Purpose. To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis. Methods. We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls. Results. Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively. Conclusions. Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions.

Purpose A case of ocular pythiosis successfully treated with surgery and intraocular and oral minocycline is reported. Summary A 30-year-old man who wore corrective contact lenses traveled to Brazil and Colombia where he swam in salt and fresh waters while wearing contact lenses. He sought treatment at an emergency department after 2 weeks of suffering with a painful corneal ulcer, redness, and loss of vision in his right eye that had been treated at other centers with ophthalmic moxifloxacin for 10 days and with fortified topical antibiotics (amikacin and vancomycin) for 2 days. Examination using a slit lamp revealed a deep central corneal ulcer with surrounding white infiltrate, endothelial plaque, and hypopyon. Due to infection severity, the patient was admitted and received empirical antibiotic therapy and i.v. and topical antifungals. During the first corneal transplantation, the patient’s original infection relapsed and was treated with voriconazole and liposomal amphotericin B intraocular injections. A subsequent infection developed, and a second keratoplasty was performed. One month after hospital admission, the patient was diagnosed with ocular pythiosis and therapy with oral minocycline was initiated. After severe infection relapse in the anterior chamber, the patient underwent a third penetrating keratoplasty, where minocycline intraocular injection was administered. After this intervention, complete infection control was achieved, and the patient was discharged 45 days after admission with oral minocycline and 1% cyclosporine and 0.3% ofloxacin eye drops. Conclusion A patient with ocular pythiosis was successfully treated with penetrating keratoplasty and 2 months of treatment with intracameral and oral minocycline.

To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

Posterior polymorphous corneal dystrophy (PPCD), a rare, bilateral, autosomal-dominant, inherited corneal dystrophy, affects the Descemet membrane and corneal endothelium. We describe an unusual presentation of PPCD associated with a previously unknown genetic alteration in the ZEB1 gene. The proband is a 64-year-old woman diagnosed with keratoconus referred for a corneal endothelium study who presented endothelial lesions in both eyes suggestive of PPCD, corectopia and iridocorneal endothelial synechiae in the right eye and intrastromal segments in the left eye. The endothelial count was 825 in the right eye and 1361 in the left eye, with typical PPCD lesions visible under specular and confocal microscopy. In the next generation sequencing genetic analysis, a heterozygous c.1A > C (p.Met1Leu) mutation was found in the ZEB1 gene (TCF8). The PPCD3 subtype is associated with corneal ectasia, and both can appear due to a pathogenic mutation in the ZEB1 gene (OMIM #189909). However, our patient had a previously unreported mutation in the ZEB1 gene, which mediates the transition between cell lines and provides a pathogenic explanation for the epithelialisation of the corneal endothelium, a characteristic of PPCD.