Research Paper Well water sources simultaneous contamination with Cryptosporidium and Acanthamoeba in East-Southeast Asia and Acanthamoeba spp. in biofilms in the Philippines Frederick R. Masangkay a, ⁎ , Giovanni D. Milanez a , Joseph D. Dionisio b , Luzelle Anne G.-L. Ormita c,d , Abel V. Alvarez d , Panagiotis Karanis e,f, ⁎⁎ a Department of Medical Technology, Faculty of Pharmacy, University of Santo Tomas, Manila 1008, Philippines b Department of Medical Technology, Far Eastern University-Manila, Manila 1015, Philippines c Department of Psychology, Far Eastern University-Manila, Manila 1015, Philippines d Community Extension Services, Far Eastern University-Manila, Manila 1015, Philippines e University of Cologne, Faculty of Medical and University Hospital Cologne, Cologne 50923, Germany f Medical School, Department of Basic and Clinical Science, University of Nicosia, Nicosia, 2417, Cyprus HIGHLIGHTS • Groundwater contamination with Crypto- sporidium and Acanthamoeba species. • Cryptosporidium spp. contamination in well water sources in Southeast Asia. • Acanthamoeba spp. contamination in well water sources in East-Southeast Asia. • Acanthamoeba spp. in biofilms in the Philippines. GRAPHICAL ABSTRACT ABSTRACT ARTICLE INFO Editor: José Virgílio Cruz Cryptosporidium is the leading agent of waterborne parasitic protozoan outbreaks and is the second leading cause of infant mortality due to diarrhoea worldwide. Acanthamoeba spp. causes Acanthamoeba keratitis (AK) and a life- threatening condition known as granulomatous amoebic encephalitis (GAE). The present study aimed to assess the water quality of an indigenous and a rural community for waterborne parasitic protozoan contamination. Aquatic sam- ples (n = 22) were processed by filtration of 500 mL portion through a 1.2 μm pore size glass microfiber filter and eluted for light microscopy, culture in non-nutrient agar, and PCR analysis. Overall, 36% (8/22) of the investigated aquatic samples were positive for either Cryptosporidium spp. oocysts (13%; 3/22) or Acanthamoeba spp., (36%; 8/ 22) or both (13%; 3/22). Cryptosporidium spp. oocysts were detected in 27% (3/11) of wet season samples only while Acanthamoeba spp. were detected in 18% (2/11) and 55% (6/11) of wet and dry season samples, respectively. Subsequently, molecular detection for Acanthamoeba species identified A. lenticulata and A. hatchetti with 98–99% BLAST similarity. This is the first report on the simultaneous contamination of Cryptosporidium and Acanthamoeba in Keywords: Acanthamoeba Cryptosporidium Well water Biofilms Indigenous Philippines Amoeba Aquifer Groundwater Science of the Total Environment 837 (2022) 155752 ⁎ Corresponding author. ⁎⁎ Correspondence to: P. Karanis, University of Cologne, Faculty of Medical and University Hospital Cologne, Cologne 50923, Germany. E-mail addresses: frmasangkay@ust.edu.ph (F.R. Masangkay), karanis.p@unic.ac.cy (P. Karanis). http://dx.doi.org/10.1016/j.scitotenv.2022.155752 Received 12 February 2022; Received in revised form 5 April 2022; Accepted 2 May 2022 Available online 06 May 2022 0048-9697/© 2022 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv