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A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi 1,2 1,2 3 4 4 Naomi W. Lucchi , Mitra Poorak , Jenna Oberstaller , Jeremy DeBarry , Ganesh Srinivasamoorthy , 2 2 2 4 2 Ira Goldman , Maniphet Xayavong , Alexandre J. da Silva , David S. Peterson , John W. Barnwell , Jessica Kissinger3,4,5, Venkatachalam Udhayakumar1,2* 1 Atlanta Research and Education Foundation, Decatur, Georgia, United States of America, 2 Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Department of Genetics, University of Georgia, Athens, Georgia, United States of America, 4 Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America, 5 Institute of Bioinformatics, University of Georgia, Athens, Georgia, United States of America Abstract Background: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR- based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more s