Currently, over 35 million people globally are affected by C. sinensis infection, but only about 1.5–2 million exhibit symptoms or complications. Among individuals with current infection, some patients present acute symptoms such as fever, jaundice, and liver function abnormalities, while others show more subtle symptoms, only testing positive for eggs in fecal examinations³². The clinical phenotypic differences of C. sinensis disease are the result of the combined effects of the host’s immune status, genetic background, characteristics of the infecting strain, infection load, and the host’s age, gender, nutritional status, and environmental factors. H. pylori infection primarily occurs in childhood, with its prevalence increasing with age, while Clonorchiasis predominantly affects adults. Poor dietary habits and unfavorable socioeconomic conditions are common risk factors for both H. pylori and Clonorchiasis infection. Additionally, both Clonorchiasis and H. pylori mediate immune responses through the balance of CD4 + T cell subsets, leading us to hypothesize potential cross-interactions between these two microorganisms. The classification of infections, particularly past infections, involves a combination of approaches. For instance, serological testing is commonly used, where IgM antibodies indicate recent infection and IgG antibodies reflect past exposure due to their prolonged presence. Moreover, advances in molecular diagnostics, including metagenomic sequencing, facilitate direct detection of pathogen nucleic acids, enhancing the identification of current or prior infections, especially in immunocompromised individuals. The integration of immunological evidence, clinical coding, symptom timing, and molecular methods thus provides a comprehensive framework for accurately categorizing infections in both clinical and research settings.

The clinical phenotypic differences of C. sinensis disease are the result of the combined effects of the host’s immune status, genetic background, characteristics of the infecting strain, infection load, and the host’s age, gender, nutritional status, and environmental factors. H. pylori infection primarily occurs in childhood, with its prevalence increasing with age, while Clonorchiasis predominantly affects adults. Poor dietary habits and unfavorable socioeconomic conditions are common risk factors for both H. pylori and Clonorchiasis infection. Additionally, both Clonorchiasis and H. pylori mediate immune responses through the balance of CD4 + T cell subsets, leading us to hypothesize potential cross-interactions between these two microorganisms.

In this study, we first employed the ELISA method to detect both H. pylori antibodies and C. sinensis antibodies in 309 civil servants undergoing health check-ups. The results indicated that the infection rate of C. sinensis among healthy individuals in this region was as high as 23.9%, likely related to local dietary habits that favor raw freshwater fish consumption. Subsequent multivariable regression analysis further confirmed that a history of consuming raw fish is one of the independent risk factors for C. sinensis infection. Our study also found that the H. pylori infection rates among individuals with and without C. sinensis infection was 45.9% and 43.0%, respectively, with no statistically significant difference. However, the H. pylori infection rate among individuals with current C. sinensis infection reached 61.5%. Although this did not show statistical significance, it suggests that H. pylori may facilitate the invasion and pathogenicity of C. sinensis in the human body through certain mechanisms, such as raw fish consumption, poor hygiene. The antibody results might reflect prior exposure or involve serological cross-reactivity rather than active synergy. Additionally, selecting hospitalized patients may introduce bias toward more severe cases. Finally, the cross-sectional design limits causal inference. Future longitudinal and mechanistic studies are needed to clarify these relationships.

H. pylori infection often occurs in childhood and, if not eradicated, can persist for a lifetime. We can’t help but wonder whether the colonization of H. pylori during childhood prepares the ground for the invasion of Clonorchiasis. H. pylori downregulates inflammation and controls the host’s immune response through various virulence factors³³. Its induced chronic active inflammation reduces the gastrointestinal mucosal barrier function, creating a suitable environment for the colonization and development of Clonorchiasis. Theoretically, if an individual has a prior H. pylori infection, especially a CagA-positive infection, this may lead to a certain degree of immunosuppression or immune tolerance. When such an individual subsequently gets infected with Clonorchiasis, the immune microenvironment changes caused by the previous H. pylori infection may facilitate the persistence of Clonorchiasis and disease progression. Furthermore, H. pylori infection may also indirectly affect the host’s susceptibility and response to Clonorchiasis by influencing the composition of the gastrointestinal microbiota. The gut microbiota is closely related to the development and function of the host immune system³⁴, and the dysbiosis caused by H. pylori infection may further impact the host’s defense against other pathogens. Therefore, we speculate that H. pylori infection could be a factor influencing the clinical heterogeneity of symptoms after Clonorchiasis infection.

To clarify the interaction between the two, we selected 73 hospitalized patients with current C. sinensis infection from another hospital and used the Western blot method to detect H. pylori antibodies. Strain identification was performed based on the presence of CagA and/or VacA regions. The results showed that the H. pylori infection rate among patients with current C. sinensis infection was 61.6%, slightly higher than the rate of 44.0% in healthy individuals, although this difference was not statistically significant. However, the infection rate of CagA + strains in patients with current C. sinensis infection was 56.2%, significantly higher than the 28.0% in healthy individuals, indicating a significant association between CagA + H. pylori strains and current C. sinensis infection.

In the hamster model established by Dangtakot et al., the colonization rate of H. pylori in the hepatobiliary system in the co-infection group with H. pylori and C. sinensis was higher than that in the single infection group, and it increased the severity of hepatobiliary abnormalities, manifested as more severe periductal fibrosis, cholangitis, and biliary hyperplasia³⁵. This may result from one or more of the following reasons: 1. The local inflammation and tissue damage in the hepatobiliary system caused by Clonorchiasis provide a more favorable environment for H. pylori colonization³⁶; 2. The urease produced by H. pylori neutralizes gastric acid, creating a more suitable weakly acidic environment for its survival³⁷. After Clonorchiasis infection, physiological changes such as bile stasis or alterations in bile duct pH facilitate the survival and proliferation of H. pylori. Additionally, bile acids can enhance the translocation of H. pylori’s CagA protein into host cells, amplifying the effects of CagA to promote inflammation and damage³⁸; 3. Co-infection by both pathogens interferes with the host’s immune response³⁹, weakening the ability to clear H. pylori; 4. Researchers speculate that H. pylori may exhibit a “ride-along” phenomenon in the lifecycle of Clonorchiasis, where L-fucose mediates H. pylori colonization in the Clonorchis intestine⁴⁰, followed by the transmission of H. pylori carried by Clonorchiasis larvae in the hepatobiliary system^41,42; 5. H. pylori may first cause damage in the stomach and then infect the biliary system via ascending duodenal infection or through the portal venous circulation ⁴³; 6. Some virulence factors of H. pylori strains (CagA and VacA) may enhance their colonization ability in the hepatobiliary system⁴⁴. Of course, the interactions between the two pathogens are not unidirectional. The inflammatory response induced by H. pylori infection may lead to changes in the biliary and intestinal mucosa, providing a suitable environment for the colonization and development of Clonorchis, while the Treg cells induced by the infection suppress the inflammatory response⁴⁵, thereby reducing the host’s defense against Clonorchis infection.

We conducted a multifactorial regression analysis on the factors that may influence current C. sinensis infection. The results suggest that CagA-positive H. pylori infection is one of the independent risk factors for current C. sinensis disease. Virulence factors like CagA enable H. pylori to activate the production of inflammatory cytokines and chemokines, leading to leukocyte recruitment. Suyapoh. W et al. observed that in patients infected with both CagA-positive H. pylori and C. sinensis, there was a significant increase in leukocyte infiltration and lymphocyte aggregation, indicating a synergistic effect of these two carcinogens in inducing severe inflammatory responses, goblet cell metaplasia, and abnormal proliferation³⁹. Notably, the polymorphism of the EPIYA sequence in the CagA protein is closely related to the severity of disease in patients with Clonorchiasis infection, with the EPIYA-AB’C type (containing the variant EPIYT B) leading to more severe periductal fibrosis, while CagA types containing multimerization (CagA multimerization, CM) sequences were significantly associated with the degree of fibrosis⁴². Other studies have shown that CagA also induces persistent advanced periductal fibrosis (APF) after praziquantel treatment, even leading to recurrence, affecting patient prognosis⁴⁶. These findings emphasize the importance of monitoring the CagA protein in the management and treatment of biliary diseases in cases of co-infection with Clonorchiasis and H. pylori.

This study has some limitations. As a cross-sectional design, it cannot establish causality or the sequence of infections. The lack of mechanistic data, such as cytokine profiles and functional assays, limits our understanding of the underlying biological interactions. In addition, potential confounders such as socioeconomic status, comorbidities, and hygiene practices were not fully accounted for and may have influenced the results. Antibody-based testing may also reflect prior rather than active infection. These factors should be addressed in future longitudinal and mechanistic studies.

In summary, the infection rate of CagA-positive H. pylori in patients with current C. sinensis infection is significantly higher than that in healthy individuals, and CagA-positive H. pylori strains are independent risk factors for current C. sinensis infection, suggesting that H. pylori carrying the CagA gene may have a synergistic effect on the occurrence and development of Clonorchiasis.

Most current studies primarily focus on the relationship between H. pylori and Opisthorchis viverrini co-infections, likely due to the distribution characteristics of Clonorchiasis in their respective locations. Our study provides supplementary insights into the synergistic pathogenic effects of C. sinensis and H. pylori. Although we have observed a potential association between CagA-positive H. pylori and current C. sinensis infection, possibly promoting more severe pathological damage and even the development of cholangiocarcinoma, there are still some research limitations. There is a lack of in-depth understanding of the molecular mechanisms by which CagA-positive H. pylori and C. sinensis co-infections synergistically affect host cells. More research is needed to clarify how these pathogens jointly influence signaling pathways and gene expression in biliary epithelial cells. The host immune response to H. pylori and C. sinensis infections is very complex and exhibits individual variability. The diversity of the host immune background may not have been adequately considered in studies, potentially affecting a comprehensive understanding of pathogen virulence. Furthermore, long-term follow-up studies on co-infected patients are crucial for understanding their relationship with the progression of C. sinensis and assessing the long-term effects of potential therapeutic interventions.