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Real-time PCR method for the detection and quantification of Acanthamoeba species in various types of water samples

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Abstract

In this study, a quantitative real-time PCR was developed to detect and quantify Acanthamoeba spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and three thermal spring recreation areas. The overall detection rate was 14.2 % (25/176) for Acanthamoeba spp. The percentages of samples containing Acanthamoeba spp. from river water, raw drinking water, and thermal spring water were 13 % (13/100), 25 % (7/28), and 10.4 % (5/48), respectively. Acanthamoeba spp. concentrations were determined according to SYBR Green quantitative real-time PCR. A plasmid-based standard curve was constructed to determine the Acanthamoeba concentration using dilution factors for achieving 1.36 × 109 gene copies per PCR for 18S rRNA gene in Acanthamoeba spp. The resulting concentrations varied by the type of water, in the range of 46–2.6 × 102 cells/l in positive raw drinking water, 2.7 × 102–1.5 × 104 cells/l in river water, and 54–1.7 × 103 cells/l in thermal spring water. The presence of Acanthamoeba spp. in the raw drinking water samples was also found to have a significant difference with heterotrophic plate count. The presence of Acanthamoeba spp. in various aquatic environments may be a potential health hazard and must be further evaluated.

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References

  • Amorn L, Chantira S, Somchai B, Yaowalark S (2005) Free-living ameba contamination in natural hot springs in Thailand. Southeast Asian J Trop Med Public Health 36:1–5

    Google Scholar 

  • Anacarso I, Guerrieri E, Bondi M, de Niederhausern S, Iseppi R, Sabia C, Contri M, Borella P, Messi P (2010) Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga. Arch Microbiol 192:877–882

    Article  PubMed  CAS  Google Scholar 

  • APHA (2005) Standard method for the examination of water and wastewater. APHA, WEF and AWWA, Washington, D.C

    Google Scholar 

  • Badirzadeh A, Niyyati M, Babaei Z, Amini H, Badirzadeh H, Rezaeian M (2011) Isolation of free-living amoebae from Sarein Hot Springs in Ardebil Province, Iran. Iran J Parasitol 6:1–8

    PubMed  CAS  Google Scholar 

  • Behets J, Declerck P, Delaedt Y, Verelst L, Ollevier F (2007) Survey for the presence of specific free-living amoebae in cooling waters from Belgian power plants. Parasitol Res 100:1249–1256

    Article  PubMed  Google Scholar 

  • Bonilla-Lemus P, Ramírez-Bautista GA, Zamora-Muñoz C, MdR I-M, Ramírez-Flores E, Hernández-Martínez MD (2010) Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City. Exp Parasitol 126:54–58

    Article  PubMed  Google Scholar 

  • Byers TJ, Hugo ER, Stewart VJ (1990) Genes of Acanthamoeba: DNA, RNA and protein sequences. J Protozool 37:17S–25S

    PubMed  CAS  Google Scholar 

  • Chang CW, Wu YC, Ming KW (2010) Evaluation of real-time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms. J Appl Microbiol 109:799–807

    Article  PubMed  CAS  Google Scholar 

  • Corsaro D, Venditti D (2010) Phylogenetic evidence for a new genotype of Acanthamoeba (Amoebozoa, Acanthamoebida). Parasitol Res 107:233–238

    Article  PubMed  Google Scholar 

  • Declerck P, Behets J, van Hoef V, Ollevier F (2007) Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments. Water Res 41:3159–3167

    Article  PubMed  CAS  Google Scholar 

  • Dhivya S, Madhavan HN, Rao CM, Rao KS, Ramchander PV, Therese KL, Malathi J (2007) Comparison of a novel semi-nested polymerase chain reaction (PCR) with a uniplex PCR for the detection of Acanthamoeba genome in corneal scrapings. Parasitol Res 100:1303–1309

    Article  PubMed  Google Scholar 

  • Edagawa A, Kimura A, Kawabuchi-Kurata T, Kusuhara Y, Karanis P (2009) Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan. Parasitol Res 105:1109–1117

    Article  PubMed  Google Scholar 

  • Ettinger M, Webb S, Harris S, McIninch S, Garman CG, Brown B (2003) Distribution of free-living amoebae in James River, Virginia, USA. Parasitol Res 89:6–15

    Article  PubMed  Google Scholar 

  • Fields BS (1993) Legionella and protozoa: interaction of a pathogen and its natural host. In: Barbaree JM, Breiman RF, Dufour AP (eds) Legionella: current status and emerging perspectives. ASM, Washington, D.C., pp 70–72

    Google Scholar 

  • Gianinazzi C, Schild M, Zumkehr B, Wuthrich F, Nuesch I, Ryter R, Schurch N, Gottstein B, Muller N (2010) Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae. Exp Parasitol 126:45–53

    Article  PubMed  Google Scholar 

  • Hoffmann R, Michel R (2001) Distribution of free-living amebae (FLA) during preparation and supply of drinking water. Int J Hyg Environ Health 203:215–219

    Article  PubMed  CAS  Google Scholar 

  • Hsu BM, Lin CL, Shih FC (2009) Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area. Water Res 43:2817–2828

    Article  PubMed  CAS  Google Scholar 

  • Hsu BM, Huang CC, Chen JS, Chen NH, Huang JT (2011) Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res 45:5171–5183

    Article  PubMed  CAS  Google Scholar 

  • Huang SW, Hsu BM (2010) Isolation and identification of Acanthamoeba from Taiwan spring recreation areas using culture enrichment combined with PCR. Acta Trop 115:282–287

    Article  PubMed  CAS  Google Scholar 

  • Huang SW, Hsu BM, Chen NH, Huang CC, Huang KH, Chen JS, Kao PM (2011) Isolation and identification of Legionella and their host amoebae from weak alkaline carbonate spring water using a culture method combined with PCR. Parasitol Res 109:1233–1241

    Article  PubMed  Google Scholar 

  • Khan NA (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 30:564–595

    Article  PubMed  Google Scholar 

  • Kuroki T, Yagita K, Yabuuchi E, Agata K, Ishima T, Katsube Y (1998) Isolation of Legionella and free-living amoebae at hot spring spas in Kanagawa, Japan. Kansenshogaku Zasshi 72:1050–1055

    PubMed  CAS  Google Scholar 

  • Lau HY, Ashbolt NJ (2009) The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol 107:368–378

    Article  PubMed  CAS  Google Scholar 

  • Leiva B, Clasdotter E, Linder E, Winiecka-Krusnell J (2008) Free-living Acanthamoeba and Naegleria spp. amebae in water sources of León, Nicaragua. Rev Biol Trop 56:439–446

    PubMed  Google Scholar 

  • Lorenzo-Morales J, Lindo JF, Martinez E, Calder D, Figueruelo E, Valladares B, Ortega-Rivas A (2005a) Pathogenic Acanthamoeba strains from water sources in Jamaica, West Indies. Ann Trop Med Parasitol 99:751–758

    Article  PubMed  CAS  Google Scholar 

  • Lorenzo-Morales J, Ortega-Rivas A, Foronda P, Martínez E, Valladares B (2005b) Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources. Parasitol Res 95:273–277

    Article  PubMed  Google Scholar 

  • Lorenzo-Morales J, Ortega-Rivas A, Martínez E, Khoubbane M, Artigas P, Periago MV, Foronda P, Abreu-Acosta N, Valladares B, Mas-Coma S (2006) Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Protozool 100:63–69

    CAS  Google Scholar 

  • Magliano AC, da Silva FM, Teixeira MM, Alfieri SC (2009) Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil. Exp Parasitol 123:231–235

    Article  PubMed  CAS  Google Scholar 

  • Pagnier I, Merchat M, La Scola B (2009) Potentially pathogenic amoeba-associated microorganisms in cooling towers and their control. Future Microbiol 4:615–629

    Article  PubMed  CAS  Google Scholar 

  • Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ (2006) Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 44:3589–3595

    Article  PubMed  CAS  Google Scholar 

  • Rivera WL, Adao DEV (2009) 18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines. Parasitol Res 105:1119–1124

    Article  PubMed  Google Scholar 

  • Stockman LJ, Wright CJ, Visvesvara GS, Fields BS, Beach MJ (2011) Prevalence of Acanthamoeba spp. and other free-living amoebae in household water, Ohio, USA—1990–1992. Parasitol Res 108:621–627

    Article  PubMed  Google Scholar 

  • Tsvetkova N, Schild M, Panaiotov S, Kurdova-Mintcheva R, Gottstein B, Walochnik J, Aspock H, Lucas MS, Muller N (2004) The identification of free-living environmental isolates of amebae from Bulgaria. Parasitol Res 92:405–413

    Article  PubMed  Google Scholar 

  • Yu HS (2004) Laboratory investigation of Acanthamoeba lugdunensis from patients with keratitis. Invest Ophthalmol Vis Sci 45:1418–1426

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a research grant from the National Science Council of Taiwan, Republic of China (NSC 100-2116-M-194-004-MY2).

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Authors and Affiliations

  1. Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, Republic of China

    Po-Min Kao, Bing-Mu Hsu, Cheng-Yu She & Shu-Min Shen

  2. Department of Surgery, Tungs’ Taichung MetroHarbor Hospital, Taichung, Taiwan, Republic of China

    Min-Che Tung

  3. Department of Emergency Medicine, Cheng Hsin Hospital, Taipei, Taiwan, Republic of China

    Hsien-Lung Tsai

  4. Department of Surgery, Mackay Memorial Hospital, Mackay Medicine, Nursing, and Management College, Taipei, Taiwan, Republic of China

    Wen-Chien Huang

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  1. Po-Min Kao
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  2. Min-Che Tung
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  3. Bing-Mu Hsu
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  4. Hsien-Lung Tsai
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  5. Cheng-Yu She
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Correspondence to Min-Che Tung or Bing-Mu Hsu.

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Hsien-Lung Tsai equally contributed with the first author.

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Kao, PM., Tung, MC., Hsu, BM. et al. Real-time PCR method for the detection and quantification of Acanthamoeba species in various types of water samples. Parasitol Res 112, 1131–1136 (2013). https://doi.org/10.1007/s00436-012-3242-x

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  • DOI: https://doi.org/10.1007/s00436-012-3242-x

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