Sains Malaysiana 41(5)(2012): 569–572 In Vitro Sensitivity Testing of Acanthamoeba Clinical Isolates from Patients with Keratitis against Polyhexamethylene biguanide (PHMB) and Chlorhexidine (Kajian Sensitiviti Isolat Klinikal Acanthamoeba daripada Pesakit Keratitis Secara In Vitro Terhadap Polyhexamethylene biguanide (PHMB) dan Chlorhexidine) NORADILAH SAMSEH ABDULLAH, MOHAMED KAMEL ABD GHANI*, ANISAH NORDIN, YUSOF SUBOH, NORAINA AB RAHIM & NORAZAH AHMAD ABSTRACT Acanthamoeba keratitis is a serious infection of the eye which can result in permanent visual impairment. Therefore this study was performed to evaluate the effectiveness of antimicrobial agents on three Acanthamoeba clinical isolates (HS 6, HKL 95 and HTH 73). Antimicrobial agents used in this study were polyhexamethylene biguanide (PHMB) and chlorhexidine and both were serially diluted. Cyst suspensions from all three strains were tested against the antimicrobial agents, respectively. After 48 h of incubation at 37°C, the suspension was filtered and the filter membrane was placed onto non- nutrient agar plate lawned with heat-killed Escherichia coli. The plates were examined daily under the inverted microscope until day 14 but were negative for Acanthamoeba trophozoites. The presence of trophozoites indicated ineffectiveness of the antimicrobial agents. Both antimicrobial agents used were found to be effective against Acanthamoeba cysts for all the strains tested. PHMB gave minimum cysticidal concentration (MCC) mean value of 4.232 µg/mL and chlorhexidine showed MCC mean value of 3.906 µg/mL. So, from this study, it can be concluded that PHMB and chlorhexidine were effective in killing the tested Acanthamoeba cysts. Keywords: Acanthamoeba; in vitro; sensitivity; polyhexamethylene biguanide; chlorhexidine ABSTRAK Keratitis Acanthamoeba merupakan infeksi mata yang serius yang boleh menyebabkan kerosakan penglihatan yang teruk. Oleh yang demikian, kajian ini dilakukan untuk mengkaji keberkesanan agen antimikrob terhadap tiga isolat klinikal Acanthamoeba (HS 6, HKL 95 and HTH 73). Agen antimikrob yang digunakan di dalam kajian ini adalah poliheksametilina biguanide (PHMB) dan kloroheksidin yang menjalani pencairan bersiri. Suspensi sista daripada ketiga-tiga strain diuji dengan kedua-dua agen antimikrob. Selepas inkubasi selama 48 jam pada suhu 37°C, campuran suspensi sista dan agen antimikrob tersebut dituras dan membran turasan diletakkan ke atas plat agar bukan nutrien yang dibanjiri Escherichia coli. Kesemua agar tersebut diperiksa setiap hari menggunakan mikroskop terbalik sehingga hari ke 14 tetapi negatif untuk trofozoit Acanthamoeba. Kehadiran trofozoit menunjukkan ketidakberkesanan agen antimikrob. Kedua-dua agen antimikrob yang digunakan didapati berkesan terhadap sista Acanthamoeba bagi kesemua strain yang diuji. PHMB memberikan nilai MCC pada kepekatan 4.232 µg/mL manakala chlorhexidine pula menunjukkan nilai MCC pada kepekatan 3.906 µg/mL. Maka, daripada kajian ini, dapat disimpulkan bahawa PHMB dan kloroheksidina berkesan membunuh sista Acanthamoeba yang diuji. Kata kunci: Acanthamoeba; in vitro; kloroheksidine; poliheksametilena biguanide; sensitivitc INTRODUCTION Acanthamoeba spp. are ubiquitous in aquatic habitats and colonize biofilms in swimming pools, hot tubs, domestic water taps, eyewash stations, contact lens disinfecting solutions, and air-conditioning systems in buildings and automobiles (Simmons et al. 1999). The trophozoite is the active motile stage that feeds by phagocytosis and pinocytosis and divides by binary fission. A cyst stage is resistant to adverse environmental conditions such as desiccation, extreme temperatures and antimicrobials (Borazjani et al. 2000). Acanthamoeba keratitis (AK) is a rare but sight- threatening ocular infection caused by Acanthamoeba. Outbreaks of AK have been associated with contaminated water and contact lens wear (Ledee et al. 2009). In recent years, the number of AK cases has been on the increase, especially among wearers of contact lenses, who make up 85 to 90% of the AK cases (Patel & Hammersmith 2008). In Malaysia, Acanthamoeba keratitis is not so rare and the incidence has increased as reported between June and December 2002, four (9.1%) of 44 cases were found to be