Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas.

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

Direct Immunofluorescence Findings and Factors Affecting Conjunctival Biopsy Positivity in Ocular Mucous Membrane Pemphigoid.

PURPOSE: The aim of this study was to describe the direct immunofluorescence (DIF) findings and factors affecting conjunctival biopsy positivity in patients clinically diagnosed with ocular mucous membrane pemphigoid (OMMP). METHODS: This retrospective observational case series included patients with clinical OMMP who underwent conjunctival biopsy for DIF in at least 1 eye between 2018 and 2021 in an institutional setting. The primary outcome measures were association of age and chronic ocular complications with biopsy positivity. RESULTS: Of 61 patients, DIF positivity was seen in 33 (54.1%) clinically suspected cases of OMMP. Of 39 patients who underwent bilateral biopsy, 23 (59%) were positive, of which 12 (52%) were positive in both eyes while 11 (48%) were positive in 1 eye. Of 22 patients who underwent unilateral biopsy, 10 (45%) were positive. Of the 100 biopsied eyes, 45 (45%) were DIF positive. Among the immunoreactants studied, linear deposition of C3 was seen in all 45 positive eyes (100%). Increasing age was significantly associated with higher likelihood of biopsy negativity ( P = 0.032), whereas a greater Sotozono chronic ocular complication score, indicative of disease severity, was associated with low likelihood of biopsy positivity ( P = 0.0042) and lower overall expression of immunoreactants on DIF ( P = 0.0007). CONCLUSIONS: Older patients and patients with more severe ocular surface disease sequelae are likely to have negative DIF results. To optimize the chances of confirming the diagnosis of OMMP by DIF, both eyes should be biopsied early in the disease course. If 1 eye is being biopsied, the less affected eye must be chosen.

Ocular surface pseudoepitheliomatous hyperplasia secondary to allergic eye disease: clinical features and management.

PURPOSE: To study the clinical characteristics and treatment outcomes of ocular surface pseudoepitheliomatous hyperplasia (PEH) associated with chronic vernal keratoconjunctivitis (VKC). METHODS: This retrospective study includes 39 eyes of 32 patients with VKC induced PEH who presented from 2016-2022. A database search was conducted for diagnosis of PEH, and data on clinical features, imaging characteristics, and treatment were analyzed. RESULTS: Of the 32 patients, 11 (34%) were children and adolescents, 21 (66%) were adults. PEH was common in males (72%) and ocular surface squamous neoplasia (OSSN) was the commonest referral diagnosis (43.7%). Mean age at presentation was 26.62 ± 10.18 (range: 6-52) years. While history of VKC was present in 21 patients, 11 were diagnosed with VKC at the time of diagnosis of PEH. The mean base/largest diameter was 5.2 ± 1.67 mm. Anterior segment optical coherence tomography (AS-OCT) showed irregular hyper-reflective epithelium, epithelial dipping, and sub-epithelial hyper-reflective lesion with shadowing in all lesions. Of the 31 eyes that received medical therapy, 21 (67%) and 10 (32%) eyes showed complete and partial resolution respectively with median time to resolution of 3(IQR:2-4) months. Eight eyes that underwent surgical excision showed complete resolution and one developed partial limbal stem cell deficiency. CONCLUSION: Ocular surface PEH is a manifestation of chronic VKC which closely mimics OSSN. Detailed history-taking, examination for signs of allergy, and AS-OCT imaging can distinguish it from OSSN. It responds well to medical therapy and should be considered first-line therapy before planning any surgical intervention.

Ocular infections associated with atypical mycobacteria: A review.

Atypical mycobacteria or non-tuberculous mycobacteria (NTM) are a group of acid-fast bacteria that are pathogenic to different parts of the eye. The organisms can cause a spectrum of ocular infections including keratitis, scleritis, uveitis, endophthalmitis and orbital cellulitis. Trauma, whether surgical or nonsurgical, has the highest correlation with development of this infection. Common surgeries after which these infections have been reported include laser in situ keratomileusis (LASIK) and scleral buckle surgery. The organism is noted to form biofilms with sequestration of the microbe at different inaccessible locations leading to high virulence. Collection of infective ocular material (corneal scraping/necrotic scleral tissue/abscess material/vitreous aspirate, etc.) and laboratory identification of the organism through microbiologic testing are vital for confirming presence of the infection and initiating treatment. In cluster infections, tracing the source of infection in the hospital setting via testing of different in-house samples is equally important to prevent further occurrences. Although the incidence of these infections is low, their presence can cause prolonged disease that may often be resistant to medical therapy alone. In this review, we describe the various types of NTM-ocular infections, their clinical presentation, laboratory diagnosis, management, and outcomes.

Metastatic retinoblastoma at presentation: Clinical presentation, treatment, and outcomes.

The aim of this study was to retrospectively determine clinical features, treatment outcomes, and overall survival in four patients with metastatic retinoblastoma at presentation. The mean age at diagnosis was 63 months (range: 24-108 months). Three patients had overt orbital disease of at least one eye and one patient had microscopic orbital disease with scleral infiltration on histopathology. Metastatic sites included regional lymph nodes (RLN) (n = 4), bone marrow (BM) (n = 2), and cerebrospinal fluid (CSF) (n = 1). The most common sites of RLN were ipsilateral preauricular nodes (two patients) and contralateral parotid gland involvement (one patient). The treatment administered included primary enucleation (n = 1), high-dose intravenous chemotherapy (n = 4), secondary enucleation (n = 2), orbital external beam radiotherapy (n = 3), and intrathecal chemotherapy (n = 1). High-risk features included massive choroidal and microscopic scleral infiltration in the eye that underwent primary enucleation. At a mean follow-up of 33 months (range, 4-68 months), one patient with CSF involvement deceased in 4 months. The remaining three patients were alive and disease-free at the last mean follow-up period of 43 months (range, 18-68 months). The results of our study showed that RLN and BM metastasis respond well to treatment while CSF metastasis is associated with poor prognosis.

Multifocal choroidal melanoma: a clinicohistopathological correlation.

A woman in her late 50s presented with on-and-off redness and diminution of vision in her left eye for 6 months. Her best corrected visual acuity was 20/40 in the right eye and hand motion in the left eye. Anterior segment examination revealed a greyish-white lesion extending from 3 to 6 o'clock hours posterior to the iris and protruding into the anterior chamber. Left eye B-scan ultrasonography showed a multifocal choroidal lesion, a smaller one involving the posterior pole, and a larger lesion involving the complete nasal quadrant and anteriorly extending to the ciliary body and iris. Fine-needle aspiration biopsy performed from the anterior lesion showed a possible neoplastic aetiology of melanocytic origin of the cells. Finally, the patient underwent left eye enucleation with a ball implant. Histopathological examination of the enucleated eye confirmed the final diagnosis of multifocal choroidal melanoma involving the adjacent ciliary body and iris.

Seeing beyond Gaucher disease: Early detection and treatment of ocular complications.

Background: Gaucher disease is a rare genetic disorder caused by a deficiency in the enzyme glucocerebrosidase, which impairs the body's ability to break down certain fats. This leads to the accumulation of glucosyl sphingosine and glucosyl ceramide in the liver, spleen, and bone marrow. Gaucher disease has two major types: nonneuropathic (Type 1) and neuropathic (Type 2 and Type 3). Gaucher disease can have various ophthalmologic manifestations, particularly in Type 3, including posterior segment abnormalities, such as vitreous opacities, condensations, and/or preretinal white dots. We present a case of a patient with Gaucher disease Type 3 who had severe bilateral vitreous and extensive retinal deposits, leading to challenges during surgery. Purpose: This video reports surgical outcomes for an uncommon ophthalmologic manifestation in a patient with Gaucher disease Type 3. We focus on the challenges and results of surgery for severe bilateral vitreous and extensive retinal deposits. Synopsis: A 16-year-old female patient with a history of Gaucher's disease since birth presented with a one-year history of blurred vision in both eyes. Her best-corrected visual acuity was 20/200 in the right eye and 20/100 in the left eye, as measured by Snellen's chart. Intraocular pressure was normal in both eyes, and anterior segment examinations were unremarkable. However, fundus evaluation revealed extensive vitreous deposits that obscured the details of the fundus. Additionally, an epiretinal membrane was observed over the macula in both eyes. Optical coherence tomography (OCT) confirmed the presence of deposits in the vitreous cavity and on the surface of the retina. The patient underwent pars plana vitrectomy with epiretinal membrane removal. A transconjunctival 23-G pars plana vitrectomy was performed to the extent possible. Multiple instruments were used to remove the fluffy vitreous deposits, as they were extremely adherent to the underlying surface of the retina, and brilliant blue dye was used to stain the internal limiting membrane. The epiretinal membrane and internal limiting membrane were removed from the macular area, and the entire cassette fluid was sent for histopathological examination to identify Gaucher cells. At one week postoperative, the patient's visual acuity improved to 20/125 in the right eye, and the fundus picture showed a cleared macular area. OCT showed a reduction in deposits over the retina. The histopathological examination revealed crumpled, barrel-like cytoplasm with an oval nucleus in a hemorrhagic background, suggestive of Gaucher cells. Highlights: Early detection and treatment of ocular manifestations of Gaucher's disease are important to prevent permanent damage to vision. An ophthalmological assessment involving a dilated fundus examination and optical coherence tomography can facilitate early diagnosis and follow-up of ocular manifestations. Timely surgery may be required to preserve functional vision in patients with severe ocular disease. Video Link: https://youtu.be/KR-kfgfDoqM.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

Purpose: To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Methods: Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Conclusions: Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

Anterior segment optical coherence tomography features of ocular surface squamous epithelial hyperplasia: A report of four cases.

Herein, we report the clinico-tomographic and histopathological features of four patients with biopsy-proven ocular surface squamous epithelial hyperplasia (OSSEH), a close mimicker of ocular surface squamous neoplasia (OSSN). The mean age at diagnosis was 58 years (median, 60 years; range, 35-77 years). All lesions were unilateral. Isolated corneal plaque was seen in 50% (n = 2) and nodular lesion at the nasal limbus in 50% (n = 2). Keratinization was seen in 75% (n = 3) of lesions and intrinsic vessels in 75% (n = 3). A clinical diagnosis of OSSN was made in all cases. Anterior segment optical coherence tomography (ASOCT) revealed mild epithelial hyperreflectivity in 100% (n = 4). The epithelium was normal in thickness in 75% (n = 3) and showed mild thickening in 25% (n = 1). Only 25% (n = 1) showed abrupt transition in epithelial thickness from the contiguous corneal epithelium. Histopathological examination revealed hyperplastic squamous epithelium and no cellular atypia in all cases. Stable ocular surface and no recurrences were noted at a mean follow-up of 17 months (median, 11 months; range, 2-43 months). Although OSSEH can mimic OSSN clinically, the presence of mild epithelial hyperreflectivity, lack of epithelial thickening, absence of abrupt transition from normal epithelium, and presence of subepithelial hyperreflectivity on ASOCT favor the diagnosis of OSSEH.

Cicatricial Entropion in Chronic Cicatrizing Conjunctivitis: Potential Pathophysiologic Mechanisms and Long-Term Outcomes of a Modified Technique.

PURPOSE: The purpose of this study was to assess the long-term outcomes of severe cicatricial entropion repair with mucous membrane grafting in patients with chronic cicatrizing conjunctivitis and report histopathological changes in the eyelid margin area. METHODS: Prospective interventional study included 19 patients with severe cicatricial entropion with trichiasis (N = 20 eyelids; 19 upper and 1 lower eyelid) who underwent anterior lamellar recession (with back cuts) and mucous membrane grafting cover for bare anterior tarsus, lid margin, and 2 mm of marginal tarsus, and had a minimum 6 months of follow-up. The anterior lamella and metaplastic eyelid margins were sent for routine Haematoxylin and Eosin and special staining with Masson trichrome stain. RESULTS: The etiologies were chronic Stevens-Johnson syndrome (N = 6), chemical injury (N = 11), and drug-induced pseudopemphigoid (N = 2). Five eyes had undergone entropion correction in the past, and 9 had electroepilation for trichiasis. Entropion was well corrected (without residual trichiasis) in 85% of eyelids with primary surgery. The etiology-wise success rates were 100% for Stevens-Johnson syndrome, 72.7% for chemical injury, and 100% for drug-induced pseudopemphigoid. Three eyelids with failure belonged to chemical injury, and trichiasis in these eyes could be managed with subsequent interventions except in 1 case. All eyelids had no entropion at a mean follow-up of 10.8 months (range, 6-18). Histopathological evaluation of anterior lamella (N = 10) and eyelid margins revealed significant fibrosis in subepithelial, perimysium (muscle of Riolan), and perifollicular areas. CONCLUSION: Anterior lamellar recession combined with mucous membrane grafting achieves good cicatricial entropion correction except in eyes with chemical injury. The eyelid margins in these eyes have persistent inflammation, and fibrosis involving lash follicles.

A review of rabbit models of meibomian gland dysfunction and scope for translational research.

Dry eye disease (DED) is an emerging global health concern with meibomian gland dysfunction (MGD) being the most common subtype of DED. Despite being quite prevalent, the pathophysiological mechanisms governing MGD are poorly understood. Animal models for MGD can be a valuable resource to advance our understanding of this entity and explore novel diagnostic and therapeutic modalities. Although a lot of literature on rodent MGD models exists, a comprehensive review on rabbit animal models is lacking. Rabbits offer a great advantage over other animals as models for studying both DED and MGD. Rabbits have a widely exposed ocular surface and meibomian gland anatomy comparable with humans, which makes performing dry eye diagnostic tests possible using clinically validated imaging platforms. The existing MGD models in rabbits can broadly be classified as pharmacologically induced and surgically induced models. Most models show keratinization of the meibomian gland orifice with plugging as the final common pathway for developing MGD. Thus, understanding the advantages and disadvantages of each rabbit MGD model can help researchers choose the appropriate experimental plan based on the objective of the study. In this review, we discuss the comparative anatomy of the meibomian glands in humans and rabbits, various rabbit models of MGD, translational applications, unmet needs, and future directions in developing MGD models in rabbits.

Ocular surface squamous neoplasia with orbital tumour extension: risk factors and outcomes.

PURPOSE: To describe the risk factors, clinical features, histopathology, treatment, and outcomes of patients with orbital tumour extension of ocular surface squamous neoplasia (OSSN). METHODS: Retrospective study of 51 patients with orbital tumour extension (cases) and 360 patients without orbital extension (controls). RESULTS: Of 1,653 patients with OSSN, orbital tumour extension was noted in 51 (3%) cases. The risk factors for orbital tumour extension included outdoor occupation (p < 0.03; Odds ratio (OR) = 1.96), Human Immunodeficiency Virus (HIV) infection (p < 0.0001; OR = 5.81), prolonged duration of symptoms (p = 0.01; OR = 1.02), tumour bilaterality (p = 0.02; OR = 2.92), forniceal and tarsal conjunctival involvement, diffuse tumour (p < 0.0001; OR = 9.13), inferior quadrantic location (p < 0.0001; OR = 7.51), increased tumour thickness (p = 0.04; OR = 1.59), higher % of ocular surface involvement (p = 0.002; OR = 1.12), nodular (p = 0.002; OR = 2.61) and nodulo-ulcerative (p < 0.0001; OR = 11.05) tumour morphology, poorly differentiated tumours (p = 0.006; OR = 4.23); invasive squamous cell carcinoma (SCC) (p < 0.0001; OR = 29.76), spindle cell and mucoepidermoid variant (p = 0.02; OR = 16.94) tumours. At a mean follow-up period of 27 months, tumour recurrence in the socket was noted in 1 (2%), locoregional lymph node metastasis (LNM) in 15 (29%) patients, and nine (18%) patients died due to systemic metastasis (SM). T4 tumour at presentation was a risk factor for LNM (p = 0.01; Hazard ratio (HR) = 5.60) and SM (p = 0.0003; HR = 5.09). CONCLUSION: Orbital extension of OSSN is rare. Outdoor occupation, HIV infection, larger and thicker tumours in the inferior quadrant with forniceal and/or tarsal conjunctival involvement with nodular or noduloulcerative morphology, poor tumour differentiation, SCC, spindle cell and mucoepidermoid variants on histopathology are at increased risk for orbital tumour extension.

Mixed B- and T-lymphocyte Vitreous Infiltrate in Multiple Sclerosis Associated Uveitis.

A 23-year-old man, under treatment for relapsing remitting multiple sclerosis, presented with sudden drop in vision in the left eye for the past 1 week. We noted optic atrophy with sclerosed vessels in multiple quadrants in both eyes, moderate vitreous haze, and active retinal vasculitis in left eye. The patient received therapeutic pars plana vitrectomy in the left eye, and the vitreous sample was analyzed for immunophenotypes by flow cytometry (T-cells) and immunohistochemistry (B-cells). 65.1% of total vitreous cells were CD3+ T-cells. These included 42.4% CD4+, and 20.6% CD8+ T-cells. Immunohistochemistry detected CD20+ B-cells (not quantifiable). Our analysis demonstrated a mixed B- and T-lymphocyte vitreous infiltrate in multiple sclerosis-associated uveitis.

Comprehensive Analysis of Serum Small Extracellular Vesicles-Derived Coding and Non-Coding RNAs from Retinoblastoma Patients for Identifying Regulatory Interactions.

The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 1011 ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.

Conjunctival Autograft for Bilateral Tarsal Keratinization in a Case of Chronic Vernal Keratoconjunctivitis.

This report describes the clinical features and management in a case of vernal keratoconjunctivitis (VKC) with bilateral tarsal conjunctival keratinization. A 32-year-old male presented with VKC since childhood that had exacerbated in the eight years prior to presentation. Examination revealed partial limbal stem cell deficiency in both eyes, with keratinization of the superior tarsal conjunctiva. The corresponding areas of the cornea exhibited punctate keratopathy in both eyes. To address this, the patient underwent excision of the conjunctival keratinization in both eyes. The resultant bare areas were covered with conjunctival autografts (CAGs). Postoperatively, the grafts were well apposed, and there was no recurrence of keratinization observed during the period of follow-up of four years. Resolution of corneal epitheliopathy was also noted. Although keratinization can occur in eyes with VKC, it is usually limited to the bulbar conjunctival areas. This is the first report of tarsal conjunctival keratinization in such cases. Milder cases may be observed or managed with scleral contact lenses. In more severe forms, there is associated corneal epitheliopathy, which may progress to corneal vascularization and scarring. Surgical excision of the lesion is recommended in these eyes. Following excision, several options exist to cover the bare area, which include a CAG, an amniotic membrane, or an oral mucous membrane. Of these, a CAG is an autologous tissue that can be harvested with a simple surgical technique and yields stable long-term results. Thus, tarsal conjunctival keratinization is a rare complication of chronic VKC. Excision of the lesion followed by a CAG is a viable approach for treatment, which reestablishes and maintains a stable ocular surface.

Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.