Dibbs Mark, Matesva Mitchelle, Theotoka Despoina, Jayaraj Christina, Metiku Beruk, Demkowicz Patrick, Heng Jacob S, Wang Yvonne, Bakhoum Christine Y, Chow Jessica, Bakhoum Mathieu F
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT, USA.
Section of Nephrology, Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA.
Transl Vis Sci Technol. 2025 Apr 1;14(4):19. doi: 10.1167/tvst.14.4.19.
This prospective observational study assesses the efficacy of using portable next-generation sequencing directly on tear samples to identify bacterial pathogens in corneal ulcers.
Tear samples were collected from ulcerated and contralateral eyes using Schirmer strips. Corneal scrapings and cultures were performed as medically indicated. The 16S rRNA gene was amplified from tear samples using polymerase chain reaction (PCR), and Nanopore sequencing was used for bacterial species identification and taxonomic classification.
Bacterial DNA was identified in 8 of 10 samples using the tear-based sequencing method. Nanopore sequencing accurately identified the causative bacteria in all four samples that exhibited bacterial growth on culture and detected bacterial pathogens in two of the four ulcers that did not show bacterial growth on culture. In two cases where cultures could not be obtained due to the ulcer's small size, tear sequencing successfully identified bacterial species. Among the nine contralateral tear samples collected, Nanopore sequencing identified commensal bacteria in four samples.
PCR amplification of 16S rRNA directly from tears followed by Nanopore sequencing is an effective, noninvasive method to identify bacterial pathogens in corneal ulcers, offering noninferior results to traditional culture methods.
By eliminating the need for corneal scrapings and nucleic acid extraction, this tear-based method improves the timing and accuracy of bacterial pathogen diagnosis in corneal ulcers, allowing for prompt detection of causative organisms and enabling earlier targeted antimicrobial therapy, thereby improving patient outcomes.
这项前瞻性观察性研究评估直接对泪液样本使用便携式下一代测序技术来鉴定角膜溃疡中细菌病原体的疗效。
使用泪液试纸从溃疡眼和对侧眼中采集泪液样本。根据医学指征进行角膜刮片和培养。使用聚合酶链反应（PCR）从泪液样本中扩增16S rRNA基因，并使用纳米孔测序进行细菌种类鉴定和分类。
使用基于泪液的测序方法在10个样本中的8个中鉴定出细菌DNA。纳米孔测序准确鉴定出在培养中显示细菌生长的所有4个样本中的致病细菌，并在培养中未显示细菌生长的4个溃疡中的2个中检测到细菌病原体。在2例因溃疡面积小而无法进行培养的病例中，泪液测序成功鉴定出细菌种类。在采集的9个对侧泪液样本中，纳米孔测序在4个样本中鉴定出共生细菌。
直接从泪液中进行16S rRNA的PCR扩增，随后进行纳米孔测序，是一种鉴定角膜溃疡中细菌病原体的有效、非侵入性方法，其结果不劣于传统培养方法。
通过无需角膜刮片和核酸提取，这种基于泪液的方法改善了角膜溃疡中细菌病原体诊断的及时性和准确性，能够迅速检测出致病生物并实现更早的靶向抗菌治疗，从而改善患者预后。
