125]	<0.001
AP [IU/L]	158	55.5 [49.6;62.1]	75.0 [60.0;88.0]	75.0 [65.0;94.0]	0.001
Bilirubin [mg/dL]	158	0.30 [0.15;0.58]	0.50 [0.30;0.60]	0.50 [0.40;0.80]	0.029
Albumin [g/dL]	157	4.40 [4.40;4.50]	4.60 [4.30;4.93]	4.40 [4.20;4.60]	0.004
INR	148	1.00 [1.00;1.00]	0.98 [0.91;1.03]	1.00 [0.90;1.00]	0.599
Creatinine [mg/dL]	159	0.81 [0.71;1.00]	0.81 [0.72;0.87]	0.85 [0.71;1.02]	0.378
Platelets [10^9/L]	150	240 [230;264]	231 [171;277]	217 [181;278]	0.496
CAP [dB/m]	99	195 [184;200]	310 [268;330]	280 [261;314]	<0.001
Stiffness [kPa]	146	4.60 [4.00;5.35]	5.50 [4.12;6.68]	6.20 [4.80;13.0]	0.003

Values are presented as median and upper and lower quartiles in brackets. The number of subjects for which data were available is indicated in the first column. Continuous variables were compared using the Wilcoxon-Whitney-Mann rank-sum test. Categorical variables were compared using the Pearson’s Chi-squared test. Bold font indicates statistical significance (p < 0.05). ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BMI, body mass index; CAP, controlled attenuation parameter; GGT, gamma-glutamyltransferase; INR, international normalized ratio.

Figure 1. The intestinal fungal microbiome differs significantly between patients with ALD and NAFLD.

(a) Principal coordinate analysis (PCoA) of mycobiome in ALD patients (n = 58), NAFLD patients (n = 78), and controls (n = 34). (b) PCoA of mycobiome in ALD patients (n = 58) and NAFLD patients (n = 78). (c-d) Linear discriminant analysis (LDA) of (c) genera and (d) species of ALD patients vs NAFLD patients. (E-H) Relative abundance of genera (e) Mucor, (f) Kluyveromyces, and of species (g) Candida albicans and (h) Malassezia restricta. A p value of equal or less than 0.05 was considered as statistically significant.

Figure 2. A fungal signature differentiates ALD from NAFLD.

(a-b) Mean decrease accuracy by random forest analysis was quantitated for (a) fungal genera and (b) species to determine their respective feature importance for detecting ALD vs NAFLD. (c) ROC curves of fungal genera and species for detecting ALD vs NAFLD (ALD patients, n = 58; NAFLD patients, n = 78). ALD, alcohol-associated liver disease; AUC, area under the curve; K, Kluyveromyces; M, Mucor; MR, Malassezia restricta; NAFLD, nonalcoholic fatty liver disease; ROC, receiver operating characteristic; SCO, Scopulariopsis/S. cordiae.

Table 2. ALD vs NAFLD predictors.

Marker	AUC	Youden	Threshold	Sens	Spec	Acc	PPV	NPV	p value DeLong’s test
Scopulariopsis/S. cordiae (SCO)	0.68	0.35	>0.0020%	0.38	0.97	0.72	0.92	0.68
Kluyveromyces (K)	0.67	0.33	>0.0022%	0.40	0.94	0.71	0.82	0.68
Mucor (M)	0.65	0.32	<0.0031%	0.95	0.37	0.62	0.53	0.91
Saccharomyces/S. cerevisiae	0.59	0.30	>91.38%	0.36	0.94	0.69	0.81	0.66
Kazachstania	0.66	0.33	<0.0024%	0.90	0.44	0.63	0.54	0.85
M. restricta (MR)	0.70	0.41	>0.00026%	0.48	0.92	0.74	0.82	0.71
C. albicans	0.64	0.30	>0.00026%	0.62	0.68	0.65	0.59	0.71
C. argentea	0.59	0.18	<0.00001%	0.97	0.22	0.54	0.48	0.90
SCO+K	0.80	0.58	SCO > 0.0020%, K > 0.0022%	0.67	0.91	0.81	0.85	0.79	<0.001 vs SCO
SCO+K+MR	0.89	0.76	SCO > 0.0020%, K > 0.0022%, MR > 0.00026%	0.93	0.83	0.88	0.81	0.94	0.007 vs SCO+K
SCO+K+MR+M	0.93	0.82	SCO >0.0020%, K > 0.0022%, MR  > 0.00026%, M < 0.0031%	0.91	0.91	0.91	0.88	0.93	0.038 vs SCO+K+MR

The best threshold was determined to maximize the Youden index (= sensitivity + specificity − 1) for each marker. N = 136. Acc, accuracy; AUC, area under the curve; C., Candida; K, Kluyveromyces; M, Mucor; MR, Malassezia restricta; NPV, negative predictive value; PPV, positive predictive value; S. cerevisiae, Saccharomyces cerevisiae; SCO, Scopulariopsis/S. cordiae; sens, sensitivity; spec, specificity.

*post-hoc p values.

	ALD F0-F1 vs ALD F2-F4	NAFLD F0-F1 vs NAFLD F2-F4	ALD F0-F1 vs NAFLD F0-F1	ALD F2-F4 vs NAFLD F2-F4
Age	0.13	0.001	0.171	0.184
BMI	0.354	0.103	<0.001	0.013
AST	0.008	<0.001	0.017	0.016
GGT	0.003	0.897	1.000	0.001
Albumin	0.018	0.011	0.022	0.813
INR	<0.001	0.004	0.556	0.043
Platelets	0.147	0.183	0.916	0.864
Stiffness	<0.001	<0.001	0.665	0.606

Table 3. Baseline demographic and laboratory data of the study population stratified by fibrosis severity.

	n	ALD F0-F1 (n = 48)	ALD F2-F4 (n = 10)	NAFLD F0-F1 (n = 43)	NAFLD F2-F4 (n = 30)	p value
Gender [male], n [%]	131	33 (68.8%)	7 (70.0%)	22 (51.2%)	14 (46.7%)	0.154
Age [years]	131	42.0 [34.0;53.0]	50.0 [43.2;62.5]	52.3 [38.5;58.6]	60.7 [55.2;66.0]	<0.001
BMI [kg/m^Citation2	131	24.2 [21.9;26.6]	25.5 [22.3;28.4]	29.6 [27.0;31.2]	31.4 [27.5;36.6]	<0.001
AST [IU/L]	131	47.5 [28.8;84.8]	107 [64.5;178]	30.0 [26.5;37.5]	50.0 [33.2;63.2]	<0.001
ALT [IU/L]	131	39.5 [23.0;77.5]	81.0 [46.8;93.5]	44.0 [30.5;58.5]	54.0 [34.0;84.0]	0.135
GGT [IU/L]	131	90.0 [40.2;212]	415 [259;896]	83.0 [42.0;120]	70.5 [49.5;124]	0.002
AP [IU/L]	130	73.0 [61.0;86.0]	88.0 [59.0;135]	73.0 [61.0;93.0]	77.5 [67.2;94.5]	0.343
Bilirubin [mg/dL]	130	0.50 [0.30;0.60]	0.55 [0.40;0.70]	0.40 [0.30;0.70]	0.60 [0.40;0.90]	0.128
Albumin [g/dL]	129	4.65 [4.43;5.00]	4.40 [4.03;4.57]	4.50 [4.30;4.65]	4.30 [4.03;4.47]	<0.001
INR	130	0.94 [0.89;1.01]	1.11 [1.04;1.21]	1.00 [0.90;1.00]	1.00 [1.00;1.10]	<0.001
Creatinine [mg/dL]	131	0.81 [0.74;0.92]	0.74 [0.71;0.81]	0.85 [0.70;1.03]	0.84 [0.70;0.98]	0.361
Platelets [10^Citation9/L]	130	240 [188;284]	169 [136;230]	250 [186;287]	208 [146;250]	0.028
CAP [dB/m]	84	308 [265;326]	328 [295;347]	279 [249;309]	291 [269;325]	0.321
Stiffness [kPa]	131	5.05 [3.98;6.12]	17.2 [12.7;24.4]	4.80 [4.40;5.55]	13.7 [11.0;17.5]	<0.001

Values are presented as median and upper and lower quartiles in brackets. The number of subjects for which data were available is indicated in the first column. Continuous variables were compared using the Wilcoxon-Whitney-Mann rank-sum test. Categorical variables were compared using the Pearson’s Chi-squared test. Bold font indicates statistical significance (p < 0.05). ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BMI, body mass index; CAP, controlled attenuation parameter; GGT, gamma-glutamyltransferase; INR, international normalized ratio.

Figure 3. Fungal subpopulations distinguish ALD and NAFLD with no or mild fibrosis.

(a-b) Linear discriminant analysis (LDA) of (a) genera and (b) species of ALD F0-F1 vs NAFLD F0-F1. (c-f) Relative abundance of genera (c) Kluyveromyces, (d) Mucor, and of species (e) Candida albicans and (f) Malassezia restricta. (g) ROC curves of fungal genera and species for detecting ALD F0-F1 vs NAFLD F0-F1 (ALD F0-F1 patients, n = 48; NAFLD F0-F1 patients, n = 43). A p value of equal or less than 0.05 was considered as statistically significant. ALD, alcohol-associated liver disease; AUC, area under the curve; K, Kluyveromyces; KAZ, Kazachstania; M, Mucor; MR, Malassezia restricta; NAFLD, nonalcoholic fatty liver disease; ROC, receiver operating characteristic; SCO, Scopulariopsis/S. cordiae.

Figure 4. A fungal signature differentiates ALD from NAFLD with significant fibrosis.

(a-b) Linear discriminant analysis (LDA) of (a) genera and (b) species of ALD F2-F4 vs NAFLD F2-F4. (c-d) Relative abundance of genera (c) Mucor and Debaryomyces, and of species (d) Malassezia restricta. (e) ROC curves of fungal genera and species for detecting ALD F2-F4 vs NAFLD F2-F4 (ALD F2-F4 patients, n = 10; NAFLD F2-F4 patients, n = 30). A p value of equal or less than 0.05 was considered as statistically significant. ALD, alcohol-associated liver disease; AUC, area under the curve; K, Kluyveromyces; M, Mucor; MR, Malassezia restricta; NAFLD, nonalcoholic fatty liver disease; ROC, receiver operating characteristic; SCO, Scopulariopsis/S. cordiae.

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