MD Alfredo Pece

MD Vincenzo Isola

MD Stefano Piermarocchi

MD Giliola Calori

Efficacy and safety of anti-VEGF therapy with intravitreal ranibizumab (LUCENTIS™) for naïve retinal vein occlusion: one-year follow-up

Efficacy and safety of anti-VEGF therapy with intravitreal ranibizumab (LUCENTIS™) for naïve retinal vein occlusion: one-year follow-up

Alfredo Pece, Vincenzo Isola, Stefano Piermarocchi, Giliola

Introduction

Branch and central retinal vein occlusions (RVO) are among the most common retinal vascular diseases, and are often associated with moderate to severe vision loss. [START_REF] Hayreh | Classification of central retinal vein occlusion[END_REF] More than 90% of RVO occur in patients older than 50 years, but they are reported in all age groups. [START_REF] Klein | The epidemiology of retinal vein occlusion: The Beaver Dam Eye Study[END_REF][START_REF] Klein | The 15-Year cumulative incidence of retinal vein occlusion. The Beaver Dam Eye Study[END_REF] Central retinal vein occlusion (CRVO) results from thrombosis in the central retinal vein within the optic nerve at variable distances posterior to the lamina cribrosa, with fundus hemorrhage involving the entire retina with edema, dilated retinal vein and disc swelling. [START_REF] Hayreh | Classification of central retinal vein occlusion[END_REF][START_REF] Klein | The epidemiology of retinal vein occlusion: The Beaver Dam Eye Study[END_REF][START_REF] Klein | The 15-Year cumulative incidence of retinal vein occlusion. The Beaver Dam Eye Study[END_REF][START_REF] Hayreh | Venous occlusive disease: management 25 years ago[END_REF] Branch retinal vein occlusion (BRVO) occurs at more distal sites and is caused by focal occlusion of a retinal vein at an arteriovenous crossing-point where compression of the vein by the artery passing anteriorly produces turbulence and thrombosis with hemorrhage and edema that usually involve the superior or inferior temporal retinal vein. [START_REF] Zhao | Eye Disease Case-Control Study Group. Arteriovenous crossing patterns in branch retinal vein occlusion[END_REF][START_REF] Duker | Anterior location of the crossing artery in branch retinal vein obstruction[END_REF][START_REF] Christoffersen | Pathophysiology and hemodynamics of branch retinal vein occlusion[END_REF] The exact pathogenesis of the occlusion is poorly understood but various local and systemic factors appear to play a role in the pathological closure of the retinal veins. [START_REF] Mcgrath | Systemic factors contributory to retinal vein occlusion[END_REF][START_REF] Cheung | Traditional and novel cardiovascular risk factors for retinal vein occlusion: The Multiethnic Study of Atherosclerosis[END_REF][START_REF]The Eye Disease Case-Control Study Group. Risk Factors for Central Retinal Vein Occlusion[END_REF][START_REF]Eye Disease Case-control Study Group. Risk factors for branch retinal vein occlusion[END_REF][START_REF] Hyreh | Intraocular pressure abnormalities associated with central and hemicentral retinal vein occlusion[END_REF][START_REF] Vine | Hyperhomocysteinemia: a new risk factor for central retinal vein occlusion[END_REF] The most common risk factors are the same as for cardiovascular disease, including systemic hypertension, atherosclerotic vascular changes, diabetes mellitus, and age over 65. [START_REF] Klein | The 15-Year cumulative incidence of retinal vein occlusion. The Beaver Dam Eye Study[END_REF][START_REF] Mcgrath | Systemic factors contributory to retinal vein occlusion[END_REF][START_REF] Cheung | Traditional and novel cardiovascular risk factors for retinal vein occlusion: The Multiethnic Study of Atherosclerosis[END_REF][START_REF]The Eye Disease Case-Control Study Group. Risk Factors for Central Retinal Vein Occlusion[END_REF][START_REF]Eye Disease Case-control Study Group. Risk factors for branch retinal vein occlusion[END_REF] Open-angle glaucoma is also considered a major risk factor and in recent years hyperhomocysteinemia and hypercoagulable risk factors have been increasingly implicated. [START_REF] Hyreh | Intraocular pressure abnormalities associated with central and hemicentral retinal vein occlusion[END_REF][START_REF] Vine | Hyperhomocysteinemia: a new risk factor for central retinal vein occlusion[END_REF] Visual acuity can be lost due to retinal ischemia or cystoid macular edema caused by capillary leakage, with endothelial cell damage combined with increased intracapillary pressure promoting exudation of extracellular proteins, intraretinal fluid, blood, and precipitation of lipids in the macula. The pathogenesis of macular edema is generally related to up-regulation of vascular endothelial growth factor A (VEGF-A) that may act on capillary hyperpermeability by increasing vesicular transport and/or lowering the content of tight-junction proteins among the endothelial cells. [START_REF] Pe'er | Vascular endothelial growth factor upregulation in human central retinal vein occlusion[END_REF][START_REF] Noma | Pathogenesis of macular edema with branch retinal vein occlusion and intraocular levels of vascular endothelial growth factor and interleukin-6[END_REF][START_REF] Ferrara | Vascular endothelial growth factor: Basic science and clinical progress[END_REF] Therefore it seems rational to inhibit VEGF-A with the intent of normalizing the inner blood-retinal barrier, reducing vascular leakage and macular edema, and improving visual function. Short-term clinical trials have shown the effectiveness and safety of intravitreal anti-VEGF-A therapy for treating CRVO. Validation of VEGF-A targeting for therapeutic intervention in CRVO and BRVO comes from clinical studies involving two different intravitreal anti-VEGF-A drugs: bevacizumab (Avastin, Genentech/Roche, Basel, Switzerland,) and pegaptanib sodium (Macugen, OSI-Eyetech Pharmaceuticals, New York, NY). [START_REF] Iturralde | Intravitreal bevacizumab (Avastin) treatment of macular edema in central retinal vein occlusion: a short-term study[END_REF][START_REF] Costa | Intravitreal bevacizumab (Avastin) for central and hemicentral retinal vein occlusions:the IBeVO study[END_REF][START_REF] Rabena | Intravitreal bevacizumab (Avastin) in the treatment of macular edema secondary to branch retinal vein occlusion[END_REF][START_REF] Pai | Clinical, anatomic, and electrophysiologic evaluation following intravitreal bevacizumab for macular edema in retinal vein occlusion[END_REF][START_REF] Ferrara | Early bevacizumab treatment of central retinal vein occlusion[END_REF][START_REF] Wu | Comparison of two doses of intravitreal bevacizumab (Avastin) for treatment of macular edema secondary to branch retinal vein occlusion: results from the Pan-American Collaborative Retina Study Group at 6 months of follow-up[END_REF][START_REF] Prager | Intravitreal bevacizumab (Avastin) for macular oedema secondary to retinal vein occlusion: 12-month results of a prospective clinical trial[END_REF][START_REF] Wroblewski | for the Pegaptanib in CRVO Study Group. Pegaptanib sodium for macular edema secondary to central retinal vein occlusion[END_REF] Ranibizumab (Lucentis, Genentech, Inc, South San Francisco, CA) is a FDA-approved anti-VEGF-A monoclonal antibody designed specifically to treat exudative age-related macular degeneration (AMD). [START_REF] Rosenfeld | Ranibizumab for neovascular agerelated macular degeneration. MARINA Study Group[END_REF] Two large clinical trials are currently in progress to investigate the efficacy and safety of ranibizumab injections in patients with macular edema secondary to CRVO (CRUISE) or BRVO (BRAVO). [START_REF] Genentech | A study of the efficacy and safety of ranibizumab injection in patients with macular edema secondary to central retinal vein occlusion (CRUISE)[END_REF][START_REF] Genentech | A study of the efficacy and safety of ranibizumab injection in patients with macular edema secondary to branch retinal vein occlusion[END_REF] The purpose of the present study was to compare visual acuity, reading performance and contrast sensitivity in patients assigned to receive intravitreal injections of ranibizumab for non-ischemic CRVO or BRVO with macular edema.

Materials and Methods

We followed in a prospective open-label study 17 eyes of 17 consecutive patients with RVO who were treated with intravitreal injection of 0.5 mg of ranibizumab. The outcome of this study was to determine the efficacy and safety of ranibizumab in the treatment of macular edema associated with RVO by investigating the following variables:

Subject Eligibility and Exclusion Criteria

To be eligible, the following inclusion criteria must be met: age ≥ 55 years; diagnosis of macular edema secondary to CRVO or BRVO at least 3 months from the time of the occlusive event; best corrected ETDRS visual acuity measurement from 20/50 to 20/400; baseline central foveal thickness 250 µm or greater using optical coherence tomography (OCT-3 Stratus; Carl Zeiss, Dublin, CA).

Exclusion criteria included ocular diseases other than CRVO and BRVO related macular edema such as age-related macular degeneration, diabetic retinopathy, uveitis, macular pucker, intraocular surgery within the past 3 months, dense cataract, glaucoma, evidence of neovascularization involving iris, retina or optic disc; brisk relative afferent pupillary defect predictive of non perfused CRVO; previous laser (grid laser or panretinal photocoagulation) within the past 3 months; anti-VEGF treatment within the past 3 months or intravitreal or periocular steroid administration in the study eye that could interfere with the treatment result; evidence of external ocular infection including blepharitis, conjunctivitis, or chalazion.

Patients were scheduled to be evaluated with retinal biomicroscopy, color fundus photography, fluorescein angiography, and OCT at baseline, at 1 week and 1, 2, 3, 6, 9 and 12 months.

The patients received an initial injection on day 0 and were then retreated as needed (pro re nata (PRN)). All patients were also examined with anterior and posterior slit-lamp biomicroscopy, Goldmann applanation tonometry, the day after each injection and were asked if they had decreased vision, eye pain, or any new symptoms.

After obtaining written informed consent, off-label anti-VEGF therapy was administered in the operating room observing a sterile protocol for intravitreal injection that included the use of 5% povidone-iodine solution, topical anesthesia, eyelid-speculum application, intravitreal injection of 0.5 mg of ranibizumab via pars plana in the infero-temporal quadrant at 4 mm from the limbus in the phakic eye and at 3.5 mm in the pseudophakic eye followed by postoperative topical antibiotic eye-drops medication. Patients were given topical netilmicin + desametason four times daily for two weeks after injection.

Retreatment criteria

Repeated intravitreal injection was performed on the basis of persistence or increase in central macular edema as determined by OCT (CMT 250 µm or greater) or decreased vision when compared to last visit; patients who were eligible for re-treatment were evaluated the day after intravitreal re-injection, and then as the protocol.

Statistical analysis

Data are shown as number (%) for categorical variables or as mean and standard deviation (SD) for continuous variables. To investigate the trend of visual function with time, one way analysis of variance (ANOVA) for repeated measures was used. When it turned out to be statistically significant the comparison of each time point with the baseline was tested, adjusting by Bonferroni's procedure. In order to investigate the association of changes among BCVA scores (distance and near vision), contrast sensitivity and CMT, a correlation analysis was run and Pearson correlation coefficient was calculated. The correlation between the maximum peak of BCVA and the CMT at the same time was also investigated. The p-value has been set at 0.05. Statistical analyses were performed by SAS v8 software (SAS Institute Inc, Cary, NC-USA).

Procedure

Testing were conducted at the Author's Institution by one vision examiner who performed all visual acuity and contrast sensitivity testing of participants. Written informed consent was obtained from all patients before the beginning of the examination session, and our study complied with the tenets of Helsinki Declaration.

Distance Visual Acuity Testing

Each patient's best corrected VA was tested with the ETDRS charts (ETDRS Illuminator Cabinets,™ Precision Vision, La Salle, IL, USA). Initially, each patient was asked to identify 10 large, individual ETDRS letters that were held by the vision examiner at a distance of 1.5 m. The distance VA of the study eye was tested at the standard distance of 4 m, using 1 of the 3 ETDRS charts. Patients were asked to identify the first 3 letters on each line. If the patient incorrectly identified 1 or more of the letters, he or she was asked to identify the remaining letters on that line.

VA was recorded as the Snellen equivalent, logMAR value, and letter score of the last line on which the patients identified 3 or more letters correctly.

Near Visual Acuity Testing

The MNREAD acuity charts (Lighthouse Low Vision Products, Long Island City, NY, USA) were used to measure reading vision. Reading acuity was determined by the smallest print size at which the patient can read an entire sentence without making significant errors. For this study, reading acuity was measured at 25-cm distance with the patient wearing an additional +4.00 DS over his distance correction and was recorded in logMAR units using the formula provided by the manufacturer: acuity = 1.4 -(sentences x 0.1) + (errors x 0.01), in which sentences are the number of sentences read correctly and errors are the number of words read incorrectly. The calculated value was adjusted for the viewing distance by adding to the measured acuity a +0.204 logMAR correction.

Contrast Sensitivity Testing

Contrast sensitivity of each patient's affected eye was measured using the commercially available Pelli-Robson contrast sensitivity chart (Clement Clark Inc, Columbus, Ohio,USA). Test distance was 1 meter, and the CS chart was front-illuminated uniformly to a luminance of 64 cd/m2 or more.

Patients were asked to make a single attempt to identify each letter on the chart, starting with discriminations of high contrast letters in the upper left corner of the chart and reading across each line. Patients were asked to guess even when they indicated that the letters were invisible. CS was recorded by the log contrast sensitivity value (log 10 1/contrast of letters at the threshold of visibility) of the last triplet on which the subject identified at least 2 of the 3 letters correctly, and also by the total number of letters identified by the patients.

Photographic Documentation Color Fundus Photography and Fluorescein Angiography

The clinical appearance of RVO was documented with color fundus photography and retinal angiography (FA) for all patients at baseline and again at 1 month, 3 months, 6-9 months and 1 year to evaluate any changes in the aspect of the retina treated with intravitreal injection of ranibizumab.

Color photography of the disc and macula of each affected eye were performed with the Topcon ImageNet Digital Imaging System (Paramus, NJ). Intravenous fluorescein angiography was performed with a confocal scanning laser ophthalmoscope (HRA, Heidelberg Retinal Angiograph, Heidelberg Enginering, Carlsbad, CA). Single angiograms were obtained in the early, middle and late phase between 2 and 5 minutes after injection to document fluorescein leakage from arteries, veins, capillary vessel walls and optic disc as well as pooling of dye in the center of the macula.

Angiography was also the gold standard to distinct about retinal capillary perfusion status, and whether the vascular occlusion was ischemic when 10 or more disc areas of hypoperfusion were detectable.

Optical Coherence Tomography

Third-generation OCT examination (Stratus Tomographer, Model 3000, Carl Zeiss Ophthalmic System Inc, Humphrey Division, Dublin, CA) was performed in all patients to measure the central macular thickness (CMT) at baseline and at each follow-up visit on the first week, and then at month 1, 2, 3, 6, 9, 12 after treatment. Patients who needed to be reinjected were examined again with OCT following the same time-point described above. The OCT map was created from six consecutive linear 6 mm scans oriented at intervals of 30° and centered on the foveal region. The fundus image generated by OCT machine during the procedure was used to center the scan at the fovea for each examination.

Results

Seventeen eyes of 17 consecutive patients were included in this cohort study. Nine patients had a CRVO and 8 patients had a BRVO. All patients completed the 1-year follow-up. Demographic and clinical characteristics, including age, gender, duration of occlusion, and associated medical conditions are displayed in Table 1.

Central Retinal Vein Occlusion (CRVO) Distance Visual Acuity

Patients with CRVO had a mean BCVA before treatment of 20/240 (1.09 ± 0.25 logMAR) and a final BCVA of 20/46 (0.36 ± 0.16 logMAR) that was significantly improved at the end of follow-up (p <.0001). The mean BCVA changes from baseline in logMAR unit and ETRDS letters scores are summarized in Figure 1 and Table 2, respectively. VA improvement was highly significant at 1 week, and 1, 2 months after the first injection. At 3, 6 and 9 months postoperatively, the mean values of BCVA in logMAR and ETDRS letter scores declined with a regression of the increase in mean logMAR BCVA and ETDRS letters. Therefore, the most important finding of this analysis was a statistical significant improvement in logMAR BCVA and ETDRS letter scores 1 week after each ranibizumab injection at 3, 6, and 9 months of follow-up. At 12 months, eyes showed a maintaining of BCVA improvement with a mean of 36.7 letters and a gain of 6.4 lines. The mean BCVA compared to baseline, improved more than 3 lines for 90% (8 eyes), remained stable for 10% (1 eye), and did not worse in any eye.

Eyes received an average number of 3 intravitreal injections (range: 2 to 4).

Near Visual Acuity

Patients with CRVO had a mean in MNREAD time ± SD (seconds) and reading fluency ± SD (words/per/minute: wpm) before treatment of 8. 3. After 1 week, the mean in MNREAD time ± SD improved significantly to 3.8 ± 0.9 seconds (p < .0001) and to 150.1 ± 29.51 wpm (p < .0001), whereas no significant (p>.05) change was observed at month 1, 2 and 3.

Therefore, the analysis indicated that a statistical significant improvement in MNREAD time and reading fluency was recorded 1 week after each ranibizumab injection at 3, 6, and 9 months of follow-up. At 12 months, the efficacy of treatment over near visual acuity was still statistically significant.

Contrast Sensitivity

Patients with CRVO had a mean log contrast sensitivity (CS) value ± SD and number of Pelli-Robson letters ± SD before treatment of 0.55 ± 0.28 and 14.3 ± 8 with a final mean log CS of 1.23 ± 0.14 (p < .0002) and 28.3 ± 2.95 letters (p < .0001) that were significantly improved at the end of follow-up. Contrast sensitivity results are given in Figure 3 for log CS and in Table 4 for number of Pelli-Robson letters correctly identified. After 1 week, the mean log CS ± SD significantly improved to 1.01 ± 0.27 log (p < .0001) and to 23.1 ± 5.41 letters (p < .0001). The efficacy of the first injection was reported between month 1 and 2 of follow-up. Three and six months postoperatively, some regression of the increase in mean log CS and Pelli-Robson letter score was recorded. Therefore, the analysis indicated that a statistical significant improvement in mean log CS value and number of Pelli-Robson letters was recorded 1 week after each ranibizumab injection at 3, 6 months of follow-up. The result of the line graph indicated that the efficacy of treatment was maintained statistically (p < .0001) significant between month 6+1week and month 12 for log CS and number of Pelli-Robson letters.

Optical Coherence Tomography (OCT)

All of the 9 eyes had a significant decrease in the mean of CMT compared to baseline.

Changes in CMT are summarized in Figure 4 and Table 5. Statistically significant reduction in CMT was evident 1 week after injection thus eliminating 52.9% of macular edema after the first injection, 60.4% after the second injection (3 month+1wk), 60.5% after the third injection (6 month+1wk), 61.1% after the fourth injection (9 month+1wk). However, the correlations between the CMT changes and the logMAR BCVA were not statistically significant (Pearson correlation: г = -0.29; p > 0.45). The improvements in BCVA, reading fluency, contrast sensitivity, and CMT recorded until two months after each injection were gradually decreased overtime during the following 2 months after each treatment owing to recurrent macular edema at month 3, 6 and 9.

Branch Retinal Vein Occlusion (BRVO) Distance Visual Acuity

Patients with BRVO had a mean BCVA before treatment of 20/126 (0.80 ± 0.29 logMAR) and a final BCVA of 20/50 (0.41 ± 0.23 logMAR) that was significantly better that the acuity at baseline (p < .0001). The mean BCVA changes from baseline in logMAR unit are summarized in Figure 1 and ETRDS letters scores are illustrated in Table 6. After 1 week, mean BCVA ± SD improved to 0.54 ± 0.20 logMAR (p < .0001) and to 58.5 ± 10.9 ETDRS letters (p < .0001). After 1, 2 and 3 months, some regression of the increase in mean BCVA and ETDRS letters was recorded. The most important findings we observed was a statistically significant improvement in logMAR BCVA and ETDRS letter score 1 week after each injection at month 6, and 9 of followup, and this gain was maintained at 12 months. Moreover, after 1-year of treatment the mean BCVA significantly improved of 25.3 ETDRS letters. Comparisons between month 12 and baseline for mean BCVA were considered indicative for improvement of more than 3 lines for 63% (5 eyes), unchanged for 37% (3), and worsened for any eye.

Eyes received a mean number of 3.6 injections of ranibizumab (range: 3 to 4).

Near Visual Acuity

Patients with BRVO had a mean in MNREAD time ± SD (seconds) and reading fluency ± SD (words/per/minute: wpm) before treatment of 7.7 ± 3.01 seconds and 82.25 ± 17.27 wpm with a final mean in MNREAD time of 4 ± 0.8 seconds (p< .006) and in reading fluency of 154.2 ± 34.12 wpm (p < .0002) that were significantly improved at the end of follow-up. The mean changes in MNREAD time and reading fluency from baseline are summarized in Figure 2 andTable 7. After 1 week, the mean in MNREAD time ± SD and reading fluency ± SD was statistically significant improved to 5.4 ± 1.7 seconds (p < .0001) and to 120.4 ± 37.8 wpm (p < .0001). After the initial improvement, the results indicated some regression of the increase in mean MNREAD time and reading fluency between month 1 and month 3. Therefore, the most important finding of this analysis was a statistical significant improvement in near visual acuity 1 week after each ranibizumab injection at 3, 6, and 9 months of follow-up. This gain was maintained at 12 months, with a highly significant improvement of MNREAD time and reading fluency.

Contrast Sensitivity

Patients with BRVO had a mean log contrast sensitivity (CS) value ± SD and number of letters ± SD before treatment of 0.86 ± 0.34 and 19.8 ± 9.39 with a final mean log CS of 1.24 ± 0.10 (p < .0002) and 27.9 ± 2.3 letters (p < .0001) that were significantly improved at the end of follow-up. Contrast sensitivity results are given in the graph illustrated in Figure 3 for log CS and in Table 8 for number of Pelli-Robson letters correctly identified. After 4 intravitreal injections occurred at time 0, month 3, month 6, month 9, the eyes showed some benefit in the mean of log CS value and number of Pelli-Robson letters that was highly significant at 9 months+1week, whereas no significant (p>.05) change was found between baseline and month 9.

Optical Coherence Tomography (OCT)

All of the 8 eyes had a significant reduction in the mean of CMT after every single injection compared to baseline. Changes in CMT are summarized in Figure 4 and percentage of eliminated macular edema were shown in Table 9. Statistically significant reduction in CMT was observed 1 week after the first injection and subsequent injections compared to pre-injection values thus eliminating 42.5% of macular edema after the first injection, 50.7% after the second injection (3 month+1wk), 54.1% after the third injection (6 month+1wk), 62.7% after the fourth injection (9 month+1wk).

Highly significant reductions of CMT when compared with baseline at all time points were recorded. However, the correlations between the CMT changes and the logMAR BCVA were not statistically significant (Pearson correlation: г = -0.41; p > .66). The improvements in BCVA, reading fluency, contrast sensitivity, and CMT recorded until two months after each injection were gradually decreased overtime during the following 2 months after each treatment owing to recurrent macular edema at month 3, 6 and 9.

Color Fundus Photography and Fluorescein Angiography

At baseline CRVO showed extensive haemorrhages in all four quadrants, macular thickening, very few or no cotton wool spots, dilated and tortuous veins, and optic disc edema. BRVO presented the same aspects but only in one quadrant with apex of hemorrhage at an arteriovenous crossing. In all eyes a gradual decrease of intraretinal blood and optic disc swelling was observed 3 months after the first ranibizumab injection, with a complete disappearance at 6-9 months after the second and third intravitreally ranibizumab injection respectively, for both CRVO (Figure 5) and BRVO (Figure 6). Consistent with a decrease in CMT as seen by OCT, fluorescein angiography showed a reduction of leakage with re-establishment in the inner blood-retinal barrier at 1 week, month 1 and 2 after the first injection, and at every three monthly + 1 week after retreatment. However, a recurrence of leakage at month 3, 6, and 9 after treatment was observed in all patients.

Safety Reporting

After 1-year of follow-up, no serious adverse ocular and systemic side-effect was reported from any of the patients in the study during the entire period of treatment; there were no IOP elevation, inflammatory reaction, endophthalmitis, retinal detachment, retinal tear, vitreous hemorrhage, traumatic cataract, cardiovascular or cerebrovascular thromboembolic events. No patient developed neovascularization involving the iris, retina and optic disc. The most common side effects were a subconjunctival hemorrhage that was observed in 6 eyes, and floaters in 3.

Discussion

The management of macular edema due to RVO has been largely dependent on the results of two large randomized collaborative prospective clinical trials by CRVO and BRVO study groups. [START_REF] Retinal | Evaluation of grid pattern photocoagulation for macular edema in central retinal vein occlusion: The Central Vein Occlusion Study Group M Report[END_REF][START_REF] Vein | Argon laser photocoagulation for macular edema in branch vein occlusion[END_REF] The CRVO Study 29 failed to show any benefit from macular grid laser for patients with CRVO. Although there was a significant reduction in the severity of macular edema in treated eyes compared to controls there was no visual acuity benefit, although a trend in favor of treatment was observed in younger patients.

In contrast, laser treatment is effective for BRVO. The BRVO Study [START_REF] Vein | Argon laser photocoagulation for macular edema in branch vein occlusion[END_REF] showed that laser photocoagulation improved visual acuity in patients whose vision was 20/40 or worse because of macular edema; however, patients with vision worse than 20/200 did not do significantly better than with observation alone. Some surgical procedures are also being tested: radial optic neurotomy and laserinduced chorioretinal anastomosis have been studied for CRVO. [START_REF] Opremcak | Radial optic neurotomy for central retinal vein occlusion: a retrospective pilot study of 11 consecutive cases[END_REF][START_REF] Fermat | Laser-induced chorioretinal anastomosis for nonischemic central or branch retinal vein occlusion[END_REF] Currently, there are no randomized clinical investigations that show these treatment modalities reproducibly and reliably improve BCVA. [START_REF] Hayreh | Radial optic neurotomy for central retinal vein occlusion (correspondence)[END_REF] Other promising approaches include off-label intravitreal injection of triamcinolone acetonide (IVTA). Small case series and presentations on its use have increased over the last eight years. An analysis of the literature showed that almost all patients had anatomical improvements, and many gained visual acuity. [START_REF] Ip | Intravitreous triamcinolone acetonide as treatment for macular edema from central retinal vein occlusion[END_REF][START_REF] Park | Intravitreal triamcinolone acetonide in eyes with cystoid macular edema associated with central retinal vein occlusion[END_REF][START_REF] Bashshur | Intravitreal triamcinolone for the management of macular edema due to nonischemic central retinal vein occlusion[END_REF] This strategy seems effective in the short term, but there is the risk of adverse side effects such as the development of lens opacities and ocular hypertension. [START_REF] Jonas | Intravitreal triamcinolone acetonide: a chance in a paradigm[END_REF] Recently have been published the results of the SCORE study (the Standard care versus corticosteroid for retinal vein occlusion). In this multicenter, randomized trial , 271 pt with central vein occlusion and macular edema have been treated with IVTA or only observed. IVTA was superior to observation and the 1-mg dose had a better safety profile. [START_REF] Ip | A randomized trial comparing the efficacy and safety of intravitreal triamcinolone with observation to treat vision loss associated with macular edema secondary to central vein occlusion: the Standard Care vs. Corticosteroid for Retinal Vein Occlusion Study( SCORE) study report 5[END_REF] The SCORE study, report 6, also investigated 411 pt with branch retinal vein occlusion and macular edema, treated with IVTA vs standard care . There was no difference in VA at 12 months in the two group and therefore, considering also the high negative side effects in IVTA group, grid laser photocoagulation should remain the standard therapy in BRVO. [START_REF] Scott | A randomized trial comparing the efficacy and safety of intravitreal triamcinolone with standard care to treat vision loss associated with macular edema secondary to branch vein occlusion: the Standard Care vs. Corticosteroid for Retinal Vein Occlusion Study( SCORE) study report 6[END_REF] Therapies for CRVO and BRVO are still limited, but several new approaches have been described. There is increasing interest in intravitreally-injected anti-VEGF drugs for macular edema due to RVO. In clinical practice, there are now three commercially available VEGF antagonists: pegaptanib sodium (Macugen, OSI/Eyetech, and Pfizer), bevacizumab (Avastin, Genentech), and ranibizumab (Lucentis, Genentech). These are used on an off-label basis, however, and their long-term efficacy and safety are still not known.

Pegaptanib sodium is a modified fragment of ribonucleic acid that inactivates VEGF 165 and is FDA-approved for the treatment of exudative AMD. [START_REF] Singerman | Pegaptanib sodium for neovascular agerelated macular degeneration: third-year safety results of the VEGF Inhibition Study in Ocular Neovascularisation (VISION) trial[END_REF] A recent phase II randomized, double-masked, multicenter trial evaluated the role of pegaptanib in CRVO patients. [START_REF] Wroblewski | for the Pegaptanib in CRVO Study Group. Pegaptanib sodium for macular edema secondary to central retinal vein occlusion[END_REF] Results were encouraging for the efficacy and safety of intravitreal injections of pegaptanib for selective inhibition of VEGF 165 in eyes with recent vision loss due to macular edema secondary to CRVO. Preliminary comparisons of Macugen doses of 0.3 and 1.0 mg as an intravitreal injection every six weeks or sham injections every six weeks for 30 weeks have shown that RVO eyes receiving Macugen gained a mean of ten ETDRS letters as compared to a mean loss of two letters in the eyes receiving sham injections.

Bevacizumab is a full-length, humanized monoclonal antibody directed against all biological isoforms of VEGF. Treatment of CRVO and BRVO with intravitreal bevacizumab achieved significant reductions in vein diameter, tortuosity, optic disc edema, and macular thickening, with some gain in vision. Iturralde et al. ran a small, prospective, non-comparative trial of 16 eyes with CRVO and found a good response to 0.05 mL of intravitreal bevacizumab, with BCVA improvement and decrease in central foveal thickness. [START_REF] Iturralde | Intravitreal bevacizumab (Avastin) treatment of macular edema in central retinal vein occlusion: a short-term study[END_REF] The drug has also shown promising early results and an excellent safety profile in the treatment of macular edema secondary to BRVO. Rebena et al. observed a positive biologic effect on macular edema in 27 eyes with BRVO that received 0.05 mL intravitreal bevacizumab injections. At three months, 44% of these eyes showed halving of the visual angle from baseline, and after a mean of 5.3 months there was a moderate correlation between decrease in retinal thickness and improvement in visual acuity. [START_REF] Rabena | Intravitreal bevacizumab (Avastin) in the treatment of macular edema secondary to branch retinal vein occlusion[END_REF] In the IbeVO study, Costa et al. examined the effect of 0.08 mL of intravitreal bevacizumab at 12-week intervals in five eyes with CRVO and two with hemi-CRVO: there was a reduction in CMT, with short-term visual acuity stabilization or improvement of three or more ETDRS lines. [START_REF] Costa | Intravitreal bevacizumab (Avastin) for central and hemicentral retinal vein occlusions:the IBeVO study[END_REF] Ranibizumab is a recombinant, humanized, monoclonal antibody antigen-binding fragment (Fab) that binds and neutralizes all the biologically active forms of VEGF. [START_REF] Rosenfeld | Ranibizumab for neovascular agerelated macular degeneration. MARINA Study Group[END_REF] Campochiaro et al. investigated the short-term effects of ranibizumab in eyes with CRVO and BRVO and found that monthly injections of 0.5 mg tended to cause more rapid decreases in central retinal thickening that lasted longer with three-monthly injections. At the final examination, OCT indicated that the excess retinal thickening was reduced by approximately 90% in all eyes. [START_REF] Campochiaro | Ranibizumab for macular edema due to retinal vein occlusions; implication of VEGF as a critical stimulator[END_REF] However, the lack of long-term outcomes did not allow any firm final conclusion on the routine use of ranibizumab for RVO. Pieramici et al. documented visual and anatomical success in ten patients with perfused CRVO treated with ranibizumab monthly for three months, then quarterly as needed. However, the benefit may have statistical significance but only limited clinical impact because improvements in BCVA and OCT-CMT appeared greatest during the initial monthly injection period (0-3 months) and were lost --with the recurrence of macular edema --in between ranibizumab injections during the quarterly treatments (3-9 months). [START_REF] Pieramici | Ranibizumab for the treatment of macular edema associated with perfused central retinal vein occlusions[END_REF] Spaide et al. reported that intravitreal ranibizumab was effective in 16 eyes with nonnaive CRVO, using a loading phase with injections at baseline then at months 1 and 2, followed by monthly clinical examinations to check whether re-treatment was necessary on the basis of persistent macular edema or new retinal hemorrhages. This study evaluated the effect of ranibizumab in eyes that had already been treated with intravitreal triamcinolone (five eyes) and bevacizumab (11 eyes); only four CRVO patients were naïve to intravitreal ranibizumab. All eyes gained in VA as a result of additional injections over one year of follow-up. [START_REF] Spaide | Prospective study of intravitreal ranibizumab as a treatment for decreased visual acuity secondary to central retinal vein occlusion[END_REF] Our prospective study found that after one year intravitreal ranibizumab for macular edema secondary to CRVO was associated with significant improvement in BCVA for 90% of patients, who gained more than three ETDRS lines from baseline (p <0.0001).

All patients showed an immediate response to these injections, with a significant reduction of CMT and an increase in acuity. OCT indicated a gradual decrease in intraretinal fluid and macular cysts. However, the reduction of cystoid edema was not statistically correlated with the change in distance, near vision, or contrast sensitivity.

The BCVA for distance vision, reading ability, and contrast sensitivity seemed to recover transiently in all eyes, especially seven days after intravitreal ranibizumab, and visual benefits were observed during the first two months. This seems to be the interval at which treated individuals report the greatest acuity benefits. However, OCT reveals re-accumulation of macular fluid and a recurrence of edema after the initial improvement in vision. This explains the decrease in VA that patients reported at 3, 6, and 9 months and is considered indicative of the loss of efficacy of the drug, that appears to be unable to maintain high levels of visual performance.

The recurrence of edema might be an indication that the disease is still active and treatment could be needed more often to counter this chronic vascular disorder. In one case, after the initial resolution of edema, there was a more pronounced increase than at the initial presentation. This rebound effect is still not completely clear, and Matsumoto et al. suggested VEGF receptors might be upregulated as a result of the inhibition of the VEGF-A pathway. [START_REF] Matsumoto | Rebound macular edema following bevacizumab (Avastin) therapy for retinal venous occlusive disease[END_REF] The mechanism inducing the gain in visual function in the early stage of ranibizumab treatment is also not entirely clear but we can speculate that changes in ocular circulation, re-establishment of normal foveal anatomy, and restoration of the inner blood-retinal barrier might be involved in the improvements in central vision.

In our series of eyes with BRVO intravitreal ranibizumab showed anatomic and functional effectiveness on macular edema, resulting in significant increases in BCVA, contrast sensitivity and reading ability, with a transient reduction of CMT. There was a significant long-term effect on CMT with a 48.5% elimination of macular edema and an improvement of more than three lines of BCVA in 63% of treated eyes at one year of follow-up. We did not use a loading phase regimen -monthly injections for three months -and considered both functional and anatomic criteria in order to guide treatment and re-injection decisions. This resulted in a smaller number of ranibizumab intravitreal injections in an attempt to maximize the benefit of visual gain and minimize the re-treatment rate. The mean number of injections from baseline was 3 for CRVO and 3.6 for BRVO. Pieramici et al. reported a mean of 4.5 injections through a ninemonth follow-up and Spaide et al. a mean of 8.5 in a one-year prospective study. [START_REF] Pieramici | Ranibizumab for the treatment of macular edema associated with perfused central retinal vein occlusions[END_REF][START_REF] Spaide | Prospective study of intravitreal ranibizumab as a treatment for decreased visual acuity secondary to central retinal vein occlusion[END_REF] This is considerably more than the injections we gave in approximately the same follow-up in our study. However, despite this difference, a similar gain of about three lines of VA, with significant elimination of macular edema, can still be obtained. It can therefore be reasonably assumed that more frequent injections are not necessarily associated with more functional and anatomic benefit than the PRN procedure.

A less-frequent-dose protocol in which ranibizumab was injected monthly for three months then quarterly for the treatment of neovascular AMD was employed in the PIER study (age-related choroidal neovascularization with or without classic CNV secondary to age-related macular degeneration) and provided VA benefit at 12 months. [START_REF] Regillo | Randomized, double-masked, shamcontrolled trial of ranibizumab for age-related macular degeneration: PIER Study year 1[END_REF] However, the two diseases are completely different in their pathogenesis and in visual and clinical outcomes so it is impossible to draw any firm conclusions.

Our results suggest that multiple intravitreal injections of ranibizumab against VEGF-A can result in early recovery of the inner blood-retinal barrier with later stabilization and remodeling of retinal microvessels after retinal venous obstruction. Unlike pegaptanib, ranibizumab binds to and neutralizes the biological activity of all isoforms of VEGF-A proteins and their active metabolic products. However, whether selective VEGF 165 blockade with pegaptanib rather than pan-VEGF inhibition with bevacizumab or ranibizumab is appropriate in RVO patients remains to be established. The finding that short-and long-term blockade of VEGF-A action has no important intraocular adverse effects may argue against it being involved in maintaining endothelial cell permeability and the circulatory changes due to CRVO.

Retinal thickening and visual acuity at 3, 6, 9 and 12 months provided information on the duration of effect. The cumulative beneficial effect resulting from multiple doses was achieved over time. Preliminary data on 17 patients in this study suggest that visual function may improve with time. These encouraging anatomic and visual benefits may be observed not only in eyes with early-onset RVO but also in those with advanced occlusion and chronic edema, as reported by Campochiaro et al. Mean CMT showed a significant reduction after every three-monthly injection in all 17 eyes. Given this information and clinical experience with off-label bevacizumab for CRVO and BRVO, our impression is that pan-VEGF inhibition with ranibizumab might last slightly longer than with bevacizumab. Besides, even if bevacizumab lowers the intraocular concentration of VEGF, other inflammatory cytokines and growth factors might be important in the recurrence of intraretinal fluid and macular edema. [START_REF] Funk | Intraocular concentrations of growth factors and cytokines in retinal vein occlusion and the effect of therapy with bevacizumab[END_REF] Furthermore, ranibizumab has potentially better retinal penetration because of its smaller molecular size and higher binding affinity for VEGF-A aminoacid sequences than bevacizumab.

Then long-term care of venous thrombosis becomes an important issue. Perhaps as many as 30% to 60% of all RVO patients will require therapy for longer than a year and for some, indefinitely. With the likelihood of protracted treatment, we need to worry about the possible negative effects of pan-VEGF inhibition on the neuroretina. In answer to this question, it is worth recalling that pan-VEGF inhibition with ranibizumab every three months for a year did not cause detectable neuroretinal toxicity.

In conclusion, intravitreal injection of ranibizumab appears safe and may offer a new therapeutic opportunity for patients with RVO and macular edema, with a significant impact on visual function that needs to be investigated in the long term. These results must be interpreted cautiously because of the small number of eyes in this study, the absence of a control group, and the short follow-up. More conclusive evidence on the utility of intravitreal ranibizumab, also in comparison with other molecules, in the management of RVO will be forthcoming from current randomized, prospective, controlled, multicenter clinical trials.

Financial Disclosure: The authors have no proprietary or commercial interest in any material discussed in this article

Licence for Publication:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in PMJ and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence. (http://group.bmj.com/products/journals/instructionsfor-authors/licence-forms)

( 1 )

 1 proportion of patients losing ≤ 15 letters or gaining > 15 letters as measured by using Early Treatment Diabetic Retinopathy Study (ETDRS) acuity charts and converted to logMAR (logarithm of the minimum angle of resolution) scale; (2) ETDRS vision determination compared to baseline, at 1 week, month 1, 2, 3 and at every three months for 1 year; (3) changing in reading acuity and contrast sensitivity (CS); (4) percentage of patients losing > 30 letters at month 1, 2, 3, 6, 9, and 12; (5) changing in central macular thickness (CMT) as measured by OCT at 1 week, and at month 1, 2, 3, 6, 9, and 12 compared to baseline. For safety concern, local and systemic adverse effects were monitored throughout the study.

  3 ± 8.5 seconds and 75.3 ± 43.8 wpm with a final mean in MNREAD time of 3.5 ± 0.4 seconds (p< .006) and in reading fluency of 173.2 ± 20.43 wpm (p < .0002) that were significantly improved at the end of follow-up. The mean change in MNREAD time and reading fluency from baseline are summarized in Figure 2 and Table

Figure 1 .

 1 Figure 1. Mean change in LogMAR visual acuity from baseline in eyes treated with

Figure 2 .

 2 Figure 2. Mean change in reading fluency (wpm) from baseline in eyes treated with

Figure 3 .

 3 Figure 3. Mean change in log contrast sensitivity from baseline in eyes treated with

Figure 4 .

 4 Figure 4. Mean change in central macular thickness from baseline in eyes treated with

Figure 5 .

 5 Figure 5. Color fundus photography (A), late-phase fluorescein angiogram (B), and

Figure 6 .

 6 Figure 6. Color fundus photography (A), late-phase fluorescein angiogram (B) and

Table 1 .

 1 Clinical Evaluation of Patients at Baseline

		CRVO	BRVO
	N° of patients	9	8
	Age in years (mean ± SD)	68.7 ± 7.5	66.5 ± 3.3
	Gender (Male/Female)	3/6	4/4
	Duration of occlusion
	(months)	3	3
	Associated Medical
	Conditions
	Hypertension	6	3
	Smoking history	5	4
	Hypercholesterolemia	5	3
	Hyperlipidemia	6	3
	Diabetes	3	0
	Glaucoma	1	0
	Initial Snellen BCVA
	(logMAR ± SD)	20/240 (1.09 ± 0.25)	20/126 (0.80 ± 0.29)
	Initial Reading Fluency
	wpm (± SD)	75.3 (± 43.08)	82.25 (± 17.3)
	Contrast sensitivity
	Log Unit (± SD)	0.55 (± 0.67)	0.86 (± 0.26)

logMAR = logarithm of minimum angle of resolution wpm = word/per/minutes SD = standard deviation


Scott C Hau
email: scott.hau@moorfields.nhs.uk

John K G Dart

Minna Vesaluoma

Dipak N Parmar

Ilse Claerhout

Kanom Bibi

Daniel F P Larkin

Diagnostic accuracy of microbial keratitis with in vivo scanning laser confocal microscopy

Keywords: Confocal microscopy, diagnostic accuracy, acanthamoeba keratitis, fungal keratitis, Microsporidia, Nocardia

Aims

To determine the accuracy of diagnosing microbial keratitis by masked medical and non-medical observers using the Heidelberg Retina Tomograph II / Rostock Cornea Module in vivo confocal microscope.

Methods

Confocal images were selected for 62 eyes with culture or biopsy proven infections.

The cases comprised of 26 Acanthamoeba, 12 fungus, 3 Microsporidia, 2 Nocardia, and 19 bacterial infections (controls). The reference standard for comparison was a positive tissue diagnosis. These images were assessed on two separate occasions by 4 observers who were masked to the tissue diagnosis. Diagnostic accuracy indices, Kappa (κ) statistic and percentage agreement values were calculated. The Spearman correlation coefficient (r s ) was calculated for the number of correct diagnoses versus duration of disease.

Results

The highest sensitivity and specificity values were 55.8% and 84.2%, and the lowest sensitivity and specificity values were 27.9% and 42.1%. The highest positive and negative likelihood ratios were 2.94 and 0.59, respectively. Agreement values were: fair to moderate (κ, 0.22-0.44) for reference standard versus observer diagnosis, moderate to good in intra-observer variability (repeatability, κ 0.56-0.88), and poor to moderate in inter-observer variability (reproducibility, κ, 0.15-0.47). The correct 4 diagnosis was associated with duration of disease for Acanthamoeba keratitis (r s = 0.60, p = 0.001).

Conclusions

The diagnostic accuracy of microbial keratitis by confocal microscopy is dependent on observer experience. Intra-observer repeatability was better than inter-observer reproducibility. Difficulty in distinguishing host cells from pathogenic organisms limits the value of confocal microscopy as a stand-alone tool in diagnosing microbial keratitis.

INTRODUCTION

Difficulties in clinical and microbiological diagnosis are one of the major problems in the management of microbial keratitis particularly when caused by protozoa (Acanthamoeba and Microsporidia), fungi or filamentary bacteria. Diagnosis of these pathogens is difficult as they often take days or weeks to grow in culture and, in any case, culture is insensitive with culture positive rates rarely exceeding 60%. [START_REF] Yeh | Analysis of bacterial cultures in infectious keratitis, 1997 to 2004[END_REF] Although culture is still the primary diagnostic tool in tertiary referral centres it is not widely available to many patients because of limited resources.

The confocal microscope allows detailed in vivo analysis of normal [START_REF] Jalbert | In vivo confocal microscopy of the human cornea[END_REF] and pathological corneas. In patients with presumed corneal infection, it is used in diagnosis and in examination of the extent of involvement of tissue by infection and associated inflammation. All published studies have been directed at diagnosis and a number have shown both white light and laser confocal microscopy to be effective in diagnosing Acanthamoeba, [START_REF] Parmar | Tandem scanning confocal corneal microscopy in the diagnosis of suspected acanthamoeba keratitis[END_REF][START_REF] Matsumoto | The application of in vivo confocal scanning laser microscopy in the management of acanthamoeba keratitis[END_REF][START_REF] Kobayashi | In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis[END_REF] fungal, [START_REF] Avunduk | Confocal microscopy of Aspergillus fumigatus keratitis[END_REF][START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF][START_REF] Tu | Recalcitrant Beauveria bassiana keratitis: confocal microscopy findings and treatment with posaconazole (Noxafil)[END_REF] Nocardia [START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF] and Microsporidia keratitis. [START_REF] Sagoo | Microsporidium stromal keratitis: in vivo confocal findings[END_REF] However, these studies only present case series or reports and there are limited published data on evaluating the diagnostic accuracy of confocal microscopy.

Two recent studies have found high sensitivity and specificity values for diagnosing fungal keratitis (FK) and Acanthamoeba keratitis (AK) with the Confoscan 3.0 (Nidek Technology, Padova, Italy). [START_REF] Kanavi | Sensitivity and specificity of confocal scan in the diagnosis of infectious keratitis[END_REF][START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF] However, factors such as observer or selection bias, the absence of masking the observers from the microbiological diagnosis, and lack of appropriate controls may have resulted in overestimates of the sensitivity and specificity values. Although experience in interpreting confocal keratitis images is essential, the accuracy of diagnosing microbial keratitis by clinicians with differing levels of confocal microscopy experience and the potential of using trained technicians in interpreting images have not previously been assessed. These are important considerations in evaluating this technique. The aim of this study was to examine the diagnostic accuracy of microbial keratitis with the Heidelberg Retina Tomograph II / Rostock Cornea Module (HRT II/RCM) in vivo confocal microscope, as a stand-alone tool, by trained medical and non-medical observers with differing confocal microscopy experience.

MATERIALS AND METHODS

Patients

This study was approved by the Research & Ethics Committee of Moorfields Eye Hospital and it adhered to the tenets of Declaration of Helsinki. We retrospectively reviewed the case notes of a consecutive series of microbial keratitis patients who had had both corneal cultures or corneal biopsy and confocal microscopy (n=105) from January 1, 2005 to January 4, 2008. These cases were both those refractory to conventional treatment and those with unusual clinical features such as perineural infiltrates and ring infiltrates. Patients were either referred from Moorfields Emergency Department or from other institutions. Of the 105 cases, 62 culture or biopsy positive cases (62 eyes) were identified: 26 Acanthamoeba, 11 fungus, 1 fungus and bacteria, 3 Microsporidia, 2 Nocardia, and 19 bacteria. Bacteria were used as controls because they are normally too small to detect with confocal microscopy, [START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF][START_REF] Petroll | Clinical confocal microscopy[END_REF] therefore the case which was culture positive for both fungus and bacteria was classified as a fungal keratitis for the purposes of the study. We did not classify Nocardia as controls because they are filamentous bacteria and can form filamentous structures that are large enough to be distinguished by confocal microscopy. [START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF] Empirical treatments started prior to assessment in this study included topical antimicrobial agents and topical steroids for presumed herpes, bacterial or keratitis of unknown cause, respectively. Irrespective of the referring diagnosis, all patients had undergone a full clinical examination by a corneal specialist and repeat corneal scraping for culture and confocal microscopy on the same day. If the scraping was culture negative, and the keratitis progressive, then a corneal biopsy was later performed. Exclusion criteria were culture or biopsy negative keratitis cases, and patients who declined to have confocal microscopy or a corneal culture as part of their clinical investigation. The reference standard for this study was a diagnosis either by isolation on culture of a corneal scraping or histological diagnosis on a corneal biopsy; other ancillary culture sources such as contact lens case and solutions were not used.

The clinical outcomes were recorded for all the patients in the study and were consistent with the diagnosis based on culture or histology therefore it is unlikely, but possible, that there was unrecognised polymicrobial infections which may have been identified on confocal but not by culture or biopsy. We followed the Standards for Reporting of Diagnostic accuracy (STARD) initiative in conducting this study. [START_REF] Bossuyt | Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. Standards for Reporting of Diagnostic Accuracy[END_REF]

Culture and biopsy methods

Corneal scrapings for microbial culture were inoculated on the following media: blood agar, Sabouraud's dextrose agar (fungi), Robertson's cooked meat (anaerobic bacteria), Escherichia coli-seeded non-nutrient agar (Acanthamoeba), brain heart infusion (fastidious organisms, fungi) and Lowenstein-Jensen (mycobacteria, Nocardia). Scrapings were smeared on sterile glass slides for Gram and Giemsa stains.

All microbiological investigations were undertaken independently in an external laboratory. For biopsy a superficial lamellar disc of the affected cornea was trephined under local anaesthetic to provide a further specimen for microbiology and histopathological staining.

Confocal microscopy measurement protocol

In vivo confocal microscopy was performed on all 62 eyes by a single experienced observer (SH) with the HRT II / RCM (Heidelberg Engineering GmbH, Dossenheim, Germany) confocal microscope following a Standard Operating Procedure as follows.

A sterile Tomocap (Heidelberg Engineering GmbH, Dossenheim, Germany) was mounted over the objective of the microscope (Zeiss, x 63), and Polyacrylic acid 0.2% (Viscotears, Novartis) was used as a coupling agent between the cap and the lens objective. Topical anaesthetic (Proxymetacaine hydrochloride 0.5%, Chauvin) and Carmellose sodium 1% (Celluvisc, Allergan) was instilled into both eyes to provide comfort and act as a coupling fluid between the front of the Tomocap and the cornea.

Options for image acquisition include section (a single image at a particular depth), volume (a series of images over 60µm depth) and sequence scans (a video sequence at a particular depth). The volume scan option was selected for image acquisition because it allowed the capturing of large number of images over a short space of time.

The central region of the corneal ulcer or corneal infiltrate was scanned first followed by the top, left, bottom and right margin of the lesion. At each point, the epithelial layer of the affected area was scanned first and the focal plane of the microscope adjusted until the whole depth of the ulcer or infiltrate had been scanned. When there was more than one infiltrate, the same scanning sequence was repeated for each infiltrate. The wavelength of the laser employed in the HRT II / RCM is 670 nm and each standard 2 dimensional image consists of 384 x 384 pixels covering an area of 400 μm x 400 μm. The axial resolution is 7.6μm; compared to other instruments such as the Tandem scanning microscope (9µm) and ConfoScan 4 (29µm). [START_REF] Zhivov | In vivo three-dimensional confocal laser scanning microscopy of corneal surface and epithelium[END_REF]

Image selection

The confocal images of all the scans were reviewed by two experienced confocal microscopist (SH and JD). In diagnosing keratitis, a considerable amount of time is often needed to find an image that would yield sufficient information to be able to identify the organism. This is due to masking of the organisms by the cellular inflammatory response and that they seldom distribute evenly within the cornea during active infection. Therefore, to ensure all our observers had the maximum likelihood in diagnosing the type of keratitis, the best quality 384 x 384 pixel resolution digital image indicating clearly the culture proven pathogen from the corneal ulcer or infiltrate was selected and exported onto Microsoft Power Point ® (Microsoft Corp., Redmond, WA, USA). These included those of Acanthamoebaround single or double walled hyper-reflective objects (~10-20 μm) consistent with Acanthamoeba cysts, [START_REF] Matsumoto | The application of in vivo confocal scanning laser microscopy in the management of acanthamoeba keratitis[END_REF][START_REF] Kobayashi | In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis[END_REF] fungus -linear irregular branching hyper-reflective objects consistent with fungal hyphae, [START_REF] Avunduk | Confocal microscopy of Aspergillus fumigatus keratitis[END_REF][START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF] Microsporidia -small round hyper-reflective deposits (~ 2 μm) located in between keratocytes, [START_REF] Sagoo | Microsporidium stromal keratitis: in vivo confocal findings[END_REF] Nocardia -small branching filamentous structures within the corneal stroma, [START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF] and bacteria (control) -a mixture of inflammatory cells. presentation before their assessment. In addition, a series of five recent articles on diagnosing keratitis with the HRT II / RCM [START_REF] Matsumoto | The application of in vivo confocal scanning laser microscopy in the management of acanthamoeba keratitis[END_REF][START_REF] Kobayashi | In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis[END_REF][START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF][START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF][START_REF] Sagoo | Microsporidium stromal keratitis: in vivo confocal findings[END_REF] were given to each observer to read 2 weeks prior to their scheduled assessment date.

Intra-and inter-observer agreement

The confocal images were viewed in random order and assigned an identification number from 1 to 62. To ensure that there was masking between observers, the order of viewing the images were randomised by computer before being assessed by the next observer on a different day. No clinical details regarding each case were made available to the observers. Each observer assessed the series of images in a masked fashion on slide show in Microsoft Powerpoint ® and recorded the diagnosis corresponding to one of the following categories: AK, FK, Microsporidia (MK), Nocardia (NK) or bacterial keratitis (BK). A reference sheet showing the range of sizes of resident and inflammatory cells including epithelium and macrophages, and pathogenic cells e.g. diameter of Acanthamoeba cysts was given to each observer.

Intra-observer variability (repeatability) was evaluated by asking each observer to reassess the images, randomised in a different order, three weeks later in the same standard fashion. Inter-observer variability (reproducibility) was assessed by determining the level of agreement in diagnosis between observers. Readings of all the digital images were collected on a standard pro-forma and analysed.

Data analysis

Data analysis was performed with SPSS V14.0 (SPSS Inc, Chicago, USA). We calculated sensitivity, specificity, positive and negative likelihood ratios (LR) for both image set for each observer. Positive LR predicts the probability of a positive test result in patients with disease compared to those who do not have the disease.

Negative LR predicts the probability of a negative test in those who have the disease compare to those who do not. The level of agreement between the reference standard and different observers, and both intra and inter-observer variability were determined using Kappa (κ) statistic. The interpretation of κ statistic is as follows: 'poor' if κ ≤ 0.20, 'fair' if κ 0.21 -0.40, 'moderate' if κ 0.41 -0.60, 'substantial' if κ 0.61 -0.80 and 'good' if κ > 0.80. [START_REF] Petrie | Medical statistics at a glance[END_REF] In addition, we also calculated percentage agreement values between reference standard and observers, within-observers, and between different observers. Spearman's rank correlation coefficient (r s ) was used to determine the relationship between the number of correct diagnoses and the duration of disease for AK, FK and BK respectively. The duration of disease was defined as the time from symptom onset to presentation to the Corneal and External Disease Service at Moorfields. A value of P < 0.05 was deemed statistically significant. MK and NK were excluded from this analysis because the numbers were too small.

RESULTS

The reference standard consisted of 52 culture positive cases from corneal scrapings and 10 histopathologically confirmed cases on corneal biopsy. Sensitivity, specificity and likelihood ratio values for each observer are shown in Table 1.

The highest sensitivity value obtained was 55.8% and the highest specificity value 84.2%. We found fair to moderate agreement between observers and reference standard (κ, 0.22-0.44), moderate to good agreement in intra-observer variability (κ, 0.56-0.88), and poor to moderate agreement in inter-observer variability (κ, 0.15-0.47), Figure 1 shows a series of images demonstrating the difference in appearance between 210 correctly diagnosed versus incorrectly diagnosed cases. Figures 1 A andF Figure 2 shows a plot between the number of correct diagnoses for Acanthamoeba, bacteria and fungal keratitis versus the duration of disease (days). The graph shows a moderate correlation between the number of correctly diagnosed cases and the duration of disease for AK (r s = 0.60, p = 0.001), but not for BK (r s = 0.17, P = 0.49) or FK (r s = -0.19, p = 0.57), respectively. Therefore, the longer the duration of AK, the higher the likelihood that a correct diagnosis was made by the observers in grading the confocal images.

DISCUSSION

Acanthamoeba and fungus are uncommon causes of corneal infection for which early

diagnosis is paramount because it yields better prognosis and reduces ocular morbidity. [START_REF] Bacon | Acanthamoeba keratitis. The value of early diagnosis[END_REF][START_REF] Duguid | Outcome of acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine[END_REF] Although the current reference standard for diagnosing microbial keratitis is corneal culture, the sensitivity varies because of numerous factors. [START_REF] Mcleod | The role of smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious keratitis[END_REF] The HRT II / RCM in vivo confocal microscope has been shown to be useful in diagnosing a range of pathogens but validation studies of this new technology are few.

A recent review has reported the efficacy of diagnosing infections keratitis with confocal microscopy to be inconclusive, with the possible exception of AK. [START_REF] Labbé | Contribution of in vivo confocal microscopy to the diagnosis and management of infectious keratitis[END_REF] Our results show moderate sensitivity and moderate to high specificity values in diagnosing microbial keratitis with the HRT II / RCM confocal microscope, whereas both Kanavi et al [START_REF] Kanavi | Sensitivity and specificity of confocal scan in the diagnosis of infectious keratitis[END_REF] and Tu et al [START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF] found very high sensitivity (>90%) in diagnosing AK and FK respectively with the Confoscan 3. Tu et al, [START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF] using multitest referencing standards, reported that when there are both clinical characteristics and objective evidence of AK, the adjunctive usage of confocal microscopy exhibited a sensitivity of 90.6% and specificity of 100%. In our study, we set out to evaluate the diagnostic accuracy of confocal microscopy as a stand alone tool rather than a supportive investigative technique, without the bias and influence of clinical findings.

Although assessing confocal images in the absence of clinical data does not reflect the use of confocal microscopy in clinical settings, it is the only way to avoid bias when analysing the images. Our inclusion criteria were based on culture positive cases irrespective of confocal classification. Although we chose only one representative image from each case this was the best available image for the organism that was cultured from each case giving the observers the best opportunity to make a correct confocal diagnosis; we believe that reviewing a series of images from each case would either have made a correct confocal diagnosis more difficult or have had no effect on the outcome. In addition, it allowed standardisation when viewing the images so that all observers assessed the same number of images consecutively. The absence of controls in the previous studies and the use of confocal 'positive' without culture confirmation as a reference standard, or for the case definition [START_REF] Kanavi | Sensitivity and specificity of confocal scan in the diagnosis of infectious keratitis[END_REF][START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF], could lead to selection bias and misdiagnosis resulting in an overestimation of sensitivity values. [START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF]21,[START_REF] Choi | Sensitivity and specificity of a single diagnostic test in the presence of work-up bias[END_REF] This is evident from our controls in which immune cells can often be confused with AK cysts and vice versa leading to erroneous diagnosis. Furthermore, 'good' confocal images have been illustrated in most published studies to present findings without discussion of difficulties in analysing equivocal images.

We found fair to moderate agreement between reference standard and observer diagnosis when a case mix of equivocal and unequivocal images were analysed by our observers. The rigorous criteria in our study design in regard to the use of masked observers and controls could explain why sensitivity values, even for the most experienced observer, were lower.

Another explanation for the very high sensitivity values reported in one previous study was the use of only one ophthalmology trained observer who, in addition to being unmasked to the clinical findings, was experienced in the use of confocal microscopy for keratitis diagnosis: this makes it difficult to extrapolate the results to what might be expected outside their centres. [START_REF] Tu | The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis[END_REF] To evaluate the potential of using this technology in clinics where an ophthalmologist with experience in confocal microscopy may not be available, our graders included 2 experienced ophthalmologists, an inexperienced ophthalmologist and an experienced technician.

We found a two-fold difference in sensitivity between the most experienced and the Intra-observer agreement (repeatability) was found to be moderate to good, indicative of good observer repeatability in grading the images irrespective of the accuracy of their diagnoses. Observer experience did not appear to improve intra-observer repeatability as the observer with the lowest sensitivity had the highest repeatability and vice versa. Inter-observer agreement (reproducibility) was poor to moderate between different observers because of factors such as observer experience and differences in techniques of classifying images by different observers. The two observers who had the highest sensitivity values also had the best inter-observer reproducibility, indicating experienced observers achieved a higher diagnostic accuracy and reproducibility than less experienced observers. Therefore, to improve reliability the same experienced operator should be employed if sequential imaging of a patient is required.

Our observers were able to diagnose AK more accurately than any other type of keratitis. The unique appearance of Acanthamoeba cysts on confocal microscopy and the higher number of AK compared to other conditions in our study might explain this outcome. However, AK was commonly confused with controls and vice versa because of the diagnostic difficulty with some of the equivocal images. There was a marked association between the accuracy of diagnosing AK and the duration of disease.

Previous case reports have mainly described the morphological features of cysts and trophozoites in the epithelium and stroma during active infection, [START_REF] Matsumoto | The application of in vivo confocal scanning laser microscopy in the management of acanthamoeba keratitis[END_REF][START_REF] Kobayashi | In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis[END_REF] but have not related the number of cysts seen and the way they distribute with the different stages of the disease process. In early disease, where the organism is mainly confined to the epithelium, the presence of large numbers of inflammatory cells made diagnosing AK more difficult because of the difficulty in distinguishing AK cysts and particularly trophozoites from inflammatory cells. [START_REF] Kobayashi | In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis[END_REF] Late presentation was associated with either a greater number of Acanthamoeba cysts seen in the images or the fact that they were easier to identify because of a reduction in the type and number of host cells seen. Our experience, therefore, suggests that AK is easier to identify with confocal microscopy in the later stages of infection.

The use of confocal microscopy in diagnosing FK has been widely reported in the literature. [START_REF] Avunduk | Confocal microscopy of Aspergillus fumigatus keratitis[END_REF][START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF][START_REF] Tu | Recalcitrant Beauveria bassiana keratitis: confocal microscopy findings and treatment with posaconazole (Noxafil)[END_REF] Filamentous fungal hyphae have characteristic linear hyper-reflective lesions branching at 45 or 90 degrees angle, [START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF] whereas candida infection produces pseudofilaments. [START_REF] Brasnu | In vivo confocal microscopy in fungal keratitis[END_REF] Despite these well described confocal appearances of FK in the literature, the percentage of correct diagnosis in our series was low possibly due to difficulties in differentiating other linear images from fungal hyphae. [START_REF] Chiou | Differential diagnosis of linear corneal images on confocal microscopy[END_REF] Nocardia and Microsporidia species are rare causes of microbial keratitis. [START_REF] Sridhar | Clinicomicrobiological review of Nocardia keratitis[END_REF] Clinically, Nocardia may be misdiagnosed as mycotic or mycobacterial keratitis, [START_REF] Vaddavalli | Confocal microscopy for Nocardia keratitis[END_REF][START_REF] Lalitha | Nocardia keratitis: species, drug sensitivities, and clinical correlation[END_REF] whilst Microsporidia can be misdiagnosed as AK or herpes simplex keratitis. Despite the rarity of these organisms, because of the unique appearance on confocal microscopy with Microsporidia, [START_REF] Sagoo | Microsporidium stromal keratitis: in vivo confocal findings[END_REF] two observers managed to identify this organism correctly in both of their assessments. Only observer B managed to obtain the correct diagnosis in both assessments for diagnosing one case of Nocardia keratitis; the unfamiliarity in interpreting confocal images of Nocardia, the similarity in appearance of fungal hyphae and Nocardia filaments, and the small number of cases in our study made diagnosing this organism difficult. The inclusion of both Nocardia and Microsporidia cases might have reduced the overall sensitivity and specificity values 20 but as confocal findings of both organisms have been reported, we believe it was appropriate to include them in the study.

In summary, to the best of our knowledge, this is the first study evaluating the diagnostic accuracy of microbial keratitis using a single reference standard for different masked observers with the HRT II / RCM confocal microscope. Although confocal microscopy is non-invasive and can provide a rapid diagnosis for microbial keratitis, (i) similarities between inflammatory and pathogenic cells, and (ii) difficulty in interpreting equivocal images, limits its usefulness as a stand-alone tool in diagnosing keratitis. Confocal microscopy is a useful adjunct in managing refractory cases and we have shown that the diagnostic accuracy improves with clinician experience. However, the diagnostic accuracy of confocal microscopy used in isolation from the clinical assessment is still too low to be a substitute for tissue diagnosis, particularly in patients with progressive disease. Improvement in clinician training and experience, greater standardization of image interpretation, and the development of new software in tandem with higher resolution imaging is likely to improve the diagnostic accuracy of this technology in diagnosing microbial keratitis in the future. Microsporidia by all the observers; small hyper-reflective granules that appear similar to Microsporidia organisms (white arrows). J, Cultured positive for Alternaria and

  All digital images were assessed prospectively in the same standard fashion in the Reading Centre at Moorfields Eye Hospital by 4 observers (3 ophthalmologists and 1 medical technician) with differing levels of experience in assessing keratitis on confocal microscopy as follows. Of the 3 ophthalmologists, observer A had 6 years of experience in assessing microbial keratitis with confocal microscopy, observer B, 10 years of experience in confocal microscopy but not keratitis, and observer C, 6 months of experience in assessing keratitis with confocal microscopy. Observer D was a medical technician who had 2 years of experience in performing confocal microscopy using the HRT II / RCM and analysing keratitis images but with no experience in the clinical appearance and treatment of different types of keratitis. To ensure each observer was familiar with the image appearance of different cell types obtained from the HRT II / RCM confocal microscope, examples of both normal cellular morphology and the standard images of different pathogens were shown in a

  show a 211 case of late diagnosed AK versus early diagnosed AK; note the presence of 212 inflammatory cells in the epithelium in early AK making distinction between host 213 cells and Acanthamoeba cysts and trophozoites difficult, whereas in delayed 214 diagnosed AK, single or clusters of cysts were seen in the stroma with minimal host difficulties in distinguishing host cells from pathogenic organisms, and Nocardia (Fig 1J) from FK because of their similarity in appearance on confocal microscopy.

  least experienced observer indicating higher diagnostic accuracy with clinicians experienced in confocal microscopy. Our results indicate the sensitivity value with a trained technician, with no experience in the clinical appearance of different types of microbial keratitis, was better than an inexperienced medical observer but with a lower specificity value and positive LR. This raises the possibility of training nonmedical personnel, in performing and analysing keratitis images. The highest positive LR and lowest negative LR was achieved by observer B who was experienced in confocal imaging of normal corneal anatomy and various pathological conditions other than microbial keratitis, indicating experience gained in other aspects of confocal microscopy improves the diagnostic outcome.

Figure 1

 1 Figure 1

Figures

  Figures E to J show a series of images of incorrectly diagnosed cases demonstrating

Table 1 . Sensitivity, specificity and likelihood ratio values for each observer

 1 95% confidence intervals; LR = likelihood ratio Positive LR = the probability of a positive test result in patients with disease compared to those who do not have the disease. Negative LR = the probability of a negative test result in patients with disease compared to those who do not have the disease.

	Observer	Sensitivity (95% CI)	Specificity (95% CI)	Positive LR (95% CI) Negative LR (95% CI)
	A
	1st assessment	55.8 (47.7-63.2)	57.9 (39.5-74.7)	1.32 (0.79-2.49)	0.76 (0.49-1.33)
	2nd assessment	55.8 (47.6-61.8)	73.7 (55.0-87.3)	2.12 (1.06-4.87)	0.60 (0.44-0.95)
	B
	1st assessment	53.4 (45.4-58.8)	78.9 (60.5-91.0)	2.53 (1.15-6.51)	0.59 (0.45-0.90)
	2nd assessment	46.5 (38.6-50.9)	84.2 (66.4-94.2)	2.94 (1.15-8.82)	0.64 (0.52-0.93)
	C
	1st assessment	27.9 (20.5-32.3)	84.2 (67.5-94.2)	1.77 (0.63-5.56)	0.86 (0.72-1.18)
	2nd assessment	27.9 (20.4-33.2)	78.9 (61.9-90.9)	1.32 (0.54-3.66)	0.91 (0.74-1.29)
	D
	1st assessment	44.2 (36.8-52.3)	42.1 (25.3-60.5)	0.76 (0.49-1.33)	1.33 (0.79-2.49)
	2nd assessment	48.8 (40.7-56.3)	57.9 (39.6-74.8)	1.16 (0.67-2.23)	0.88 (0.59-1.50)
	95% CI =

Table 2 .

 2 One observer (observer B) obtained the highest positive and lowest negative LR for diagnosing microbial keratitis. This observer also achieved the best overall kappa and percentage agreement values in diagnoses compared to reference standard,

Table 3 .

 3 The best inter-observer agreement (percentage agreement, 61.3-66.1%; κ,

	0.43-0.47) was between observer A and B, the two most experienced observers in the
	study. Observer C was the most repeatable (percentage agreement, 93.5%; κ, 0.88)
	despite having the lowest kappa and percentage agreement values compared to
	reference standard, Tables 2 &3.

Table 2 . Kappa values: reference standard versus observers, intra- observer and inter-observer variability

 2

	Observer Reference standard A	B	C	D
	A	0.36 (0.42)	0.56	0.43 (0.47) 0.23 (0.15) 0.28 (0.29)
	B	0.44 (0.40)	0.43 (0.47) 0.76	0.40 (0.32) 0.18 (0.32)
	C	0.24 (0.22)	0.23 (0.15) 0.40 (0.32) 0.88	0.24 (0.33)
	D	0.27 (0.36)	0.28 (0.29) 0.18 (0.32) 0.24 (0.33) 0.65
	Reference standard = culture or biopsy proven cases
	Values in parentheses = observers' second assessment

Table 3 . Percentage (%) agreement values between reference standard and observers, within-observers, and between different observers.

 3

	Observer Reference standard A	B	C	D
	A	56.5 (61.3)	70.9	61.3 (66.1) 45.2 (43.5) 45.2 (50)
	B	61.3 (58.1)	61.3 (66.1) 85.5	64.5 (61.3) 37.1 (53.2)
	C	45.2 (43.5)	45.2 (43.5) 64.5 (61.3) 93.5	40.3 (53.2)
	D	43.5 (51.6)	45.2 (50)	37.1 (53.2) 40.3 (53.2) 72.6
	Reference standard = culture or biopsy proven cases
	Values in parentheses = observers' second assessment

Table 4 . Percentage of correct diagnoses of the different causes of keratitis for different observers Reference standard Diagnosis

 4

			Observer, n (%)
		Observer A	Observer B	Observer C	Observer D
	Acanthamoeba (n=26)	19 (73.1)	11 (42.3)	4 (15.4)	(46.2)
	Bacteria* (n=19)	10 (52.6)	15 (78.9)	15 (78.9)	(42.1)
	Fungus † (n=12)	1 (8.3)	5 (41.2)	5 (41.2)	(41.2)
	Microsporidia (n=3)	1 (33.3)	2 (66.7)	2 (66.7)	(33.3)
	Nocardia (n=2)	0	1 (50)	0
	Percentage correct diagnosis was calculated by taking into account only those cases where each
	observer had made the correct diagnosis on both assessments.

* 2 Enterobacter, 1 Haemophilus, 1 Moraxella, 1, Mycobacteria, 1 Pasteurella, 3 Pseudomonas, 1 Serratia, 7 Staphylococcus, 2 Streptococcus. † 1 Alternaria, 1 Candida, 10 Fusarium. 209

immune and resident cells seen. The incorrectly diagnosed cases demonstrate the

ACKNOWLEDGEMENTS

The authors would like to thank Dr Catey Bunce for her statistical advice and support.

COMPETING INTERESTS

None declared

Staphylococcus aureus but diagnosed as Nocardia by observers A, B and D; hyphae type lesions that appear similar to Nocardia filaments (black arrows).

Figure 2

Scattered plot showing the relationship between number of correctly diagnosed cases and duration of disease (days) for Acanthamoeba, bacteria and fungal keratitis.