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
medRxiv. 2024 Sep 28:2024.09.26.24314375. doi: 10.1101/2024.09.26.24314375.
Corneal ulcers pose a significant threat to vision, with the need for prompt and precise pathogen identification being critical to effective treatment. This study assesses the efficacy of using next-generation portable sequencing (Nanopore Technology) to detect and identify bacterial pathogens directly from tear samples, providing a non-invasive alternative to traditional corneal scraping and culture, which are limited by high false-negative rates.
Prospective observational study.
Ten participants diagnosed with corneal ulcers.
Tear samples were collected from the ocular surface using Schirmer strips. Corneal scrapings and cultures were performed as medically indicated. The 16S rRNA gene was amplified directly from the tear samples using polymerase chain reaction (PCR), and Nanopore sequencing was used for bacterial species identification and taxonomic classification. Comparative analysis was conducted to evaluate the concordance between Nanopore sequencing results and traditional culture methods.
Comparison of bacterial species detected via Nanopore sequencing with those identified through traditional culture methods.
Bacterial DNA was identified in 8 of the 10 samples analyzed using the tear-based sequencing method. Notably, Nanopore sequencing accurately identified the causative bacteria in all 4 samples that exhibited bacterial growth on culture. Additionally, it detected bacterial pathogens in 2 of the 4 ulcers that did not show bacterial growth on culture. In 2 cases where cultures could not be obtained due to the small size of the ulcer, tear sequencing successfully identified bacterial species, highlighting potentially overlooked pathogens in corneal ulcers.
PCR amplification of 16S RNA directly from tears followed by Nanopore sequencing is an effective, non-invasive method to identify bacterial pathogens in corneal ulcers, offering non-inferior results to traditional culture methods. This technique not only allows for the detection of traditionally hard-to-culture organisms, providing immediate diagnostic value to guide treatment, but also enhances our understanding of the microbiological landscape of corneal ulcers, thereby informing more effective treatment strategies.
角膜溃疡对视力构成重大威胁，迅速且准确地鉴定病原体对于有效治疗至关重要。本研究评估了使用新一代便携式测序技术（纳米孔技术）直接从泪液样本中检测和鉴定细菌病原体的效果，为传统角膜刮片和培养提供了一种非侵入性替代方法，传统方法受高假阴性率限制。
前瞻性观察性研究。
10名被诊断为角膜溃疡的患者。
使用泪液试纸从眼表采集泪液样本。根据医学指征进行角膜刮片和培养。使用聚合酶链反应（PCR）直接从泪液样本中扩增16S rRNA基因，并使用纳米孔测序进行细菌种类鉴定和分类。进行比较分析以评估纳米孔测序结果与传统培养方法之间的一致性。
通过纳米孔测序检测到的细菌种类与通过传统培养方法鉴定的细菌种类的比较。
在使用基于泪液的测序方法分析的10个样本中，有8个鉴定出细菌DNA。值得注意的是，纳米孔测序准确鉴定出了所有4个在培养中显示细菌生长的样本中的致病细菌。此外，它还在4个培养中未显示细菌生长的溃疡中的2个中检测到了细菌病原体。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，凸显了角膜溃疡中可能被忽视的病原体。
直接从泪液中进行16S RNA的PCR扩增，随后进行纳米孔测序是一种有效、非侵入性的方法，可用于鉴定角膜溃疡中的细菌病原体，其结果不逊色于传统培养方法。该技术不仅能够检测传统上难以培养的生物体，为指导治疗提供即时诊断价值，还能增强我们对角膜溃疡微生物群落的理解，从而为更有效的治疗策略提供依据。
