Clinical characteristics of participants
65 subjects were recruited in this study, including 33 patients with chronic S. japonicum infection, 15 patients with advanced S. japonicum infection, and 17 healthy volunteers. The diagnosis of S. japonicum infection was based on medical history, stool examination, serum antibody test and imaging examination. No significant differences in age or sex were observed among the three groups (Table 1). The total bile acid level in S. japonicum infection patients was higher than that in healthy individuals, and patients with advanced S. japonicum infection had a higher level than those with chronic S. japonicum infection (Table 1). Compared to the healthy control group, patients with S. japonicum infection showed an increased level of blood glucose, total cholesterol, and low-density lipoprotein (Table 1). Nevertheless, there was no significant differences between patients with advanced S. japonicum infection and those with chronic S. japonicum infection. The clinical characteristics of the participants were shown in Table 1.
Metabolomic analysis of S. japonicum infection serum
The overlaid base peak intensity chromatograms of the QC samples in positive and negative ion modes verified the reliability and stability of the system (Figure. 1a). PCA indicates variability between healthy controls, patients with chronic S. japonicum infection and advanced S. japonicum infection (Figure. 1b). The observed differences among the samples, as depicted in the figure, can be attributed to variations in metabolite levels. Then PLS-DA was applied to these three groups of biological samples to establish the relationship model between metabolite expression and sample class, which can realize the modeling and prediction of sample class (Figure. 1c). To further identify variations in serum metabolites among healthy controls, chronic S. japonicum infection patients and advanced S. japonicum infection patients, OPLS-DA was performed on the collected data. 200 iterations of the response permutation test showed negative Q2 intercept values (Figure 1d), indicating that the OPLS-DA model was not overfit. OPLS-DA score plots showed great separation, confirming significant differences between the three groups (Figure. 1e).
Differential metabolites in different stages of S. japonicum infection
In this study, we conducted a differential analysis of high-quality metabolites to gain quantitative insights into their relative levels among samples from healthy controls, patients with chronic S. japonicum infection and patients with advanced S. japonicum infection. Differential metabolites were identified based on specific criteria: FC ≥ 1.2 or FC ≤ 0.83, p-value<0.05, and VIP ≥ 1.0. Comparing samples of chronic S. japonicum infection with healthy controls, we found 185 up-regulated metabolites and 297 down-regulated metabolites. The significant metabolites mainly belonged to categories such as benzene and derivatives, organoheterocyclic compounds, glycerophospholipids, bile acids, alcohols and derivatives, and fatty acyls (Figure. 2a and Figure S1). Furthermore, comparing the advanced S. japonicum infection group to the chronic S. japonicum infection group, we identified 199 up-regulated metabolites and 207 down-regulated metabolites in advanced S. japonicum infection group. These differentially abundant metabolites were categorized as steroids and derivatives, glycerophospholipids, amino acids, peptides and analogues, organoheterocyclic compounds, organic acids, and fatty acyls (Figure. 2b and Figure S2). In the comparison between the advanced S. japonicum infection group and the healthy control group, we found 187 up-regulated metabolites and 383 down-regulated metabolites in advanced S. japonicum infection group. These metabolites were classified in categories such as benzene and derivatives, steroids and derivatives, organoheterocyclic compounds, fatty acyls, glycerophospholipids, terpenoids and carbohydrates (Figure. 2c and Figure S3).
Functional enrichment analysis of the differentially expressed metabolites
To investigate potential metabolite pathways involved in the progression of S. japonicum infection, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the different expressed metabolites in different stages of S. japonicum infection. The result indicated that there were 10 pathways significantly enriched, including steroid hormone biosynthesis, cholesterol metabolism and bile secretion (Table 2 and Figure. 3a). Differential abundance score (DA score) is a pathway-based metabolite change analysis method, which could help to understand the overall changes in metabolite pathways22. Compared with the chronic S. japonicum infection group, we found that the steroid hormone biosynthesis pathway was significantly down-regulated in advanced S. japonicum infection patients, pathways such as cholesterol metabolism, bile secretion, ATP-binding cassette (ABC) transporters, amino acid biosynthesis and primary bile acid biosynthesis were significantly up-regulated (Figure. 3b).
Metabolites associated with the progression of S. japonicum infection
To identify metabolites that have potential in assisting diagnosis of different stages of S. japonicum infection, we performed further analysis on the different expressed metabolites. We noticed that GCA, GCDCA, and TCDCA exhibited a gradual rise in concentration as the disease progression (Figure. 4a). To evaluate the predictive ability of the potential biomarkers for advanced S. japonicum infection, we performed ROC analysis on different expressed metabolites. Our study identified 3 metabolites with area under curve (AUC) higher than 0.8, including GCA, GCDCA, and TCDCA, concentrated in cholesterol metabolism, biliary secretion, and primary bile acid biosynthesis. Thus, ROC analysis indicated that GCA, GCDCA, and TCDCA seemed to have potential to act as candidate biomarkers for diagnosis of advanced S. japonicum infection (Figure. 4b).