Severe hepatobiliary morbidity is associated with Clonorchis sinensis infection: The evidence from a cross-sectional community study

Clonorchis sinensis infection is highly prevalent in Asia. Diverse hepatobiliary morbidity has been documented for C. sinensis infection. This study aimed to assess the association between C. sinensis infection and hepatobiliary morbidity, taking into consideration of the control, confounders and infection intensity. A cross-sectional community survey was implemented in Hengxian county, southeastern China. Helminth infections were detected by fecal examination. Physical examination and abdominal ultrasonography were then conducted. After excluding confounding effects from gender, age and alcohol drinking, quantitative association between C. sinensis infection and hepatobiliary morbidity was assessed, and the effect from infection intensity was also evaluated, through adjusted odds ratio (aOR) and 95% confidence intervals (95% CI). 696 villagers older than 10 years were enrolled. The prevalence and infection intensity of C. sinensis were higher in male, elder people and the individuals consuming alcohol. Light C. sinensis infection was associated with the increase of diarrhoea (aOR: 2.2, 95% CI: 1.1–4.5). C. sinensis infection was associated with the increase of fatty liver (aOR: 2.7, 95% CI: 1.4–5.2), and the effect was similar in different infection intensities. Moderate C. sinensis infection was associated with the increase of gallbladder stone (aOR: 3.0, 95% CI: 1.1–8.6), while moderate and heavy infections with the increase of intrahepatic bile duct dilatation (aOR: 2.2, 95% CI: 1.0–4.9 and aOR: 4.3, 95% CI: 1.9–9.9, respectively). C. sinensis infection had an effect on the development of periductal fibrosis (aOR: 3.2, 95% CI: 2.1–4.9), which showed increasing trend by infection intensity. The length and width of gallbladder in those with C. sinensis infection were enlarged, especially in those over 30 years old. C. sinensis infection is significantly associated with hepatobiliary morbidity. The occurrence of some morbidity was strongly related to the infection intensity. Awareness on harm of clonorchiasis should be raised both for policy-makers and villagers to adopt effective interventions.


Introduction
Clonorchiasis is caused by the ingestion of raw freshwater fish, which contains the infective larvae of Clonorchis sinensis [1,2].Clonorchiasis is one important food-borne trematodiasis [3].An estimation of 15 million cases with clonorchiasis distribute in China, the Republic of Korea, northern Vietnam and parts of Russia, where habit of ingesting raw freshwater fish is deeply rooted [1,2,4].Particularly, about 13 million cases are estimated in China [1,2].
Adult worms of C. sinensis parasitize in intrahepatic bile ducts [5,6].In some cases with high number of worms, the adults may migrate to the gallbladder [5].C. sinensis eggs are expelled with the faeces, and thus fecal examination for eggs is usually applied for definite diagnosis [2].The number of eggs in faeces is directly related to the worm burden in hosts and thus the indicator of infection intensity by quantitating eggs is usually used to refer to the degree of infection [7].
C. sinensis infection predominantly leads to hepatobiliary abnormalities [8].In early stage of infection, symptoms are usually mild, while some cases present diverse symptoms, especially diarrhoea and right upper quadrant pain [2].Chronic C. sinensis infection could lead to various liver and biliary morbidity, among which cholelithiasis is frequently reported [9][10][11][12].Particularly, C. sinensis is classified as definite carcinogen, causing fatal cholangiocarcinoma [13,14].Although diverse morbidity has been reported to be associated with C. sinensis infection, most was documented in cases studies without control.Cofounding factors as gender and age are usually neglected.Especially, alcohol also causes liver conditions, and it is frequently consumed when people enjoy raw freshwater fish [15,16].Furthermore, the impact of infection intensity on morbidity deserves to be explored.Thus, we carried out a cross-sectional community survey in southeastern China to assess the hepatobiliary morbidity associated with C. sinensis infection.

Ethics statement
The study was approved by the Ethics Committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention in Shanghai, China (reference no.2011-005).Written informed consent was obtained from all participants or their guardians for those aged <18 years.People infected with C. sinensis or soil-transmitted helminths were administrated free of charge with praziquantel (25 mg/kg, t.i.d, 2 days) and albendazole (400 mg, single oral dose), respectively.

Study area and participants
This study was conducted between March and May 2012 in Hengxian county, Guangxi, China.Hengxian county is located in the southeast of China, where increasing prevalence of C. sinensis was documented [17].Two villages named Xiaoling and Shangcha were included.Villagers over 5 years old were firstly invited to participate in fecal examination, and then those over 10 years old were again invited to join the ultrasound examination.

Diagnosis of helminth infections
One stool sample was collected from each participant.Then both the Kato-Katz method and washing sedimentation technique were employed.Three Kato-Katz thick smears with a 41.7 mg template were prepared for each sample [18], in which C. sinensis eggs were recorded quantitatively and other helminth eggs qualitatively.One smear was examined for each sample in washing sedimentation technique [19], in which all helminth eggs were recorded qualitatively.

Questionnaire and abdominal ultrasonography examination
The information on alcohol drinking, diarrhoea and right upper quadrant pain was obtained by a trained interviewer.Diarrhoea was defined as the occurrence of watery stool or over three times defecation per day during past one month.The timing of right upper quadrant pain also referred to the past one month.Three categories were provided for alcohol drinking, namely none, 1-7 times per week and over 7 times per week.Hepatomegaly and splenomegaly were detected through physical examination.Abdominal ultrasonographical examination (SSC-218, ALOKA, Tokyo, Japan) with a 3.5MHz probe was performed by an experienced radiologist.The size of gallbladder was measured, and the change of gallbladder wall (thickening or blur), sludge or polyps of gallbladder, cholecystectomy were recorded.Cholelithiasis was evaluated separately for intrahepatic bile duct, extrahepatic bile duct, and gallbladder.Duct dilation was assessed for intrahepatic bile duct (over 0.3 cm in internal diameter) and extrahepatic bile duct (over 0.8 cm in internal diameter) [20].Periductal fibrosis was defined by periductal echoes (more than 3 mm) around second order intrahepatic bile duct [21].Grading of the fibrosis included grade 0 = normal, grade I = echoes in 1 segment of the liver, grade II = 2-3 segments, and grade III = more than 3 segments [21].Fatty liver, liver cyst and other abnormalities were also evaluated.

Statistical analysis
Age was grouped into four categories, namely 10-29 years, 30-44 years, 45-59 years and 60 + years.Alcohol drinking was grouped into two categories, namely negative and positive (combination of those drinking 1-7 times per week and over 7 times per week), because those over 7 times per week were all infected with C. sinensis in this study.Helminth infection was classified as positive if one egg was detected in any of the three Kato-Katz thick smears and one washing sedimentation smear.In C. sinensis, eggs per gram of feces (EPG) was calculated by multiplying the average of eggs in three Kato-Katz thick smears by 24.Infection intensity of C. sinensis was then categorized as light (1-999 EPG), moderate (1000-9999 EPG), and heavy (�10 000 EPG) [22].If C. sinensis egg was detected in washing sedimentation technique other than the Kato-Katz method, then the intensity was classified as light infection, because EPG is usually very low in false negative individuals in the Kato-Katz method [23].Periductal fibrosis was grouped into two categories, namely negative (grade 0) and positive (combination of grade I, II and III).
Multivariable logistic regression model was used to analyze the odds of infection, in which C. sinensis infection was set as outcome, and gender, age groups and alcohol drinking were all included as predictors.To analyze the odds of morbidity, univariable logistic regression model was first applied, in which each morbidity indicator was considered as outcome, and gender, age groups, alcohol drinking, C. sinensis infection (dichotomous variable including negative and positive categories) or C. sinensis intensity (polytomous variable including four categories, namely negative, light infection, moderate infection and heavy infection) as predictors, respectively.Then, one multivariable logistic regression model was used in which each morbidity indicator was set as outcome, and gender, age groups, alcohol drinking and C. sinensis infection simultaneously as predictors.Finally, another multivariable logistic regression model was employed in which single morbidity was set as outcome, and gender, age groups, alcohol drinking and C. sinensis intensity all as predictors.In univariable logistic regression, crude odds ratio (cOR) and 95% confidence intervals (95% CI) were provided, while adjusted odds ratio (aOR) and corresponding 95% CI were provided in multivariable logistic regression.General linear model was employed to compare the size of gallbladder, in which the length and width was set as dependent variable respectively while gender, age groups, alcohol drinking and C. sinensis infection as factors.If there existed interaction, further comparisons by C. sinensis infection stratified by gender, age groups and/or alcohol drinking were done.

Self-report morbidity and physical examination
The prevalence of diarrhoea was 15.4% and 5.9% in those with and without C. sinensis infection (cOR: 2.9, 95% CI:1.6-5.3)(Tables 2 and S1).Overall, the aOR was 2.0 (95% CI: 1.0-4.0) in those with the infection compared to those without (Table 3).The aOR was also significant in those with light infection (2.2, 95% CI: 1.1-4.5)compared to non-infected individuals, but there existed no significant differences in moderate and heavy infections compared to those without C. sinensis.
Although the prevalence of right upper quadrant pain and hepatomegaly in individuals with C. sinensis infection was slightly higher than those without infection, the differences were nonsignificant in both univariable and multivariable logistic regression analysis (S2 Table ).Splenomegaly was not detected in any participant.

C. sinensis infection
No. with morbidity (%) participants.The association of C. sinensis infection with intrahepatic duct dilatation was similar to that in any bile duct dilatation (Tables 3 and S7).
Although the changes in gallbladder wall was significantly higher in those with C. sinensis infection compared to those without infection in univariate logistic regression analysis (cOR: 1.7, 95% CI: 1.1-2.8), the difference was not significant in multivariate logistic regression model (aOR: 1.0, 95% CI: 0.6-1.8)(Tables 3 and S9).
Although such morbidity indicators including liver cyst, intrahepatic bile duct stone, extrahepatic bile duct dilatation, cholecystectomy, polyps of gallbladder and sludge of gallbladder was detected, they showed no association with C. sinensis infection in both univariable and multivariable logistic regression analysis (Tables 2 and S2).

Size of gallbladder
In female, the length and width of gallbladder in individuals without C. sinensis infection were 4.6 cm and 2.0 cm in aged 10-29 years, 4.9 cm and 2.2 cm in aged 30-44 years, 4.8 cm and 2.0 cm in aged 45-59 years, and 4.7 cm and 2.1 cm in aged 60+ years (Table 4).The corresponding figures in infected individuals were 4.9 cm and 2.3 cm, 5.3 cm and 2.4 cm, 5.7 cm (P = 0.003) and 2.5 cm (P = 0.002), 5.9 cm (P = 0.001) and 2.7 cm (P = 0.002).
In male, the length and width of gallbladder in those non-infected persons were 4.9 cm and 2.0 cm in aged 10-29 years, 5.1 cm and 2.2 cm in aged 30-44 years, 6.5 cm and 2.0 cm in aged 45-59 years, and 5.3 cm and 2.5 cm in aged 60+ years (Table 4).The corresponding figures in individuals with C. sinensis infection were 5.3 cm and 2.2 cm, 7.0 cm (P = 0.008) and 3.2 cm (P = 0.007), 7.5 cm and 3.4 cm (P = 0.006), 6.4 cm and 3.1 cm.

Discussion
Compared to the low endemicity of soil-transmitted helminthiases, clonorchiasis was highly endemic in the two study villages, which was consistent to the county-level epidemiological survey [17].The epidemiological profiles were characterized by a higher prevalence and infection intensity in male and elder people, which was same to other surveys [17,[24][25][26][27][28].Differences in the ingestion of raw freshwater fish determine the characteristics.There existed a strong relationship between C. sinensis infection and alcohol drinking.Several potential reasons might be responsible.Firstly, alcohol could help remove the fishy smell of raw fish.Secondly, ingestion of raw freshwater fish occurs frequently when people get together, and on this occasion alcohol is usually consumed.Thirdly, there exists a misunderstanding in some persons that alcohol could help kill the larvae of C. sinensis in raw fish [15].Thus, it is necessary to exclude potential confounding effects from gender, age and alcohol drinking, to accurately assess the morbidity associated with C. sinensis infection, which was also justified in this study.This study firstly establishes the relationship between C. sinensis infection and fatty liver.High proportion (11.9%) of participants developed fatty liver, and it was higher in those with C. sinensis infection (15.0%) compared to non-infected individuals (5.9%).Even the effects from other confounders (gender, age and alcohol drinking) were eliminated, the association
b Data on length were not provided in five cases and data on width were not provided in six cases.was still significant in any infection intensity.High occurrence of fatty liver had also been documented in persons infected with Opisthorchis viverrini, e.g.10.6% in Thailand [29] and 12.1% in Laos [30].However, no control was included in those studies.In the genome of C. sinensis, the pathway for fatty acid metabolism is complete, while key genes involved in fatty acid biosynthesis are missing [31,32].Thus, the worms need to receive lipids from the bile of the hosts.C. sinensis fatty acid binding protein is highly expressed in the worms, and it plays an important role in the intracellular transport of long-chain fatty acids obtained from the hosts [33,34].In mouse model, many genes involved in fatty acid metabolism are downregulated in the livers of mouse infected with C. sinensis, which is argued to inhibit the use of fatty acids by hosts and thus increase the use by the worms [35].It has also been observed that fatty drops appear in the liver of guinea-pigs after infection with C. sinensis [36].These evidences support our finding that C. sinensis infection is associated with the development of fatty liver.However, more researches are needed to further verify the etiological association and illuminate the mechanism.
Cholelithiasis is frequently presented in individuals infected with C. sinensis [9][10][11][12].In one case-control study, the occurrence of intrahepatic bile duct stone increased in those with C. sinensis infection, which was diagnosed based on radiological evidence other than fecal examination [9].However, in the same study, the occurrence of extrahepatic bile duct stone and gallbladder stone was not associated with C. sinensis infection.In another case study, C. sinensis infection was argued to cause gallbladder stone, because C. sinensis infection weakens the function of gallbladder, and causes the precipitation of bilirubinate, calcium carbonate crystals, and mucin on C. sinensis eggs [10].Our study adds further evidence on the association between C. sinensis infection and cholelithiasis.
Bile duct dilatation is important imaging appearance in clonorchiasis [37,38].Adult worms of C. sinensis usually parasitize in intrahepatic bile duct [5,6].Thus, it is reasonable that high occurrence of dilatation was recorded in intrahepatic other than extrahepatic bile duct.High infection intensities indicate more worm burden in bile duct, and thus the increase of dilatation in moderate and heavy infections is sound.On comparison, dilatation has not yet developed in individuals with light infection.
The development of periductal fibrosis is complex.The mechanical obstruction and injury of bile duct by the worms, toxic effects of the worms' excretory-secretory products, and subsequent immunopathology by inflammation comprehensively contribute to the development of periductal fibrosis in hosts infected by C. sinensis [2].Thus, the worm burden is directly related to the damage, which explains the increasing trend of periductal fibrosis by infection intensity.Periductal fibrosis is early indicator in the development of cholangiocarcinoma [39].However, this study didn't detect liver mass lesions suspected for cholangiocarcinoma.That is probably attributable to the relative low incidence of cholangiocarcinoma [1] and limited sample size in this study.
Diarrhoea is a frequently reported symptom in those individuals with clonorchiasis [2,[40][41][42].In this study, the relationship between C. sinensis infection and diarrhoea was also established.However, the effect was only demonstrated significantly in light infection.Although right upper quadrant pain is also another frequent symptom in clonorchiasis [2,40,42], the association was not demonstrated in this study.
This study demonstrated the association between C. sinensis infection and enlargement of gallbladder.The individuals with C. sinensis infection showed higher length and width in gallbladder regardless of gender and age, compared to those without the infection.However, the difference was only significant in those aged over 45 years in female and over 30 years in male.The insignificance in low age and later occurrence in female could be attributable to the low infection intensity in these populations.The insignificance in male aged over 45 years is argued to be caused by the limited sample size in negative cases.The enlargement of gallbladder weakens its function, which could also promote the formation of cholelithiasis [10].This is consistent to the finding on the association between C. sinensis infection and cholelithiasis mentioned above.
Severe A. lumbricoides infection could cause hepatic ascariasis and biliary ascariasis, in which diverse hepatobiliary damages are also caused [43].However, the prevalence of ascariasis was very low in this study, which avoided the potential interference on assessment on the association between C. sinensis infection and morbidity.
This study had several limitations.Firstly, the low prevalence of some morbidity indicators and limited sample size might lead to false non-association between these morbidity indicators and C. sinensis infection.Additionally, the sample size of control (without clonorchiasis) in male older than 45 years was quite small, which probably caused the nonsignificant results in the gallbladder size between those with and without infections.Secondly, C. sinensis infection is associated with severe morbidity as demonstrated in this study and thus some individuals in control probably had been infected by C. sinensis and then treated.Although C. sinensis infection had been cured, the hepatobiliary damages might still persist in imaging [44].In this case, the effect between C. sinensis infection and hepatobiliary morbidity is underestimated.Thirdly, the subjective symptoms have not been evaluated in this study.They are difficult to be measured and thus we only assessed the objective presentations and sonographic appearances.Fourthly, follow-up studies are expected in future, which could observe the long-term outcomes, especially the association with cholangiocarcinoma.
Overall, it is demonstrated that significant hepatobiliary morbidity is associated with C. sinensis infection in this study.There also exist increasing trends by infection intensity in some morbidity.Our study justifies the reasonability of morbidity control for clonorchiasis in current stage because of high infection and intensity as well as subsequent morbidity.Intervention measurements are needed in clonorchiasis endemic areas.The association between C. sinensis infection and diverse morbidity could be used to persuade those individuals eating raw freshwater fish to give up the practice and inform the policy-makers to implement interventions.

Table 1 . The prevalence of Clonorchis sinensis infection by gender, age groups and alcohol drinking.
a Gender, age groups and alcohol drinking were all included in multivariable logistic regression model.b Data were not provided in two persons.https://doi.org/10.1371/journal.pntd.0009116.t001