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.

Impression membrane for the diagnosis of microbial keratitis.

PURPOSE: To evaluate a corneal impression membrane (CIM) for isolation of bacteria, fungi and acanthamoeba in suspected microbial keratitis. METHODS: Consecutive patients presenting with suspected microbial keratitis were included. For each patient, samples were collected in a random order using a surgical blade and a 4-mm-diameter polytetrafluoroethylene CIM disc, and transported in brain heart infusion broth. Risk factors, best corrected visual acuity (BCVA), size, location, depth and healing time of the ulcer were recorded. The microbial isolation rate was used to compare sampling methods. RESULTS: 130 patients were included (mean age 62.6 years, SD 19.0). An antimicrobial had been used prior to presentation in 36 (27.7%) patients. Mean major and minor ulcer diameters were 2.1 mm (SD 2.0) and 1.6 mm (SD 1.7). Mean healing time was 12.4 days (SD 13.6). BCVA at presentation and following healing was 0.7 (SD 0.7) and 0.62 (SD 0.7) (p=0.34). There were 66 isolates (50.8%); 53 (40.8%) using a CIM and 35 (26.9%) using a blade (p=0.02). ITALIC! Staphylococcus aureus and coagulase-negative staphylococci were the commonest isolates. Isolation rate was not influenced by organism type, although in four cases ITALIC! Acanthamoeba spp. were isolated; three using CIM and one a blade. CONCLUSIONS: In this study, the isolation of microorganisms from cases of suspected microbial keratitis was significantly higher using a CIM than a surgical blade. A CIM may be a useful alternative or addition for sample collection in microbial keratitis.

Pharmacokinetics of Meropenem for Use in Bacterial Keratitis.

PURPOSE: To investigate the toxicity and corneal pharmacokinetics of meropenem as a potential antimicrobial for bacterial keratitis. METHODS: Corneal epithelial cell and keratocyte toxicity was investigated using methyl thiazolyl tetrazolium (MTT) and LIVE/DEAD assays. The penetration of meropenem through the human cornea was measured using an artificial anterior chamber. In one group of corneas, the epithelial and endothelial layers were removed and in a second group these layers were left intact. We applied 50 µL (10 mg/mL) meropenem to the corneal surface and collected samples in the anterior chamber from 45 minutes up to 24 hours. Meropenem concentrations were estimated with a bioassay and HPLC. RESULTS: Meropenem had significantly higher cellular metabolic activity (MTT assay) at both 5 mg/mL and 2.5 mg/mL compared with moxifloxacin (P = 0.029 and P = 0.018, respectively), with 96% cell viability (LIVE/DEAD assay). The measured values for meropenem concentrations in corneal and aqueous samples were significantly higher using a bioassay than with HPLC (P = 0.004). For both intact and denuded corneas, the concentrations in the anterior chamber increased from 0.48 µg/mL (SD 0.89) and 0.89 µg/mL (SD 0.81) to 6.35 µg/mL (SD 0.81) and 13.48 µg/mL (SD 14.82) using HPLC, and from 0.68 µg/mL (SD 1.50) and 1.31 µg/mL (SD 1.55) to 47.03 µg/mL (SD 5.51) and 43.69 µg/mL (SD 27.22) measured with a bioassay. CONCLUSIONS: Meropenem has very low toxicity in vitro. It has good corneal penetration, achieving anterior chamber concentrations above MIC90 for bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, streptococci, coagulase-negative staphylococci, and the Enterobacteriaceae.

Recurrent bacterial keratitis.

PURPOSE: To investigate the rate of recurrent bacterial keratitis, associated bacteria, and surgical intervention. METHODS: Patients with suspected bacterial keratitis were identified from microbiological requests over a 16-year period between 1995 and 2010. Recurrences and number of surgical interventions were analyzed according to bacterial type. RESULTS: A total of 2418 patients were included, of whom 2124 (87.84%) had only one episode of keratitis, 294 (12.15%) at least two, 88 (3.63%) at least three, 40 (1.65%) at least four, and 22 (0.91%) five or more episodes. The bacterial isolation rate was 35.74% (SD 9.41%), increasing to 56.01% in patients with two or more episodes. There was an increase in the isolation of Staphylococcus aureus with increasing number of episodes (P = 0.008), and S. aureus occurred more commonly in patients with recurrent disease due to the same bacterial group (P = 0.04). Patients whose recurrent keratitis was associated with S. aureus had a higher rate of requiring subsequent corneal transplantation (7 of 10) compared to those with Enterobacteriaceae (2 of 7), Pseudomonas aeruginosa (2 of 4), streptococci (2 of 5), or coagulase-negative staphylococci (none of 8) (P = 0.02). CONCLUSIONS: S. aureus is particularly associated with recurrent keratitis. Identification and treatment of the possible source of the infection may be necessary to reduce the risk of recurrent disease. The potential for the autocthonous S. aureus colonizing the nasopharynx or conjunctiva or lid margin to be a reservoir for recurrent keratitis suggests that decolonization of S. aureus could be considered as a potential intervention in those patients with recurrent disease.

lukSF-PV in Staphylococcus aureus keratitis isolates and association with clinical outcome.

PURPOSE: To determine the prevalence, genetic diversity, and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton-Valentine leukocidin, in Staphylococcus aureus isolates from cases of bacterial keratitis in the United Kingdom. METHODS: Multiplex PCRs investigating carriage of lukSF-PV and mecA were performed on S. aureus isolates from patients. The lukSF-PV operon was sequenced to investigate its diversity, and multilocus sequence typing to test for a clonal relationship between lukSF-PV isolates. Antimicrobial minimum inhibitory concentrations (MICs) and clinical outcome data were compared for isolates characterized as lukSF-PV+ve, mecA+ve, and lukSF-PV/mecA-ve. RESULTS: Of 95 isolates, 9 (9.5%) were lukSF-PV+ve, 9 (9.5%) mecA+ve, and 1 was positive for both. Five single nucleotide polymorphisms were found in lukSF-PV genes of seven strains. There was no significant difference between the MICs of lukSF-PV/mecA-ve and lukSF-PV+ve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecA+ve isolates had significantly higher mean MICs to meropenem and fluoroquinolones (P < 0.05). There were nonsignificant trends for healing and treatment times, ulcer and scar size, and overall clinical score to be greater in the lukSF-PV+ve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PV+ve isolates with an odds ratio of 7.8 (95% CI 1-42, P = 0.018) for patients requiring surgery. CONCLUSIONS: lukSF-PV+ve isolates were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus-associated keratitis.

Quantifying changes in corneal neovascularization using fluorescein and indocyanine green angiography.

PURPOSE: To quantify changes in corneal neovascularization in patients with active keratitis after treatment using color imaging, fluorescein angiography (FA), and indocyanine green angiography (ICGA). DESIGN: Prospective, interventional case series. METHODS: Twelve consecutive patients were studied. A comparison of corneal neovascularization parameters was undertaken before and after resolution of the keratitis. A slit-lamp digital camera acquired images of the neovascularization using color imaging, FA, and ICGA. The best-quality images were selected using a grading system, and the neovascular regions of interest were analyzed using automated in-house software. The parameters of analysis were vessel area, diameter, tortuosity, and FA dye leakage. RESULTS: There was a significant reduction in the area of neovascularization after treatment on color imaging (0.78 mm(2); P < .05), FA (2.33 mm(2); P < .01), and ICGA (2.07 mm(2); P < .01). There was also a significant reduction in mean vessel diameter across the region of interest for each patient, more marked on FA (42.74 to 32.52 µm; P < .01) and ICGA (44.77 to 33.29 µm; P < .01) than on color imaging (29.10 to 25.17 µm; P < .01). A significant change in vessel tortuosity was not observed. There was a significant increase in FA dye leakage time (12.41 seconds; P < .05) after treatment. CONCLUSIONS: We demonstrate application of an objective method for analyzing changes in corneal neovascularization. The excellent vessel delineation with ICGA even in the presence of stromal scars makes it an ideal agent for measurement of vessel parameters. FA is useful at detecting vessel leakage, and the time to leakage provides a possible measure of vessel staging.

Genotypic analysis of UK keratitis-associated Pseudomonas aeruginosa suggests adaptation to environmental water as a key component in the development of eye infections.

To examine temporal dynamics of corneal infection (keratitis)-associated Pseudomonas aeruginosa, we compared the genetic characteristics of isolates collected during two different time periods (2003-2004 and 2009-2010) using an ArrayTube genotyping system. The distribution of keratitis-associated isolates from the two studies (n = 123) among a database of P. aeruginosa strains of non-ocular origin (n = 322) indicated that 71% of UK keratitis-associated P. aeruginosa isolates clustered together, and there was no evidence for major variations in the distribution of clone types between the two collections. Our analysis indicates the presence of a 'core keratitis cluster', associated with corneal infections, that is related to the P. aeruginosa eccB clonal complex, which is associated with adaptation to survival in environmental water. This suggests that adaptation to environmental water is a key factor in the ability of P. aeruginosa to cause eye infections.

Imaging and evaluation of corneal vascularization using fluorescein and indocyanine green angiography.

PURPOSE: To evaluate indocyanine green angiography (ICGA) and fluorescein angiography (FA) in imaging and quantifying corneal neovascularization (CNV). METHODS: Patients with CNV were studied using a standardized protocol of color digital photography, FA, and ICGA. Images were graded independently by two observers and assessed for quality, phases of fluorescence, and leakage. Areas of CNV and vasculature geometric properties were analyzed and quantified by an automated program. RESULTS: Twenty-three patients with good quality images were included. Mean times to appearance of ICG and fluorescein were 17 and 20 seconds (P = 0.10). Best images for analysis were obtained at 64 seconds for ICGA and 47 seconds for FA. CNV not apparent on color or FA, particularly in the presence of scarring, was well delineated by ICGA. Leakage of ICGA did not occur. Fluorescein leakage from apical CNV images occurred significantly earlier (32 seconds) in patients with CNV of <6-month duration than those of >1-year (50 seconds) duration (P = 0.04). Mean area of CNV and vessel diameter were similar with ICGA (8.79 mm(2), 0.058 mm) or FA (7.74 mm(2), 0.054 mm) but significantly larger than on color (1.94 mm(2), 0.026 mm) images (P < 0.01). Vessel tortuosity was similar on ICGA (1.16), FA (1.17), and color (1.15) (P = 0.27). CONCLUSIONS: Combined use of FA and ICGA are valuable tools with which to assess CNV and provide better vessel delineation than can be obtained with only color images. Parameters used to assess CNV, such as leakage, area, diameter, and tortuosity, may be useful measures for evaluating treatment. Videography is useful for detecting early leakage.

Comparison of Damato campimetry and Humphrey automated perimetry results in a clinical population.

BACKGROUND/AIMS: The purpose of the study was to evaluate patients' ease of understanding of Damato campimetry assessment and to determine the sensitivity and specificity of the results compared with Humphrey automated perimetry. METHODS: Patients underwent Humphrey perimetry and Damato campimetry on the same day. Patients were excluded if they were unable to undergo Humphrey perimetry. Results were graded as matched, partially matched and not matched with those of Humphrey perimetry. RESULTS: One hundred patients (197 eyes) were assessed: 62 women and 38 men with mean age of 62.8 (SD 15.98) years. It was not possible to plot Damato campimetry in 19 eyes (6.5%): 13 due to lack of understanding and six due to low vision. In total 178 eyes were tested with both methods. Results showed 94 eyes as true positives, 45 as true negatives, 22 as false negatives and 17 as false positives. Ninety-five eyes had matched visual field results, five eyes had partial matches and 78 eyes (36%) were not matched. The extent of agreement was 0.216 (95% CI 0.073 to 0.36) with kappa analysis. CONCLUSIONS: We found Damato campimetry to be a useful portable device to assess the visual field, with an optimal sensitivity of 81% and a specificity of 72% based on comparison with a Humphrey 24-2 programme. Of the patients studied, 6.5% were unable to do the test and 64% had matched or partially matched results from both assessments. Further study is required to compare complete results with a Humphrey 30-2 programme and also to study populations where patients do not have access to outpatient formal visual field assessments.

An in vitro investigation of synergy or antagonism between antimicrobial combinations against isolates from bacterial keratitis.

PURPOSE: To investigate antimicrobial combinations for synergy or antagonism against isolates of Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: Isolates were collected from cases of microbial keratitis from six centers in the United Kingdom. Minimum inhibitory concentrations (MICs) were determined by using E-test strips for 16 antimicrobials, including both current and potentially available agents. E-test strips were used to test selected antimicrobials in combination against a representative set of 10 S. aureus and 10 P. aeruginosa isolates. E-tests of the two antimicrobials were placed on sensitivity agar at right angles intersecting at their respective MICs. Antimicrobial combinations were classified as synergistic, additive, indifferent, or antagonistic, according to their fractional inhibitory concentration (FIC). RESULTS: The combinations meropenem and ciprofloxacin, meropenem and teicoplanin, moxifloxacin and teicoplanin, and ciprofloxacin and teicoplanin, gave the lowest mean FICs for S. aureus, with synergy or additivity being seen in 60% to 80% of isolates. The meropenem/ciprofloxacin combination gave the lowest mean FIC for P. aeruginosa isolates, with 90% showing an additive or synergistic effect. The other combinations elicited a predominantly indifferent response. No consistent antagonistic effect was observed with the combinations used. CONCLUSIONS: The combination of meropenem and ciprofloxacin was predominantly additive or synergistic for both S. aureus and P. aeruginosa. Teicoplanin combined with meropenem, ciprofloxacin, or moxifloxacin was also predominantly additive or synergistic against S. aureus.

Minimum inhibitory concentrations of standard and novel antimicrobials for isolates from bacterial keratitis.

PURPOSE: To determine the minimum inhibitory concentrations (MICs) of 12 antimicrobials in current ophthalmic use and 4 potentially new alternatives against isolates from bacterial keratitis. METHODS: Bacteria were collected from cases of bacterial keratitis in six centers in the United Kingdom between 2003 and 2006. MICs were measured by using susceptibility strips containing a concentration gradient of the antimicrobials penicillin, cefuroxime, ceftazidime, chloramphenicol, gentamicin, amikacin, vancomycin, teicoplanin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, meropenem, linezolid, tigecycline, and daptomycin. RESULTS: Isolates (n = 772) were collected including coagulase negative Staphylococcus (CNS) (30%), Pseudomonas aeruginosa (23%), Staphylococcus aureus (14%), Enterobacteriaceae (14%), and streptococci (13%). Meropenem had low MICs for most isolates. All isolates except P. aeruginosa were susceptible to tigecycline. Linezolid was active against the majority of Gram-positive pathogens. Ten percent of S. aureus and 20% of CNS isolates were methicillin resistant. When systemic breakpoints were used, 84% of S. aureus isolates were susceptible to ciprofloxacin and 98% to moxifloxacin. Of the P. aeruginosa isolates, 99% were susceptible to ceftazidime, 96% to gentamicin, 99% to ciprofloxacin and 100% to moxifloxacin. More than 97% of Enterobacteriaceae isolates were susceptible to ceftazidime, gentamicin, ciprofloxacin, and moxifloxacin. CONCLUSIONS: Based on systemic breakpoint data, resistance to commonly used antimicrobials was apparent. Meropenem is a potentially effective agent for ophthalmic use, with low MICs throughout all the bacterial subgroups. Tigecycline and linezolid showed good activity against particular groups and may be useful for treating bacterial keratitis resistant to current antimicrobials. Of the fluoroquinolones, moxifloxacin showed the lowest MICs and resistance for both Gram-positive and -negative bacteria.