Table 1. The detection of eggs in three types of gallbladder stones with light microscopy. Type of stones Pigment stones cholesterol stones 4 mixed stones Total 97 21 122 doi:10.1371/journal.pone.0042471.t001 18 19 24 61 115 23 45 183 84.3% 17.4% 46.7% 66.7% Detection Pos. No. Neg. no. total rate P value ,0.0001 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). Cholecystolithiasis Is Associated with Clonorchis Discussion Clonorchiasis, a zoonotic disease, is caused by C. sinensis that parasitizes the human intrahepatic bile duct. Humans are often infected with C. sinensis due to the consumption of undercooked freshwater fish or shrimp containing metacercariae [27–29]. Clonorchiasis can produce complications including cholangitis, gallstones, cholangiohepatitis, recurrent pyogenic cholangitis, cirrhosis and cholangiocarcinoma [13,14,17,30–38]. In addition, C. sinensis infection may promote gallstone formation as deter- mined previously using radiological examination, ultrasonic inspection, serologic examination and microscopic examination of feces and bile [17–21,39,40]. Furthermore, other researchers have confirmed that C. sinensis infection is involved in the formation of bile duct stones based on the presence of C. sinensis eggs or genes in the bile duct stones [21,22]. Nevertheless, there have been few reports investigating the association of C. sinensis infection and cholecystolithiasis through analysis of gallbladder stones. 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 cholecys- tolithiasis are infected with C. sinensis. Furthermore, the results of microscopic examination revealed that a large portion of the eggs Figure 4. Gene identification of eggs in gallbladder stones. a, DNA of adults; b, DNA of egg-positive gallbladder stones; c, DNA of egg- negative gallbladder stones. The cycle threshold (Ct) for C. sinensis adult and egg-positive gallbladder stone was 26.49 and 29.21 respectively. doi:10.1371/journal.pone.0042471.g004 PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42471