Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification

Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.

Detection methods for milk pathogenic bacteria by loop-mediated isothermal amplification

“…LAMP has been widely used as a tool in disease diagnosis (12,13), detection of food-borne Pathogenic microorganisms (14) and rapid authentication of ingredients (15). In the present study, the LAMP method developed was specific for ETEC strain and had no amplified product for 9 non-E.coli strains.…”

A Rabbit Model of Acanthamoeba Keratitis: Use of Infected Soft Contact Lenses After Corneal Epithelium Debridement With a Diamond Burr

“…Group 2L-3d showed the highest clinical scores of epithelial defect, corneal edema, neovascularization, and opacity/infiltration, with the only two cases of keratomalacia (lower right corner) and endophthalmitis noted. 4,5,9,10,12,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] In the majority of them, the cornea was partially or completely abraded with a sterile cotton applicator, scratched with a syringe needle or scraped with a blade before placing the contact lens, 4,5,9,10,16-30 because van Klink et al 12 and Ledbetter et al demonstrated that this lesion was essential to allow the penetration of Acanthamoeba into the cornea (Ledbetter EC, et al IOVS 2012;53:ARVO E-Abstract 6146). In this study, the epithelial debridement was performed with a diamond burr unit, used for removing the nonadherent epithelial tissue in the management of spontaneous chronic corneal epithelial defects in dogs and horses.…”

“…19,23,24,[26][27][28]30 However, good results have been shown using exposure times of 24 hours in rats and mice, 4 to 5 days in pigs and 3 to 6 days in hamsters, but not with 4 to 5 days in rabbits. 4,5,9,10,[16][17][18][20][21][22]25,29 Contact lenses serve as a mechanical vector for transmitting trophozoites to the corneal surface, facilitating parasite binding to the epithelium. The adhesion is regulated mainly by mannosylated glycoproteins, and the use of contact lenses and presence of a previous abrasion or mild trauma have been correlated with an increased expression of these proteins on the epithelium, promoting the adhesion.…”

“…6,9,12,13 Nevertheless, considering the contact lens wear as the main risk factor, animal models induced through contaminated contact lenses over previously abraded or scratched corneas seem to be the most realistic method and this has been widely used (Ledbetter EC, et al IOVS 2012;53:ARVO E-Abstract 6146). 4,5,9,10,12,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] The aim of this study was to develop a rabbit model of AK as the best method to reproduce the natural course of this disease, allowing further pathogenicity studies and assess new therapeutic strategies. Bandage contact lenses infected with Acanthamoeba trophozoites and cysts were placed onto the ocular surface after epithelial debridement with a diamond burr unit.…”

Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

“…Even though detection limits for diagnostic methods are usually determined using pure pathogen DNA, 183,184 in a real world situation DNA extracted from an infected plant contains a complex mixture of genomic DNA from the plant as well as the pathogen. The ratio of plant DNA to pathogen DNA in a given sample is variable as it depends on the pathogen and disease progression, but even in advanced stages of infection, plant DNA will always be present in vast excess over pathogen DNA.…”