Cholecystolithiasis Is Associated with Clonorchis sinensis Infection

Background The objective of this study was to analyze gallbladder stones for direct evidence of a relationship between Clonorchis sinensis infection and gallbladder stones formation. Methodology We investigated one hundred eighty-three gallbladder stones for the presence of Clonorchis sinensis eggs using microscopy, and analyzed their composition using Fourier transform infrared spectroscopy. We confirmed the presence of Clonorchis sinensis eggs in the gallbladder stones using real-time fluorescent PCR and scanning electron microscopy. Principal Findings Clonorchis sinensis eggs were detected in 122 of 183 gallbladder stones based on morphologic characteristics and results from real-time fluorescent PCR. The proportion of pigment stones, cholesterol stones and mixed gallstones in the egg-positive stones was 79.5% (97/122), 3.3% (4/122) and 17.2% (21/122), respectively, while 29.5% (18/61), 31.1% (19/61) and 39.3% (24/61) in the egg-negative stones. The proportion of pigment stone in the Clonorchis sinensis egg-positive stones was higher than in egg-negative stones (P<0.0001). In the 30 egg-positive stones examined by scanning electron microscopy, dozens or even hundreds of Clonorchis sinensis eggs were visible (×400) showing a distinct morphology. Many eggs were wrapped with surrounding particles, and in some, muskmelon wrinkles was seen on the surface of the eggs. Also visible were pieces of texture shed from some of the eggs. Some eggs were depressed or without operculum while most eggs were adhered to or wrapped with amorphous particles or mucoid matter (×3000). Conclusion Clonorchis sinensis eggs were detected in the gallbladder stones which suggests an association between Clonorchis sinensis infection and gallbladder stones formation, especially pigment stones.


Introduction
Cholecystolithiasis or the presence of gallstones in the gallbladder is a common disease with a mean prevalence rate of 10% based on epidemiological studies [1]. The incidence rate increases with changes in dietary patterns and with the development of an aging population [2][3][4][5][6]. In spite of its high incidence rate, the etiology and pathogenesis of gallbladder stones is still not well understood [7][8][9][10][11][12].
Clonorchiasis, also known as liver fluke disease, is an important amphixenosis. It is mainly distributed in East Asia and Southeast Asia, including China, DPR.Korea, the Republic of Korea, Vietnam, and the Philippines [13]. Based on the second national survey on parasitic diseases between 2001 and 2004 in China, the overall C. sinensis infection rate of the surveyed population was 0.58% [14,15]. More surveys at known endemic areas in China found 2.4% people infected with C. sinensis [16]. Guangdong had the highest infection rate (16.4%) among provinces, followed by Guangxi (9.8%) [16].
Many studies based on epidemiology and imaging have shown that gallstones are associated with C. sinensis infection [17][18][19][20][21][22][23][24], many researchers considered that C. sinensis eggs may become the nucleus of gallstones and then promote the formation of gallstones, but there has been no direct evidence for the association of C. sinensis infection and gallbladder stone formation. We found C. sinensis eggs in gallbladder stones by light microscopy incidentally. To test a possible link between C. sinensis infection and gallbladder stone formation, we analyzed gallbladder stones by microscopy as well as real-time PCR for the presence of C. sinensis adult worm and/or eggs. C. sinensis eggs were indeed present in a significant number of gallbladder stones, providing the first direct evidence for an association between C. sinensis infection and gallbladder stone formation.

Ethics Statement
A written informed consent was obtained from all subjects. This research was approved by the Medical Ethics Committee of The Second People's Hospital of Panyu, Guangzhou.

Subjects and Specimens
Gallbladder stones from one hundred eighty-three cholecystolithiasis patients that had received gallbladder-preserving cholelithotomies in the Department of General Surgery of The Second People's Hospital during the period of March 2011 to August 2011 were obtained. All patients were from Guangdong province, and consist of 101 males and 82 females. The mean age was 46.6612.8 (Mean 6SD) years old. The BMI of male and female patients was  During the operation, no indications such as bile leakage and common bile duct injury occurred. The number of stones from each patient ranged from one to hundreds; single stones and multiple stones was found in 21.9% (40/183) and 78.1% (143/ 183) of patients respectively. All stones from one patient were counted as one case; all the 183 cases of stones were analyzed in the present study.
Microscopic Examination of Gallbladder Stones (Chinese National Inventive Patent Number: ZL201010123552.3) Gallbladder stones were washed twice with distilled water, and then dried at 60uC for 12 hours. Subsequently, stones were split, and about 10 mg of each layer was weighed if the hierarchical structure of the profile was clear. Otherwise, about 10 mg was weighed directly, put in a mortar and 300 ml of 0.9% NaCl was added. The stones were ground thoroughly and then filtered with a 260 mesh nylon yarn. Each filtrate was then smeared onto 3-4 labeled slides and scored for the presence or absence of C. sinensis eggs under an Olympus System Microscope (BX51, Japan).

Detection of C. sinensis DNA in the Gallbladder Stones by Real-time Fluorescent PCR
Gallbladder stones that were scored positive or negative for eggs (thirty each) based on morphology were chosen randomly from the pool of stone samples, for confirmation of the presence of C. sinensis DNA using real-time fluorescent PCR. DNA from a C. sinensis adult was used as a positive control.
Extraction of DNA. Adult of C. sinensis: The adult worm of C. sinensis obtained from clinical patients was first washed twice with 0.9% NaCl, then dried on the filter paper and ground thoroughly in a mortar by adding liquid nitrogen constantly. Only a single specimen was used and DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's instructions. Briefly, the grinding powder was suspended into 180 ml of ATL buffer, and then 20 ml proteinase-K was added and incubated at 58uC for 4 h with brief vortexing every 30 min. Thereafter, 200 ml AL buffer and 200 ml absolute alcohol was added successively and mixed by vortexing for 15-20 s. Finally, the genomic DNA was collected using DNeasy Mini spin column and eluted in 100 ml elution buffer (AE) and stored at 220uC until use. Gallbladder stones: For mechanical breaking, 20 mg of the stones were weighed and frozen in liquid nitrogen gas for 10 min, and then ground thoroughly in a mortar. After adding 1 ml 0.9% NaCl, the suspension was transferred to a new 2.0 Ep tube and centrifuged at 12000 rpm for 10 min. The pellet was treated and used for DNA extraction with a DNeasy Blood Tissue Kit (Cat.No. 69504, Qiagen, Germany) in accordance with the manufacturer's instructions.
Fluorescent PCR. The primers and Taqman probe were designed to detect the cytochrome C oxidase subunit 1 gene of C. sinensis (GenBank: FJ965388.1) using the Beacon Designer-v 7.51 software. It was then submitted to the BLAST program of NCBI for specific analysis. These primers Cs-F (59-GGTTTGGTAT-GATTAGTCACATTTG-39) and Cs-R.
(59-JOE-AGCAAACATAGCCAACACCAAGCCC-BHQ1-39) was used to detect the C. sinensis specific product. The real-time PCR assay was run in a total reaction volume of 50 ml. The final concentration of the reaction solution included 0.2 mM of both forward and reverse primers, 0.2 mM of the TaqMan probe, 25 ml of Premix Ex Taq TM (Takara), 1 ml ROX buffer and 2 ml templates. The real-time PCR cycling parameters were set as follows: initial 95uC for 30 s, then 45 cycles of 15 s at 95uC and 31 s at 60uC. PCR amplification, detection, and data analysis were performed with an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA).

Composition Analysis of Gallbladder Stones by Fourier Transform Infrared (FTIR) Spectroscopy
Gallbladder stones samples were analyzed with a Bruker (TENSOR27, Germany) FTIR spectrometer in the frequency range 400-4000 cm-1 at 4 cm-1 resolution. For each measurement, 2 mg of finely powdered stone sample mixed with 300 mg KBr was used to make discs. Control spectrums were acquired from standard substances, which were of high quality (99% pure) and purchased from Sigma Chemical Company (St. Louis, MO, USA). Through comparisons of the spectra between gallbladder stones and standard controls, gallbladder stones were grouped as cholesterol stones, pigment stones or mixed stones [25,26].

Observation of Gallbladder Stones with Scanning Electron Microscopy (SEM)
Thirty egg-positive gallbladder stones were chosen for SEM analysis. Stones were washed thoroughly with distilled water and dried at 60uC. Then, they were split and one piece was taken from each layer if the hierarchical structure of the profile was clear; otherwise, one piece from the surface and one piece from the core were taken and then fixed on the sample table and dried at 60uC overnight. Then, the stones were sputter-coated with gold (ETD-2000, China) and observed under a ZEISS (EVO LS10, Germany) SEM and photographed. The EHT was 20 kv.

Statistical Analysis
Age was analyzed as mean 6SE, the detection rate was analyzed using a chi square test and the partitions of the chi square method were used for multiple comparisons using SPSS v.11.5 software. P,0.05 was regarded as statistically significant.

Results
C. sinensis Eggs were Detected in the Gallbladder Stones using Light Microscopy and Egg-positive Gallbladder Stones were Mainly Pigment Stones C. sinensis eggs are yellow-brown in color, resemble sesame seeds, have a thick shell and a small operculum in the front end, shouldering in the junction of the shell and operculum, with occasional visible small protrusions in the obtuse posterior end. Through microscopic examination, C. sinensis eggs were found in 122 of 183 gallbladder stones, providing a detection rate of 66.7%. Meanwhile, large portions of the eggs were adhered to or wrapped by bilirubinate granules and/or mucoid matter. Based on these observations, eggs in the gallbladder stones were preliminarily judged as C. sinensis eggs (as shown in Figure 1).
To determine the main composition of the stones, we analyzed the stones using FTIR spectroscopy. Results showed that the 183 gallbladder stones were composed of 115 pigment stones, 45 mixed stones and 23 cholesterol stones. The characteristic spectrum of the three types of stones is shown in Figure 2 and the spectrum of the standard controls is shown in Figure 3. The FTIR spectroscopy analysis revealed that 122 egg-positive gallbladder stones were composed of 97 pigment stones, 21 mixed stones and 4 cholesterol stones, while the 61 egg-negative stones were composed of 18 pigment stones, 24 mixed stones and 19 cholesterol stones. The proportion of pigment stone in the eggpositive gallbladder stones was higher than those in egg-negative stones (P,0.0001); in other words, egg-positive gallbladder stones were mainly pigment stones, as shown in Table 1.

C. sinensis Eggs in the Gallbladder Stones were Further Confirmed by Real-time Fluorescent PCR
In order to confirm that the eggs detected in the gallbladder stones were C. sinensis eggs, we analyzed the gallbladder stones for the presence of C. sinensis DNA using real-time fluorescent PCR. A positive amplification curve emerged in the DNA of the positive control and egg-positive stones (Figure 4), but no positive amplification curve (a straight line) in the DNA of egg-negative stones. These results confirm that the eggs detected in the gallbladder stones were C. sinensis eggs, and it was consistent with that of the microscopy.

Many C. sinensis Eggs were Discovered in the Gallbladder Stones Under SEM
To investigate the association between C. sinensis eggs and gallbladder stones more accurately, while preserving the ultrastructural histology, we analyzed 30 stones that scored positive for C. sinensis using previous methods for the presence of C. sinensis eggs using SEM. To our surprise, dozens or even hundreds, in some cases, of C. sinensis eggs were visible in the stones ( Figure 5, original magnification, 6400). Several C. sinensis eggs were adhered to or wrapped with surrounding particles and/or mucoid matter (original magnification, 61000). Muskmelon wrinkles was seen on the surface of the eggs, which interrupted the edge of the   , 1703, 1663, 1627, 1573, 1308, 1250, 1105, 1050, 990, 936, 756, 699  operculum. The junction of the shell and operculum was loose and appeared shouldering. Part of the eggs had visible small protrusions in the posterior end and part of the eggs were depressed or without operculum. Pieces of texture shed from some eggs, and amorphous particles or mucoid matter were adhered to the surface, surrounding the eggs as shown in Figure 6 (original magnification, 63000).
In the present study, we detected C. sinensis eggs in gallbladder stones from 66.7% of the patients with cholecystolithiasis through light microscopy. In other words, 66.7% patients with cholecystolithiasis are infected with C. sinensis. Furthermore, the results of microscopic examination revealed that a large portion of the eggs  were adhered to or wrapped by bilirubinate granules, mucoid matter and calcium carbonate crystals, which indicate that C. sinensis eggs might be the important nuclear factor involved in the formation of the gallbladder stones, especially pigment gallbladder stones. Coincidentally, results from FTIR spectroscopy analysis showed that the egg-positive stones were mainly pigment stones. However, some researchers have proposed that there is no relationship between C. sinensis infection and cholecystolithiasis from a comparison of patients with and without C. sinensis infection [18][19][20]. We think that the two opposing conclusions may be due to the difference in the types of samples analyzed and in the experimental methods employed (e.g. diagnosis of C. sinensis infection). Remarkably, the egg-positive stones were mainly pigment stones. These results suggest that although C. sinensis infection may not necessary lead to increased incidence of cholecystolithiasis [18][19][20], it is positively associated with high proportion of pigment stones. It remains to be investigated  whether C. sinensis infection is the etiological factor for cholecystolithiasis.
Due to the subjectivity inherent in morphological analysis by light microscopy, we used real-time fluorescence PCR to detect the presence of C. sinensis eggs. The cytochrome C oxidase subunit 1 gene, a mitochondrial gene in C. sinensis present in hundreds or thousands of copies per cell, is more highly conserved than the ITS-2 sequences of C. sinensis and thus may be a good target for the development and evaluation with real-time PCR [41,42]. A realtime PCR assay based on the cytochrome coxidase subunit 1 gene of C. sinensis was previously used to successfully detect C. sinensis DNA in stool samples of rats [43,44]. Therefore, in the present study, we chose the cytochrome C oxidase subunit 1 gene as the target gene for real-time PCR detection, and the results confirmed that the eggs in the gallbladder stones were indeed C. sinensis eggs.
To further explore the relationship between C. sinensis eggs and gallbladder stones formation without destroying the original structure of the stones, we analyzed the stones by SEM. SEM analysis revealed the presence of a large number of bilirubinate granules and a small amount of calcium carbonate crystals in the egg-positive gallbladder stones. Meanwhile, many of the C. sinensis eggs with a textured surface were discovered in gallbladder stones, and many tiny bilirubinate granules were adhered to the muskmelon wrinkles. Some eggs were wrapped with mucoid matter, while some were depressed or with thickened shell and/or without operculum. Other eggs displayed a shedding texture. The morphologic changes and diversity in the eggs might result from the extended periods that they had existed in the stones, or from nutritional deficiencies, calcification, nucleation and other factors. Based on the morphological study results, we conclude a possible mechanism for the formation of gallbladder stones involving C. sinensis eggs. The inherent texture of C. sinensis eggs is cancellate and uneven, and it is easy for particles and crystals to adhere to its surface. Also, the eddy effect of the bile during gallbladder contraction may lead to the deposition and aggregation of many eggs. Moreover, because of the intermediary role of mucus (mucin) secreted by the gallbladder and the stimulation of C. sinensis eggs and/or worms metabolic product, C. sinensis eggs adhere to or become enveloped by surrounding particles, mucoid matter and crystals. This becomes the nucleus of the gallbladder stone, and consolidation of these nuclei occurs to promote the formation of gallbladder stones.

Conclusion
Our data indicate that C. sinensis eggs may directly participate in the formation of gallbladder stones, especially pigment gallbladder stones. Results reveal a possible association between C. sinensis eggs and formation of gallbladder stones, which contributes to our knowledge on the etiology of clonorchiasis and gallbladder stone formation. Also, our results suggest that blocking water pollution, preventing the intake of water and food contaminated by C. sinensis, changing eating habits (eating raw freshwater fish or shrimp), or applying anti-C. sinensis treatment in patients with C. sinensis eggs would reduce not only the prevalence of clonorchiasis but also the incidence of pigment gallstones in the epidemic areas.