Microbial keratitis in Southern Malawi: a microbiological pilot study.

OBJECTIVE: Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi. METHODS AND ANALYSIS: Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for Acanthamoeba, herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise Staphylococcus aureus isolates. RESULTS: 71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative Staphylococcus 31.8% and Streptococcus species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and Acanthamoeba were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with S. aureus keratitis. CONCLUSIONS: In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.

Potential new fluoroquinolone treatments for suspected bacterial keratitis.

Topical fluoroquinolones (FQs) are an established treatment for suspected microbial keratitis. An increased FQ resistance in some classes of bacterial pathogens is a concern. Some recently developed FQs have an extended spectrum of activity, making them a suitable alternative for topical ophthalmic use. For example, the new generation FQs, avarofloxacin, delafloxacin, finafloxacin, lascufloxacin, nadifloxacin, levonadifloxacin, nemonoxacin and zabofloxacin have good activity against the common ophthalmic pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae and several of the Enterobacteriaceae However, because there are no published ophthalmic break-point concentrations, the susceptibility of an isolated micro-organism to a topical FQ is extrapolated from systemic break-point data and wild type susceptibility. The purpose of this review is to compare the pharmacokinetics and pharmacodynamics of the FQs licensed for topical ophthalmic use with the same parameters for new generation FQs. We performed a literature review of the FQs approved for topical treatment and the new generation FQs licensed to treat systemic infections. We then compared the minimum inhibitory concentrations (MIC) of bacterial isolates and the published concentrations that FQs achieved in the cornea and aqueous. We also considered the potential suitability of new generation FQs for topical use based on their medicinal properties. Notably, we found significant variation in the reported corneal and aqueous FQ concentrations so that reliance on the reported mean concentration may not be appropriate, and the first quartile concentration may be more clinically relevant. The provision of the MIC for the microorganism together with the achieved lower (first) quartile concentration of a FQ in the cornea could inform management decisions such as whether to continue with the prescribed antimicrobial, increase the frequency of application, use a combination of antimicrobials or change treatment.

Bacterial keratitis: identifying the areas of clinical uncertainty.

Bacterial keratitis is a common corneal infection that is treated with topical antimicrobials. By the time of presentation there may already be severe visual loss from corneal ulceration and opacity, which may persist despite treatment. There are significant differences in the associated risk factors and the bacterial isolates between high income and low- or middle-income countries, so that general management guidelines may not be appropriate. Although the diagnosis of bacterial keratitis may seem intuitive there are multiple uncertainties about the criteria that are used, which impacts the interpretation of investigations and recruitment to clinical studies. Importantly, the concept that bacterial keratitis can only be confirmed by culture ignores the approximately 50% of cases clinically consistent with bacterial keratitis in which investigations are negative. The aetiology of these culture-negative cases is unknown. Currently, the estimation of bacterial susceptibility to antimicrobials is based on data from systemic administration and achievable serum or tissue concentrations, rather than relevant corneal concentrations and biological activity in the cornea. The provision to the clinician of minimum inhibitory concentrations of the antimicrobials for the isolated bacteria would be an important step forward. An increase in the prevalence of antimicrobial resistance is a concern, but the effect this has on disease outcomes is yet unclear. Virulence factors are not routinely assessed although they may affect the pathogenicity of bacteria within species and affect outcomes. New technologies have been developed to detect and kill bacteria, and their application to bacterial keratitis is discussed. In this review we present the multiple areas of clinical uncertainty that hamper research and the clinical management of bacterial keratitis, and we address some of the assumptions and dogma that have become established in the literature.

Long-term outcomes of viscocanalostomy and phaco-viscocanalostomy in patients with narrow angle glaucoma.

PURPOSE: To assess the outcomes of viscocanalostomy and phaco-viscocanalostomy in patients with narrow angle glaucoma at a single tertiary eye centre. METHOD: All patients undergoing viscocanalostomy for narrow angle glaucoma between June 2010 and June 2017 with a minimum follow-up of 12 months were included. Data was analysed from a prospectively maintained surgical outcome database. Primary outcome was a change in intraocular pressure (IOP). Secondary outcomes were changes in LogMAR visual acuity, number of eye drops, post-operative complications and further surgical interventions. Success was defined at two IOP cut-off points: IOP ≤ 21 mmHg and IOP ≤ 15 mmHg with (qualified success) or without (complete success) drops. Failure was any repeat glaucoma surgery or loss of light perception. RESULTS: Seventy eyes of 46 patients with a mean follow-up of 41.31 months (range 12-60 months) were included. Mean IOP changed from 25.7 ± 9.6 to 15.2, 15.6, 14.6, 13.8 and 14.0 mmHg at 1, 2, 3, 4 and 5 years post-operatively. Drops reduced from 3.2 ± 1.1 pre-operatively to 0.5 at 1 year and 1.1 at all time points thereafter. Qualified success for an IOP ≤ 21 mmHg was achieved in 94.2%, 88.1%, 92.5%, 91.1% and 92.0% and complete success in 63.8%, 37.3%, 30.2%, 22.2% and 24.0% in years 1 to 5, respectively. Qualified success for an IOP ≤ 15 mmHg was achieved in 53.6%, 60.9%, 69.8%, 68.9% and 64.0% and complete success in 39.1%, 26.9%, 22.6%, 20.0% and 8.0% in years 1 to 5, respectively. IOP was significantly lower at all examined post-operative time points (41.1%, 39.3%, 43.3%, 46.4% and 45.3% at years 1 to 5, respectively, p < 0.001 at all time points). Four eyes (5.7%) failed to meet any of the success criteria. Of these, 3 eyes (4.3%) required further glaucoma surgery and one eye (1.4%) progressed to no perception of light at 48 months. No patients had an IOP ≤ 5 mmHg on two consecutive occasions after 3 months. CONCLUSION: Viscocanalostomy and phaco-viscocanalostomy are a safe and effective surgical option in the management of chronic narrow angle glaucoma.

An Evaluation of a Simplified Impression Membrane Sampling Method for the Diagnosis of Microbial Keratitis.

The purpose of this study was to compare bacterial isolation rate using a corneal impression membrane (CIM) and a sharp instrument for obtaining corneal samples from patients with suspected microbial keratitis (MK). Data was retrospectively collected for all patients that had corneal samples taken for presumed MK between May 2014 and May 2020. Prior to May 2017 samples were collected by scraping the edges of the ulcer with a blade. From May 2017, samples were collected by placing a CIM (Millicell cell culture insert) against the ulcer. All corneal samples were processed using the same conventional diagnostic culture method. A total of 3099 corneal samples were included, of which 1214 (39.2%) were corneal scrapes and 1885 (60.9%) CIMs. Microorganisms were isolated from 235 (19.4%) and 1229 (65.2%) cases using a corneal scrape and CIM, respectively (p < 0.001). Of routinely described pathogenic microorganisms, there were significant increases in the isolations of S. aureus (2.4% to 11.3%) and Serratia (0.5% to 1.7%) using the CIM and no significant changes in the isolations of S. pneumoniae and P. aeruginosa. No significant differences were seen between the isolation rates of fungi or Acanthamoeba species. There was a significant increase in the isolation rates of other Streptococcal species (0.7% to 6.9%) and CNS species, specifically, S. epidermidis (2.1% to 26.2%), S. capitis (0.4% to 2.6%) and S. warneri (0.3% to 1.6%) using the CIM. The simplified CIM sampling method is an effective method for collecting corneal samples from patients with presumed MK in clinical practice.

Standard automated perimetry using size III and size V stimuli in advanced stage glaucoma: an observational cross-sectional comparative study.

OBJECTIVES: In this study, we sought to evaluate the extent of further visual field that could be assessed when using stimulus size V in standard automated perimetry compared with size III in advanced stage glaucoma and whether cut-off values could be determined for when to switch from size III to size V. DESIGN: Prospective cross-sectional study. SETTING: Single-centre outpatient eye clinic in India (New Delhi). PARTICIPANTS: Advanced stage glaucoma defined as stages 3-4. INTERVENTION: Central static perimetry with Octopus 900 G programme (size III stimulus dynamic strategy) and low vision central programme (size V stimulus dynamic strategy). PRIMARY AND SECONDARY OUTCOME MEASURES: Visual field assessment for right and left eyes with both sizes III and V were undertaken within one clinic visit. RESULTS: We recruited 126 patients (170 eyes). Mean patient age at assessment was 55.86 years (SD 15.15). Means (SD) for size III versus size V, respectively, were 6.94 dB (5.58) and 12.98 dB (7.77) for mean sensitivity, 20.02 dB (5.67) and 19.22 dB (7.74) for mean deviation, 5.89 dB (2.29) and 7.69 dB (2.78) for standard loss variance and 3.32 min (1.07) and 6.40 min (1.43) for test duration. All except mean deviation were significantly different between size III and V tests. CONCLUSION: Useful visual field information was obtained with size V stimuli which allowed continued monitoring of these patients that was not possible with size III. Increased test duration, standard loss variance and mean sensitivity were found with size V, as expected, given that more visual responses were obtained with the increased target size. A switch from size III to V may be considered when mean sensitivity reaches 10 dB and/or mean deviation reaches 18 dB.

Staphylococcus aureus Keratitis: Incidence, Pathophysiology, Risk Factors and Novel Strategies for Treatment.

Bacterial keratitis is a devastating condition that can rapidly progress to serious complications if not treated promptly. Certain causative microorganisms such as Staphylococcus aureus and Pseudomonas aeruginosa are notorious for their resistance to antibiotics. Resistant bacterial keratitis results in poorer outcomes such as scarring and the need for surgical intervention. Thorough understanding of the causative pathogen and its virulence factors is vital for the discovery of novel treatments to avoid further antibiotic resistance. While much has been previously reported on P. aeruginosa, S. aureus has been less extensively studied. This review aims to give a brief overview of S. aureus epidemiology, pathophysiology and clinical characteristics as well as summarise the current evidence for potential novel therapies.

Deep sclerectomy and trabeculectomy augmented with Mitomycin C: 2-year post-operative outcomes.

PURPOSE: Two-year post-operative outcomes of both deep sclerectomy (DS) and trabeculectomy surgery (Trab) augmented with Mitomycin C (MMC) at a single tertiary eye centre. METHODS: Retrospective review of DS + MMC and trabeculectomy + MMC at a single centre between February 2015 and March 2018. Patients with a minimum of 12-month follow-up were included. Post-operative follow-up: day 1, week 1, months 1/3/6/12/18/24. Primary outcomes: changes in intraocular pressure (IOP) and changes in LogMAR visual acuity (BCVA) pre- and post-procedure. SECONDARY OUTCOMES: changes in number of eye drops, number of follow-up clinic visits, post-operative complications and further surgical interventions. Complete success: IOP ≤ 21 mmHg off all IOP-lowering medications. Qualified success: IOP ≤ 21 mmHg on medication. Failure: IOP > 21 mmHg at 24 months or ≤ 5 mmHg on 2 consecutive follow-up visits after 3 months +/- additional incisional glaucoma surgery +/- loss of light perception. Statistical analysis performed using Microsoft Excel + SPSS. RESULTS: 90 eyes: DS + MMC = 46 eyes, Trab + MMC = 44 eyes. DS + MMC v Trab + MMC: mean pre-op IOP = 19.57 mmHg v 18.89 mmHg, significantly reduced at all post-operative time-points for both groups (p < 0.001). Mean IOP reduction from baseline = 33.94% v 38.39%; > 30% IOP reduction = 54.35% v 68.18%. IOP ≤ 16 mmHg = 82.61% (38/46) v 95.46% (42/44), IOP ≤ 12 mmHg = 52.17% (24/46) v 72.72% (32/44). Complete success = 67.39% v 61.36%, qualified success = 26.09% v 29.55%, failure = 6.52% v 9.09%. Post-op BCVA: no statistically significant differences between two groups (p = 0.09). Mean pre-op drops v post-op drops = 2.98 v 0.38 (DS + MMC; p < 0.001); 2.68 v 0.39 (Trab + MMC; p < 0.001). Further surgical intervention = 13% v 29.55%. Mean number of post-op clinic visits DS + MMC v Trab + MMC = 10.09 v 13.02 (p = 0.005). CONCLUSION: Both procedures achieve sustained intraocular pressure and drop reduction at 2 years post-op. DS + MMC has lower complication rates requiring less intervention and significantly fewer clinic visits, which may be an important factor for deciding surgical management of glaucoma patients in the era of Covid-19 to reduce patient/clinician exposure to the virus.

Octopus 900 Automated Kinetic Perimetry versus Standard Automated Static Perimetry in Glaucoma Practice.

Purpose: The presence of central visual field loss does not infer the extent of peripheral visual field loss. In advanced stage glaucoma, we evaluated whether automated kinetic perimetry provided additional visual field information to that of central static perimetry. Materials and Methods: We undertook a prospective cross-sectional study of advanced-stage glaucoma defined as stages 3-4. Visual field assessment for right and left eyes was undertaken within one clinic visit using the Octopus 900 G programme and kinetic strategy. Results: We recruited 126 patients (170 eyes). Mean patient age at assessment was 55.86 years (SD 15.15). Mean kinetic reaction time was 1503.96 ms (SD 801.68). Kinetic I4e was plotted in 71% of eyes with an unadjusted area of 2513.68 degrees2 (SD 2397.91) and mean isopter radius of 23.16 degrees (SD 13.07). Kinetic I2e was plotted in 53.5% of eyes with an unadjusted area of 627.07 degrees2 (SD 1291.94) and mean isopter radius of 7.47 degrees (SD 10.59). Increased reaction time was associated with a poorer visual field (p = .001). Mean sensitivity, mean deviation and standard loss variance values on static perimetry were higher in patients who had a defined kinetic field boundary than in patients with no kinetic response to I4e stimulus (p = .0001). However, this corresponded to only small-to-medium correlation between static fields and existent kinetic fields: the presence of poor static fields did not always infer a poor kinetic visual field as poor static fields could also have good kinetic visual fields. Conclusions: Although we confirmed a lack of agreement and only a small to medium correlation between the extents of central versus peripheral visual field loss, automated kinetic perimetry did provide additional peripheral (outside the static 30-degree central field) visual field information which was clinically useful in the presence of non-informative severely defected central visual fields.

16S Ribosomal RNA PCR Versus Conventional Diagnostic Culture in the Investigation of Suspected Bacterial Keratitis.

Purpose: The purpose of this study was to compare conventional diagnostic culture (CDC) to 16S ribosomal RNA polymerase chain reaction (PCR) analysis for diagnosing bacterial keratitis. Methods: Samples collected from 100 consecutive patients presenting to the Royal Liverpool University Hospital with bacterial keratitis were processed using CDC and 16S PCR analysis. Results: The overall detection rate using both methods was 36%. Of these, 72.2% (26/36) were detected by PCR and 63.9% (23/36) isolated by CDC (P = 0.62). Using a combination of both PCR and CDC increased the detection rate for pathogenic bacteria by 13% compared to using CDC alone (P = 0.04). In CDC negative samples, 16S PCR identified more pathogens than CDC in 16S PCR negative samples. Neither order of sample collection nor prior antimicrobial use affected the detection rate. Conclusions: 16S rRNA gene PCR performed in addition to CDC on corneal samples from patients with clinically suspected bacterial keratitis led to additional pathogen detection. Translational Relevance: 16S rRNA gene PCR should be developed to become an additional part of clinical service for patients with bacterial keratitis rather than used in isolation.

Recurrent microbial keratitis and endogenous site Staphylococcus aureus colonisation.

This study investigated Staphylococcus aureus carriage in patients with microbial keratitis (MK). 215 patients with MK, 60 healthy controls and 35 patients with rheumatoid arthritis (RA) were included. Corneal scrapes were collected from patients with MK. Conjunctival, nasal and throat swabs were collected from the non-MK groups on a single occasion and from the MK group at presentation and then at 6 and 12 weeks. Samples were processed using conventional diagnostic culture. 68 (31.6%) episodes of clinically suspected MK were classed as recurrent. Patients with recurrent MK had a higher isolation rate of S. aureus from their cornea than those with a single episode (p < 0.01) and a higher isolation rate of S. aureus from their conjunctiva compared to control participants, 20.6% (14/68) versus 3% (5/60) respectively (p = 0.01). Significantly more patients with recurrent MK (12/68, 17.6%) were found to have S. aureus isolated from both their conjunctiva and nose than those with a single episode of MK (7/147, 4.8% p = 0.002) and compared to patients in the control group (3/60, 5.0% p = 0.03). The results indicate that patients with recurrent MK have higher rates of carriage of S. aureus suggesting endogenous site colonisation as a possible source of recurrent infection.

Metagenomics in ophthalmology: current findings and future prospectives.

Less than 1% of all microorganisms of the available environmental microbiota can be cultured with the currently available techniques. Metagenomics is a new methodology of high-throughput DNA sequencing, able to provide taxonomic and functional profiles of microbial communities without the necessity to culture microbes in the laboratory. Metagenomics opens to a 'hypothesis-free' approach, giving important details for future research and treatment of ocular diseases in ophthalmology, such as ocular infection and ocular surface diseases.

Use of a corneal impression membrane and PCR for the detection of herpes simplex virus type-1.

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.

Effect of storage time and temperature on the detection of Pseudomonas aeruginosa, Acanthamoeba and Herpes Simplex Virus from corneal impression membranes.

The effect of storage time and temperature on the recovery of pathogen DNA from polytetrafluorethylene (PTFE) was investigated. PTFE impression membranes were inoculated with Pseudomonas aeruginosa, Herpes Simplex Virus-1 (HSV-1) or Acanthamoeba and stored at -70 °C, -20 °C, +4 °C or +35 °C. PCR was performed on days 0, 1, 2, 3, 7 and months 1, 3 and 10 post-inoculation. We found no reduction in the DNA recovery of any of the studied microorganisms for the first 3 days of storage up to +35 °C. For HSV-1 and P. aeruginosa, storage for 3 months at +35 °C was associated with a significant reduction in DNA recovery (P<0.001), but not at +4 °C, -20 °C or -70 °C for 1 month for P. aeruginosa and for 10 months for HSV-1. Acanthamoeba DNA recovery was not affected by any storage parameters (P=0.203). These results will inform the investigation of microbial keratitis where access to microbiological testing is not readily available.