Original Article

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay


Background: The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene.

Methods: Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique.  Sensitivity, specificity, accuracy and likelihood ratio were evaluated.

Results: Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype.

Conclusion: Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

1. Aboul-Magd LA, Abaza BE, Nada WM, et al. Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis. Parasitol United J. 2016;9(2):87.
2. Grün A-L, Stemplewitz B, Scheid P. First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans. Parasitol Res. 2014;113(6):2395-400.
3. Lorenzo-Morales J, Khan NA, Walochnik J. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite. 2015;22:10.
4. El-Sayed NM, Younis MS, Elhamshary AM, et al. Acanthamoeba DNA can be directly amplified from corneal scrapings. Parasitol Res. 2014;113(9):3267-72.
5. Khan NA. Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev. 2006;30(4):564-95.
6. Hajialilo E, Behnia M, Tarighi F, et al. Isolation and genotyping of Acanthamoeba strains (T4, T9, and T11) from amoebic keratitis patients in Iran. Parasitol Res. 2016;115(8):3147-51.
7. Siddiqui R, Khan NA. Biology and pathogenesis of Acanthamoeba. Parasit Vectors. 2012;5:6.
8. Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev. 2003;16(2):273-307.
9. Tay‐Kearney ML, McGhee CN, Crawford GJ, et al. Acanthamoeba keratitis: a masquerade of presentation in six cases. Acanthamoeba keratitis: a masquerade of presentation in six cases. Aust N Z J Ophthalmol. 1993;21(4):237-45.
10. Orosz E, Farkas Á, Kucsera I. Laboratory diagnosis of Acanthamoeba keratitis in Hungary. Acta Microbiol Immunol Hung. 2016;63(3):293-9.
11. Hammersmith KM. Diagnosis and management of Acanthamoeba keratitis. Curr Opin Ophthalmol. 2006;17(4):327-31.
12. Boggild AK, Martin DS, Lee TY, et al. Laboratory diagnosis of amoebic keratitis: comparison of four diagnostic methods for different types of clinical specimens. J Clin Microbiol. 2009;47(5):1314-8.
13. Bhosale NK, Parija SC, Mandal J, et al. Utility of Polymerase chain reaction in diagnosis of Acanthamoeba and Microsporidial keratitis. Int J Curr Microbiol App Sci. 2016;5(2):854-60.
14. Lehmann OJ, Green SM, Morlet N, et al. Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis. Invest Ophthalmol Vis Sci. 1998;39(7):1261-5.
15. Niyyati M, Lorenzo-Morales J, Mohebali M, et al. Comparison of a PCR-based method with culture and direct examination for diagnosis of Acanthamoeba keratitis. Iran J Parasitol. 2009;4(2):38-43.
16. Yera H, Zamfir O, Bourcier T, et al. 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. 2007;26(3):221-4.
17. Pasricha G, Sharma S, Garg P, et al. Use of 18S rRNA gene-based PCR assay for diagnosis of Acanthamoeba keratitis in non-contact lens wearers in India. J Clin Microbiol. 2003;41(7):3206-11.
18. Khoushzaban F, Deylamiasl A, Jabarvand M, et al. Diagnosis of Acanthamoeba Keratitis by Polymerase Chain Reaction. 2004; J Curr Ophthalmol. 2004:17(1);30- 36.
19. Niyyati M, Lorenzo-Morales J, Rezaie S, et al. Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran. Exp Parasitol. 2009;121(3):242-5.
20. Hajialilo E, Niyyati M, Solaymani M, et al. Pathogenic Free-Living Amoebae Isolated From Contact Lenses of Keratitis Patients. Iran J Parasitol. 2015;10(4):541-6.
21. Rezeaian M, Farnia S, Niyyati M, et al. Amoebic keratitis in Iran (1997-2007). Iran J Parasitol. 2012;2(3):1-6.
22. Schuster FL. Cultivation of pathogenic and opportunistic free-living amoebas. Clin Microbiol Rev. 2002;15(3):342-54.
23. Schroeder JM, Booton GC, Hay J, et al. 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. 2001;39(5):1903-11.
24. Khairnar K, Tamber GS, Ralevski F, et al. Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis. Diagn Microbiol Infect Dis. 2011;70(4):499-506.
25. Lasjerdi Z, Niyyati M, Haghighi A, et al. Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran. Parasitol Res. 2011;109(3):575-80.
26. Khan NA. Pathogenesis of Acanthamoeba infections. Microb Pathog. 2003;34(6):277-85.
27. Khan NA, Jarroll EL, Paget TA. Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays. Curr Microbiol. 2001;43(3):204-8.
28. Mirjalali H, Niyyati M, Abedkhojasteh H, et al. Pathogenic assays of Acanthamoeba belonging to the t4 genotype. Iran J Parasitol. 2013;8(4):530-5.
29. Walochnik J, Obwaller A, Aspöck H. Correlations between morphological, molecular biological, and physiological characteristics in clinical and nonclinical isolates of Acanthamoeba spp. Appl Environ Microbiol. 2000;66(10):4408-13.
30. Garate M, Cubillos I, Marchant J, et al. Biochemical characterization and functional studies of Acanthamoeba mannose-binding protein. Infect Immun. 2005;73(9):5775-81.
31. Niyyati M, Lorenzo-Morales J, Rahimi F, et al. Isolation and genotyping of potentially pathogenic Acanthamoeba strains from dust sources in Iran. Trans R Soc Trop Med Hyg. 2009;103(4):425-7.
32. Maghsood AH, Sissons J, Rezaian M, et al. Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates. J Med Microbiol. 2005;54(Pt 8):755-9.
IssueVol 16 No 1 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v16i1.5530
PCR Genotype Acanthamoeba keratitis Diagnosis Iran

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KHOSRAVINIA N, FATA A, MOGHADDAS elham, HOSSEINI FARASH BR, SADAGHAT MR, ESLAMPOUR AR, JARAHI L. Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay. Iran J Parasitol. 16(1):111-121.