Browsing by Author "Ferrari, Stefano"

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    Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium
    (2019-04-01) Parekh, Mohit; Borroni, Davide; Romano, Vito; Kaye, Stephen B.; Camposampiero, Davide; Ponzin, DIego; Ferrari, Stefano
    Objective To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.
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    Shotgun sequencing to determine corneal infection
    (2020-09) Parekh, Mohit; Romano, Vito; Franch, Antonella; Leon, Pia; Birattari, Federica; Borroni, Davide; Kaye, Stephen B.; Ponzin, Diego; Ahmad, Sajjad; Ferrari, Stefano
    Purpose: To investigate if shotgun-sequencing method could be useful in detailed diagnosis of herpes simplex virus (HSV) infection and compare it with the conventional diagnostic method. Observations: Using a sterile scraper, the infectious part of the ocular surface was scraped gently and placed on a glass slide for conventional diagnosis using PCR and histology and in RNA stabilizing reagent for shotgun sequencing respectively. Concentration of the DNA was determined using a sensitive fluorescence dye-based Qubit dsDNA HS Assay Kit. Shotgun-sequencing libraries were generated using the NEBNext DNA ultra II protocol. The samples were sequenced on the Illumina NextSeq 500 in high output mode with 2X150 bp paired-end sequencing. Taxonomic and functional profiles were generated. Conventional diagnostic method suspected herpetic keratitis. The results indicated presence of an amplified product of 92 bp positive HSV-DNA. Conventional diagnostic method detected the presence of Herpes Simplex Virus DNA (type 1). Shotgun sequencing confirmed the diagnosis of HSV along with the taxonomical profiling of the virus. These results were achieved using 1.9 ng/μL of DNA concentration (114 ng in 60 μL) of the total sample volume. Conclusions and importance: Shotgun sequencing is a hypothesis-free approach that identifies full taxonomic and functional profile of an organism. This technology is advantageous as it requires smaller sample size compared to conventional diagnostic methods.
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    The "yogurt" Technique for Descemet Membrane Endothelial Keratoplasty Graft Preparation : A Novel Quick and Safe Method for Both Inexperienced and Senior Surgeons
    (2020-09-01) Tzamalis, Argyrios; Vinciguerra, Riccardo; Romano, Vito; Arbabi, Esmaeil; Borroni, Davide; Wojcik, Gabriela; Ferrari, Stefano; Ziakas, Nikolaos; Kaye, Stephen
    Purpose:To describe and evaluate the efficacy and safety of a novel technique to prepare Descemet membrane endothelial keratoplasty (DMEK) donor grafts using a newly designed partial-thickness hinge punch.Methods:The novel punch has a circular guarded blade missing 1 clock hour, creating an uncut hinge on the donor cornea. In addition, 2 straight cuts are made by the punch perpendicular to the edge of trephination toward the trabecular meshwork in the hinge area. After the donor corneoscleral rim is positioned endothelial side up, a partial-thickness trephination is performed avoiding any rotational movements. Descemet membrane is lifted from Schwalbe line in the hinge area, and DMEK graft is peeled after desired marking without further preparation.Results:Three surgeons of different experience levels on DMEK (senior/independent/fellow) initially applied the new technique in 18 research corneas, divided into equal groups. Two failures in graft preparation were noted, defined as radial tears extending ≥0.5 mm. The mean preparation time was 6.21 ± 1.45 minutes. No statistically significant differences were noted in success rate, duration, and endothelial cell loss (ECL) between surgeons (P > 0.05). ECL was evaluated as an average of 5 readings on randomly selected graft areas, not including graft periphery. Fifteen additional research corneas were stripped by 1 single user in an eye bank setting. No tissue loss was recorded, whereas ECL and mortality rate remained unaffected after preparation (P = 0.64 and P = 0.72, respectively).Conclusions:This new DMEK graft preparation technique, simulating the opening of a yogurt cup, seems to be a safe and an efficient method, providing shorter preparation time and low failure rates independent of surgeon's experience level.