Comparison of UVA- and UVA/riboflavin-induced growth inhibition of Acanthamoeba Castellanii

  • Karim Makdoumi
  • Anders Bäckman
  • Jes Mortensen
  • Anders Magnuson
  • Sven Crafoord
Basic Science



To investigate whether ultraviolet light (UVA) at 365 nm can inhibit/eliminate Acanthamoeba growth and if riboflavin would potentiate such an association.


Acanthamoeba castellanii in a fluid medium with a concentration of approximately 1.7 × 104 protozoa/ml were prepared with (0.01 %) and without riboflavin. Exposure of UVA (dose 5.475 J/cm2) took place twice, with each illumination period followed by culturing of 10 μl in peptone yeast-extract glucose (PYG) medium for 7 days. Every suspension prepared had a non-exposed control solution. Determination of Acanthamoeba was conducted daily, by count in Burker chamber days 4 through 7 after exposure. Statistical analysis was done by repeated-measurement ANOVA and post-hoc analysis for unpaired samples.


The exposure of ultraviolet light resulted in an inhibited growth of Acanthamoeba compared to the non-exposed solutions, with a statistically significant reduction over time (p = 0.0003). The addition of riboflavin did not amplify the effect, and there were no tendencies for an interaction effect between UVA and riboflavin.


The antiprotozoal effect of the UVA wavelength, utilized in CXL, is solely mediated by ultraviolet light, and riboflavin does not seem to amplify the antimicrobial efficacy.


Acanthamoeba Acanthamoeba castellanii UV UVA Riboflavin Keratitis Growth inhibition 


  1. 1.
    Yaeger RG (1996) Protozoa: structure, classification, growth, and development. In: Baron S (ed) Medical microbiology. The University of Texas Medical Branch at Galveston, GalvestonGoogle Scholar
  2. 2.
    Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1–26PubMedCrossRefGoogle Scholar
  3. 3.
    Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16:273–307PubMedCrossRefGoogle Scholar
  4. 4.
    Brindley N, Matin A, Khan NA (2009) Acanthamoeba castellanii: high antibody prevalence in racially and ethnically diverse populations. Exp Parasitol 121:254–256PubMedCrossRefGoogle Scholar
  5. 5.
    Alizadeh H, Apte S, El-Agha MS, Li L, Hurt M, Howard K, Cavanagh HD, McCulley JP, Niederkorn JY (2001) Tear IgA and serum IgG antibodies against Acanthamoeba in patients with Acanthamoeba keratitis. Cornea 20:622–627PubMedCrossRefGoogle Scholar
  6. 6.
    Khan NA (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 30:564–595PubMedCrossRefGoogle Scholar
  7. 7.
    Kohsler M, Leitner B, Blaschitz M, Michel R, Aspock H, Walochnik J (2006) ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa). Parasitol Res 98:86–93PubMedCrossRefGoogle Scholar
  8. 8.
    Nuprasert W, Putaporntip C, Pariyakanok L, Jongwutiwes S (2010) Identification of a novel t17 genotype of acanthamoeba from environmental isolates and t10 genotype causing keratitis in Thailand. J Clin Microbiol 48:4636–4640PubMedCrossRefGoogle Scholar
  9. 9.
    Seal DV (2003) Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment. Eye (Lond) 17:893–905CrossRefGoogle Scholar
  10. 10.
    Ku JY, Chan FM, Beckingsale P (2009) Acanthamoeba keratitis cluster: an increase in Acanthamoeba keratitis in Australia. Clin Experiment Ophthalmol 37:181–190PubMedCrossRefGoogle Scholar
  11. 11.
    Thebpatiphat N, Hammersmith KM, Rocha FN, Rapuano CJ, Ayres BD, Laibson PR, Eagle RC Jr, Cohen EJ (2007) Acanthamoeba keratitis: a parasite on the rise. Cornea 26:701–706PubMedCrossRefGoogle Scholar
  12. 12.
    Dart JK, Saw VP, Kilvington S (2009) Acanthamoeba keratitis: diagnosis and treatment update 2009. Am J Ophthalmol 148:487–499PubMedCrossRefGoogle Scholar
  13. 13.
    Sun X, Zhang Y, Li R, Wang Z, Luo S, Gao M, Deng S, Chen W, Jin X (2006) Acanthamoeba keratitis: clinical characteristics and management. Ophthalmology 113:412–416PubMedCrossRefGoogle Scholar
  14. 14.
    Illingworth CD, Cook SD, Karabatsas CH, Easty DL (1995) Acanthamoeba keratitis: risk factors and outcome. Br J Ophthalmol 79:1078–1082PubMedCrossRefGoogle Scholar
  15. 15.
    Butler TK, Males JJ, Robinson LP, Wechsler AW, Sutton GL, Cheng J, Taylor P, McClellan K (2005) Six-year review of Acanthamoeba keratitis in New South Wales, Australia: 1997–2002. Clin Experiment Ophthalmol 33:41–46PubMedCrossRefGoogle Scholar
  16. 16.
    Seal D (2003) Treatment of Acanthamoeba keratitis. Expert Rev Anti Infect Ther 1:205–208PubMedCrossRefGoogle Scholar
  17. 17.
    Lindquist TD (1998) Treatment of Acanthamoeba keratitis. Cornea 17:11–16PubMedCrossRefGoogle Scholar
  18. 18.
    Iovieno A, Ledee DR, Miller D, Alfonso EC (2010) Detection of bacterial endosymbionts in clinical acanthamoeba isolates. Ophthalmology 117:445–452PubMedCrossRefGoogle Scholar
  19. 19.
    Fritsche TR, Gautom RK, Seyedirashti S, Bergeron DL, Lindquist TD (1993) Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J Clin Microbiol 31:1122–1126PubMedGoogle Scholar
  20. 20.
    Horn M, Wagner M (2004) Bacterial endosymbionts of free-living amoebae. J Eukaryot Microbiol 51:509–514PubMedCrossRefGoogle Scholar
  21. 21.
    Paterson GN, Rittig M, Siddiqui R, Khan NA (2011) Is Acanthamoeba pathogenicity associated with intracellular bacteria? Exp Parasitol 129:207–210PubMedCrossRefGoogle Scholar
  22. 22.
    Martins SA, Combs JC, Noguera G, Camacho W, Wittmann P, Walther R, Cano M, Dick J, Behrens A (2008) Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci 49:3402–3408PubMedCrossRefGoogle Scholar
  23. 23.
    Schrier A, Greebel G, Attia H, Trokel S, Smith EF (2009) In vitro antimicrobial efficacy of riboflavin and ultraviolet light on Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. J Refract Surg 25:S799–S802PubMedCrossRefGoogle Scholar
  24. 24.
    Makdoumi K, Backman A, Mortensen J, Crafoord S (2010) Evaluation of antibacterial efficacy of photo-activated riboflavin using ultraviolet light (UVA). Graefes Arch Clin Exp Ophthalmol 248:207–212PubMedCrossRefGoogle Scholar
  25. 25.
    Iseli HP, Thiel MA, Hafezi F, Kampmeier J, Seiler T (2008) Ultraviolet A/riboflavin corneal cross-linking for infectious keratitis associated with corneal melts. Cornea 27:590–594PubMedCrossRefGoogle Scholar
  26. 26.
    Micelli Ferrari T, Leozappa M, Lorusso M, Epifani E, Micelli Ferrari L (2009) Escherichia coli keratitis treated with ultraviolet A/riboflavin corneal cross-linking: a case report. Eur J Ophthalmol 19:295–297PubMedGoogle Scholar
  27. 27.
    Moren H, Malmsjo M, Mortensen J, Ohrstrom A (2010) Riboflavin and ultraviolet a collagen crosslinking of the cornea for the treatment of keratitis. Cornea 29:102–104PubMedCrossRefGoogle Scholar
  28. 28.
    Makdoumi K, Mortensen J, Crafoord S (2010) Infectious keratitis treated with corneal crosslinking. Cornea 29:1353–1358PubMedCrossRefGoogle Scholar
  29. 29.
    Al-Sabai N, Koppen C, Tassignon MJ (2010) UVA/riboflavin crosslinking as treatment for corneal melting. Bull Soc Belge Ophtalmol 315:13–17PubMedGoogle Scholar
  30. 30.
    Wollensak G, Spoerl E, Seiler T (2003) Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 135:620–627PubMedCrossRefGoogle Scholar
  31. 31.
    Sauer A, Letscher-Bru V, Speeg-Schatz C, Touboul D, Colin J, Candolfi E, Bourcier T (2010) In vitro efficacy of antifungal treatment using riboflavin/UV-A (365 nm) combination and amphotericin B. Invest Ophthalmol Vis Sci 51:3950–3953PubMedCrossRefGoogle Scholar
  32. 32.
    Khan YA, Kashiwabuchi RT, Martins SA, Castro-Combs JM, Kalyani S, Stanley P, Flikier D, Behrens A (2011) Riboflavin and ultraviolet light A therapy as an adjuvant treatment for medically refractive Acanthamoeba keratitis: report of 3 cases. Ophthalmology 118:324–331PubMedCrossRefGoogle Scholar
  33. 33.
    Garduno-Vieyra L, Gonzalez-Sanchez CR, Hernandez-Da Mota SE (2011) Ultraviolet-a light and riboflavin therapy for acanthamoeba keratitis: a case report. Case Report Ophthalmol 2:291–295PubMedGoogle Scholar
  34. 34.
    Hijnen WA, Beerendonk EF, Medema GJ (2006) Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: a review. Water Res 40:3–22PubMedCrossRefGoogle Scholar
  35. 35.
    Sokmen M, Degerli S, Aslan A (2008) Photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in water. Exp Parasitol 119:44–48PubMedCrossRefGoogle Scholar
  36. 36.
    Del Buey MA, Cristobal JA, Casas P, Goni P, Clavel A, Minguez E, Lanchares E, Garcia A, Calvo B (2012) Evaluation of in vitro efficacy of combined riboflavin and ultraviolet a for acanthamoeba isolates. Am J Ophthalmol 153:399–404PubMedCrossRefGoogle Scholar
  37. 37.
    Kashiwabuchi RT, Carvalho FR, Khan YA, de Freitas D, Foronda AS, Hirai FE, Campos MS, McDonnell PJ (2011) Assessing efficacy of combined riboflavin and UV-A light (365 nm) treatment of Acanthamoeba trophozoites. Invest Ophthalmol Vis Sci 52:9333–9338PubMedCrossRefGoogle Scholar
  38. 38.
    Marschner S, Goodrich R (2011) Pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and UV light. Transfus Med Hemother 38:8–18PubMedCrossRefGoogle Scholar
  39. 39.
    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
  40. 40.
    Cariello AJ, de Souza GF, Foronda AS, Yu MC, Hofling-Lima AL, de Oliveira MG (2010) In vitro amoebicidal activity of S-nitrosoglutathione and S-nitroso-N-acetylcysteine against trophozoites of Acanthamoeba castellanii. J Antimicrob Chemother 65:588–591PubMedCrossRefGoogle Scholar
  41. 41.
    Beattie TK, Seal DV, Tomlinson A, McFadyen AK, Grimason AM (2003) Determination of amoebicidal activities of multipurpose contact lens solutions by using a most probable number enumeration technique. J Clin Microbiol 41:2992–3000PubMedCrossRefGoogle Scholar
  42. 42.
    Oldenburg CE, Keenan JD, Cevallos V, Chan MF, Acharya NR, Gaynor BD, McLeod SD, Esterberg EJ, Porco TC, Lietman TM (2011) Microbiological cure times in acanthamoeba keratitis. Eye (Lond) 25:1155–1160CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Karim Makdoumi
    • 1
    • 4
  • Anders Bäckman
    • 2
    • 4
  • Jes Mortensen
    • 1
  • Anders Magnuson
    • 3
    • 4
  • Sven Crafoord
    • 1
    • 4
  1. 1.Department of OphthalmologyÖrebro University HospitalÖrebroSweden
  2. 2.Clinical Research CentreÖrebro University HospitalÖrebroSweden
  3. 3.Clinical Epidemiology and Biostatistic UnitÖrebro University HospitalÖrebroSweden
  4. 4.School of Health and Medical SciencesÖrebro UniversityÖrebroSweden

Personalised recommendations