A single eye infection sample, placed in a portable nanopore sequencing device, could reveal every bacterium, virus, or fungus present—and whether those microbes are actively causing disease—within hours rather than days. Current diagnostic methods for severe eye infections like microbial keratitis and endophthalmitis are slow or narrow. Culture takes days and often misses pathogens; PCR can only detect what it is designed to look for, leaving unexpected or drug-resistant organisms undetected. This project addresses that gap by developing a combined DNA/RNA nanopore sequencing protocol that captures the full genetic picture of an infection in real time. By detecting both microbial DNA and RNA, the method can distinguish active infection from harmless contamination, and identify antimicrobial resistance genes early. If successful, this approach could transform how hospitals diagnose severe eye infections. Faster, more comprehensive pathogen identification would allow clinicians to target antibiotics precisely, reducing misuse and preventing vision loss. The researcher will first optimise the protocol using known pathogens, then test it in a clinical feasibility study with 24 patients at the Birmingham and Midland Eye Centre, comparing its speed and accuracy against standard culture and PCR.
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Severe eye infections, such as microbial keratitis and endophthalmitis, require rapid and precise diagnosis to guide appropriate treatment and prevent complications, including vision loss. However, culture-based methods are slow, taking several days, and have low sensitivities, whilst PCR is limited to predefined targets, potentially missing unexpected or resistant pathogens. My project aims to develop and optimise a nanopore sequencing-based method that can detect both DNA and RNA from all pathogens present in an eye infection sample, providing a more comprehensive and real-time approach to pathogen identification. Unlike traditional methods, this approach will not only identify microbial species but also determine their transcriptional activity, distinguishing contamination from active infection. Additionally, it will enable the detection of antimicrobial resistance genes, allowing for early identification of drug-resistant pathogens. This builds upon work done in my PhD on the novel use of full-length 16S and whole genome DNA nanopore sequencing in microbial keratitis and endophthalmitis samples. Firstly, I will optimise the combined DNA/RNA nanopore sequencing protocol and benchmark the bioinformatics workflow, using known bacterial, viral, and fungal pathogens. Next, the optimised sequencing method will be evaluated through a clinical feasibility study of 24 prospective patients presenting to the Birmingham and Midland Eye Centre Emergency Department with severe eye infections, comparing the nanopore sequencing results against culture and PCR in terms of sensitivity, specificity, turnaround time, and antimicrobial resistant gene detection. This combined DNA/RNA nanopore sequencing approach could revolutionise rapid infectious disease diagnostics, reducing antibiotic misuse, and improving patient outcomes.
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