Cholecystolithiasis Is Associated with Clonorchis sinensis Infection Tie Qiao1,2*, Rui-hong Ma1,2, Xiao-bing Luo1,2, Zhen-liang Luo1,2, Pei-ming Zheng1,2 1 Laboratory of Gallbladder Diseases, Institute of Gallbladder Disease of Panyu, Guangzhou, People’s Republic of China, 2 Laboratory of Gallbladder Diseases, The Second People’s Hospital of Panyu, Panyu, Guangzhou, People’s Republic of China Abstract 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 (6400) 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 (63000). 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. Citation: Qiao T, Ma R-h, Luo X-b, Luo Z-l, Zheng P-m (2012) Cholecystolithiasis Is Associated with Clonorchis sinensis Infection. PLoS ONE 7(8): e42471. doi:10.1371/journal.pone.0042471 Editor: Ana Paula Arez, Instituto de Higiene e Medicina Tropical, Portugal Received May 10, 2012; Accepted July 5, 2012; Published August 8, 2012 Copyright: 2012 Qiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funds for this study. Competing Interests: The authors have declared that no competing interests exists. * E-mail: fqj1958@163.com Introduction Cholecystolithiasis or the presence of gallstones in the gallblad- der 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 [2–6]. In spite of its high incidence rate, the etiology and pathogenesis of gallbladder stones is still not well understood [7–12]. 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 [17–24], 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 inciden- tally. 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. Materials and Methods 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. PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42471