a comparisons were derived by one-way ANOVA using SPSS 13.0 software. We used a one-way ANOVA test to compare any significant differences among all kinds of groups for the same time point. Student’s t-test was used to compare differences between two unpaired samples and statistical differences were designated by , and . Acknowledgments The authors are thankful to Drs. D. Sibley and J. Boothroyd for critical comments and to Dr. X.N. Xuan for providing Toxoplasma gondii RH-GFP strain. In Memory: To the memory of one of the authors, Mr. Qing-Shi Ren, who passed away in January 27, 2012 at age of 28. Mr. Ren did an excellent job in this project and we all will never forget his significant contribution to this field. New Approaches for Enhanced Detection of Enteroviruses from Hawaiian Environmental Waters Christina Connell, Hsin-I Tong, Zi Wang, Erin Allmann, Yuanan Lu Departments of Public Health 
Sciences and Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America Abstract Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the environment. Reported here are novel and effective laboratory protocols for viral concentration and highly sensitive and optimized RT-PCR for the efficient detection of enteroviruses, an important enteric virus subset, in Hawaiian environmental waters. Eighteen published enterovirus primer pairs were comparatively evaluated for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites (S)-(-)-Blebbistatin tested positive for enterovirus, indicating fecal contamination at these locations. As an additional means of viral concentration, shellfish were collected from 9 sample sites and subjected to dissection, RNA extraction, and subsequent RT-PCR. Shellfish tissue from 6 of 9 sites tested positive for enterovirus. The techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii’s environmental waters. Citation: Connell C, Tong H-I, Wang Z, Allmann E, Lu Y New Approaches for Enhanced Detection of Enteroviruses from Hawaiian Environmental Waters. PLoS ONE 7: e32442. Editor: Niyaz Ahmed, University of Hyderabad, India Received November 25, 2011; Accepted January 31, 2012; Published May 2, 2012 Copyright: 2012 Connell 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: This work was supported in part by grants from the Centers for “9357531 Oceans and Human Health program, the National Institutes of Environmental Health Sciences and the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests e
