Cholecystolithiasis Is Associated with Clonorchis Figure 2. The characteristic spectrum of three types of gallbladder stones. a, characteristic spectrum of cholesterol stones; 1465, 1368, 1196, 1135, 1053, 1023, 985, 955, 883, 839, 798, 739, 592 are the peaks of cholesterol, 1650, 1253, 1196, 1109, 985, 926, 625 are the peaks of bilirubin, so the main component is cholesterol, containing a small amount of bilirubin; b, characteristic spectrum of pigment stones; 1663, 1627, 1572, 1496, 1309, 1250, 1040, 991, 937, 849, 756, 700 are the peaks of the bilirubin and bilirubinate, 1050, 991, 952, 849 are the peaks of cholesterol, 1800, 1422, 875 are the peaks of calcium carbonate, 1627, 1572, 1422 are peaks of calcium stearate, 1040, 608, 568 are the peaks of hydroxyapatite, so the main components are bilirubin and bilirubinate, containing a small amount of calcium stearate, calcium carbonate, hydroxyapatite and cholesterol; c, d, spectrum of the outer layer and core of mixed stones; c, the outer layer of the mixed stones, 1800, 1492, 855, 712, 699 are the peaks of calcium carbonate, 1660, 1627, 1250 are the peaks of bilirubin and bilirubinate, so the main component of the outer layer is calcium carbonate, containing a small amount of bilirubin and bilirubinate, d, the core of the mixed stones, 1703, 1663, 1627, 1573, 1308, 1250, 1105, 1050, 990, 936, 756, 699, 555, 434 are the peaks of bilirubin and bilirubinate, 1800, 1492, 855, 712, 699 are the peaks of calcium carbonate, 1050, 991, 952 are the peaks of cholesterol, 1627, 1573, 1105, 712 are the peaks of calcium stearate, so the main components of the core are bilirubin and bilirubinate, containing calcium carbonate, cholesterol and small amounts of calcium stearate. doi:10.1371/journal.pone.0042471.g002 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 egg- positive 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 ultra- structural 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 Figure 3. The spectrum of the standard controls. a, spectrum of cholesterol standard; b, spectrum of bilirubin standard; c, spectrum of calcium carbonate standard. doi:10.1371/journal.pone.0042471.g003 PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42471