Antonio Ortega-Rivas

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.

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. RID=”” ID=”” Correspondence to: A. Ortega-Rivas; email: aortega@ull.es

A SPECIFIC PRIMER PAIR FOR THE DIAGNOSIS AND IDENTIFICATION OF ACANTHAMOEBA ASTRONYXIS BY RANDOM AMPLIFIED POLYMORPHIC DNA– POLYMERASE CHAIN REACTION

Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.

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.