Genotyping of Acanthamoeba Isolated From Surface and Stagnant Waters of Qazvin, Central Iran

The free living amoeba, Acanthamoeba spp are found and distributed in a wide variety of environmental sources including air, water, soil dust, vegetables as well as the skin, cornea, lung and brain of human (1). Isolation of Acanthamoeba cyst from environmental sources such as soil, fresh surface water and stagnant water have been showed that this environmental sources have potential importance for transmission of this amoeba in human and others mammals (2). The resistant double-layered coat cyst of this amoeba can survive in water in the presence of chlorine and other disinfectants. Several species of Acanthamoeba have been known to cause life-threatening granulomatus amoebic encephalitis (GAE), pneumonitis, skin serious lesion in immune compromised individuals, and amoebic keratitis in contact lens wearers. Some studies showed that the incidence of human Acanthamoeba infections especially amoebic keratitis increased through worlds during that last 30 years (1). The identification of Acanthamoeba can be easily accomplished by the morphological characteristic of cysts; however the morphological feature of cysts can be change with the culture condition and be variable within the same strain (1). The most recently proposed method for molecular identification and taxonomy of Acanthamoeba is sequence analysis of variable region of 18s rRNA gene. To date, based on 18s rRNA sequencing, the Acanthamoeba genus classified into 17 (T1-T17) different genotypes (1). Studies showed T4 genotype of Acanthamoeba is most important causative agent of amoebic keratitis and GAE. In Iran, genotypes of T2, T4, T6 were previously reported from some environmental sources such as fresh and stagnant water, soil and hot water (3-5). Since few studies regarding Acanthamoeba genotypes isolated from stagnant water have been previously reported in Iran and important of it to separate of Acanthamoeba cysts, This study was conducted to determine the presence and identify Acanthamoeba in stagnant water of Qazvin, central Iran, using culture and molecular methods. This study was conducted as a descriptive cross-sectional study. From September to December 2010 a total of 40 water samples were collected randomly from 20 stagnant water of square and parks in different region of Qazvin. All samples put in to 250-400 ml plastic bottles and were carried to Parasitology laboratory in Qazvin University of medical sciences. Water samples were filtered using sterile cellulose nitrate membrane filter (Millipore, Pore size 0.45 μm). Each filter was separated and cultured on 1.5% non-nutrient agar medium was prepared with amoeba page saline that overlaid with heat-killed Escherichia coli (5). The plates were incubated at 25-30 ̊c and monitored for present of trophozoite or cyst of amoeba daily until 30 days. The trophozoites and cyst were harvested by phosphate buffered saline (PBS) and using a cell scraper. For washing, centrifugation of amoeba in PBS (PH = 7.2) three times at 4000 rpm for 5 minute was performed. The supernatants were then discarded and cell pellets were re-suspended in DNG lysis buffer and DNA extraction was performed by DNGTM – PLUS kit (cinna gen, Iran). PCR reaction was performed using a pair of primer (JDP1-JDP2) that amplified a fragment of 423 to 551 bp of Acanthamoeba specific 18s rDNA (6). The sequence of primer in this study were: