Purpose: To study the outcome of ocular nocardiosis following intraocular surgery. Materials and Methods: A retrospective review of medical records of all postoperative cases of culture proven Nocardia infection over a period of 3 years, from October 2010 to September 2013, was performed. Microbiological analysis was performed for all cases and included smears and cultures. Fortified 2% amikacin eye drops were the mainstay of treatment. Surgical intervention was performed in case of nonresponse to medical therapy or suspected endophthalmitis. Results: Seven cases of culture proven Nocardia infection were seen. All cases had been operated in a hospital surgical facility. Six followed phacoemulsification, and one followed a secondary intraocular lens implantation. Four patients were part of a cluster infection. The mean duration between the primary surgical procedure and presentation was 16.14 ± 9.82 days. Five patients had infiltrates at the site of the surgical incision. One each had endophthalmitis and panophthalmitis. Six eyes required surgical intervention. Infection was seen to resolve in four eyes. Two eyes went into phthisis, and one was eviscerated. Only two of the six eyes, where in surgical intervention was performed early, obtained a final visual acuity of 20/60. Conclusion: Early surgical intervention, before the involvement of the anterior chamber, may help preserve the anatomic and functional integrity of the eye.
Congenital aniridia is a pan ocular disorder characterized by partial or total loss of iris tissue as the defining feature. Classic aniridia, however, has a spectrum of ocular findings, including foveal hypoplasia, optic nerve hypoplasia, nystagmus, late-onset cataract, glaucoma, and keratopathy. The latter three are reasons for further visual compromise in such patients. This entity is often due to mutations in the PAX6 (Paired box protein Pax-6) gene. Recently, aniridia-like phenotypes have been reported due to non- PAX6 mutations as in PITX2, FOXC1, FOXD3, TRIM44 , and CYP1B1 as well wherein there is an overlap of aniridia, such as iris defects with congenital glaucoma or anterior segment dysgenesis. In this review, we describe the various clinical features of classic aniridia, the comorbidities and their management, the mutation spectrum of the genes involved, genotype-phenotype correlation of PAX6 and non- PAX6 mutations, and the genetic testing plan. The various systemic associations and their implications in screening and genetic testing have been discussed. Finally, the future course of aniridia treatment in the form of drugs (such as ataluren) and targeted gene therapy has been discussed.
Purpose: Failure of rapid re-epithelialization within 10-14 days after corneal injury, even with standard supportive treatment, is referred to as persistent corneal epithelial (CE) defect (PED). Though an array of genes regulates reepithelization, their mechanisms are poorly understood. We sought to understand the network of genes driving the re-epithelialization in PED. Method: After obtaining informed consent, patients underwent an ophthalmic examination. Epithelial scrapes and tears samples of six PED patients and six individuals (control) undergoing photorefractive keratectomy (PRK) were collected. RNA isolation and quantification were performed using either the epithelial scrape taken from PED patients or from HCLE cells treated with control tears or tears of PED patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of a few important genes in CE homeostasis, inflammation, and cell-cell communication, viz., Kruppel-like factor 4 (KLF4), GPX4, IL6, TNFα, STING, IL8, desmoglein, and E-cadherin, among others. Their expressions were normalized with their respective housekeeping genes and fold changes were recorded. KLF4 localization and MMPs activity was carried out via immunofluorescence and zymography, respectively. Results: KLF4, a transcription factor important for CE homeostasis, was upregulated in tears-treated HCLE cells and downregulated in PED patients compared to the healthy PRK group. Cell-cell communication genes were also upregulated in tears-treated cells, whereas they were downregulated in the PED tissue group. Genes involved in proinflammation (IL6, 282-fold; TNFα, 43-fold; IL8, 4.2-fold) were highly upregulated in both conditions. MMP9 activity increased upon tears treatment. Conclusions: This study suggests that tears create an acute proinflammatory milieu driving the PED disease pathology, whereas the PED patients scrapes are an indicator of the chronic stage of the disease. Interferons, pro-inflammatory genes, and their pathways are involved in PED, which can be a potential target for inducing epithelialization of the cornea.
Simple limbal epithelial transplantation (SLET) has emerged as an effective treatment option for limbal stem cell deficiency (LSCD). However, the precise molecular mechanisms underlying its success remain incompletely understood. This review delves into the proposed mechanisms involving the donor limbus, host microenvironment, and the amniotic membrane as a scaffold in SLET. The donor limbus contributes to SLET efficacy through various factors secreted by limbal epithelial stem cells, including hepatocyte growth factor (HGF), soluble Fms-like tyrosine kinase-1 (sFLT-1), and pigment epithelium-derived factor (PEDF), which support corneal healing and transparency. Additionally, the presence of melanocytes, immune cells, limbal fibroblasts, and adhesion molecules within the donor tissue helps preserve the integrity of the limbal niche. The host environment plays a critical role in supporting the transplanted stem cells, with mesenchymal stem cell-secreted factors promoting proliferation and differentiation. Although the amniotic membrane has traditionally been used as a scaffold, emerging evidence suggests that it may not always be necessary. Further studies are needed to validate this scaffold-free approach and to evaluate the vitality and functional contributions of individual components used in SLET. Understanding these complex interactions and molecular mechanisms sheds light on the importance of the donor tissue, host microenvironment, and scaffold in SLET, paving the way for the optimization of this technique for the effective treatment of LSCD.
Congenital aniridia is characterized by partial or complete absence of the iris.[1] The disease manifestations extend to almost every part of the eye, including the cornea, anterior chamber angle, lens, fovea, and the optic nerve.[2] It has a global prevalence of approximately one in 40,000–100,000 live births.[2] Some of the clinical features like congenital nystagmus and foveal hypoplasia are present since birth, while others like cataract, aniridia-associated keratopathy (AAK), and glaucoma may develop later in life.[123] Two-third of the aniridia cases are familial and one-third are a result of de novo (sporadic) germline mutations in the parent of the affected individual.[1] The most common inheritance pattern is autosomal dominant (AD) with high penetrance and variable expression.[123] However, rare autosomal recessive forms have been reported.[4] Aniridia might present as a part of the WAGR syndrome (OMIM 194072; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) due to a contiguous gene deletion encompassing the PAX6 and WT1 genes.[1] Around 30% of individuals detected with sporadic aniridia are likely to develop symptoms of WAGR syndrome, making abdominal ultrasound screening among children mandatory up to 7 years of age.[56] Another phenotypically distinct disease entity called the Gillespie syndrome presents itself as a triad of partial aniridia, non-progressive cerebellar ataxia and intellectual disability.[7] However, a causative gene has recently been identified as ITPR1, an inosine triphosphate receptor with calcium channel activity which is genotypically distinct from PAX6-associated variations.[7] Aniridia can broadly be divided into two subgroups: classic aniridia, encompassing mutations in the PAX6; and aniridia-like, which includes mutations in other genes like FOXC1, PITX2, CYP1B1, FOXD3 and TRIM44.[3] Although, non-PAX6 genes are increasingly being identified to be linked with aniridia, PAX6 defects remain the leading cause of congenital aniridia. Majority of aniridia cases (90%) result from haploinsufficiency in the PAX6, either due to mutations or chromosomal rearrangements and are inherited in AD manner.[26] Genotype–phenotype correlations have been described based on the type of intragenic mutations in the PAX6 gene, wherein the premature termination codon and C-terminal extensions (CTE) lead to a more severe phenotype as compared to the missense mutations.[89] The present case report describes a rare run-on mutation in the PAX6 gene and the specific clinical phenotype of congenital aniridia linked with it. Ethical approval was obtained from the institutional review board of Dr Shroff's Charity Eye Hospital and was consistent with the provisions of the Declaration of Helsinki. A 21-year-old female presented at Dr Shroff's Charity Eye Hospital in October 2018 with complaints of gradually increasing whitish lesion over the black part of both eyes (B/E) associated with photophobia since past few years. The proband (III: 5), her mother (II: 6), younger sister (III: 6), and younger half-brother (III: 3) [Fig. 1] had shaky eyeballs and poor vision in both the eyes since birth. The best corrected visual acuity (BCVA) of the proband was finger counting at 20 cm in right eye (R/E) and finger counting one meter in the left eye (L/E). Media opacity precluded refraction in the R/E. The refractive error was −18.0 D/−8.0 D at 180° in the L/E. Anterior segment examination revealed complete aniridia with only a thin stump of iris tissue in both eyes (B/E). There was centripetal growth of superficial vascularization from the limbus over the cornea, and adjacent stromal opacification. The severity of this AAK was more in the R/E, consequently involving the visual axis [Fig. 2a]. In the L/E, a central island of relatively clear cornea was present [Fig. 2b]. There was superior subluxation of the lens with zonular weakness in meridians 4–8 o'clock in the R/E and 4–7 o' clock in the L/E [Fig. 2c and 2d]. There was diffuse lenticular haze and dot-like lenticular opacities in B/E. Gonioscopy (Ocular Sussman four mirror handheld gonioscope, Ocular Instruments, USA) showed open angles with increased pigmentation, and multiple thin strands of iris tissue bridging the angle [Fig. 2e and 2f]. The intraocular pressure (IOP) measured with rebound tonometry (IC 200, iCare, Finland) was 10 mmHg and 9 mmHg in the R/E and L/E, respectively. The details of the fundus were hazy in the R/E and only mild pallor could be appreciated. The retina findings of the L/E revealed disc pallor, normal cup/disc ratio and absent foveal reflex [Fig. 3]. The corneal thickness was 784 μm and 720μm in R/E and L/E, respectively. The younger sister had similar phenotype in B/E except that the AAK was less severe than the proband. The mother was not available for evaluation.Figure 1: Pedigree of an aniridia family of Indian origin harboring a heterozygous c.1268A>T; p.*423L variant in PAX6. Proband (III: 5) is highlighted with an arrow. Star is used to denote members who gave their blood samples and participated in genetic testingFigure 2: Clinical photographs of anterior segment of proband. (a and b) show the aniridia-associated keratopathy, (c and d) show superior subluxation of the lens (arrow), and (e and f) show the gonioscopic view of the inferior angleFigure 3: Fundus photograph of the left eye of proband (III: 5) showing average-sized disc with pallor, a normal cup/disc ratio and the absence of foveal reflexThe proband was visually rehabilitated with photochromatic glasses. Preservative-free artificial tears eye drops (carboxymethylcellulose, Allergan) were prescribed for the AAK. She was advised for simple limbal stem cell transplantation followed by penetrating keratoplasty for the right eye. However, she denied any surgery considering the high rates of recurrences. She was referred for low vision and assistive devices. Given the unique aniridia phenotype and positive family history, members of the family (III: 5, III: 6, II: 10) were counselled about importance of molecular diagnosis and assessing recurrence risk to offer prenatal diagnostic options. The proband was a young, married individual planning to start a family and her younger sister was unmarried. Pre-conceptional counselling for the proband, and pre-marital counselling for her sister was carried out as they were keen to have unaffected children. Post counselling, they consented for the genetic investigation. Subsequently, molecular genetic investigations were carried out for individuals III: 5, III: 6 and II: 10. Blood samples were collected in EDTA solution. Clinical exome analysis was carried out for the proband (III: 5) by next generation sequencing.[10] Genomic DNA was isolated and in-solution hybridization of the coding exons and flanking intronic regions within the genes tested was performed on the DNA. Illumina NextSeq system was used for paired read sequencing of the amplified captured regions. Sequence obtained was aligned to the human reference genome (GRCh37/hg19) and variant calling was performed using BWAGATK. The analyzed region included all the coding exons and ±10 base pairs (bp) of flanking intronic region on both sides. Only variants in the coding region and the flanking intronic regions with a minor allele frequency of <5% were evaluated. The mutation annotation was carried out using published variants in literature and set of disease databases such as ClinVar, OMIM, GWAS, HGMD and SwissVar. Based on results of the above-mentioned bioinformatics analysis, the mutation identified in the proband was a heterozygous variant (c.1268A>T; p.*423L) causing a stop loss change in PAX6. Sanger sequencing was performed for individuals II: 10 and III: 6. High-fidelity polymerase chain reaction (PCR) was performed on their genomic DNA for amplification of PAX6 gene covering the variant identified in individual III: 5 using separate primer sets. The amplified product was sequenced bi-directionally as described elsewhere by Vanita et al. in 2006.[11] The nucleotide sequence was extracted in FASTA format and aligned using CodonCode Aligner to identify variants and was verified for quality using ChromasLite software. The final result confirmed c.1268A>T; p.*423L in the affected sister (III: 6) too and the unaffected maternal uncle (II: 10) showed wild-type (AA) genotype. Discussion PAX6 (OMIM: 607108) is a crucial transcription factor gene for oculogenesis that is expressed during developmental stages of eye.[12] Several types of mutations in the PAX6 gene such as frameshifts, nonsense, splice site, insertions, deletions, and CTE have been reported for aniridia with other co-occurring ocular anomalies (PAX6 webpage - MRC Human Genetics Unit LOVD at MRC IGMM - Leiden Open Variation Database, 2022)).[13] C-terminal extensions in PAX6 cause the reading frame to continue protein coding into the 3' UTR and leads to gain-of-function and severe aniridic phenotype.[2] The c.1268A>T substitution observed in present study is a CTE mutation leading to loss of the stop codon at the 3' end and leading to addition of 21 to 24 Poly-dAs, as also previously reported by Singh et al.[14] c.1268A>T has previously been reported in few individuals with aniridia in different studies across the globe [Table 1]. Compared to the earlier reports, the unique clinical features of the affected individuals with aniridia in the current study are superior subluxation of the lens, high myopia and optic atrophy. These features are similar to those reported by Souzeau et al. in a Cambodian family.[15]Table 1: Aniridia cases reported to be linked with c.1268A>T; p.*423L in PAX6The minor allele frequency for c.1268A>T substitution is not reported in the general population. In silico analysis of c.1268A>T supports its deleterious effect. Mutation Taster, CADD, DANN, FATHMM, Eigen and GenoCanyon predicted it to be disease causing/damaging. Validation by Sanger sequencing confirmed c.1268A>T substitution in PAX6 in the proband as well her sister in heterozygous form, while their unaffected maternal uncle had wild-type genotype. Affected mother of proband could not be tested for c.1268A>T in PAX6 due to geographical limitations. Based on the segregation of variant with the phenotype in the present study and previously published reports [Table 1], c. 1268A in PAX6 seems to be a mutation hot-spot for aniridia. The treatment of aniridia entails treating its various clinical manifestations individually.[13] The foveal hypoplasia and the nystagmus do not have any definitive treatment. Cataract surgery may benefit those with significant lenticular opacities or lens subluxation.[20] Since the lenticular opacity and subluxation were mild in this patient, they did not warrant any treatment. Cataract extraction, when indicated, may be combined with iris reconstruction with various iris prosthetic devices.[21] AAK may benefit in early stages with artificial tears and autologous serum.[1] However, in advanced stages when the visual axis is affected, restoration of the limbal stem cell niche followed by keratoplasty has been shown to have limited success.[1] To conclude, through this case report, we have described the detailed phenotype of the c.1268A>T; p.*423L run-on mutation in the PAX6 gene in an Indian family. We have demonstrated the likely pathogenic modality of this mutation by segregation analysis among affected and unaffected members of this family, as well as in silico analysis. To the best of our knowledge, this mutation has not been reported previously in the Indian population. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
We read with great interest the article by Ağca et al.1 This study is commendable in comparing 2 different protocols with analysis of 5-year follow-up after intervention in the pediatric population. We feel that there are few points requiring further clarity in this article. There is a disparity in description of both groups at 3 different points in the article. In the Abstract, it is mentioned that they divided the patients into 2 groups and that Group 1 received 4 minutes illumination at 30 mW/cm2 and Group 2 received 5 minutes illumination at 18 mW/cm2. However, in the Methods section, it is stated that Group 1 received 5 minutes illumination at 18 mW/cm2 and Group 2 received 4 minutes illumination at 30 mW/cm2. The surgical technique description of 2 groups was that Group 1 received 30 mW/cm2 and Group 2 received 18 mW/cm2 irradiances at 5 minutes each with no difference in the amount of time taken in the technique. We found a disparity in the mention of study designs in the Abstract and the Methods. The Abstract states the study design was a retrospective case–control study, and the Methods section states it was a retrospective interventional case series. We feel the study design would be a retrospective nonrandomized interventional case series because there are no well-defined cases and controls in the study. Although this study reported that keratometric progression rates were similar over the 3-year and 5-year follow-up period, 23.3% vs 16.8% in Groups 1 and 2, respectively (P = .411), there is no mention of the use of anterior segment optical coherence tomography to look for the demarcation line to assess the effectiveness of the crosslinking (CXL) procedure in both groups. So, it is difficult to prove whether the CXL procedure alone could explain this effect. There are several confounders related to the progression of keratoconus significant in the pediatric population, for example, history of allergy or atopy and the stage of keratoconus when the intervention was performed, which have not been mentioned by the authors.2 The authors reported that there were no complications, but the endothelial cell density was not documented at any visit. This parameter is important considering the long-term effects of CXL with higher irradiances. Cingü et al. found significant endothelial cell changes in both density and morphology (coefficient of variation and 6A) after accelerated CXL (18 mW/cm2 for 5 minutes), which returned to baseline at 6 months.3 Bhandari et al. used more intensive ultraviolet A irradiance (30 mW/cm2 for 3 minutes) and reported endothelial changes that did not return to baseline.4 As mentioned by the authors as one of the study limitations, we also feel that the unequal distribution of children in the 2 groups could also influence the comparative results.
Cornea-related injuries are the most common cause of blindness worldwide. Transplantation remains the primary approach for addressing corneal blindness, though the demand for donor corneas outmatches the supply by millions. Tissue adhesives employed to seal corneal wounds have shown inefficient healing and incomplete vision restoration. We have developed a biodegradable hydrogel - Kuragel, with the ability to promote corneal regeneration. Functionalized gelatin and hyaluronic acid form photo-crosslinkable hydrogel with transparency and compressive modulus similar to healthy human cornea. Kuragel composition was tuned to achieve sufficient adhesive strength for sutureless integration to host tissue, with minimal swelling post-administration. Studies in the New Zealand rabbit mechanical injury model affecting corneal epithelium and stroma demonstrate that Kuragel efficiently promotes re-epithelialization within 1 month of administration, while stroma and sub-basal nerve plexus regenerate within 3 months. We propose Kuragel as a regenerative treatment for patients suffering from corneal defects including thinning, by restoration of transparency and thickness.
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