Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms

Alternaria fungus growing on top of cyanoacrylate glue in a patient with perforated corneal ulcer

Purpose: To demonstrate a case where Alternaria fungus grew on top of cyanoacrylate glue used to seal a perforated corneal ulcer. Observations: We document the clinical course of a rare case of Alternaria keratitis over the course of 6 months. Despite the purported antifungal properties of cyanoacrylate glue demonstrated in vitro, this case provides in vivo evidence that this substance can serve as a scaffold on which pathogenic fungi may grow. Conclusion: This report demonstrates the importance of close follow up of patients with corneal glue patches in place. Ophthalmologists should continue to inspect the cornea and glue for possible development of secondary infection, particularly with concomitant contact lens and/or steroid use

Very late onset LASIK flap Acremonium fungal keratitis confirmed by metagenomic deep sequencing.

Purpose: To describe a unique case of LASIK flap fungal keratitis confirmed by next generation sequencing. Observations: A 56-year-old female presented with refractory keratitis involving her LASIK flap 21 years after surgery. Confocal was positive for filamentous structures. The patient underwent immediate flap amputation followed by topical antifungal treatment. Corneal culture was positive for Acremonium sp. Metagenomic deep sequencing confirmed Acremonium as the primary source of infection and also identified Fusarium as a likely contributor of a mixed fungal infection. Sequencing also identified hay as the likely source of the infection. Treatment resulted in eradication of the infection. The patients final best corrected visual acuity was 20/30 with rigid contact lens overrefraction. Conclusions: Metagenomic deep sequencing is a novel diagnostic tool that is increasingly being utilized for diagnosis of refractory keratitis. This case demonstrates the diagnostic potential of deep sequencing for identifying post-LASIK keratitis and reinforces the utility of LASIK flap amputation in the setting of tectonic flap instability due to keratolysis. Importance: This case highlights several important clinical points for treating LASIK flap keratitis and highlights the emerging role metagenomic sequencing has in the diagnosis of infectious keratitis. This is first known case using next generation sequencing to diagnose a post-LASIK infectious keratitis

Neonatal corneal ulcer secondary to congenital entropion.

PurposeTo describe a case of central corneal ulceration in a newborn secondary to congenital entropion.ObservationsCorneal ulcers during infancy are rare and may occur secondary to congenital structural anomalies, including congenital entropion. Correct anatomic eyelid position in newborns is challenging to determine with closed eyelids, and eyelid squeezing during crying and discomfort adds to this challenge.Conclusions and importanceThis report reinforces the importance of careful examination of the adnexa in infants with corneal ulcers while they are most comfortable, usually after topical anesthesia and prior to placement of eyelid speculum. Ophthalmologists caring for infants must be able to detect this condition because prompt entropion repair is necessary for corneal ulcer resolution and prevention of permanent vision loss

In Vitro Comparison of the Acanthamoeba Cysticidal Activity of Povidone Iodine, Natamycin, and Chlorhexidine.

PurposeAcanthamoeba keratitis often is refractory to medical and surgical therapy, primarily because of the remarkable resilience of Acanthamoeba cysts. In this study, we directly compared the cysticidal activity and potency of several candidate medical therapies in vitro.DesignExperimental study.ParticipantsIn vitro Acanthamoeba specimens obtained from 9 patients with keratitis seen at the Francis I. Proctor Foundation from 2008 through 2012.MethodsThe minimum cysticidal concentration (MCC) of povidone iodine, natamycin, and chlorhexidine was investigated using an established assay technique. The relative potency of each agent was estimated starting with concentrations commonly used in clinical practice and determining the number of two-fold dilutions required to reach the MCC. Statistical comparisons of relative potency were performed using bootstrap simulations and permutation tests.Main outcome measuresMinimum cysticidal concentration and the number of two-fold dilutions required to reach the MCC.ResultsThe MCC for chlorhexidine ranged from 3.1 to 25 μg/ml (median, 12.5 μg/ml; interquartile range [IQR], 6.25-12.5 μg/ml), the MCC for natamycin ranged from 390.6 to 3125 μg/ml (median, 390.6 μg/ml; IQR, 390.6-781.2 μg/ml), and the MCC for povidone iodine ranged from 0.3 to 78.1 μg/ml (median, 2.4 μg/ml; IQR, 0.6-9.8 μg/ml). Doses commonly used in clinical practice (povidone iodine 1%, natamycin 5%, and chlorhexidine 0.04%) were approximately 12, 7, and 5 two-fold dilutions higher than the drug's corresponding median MCC, respectively (P < 0.001, comparing 3 drugs). Povidone iodine 1% had the highest potency of the 3 medications tested, requiring more dilutions than natamycin 5% (P < 0.001) and chlorhexidine 0.04% (P < 0.001) to reach the MCC.ConclusionsAll 3 medications demonstrated in vitro cysticidal activity in each of the 9 isolates. The potency of 1% povidone iodine was greater than standard formulations of natamycin or chlorhexidine. Although its clinical efficacy is yet to be determined, povidone iodine may be considered as a potential adjuvant treatment in cases of recalcitrant Acanthamoeba keratitis

Application of Corneal Optical Coherence Tomography Angiography for Assessment of Vessel Depth in Corneal Neovascularization

Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Purpose:To map and measure the depths of corneal neovascularization (NV) using 3-dimensional optical coherence tomography angiography (OCTA) at 2 different wavelengths.Methods:Corneal NV of varying severity, distribution, and underlying etiology was examined. Average NV depth and vessel density were measured using 840-nm spectral-domain OCTA and 1050-nm swept-source OCTA. The OCTA results were compared with clinical slit-lamp estimation of NV depth.Results:Twelve eyes with corneal NV from 12 patients were imaged with OCTA. Clinically "superficial," "midstromal," and "deep" cases had an average vessel depth of 23%, 39%, and 66% on 1050-nm OCTA, respectively. Average vessel depth on OCTA followed a statistically significant ordinal trend according to the clinical classification of vessel depth (Jonckheere-Terpstra test, P < 0.001). In 8 cases where both 840-nm OCTA and 1050-nm OCTA were acquired, there was excellent agreement in the mean vessel depth between the 2 systems (concordance correlation coefficient = 0.94, P < 0.001). The average vessel density measured by 840-nm OCTA was higher (average 1.6-fold) than that measured by 1050-nm OCTA.Conclusions:Corneal OCTA was able to map corneal NV in 3 dimensions and measure vessel depth and density. The depth of corneal NV varied between different pathologies in a manner consistent with previous pathologic studies. The measured vessel density appeared to be affected by the interscan time, which affects blood flow velocity sensitivity, and the wavelength, which affects the ability to penetrate through opacity. These findings suggest possible clinical applications of OCTA for the diagnosis of corneal pathology and quantitative monitoring of therapeutic response in patients with corneal NV