Acta Tropica 132 (2014) 45–50 Contents lists available at ScienceDirect Acta Tropica journal h om epa ge: www.elsevier.com/locate/actatropica Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques Po-Min Kao a , Bing-Mu Hsu a,∗ , Chen-Te Chen b,1 , Shih-Wei Huang c , Erl-Shyh Kao d , Jyh-Larng Chen e , Nan-Min Wu e , Wen-Tsai Ji a a Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC b Department of Emergency Medicine, Cheng Hsin General Hospital, Taipei, Taiwan, ROC c Center for General Education, Cheng-Shiu University, Kaohsiung, Taiwan, ROC d Department of Beauty Science and Graduate Institute of Beauty Science Technology, Chienkuo Technology University, Changhua, Taiwan, ROC e Department of Environmental Engineering and Health, Yuanpei University of Science and Technology, Hsinchu, Taiwan, ROC a r t i c l e i n f o Article history: Received 16 September 2013 Received in revised form 19 December 2013 Accepted 23 December 2013 Available online 31 December 2013 Keywords: Acanthamoeba Acanthamoeba keratitis PCR TaqMan real-time qPCR a b s t r a c t The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acan- thamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acan- thamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0–1.8 × 10 3 cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Free-living amoebae are widely distributed protozoa in the various ecological environments (Visvesvara and Schuster, 2008a, 2008b). Some genera of Acanthamoeba, Balamuthia, Hartmannella, Naegleria, Paravhalkampfia and Sappinia have been related to path- ogenicity in animals and humans (Visvesvara et al., 2007, 2009). Acanthamoeba cause different infections, which produces Acan- thamoeba keratitis, subacute or chronic granulomatous amoebic encephalitis, and skin infections (Marciano-Cabral and Cabral, 2003; Khan, 2006; Marciano-Cabral, 2009). Acanthamoeba are distributed throughout different aquatic environments, climatic regions and water quality conditions all over the world. Historically, Acanthamoeba were classified into three subgeneric groups (I–III) by morphology of the cysts that included more than 25 nominal species (Pussard and Pons, 1977). Current developments in the subgenus classification and taxonomy of Acanthamoeba has identified 17 different genotypes (T1–T17) ∗ Corresponding author. Tel.: +886 95 2840868/+886 5 2720411; fax: +886 5 2720807. E-mail addresses: bmhsu@eq.ccu.edu.tw, bmhsu@ccu.edu.tw (B.-M. Hsu). 1 Equal contribution to first author: Chen-Chen Te. based on 18S rRNA gene sequencing by molecular biology tech- niques (Stothard et al., 1998; Horn et al., 1999; Gast, 2001; Hewett et al., 2003; Corsaro and Venditti, 2010; Nuprasert et al., 2010). Acanthamoeba genotype T4 is the most prevalent type causing dis- ease in human, the main Acanthamoeba keratitis-related genotype worldwide (Corsaro and Venditti, 2010). In addition, Acanthamoeba has also been shown to support the intracellular replication and survival of human pathogens such as Campylobacter jejuni, Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa adenoviruses and fungi (Simmons et al., 1998; Axelsson-Olsson et al., 2005; Anacarso et al., 2010; Ben Salah and Drancourt, 2010; Scheid and Schwarzenberger, 2012). Acanthamoeba is an important reservoir of pathogenic microorgan- isms, protecting them from adverse conditions, disinfectants and therapeutic agents, while also enhancing their virulence in humans. Moreover, these bacterial endosymbionts enhance the pathogeni- city of the Acanthamoeba that hosts them (Fritsche et al., 1993). Because of all these threats and their potential impact on human health, it is crucial to detect the pathogenic Acanthamoeba in aquatic environments. Climatology and geographical location made the collection of water in reservoirs an important source for drinking water in Taiwan. In addition, reservoirs are used for drink- ing water, recreational and sports activities. Therefore, the study of pathogenic Acanthamoeba is important in the reservoir. 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.12.020