Preliminary Exploration of Hybridization Capture-Based Next-Generation Sequencing for the Detection of Ocular Infection Pathogens
Objective:To initially investigate the application value of hybridization capture-based next-generation sequencing in the detection of ocular infection pathogens.Methods:This was a retrospective series case study.From July 2021 to November 2023,totally 17 patients(17 eyes),randomly selected,who were admitted to the Eye Hospital,Wenzhou Medical University and diagnosed with ocular infections with positive results from traditional pathogenic detection were included in this study.Samples of conjunctival sac secretions,corneal scrapings,or vitreous humor were collected from all patients at admission,based on the site of infection.These samples were subjected to smear microscopy,culture,and identification.The remaining samples underwent metagenomics next-generation sequencing(mNGS),and hybridization capture-based next-generation sequencing(HC-NGS).A comprehensive analysis was conducted on the types and quantities of pathogens detected by these three methodologies,focusing on factors such as pathogen detection rate,mixed infection rate,and consistency in pathogen types.Additionally,a comparison was made between mNGS and HC-NGS regarding differences in normalized sequence counts and drug resistance genes.The detection rates and mixed infection rates of the three methods were compared using Fisher's exact test,while the normalized sequence counts of mNGS and HC-NGS were compared using the rank sum test.Results:This study identified 18,31 and 39 pathogens using smear microscopy and culture,HC-NGS and mNGS respectively.The bacterial detection rates were 35％,41％,and 47％,respectively.The fungal detection rates were 59％,41％,and 41％,respectively.And the virus detection rates were 6％,53％,and 65％,respectively.Compared with smear microscopy and culture,HC-NGS and mNGS significantly improved the virus detection rate(x2=7.70,P=0.008;x2=12.88,P=0.001).The detection rates of mixed infections using smear microscopy and culture,mNGS,and HC-NGS were 6％,47％,and 53％,respectively.The detection rates of the latter two methods were significantly higher than those of the smear microscopy and culture(x2=7.40,P=0.017;x2=9.07,P=0.007).The pathogen types detected by smear microscopy and culture,mNGS and HC-NGS were 14,23,and 29,respectively.In terms of pathogen species consistency,18％of the samples were completely consistent across all three methods.And 35％of the samples showed partial consistency among the three methods,and the pathogens detected by HC-NGS and mNGS included those detected by smear microscopy and culture.Additionally,35％of the samples showed complete consistency between HC-NGS and mNGS,while 12％of the samples showed partial consistency between HC-NGS and mNGS,with HC-NGS encompassing all the pathogens detected by mNGS.In addition,HC-NGS had more normalized sequence counts than mNGS(W=496.00,P＜0.001)and detected more drug resistance genes.Conclusion:The HC-NGS has a high detection rate and wide coverage of pathogens in ocular infection pathogens,and it can detect more drug resistance genes,suggesting substantial application potential.
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