Molecular and morphological identification of pathogen free-living amoebae in superficial water in Shiraz, Iran

Among the many genera of free-living amoeba that exist in nature, members of only four genera (Negleria , Acanthamoeba, Balamuthia, Sappinia) have an association with human infection. These genera are known as causative agents of many clinical manifestations such as granulomatous amoebic encephalitis, amoebic keratitis, cutaneous lesions and primary amoebic meningoencephalitis. Infective forms of these pathogenic organisms distribute in wide geographical regions of the world and consider in water, soil and air. There are only a few studies have done about these protozoa in Iran. 30 samples were collected from superficial water of shiraz during July and August of 2009. The samples were filtered and their sediments were cultured on non-nutrient agar medium that were seeded with Escherichia coli and were incubated at three different temperatures 22˚C ,37˚C ,44˚C . The media were inspected with Invert microscopy and ameboa were identified by phase–contrast microscopy and were evaluated by light microscopy after trichrom staining. Polymerase chain reaction was performed for molecular detection of amoebae. Among 30 samples, 29 out of them were identified as Acanthamoeba and 20 out of 29 positive samples were confirmed by PCR. The growth rate of amoeba in 22˚C was more than 37˚C. Eight of 30 samples grew at 44˚C, but flagellate forming test and PCR were negative for them. Two of samples were identified morphologically as Balamuthia and Sappinia. As regard Fars province is subtropical region and there are a lot of parks and green area with superficial waters, the potential risk of diseases caused by free-living amoeba should be considered also further investigations about various aspects of this important opportunistic protozoa is recommended especially for establishment of appropriate prevention tools