Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.
Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/ beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.
Understanding the most efficacious products will allow clinicians to best communicate to patients and consumers the safest products on the market to reduce adverse events, including microbial keratitis, during contact lens use.
Microbial keratitis (MK) is an eye infection caused by opportunistic bacteria or fungi, which may lead to sight-threatening corneal ulcers. These microorganisms can be introduced to the eye via improper contact lens usage or hygiene, or ineffective multipurpose solutions (MPSs) to disinfect daily wear contact lenses. Thus, the patient’s choice and use of these MPSs is a known risk factor for the development of MK. It is then critical to determine the efficacy of popular MPSs against ubiquitous ocular microorganisms. Therefore, we compare the efficacy of nine major MPSs on the global market against four different microorganism species, and with four different common contact lenses. In accordance with International Standards Organization protocol 14729 and 18259, the microorganisms were inoculated into each MPS with and without contact lenses, and held for the manufacturer’s disinfection time, 24 h, and 7 days after challenge with Serratia marcescens or Fusarium spp. Plates were incubated for 2–7 days and plate counts were conducted to determine the number of surviving microorganisms. The majority of MPSs demonstrated significantly higher disinfection efficacies without contact lenses. Broadly, among the microorganisms tested, the OPTI-FREE products (Puremoist, Express, and Replenish) maintained the highest disinfection efficacies at the manufacturer’s stated disinfection time when paired with any contact lens, compared with other MPSs. These were followed closely by RevitaLens and renu Advanced. MPSs containing dual biocides polyquaternium-1 and myristamidopropyl dimethylamine possessed the highest disinfection efficacy against multiple ocular pathogens.
Acanthamoeba keratitis is a serious ocular infection which is challenging to treat and can lead to blindness. While this pathogen is ubiquitous and can contaminate contact lenses after contact with water, its habits remain elusive. Understanding this organism’s natural behavior will better inform us on how Acanthamoeba colonize contact lens care systems. Acanthamoeba trophozoites were allowed to adhere to either a glass coverslip or non-nutrient agar (NNA) within a flow cell with nutrients (Escherichia coli or an axenic culture medium (AC6)) or without nutrients (Ringer’s solution). Images were taken once every 24 s over 12 h and compiled, and videos were analyzed using ImageJ Trackmate software. Acanthamoeba maintained continuous movement for the entire 12 h period. ATCC 50370 had limited differences between conditions and surfaces throughout the experiment. Nutrient differences had a noticeable impact for ATCC 30461, where E. coli resulted in the highest total distance and speed during the early periods of the experiment but had the lowest total distance and speed by 12 h. The Ringer’s and AC6 conditions were the most similar between strains, while Acanthamoeba in the E. coli and NNA conditions demonstrated significant differences between strains (p < 0.05). These results indicate that quantifiable visual tracking of Acanthamoeba may be a novel and robust method for identifying the movement of Acanthamoeba in relation to contact lens care products. The present study indicates that Acanthamoeba can undertake sustained movement for at least 12 h with and without nutrients, on both rough and smooth surfaces, and that different strains have divergent behavior.
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