Prevalence, Anatomic Distribution, and Correlates of Chlamydia trachomatis and Neisseria gonorrhoeae Infections Among a Cohort of Men Who Have Sex With Men in Hanoi, Vietnam.

BACKGROUND: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) disproportionately affect men who have sex with men (MSM). Data on the prevalence, anatomical distribution, and correlates of NG and CT infections among MSM in Vietnam are limited. METHODS: Between July 2017 and April 2019, MSM 16 years or older without HIV were enrolled into an observational cohort study. Baseline data, including sociodemographics, sexual behavior, and HIV status, were collected. Testing for NG and CT were performed on urine, rectal, and pharyngeal specimens. Multivariate logistic regression models identified factors associated with NG and CT infections at baseline. RESULTS: In total, 1489 participants underwent NG/CT testing. The median age was 22 years (interquartile range, 20-26 years). There were 424 (28.5%) NG or CT infections: 322 (21.6%) with CT and 173 (11.6%) with NG. Rectal infections were most common for CT (73.9%), whereas pharyngeal infections were most common for NG (70.5%). Independent risk factors for CT or NG infection included ≥2 sex partners in the prior month (adjusted odds ratio [aOR], 2.04; 95% confidence interval [CI], 1.44-2.91), condomless anal sex (aOR, 1.44; 95% CI, 1.12-1.86), and meeting sex partners online (aOR, 1.35; 95% CI, 1.03-1.76). Recent genitourinary or rectal symptoms were not associated with infections. CONCLUSIONS: The overall and extragenital prevalences of NG and CT infections were high within this sample of young MSM without HIV in Hanoi. Testing limited to urethral specimens would have missed nearly three-quarters of CT and NG infections, supporting the need for routine testing at multiple anatomic sites.

A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana.

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

Light scattering measurements in electron-beam sterilized corneas stored in recombinant human serum albumin.

Irradiated corneal tissues have been used for a variety of ophthalmic procedures including glaucoma drainage device covers and lamellar grafts. The maintenance of corneal clarity is important, as light obstructions resulting from processing or long-term storage of irradiated corneas may negatively affect vision and postoperative cosmesis. It has been reported that corneal tissues can be preserved in human serum albumin (HSA), however, the clarity of corneas after long-term storage in HSA has not been well described. Furthermore, the use of donor-pooled serum increases the risk for transmission of blood-borne diseases and may induce an immune response in the recipient. Here, we examined changes in corneal clarity due to electron-beam (e-beam) irradiation and storage in a recombinant human serum albumin (rHSA). Dark-field microscopy was employed to examine the light scattering effects of fresh and irradiated corneas. Compared to measurements taken prior to tissue preparation and e-beam treatment, irradiated corneas showed an average 2.6% increase in light scattering (P = 0.002). Irradiated corneas stored in rHSA at room-temperature for 20 months showed an average increase of 11.6% light scattering compared to fresh corneas (P â‰ª 0.01), but did not negatively affect the visualization of printed text, and were deemed suitable for transplant use. Therefore, the slight increase in cornea light scattering, and resulting reduction in corneal clarity, after e-beam treatment and long-term storage in rHSA may not be clinically significant. These results suggest that e-beam sterilized corneal grafts may be used as an alternative to fresh tissue for certain ophthalmic applications.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

Flagellar membrane proteins in kinetoplastid parasites.

All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

Both sequence and context are important for flagellar targeting of a glucose transporter.

Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins.

Long-Term Results of Sterile Corneal Allograft Ring Segments Implantation in Keratoconus Treatment.

PURPOSE: To evaluate the efficacy and safety of sterile corneal allograft ring segments implantation for the treatment of keratoconus by analyzing long-term visual, refractive, and tomographic clinical outcomes. METHODS: This prospective study included 62 eyes of 49 patients with keratoconus who underwent corneal allograft ring segments implantation at Istanbul Medipol University Faculty of Medicine between February 2020 and August 2022. Surgical outcomes using the Istanbul nomogram were evaluated in patients preoperatively and postoperatively at 1 month, 6 months, 1 year, and 3 years. Outcomes measured were uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), spherical equivalent (SE), spherical refraction (SR), cylindrical refraction (CR), topographic keratometric values, and corneal thickness at the thinnest point. RESULTS: Preoperative mean UDVA and CDVA (LogMAR) were 0.96 ± 0.50 and 0.72 ± 0.47, respectively, and increased to 0.41 ± 0.34 and 0.22 ± 0.19 at the last visit (P < 0.001). There was a significant decrease in SE, SR, and keratometric values postoperatively (P < 0.001). There was no difference in CR and thinnest corneal thickness values (P = 0.333 and 0.154, respectively). The stromal and epithelial thicknesses measured by anterior segment optical coherence tomography were stabilized at 6 months and 1 year, respectively. No major complications or side effects were observed intraoperatively or postoperatively. CONCLUSIONS: This study demonstrated that sterile corneal allograft ring segments implantation is a safe and feasible treatment for keratoconus, yielding notable long-term visual outcomes with minimal implant-related complications.

Simple Modification of a DSAEK Carrier Enables Graft Insertion by Injection Without Compromising Endothelial Cell Viability.

PURPOSE: We evaluate the quality and feasibility of preloading Descemet stripping automated endothelial keratoplasty (DSAEK) grafts into a modified EndoGlide Ultrathin system for graft injection. METHODS: DSAEK grafts were prepared by experienced processing technicians at 2 separate locations, loaded into a modified EndoGlide Ultrathin, and placed in storage media. Grafts processed at one location were shipped cross-country overnight to the other location and were examined on arrival for positioning within the modified EndoGlide Ultrathin. All grafts were ejected and analyzed for endothelial cell loss (ECL) with calcein acetoxymethyl staining and FIJI segmentation. A subset of grafts was measured by optical coherence tomography for graft thickness 1 hour after cut, 1 hour after loading, and 1 day after loading. RESULTS: No grafts were displaced from the modified carrier over 3 shipping events (n = 9), and all grafts (n = 18) were successfully ejected. Grafts loaded into the modified carrier and ejected exhibited no more cell loss than grafts loaded into the standard carrier and removed by pull-through (14.0% ± 2.8% vs. 12.2% ± 3.4%, respectively, P = 0.24). Carrier modification skills can be successfully transferred as grafts loaded by a processing technician new to carrier modification were within the acceptable limit of 25% ECL for transplant DSAEK grafts. Graft thickness increased significantly (P < 0.05) between the postcut and 1-hour postload measurement and the postcut and 24-hour postload measurement. CONCLUSIONS: The EndoGlide Ultrathin can be modified to enable its use for graft injection while not compromising the ability to use the pull-through method for graft delivery. Preloaded DSAEK grafts swell significantly during the 24-hour storage period, and patterns of ECL may be linked to swelling.

E2-2 protein and Fuchs's corneal dystrophy.

BACKGROUND: Fuchs's corneal dystrophy (FCD) is a leading cause of corneal transplantation and affects 5% of persons in the United States who are over the age of 40 years. Clinically visible deposits called guttae develop under the corneal endothelium in patients with FCD. A loss of endothelial cells and deposition of an abnormal extracellular matrix are observed microscopically. In advanced disease, the cornea swells and becomes cloudy because the remaining endothelial cells are not sufficient to keep the cornea dehydrated and clear. Although rare genetic variation that contributes to both early-onset and typical late-onset forms of FCD has been identified, to our knowledge, no common variants have been reported. METHODS: We performed a genomewide association study and replicated the most significant observations in a second, independent group of subjects. RESULTS: Alleles in the transcription factor 4 gene (TCF4), encoding a member of the E-protein family (E2-2), were associated with typical FCD (P=2.3x10(-26)). The association increased the odds of having FCD by a factor of 30 for persons with two copies of the disease variants (homozygotes) and discriminated between case subjects and control subjects with about 76% accuracy. At least two regions of the TCF4 locus were associated independently with FCD. Alleles in the gene encoding protein tyrosine phosphatase receptor type G (PTPRG) were associated with FCD (P=4.0x10(-7)), but the association did not reach genomewide significance. CONCLUSIONS: Genetic variation in TCF4 contributes to the development of FCD. (Funded by the National Eye Institute and others.)

Recombineering Hunchback identifies two conserved domains required to maintain neuroblast competence and specify early-born neuronal identity.

The Hunchback/Ikaros family of zinc-finger transcription factors is essential for specifying the anterior/posterior body axis in insects, the fate of early-born pioneer neurons in Drosophila, and for retinal and immune development in mammals. Hunchback/Ikaros proteins can directly activate or repress target gene transcription during early insect development, but their mode of action during neural development is unknown. Here, we use recombineering to generate a series of Hunchback domain deletion variants and assay their function during neurogenesis in the absence of endogenous Hunchback. Previous studies have shown that Hunchback can specify early-born neuronal identity and maintain 'young' neural progenitor (neuroblast) competence. We identify two conserved domains required for Hunchback-mediated transcriptional repression, and show that transcriptional repression is necessary and sufficient to induce early-born neuronal identity and maintain neuroblast competence. We identify pdm2 as a direct target gene that must be repressed to maintain competence, but show that additional genes must also be repressed. We propose that Hunchback maintains early neuroblast competence by silencing a suite of late-expressed genes.

Double-Scroll Formation by Fluid Column Manipulation in Preloaded DMEK Grafts Prepared From Younger and Older Donor Tissue.

PURPOSE: The purpose of this study was to determine whether manipulation of preloaded single-scroll Descemet membrane endothelial keratoplasty (DMEK) grafts within the fluid column of an injector can safely and reliably result in formation of double-scroll DMEK grafts and whether there are differential effects on younger versus older donor tissue. METHODS: Pairs of DMEK grafts prepared from older (65-80 years) and younger (48-64 years) donors were preloaded into a Straiko modified Jones tube. One member of the pair was manipulated within the fluid column to form a double-scroll graft, and the other remained unmanipulated. Outcomes measured include success rate for double-scroll formation, endothelial cell loss (ECL), and relative scroll width. RESULTS: Older donor grafts formed double scrolls with a 100% success rate. ECL of older donor manipulated grafts was statistically higher than that of unmanipulated mate grafts (17.4% ± 3.5% vs. 13.0% ± 4.2%, P = 0.03), but was still within the acceptable range for transplant. Younger donor grafts were successfully manipulated into double scrolls with a 67% success rate, and there was no difference in the ECL of manipulated and unmanipulated grafts (15.5% ± 4.4% vs. 13.0% ± 4.5%, P = 0.24). For all grafts and conformations, there was a significant relationship between relative scroll width and ECL ( P < 0.01). CONCLUSIONS: Fluid column manipulation can be used reliably to form double-scroll DMEK grafts. For younger donor grafts, manipulation yields a double scroll without increasing ECL. For older donor grafts, manipulation results in a minimal, acceptable increase in ECL. Surgeons should weigh the advantage of an easily opened graft against the risk of increased ECL when considering this technique.

Prospective In Vitro Comparison of Kerasave and Optisol-GS Corneal Storage Solutions.

PURPOSE: The aim of this study was to compare the performance of Kerasave and Optisol-GS for hypothermic corneal storage for 14 days. METHODS: This study was a prospective laboratory investigation. Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2°C to 8°C for 14 days. Corneas were evaluated by trained eye bank technicians, and study parameters were compared between the initial and final evaluations. Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by optical coherence tomography after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit lamp examination at days 1 and 14. RESULTS: Average ECD, HEX, and CV for the Kerasave (2653 ± 303 cells/mm 2 , 57 ± 4%, and 36 ± 3%) and Optisol-GS (2623 ± 306 cells/mm 2 , 57 ± 5%, and 36 ± 4%) groups were not significantly different at day 1. There was also no difference at any other study time points (all P > 0.05). ECD did not significantly change from day 1 to day 14 in either group ( P > 0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups ( P < 0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622 ± 49 µm and those stored in Optisol-GS was 580 ± 35 µm ( P < 0.01). The difference in CCT measurements was not significantly different at day 14 (Kerasave: 674 ± 46 µm vs. Optisol-GS: 647 ± 58 µm, P > 0.05). Corneal transparency was not significantly different between the 2 groups at day 1 or day 14. CONCLUSIONS: The corneal quality and clinically relevant parameters including ECD, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days. The initial difference in CCT between the 2 groups decreased at day 14. These results demonstrated that Kerasave corneal storage solution preserves the corneal endothelium similarly to Optisol-GS.

Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology.

Crustaceans possess remarkably diverse appendages, both between segments of a single individual as well as between species. Previous studies in a wide range of crustaceans have demonstrated a correlation between the anterior expression boundary of the homeotic (Hox) gene Ultrabithorax (Ubx) and the location and number of specialized thoracic feeding appendages, called maxillipeds. Given that Hox genes regulate regional identity in organisms as diverse as mice and flies, these observations in crustaceans led to the hypothesis that Ubx expression regulates the number of maxillipeds and that evolutionary changes in Ubx expression have generated various aspects of crustacean appendage diversity. Specifically, evolutionary changes in the expression boundary of Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs of thoracic maxillipeds. Here we test this hypothesis by altering the expression of Ubx in Parhyale hawaiensis, a crustacean that normally possesses a single pair of maxillipeds. By reducing Ubx expression, we can generate Parhyale with additional maxillipeds in a pattern reminiscent of that seen in other crustacean species, and these morphological alterations are maintained as the animals molt and mature. These results provide critical evidence supporting the proposition that changes in Ubx expression have played a role in generating crustacean appendage diversity and lend general insights into the mechanisms of morphological evolution.

Allograft corneal ring segment for keratoconus management: Istanbul nomogram clinical results.

PURPOSE: To evaluate the clinical feasibility and visual outcomes of allograft corneal ring segment implantation for the treatment of keratoconus. METHODS: This case series, included forty-four eyes of 32 patients with a 6-month follow-up. All cases were treated according to the Istanbul nomogram. In the Istanbul Nomogram, corneal tunnels of 4 × 7.5 mm diameters are created at depth of 200 µm and implanted with sterile allograft corneal rings (KeraNaturalTM, Lions VisionGift, Portland, OR, USA) at the cone location. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), spherical equivalent (SE) and keratometric values were compared preoperatively versus postoperatively. RESULTS: There was significant improvement in UDVA, CDVA, SE and topographic keratometric values. The mean preoperative CDVA (Snellen, decimal) increased from 0.29 ± 0.20, to 0.56 ± 0.26 (P < 0.001), at the last visit. There was no statistically significant difference between preoperative and postoperative thinnest pachymetry values (P = 0.509). No major complications or adverse event were observed during and after the operation. CONCLUSIONS: The results of this pilot study show that sterile allograft corneal ring segments may be safe, effective and enhance the visual performance of keratoconus patients. Larger clinical studies are needed to demonstrate the effectiveness and safety with long term follow-up.

Modified procedure for Loading "Flat" DMEK Grafts Into an Injector.

PURPOSE: The aim of this study was to determine whether loading a Descemet membrane endothelial keratoplasty (DMEK) graft using a drop-in procedure results in more endothelial cell loss (ECL) than the standard suction procedure. METHODS: Pairs of donor corneas with equivalent preprocessing endothelium were prepared using the standard protocol of our eye bank. One member of each pair was loaded into an injector using the standard suction protocol. The mate graft was loaded using a drop-in protocol, in which the edge of the graft was gently grasped with a forceps, lifted to the edge of the injector, and dropped inside. Grafts were evaluated for ECL and examined for grab marks or other loading-associated damage. RESULTS: There was no difference in mean ECL of grafts prepared for DMEK using the standard protocol (20.6% ± 4.5%) compared with that of mate grafts prepared using the drop-in loading protocol (19.5% ± 4.8%, P = 0.59). There was no consistent pattern of damage in the drop-in-loaded grafts, as grab marks or other tissue damage associated with the drop-in loading protocol were not consistently identified by a trained corneal surgeon. CONCLUSIONS: ECL was not significantly different in grafts prepared using a drop-in loading procedure compared with grafts prepared using the standard suction protocol. The drop-in loading protocol may be particularly useful to surgeons who load their own grafts and eye bank processing technicians who encounter a "flat" DMEK graft that does not scroll or a loosely scrolled DMEK graft.

Examination of a Modified Graft Preparation Technique to Induce Double-Scroll Formation and Promote the Use of Younger Descemet Membrane Endothelial Keratoplasty Donor Tissue.

PURPOSE: The purpose of this study was to determine whether controlled balanced salt solution (BSS) bursts during graft preparation can safely promote formation of a double-scrolled Descemet membrane endothelial keratoplasty (DMEK) graft in younger donor tissue. METHODS: DMEK grafts prepared from young donor tissue (average age, 55 years; range, 39-66 years) were floated in BSS to spontaneously form scrolls (N = 10 pairs). Controlled BSS bursts were used to promote double-scroll (DS) formation in 1 member of each pair. Grafts were stained, preloaded, and shipped before cell viability analysis. After appropriate training, a less experienced technician performed this technique on 10 additional corneas. Outcomes measured for both technicians include the success rate for obtaining a DS, scroll conformation after shipping, and endothelial cell loss (ECL). RESULTS: There was no difference in ECL between grafts subjected to additional manipulation compared with unmanipulated mate grafts (observer 1: 15.2% ± 3.3% vs. 15.2% ± 4.4%, P = 0.99; observer 2: 16.3% ± 2.9% vs. 15.9% ± 4.5%, P = 0.8). A technician experienced with this technique had a 90% success rate, whereas a less experienced technician had a 70% success rate. The mean ECL of the 10 grafts manipulated by the less experienced technician was not significantly different from results obtained from the experienced technician (observer 1: 18.5% ± 6.0% vs. 15.2% ± 3.3%, P = 0.15; observer 2: 18.1% ± 5.6% vs. 16.3% ± 2.9%, P = 0.34). Scrolls maintained their conformation during shipping events. CONCLUSIONS: Double-scroll graft formation using controlled BSS bursts is a reliable technique that can be performed without causing additional damage to DMEK grafts. This technique may make graft unscrolling easier and can promote the use of younger donor tissue for DMEK.

Tissue Loss, Processing Time, and Primary Graft Failure in Eye Bank-Prepared Descemet Membrane Endothelial Keratoplasty Grafts Before and After Prestripped to Preloaded Graft Transition.

PURPOSE: To examine tissue loss rates, processing time, and primary graft failure (PGF) of "prestripped-only" Descemet membrane endothelial keratoplasty (DMEK) grafts at a single eye bank and how these parameters changed after the introduction of steps to preload tissue among experienced processors. METHODS: Tissue loss and processing time during DMEK graft preparation as well as PGF were analyzed retrospectively at a single eye bank between 2012 and 2018. Outcomes were assessed in consecutive grafts before and after the introduction of preloading to the eye bank's standard operating procedure. RESULTS: A total of 1326 grafts were analyzed, composed of the first 663 preloaded DMEK grafts and, for comparison, the 663 DMEK grafts processed immediately before starting the preloaded service. Mean processing time increased from 17.0 ± 3.9 minutes to 26.0 ± 5.4 minutes with the advent of preloading (P < 0.01). Initially, average processing time increased dramatically, with a maximum processing time of 51 minutes, before regressing to the average. No significant difference in the rate of tissue wastage was observed before versus after the implementation of preloaded DMEK (1.2% vs. 1.7%, P = 0.48). PGF occurred in 7 grafts before the preloaded service and 10 grafts after starting the service (1.6% vs. 2.3%, P = 0.47). CONCLUSIONS: Preloading does not affect tissue wastage for experienced technicians or the PGF rate but increases processing time. Eye banks that are considering adding preloading to their standard operating procedure may need to account for longer processing times in their daily operations.

Primary Graft Failure, Infection, and Endothelial Cell Density in Corneal Transplants With Increased Death-to-Preservation Time.

PURPOSE: To ascertain whether death-to-preservation time (DPT) is associated with donor endothelial cell density (ECD), primary graft failure (PGF), and infection. METHODS: Donor corneas aged older than 10 years with ECD 2000 to 4500 cells/mm2 were procured between 2011 and 2018 by a single eye bank. Donor corneas were analyzed retrospectively for the main outcome measures of PGF, infection, and ECD. Means and proportions of study parameters were compared between corneas with long and short DPT, defined as greater or less than 14 hours, respectively, excluding corneas with a history of intraocular surgery or diabetes. Multivariate analyses were performed using logistic regression, adjusting for donor age at time of death, history of diabetes mellitus, and history of cataract surgery. RESULTS: Among 12,015 corneas, those with long DPT had a statistically but not clinically significant higher ECD than that of corneas with short DPT (2754 vs. 2724 cells/mm2, P < 0.01). There was no difference in PGF and infections in corneas with long versus short DPT (0.28% vs. 0.26%, P = 0.86; 0.43% vs. 0.29%, P = 0.51, respectively). CONCLUSIONS: Longer DPT is not associated with a clinically meaningful reduction in donor ECD, PGF, or infection.

Ink Retention and Endothelial Cell Viability After the Application of an Orientation Stamp Over an Air Bubble During Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: To investigate stamp visibility and endothelial cell loss (ECL) after the application of an orientation mark to Descemet membrane endothelial keratoplasty (DMEK) grafts supported by an air bubble. METHODS: Eighteen DMEK grafts were prepared at an eye bank using a technique where an orientation mark was applied to the stromal surface of a DMEK graft that was supported by a small air bubble placed at the edge of the 2 endothelial surfaces of the graft. Grafts were evaluated at 2 and 5 days for stamp visibility and at 5 days with calcein-AM staining for ECL. Nine grafts underwent cross-country shipping, and the ECL of shipped and nonshipped grafts was compared using unpaired t test. RESULTS: All 18 DMEK grafts exhibited a single, solid, readily visible orientation mark 2 and 5 days after preparation with a mean ECL of 13.5% ± 4.9%. Shipping conditions had no effect on stain retention or ECL. CONCLUSIONS: The application of an orientation stamp to a DMEK graft over an air bubble in an eye bank setting results in a single, solid orientation mark that is readily visible within the period in which most eye bank-prepared tissue is used. This technique produces no further ECL compared with the methods where the orientation stamp is applied through a stromal window. Eye bank technicians and surgeons can be confident that this modified preparation technique results in transplant-quality DMEK grafts with the additional benefit of conserving the stromal cap for use in other anterior lamellar procedures, thereby making efficient use of donor tissue.