Parasitology Research

, Volume 112, Issue 12, pp 4087–4095 | Cite as

In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites

  • I. Heredero-Bermejo
  • J. L. Copa-Patiño
  • J. Soliveri
  • R. Gómez
  • F. J. de la Mata
  • J. Pérez-Serrano
Original Paper

Abstract

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

References

  1. Bénéré E, da Luz RA, Vermeersch M, Cos P, Maes L (2007) A new quantitative in vitro microculture method for Giardia duodenalis trophozoites. J Microbiol Methods 71:101–106PubMedCrossRefGoogle Scholar
  2. Borazjani RN, May LL, Noble JA, Avery SV, Ahearn DG (2000) Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp. Appl Environ Microbiol 66:1057–1061PubMedCrossRefGoogle Scholar
  3. Cedillo-Rivera R, Ramírez A, Muñoz O (1992) A rapid colorimetric assay with the tetrazolium salt MTT and phenazine methosulfate (PMS) for viability of Entamoeba histolytica. Arch Med Res 23:59–61PubMedGoogle Scholar
  4. Clarke B, Sinha A, Parmar DN, Sykakis E (2012) Advances in the diagnosis and treatment of acanthamoeba keratitis. J Ophthalmol 2012:484892PubMedGoogle Scholar
  5. Cohen EJ, Buchanan HW, Laughrea PA, Adams CP, Galentine PG, Visvesvara GS, Folberg R, Arentsen JJ, Laibson PR (1985) Diagnosis and management of Acanthamoeba keratitis. Am J Ophthalmol 100:389–395PubMedGoogle Scholar
  6. Conza L, Pagani SC, Gaia V (2013) Presence of legionella and free-living amoebae in composts and bioaerosols from composting facilities. PLoS One 8(7):e68244. doi:10.1371/journal.pone.0068244 PubMedCrossRefGoogle Scholar
  7. Degerli S, Tepe B, Celiksoz A, Berk S, Malatyali E (2012) In vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites. Exp Parasitol 131:20–24PubMedCrossRefGoogle Scholar
  8. Fürnkranz U, Nagl M, Gottardi W, Köhsler M, Aspöck H, Walochnik J (2008) Cytotoxic activity of N-chlorotaurine on Acanthamoeba spp. Antimicrob Agents Chemother 52:470–476PubMedCrossRefGoogle Scholar
  9. Heredero-Bermejo I, San Juan Martin C, Soliveri de Carranza J, Copa-Patiño JL, Pérez-Serrano J (2012) Acanthamoeba castellanii: in vitro UAH-T17c3 trophozoite growth study in different culture media. Parasitol Res 110:2563–2567PubMedCrossRefGoogle Scholar
  10. Heredero-Bermejo I, Copa Patiño JL, Soliveri J, García-Gallego S, Rasines B, Gomez R, De la Mata FJ, Pérez-Serrano J (2013) In vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against Acanthamoeba castellanii UAH-T17c3 trophozoites. Parasitol Res 112:961–969PubMedCrossRefGoogle Scholar
  11. Hutchinson K, Apel A (2002) Infectious keratitis in orthokeratology. Clin Experiment Ophthalmol 30:49–51PubMedCrossRefGoogle Scholar
  12. John DT, John RA (1996) Viability of pathogenic Acanthamoeba and Naegleria and virulence of N. fowleri during long-term criopreservation. Folia Parasitol 43:43–46PubMedGoogle Scholar
  13. Khan NA (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 30:564–595PubMedCrossRefGoogle Scholar
  14. Khan NA, Paget TA (2002) Molecular tools for speciation and epidemiological studies of Acanthamoeba. Curr Microbiol 44:444–449PubMedCrossRefGoogle Scholar
  15. Khunkitti W, Avery SV, Lloyd D, Furr JR, Russell AD (1997) Effects of biocides on Acanthamoeba castellanii as measured by flow cytometry and plaque assay. J Antimicrob Chemother 40:227–233PubMedCrossRefGoogle Scholar
  16. Kilvington S, White DG (1994) Acanthamoeba: biology, ecology and human disease. Rev Med Microbiol 5:12–26CrossRefGoogle Scholar
  17. Lau HY, Ashbolt NJ (2009) The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol 107:368–378PubMedCrossRefGoogle Scholar
  18. Malatyali E, Tepe B, Degerli S, Berk S, Akpulat HA (2012a) In vitro amoebicidal activity of four Peucedanum species on Acanthamoeba castellanii cysts and trophozoites. Parasitol Res 110:167–174PubMedCrossRefGoogle Scholar
  19. Malatyali E, Tepe B, Degerli S, Berk S (2012b) In vitro amoebicidal activities of Satureja cuneifolia and Melissa officinalis on Acanthamoeba castellanii cysts and trophozoites. Parasitol Res 110:2175–2180PubMedCrossRefGoogle Scholar
  20. Martín-Navarro CM, Lorenzo-Morales J, Cabrera-Serra MG, Rancel F, Coronado-Alvarez NM, Piñero JE, Valladares B (2008) The potential pathogenicity of chlorhexidine-sensitive Acanthamoeba strains isolated from contact lens cases from asymptomatic individuals in Tenerife, Canary Islands, Spain. J Med Microbiol 57:1399–1404PubMedCrossRefGoogle Scholar
  21. Martín-Navarro CM, López-Arencibia A, Lorenzo-Morales J, Oramas-Royo S, Hernández-Molina R, Estévez-Braun A, Ravelo AG, Valladares B, Piñero JE (2010) Acanthamoeba castellanii Neff: In vitro activity against the trophozoite stage of a natural sesquiterpene and a synthetic cobalt (II)-lapachol complex. Exp Parasitol 126:106–108PubMedCrossRefGoogle Scholar
  22. Mattana A, Biancu G, Alberti L, Accardo A, Delogu G, Fiori PL, Cappuccinelli P (2004) In vitro evaluation of the effectiveness of the macrolide rokitamycin and chlorpromazine against Acanthamoeba castellanii. Antimicrob Agents Chemother 48:4520–4527PubMedCrossRefGoogle Scholar
  23. McBride J, Ingram PR, Henriquez FL, Roberts CW (2005) Development of colorimetric microtiter plate assay for assessment of antimicrobials against Acanthamoeba. J Clin Microbiol 43:629–634PubMedCrossRefGoogle Scholar
  24. McBride J, Mullen AB, Carter KC, Roberts CW (2007) Differential cytotoxicity of phospholipid analogues to pathogenic Acanthamoeba species and mammalian cells. J Antimicrob Chemother 60:521–525PubMedCrossRefGoogle Scholar
  25. Mikus J, Steverding D (2000) A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48:265–269PubMedCrossRefGoogle Scholar
  26. Monteiro-Riviere NA, Inman AO, Zhang LW (2009) Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. Toxicol Appl Pharmacol 234:222–235PubMedCrossRefGoogle Scholar
  27. Mosmann T (1983) A rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  28. Nociari MM, Shalev A, Benias P, Russo C (1998) A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods 15:157–167CrossRefGoogle Scholar
  29. Ondarza RN, Iturbe A, Hernández E (2006) In vitro antiproliferative effects of neuroleptics, antimycotics and antibiotics on the human pathogens Acanthamoeba polyphaga and Naegleria fowleri. Arch Med Res 37:723–729PubMedCrossRefGoogle Scholar
  30. O’Toole SA, Sheppard BL, McGuinness EP, Gleeson NC, Yoneda M, Bonnar J (2003) The MTS assay as an indicator of chemosensitivity/resistance in malignant gynaecological tumours. Cancer Detect Prev 27:47–54PubMedCrossRefGoogle Scholar
  31. Ponce-Macotela M, Navarro-Alegría I, Martínez-Gordillo MN, Alvarez-Chacón R (1994) In vitro effect against Giardia of 14 plant extracts. Rev Invest Clin 46:343–347PubMedGoogle Scholar
  32. Rolón M, Vega C, Escario J, Gómez-Barrio A (2006) Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes. Parasitol Res 99:103–107PubMedCrossRefGoogle Scholar
  33. Schuster FL, Visvesvara GS (2004) Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34:1001–1027PubMedCrossRefGoogle Scholar
  34. Vega-Avila E, Pugsley MK (2011) An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Weat Pharmacol Soc 54:10–14Google Scholar
  35. Wang QJ, Knezetic JA, Schally AV, Pour PM, Adrian TE (1996) Bombesin may stimulate proliferation of human pancreatic cancer cells through an autocrine pathway. Int J Cancer 15:528–534CrossRefGoogle Scholar
  36. Wang YB, Hu Y, Li Z, Wang P, Xue YX, Yao YL, Yu B, Liu YH (2013) Artemether combined with shRNA interference of vascular cell adhesion molecule-1 significantly inhibited the malignant biological behaviour of human glioma cells. PLoS One 8(4):e60834PubMedCrossRefGoogle Scholar
  37. Yu HG, Chung H, Yu YS, Seo JM, Heo JW (2003) A new rapid and non-radioactive assay for monitoring and determining the proliferation of retinal pigment epithelial cells. Korean J Ophthalmol 17:29–34PubMedGoogle Scholar
  38. Zhao QF, Gao XL, Qian M (2003) Study on the effect of H2O2 against Acanthamoeba in vitro. Zhonghua Zhong Liu Za Zhi 21:218–220Google Scholar
  39. Zhi-Jun Y, Sriranganathan N, Vaught T, Arastu SK, Ahmed SA (1997) A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J Immunol Methods 210:25–39PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • I. Heredero-Bermejo
    • 1
  • J. L. Copa-Patiño
    • 1
  • J. Soliveri
    • 1
  • R. Gómez
    • 2
  • F. J. de la Mata
    • 2
  • J. Pérez-Serrano
    • 1
  1. 1.Department of Biomedicine and Biotechnology, School of PharmacyUniversity of AlcaláAlcalá de HenaresSpain
  2. 2.Department of Organic Chemistry and Inorganic Chemistry, School of PharmacyUniversity of Alcalá, CIBER-BBNAlcalá de HenaresSpain

Personalised recommendations