Prospective study of serum and aqueous humour anti-Hsp70.1 IgG antibody levels in ocular toxoplasmosis.

AIMS: We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis. METHODS AND RESULTS: In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis. CONCLUSION: While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis.

Occurrence and risk factors for retinal detachment after pars plana vitrectomy in acute postcataract bacterial endophthalmitis.

BACKGROUND/AIMS: To report the incidence, risk factors and prognosis of retinal detachment (RD) in patients who had vitrectomy for acute bacterial endophthalmitis after cataract surgery. METHODS: 123 patients with acute postcataract endophthalmitis, consecutively treated with pars plana vitrectomy (PPV) were included by the French Institutional Endophthalmitis Study group, in a prospective multicentre cohort study. Risk factors of RD were analysed using logistic regression. RESULTS: At the 6-month follow-up, the rate of post-PPV RD was 13% (n=16). The risk factors of post-PPV RD were diabetes (OR=4.7 (1.4-15.4), p=0.01) and visualisation of retinal vasculitis on the posterior pole (OR=3.8 (1.1-13.9), p=0.03) at the time of PPV. Postoperative RD occurred in 56% (n=9) of cases in the first month, in 31% (n=5) in the second month and in 6% (n=1) in the third month, with a mean delay of 47±71 days after PPV. The macula was detached in 12 cases (75%) and proliferative vitreoretinopathy grade C was present in seven cases. Final successful reattachment of the retina was obtained in 60% (n=9/15) of cases, with one (7/9) or two surgeries (2/9). Final visual acuity after surgical repair was ≥20/40 in 19% of cases, compared with 43% in patients without RD (p=0.05). CONCLUSIONS: RD is a major and severe complication of PPV performed in patients with acute postcataract endophthalmitis. Retinal vasculitis is a major risk factor of RD after PPV. Anatomical and functional outcome remain poor.

Handheld reflectance confocal microscopy for the diagnosis of conjunctival tumors.

PURPOSE: To evaluate whether the handheld in vivo reflectance confocal microscopy that has been recently developed for the study of skin tumors is suitable for the diagnosis of conjunctival tumors. DESIGN: Prospective study, observational case series. METHODS: We prospectively evaluated the reflectance confocal microscopy features of 53 conjunctival lesions clinically suspicious for tumors of 46 patients referred to the University Hospital of Saint-Etienne (France) by using the handheld device. Twenty-three lesions were excised (3 nevi, 10 melanomas, 5 squamous cell carcinoma, 2 lymphomas, and 3 pinguecula/pterygium) while the other 30, presenting no reflectance confocal microscopy malignant features, were under follow-up for at least 1 year. Clinical reflectance confocal microscopy and histologic diagnosis were compared. RESULTS: In vivo reflectance confocal microscopy diagnosis was in agreement with the histologic diagnosis in all cases and none of the lesions that were not excised show any clinical progression under follow-up. CONCLUSION: In vivo reflectance confocal microscopy with a handheld dermatology-dedicated microscope can play a role in the noninvasive diagnosis of conjunctival lesions. Further studies should be performed to better define the diagnostic ability of this technique.

The role of in vivo confocal microscopy in the diagnosis of eyelid margin tumors: 47 cases.

BACKGROUND: Handheld in vivo reflectance confocal microscopy (IVCM) is a new imaging method that allows noninvasive diagnosis of cutaneous tumors but to date it has not been used in the study of eyelid tumors. OBJECTIVE: We sought to evaluate the suitability of IVCM for eyelid margin tumors. METHODS: We prospectively evaluated the IVCM features of 47 eyelid margin lesions, clinically suspicious of malignancy; 35 of these were excised whereas the other 12, with no IVCM malignant features, were followed up for at least 1 year. Clinical, IVCM, and histologic diagnoses were compared. RESULTS: IVCM showed sensitivity and specificity of 100% and 69.2%, respectively, for malignancy (basal cell carcinoma, squamous cell carcinoma, and melanoma). The follow-up of the 12 nonexcised lesions did not show any clinical progression. LIMITATIONS: The lesions showing neither clinical nor IVCM features for malignancies were not biopsied in view of the potential functional and aesthetic consequences of eyelid margin surgery. CONCLUSION: Used with a handheld dermatology-specific microscope, IVCM can play a role in the noninvasive diagnosis of eyelid margin lesions. Further studies are needed to better define diagnostic criteria of eyelid tumors and improve the specificity of this technique.

Comparison of endothelial cell density of organ cultured corneas with cornea donor study.

PURPOSE: Determination of the endothelial cell density (ECD) by eye banks is paramount in donor cornea qualification. Unbiased measurement avoids wastage and grafts with an increased risk of premature failure. Internal calibration of the counting method is essential, but external validation would add an extra stage in the assessment of reliability. In this respect, data published by the multicenter Cornea Donor Study (CDS) in 2005 is a reference. The aim of the study was to compare ECD determined within a single eye bank, which uses calibrated image analysis software designed for transmitted light microscopy images of organ cultured corneas, with the CDS data determined on specular microscopy images of corneas stored at 4°C. METHODS: ECD of consecutive corneas retrieved between 2005 and 2013 was determined after exposure to 0.9% NaCl. More than 300 ECs were counted on 3 fields of the central 8 mm. Endothelial cell boundaries were automatically drawn and verified by a skilled technician who performed all necessary corrections. RESULTS: Three thousand fifty-two corneas were analyzed, of which 48.5% donors were >75 years (CDS upper age limit). Between 10 and 75 years, the ECD varied according to donor age exactly in the same manner as in the CDS, but were consistently higher of 100 ± 25 cells per square millimeter (P < 0.001). CONCLUSIONS: ECD determined by a computer-aided method from transmitted light microscopy images compares favorably with the American CDS reference series. The slight systematic difference on either side of the Atlantic Ocean could be due to (1) differences in counting principles and/or (2) higher shrinkage of the cornea caused by stromal edema in organ culture.

Anterior segment biometry using spectral-domain optical coherence tomography.

PURPOSE: To compare the anterior chamber and anterior chamber angle measurements obtained with spectral-domain anterior segment optical coherence tomography (AS-OCT) and time-domain AS-OCT. METHODS: The anterior chamber of healthy participants was imaged with spectral-domain AS-OCT (Casia SS-1000; Tomey, Nagoya, Japan) and time-domain AS-OCT (Visante; Carl Zeiss Meditec, Dublin, CA). Central corneal thickness (CCT), anterior chamber depth (ACD), angle opening distance at 500 and 750 µm (AOD 500 and AOD 750), trabecular iris space area at 500 and 750 µm (TISA 500 and TISA 750), and scleral spur angle were measured. The intraclass correlation coefficients (ICCs) were calculated. The Pearson correlation test was used to evaluate the correlation between the measurements and Bland-Altman plots to evaluate the agreement. RESULTS: One hundred one eyes of 101 healthy participants were analyzed. Excellent repeatability was found with both devices for CCT, AOD 500, AOD 750, TISA 750, and scleral spur angle (ICC = 0.90 to 0.98 and 0.89 to 0.97, respectively) and excellent to moderate repeatability was found for TISA 500 (ICC = 0.68 to 0.97 and 0.70 to 0.93, respectively). For all parameters, Casia and Visante measurements were significantly correlated (r = 0.76 to 0.98; P < .02). ACD measured with the Casia was significantly larger (mean difference = 0.12 ± 0.08 mm; P < .0001), and the scleral spur angle measured with the Casia was significantly lower (mean difference = 4.85° ± 5.30°; P < .01). There were nonsignificant differences between the devices for the other parameters (P > .06). CONCLUSIONS: Both Casia and Visante AS-OCT demonstrate high repeatability. Except for the ACD and scleral spur angles, the two devices show good agreement.

Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells.

Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

CorneaJ: an imageJ Plugin for semi-automated measurement of corneal endothelial cell viability.

PURPOSE: A reliable experimental measurement of endothelial cell (EC) viability is paramount in the assessment of new drugs, devices, and surgical processes liable to damage the corneal endothelium, as well as during endothelial bioengineering. We previously used triple Hoechst-Ethidium-Calcein-AM labeling coupled with image analysis to determine the viability and mortality of ECs on the whole cornea, thus defining the new notion of viable EC density. To make it accessible to all, and for improved reproducibility, we have now developed an ImageJ plugin with improved thresholding algorithms. METHODS: The CorneaJ plugin comprised contrast improvement, regional selection of pixels with similar gray levels, simplified thresholding facilitated by a user-friendly images display, and the option of manual touch-up to increase accuracy. After Hoechst-Ethidium-Calcein-AM labeling, the endothelium of 10 human corneas was observed with a fluorescent microscope with motorized stage. The performance of CorneaJ was compared with standard manual thresholding: accuracy was determined by comparison with fully manual selection of viable ECs by an expert; and reproducibility, by calculating the intraclass coefficient and coefficient of variation (100 × SD/mean) of 7 independent observers. RESULTS: CorneaJ was more accurate than the standard thresholding, with a deviation from the expected value of -1.8% [95% confidence interval (CI), -2.7 to -0.9] versus 6.0% (95% CI, 2.8-9.3), respectively, P < 0.001. It was also more reproducible, with an intraclass coefficient of 0.98 (95% CI, 0.954-0.994) versus 0.81 (95% CI, 0.628-0.937) and a mean coefficient of variation of 2.6 (1.4-7.4) and 5.7 (3.4-19.8), P = 0.005. CONCLUSIONS: CorneaJ is a new, fast, and reproducible free image analysis tool that could help standardize experimental measurement of corneal EC viability.

Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks.

The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm(2) by steps of 400 cells/mm(2)) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 µm(2) (incremental steps of 25 µm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 µm(2) grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 µm(2) grid counts. Digital image analysis with a variable frame counting method would be the best alternative.

Very early endothelial cell loss after simultaneous corneal autograft and allograft.

PURPOSE: For a better understanding of the very early endothelial cell (EC) loss universally described after all types of keratoplasty, we compared the EC decrease after performing a simultaneous autograft and organ-cultured allograft. METHODS: A 71-year-old woman presented with a central corneal opacity in her left eye and a profoundly amblyopic right eye with a transparent cornea. Both corneas had a normal EC density (ECD). She underwent a left autograft and a right allograft procedure with an organ-cultured cornea, in which the ECD was determined using a calibrated light microscope with image analysis 48 hours before the surgery, that is, just before the final deswelling step with dextran. The postoperative central ECD was determined using specular microscopy on days (D) 1, 2, 3, 4, 5, 15, 20, 30, 60, 90, 120, and 180. RESULTS: Both grafts were uneventful. For the autograft, the pregraft ECD was 2303 cells per square millimeter, and the cell loss was very low, from 4% (D1) to 3% (D180). For the allograft, the pregraft eye bank ECD was 2787, and this decreased by 32% on D5. On D180, the ECD decrease was almost stabilized at 38%. CONCLUSIONS: This difference between the autograft and allograft, both performed in corneas with a normal peripheral endothelial reserve, indicates that the typical very early postoperative decrease in the EC is not caused mainly by surgery-dependent overmortality. It may be mostly artificial, revealing the overestimation of eye bank ECD caused by the technical unfeasibility of strictly considering living ECs and by measuring the ECD several days before grafting. This exceptional case suggests a new paradigm: surgeons graft fewer ECs than they think.

Corneal perforation: another side effect of nicorandil.

CONTEXT: Nicorandil is an antianginal drug used for 20 years in Japan and introduced in France in 1994. Since 1997, side effects such as mucocutaneous ulcerations have regularly been reported. OBJECTIVE: To describe the first case of a patient with a spontaneous corneal perforation associated with mucocutaneous ulcerations while taking Nicorandil. MATERIALS AND METHODS: A 81-year-old patient, with no past history of ocular disease but a long past history of cardiovascular disease, presented with a spontaneous paracentral corneal perforation. This was consecutive to 5 months of recurrent keratoconjunctivitis and mucocutaneous ulcerations resistant to conventional therapy. (He was taking nicorandil for 5 years.) A penetrating keratoplasty was performed in emergency. RESULTS: Inflammatory and infectious causes of spontaneous corneal perforation were ruled out. After initial uneventful post-operative wound healing, an epithelial ulcer appeared on the graft. Dermatologists suggested the iatrogenic role of nicorandil and the drug was discontinued. Both mucocutaneous and corneal ulcerations resolved rapidly. DISCUSSION: Although mucocutaneous ulcerations have been attributed several times to nicorandil, this is, to our knowledge, the first major corneal damage due to this antianginal drug. Timing, pattern of illness, absence of other aetiology, recurrence of epithelial ulceration on the corneal graft and its spontaneous healing after nicorandil discontinuation make it highly apparent probable that nicorandil was directly involved in this corneal perforation. CONCLUSION: Ophthalmologists and dermatologists should be aware of the risk of severe but reversible corneal ulcerations in patients treated with nicorandil. A pharmacovigilance warning statement should be compulsory.

Non-invasive measurement of transparency, arcus senilis, and scleral rim diameter of corneas during eye banking.

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0-33.6), 38.3 (24.3-75.4) and 57.9 (33.9-90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3-10.6), 10.5 (8.2-11.5), and 11.1 (9.9-12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.

Baseline factors predictive of visual prognosis in acute postoperative bacterial endophthalmitis in patients undergoing cataract surgery.

IMPORTANCE: Although rare, postoperative endophthalmitis in patients undergoing cataract surgery can lead to anatomical or functional loss of the eye. Therapeutic strategies such as antibiotic prophylaxis and microbiological diagnosis are more effective with a target patient population. New prospective data are needed to identify prognostic factors. OBJECTIVE: To identify baseline factors of visual prognosis in patients with acute bacterial endophthalmitis after cataract surgery. DESIGN: Prospective study of consecutive patients undergoing cataract surgery, enrolled from March 1, 2004, through December 31, 2005. We analyzed outcomes to determine the effect on the final visual outcome, defined as poor (visual acuity [VA] worse than 20/100) or good (VA 20/40 or better) using univariate and multivariate analysis. SETTING: Four academic hospitals. PARTICIPANTS: Ninety-nine consecutive patients with cataract. INTERVENTION: Corneal phacoemulsification. MAIN OUTCOMES AND MEASURES: Factors related to the cataract surgery (complications), initial clinical presentation, and microbiological diagnosis and the final VA. RESULTS: The significant baseline factors (at presentation) for good visual outcome (45% of the series) were the winter season, absence of complications during cataract surgery, initial VA, microbiological investigations revealing no microorganism or a coagulase-negative Staphylococcus species (CNSP), and fundus visibility. Quantitative factors associated with a good clinical prognosis were shorter duration of cataract surgery, younger age, and a hypopyon no greater than 1.5 mm. Significant factors associated with poor visual outcome were infection of the right eye, initial VA, corneal edema, a hypopyon larger than 1.5 mm, detection of bacterial species other than a CNSP, and the absence of fundus visibility. Multiple logistic regression analysis showed that high bacterial virulence was the only independent factor (odds ratio, 14.0 [95% CI, 2.7-71.0]; P = .001) for poor visual outcome. On the other hand, low bacterial virulence (odds ratio, 0.2 [95% CI, 0.03-0.6]; P = .01) and the absence of complications during cataract surgery (0.1 [0.01-0.4]; P = .003) were independent factors for good VA. CONCLUSIONS AND RELEVANCE: Most clinical outcome factors in acute postoperative endophthalmitis can be identified at presentation. The bacterial virulence level is the main factor predictive of the final visual prognosis.

Optical diagnosis of a metabolic disease: cystinosis.

Nephropathic cystinosis (NC) is a rare autosomal recessive storage disease characterized by the lysosomal accumulation of cystine crystals throughout the body, particularly in blood cells, the cornea, skin, kidneys, the central nervous system, and the muscles. The skin and the cornea are the most accessible sites to explore, and in vivo reflectance confocal microscopy (IVCM) helps identify crystals in both but does not provide any information to help define their composition. Raman spectroscopy (RS) allows cystine to be easily recognized thanks to its characteristic signature with a band at 499 cm⁻¹. Two dermatology confocal microscopes were used to visualize crystals in both the skin and the ocular surface of a cystinosis patient, and an ex vivo Raman examination of a skin biopsy and of the cornea was performed and removed during a corneal graft to confirm the cystine composition of the crystals. Recently, RS has been performed in vivo and coupled with IVCM. In the future, it is suggested that crystals in NC and other deposits in storage diseases could be identified with this noninvasive in vivo technique that combines IVCM to recognize the deposits and RS to confirm their chemical nature.

Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans.

The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat-mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 µm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 µm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches.

Fabrication of optical mosaics mimicking human corneal endothelium for the training and assessment of eye bank technicians.

The determination of endothelial cell density (ECD) is a crucial activity in eye banks for the assessment of corneal tissue quality. These cells are responsible for corneal transparency, and ECD correlates with graft survival. ECD is mainly assessed with a manual "naked-eye" procedure under a transmitted light microscope in Europe and using a specular microscope in the United States. Interbank and intrabank variability has been previously demonstrated. In order to facilitate training and continuing education of technicians and reliability assessment of eye banks' ECD determination, we use micro-optics technologies to fabricate test mosaics that exactly reproduce the image of human corneal endothelium. The description of the fabrication process is detailed, and comparisons are made between amplitude and phase mosaics.