Acanthamoeba castellanii: A new high-throughput method for drug screening in vitro.

Despite significant public health impact, there is no specific antiprotozoal therapy for prevention and treatment of Acanthamoeba castellanii infection. There is a need for new and efficient anti-Acanthamoeba drugs that are less toxic and can reduce treatment duration and frequency of administration. In this context a new, rapid and sensitive assay is required for high-throughput activity testing and screening of new therapeutic compounds. A colorimetric assay based on sulforhodamine B (SRB) staining has been developed for anti-Acanthamoeba drug susceptibility testing and adapted to a 96-well microtiter plate format. Under these conditions chlorhexidine was tested to validate the assay using two clinical strains of A. castellanii (Neff strain, T4 genotype [IC50 4.68±0.6µM] and T3 genotype [IC50 5.69±0.9µM]). These results were in good agreement with those obtained by the conventional Alamar Blue assay, OCR cytotoxicity assay and manual cell counting method. Our new assay offers an inexpensive and reliable method, which complements current assays by enhancing high-throughput anti-Acanthamoeba drug screening capabilities.

Molecular identification of Hartmannella vermiformis and Vannella persistens from man-made recreational water environments, Tehran, Iran.

A survey was conducted on man-made recreational water located in different regions of Tehran, Iran to detect the free-living amoebae present in ponds and fountains of parks and squares. Fifty water samples from 22 municipal districts of Tehran were screened for free-living amoebae and identified by morphological characters and polymerase chain reaction amplification. Amoebae detected were identified as Hartmannella vermiformis (12 %) and Vannella persistens (4 %), which are the first reports of these two amoebas in recreational water environments of Iran. Since, H. vermiformis, which is highly similar to strains serving as hosts for Legionella pneumophila, is a common component of the microbial community in fresh surface water. Although Vannella spp. is not proved to be pathogenic itself, they are capable of harboring pathogenic intracellular organisms. Due to some reports related to pathogenicity of these amoebas, the particular hazard related to these microorganisms should be taken into account in the encounter with drinking and washing in these waters. We recommend control strategies based on physical removal rather than on disinfection to be adopted where necessary.

Balamuthia mandrillaris resistance to hostile conditions.

The resistance of Balamuthia mandrillaris to physical, chemical and radiological conditions was tested. Following treatments, viability was determined by culturing amoebae on human brain microvascular endothelial cells for up to 12 days. B. mandrillaris cysts were resistant to repeated freeze-thawing (five times), temperatures of up to 70 degrees C, 0.5 % SDS, 25 p.p.m. chlorine, 10 microg pentamidine isethionate ml(-1) and 200 mJ UV irradiation cm(-2).

Clinical efficacy of antiparasite treatments against intestinal helminths and haematic protozoa in Gallotia caesaris (lizards).

Intestinal helminths and blood protozoa are common parasites of lizards. In captivity they can be a serious problem, but no information on treatments is available. In this study, several antiparasitic drugs were studied in Gallotia caesaris (Lacertida) from La Gomera, the Canary Islands, Spain. A lack of efficacy of chloroquine, and a decrease in haemogregarine (Apicomplexa) infection by atovaquone-proguanil was demonstrated. Furthermore, a positive effect of fenbendazole against intestinal nematode infection was observed. In the Canary Islands, Gallotia bravoana and Gallotia simonyi (Lacertida) are included in a captive breading recovery plan. The present results are useful in order to keep these animals in good condition and to control their parasites. Moreover, these results are important for reptiles kept as pets, due to the previous lack of knowledge of parasite management.

Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt.

The free-living amoebae of the genus Acanthamoeba include non-pathogenic and pathogenic species and has been recently classified into 15 different genotypes, T1-T15. In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in freshwater sources associated with human activities in the Nile Delta region, Egypt. Identification of Acanthamoeba was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized using temperature and osmotolerance assays and PCR reactions with two primer pairs specific to Acanthamoeba pathogenesis. Isolates genotypes were also determined after ribosomal DNA sequencing. These data revealed that isolates belong to T1, T2, T3, T4 and T7 genotypes. As expected, T4 isolates exhibited the most pathogenic traits and were osmotolerant, temperature tolerant and expressed extracellular serine proteases. This is the first report presenting environmental distribution of Acanthamoeba genotypes in Egypt.

RNA interference (RNAi) for the silencing of extracellular serine proteases genes in Acanthamoeba: molecular analysis and effect on pathogenecity.

Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.

Acanthamoeba infection as a cause of severe keratitis in a soft contact lens wearer in Jamaica.

We report the case of a 29-year-old Jamaican patient who presented with severe pain, redness, and swelling of both eyes. She was a regular soft contact lens wearer who did not maintain standard lens care. She was treated for a possible microbial/viral keratitis using topical ciprofloxacin drops, topical acyclovir ointment, and topical atropine drops. The response was inadequate, and scrapings from her cornea, contact lens cases, and both lenses revealed Acanthamoeba on microscopy, which was shown to be Acanthamoeba polyphaga using polymerase chain reaction. She was treated using chlorhexidine 0.02% hourly, ciprofloxacin every 4 hours, and atropine 1% every 12 hours, along with oral ketoconazole 200 mg twice daily with a dramatic response. However, she subsequently suffered slow corneal epithelial regrowth with severe scarring, vascularization, and cortical lens opacification and was referred for penetrating keratoplasty and cataract surgery. This is the first case of severe keratitis caused by Acanthamoeba to be reported from Jamaica and demonstrates that this emerging pathogen can be a cause of severe keratitis in the tropics.

Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources.

A comprehensive survey to document the presence of free-living amoebae of the genus Acanthamoeba was conducted in tap water and sea water sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus-specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potentially pathogenic strains in both sources. Thus, some of the amoebae in these aquatic habitats can act as opportunistic pathogens, could play a role in the diseases of aquatic organisms, and may present a risk to human health.

Evaluation of Acanthamoeba isolates from environmental sources in Tenerife, Canary Islands, Spain.

Tests for potentially pathogenic amoebae were carried out in order to determine the presence of free-living amoebae of genus Acanthamoeba in soil and beach sand sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus specific primer pair. The pathogenical potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potential pathogenic strain in both sources. Thus, some of the amoebae found in these habitats could act as opportunistic pathogens and may present a risk to human health.

Design and evaluation of a specific primer pair for the diagnosis and identification of Acanthamoeba polyphaga.

Random amplified polymorphism DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that may be useful for species diagnosis. In this study, a distinctive a 962-bp band in A. polyphaga band patterns was found, by using the OPC20 primer (ACTTCGCCAC). The DNA fragment was used to design a specific primer pair that was useful for the identification of different isolates as A. polyphaga species. A case of A. polyphaga in disseminated acanthamoebiasis affecting mesenteric nodes is also reported.

Random amplified polymorphic DNA profiles as a tool for the identification of Acanthamoeba divionensis.

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates.