الفهرس 
Historical ReviewOnly 14 pages are availabe for public view
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Abstract
Acanthamoebae are commonly found in soil and aquatic environments worldwide. These amoebae have been isolated from very diverse habitats, including bottled water, swimming pools, dental units, eye wash stations and even from dust in the atmosphere. Acanthamoeba spp. have been known to cause life-threatening granulomatous amoebic encephalitis (GAE) in immunocompromised individuals and sight-threatening amoebic keratitis (AK) in contact lens wearers. In addition, these amoebae can act as vehicles for many pathogenic microorganisms with high virulence.
The present study aimed to determine the presence of Acanthamoeba in different environmental sources: water, soil and dust from some districts in Cairo Governorate and to characterize the pathogenic potential of the isolated Acanthamoeba using physiological and biochemical assays in correlation to Acanthamoeba isolated from keratitis patients. In addition, the study aimed to determine the genotypes of the potentially pathogenic environmental isolates by partial sequencing the genus-specific Rns amplicon (including DF3), in an attempt to correlate pathogenicity with genotypes. Finally, to establish a group of physiological, biochemical and genetic features that might be pathogenicity predictive markers of Acanthamoeba.
This cross-sectional study included the collection of 22 corneal scrapings from clinically diagnosed AK patients and 75 environmental samples including 35 water, 30 soil and 10 dust samples.
• Acanthamoeba spp. were isolated by cultivation of the obtained samples on non-nutrient agar plates pre-seeded with E. coli (NNA- E. coli), incubation at 25-28°C and daily monitoring for Acanthamoeba trophozoite growth.
• Determination of the pathogenic potential of Acanthamoeba environmental isolates in comparison to pathogenic Acanthamoeba isolates from AK patients (positive control group) was assessed through Osmotolerance assay, Temperature tolerance assay and detection of proteolytic activity in Zymography analysis.
• Potentially pathogenic environmental Acanthamoeba isolates were subjected to DNA extraction and PCR amplification of Acanthamoeba specific amplimer-S1 (ASA-S1) using Acanthamoeba genus specific primers JDP1 and JDP2. DNA sequencing for genotype determination was done followed by analysis of sequencing data using blasting programs.
Results of the present work showed the following:
• Five (22.7%) of the keratitis cases with symptoms suggestive of AK were positive for Acanthamoeba.
• Frequency of Acanthamoeba from environmental samples from different Cairo districts was 33.3% (25 out of 75 environmental samples). They included 11(31.4%) of 35 tap water samples, 12 (40%) of 30 soil samples and 2 (20%) of 10 dust samples.
• It was found that 70.5% of the environmental Acanthamoeba isolates demonstrated pathogenic potential by osmotolerance assay in comparison to 60% of the AK isolates. 62.5% of Acanthamoeba isolates from water samples, 71.4% of soil isolates and 100% of dust isolates were osmotolerant.
• It was also found that 76.5% of the environmental Acanthamoeba isolates demonstrated pathogenic potential by temperature tolerance assay in comparison to 100% of the AK isolates. 75% of Acanthamoeba isolates from water samples, 85.7% of soil isolates and 50% of dust isolates showed growth at 37oC.
• Most of the environmental Acanthamoeba isolates showed gelatin digestion bands ranging in MW between 100 and 43 kDa.
• Acanthamoeba environmental isolates (5 isolates) that expressed proteolytic activity common with all Acanthamoeba isolates from keratitis cases and showed growth at high temperature and high osmolarity were considered potential pathogens. Environmental isolates (12 isolates) that did not exhibit the full range of pathogenic trait were considered weak potential pathogens.
• The potentially pathogenic (both potential pathogens and weak potential pathogens) environmental Acanthamoeba isolates were introduced to molecular typing.
• PCR of Acanthamoeba isolates from all keratitis cases and only 10 out of 17 (58.8%) samples from water and soil showed bands between 450 and 500 bp confirming the presence of Acanthamoeba amplification product.
• The genotype analysis showed that 70% of the genotyped environmental Acanthamoeba isolates from studied Cairo districts were T4 genotype, 20% were T5 and 10% were T3.
• Environmental Acanthamoeba isolate(s) belonging to:
- T5 genotype (100%) were potential pathogens isolated from soil.
- T4 genotype were either potential pathogens isolated from water (3 out of 7, 42.9%) or weak potential pathogens isolated from soil and water (4 out of 7, 57.1%).
- T3 genotype was weak potential pathogen isolated from water.
• The 2 Acanthamoeba isolates that were genotyped as representatives from keratitis cases belong to T7 genotype.
• Osmotolerance and temperature tolerance as simple plating assays could be used as pathogenicity predictive markers for Acanthamoeba isolates, rather than Zymography.
• Pathogenic Acanthamoeba isolates present in habitats related directly to human populations which could represent a risk for human health.
It is recommended to perform further studies with larger environmental sample size from diverse sources and representing diverse geographic locations. Further work is also recommended to determine if there is a particular correlation between certain T4 subtypes and human disease. In addition, further analysis of the virulence factors of isolates belonging toT5 and T7 genotypes is needed in order to confirm their pathogenic potential.