Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method Usa Lek-Uthai a, * ,1 , Rangsima Passara b,1 , Kosol Roongruangchai c , Prayute Buddhirakkul a , Nitaya Thammapalerd d a Department of Parasitology, Faculty of Public Health, Mahidol University, 420/1 Rajavithi Road, Rajathewee District, Bangkok 10400, Thailand b Faculty of Graduate Studies, Mahidol University, Thailand c Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand d Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Thailand article info Article history: Received 23 September 2008 Received in revised form 8 December 2008 Accepted 15 December 2008 Available online 25 December 2008 Keywords: Protozoa Acanthamoeba keratitis Rapid identification Contact lens Loop-mediated isothermal amplification SYBR Green quantitative PCR abstract A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sen- sitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Opportunistic infection in humans with the free-living Acantha- moeba can lead to a variety of clinical symptoms including granu- lomatous amoebic encephalitis (GAE) (Matinez et al., 1980), a disease of the central nervous system, Acanthamoeba keratitis (Nagington et al., 1974), a sight-threatening eye disease and skin lesions associated with immunocompromised individuals, such as AIDS patients (Tan et al., 1993). Contact lens wearers are most at risk from Acanthamoeba keratitis and account for 62–71% of cases (Radford et al., 2002). Due to very frequent misdiagnosis and complicated treatment, Acanthamoeba keratitis often pro- gresses to a state that may cause serious visual loss and perforating keratoplasty. Acanthamoeba keratitis is easily confused with atyp- ical Herpes simplex keratitis or fungal keratitis. Therefore, a fast and reliable diagnosis of Acanthamoeba keratitis is of crucial impor- tance. Detection of cysts or trophozoites on contact lenses or on the inside of lens cases, are not confirmatory for Acanthamoeba kerati- tis. Agar culture is the mainstay for laboratory detection of Acan- thamoeba, which requires familiarity with morphology of cysts and trophozoites of Acanthamoeba, and may take 1–10 days, delay- ing instituting specific therapy (Berger et al., 1990). Savitri et al. (2001) described a simple immunoperoxidase (IP) technique to screen corneal scrapings for Acanthamoeba cysts and trophozoites, which has a sensitivity of 100% and a specificity of 94% in compar- ison with calcofluor white staining and culture method. Pasricha et al. (2003) using 18S rRNA gene-based PCR assay for diagnosis of Acanthamoeba keratitis in microbial keratitis patients obtained similar sensitivity as the smear ‘‘gold standard”, but no improve- ment in specificity or negative predictive value. Loop-mediated isothermal amplification (LAMP) method recently developed by Notomi et al. (2000) relies on autocycling strand displacement DNA synthesis by Bst DNA polymerase. LAMP requires two spe- cially designed inner and two outer DNA primers. As the LAMP reaction is performed under isothermal conditions, a simple incu- bator is sufficient for the DNA amplification process. LAMP synthe- sizes 10–20 lg of target DNA within 30–60 min and the reaction appears to be limited only by amounts of deoxynucleoside triphos- phates and primers provided. As a consequence, a large amount of pyrophosphate is produced, which reacts with magnesium ions to form magnesium pyrophosphate, a white precipitate by-product. Thus results can be conducted by visual inspection. This technique has been successfully applied to the diagnosis of a range of infec- tious diseases (Poon et al., 2006; Notomi et al., 2000; Han et al., 2007). Unlike other PCR methods and the standard culture tech- 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.12.009 * Corresponding author. Fax: +66 26445130. E-mail address: phulu@mahidol.ac.th (U. Lek-Uthai). 1 These two authors contributed equally to this work. Experimental Parasitology 121 (2009) 342–345 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr