Corneal Ulcer

Globally, infectious keratitis is the fifth leading cause of blindness. The main predisposing factors include contact lens wear, ocular injury and ocular surface disease. Staphylococcus species, Pseudomonas aeruginosa, Fusarium species, Candida species and Acanthamoeba species are the most common causal organisms. Culture of corneal scrapes is the preferred initial test to identify the culprit organism. Polymerase chain reaction (PCR) tests and in vivo confocal microscopy can complement the diagnosis. Empiric therapy is typically commenced with fluoroquinolones, or fortified antibiotics for bacterial keratitis; topical natamycin for fungal keratitis; and polyhexamethylene biguanide or chlorhexidine for acanthamoeba keratitis. Herpes simplex keratitis is mainly diagnosed clinically; however, PCR can also be used to confirm the initial diagnosis and in atypical cases. Antivirals and topical corticosteroids are indicated depending on the corneal layer infected. Vision impairment, blindness and even loss of the eye can occur with a delay in diagnosis and inappropriate antimicrobial therapy.

PurposeWe test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis.MethodsThis retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool.ResultsSequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge.ConclusionsNext generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

AimsTo evaluate the sensitivity and specificity of polymerase chain reaction (PCR), in vivo confocal microscopy (IVCM) and culture for microbial keratitis (MK) diagnosis.MethodsRetrospective review of PCR, IVCM and culture results for MK diagnosis at Moorfields Eye Hospital between August 2013 and December 2014.ResultsPCR results were available for 259 MK patients with concurrent culture for 203/259 and IVCM for 149/259. Sensitivities and specificities with 95% confidence intervals [95% CI] were calculated for Acanthamoeba keratitis (AK) and fungal keratitis (FK), by comparison with culture, for both IVCM and PCR. For AK, FK and bacterial keratitis (BK) sensitivities were calculated, for each diagnostic method, by comparison with a composite reference standard (a positive result for one or more of culture, PCR or IVCM having a specificity of 100% by definition). For the latter, sensitivities with [95% CI] were: for AK, IVCM 77.1% [62.7-88.0%], PCR 63.3% [48.3-76.6%], culture 35.6 [21.9-51.2]; for FK, IVCM 81.8% [48.2-97.7%], PCR 30.8% [9.09-61.4%], culture 41.7% [15.2-72.3%]; for BK, PCR 25.0% [14.7-37.9%], culture 95.6% [87.6-99.1%].ConclusionIVCM was the most sensitive technique for AK and FK diagnosis but culture remains our gold standard for BK. These findings reflect results to be expected from service providers to UK ophthalmology units and demonstrates the need at our centre for ongoing diagnostic result audit leading to the potential to improve PCR diagnosis. Both FK and AK are now common in the UK; ophthalmology units need to have all these techniques available to optimise their MK management.

PurposeBacterial keratitis (BK) is a leading cause of corneal blindness globally, with an alarming rise in resistant infections. Current standard-of-care antibiotic eye drops or ointments suffer from low bioavailability and often fail to achieve therapeutic concentrations in cornea. This study evaluated a therapeutic contact lens (M-TCL) designed to provide sustained, localized high drug concentrations of moxifloxacin-a commonly used broad-spectrum antibiotic-as a potential treatment for drug-resistant BK.MethodsM-TCLs were engineered by encapsulating a thin moxifloxacin-polymer film within periphery of a contact lens hydrogel. Pharmacokinetics were assessed in rabbit ocular tissues and serum. Efficacy was evaluated using a rabbit keratitis model induced by clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates that displayed in vitro resistance to moxifloxacin (minimum inhibitory concentration = 8 µg/mL). Biocompatibility was assessed through Draize testing and histological analysis.ResultsIn vitro, M-TCLs retained physical properties comparable to commercial contact lenses, sustained moxifloxacin release over 24 hours, and remained stable in a hydrated state for 12 months, enabling on-demand use. In rabbits, M-TCLs maintained higher corneal drug concentrations at all time points over 24 hours than the peak concentration from moxifloxacin drops (P = 0.03), with a 32-fold higher peak drug concentration and at least 11.3-fold greater area under the concentration-time curve (AUC(0-24 h)) in cornea. M-TCLs demonstrated bactericidal efficacy against MRSA keratitis with bacterial reduction of 3.86-log CFU/cornea (P ConclusionsM-TCLs offer a promising therapy for BK and may serve as a potential therapy for drug-resistant BK.