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BMJ Open Ophthalmol. 2019 Apr 20;4(1):e000246. doi: 10.1136/bmjophth-2018-000246. eCollection 2019.

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium.

Author information

1
Institute of Ophthalmology, University College London, London, UK.
2
International Center for Ocular Physiopathology, Fondazione Banca degli Occhi del Veneto Onlus, Venice, Italy.
3
St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, UK.
4
Department of Eye and Vision Science, Institute of Ageing andChronic Disease, University of Liverpool, Liverpool, UK.
5
Instituto Universitario Fernandez-Vega, Universidad de Oviedo and Fundacion de Investigacion on Oftalmologica, Oviedo, Spain.
#
Contributed equally

Abstract

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.

KEYWORDS:

16S; 18S; NGS; bacteria; cornea; eye bank; fungus; media; microbiology; preservation; storage

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