[Microscopic isolation and characterization of free living amoebae [FLA] from surface water sources.in Birjand, the capital city of the South Khorasan]

Background and Aim: Free living amoebae [FLA] are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious infections in human and animal nervous systems. The aim of the current study was to determine the presence of Acanthamoeba spp in surface water sources in Birjand city employing microscopic culture analysis based on morphology features of the amoebae

Materials and Methods: In this cross-sectional study, 50 samples of surface water sources in Birjand city including parks pools, public squares, waterfronts, and water stations were collected and transferred to the laboratory and were passed through nitrocellulose filter paper. The remained elements in the filters were cultured on non-nutrient agar [NNA] with 100microl Escherichia coli suspension. After a few weeks of using morphological features, the amoeba grown were identified

Results: Out of the total of 50 samples cultured on non-nutrient agar [NNA], 19 [38%] samples were morphologically polluted with Acanthamoeba spp,. In 2 samples [4%] a colony of Vahlkampfiidae were observed

Conclusion: The results indicated that a significant percentage of surface water sources in Birjand city was contaminated with Acanthamoeba spp. It is necessary for physicians, therefore, to take into account the diseases caused by these infectious agents. Besides, local regional health professionals should take into consideration the potential role of surface stagnant water sources in transferring these infectious agents. Placing warning signs in areas contaminated with these infectious agents seems a useful measure

Reporting of T4 genotype of acanthamoeba isolates in recreational water sources of Gilan province, northern Iran

Acanthamoeba spp. is the causative agent of blindness keratitis and fatal encephalaitis. Presence of Acanthamoeba spp. in a wide variety of niches such as different water types can lead to exposure of high risk people such as contact lens wearers. The main aim of the present study was to explore the occurrence of Acanthamoeba genotypes in the recreational water sources using both morphological and molecular approaches in Gilan province, Iran. Overall, 50 samples were collected from recreational water sources including man- made and natural waters in Gilan province. Filtration and cultivation of samples was performed using non-nutrient agar. Cloning of Acanthamoeba spp. was done to eliminate bacterial and fungi contamination. PCR amplification and sequencing were performed using genus-specific primer pair. Genotype identification was based on homology analysis of 18S rRNA gene [DF3] of the obtained sequences with the available genes in the gene bank data base. Out of 50 water samples, 15 [30%] were positive for Acanthamoeba trophozoites and cysts according to morphological criteria. Cloning of 13 isolates [26%] was done successfully. Molecular analysis of 13 Acanthamoeba strain revealed that all isolates were belonged to potentially pathogenic T4 genotype. T4 genotype is the main cause of Acanthamoeba-related infections. Presence of Acanthamoeba belonged to T4 genotype in recreational water sources is of concern for high risk people. Alarming sign and education to high risk people is of utmost importance to prevent such infections

Application of multiplex PCR for detection and differentiation of entamoeba histolytica, entamoeba dispar and entamoeba moshkovskii

Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction [Multiplex PCR] is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys