Clinical utility of genomic sequencing for ocular inflammatory and infectious diseases

Low, Liying ORCID: 0000-0002-0631-8891 (2021). Clinical utility of genomic sequencing for ocular inflammatory and infectious diseases. University of Birmingham. Ph.D.

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Abstract

The human body is thought to be an ecosystem of mutually beneficial interactions between the human cells and the microorganisms that live in and on our bodies. The largest and most diverse microbial community reside in the gut. Collectively, the genomes of these microbes are known as the gut microbiome. The gut microbiome is important in the development and maintenance of a healthy immune system. Additionally, the gut microbiome regulation of secretory immunoglobulin A and polymorphonuclear cells recruitment on the ocular surface contributes to immune protection against ocular infections. Perturbations in the gut microbiome, also known as gut dysbiosis, has been linked to systemic inflammation elsewhere in the body (extraintestinal sites) and may play a role in ocular inflammation and increased susceptibility to ocular infections. The current gold standard for diagnosis of most ocular infections is microbial culture, however, it is time-consuming and approximately 40% of clinically suspected cases are culture-negative.

Next generation sequencing technologies have revolutionised genomic research and has the potential to provide comprehensive microbial and host data with a wide range of translational clinical applications, from microbiome analysis in diseased and healthy states, to rapid identification of pathogens in infectious diseases. However, these sequencing methodologies have yet to be adopted into routine clinical practice, in part due to the highly contextual and bidirectional host-microbiome interactions, as exemplified by the role of the gut microbiome in modulating host immune homeostasis (i.e. commensal versus pathogenic organisms), and the lack of optimised metagenomic protocols for technically-challenging clinical samples such as ocular samples that are often minute and have high background host DNA contamination.

The general hypothesis investigated in this thesis was the contribution of the microbiomes to ocular disease and the potential application of novel sequencing technologies for rapid identification of these microbiomes, both pathogenic and commensals, in ocular inflammatory diseases and infections. I applied Illumina 16S rRNA gene sequencing to characterise the gut microbiome profile of patients with ocular mucous membrane pemphigoid (OcMMP), a devastating mucosal scarring autoimmune condition of unknown cause. I found that patients with OcMMP had lower alpha diversity compared to healthy controls, and that gut dysbiosis correlated with ocular inflammation, and treatment with immunosuppressive therapies. This broad sampling of the gut microbiome provides a framework for the integration of deeper multi-omic datasets to unravel the complex host immune-microbiome interactions in autoimmunity.

Using the novel nanopore sequencing technology, I extended the research to identify specific microbial species causing ocular infections. Microbial keratitis, infection of the cornea, is a leading cause of preventable blindness worldwide. Using a defined mock community and ex- vivo porcine eye model, I optimised the sampling method and developed a bioinformatics pipeline for full-length 16S rRNA nanopore sequencing, and was successful in implementing this sequencing workflow directly onto clinical samples of patients presenting with microbial keratitis.

Next, I evaluated the clinical utility of nanopore sequencing in endophthalmitis, a severe, sight-threatening intraocular infection. The sensitivity of metagenomic sequencing in detecting pathogens decreases with high host DNA contamination. Therefore, I examined the effect of saponification on host DNA depletion by qPCR using murine peripheral blood mononuclear cells spiked with serial dilutions of E.coli cells and found that there was a 1500- fold depletion of host DNA with very minimal bacterial DNA loss between saponified and un- saponified samples. I compared the effects of saponification on human DNA depletion by whole genome nanopore sequencing using intraocular fluid biopsy samples from a subset of patients with endophthalmitis and found that saponification increased the proportion of bacterial to human reads. Finally, I performed full-length 16S rRNA nanopore sequencing (16S nanopore), whole genome nanopore sequencing (WGS nanopore) and Illumina whole genome sequencing (Illumina WGS) directly on intraocular fluid biopsy samples of patients with clinically suspected endophthalmitis, showing that whole genome nanopore sequencing performed better than 16S nanopore and Illumina WGS in detecting the predominant organism in endophthalmitis. Taken together, this suggests that with further optimisation, the future use of the portable, real-time nanopore sequencing technology as a point-of-care testing for microbial identification and quantification may be beneficial in ocular infections.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):