Preliminary identification and typing of pathogenic and toxigenic fusarium species using restriction digestion of ITS1-5.8S rDNA-ITS2 region

Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 universal primers followed by sequencing and restriction enzyme digestion of PCR products. The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS1-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species

Contact lens-associated acanthamoeba keratitis in Iran

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. In this study, 13 Acanthamoeba keratitis cases were diagnosed among 52 keratitis patients. To confirm the identity of Acanthamoeba at the genus level, a PCR-based method was used, and their pathogenic potential was determined using in vitro cytotoxicity assays on human corneal epithelial cells. Twelve [92.3%] of Acanthamoeba keratitis patients were contact lens wearers; among them eleven [91.7%] wore soft contact lenses. 11/13 [84.6%] isolates were axenised in liquid culture medium, of which 10 [90.9%] isolates disrupted corneal cells. Nine [69.2%] isolates showed Acanthamoeba sp. group II, and four [30.8%] showed group III morphology. To our knowledge this is the first report of determination of Acanthamoeba pathogenicity in Iran. This study confirms the importance of determination of pathogenic potential of Acanthamoeba isolates for clinical purposes