Isolation and genotyping of Acanthamoeba strains (T4, T9, and T11) from amoebic keratitis patients in Iran

Abstract

The continuous increase in Acanthamoeba keratitis, a severe corneal infection, worldwide is mainly due to the increase in the number of soft contact lens users. In the present study, which involves a 5-year study, a total of 138 corneal scraps and contact lenses together with their paraphernalias were obtained from suspected amoebic keratitis patients. All samples were cultured using culture-enrichment method. Pathogenic assay, using thermotolerance and osmotolerance tests were also performed on the positive strains. Sequencing of the isolated strains was done by targeting the DF3 region of 18s rRNA gene. The results revealed that 18 (13 %) of patients were infected with Acanthamoeba spp. As expected, T4 genotype was the most common genotype among the clinical samples; however, in three cases, Acanthamoeba belonging to T11 and T9 were detected. Interestingly, T9 genotype, commonly classified as non-pathogenic amoebae, was identified as a causal agent of a patient with amoebic keratitis. From the pathogenic assay, four strains belonging to T4 genotypes were highly pathogenic. This is the first report of Acanthamoeba T9 genotypes isolated in Iran and the first report of T9 type occurring in amoebic keratitis patients worldwide. Due to the increasing trend of amoebic keratitis (AK) and the identification of new genotypes, such as T9 as the causative agent of AK, more researches in this field are necessary in the region and the world at large.

First report of Acanthamoeba genotype T8 human keratitis

“…Acanthamoeba identification can be rapidly achieved using real-time molecular methods. For diagnostic purposes, the detection of Acanthamoeba at the genus level is sufficient to recognize whether an individual is infected [18,19]. A more precise differentiation of genotype is beneficial for better understanding of Acanthamoeba taxonomy and also facilitates the correct identification.…”

Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China

“…Eighteen species of Acanthamoeba have been classified into three groups according to the shape and size of cysts. Species in group I are nonpathogenic except A. astronyxis , A. byersi and A. comandoni [ 22 , 23 ]. Most of the pathogenic Acanthamoeba species belong to group II.…”

Isolation and identification of Acanthamoeba from pond water of parks in a tropical and subtropical region in the Middle East, and its relation with physicochemical parameters

“…In Iran, different Acanthamoeba was isolated from soil and water resources in different areas. The isolates belonged to different genotypes especially T4 [ 12 ]. A research was conducted by Armand et al showed that the most prevalent genotype of Acanthamoeba in Shiraz water supplies is T4 [ 21 ].…”

“…The PCR reaction was performed using JDP primers including JDP1 5′-GGCCCAGAT CGTTTACCGTGAA-3′ and JDP2 5′- TCTCACAAGCTGCTAGGGGAGTCA-3 [ 12 ].…”

“…Every PCR run contained a tube with distilled water instead of template DNA as a negative control. The cycles of PCR were set up as following: first denaturation step at 95 °C for 5 min and 35 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 1 min and a final extension step at 72 °C for 5 min [ 12 ].…”