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Parasitology Research

, Volume 116, Issue 4, pp 1339–1344 | Cite as

Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases

  • Adriana Oliveira CostaEmail author
  • Cinthia Furst
  • Lucas Oliveira Rocha
  • Cecília Cirelli
  • Carolina Neris Cardoso
  • Fagner Salmazo Neiva
  • Cynara Oliveira Possamai
  • Daniel de Assis Santos
  • Vanete Thomaz-Soccol
Original Paper

Abstract

Acanthamoeba keratitis (AK) is a progressive corneal infection that demands rapid and sensitive techniques for diagnosis to avoid risk of visual impairment. We evaluated two DNA extraction techniques and a semi-nested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. The most effective protocol was evaluated in samples of corneal scrapings and biopsies from an AK rat model and applied to diagnosis of human cases of AK. DNA extraction performed with a commercial kit based on DNA binding to magnetic beads was more efficient than a method based on alkaline lysis, allowing the detection of one trophozoite and one cyst of Acanthamoeba in samples prepared from cultures. This technique and sn-PCR were applied in corneal scrapings of rats experimentally infected with Acanthamoeba (n = 6), resulting in 100% of positivity, against 16.7% (n = 6) of positive identification in culture method using non-nutrient agar (NNA) with Escherichia coli. Corneal biopsies from rats were also tested (n = 6) and resulted in positivity in all samples in both molecular and culture methods. Eight out of ten presumptive human cases of Acanthamoeba keratitis were also confirmed by sn-PCR of scrapping samples, while the culture method was positive in only four cases. We discuss that animal model of AK can be an efficient tool to validate diagnostic methods and conclude that DNA extraction with the kit and snPCR protocol described here is an effective alternative for diagnosis of AK.

Keywords

Acanthamoeba keratitis Rat model Diagnosis Semi-nested PCR Culture 

Notes

Acknowledgements

The authors thank CNPq, Fundação Araucária, and PRPq/UFMG for financial support. We also thank Ophthalmology Sector staff from Cassiano Antônio de Moraes University Hospital for providing the human corneal scrapings used in this study.

Compliance with ethical standards

All procedures with animal were conducted under accepted guidelines for the care and use of laboratory animals for research and approved by the Ethical Committee for Animal Experimentation of the Biological Sciences Sector of Federal University of Paraná, Brazil, protocol number 420b.

The use of information on the diagnosis of patients suspected of Acanthamoeba keratitis was approved by the Ethical Committee of Health Sciences Center of Federal University of Espírito Santo, Vitória, ES, Brazil (protocol number 006/07), and followed the tenets of Declaration of Helsinki.

References

  1. Bacon AS, Frazer DG, Dart JKG, Matheson M, Ficker LA, Wright P (1993) A review of 72 consecutive cases of Acanthamoeba keratitis, 1984–1992. Eye 7:719–725. doi: 10.1038/eye.1993.168 CrossRefPubMedGoogle Scholar
  2. Claerhout I, Goegebuer A, Van Den Broecke C, Kestelyn P (2004) Delay in diagnosis and outcome of Acanthamoeba keratitis. Graefes Arch Clin Exp Ophthalmol 242:648–653. doi: 10.1007/s00417-003-0805-7 CrossRefPubMedGoogle Scholar
  3. Costa AO, Castro EA, Ferreira GA, Furst C, Crozeta MA, Thomaz-Soccol V (2010) Characterization of Acanthamoeba isolates from dust of a public hospital in Curitiba, Paraná, Brazil. J Eukaryot Microbiol 57:70–75. doi: 10.1111/j.1550-7408.2009.00453.x CrossRefPubMedGoogle Scholar
  4. Dart JKG, Saw VPJ, Kilvington S (2009) Acanthamoeba keratitis: diagnosis and treatment update 2009. Am J Ophthalmol 148:487–499.e2. doi: 10.1016/j.ajo.2009.06.009 CrossRefPubMedGoogle Scholar
  5. Dhivya S, Madhavan HN, Rao CM, 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. doi: 10.1007/s00436-006-0413-7 CrossRefPubMedGoogle Scholar
  6. Goldschmidt P, Degorge S, Saint-Jean C, Yera H, Zekhnini F, Batellier L, Laroche L, Chaumeil C (2008) Resistance of Acanthamoeba to classic DNA extraction methods used for the diagnosis of corneal infections. Br J Ophthalmol 92:112–115. doi: 10.1136/bjo.2007.125898 CrossRefPubMedGoogle Scholar
  7. Khairnar K, Tamber GS, Ralevski F, Pilla DR (2011) Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis. Diagn Microbiol Infect Dis 70:499–506. doi: 10.1016/j.diagmicrobio.2011.03.019 CrossRefPubMedGoogle Scholar
  8. Khan NA (2001) Pathogenicity, morphology, and differentiation of Acanthamoeba. Curr Microbiol 43:391–395. doi: 10.1007/s002840010325 CrossRefPubMedGoogle Scholar
  9. Lasman M (1977) Light and electron microscopic observations on encystment of Acanthamoeba palestinensis, Reich. J Protozool 24:244–248CrossRefPubMedGoogle Scholar
  10. Laummaunwai P, Ruangjirachuporn W, Boonmars T (2012) A simple PCR condition for detection of a single cyst of Acanthamoeba species. Parasitol Res 110:1569–1572. doi: 10.1007/s00436-011-2662-3 CrossRefPubMedGoogle Scholar
  11. Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16:273–307. doi: 10.1128/CMR.16.2.273-307.2003 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Maycock NJR, Jayaswal R, Ed F (2016) Update on Acanthamoeba Keratitis: diagnosis, treatment and outcomes. Cornea 35:713–720. doi: 10.1097/ICO.0000000000000804 CrossRefPubMedGoogle Scholar
  13. Neff R, Ray S, Benton W, Wilborn M (1964) Induction of synchronous encystment (differentiation) in Acanthamoeba sp. Methods Cell Physiol 55–83Google Scholar
  14. Radford CF, Minassian DC, Dart JKG (2002) Acanthamoeba keratitis in England and Wales: incidence, outcome, and risk factors. Br J Ophthalmol 86:536–542CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ren M, Wu X (2010) Evaluation of three different methods to establish animal models of Acanthamoeba keratitis. Yonsei Med J 51:121–127. doi: 10.3349/ymj.2010.51.1.121 CrossRefPubMedGoogle Scholar
  16. Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, Fuerst PA, Byers TJ (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 39:1903–1911. doi: 10.1128/JCM.39.5.1903-1911.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Schuster FL, Visvesvara GS (2004) Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease. Vet Parasitol 126:91–120. doi: 10.1016/j.vetpar.2004.09.019 CrossRefPubMedGoogle Scholar
  18. Sharma S, Garg P, Rao GN (2000) Patient characteristics, diagnosis, and treatment of non-contact lens related Acanthamoeba keratitis. Br J Ophthalmol 84:1103–1108CrossRefPubMedPubMedCentralGoogle Scholar
  19. Siddiqui, Khan (2012) Biology and pathogenesis of Acanthamoeba. Parasites & Vectors 2012 5:6. doi: 10.1186/1756-3305-5-6 CrossRefGoogle Scholar
  20. 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 113:412–416. doi: 10.1016/j.ophtha.2005.10.041 CrossRefPubMedGoogle Scholar
  21. Thompson PP, Kowalski RP, Shanks RMQ, Gordon YJ (2008) Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol 46:3232–3236. doi: 10.1128/JCM.00908-08 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Vianna EN, Costa JO, Santos CKS, Cury MC, Silva EF, Costa AO, Gomes MA (2009) An alternative method for DNA extraction and PCR identification of Entamoeba histolytica and E. dispar in fecal samples. Parasitology 136:765. doi: 10.1017/S0031182009006167 CrossRefPubMedGoogle Scholar
  23. Visvesvara GS, Balamuth W (1975) Comparative studies on related free-living and pathogenic amebae with special reference to Acanthamoeba. J Protozool 22:245–256CrossRefPubMedGoogle Scholar
  24. Yera H, Zamfir O, Bourcier T, Viscogliosi E, Noel C, Dupouy-Camet J, Chaumeil C (2007) Comparison of PCR, microscopic examination and culture for the early diagnosis and characterization of Acanthamoeba isolates from ocular infections. Eur J Clin Microbiol Infect Dis 26:221–224. doi: 10.1007/s10096-007-0268-6 CrossRefPubMedGoogle Scholar
  25. Younger JR, Johnson RD, Holland GN, Page JP, Nepomuceno RL, Glasgow BJ, Aldave AJ, Yu F, Litak J, Mondino BJ, UCLA Cornea Service (2012) Microbiologic and histopathologic assessment of corneal biopsies in the evaluation of microbial keratitis. Am J Ophthalmol 154:512–519.e2. doi: 10.1016/j.ajo.2012.03.014 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Adriana Oliveira Costa
    • 1
    Email author
  • Cinthia Furst
    • 2
  • Lucas Oliveira Rocha
    • 3
  • Cecília Cirelli
    • 1
  • Carolina Neris Cardoso
    • 1
  • Fagner Salmazo Neiva
    • 3
  • Cynara Oliveira Possamai
    • 2
  • Daniel de Assis Santos
    • 4
  • Vanete Thomaz-Soccol
    • 3
  1. 1.Departamento de Análises Clínicas e Toxicológicas, Faculdade de FarmáciaUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  2. 2.Departamento de PatologiaUniversidade Federal do Espírito SantoVitóriaBrazil
  3. 3.Departamento de Patologia Básica, Setor de Ciências BiológicasUniversidade Federal do ParanáCuritibaBrazil
  4. 4.Departamento de Microbiologia, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil

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