The association between Clonorchis sinensis infection and hepatocellular carcinoma in an endemic area: a study in Guangxi, China

The association between Clonorchis sinensis infection and hepatocellular carcinoma in an endemic area: a study in Guangxi, China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The association between Clonorchis sinensis infection and hepatocellular carcinoma in an endemic area: a study in Guangxi, China Qing-Li Yang, Xi-Wei Lu, Zhong-Liao Fang, Yu-Qiu Gao, Yi-Ning He, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4447355/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 19 You are reading this latest preprint version Abstract Background Chronic infection with Clonorchis sinensis ( C . sinensis ) has been associated with the development of intrahepatic cholangiocarcinoma (ICC); however, the relationship between C . sinensis and hepatocellular carcinoma (HCC) remains uncertain. Methods This study examined 120 patients with liver cancer in the clonorchiasis endemic area of Hengzhou, Guangxi, China. The type of cancer, the differentiation grade according to Edmondson Steiner's classification, and the pathological characteristics of HCC were determined through postoperative tissue biopsy. C . sinensis infection was detected by measuring serum specific IgG antibody, and hepatitis B virus (HBV) infection was determined by detecting serum HBsAg and HBV DNA in HCC tissues. Results Of the patients evaluated, 98 (81.7%) had HCC, 21 (17.5%) had ICC, and 1 (0.8%) had comorbidity of HCC/ICC. Among the HCC patients, 24 (24.5%) were solely infected with HBV, 71 (72.4%) were infected with C. sinensis, and 3 (3.1%) showed no evidence of infection. C. sinensis infection rates in HCC patients are much higher than in the general population, as well as in general outpatient and non-liver cancer inpatients in the local area (χ 2 = 82.61/141.92, P 0.05). Among the patients with C . sinensis -related HCC, 8 (8.2%) were solely infected with C . sinensis , while 63 (64.3%) were co-infected with HBV. Infection with C. sinensis and HBV has a significant impact on the kind of liver cancer (χ 2 = 22.858, cramer’V/φ = 0.444, contingency coefficient = 0.406, P < 0.001). Conclusions These findings indicate that HCC still accounts for the majority of liver cancer in this region. In addition to being most commonly related with HBV infection, HCC is also related with C. sinensis infection. The co-infection of HBV and C. sinensis is the leading cause of HCC in this region. Clonorchis sinensis hepatitis B virus hepatocellular carcinoma association Figures Figure 1 Figure 2 Figure 3 Background The adult worms of Clonorchis sinensis ( C . sinensis ) reside within the intrahepatic bile ducts of humans and animals, leading to the development of clonorchiasis and intrahepatic cholangiocarcinoma (ICC) [ 1 , 2 ]. Clonorchiasis is predominant in East Asia [ 3 ], with over 15 million individuals estimated to be infected globally [ 4 ]. Guangxi, China, is recognized as a major region affected by clonorchiasis. According to the 2015 surveillance of major human parasitic diseases, the weighted infection rate of C . sinensis in Guangxi was 6.68%, with approximately 2.9163 million people affected, ranking it the highest in China [ 5 , 6 ]. Additionally, Guangxi is also known as a high prevalence area for hepatitis B. Hepatitis B virus (HBV) infection is the primary cause of hepatocellular carcinoma (HCC) in this region [ 7 , 8 ]. In recent decades, there have been individual HCC cases reported after C . sinensis infection, which suggests that C . sinensis infection not only triggers ICC but may also be associated with the occurrence of HCC. Between 2014 and 2015, we identified a total of 10 cases of C . sinensis -related HCC in the clonorchiasis epidemic zone of Hengzhou City, Guangxi, including six cases of C . sinensis mono-infection and four cases of co-infection with HBV [ 9 ]. To further establish the correlation between C . sinensis infection and HCC, a larger cohort survey of liver cancer patients in Hengzhou, Guangxi was subsequently conducted. Methods Cases and specimens The study included a cohort of 120 patients diagnosed with liver cancer who underwent surgical treatment at People's Hospital of Hengzhou (PHH) between August 2020 and May 2023. Prior to the surgery, blood samples were obtained from each patient and the serum was separated and stored at -80°C. Liver cancer tissues were collected during the operation and also stored at -80°C. Additional tissue samples were fixed with a 4% paraformaldehyde solution and stored at room temperature. ELISA The qualitative detection of the IgG antibody against C . sinensis and hepatitis B virus surface antigen (HBsAg) in serum samples was conducted using the enzyme-linked immunosorbent assay (ELISA) method. A liver fluke IgG antibody detection kit (Shenzhen Huakang Biomedical Engineering Co., Ltd., Shenzhen) was used to detect the C . sinensis IgG antibody, while a diagnostic kit (Shanghai Rongsheng Biopharmaceutical Co., Ltd., Shanghai) was used to detect HBsAg. For the detection of C . sinensis IgG antibody, the following steps were performed: The serum was diluted 1:10 with dilution buffer and added to a microplate coated with C . sinensis antigen. The mixture was incubated at 37°C for 1 hour, followed by washing the plate 5 times for 1 minute each. After drying, 50 µL of enzyme conjugate was added and incubated at 37°C for 30 minutes. The plate was washed again 5 times for 1 minute each. The double antibody sandwich method was used for the detection of HBsAg. 50 µL of the serum to be tested was added to a microplate coated with anti-HBs and incubated at 37°C for 1 hour. Then, 50 µL of enzyme conjugate was added and incubated at 37°C for 30 minutes. The plate was washed 5 times for 10 seconds each. Finally, substrates were added for color development and the reaction was terminated. The experiment included blank, positive, and negative controls. The OD 450 value was measured using the Bio Tek Epoch microplate reader, and the result was determined based on the cut-off value. Detection of HBV DNA HCC tissue samples weighing between 30 and 50 mg were homogenized in a heating block at 56°C for 6 hours. During this process, intermittent grinding was performed using 1 mL of lysis buffer containing 100 mM Tris, 50 mM EDTA, 2% SDS, and 500 g/ml proteinase K at a pH of 8.0. Subsequently, Tris-saturated phenol and chloroform-isopentanol (at a ratio of 24:1) were used to extract and purify the entire DNA content. The DNA was then precipitated using ethanol and subsequently dissolved in 200 µL of 8 mM NaOH solution (pH 8.0). The DNA concentration was measured using spectrophotometry at a wavelength of 260 nm using the NanoDrop One device (Thermo Scientific). For the quantification of HBV DNA, primers based on the HBV PreC/C gene were created using Primer-BLAST (available at nih.gov). The primer sequences were as follows: forward 5’- ACTTTTTCACCTCTGCCTAA-3’ and reverse 5’- AGCTCCAAATTCTTTATA-3’. These primers were synthesized by Sangon Biotech Co., Ltd. in Shanghai, China. Real-time quantitative PCR (qPCR) was used to detect HBV DNA with the TB Green® Premix Ex Taq qPCR Mix. Specifically, 20.0 µL of the TB Green® Premix Ex Taq qPCR Mix (2 ×), 0.8 µL of each primer (10 µM), and 1.0 µL of the DNA template were combined to achieve a final volume of 40 µL. The amplification and data acquisition steps were carried out using an Analytik Jena qTOWER 3 /G touch Real-Time PCR system with V4.0 software. The cycling parameters included a pre-denaturation step at 95°C for 30 seconds, followed by 50 cycles of amplification at 95°C for 5 seconds, and annealing and extension at 60°C for 30 seconds. Subsequently, a melt curve analysis ranging from 65 to 95°C was performed to determine the melting temperature (Tm) for the specific DNA product populations that were identified. The HBV DNA copy number was standardized using 100 ng of tissue DNA. Postoperative tissue biopsy The specimens underwent fixation, paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E). The Zeiss Apotome.2 imaging system was used to observe the pathological types of HCC. Additionally, the differentiation of HCC was assessed using the grading system I ~ IV based on Edmondson Steiner's criteria [ 10 , 11 ]. Furthermore, the presence of inflammatory cell infiltration, fibrosis, and necrosis in the tissue sections was documented. Statistical analysis The Pearson χ 2 test was used to compare the influence and correlation of C . sinensis and HBV infection on different types of liver cancer, and the pathological type, differentiation degree, and ratio of pathological constituents in HCC following C . sinensis , HBV mono-infection, and HBV co-infection. The statistical analysis was conducted using IBM SPSS Statistics 19.0 software, and a P -value of 0.05 or less was considered statistically significant. Results The state and relationship between HBV and C. sinensis infection in patients with liver cancer. A comprehensive investigation was conducted of a cohort of 120 patients suffering from liver cancer, ranging in age from 33 to 76 years. Of these patients, 104 were male and 16 were female. The postoperative liver tissue specimens of 115 cases were examined by pathology at a local hospital, PHH, while 108 cases were examined in our laboratory, the Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine (LHIID). Among the cases, 103 underwent simultaneous pathological examination by both institutions, resulting in a high coincidence rate of 98.1% (101/103). Additionally, 17 cases were examined exclusively by one institution. In cases where there were inconsistent pathological results, preference was given to the results obtained from our laboratory (refer to Table 1). In terms of the specific diagnoses, of all the patients with liver cancer, 98 cases were diagnosed as HCC, 21 cases as ICC, and 1 case as a comorbidity of HCC/ICC. These diagnoses accounted for 81.7%, 17.5%, and 0.8% of the cases, respectively. Serum specific IgG antibodies were detected in order to determine past or current Clonorchis sinensis infection in patients with liver cancer [12]. Among the HCC patients, 71 cases (72.5%) tested positive for the C . sinensis IgG antibody. Of these, 60 cases were also positive for HBsAg, and the HBV DNA content in the HCC tissues ranged from 6 to 23,270,015 copies/100 ng DNA. On the other hand, 11 cases were negative for HBsAg, but in 3 of them HBV DNA was detected in the HCC tissues, with contents ranging from 1 to 124 copies/100 ng DNA. Notably, HCC tissues and serum from 8 patients with C . sinensis infection did not yield any detectable HBV DNA. The infection rate of C . sinensis in HCC patients is significantly higher than that in the general population (28.8%) [13], as well as in general outpatient and non-liver cancer inpatients (22.1%) [14] in the local area (χ 2 =82.61/141.92, P <0.001). All patients diagnosed with ICC tested positive for C . sinensis IgG antibody (refer to Table 2). The infection of C . sinensis and HBV has a significant impact and correlation on the occurrence of different types of liver cancer (χ 2 =22.858, cramer’V/φ=0.444, contingency coefficient=0.406, P <0.001). HBV mono-infection is only associated with HCC, while C . sinensis mono-infection and co-infection with HBV are both associated with ICC and HCC. The occurrence of ICC is solely associated with C . sinensis infection, wheres the occurrence of HCC is not only commonly associated with HBV, but also with C . sinensis infection. Histopathological analysis of HCC associated with C . sinensis and HBV infection A total of 88 cases of HCC associated with C . sinensis and HBV infection were subjected to pathological analysis in our laboratory. Pathological examination revealed that the morphology of the cancer cells in 54 patients resembled normal hepatocytes, exhibiting solid growth, adenoid growth, trabecular growth, and fiberboard growth patterns [15]. These differentiation patterns were classified as hepatocellular carcinoma (HCC) and accounted for 61.4% of the cases. In 28 patients, the cytoplasm of their cancer cells exhibited clear cell hepatocellular carcinoma (CC-HCC) characterized by massive storage of α-glycogen particles [16, 17]; these accounted for 31.8% of the cases. Two cases (2.3%) presented as steatohepatic hepatocellular carcinoma (SH-HCC) with multiple lipid droplets in the cytoplasm [15, 18, 19]. The remaining 4 cases (4.5%) were spindle cell hepatocellular carcinoma (Sp-HCC) consisting mainly of spindle-shaped cells [20]. According to Edmondson Steiner's grading, differentiation grades I~IV were observed in 5 (5.7%), 37 (42.0%), 18 (20.5%), and 28 cases (31.8%), respectively. The proportions of inflammatory cell infiltration, fibrosis, and necrosis in HCC tissue sections were 36.8%, 37.9%, and 25.3%, respectively. In the case of C . sinensis mono-infection, 8 cases of HCC were identified through pathological examination. Among these cases, 4 displayed HCC with trabecular growth, solid growth, and fiberboard growth patterns, 3 were classified as CC-HCC, and 1 was identified as SH-HCC (see Fig. 1). The pathological types of HCC after co-infection with C . sinensis and HBV were found to be diverse, including the majority of HCC, CC-HCC, and a few cases of SH-HCC and Sp-HCC (see Fig. 2). Statistical analysis revealed no significant difference in the proportion of pathological types between C . sinensis mono-infection, HBV mono-infection, and co-infection related HCC. Similarly, there was no significant difference in the distribution of Edmondson Steiner I~IV differentiation among these groups. Furthermore, no significant difference was observed in the frequency of inflammatory cell infiltration, fibrosis, and necrosis in HCC tissue sections (see Fig. 3). Discussion The primary focus of this study is to examine the connection between C . sinensis infection and HCC. In a study conducted by Strauss sixty years ago, it was observed that, of five cases of HCC found in patients infected with C . sinensis , four were accompanied by Laennec's cirrhosis. However, it should be noted that HBV infection was not excluded in these cases [ 21 ]. Nakashima et al. reported two cases of elderly patients with HCC after C . sinensis infection forty-five years ago. One patient had a mild infestation with hepatic changes indicative of posthepatitic cirrhosis, while the other had a heavy infestation that exhibited secondary biliary cirrhosis with dilated intrahepatic bile ducts and periductal fibrosis. In both cases, the tumor nodule was solitary, and the cells were differentiated and classified as Grade I of Edmondson-Steiner's scale of anaplasia. HBsAg was determined to be negative in both cases through radioimmunoassay [ 22 ]. These early studies suggest a potential relationship between C . sinensis infection and the development of HCC. In a more recent investigation conducted by Tan et al. fifteen years ago, it was found that the infection rate of C . sinensis in HCC patients at the First Affiliated Hospital of Guangxi Medical University and Cancer Hospital Affiliated to Guangxi Medical University was 16.44%, while the infection rate in non-HCC patients was only 2.40%. HBsAg was examined using ELISA to determine HBV infection. Based on these findings, researchers suggest that clonorchiasis may be an important risk factor for HCC [ 23 ]. In the past decade, we have conducted an ongoing investigation into liver cancer patients in the region of Hengzhou, Guangxi, which is known for its high prevalence of clonorchiasis. The purpose of this study is to clarify the relationship between C. sinensis infection and HCC in this particular demographic. We identified six cases of C . sinensis infection in a group of 20 liver cancer patients. To rule out the possibility of HBV infection, we conducted tests for HBsAg, which yielded negative results [ 9 ]. Over the course of the past three years, we have expanded our research to include a larger sample size of liver cancer patients in Hengzhou City. Our findings revealed eight cases of HCC following C . sinensis infection.Among the liver cancer patients surveyed, we observed that HCC was the predominant type of malignancy, with ICC being less common and is only associated with C . sinensis infection. Previous surveillance revealed that the infection rates of C . sinensis in the local population and outpatient/non-liver cancer inpatients are 28.8% [ 13 ] and 22.1% [ 14 ], respectively. In this investigation, we discovered that 72.5% of HCC patients were infected with C. sinensis , which was much higher than the infection rate in the general population in this location. The findings of this survey in a high-clonorchiasis-endemic area revealed a specific connection between C. sinensis infection and HCC. Guangxi is also a hepatitis B epidemic area. HBV infection is the leading cause of HCC in this region [ 7 , 8 ]. In the survey, we determined that HBV infection by testing for serum HBsAg and HBV DNA in liver cancer tissues [ 24 ]. The study discovered that the majority of patients with HCC in this location were infected with both HBV and C. sinensis , complicating the causes and risk of HBV-induced HCC [ 25 ]. Co-infection with HBV and C. sinensis , as well as the resulting HCC, are unavoidable difficulties in this area. Co-infection with HBV and C. sinensis is the leading cause of HCC, and it is an inescapable public health hazard in this region. This suggests that C . sinensis infection on its own may directly induce HCC, while co-infection with HBV may contribute to the higher incidence of HCC. There were no significant differences observed in the pathological type, differentiation degree, or detection frequency of inflammatory cell infiltration, fibrosis, and necrosis in HCC after C . sinensis infection alone or co-infection with HBV, when compared to HBV infection alone. During the investigation, one case of steatohepatic HCC was found in patients with C . sinensis infection, as well as one case with HBV co-infection. Further confirmation is required to determine the effect and mechanism of C . sinensis infection on lipid metabolism in HCC [ 26 , 27 ]. The mechanisms by which C . sinensis infection induces HCC are not yet to be determined. Initial investigations have revealed that the excretory-secretory products (ESPs) of C . sinensis can enhance the replication of HBV and elicit a Th2 immune response. It has been observed that patients co-infected with C . sinensis and HBV exhibit higher levels of serum HBV DNA than patients infected with HBV alone [ 28 , 29 ]. Clinical data have demonstrated that patients with co-infection of C . sinensis and HBV have significantly elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and hyaluronic acid (HA) compare to those with HBV infection alone [ 30 ]. A survey conducted in the high incidence area of clonorchiasis in the Lalin River, Northeast China, reported that out of 854 cases of clonorchiasis, 46 cases were co-infected with HBV (HBsAg positive). However, no significant differences were observed in 16 clinical indicators, including ALT, AST, and TB, between the C . sinensis infection, HBV infection, and co-infection groups [ 31 ]. In vitro experiments have revealed that co-infection of C . sinensis and HBV can promote liver fibrosis and chronic inflammation. Co-stimulation of the hepatic stellate cell line LX-2 with the total proteins from C.sinensis adult worms (CsTPs) and HBV positive serum leads to increased transcription of alpha-smooth muscle actin and types I and III collagen, as well as pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 [ 30 ]. Recent studies have highlighted the ability of parasite extracellular vesicles (EVs) to mediate the host competing endogenous RNA (ceRNA) regulatory network across species through their own non-coding RNA (ncRNA) with regulatory function [ 32 – 34 ]. These EVs have a profound impact on the occurrence of HCC [ 35 , 36 ]. Specifically, EVs derived from C . sinensis (CsEVs) [ 37 ] have been found to activate the TLR9 signal, promoting an inflammatory response characterized by the production of IL-6 and TNF-α in mouse biliary epithelial cells (BECs) [ 38 ]. The miRNA csi-let-7a-5p, which is enriched in CsEVs, negatively regulates the expression of SOCS1 and clec7a genes in the NF-κB signaling pathway, thereby enhancing the production of TNF-α, IL-6, IL-1β, iNOS, and promoting the differentiation of M1-like macrophages. These processes play a crucial role in the development of bile duct injury and fibrosis following infection [ 32 ]. However, the precise role and mechanism of CsEVs in interfering with the miRNA-mediated ceRNA regulatory network in host hepatocytes, and their impact on hepatocellular carcinogenesis, remain unclear. Clonorchiasis poses considerable public health risks [ 39 ], and the Hepatitis B virus is the leading cause of HCC in China [ 40 , 41 ]. However, chronic alcohol consumption [ 42 ], non-alcoholic fatty liver disease (NAFLD) [ 43 ] and hepatitis C virus infection [ 44 , 45 ] are not the primary hazards or burdens of HCC in this region. This investigation has provided further clarification of the correlation between Clonorchis sinensis infection and the occurrence of HCC. In populations with a high incidence of HBV infection and related HCC, the role of C . sinensis in inducing HCC may be overshadowed. In China, hepatitis B virus is the primary risk factor for HCC [ 40 , 41 ]. However, with the implementation of the “hepatitis B elimination” strategy in 2030 [ 46 , 47 ], the role of C . sinensis in inducing HCC may gradually become more evident. Furthermore, this investigation discovered that three patients with functional cured (FC) hepatitis B [ 48 , 49 ], who had negative HBsAg but low liver HBV DNA load, were all co-infected with C . sinensis and suffering from HCC. Therefore, the potential threat of C . sinensis infection cannot be disregarded in the clinical treatment of hepatitis B and the prevention and treatment of HBV-related HCC. In areas with a high prevalence of C . sinensis , effective control of human HCC should not only focus on the “hepatitis B elimination” strategy but also actively implement comprehensive prevention and control measures for clonorchiasis. Conclusions These results indicate that HCC still dominates the composition of liver cancer in this region, while ICC only accounts for a small number and is all associated with C . sinensis infection. C. sinensis infection was prevalent in HCC patients, and the occurrence of HCC was not only commonly associated with HBV, but also with C . sinensis infection. HBV mono-infection is only associated with HCC, but C . sinensis mono-infection and co-infection with HBV are both associated with ICC and HCC. The pathogenic type, differentiation, and basic pathological content of HCC were all unaffected by C. sinensis infection. The co-infection of HBV and C . sinensis is the leading cause of HCC in this region. Declarations Acknowledgements We appreciate Dr. Harrison TJ’s meticulous revision of the manuscript. Author contributions Q-L Y wrote original draft; Z-H J, X-W L and M-Y L conducted case investigation; Q-L Y, Y-Q G, Y-N H, YH and YD performed the laboratory works, and prepared figures 1-3 and tables 1-2; Z-H J, Z-L F and CHFC reviewed and edited the manuscript. All authors reviewed the manuscript. Funding This study was supported by the Natural Science Foundation of Guangxi (No. 2020GXNSFAA238016), the Global Health Research Seed Grant from the Carver College of Medicine at the University of Iowa, U.S.A. (No. XZ2020002), and the Research project of Guangxi Zhuang Autonomous Region Bureau of Disease Prevention and Control. Availability of data and materials The original contributions presented in the study are included in the article. Ethics approval and consent to participate The investigation and specimen collection procedures adhered to the principles of informed consent and were reviewed and approved by the Guangxi Ethics Review Committee (approval No. GXIRB 2020-0022). Consent for publicatio n All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare that they have no competing interests. Author details 1 Guangxi University of Chinese Medicine, Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine, Nanning 530200, Guangxi, People’s Republic of China. 2 People's Hospital of Hengzhou, Nanning 530300, Guangxi, People’s Republic of China. 3 Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for Viral Hepatitis Prevention and Control, Nanning 530028, Guangxi, People’s Republic of China. 4 University of Iowa Carver College of Medicine, 200 Hawkins Drive, JCP 4642, Iowa City, IA 52242, USA References Hong ST, Fang Y. Clonorchis sinensis and clonorchiasis, an update. Parasitol Int. 2012;61:17–24. Sithithaworn P, Yongvanit P, Duenngai K, Kiatsopit N, Pairojkul C. Roles of liver fluke infection as risk factor for cholangiocarcinoma. J Hepatobiliary Pancreat Sci. 2014;21:301–8. Qian MB, Utzinger J, Keiser J, Zhou XN, Clonorchiasis. Lancet. 2016;387:800–10. 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Infect Dis Poverty. 2013;2:4. McGlynn KA, Petrick JL, El-Serag HB. Epidemiol Hepatocellular Carcinoma Hepatol. 2021;73(Suppl 1):4–13. Xie Y, Hepatitis B. Virus-Associated Hepatocellular Carcinoma. Adv Exp Med Biol. 2017;1018:11–21. Åberg F, Byrne CD, Pirola CJ, Männistö V, Sookoian S. Alcohol consumption and metabolic syndrome: Clinical and epidemiological impact on liver disease. J Hepatol. 2023;78:191–206. Pouwels S, Sakran N, Graham Y, Leal A, Pintar T, Yang W, et al. Non-alcoholic fatty liver disease (NAFLD): a review of pathophysiology, clinical management and effects of weight loss. BMC Endocr Disord. 2022;22:63. Yang J, Qi JL, Wang XX, Li XH, Jin R, Liu BY, et al. The burden of hepatitis C virus in the world, China, India, and the United States from 1990 to 2019. Front Public Health. 2023;11:1041201. Zhou Q, Liu A, Wang S, Li J, He M, Chen L. Hepatitis C virus screening reactive among blood donors in mainland China: A systematic review and meta-analysis. Transfus Med. 2023;33:147–58. Liu J, Liang W, Jing W, Liu M. Countdown to 2030: eliminating hepatitis B disease, China. Bull World Health Organ. 2019;97:230–8. Block TM, Chang KM, Guo JT. Prospects for the Global Elimination of Hepatitis B. Annu Rev Virol. 2021;8:437–58. Leowattana W, Leowattana T. Chronic hepatitis B: New potential therapeutic drugs target. World J Virol. 2022;11:57–72. Phillips S, Jagatia R, Chokshi S. Novel therapeutic strategies for chronic hepatitis B. Virulence. 2022;13:1111–32. Tables Tables 1-2 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Published Journal Publication published 25 Feb, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 09 Dec, 2024 Reviews received at journal 08 Dec, 2024 Reviewers agreed at journal 08 Dec, 2024 Reviewers agreed at journal 07 Dec, 2024 Reviews received at journal 07 Dec, 2024 Reviewers agreed at journal 07 Dec, 2024 Reviewers agreed at journal 07 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviews received at journal 16 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers invited by journal 18 Jun, 2024 Editor invited by journal 23 May, 2024 Editor assigned by journal 22 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4447355","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308248655,"identity":"39fdf4d1-8e53-4fb1-9a30-866ca7f3b300","order_by":0,"name":"Qing-Li Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qing-Li","middleName":"","lastName":"Yang","suffix":""},{"id":308248656,"identity":"22d6d7d2-baa3-4864-a2f2-6398fae5ea66","order_by":1,"name":"Xi-Wei Lu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xi-Wei","middleName":"","lastName":"Lu","suffix":""},{"id":308248657,"identity":"94ec25f2-2eca-4cda-ab13-779b87c51684","order_by":2,"name":"Zhong-Liao Fang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhong-Liao","middleName":"","lastName":"Fang","suffix":""},{"id":308248658,"identity":"c5cbbc8d-2387-4115-bb50-ad29583a00f8","order_by":3,"name":"Yu-Qiu Gao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yu-Qiu","middleName":"","lastName":"Gao","suffix":""},{"id":308248659,"identity":"22b9beb6-8acb-48d9-b7b3-4d2a3f071616","order_by":4,"name":"Yi-Ning He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi-Ning","middleName":"","lastName":"He","suffix":""},{"id":308248661,"identity":"36d126db-ac48-4170-85de-1b85ec4ee9b3","order_by":5,"name":"Yan Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Huang","suffix":""},{"id":308248663,"identity":"ef027451-31f8-4ea0-8338-8887481d2a83","order_by":6,"name":"Yue Dai","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Dai","suffix":""},{"id":308248664,"identity":"88cf53ec-a8f1-476a-9e46-e9efdcc501a3","order_by":7,"name":"Ming-Yong Liang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ming-Yong","middleName":"","lastName":"Liang","suffix":""},{"id":308248665,"identity":"ad37b2e6-6234-4fc7-b96d-14144789fb3b","order_by":8,"name":"Carlos H. F. Chan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"H. F.","lastName":"Chan","suffix":""},{"id":308248666,"identity":"249b411c-ae31-46ff-ba2d-3c030cc1471c","order_by":9,"name":"Zhi-Hua Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYJACCQYehgR+ZubDD0jTItnOlmZAghYGhgSD8zwKEkQpl49Ifnjjh0xdnvFhHgYDhhqbaIJaDM8cM7bs4WErNjvMe+ABw7G03AaCWtp72KQZeHgStx3mSzBgbDhMhJZmHpAWicTNzTwGEkRpkWcH22KQuIGZWC0GPGC/JCTOOAwM5ARi/CI/AxhiP3vqEvv7Dx9+8KHGhghbDgAJxh4oL4GQcrAtYEN/EKN0FIyCUTAKRiwAAEp6Onns/IEeAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Hua","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-05-20 07:31:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4447355/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4447355/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-10675-2","type":"published","date":"2025-02-25T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57948306,"identity":"7955101a-f0b8-4f3d-b700-4e53ff9753a2","added_by":"auto","created_at":"2024-06-07 20:33:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5664478,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology of the liver in HCC patients with \u003cem\u003eC. sinensis\u003c/em\u003e mono-infection\u003c/p\u003e\n\u003cp\u003eA, B, HCC with trabecular growth and solid growth pattern; C, D, HCC with fibrolamellar growth pattern; E~G, CC-HCC; H, SH-HCC. H\u0026amp;E stain, ×100\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/ae4ba8c0f36e2d3d41804eaf.jpg"},{"id":57948308,"identity":"a9517010-ca19-4711-9314-6f9fb9c6b30c","added_by":"auto","created_at":"2024-06-07 20:33:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6192113,"visible":true,"origin":"","legend":"\u003cp\u003eTypical pathological changes in HCC patients with co-infection of\u003cem\u003e C. sinensis\u003c/em\u003e and HBV\u003c/p\u003e\n\u003cp\u003eA~C, HCC with solid growth, adenoid growth and fibrolamellar patterns; D~F, CC-HCC; G, SH-HCC; H, Sp-HCC; I, HCC/ICC comorbidity. H\u0026amp;E stain, ×100\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/a80ea76c6abe23b1b4aafdfd.jpg"},{"id":57948307,"identity":"83ad4b8c-1321-412e-9dbb-d046a5c7c227","added_by":"auto","created_at":"2024-06-07 20:33:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":382264,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the pathological composition of \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e sinensis\u003c/em\u003e and HBV infection related HCC\u003c/p\u003e\n\u003cp\u003eA, Proportion of pathological types (%); B, Proportion of Edmondson-Steiner's grade I~IV differentiation (%); C, Frequency of inflammatory cell infiltration, fibrosis and necrosis in HCC tissue sections (%).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/20d8da19ac96b84854d81e75.jpg"},{"id":77622419,"identity":"bcd1d1e7-4a14-4cfc-9deb-3633daf16157","added_by":"auto","created_at":"2025-03-03 16:06:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12934060,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/b9f2cf91-706e-4f33-80ab-9ff94137b593.pdf"},{"id":57948305,"identity":"22285bfe-24a8-4f4d-b78e-3639dca38725","added_by":"auto","created_at":"2024-06-07 20:33:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15400,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/2fc7c1dbe1a7326bc3cd85bf.docx"},{"id":57948304,"identity":"65d73b2f-47cf-4524-ad88-8fb98b3062a6","added_by":"auto","created_at":"2024-06-07 20:33:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15456,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4447355/v1/7372580668b3ab0553ceda72.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The association between Clonorchis sinensis infection and hepatocellular carcinoma in an endemic area: a study in Guangxi, China","fulltext":[{"header":"Background","content":"\u003cp\u003eThe adult worms of \u003cem\u003eClonorchis sinensis\u003c/em\u003e (\u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e) reside within the intrahepatic bile ducts of humans and animals, leading to the development of clonorchiasis and intrahepatic cholangiocarcinoma (ICC) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clonorchiasis is predominant in East Asia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], with over 15\u0026nbsp;million individuals estimated to be infected globally [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Guangxi, China, is recognized as a major region affected by clonorchiasis. According to the 2015 surveillance of major human parasitic diseases, the weighted infection rate of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e in Guangxi was 6.68%, with approximately 2.9163\u0026nbsp;million people affected, ranking it the highest in China [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, Guangxi is also known as a high prevalence area for hepatitis B. Hepatitis B virus (HBV) infection is the primary cause of hepatocellular carcinoma (HCC) in this region [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent decades, there have been individual HCC cases reported after \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection, which suggests that \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection not only triggers ICC but may also be associated with the occurrence of HCC. Between 2014 and 2015, we identified a total of 10 cases of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e-related HCC in the clonorchiasis epidemic zone of Hengzhou City, Guangxi, including six cases of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e mono-infection and four cases of co-infection with HBV [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To further establish the correlation between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection and HCC, a larger cohort survey of liver cancer patients in Hengzhou, Guangxi was subsequently conducted.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCases and specimens\u003c/h2\u003e \u003cp\u003eThe study included a cohort of 120 patients diagnosed with liver cancer who underwent surgical treatment at People's Hospital of Hengzhou (PHH) between August 2020 and May 2023. Prior to the surgery, blood samples were obtained from each patient and the serum was separated and stored at -80\u0026deg;C. Liver cancer tissues were collected during the operation and also stored at -80\u0026deg;C. Additional tissue samples were fixed with a 4% paraformaldehyde solution and stored at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe qualitative detection of the IgG antibody against \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and hepatitis B virus surface antigen (HBsAg) in serum samples was conducted using the enzyme-linked immunosorbent assay (ELISA) method. A liver fluke IgG antibody detection kit (Shenzhen Huakang Biomedical Engineering Co., Ltd., Shenzhen) was used to detect the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e IgG antibody, while a diagnostic kit (Shanghai Rongsheng Biopharmaceutical Co., Ltd., Shanghai) was used to detect HBsAg. For the detection of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e IgG antibody, the following steps were performed: The serum was diluted 1:10 with dilution buffer and added to a microplate coated with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e antigen. The mixture was incubated at 37\u0026deg;C for 1 hour, followed by washing the plate 5 times for 1 minute each. After drying, 50 \u0026micro;L of enzyme conjugate was added and incubated at 37\u0026deg;C for 30 minutes. The plate was washed again 5 times for 1 minute each. The double antibody sandwich method was used for the detection of HBsAg. 50 \u0026micro;L of the serum to be tested was added to a microplate coated with anti-HBs and incubated at 37\u0026deg;C for 1 hour. Then, 50 \u0026micro;L of enzyme conjugate was added and incubated at 37\u0026deg;C for 30 minutes. The plate was washed 5 times for 10 seconds each. Finally, substrates were added for color development and the reaction was terminated. The experiment included blank, positive, and negative controls. The OD\u003csub\u003e450\u003c/sub\u003e value was measured using the Bio Tek Epoch microplate reader, and the result was determined based on the cut-off value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetection of HBV DNA\u003c/h2\u003e \u003cp\u003eHCC tissue samples weighing between 30 and 50 mg were homogenized in a heating block at 56\u0026deg;C for 6 hours. During this process, intermittent grinding was performed using 1 mL of lysis buffer containing 100 mM Tris, 50 mM EDTA, 2% SDS, and 500 g/ml proteinase K at a pH of 8.0. Subsequently, Tris-saturated phenol and chloroform-isopentanol (at a ratio of 24:1) were used to extract and purify the entire DNA content. The DNA was then precipitated using ethanol and subsequently dissolved in 200 \u0026micro;L of 8 mM NaOH solution (pH 8.0). The DNA concentration was measured using spectrophotometry at a wavelength of 260 nm using the NanoDrop One device (Thermo Scientific). For the quantification of HBV DNA, primers based on the HBV \u003cem\u003ePreC/C\u003c/em\u003e gene were created using Primer-BLAST (available at nih.gov). The primer sequences were as follows: forward 5\u0026rsquo;- ACTTTTTCACCTCTGCCTAA-3\u0026rsquo; and reverse 5\u0026rsquo;- AGCTCCAAATTCTTTATA-3\u0026rsquo;. These primers were synthesized by Sangon Biotech Co., Ltd. in Shanghai, China. Real-time quantitative PCR (qPCR) was used to detect HBV DNA with the TB Green\u0026reg; Premix Ex Taq qPCR Mix. Specifically, 20.0 \u0026micro;L of the TB Green\u0026reg; Premix Ex Taq qPCR Mix (2 \u0026times;), 0.8 \u0026micro;L of each primer (10 \u0026micro;M), and 1.0 \u0026micro;L of the DNA template were combined to achieve a final volume of 40 \u0026micro;L. The amplification and data acquisition steps were carried out using an Analytik Jena qTOWER\u003csup\u003e3\u003c/sup\u003e/G touch Real-Time PCR system with V4.0 software. The cycling parameters included a pre-denaturation step at 95\u0026deg;C for 30 seconds, followed by 50 cycles of amplification at 95\u0026deg;C for 5 seconds, and annealing and extension at 60\u0026deg;C for 30 seconds. Subsequently, a melt curve analysis ranging from 65 to 95\u0026deg;C was performed to determine the melting temperature (Tm) for the specific DNA product populations that were identified. The HBV DNA copy number was standardized using 100 ng of tissue DNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePostoperative tissue biopsy\u003c/h2\u003e \u003cp\u003eThe specimens underwent fixation, paraffin embedding, sectioning, and staining with hematoxylin and eosin (H\u0026amp;E). The Zeiss Apotome.2 imaging system was used to observe the pathological types of HCC. Additionally, the differentiation of HCC was assessed using the grading system I\u0026thinsp;~\u0026thinsp;IV based on Edmondson Steiner's criteria [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, the presence of inflammatory cell infiltration, fibrosis, and necrosis in the tissue sections was documented.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Pearson χ\u003csup\u003e2\u003c/sup\u003e test was used to compare the influence and correlation of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and HBV infection on different types of liver cancer, and the pathological type, differentiation degree, and\u003c/p\u003e \u003cp\u003eratio of pathological constituents in HCC following \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e, HBV mono-infection, and HBV co-infection. The statistical analysis was conducted using IBM SPSS Statistics 19.0 software, and a \u003cem\u003eP\u003c/em\u003e-value of 0.05 or less was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe state and relationship between HBV and \u003cem\u003eC. sinensis\u003c/em\u003e infection in patients with liver cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comprehensive investigation was conducted of a cohort of 120 patients suffering from liver cancer, ranging in age from 33 to 76 years. Of these patients, 104 were male and 16 were female. The postoperative liver tissue specimens of 115 cases were examined by pathology at a local hospital,\u0026nbsp;PHH, while 108 cases were examined in our laboratory, the Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine (LHIID). Among the cases, 103 underwent simultaneous pathological examination by both institutions, resulting in a high coincidence rate of 98.1% (101/103). Additionally, 17 cases were examined exclusively by one institution. In cases where there were inconsistent pathological results, preference was given to the results obtained from our laboratory (refer to Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of the specific diagnoses, of all the patients with liver cancer, 98 cases were diagnosed as HCC, 21 cases as ICC, and 1 case as a comorbidity of HCC/ICC. These diagnoses accounted for 81.7%, 17.5%, and 0.8% of the cases, respectively. Serum specific IgG antibodies were detected in order to determine past or current \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection in patients with liver cancer [12]. Among the HCC patients, 71 cases (72.5%) tested positive for the \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e IgG antibody. Of these, 60 cases were also positive for HBsAg, and the HBV DNA content in the HCC tissues ranged from 6 to 23,270,015 copies/100 ng DNA. On the other hand, 11 cases were negative for HBsAg, but in 3 of them HBV DNA was detected in the HCC tissues, with contents ranging from 1 to 124 copies/100 ng DNA. Notably, HCC tissues and serum from 8 patients with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e infection did not yield any detectable HBV DNA.\u0026nbsp;The infection rate of \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e in HCC patients is significantly higher than that in the general population (28.8%) [13], as well as in general outpatient and non-liver cancer inpatients (22.1%) [14] in the local area (\u0026chi;\u003csup\u003e2\u003c/sup\u003e=82.61/141.92, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u0026nbsp;All patients diagnosed with ICC tested positive for \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e IgG antibody (refer to Table 2).\u003c/p\u003e\n\u003cp\u003eThe infection of \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e and HBV has a significant impact and correlation on the occurrence of different types of liver cancer (\u0026chi;\u003csup\u003e2\u003c/sup\u003e=22.858, cramer\u0026rsquo;V/\u0026phi;=0.444, contingency coefficient=0.406, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). HBV\u0026nbsp;mono-infection is only associated with HCC, while \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e mono-infection and co-infection with HBV are both associated with ICC and HCC. The occurrence of ICC is solely associated with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e infection, wheres the occurrence of HCC is not only commonly associated with HBV, but also with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological analysis of HCC associated with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e and HBV infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 88 cases of HCC associated with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e and HBV infection were subjected to pathological analysis in our laboratory. Pathological examination revealed that the morphology of the cancer cells in 54 patients resembled normal hepatocytes, exhibiting solid growth, adenoid growth, trabecular growth, and fiberboard growth patterns\u0026nbsp;[15]. These differentiation patterns were classified as hepatocellular carcinoma (HCC) and accounted for 61.4% of the cases. In 28 patients, the cytoplasm of their cancer cells exhibited clear cell hepatocellular carcinoma (CC-HCC) characterized by massive storage of \u0026alpha;-glycogen particles\u0026nbsp;[16, 17]; these accounted for 31.8% of the cases. Two cases (2.3%) presented as steatohepatic hepatocellular carcinoma (SH-HCC) with multiple lipid droplets in the cytoplasm\u0026nbsp;[15, 18, 19]. The remaining 4 cases (4.5%) were spindle cell hepatocellular carcinoma (Sp-HCC) consisting mainly of spindle-shaped cells\u0026nbsp;[20]. According to Edmondson Steiner\u0026apos;s grading, differentiation grades I~IV were observed in 5 (5.7%), 37 (42.0%), 18 (20.5%), and 28 cases (31.8%), respectively. The proportions of inflammatory cell infiltration, fibrosis, and necrosis in HCC tissue sections were 36.8%, 37.9%, and 25.3%, respectively.\u003c/p\u003e\n\u003cp\u003eIn the case of \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e mono-infection, 8 cases of HCC were identified through pathological examination. Among these cases, 4 displayed HCC with trabecular growth, solid growth, and fiberboard growth patterns, 3 were classified as CC-HCC, and 1 was identified as SH-HCC (see Fig. 1). The pathological types of HCC after co-infection with \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e and HBV were found to be diverse, including the majority of HCC, CC-HCC, and a few cases of SH-HCC and Sp-HCC (see Fig. 2). Statistical analysis revealed no significant difference in the proportion of pathological types between \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e\u0026nbsp;sinensis\u003c/em\u003e mono-infection, HBV mono-infection, and co-infection related HCC. Similarly, there was no significant difference in the distribution of Edmondson Steiner I~IV differentiation among these groups. Furthermore, no significant difference was observed in the frequency of inflammatory cell infiltration, fibrosis, and necrosis in HCC tissue sections (see Fig. 3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary focus of this study is to examine the connection between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection and HCC. In a study conducted by Strauss sixty years ago, it was observed that, of five cases of HCC found in patients infected with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e, four were accompanied by Laennec's cirrhosis. However, it should be noted that HBV infection was not excluded in these cases [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nakashima et al. reported two cases of elderly patients with HCC after \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection forty-five years ago. One patient had a mild infestation with hepatic changes indicative of posthepatitic cirrhosis, while the other had a heavy infestation that exhibited secondary biliary cirrhosis with dilated intrahepatic bile ducts and periductal fibrosis. In both cases, the tumor nodule was solitary, and the cells were differentiated and classified as Grade I of Edmondson-Steiner's scale of anaplasia. HBsAg was determined to be negative in both cases through radioimmunoassay [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These early studies suggest a potential relationship between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection and the development of HCC. In a more recent investigation conducted by Tan et al. fifteen years ago, it was found that the infection rate of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e in HCC patients at the First Affiliated Hospital of Guangxi Medical University and Cancer Hospital Affiliated to Guangxi Medical University was 16.44%, while the infection rate in non-HCC patients was only 2.40%. HBsAg was examined using ELISA to determine HBV infection. Based on these findings, researchers suggest that clonorchiasis may be an important risk factor for HCC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past decade, we have conducted an ongoing investigation into liver cancer patients in the region of Hengzhou, Guangxi, which is known for its high prevalence of clonorchiasis. The purpose of this study is to clarify the relationship between \u003cem\u003eC. sinensis\u003c/em\u003e infection and HCC in this particular demographic. We identified six cases of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection in a group of 20 liver cancer patients. To rule out the possibility of HBV infection, we conducted tests for HBsAg, which yielded negative results [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Over the course of the past three years, we have expanded our research to include a larger sample size of liver cancer patients in Hengzhou City. Our findings revealed eight cases of HCC following \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection.Among the liver cancer patients surveyed, we observed that HCC was the predominant type of malignancy, with ICC being less common and is only associated with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003ePrevious surveillance revealed that the infection rates of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e in the local population and outpatient/non-liver cancer inpatients are 28.8% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and 22.1% [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], respectively. In this investigation, we discovered that 72.5% of HCC patients were infected with \u003cem\u003eC. sinensis\u003c/em\u003e, which was much higher than the infection rate in the general population in this location. The findings of this survey in a high-clonorchiasis-endemic area revealed a specific connection between \u003cem\u003eC. sinensis\u003c/em\u003e infection and HCC. Guangxi is also a hepatitis B epidemic area. HBV infection is the leading cause of HCC in this region [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the survey, we determined that HBV infection by testing for serum HBsAg and HBV DNA in liver cancer tissues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The study discovered that the majority of patients with HCC in this location were infected with both HBV and \u003cem\u003eC. sinensis\u003c/em\u003e, complicating the causes and risk of HBV-induced HCC [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Co-infection with HBV and \u003cem\u003eC. sinensis\u003c/em\u003e, as well as the resulting HCC, are unavoidable difficulties in this area. Co-infection with HBV and \u003cem\u003eC. sinensis\u003c/em\u003e is the leading cause of HCC, and it is an inescapable public health hazard in this region.\u003c/p\u003e \u003cp\u003eThis suggests that \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection on its own may directly induce HCC, while co-infection with HBV may contribute to the higher incidence of HCC. There were no significant differences observed in the pathological type, differentiation degree, or detection frequency of inflammatory cell infiltration, fibrosis, and necrosis in HCC after \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection alone or co-infection with HBV, when compared to HBV infection alone. During the investigation, one case of steatohepatic HCC was found in patients with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection, as well as one case with HBV co-infection. Further confirmation is required to determine the effect and mechanism of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection on lipid metabolism in HCC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanisms by which \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection induces HCC are not yet to be determined. Initial investigations have revealed that the excretory-secretory products (ESPs) of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e can enhance the replication of HBV and elicit a Th2 immune response. It has been observed that patients co-infected with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and HBV exhibit higher levels of serum HBV DNA than patients infected with HBV alone [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Clinical data have demonstrated that patients with co-infection of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and HBV have significantly elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and hyaluronic acid (HA) compare to those with HBV infection alone [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A survey conducted in the high incidence area of clonorchiasis in the Lalin River, Northeast China, reported that out of 854 cases of clonorchiasis, 46 cases were co-infected with HBV (HBsAg positive). However, no significant differences were observed in 16 clinical indicators, including ALT, AST, and TB, between the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection, HBV infection, and co-infection groups [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e experiments have revealed that co-infection of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and HBV can promote liver fibrosis and chronic inflammation. Co-stimulation of the hepatic stellate cell line LX-2 with the total proteins from \u003cem\u003eC.sinensis\u003c/em\u003e adult worms (CsTPs) and HBV positive serum leads to increased transcription of alpha-smooth muscle actin and types I and III collagen, as well as pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Recent studies have highlighted the ability of parasite extracellular vesicles (EVs) to mediate the host competing endogenous RNA (ceRNA) regulatory network across species through their own non-coding RNA (ncRNA) with regulatory function [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These EVs have a profound impact on the occurrence of HCC [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Specifically, EVs derived from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e (CsEVs) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] have been found to activate the TLR9 signal, promoting an inflammatory response characterized by the production of IL-6 and TNF-α in mouse biliary epithelial cells (BECs) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The miRNA csi-let-7a-5p, which is enriched in CsEVs, negatively regulates the expression of \u003cem\u003eSOCS1\u003c/em\u003e and \u003cem\u003eclec7a\u003c/em\u003e genes in the NF-κB signaling pathway, thereby enhancing the production of TNF-α, IL-6, IL-1β, iNOS, and promoting the differentiation of M1-like macrophages. These processes play a crucial role in the development of bile duct injury and fibrosis following infection [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, the precise role and mechanism of CsEVs in interfering with the miRNA-mediated ceRNA regulatory network in host hepatocytes, and their impact on hepatocellular carcinogenesis, remain unclear.\u003c/p\u003e \u003cp\u003eClonorchiasis poses considerable public health risks [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and the Hepatitis B virus is the leading cause of HCC in China [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, chronic alcohol consumption [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], non-alcoholic fatty liver disease (NAFLD) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and hepatitis C virus infection [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] are not the primary hazards or burdens of HCC in this region. This investigation has provided further clarification of the correlation between \u003cem\u003eClonorchis sinensis\u003c/em\u003e infection and the occurrence of HCC. In populations with a high incidence of HBV infection and related HCC, the role of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e in inducing HCC may be overshadowed. In China, hepatitis B virus is the primary risk factor for HCC [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, with the implementation of the \u0026ldquo;hepatitis B elimination\u0026rdquo; strategy in 2030 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], the role of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e in inducing HCC may gradually become more evident. Furthermore, this investigation discovered that three patients with functional cured (FC) hepatitis B [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], who had negative HBsAg but low liver HBV DNA load, were all co-infected with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and suffering from HCC. Therefore, the potential threat of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection cannot be disregarded in the clinical treatment of hepatitis B and the prevention and treatment of HBV-related HCC. In areas with a high prevalence of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e, effective control of human HCC should not only focus on the \u0026ldquo;hepatitis B elimination\u0026rdquo; strategy but also actively implement comprehensive prevention and control measures for clonorchiasis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThese results indicate that HCC still dominates the composition of liver cancer in this region, while ICC only accounts for a small number and is all associated with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection. \u003cem\u003eC. sinensis\u003c/em\u003e infection was prevalent in HCC patients, and the occurrence of HCC was not only commonly associated with HBV, but also with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection. HBV mono-infection is only associated with HCC, but \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e mono-infection and co-infection with HBV are both associated with ICC and HCC. The pathogenic type, differentiation, and basic pathological content of HCC were all unaffected by \u003cem\u003eC. sinensis\u003c/em\u003e infection. The co-infection of HBV and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e is the leading cause of HCC in this region.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate Dr. Harrison TJ\u0026rsquo;s meticulous revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ-L Y wrote original draft; Z-H J, X-W L and M-Y L conducted case investigation; Q-L Y, Y-Q G, Y-N H, YH and YD performed the laboratory works, and prepared figures 1-3 and tables 1-2; Z-H J, Z-L F and CHFC reviewed and edited the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Guangxi (No. 2020GXNSFAA238016), the Global Health Research Seed Grant from the Carver College of Medicine at the University of Iowa, U.S.A. (No. XZ2020002), and the\u0026nbsp;Research project of Guangxi Zhuang Autonomous Region Bureau of Disease Prevention and Control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigation and specimen collection procedures adhered to the principles of informed consent and were reviewed and approved by the Guangxi Ethics Review Committee (approval No. GXIRB 2020-0022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publicatio\u003c/strong\u003en\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eGuangxi University of Chinese Medicine, Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine, Nanning 530200, Guangxi, People\u0026rsquo;s Republic of China. \u003csup\u003e2\u003c/sup\u003ePeople\u0026apos;s Hospital of Hengzhou, Nanning 530300, Guangxi, People\u0026rsquo;s Republic of China. \u003csup\u003e3\u003c/sup\u003eGuangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for Viral Hepatitis Prevention and Control, Nanning 530028, Guangxi, People\u0026rsquo;s Republic of China. \u003csup\u003e4\u003c/sup\u003eUniversity of Iowa Carver College of Medicine, 200 Hawkins Drive, JCP 4642, Iowa City, IA 52242, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHong ST, Fang Y. \u003cem\u003eClonorchis sinensis\u003c/em\u003e and clonorchiasis, an update. 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Competing Endogenous RNAs in Hepatocellular Carcinoma-The Pinnacle of Rivalry. Semin Liver Dis. 2019;39:463\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Duan S, Li X, Ding J, Zuo L, Sun B, et al. Differences in the secretory exosomes of \u003cem\u003eClonorchis sinensis\u003c/em\u003e adults at different incubation times. Acta Trop. 2022;234:106604.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wang X, Zhang N, Yu Y, Bao P, Ma Y, et al. Extracellular vesicles of \u003cem\u003eClonorchis sinensis\u003c/em\u003e promote IL-6 and TNF-α secretion via the Toll-like receptor 9-mediated ERK pathway in biliary epithelial cells. Dev Comp Immunol. 2023;139:104555.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian MB, Chen YD, Yan F. Time to tackle clonorchiasis in China. Infect Dis Poverty. 2013;2:4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcGlynn KA, Petrick JL, El-Serag HB. Epidemiol Hepatocellular Carcinoma Hepatol. 2021;73(Suppl 1):4\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Hepatitis B. Virus-Associated Hepatocellular Carcinoma. Adv Exp Med Biol. 2017;1018:11\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Aring;berg F, Byrne CD, Pirola CJ, M\u0026auml;nnist\u0026ouml; V, Sookoian S. Alcohol consumption and metabolic syndrome: Clinical and epidemiological impact on liver disease. J Hepatol. 2023;78:191\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePouwels S, Sakran N, Graham Y, Leal A, Pintar T, Yang W, et al. Non-alcoholic fatty liver disease (NAFLD): a review of pathophysiology, clinical management and effects of weight loss. BMC Endocr Disord. 2022;22:63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Qi JL, Wang XX, Li XH, Jin R, Liu BY, et al. The burden of hepatitis C virus in the world, China, India, and the United States from 1990 to 2019. Front Public Health. 2023;11:1041201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Q, Liu A, Wang S, Li J, He M, Chen L. Hepatitis C virus screening reactive among blood donors in mainland China: A systematic review and meta-analysis. Transfus Med. 2023;33:147\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Liang W, Jing W, Liu M. Countdown to 2030: eliminating hepatitis B disease, China. Bull World Health Organ. 2019;97:230\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlock TM, Chang KM, Guo JT. Prospects for the Global Elimination of Hepatitis B. Annu Rev Virol. 2021;8:437\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeowattana W, Leowattana T. Chronic hepatitis B: New potential therapeutic drugs target. World J Virol. 2022;11:57\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips S, Jagatia R, Chokshi S. Novel therapeutic strategies for chronic hepatitis B. Virulence. 2022;13:1111\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Clonorchis sinensis, hepatitis B virus, hepatocellular carcinoma, association","lastPublishedDoi":"10.21203/rs.3.rs-4447355/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4447355/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChronic infection with \u003cem\u003eClonorchis sinensis\u003c/em\u003e (\u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e) has been associated with the development of intrahepatic cholangiocarcinoma (ICC); however, the relationship between \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e and hepatocellular carcinoma (HCC) remains uncertain.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study examined 120 patients with liver cancer in the clonorchiasis endemic area of Hengzhou, Guangxi, China. The type of cancer, the differentiation grade according to Edmondson Steiner's classification, and the pathological characteristics of HCC were determined through postoperative tissue biopsy. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e infection was detected by measuring serum specific IgG antibody, and hepatitis B virus (HBV) infection was determined by detecting serum HBsAg and HBV DNA in HCC tissues.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the patients evaluated, 98 (81.7%) had HCC, 21 (17.5%) had ICC, and 1 (0.8%) had comorbidity of HCC/ICC. Among the HCC patients, 24 (24.5%) were solely infected with HBV, 71 (72.4%) were infected with C. sinensis, and 3 (3.1%) showed no evidence of infection. \u003cem\u003eC. sinensis\u003c/em\u003e infection rates in HCC patients are much higher than in the general population, as well as in general outpatient and non-liver cancer inpatients in the local area (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;82.61/141.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There were no significant differences in the pathological type, differentiation grade, and lesion composition between the tumor associated with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e/HBV mono- and co-infection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Among the patients with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e-related HCC, 8 (8.2%) were solely infected with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003esinensis\u003c/em\u003e, while 63 (64.3%) were co-infected with HBV. Infection with \u003cem\u003eC. sinensis\u003c/em\u003e and HBV has a significant impact on the kind of liver cancer (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;22.858, cramer\u0026rsquo;V/φ\u0026thinsp;=\u0026thinsp;0.444, contingency coefficient\u0026thinsp;=\u0026thinsp;0.406, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings indicate that HCC still accounts for the majority of liver cancer in this region. In addition to being most commonly related with HBV infection, HCC is also related with \u003cem\u003eC. sinensis\u003c/em\u003e infection. The co-infection of HBV and \u003cem\u003eC. sinensis\u003c/em\u003e is the leading cause of HCC in this region.\u003c/p\u003e","manuscriptTitle":"The association between Clonorchis sinensis infection and hepatocellular carcinoma in an endemic area: a study in Guangxi, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 20:33:00","doi":"10.21203/rs.3.rs-4447355/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-09T15:42:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-08T12:40:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135304903521580472949836847042343780000","date":"2024-12-08T08:55:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197230137408345955601323849179157068999","date":"2024-12-07T22:46:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-07T20:58:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138808094461184808706663982330744789135","date":"2024-12-07T15:33:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184308317387456224998814282011360095763","date":"2024-12-07T15:33:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290802006039665953538855864687936496838","date":"2024-12-05T16:21:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29898877134887725593825358448126200105","date":"2024-12-05T16:15:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175410263041617355160446503110923598810","date":"2024-12-05T16:14:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264017649507687009097297796521917439649","date":"2024-12-05T15:48:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46512173263127342271572782368759646700","date":"2024-12-05T15:48:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-16T13:33:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194162475578038730456266758298140594605","date":"2024-07-09T02:13:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-18T13:54:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-23T08:47:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T09:24:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T09:24:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-05-20T07:29:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7ba2e3a-3e3d-4f3f-837c-c5457e6581f3","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:00:20+00:00","versionOfRecord":{"articleIdentity":"rs-4447355","link":"https://doi.org/10.1186/s12879-025-10675-2","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-02-25 15:57:15","publishedOnDateReadable":"February 25th, 2025"},"versionCreatedAt":"2024-06-07 20:33:00","video":"","vorDoi":"10.1186/s12879-025-10675-2","vorDoiUrl":"https://doi.org/10.1186/s12879-025-10675-2","workflowStages":[]},"version":"v1","identity":"rs-4447355","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4447355","identity":"rs-4447355","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}