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Detection and Identification of Acanthamoeba and Other Nonviral Causes of Infectious Keratitis in Corneal Scrapings by Real-Time PCR and Next-Generation Sequencing-Based 16S-18S Gene Analysis
Journal of Clinical Microbiology ( IF 9.4 ) Pub Date : 2021-01-21 , DOI: 10.1128/jcm.02224-20
Dennis Back Holmgaard 1 , Celine Barnadas 2, 3 , Seyed Hossein Mirbarati 4 , Lee O'Brien Andersen 5 , Henrik Vedel Nielsen 5 , Christen Rune Stensvold 6
Affiliation  

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including Acanthamoeba.

中文翻译:

实时PCR和基于下一代测序的16S-18S基因分析检测和鉴定角膜刮片中棘阿米巴和其他非病毒性感染性角膜炎原因

棘阿米巴是一种具有广泛遗传多样性的自由生存的变形虫。它可能引起感染性角膜炎(IK),也可能由细菌,真菌和病毒引起。高的诊断敏感性对于早期诊断棘阿米巴相关性角膜炎至关重要。在这里,我们调查了基于下一代测序(NGS)的核糖体基因检测和分化(16S-18S)与特定的实时PCR相比,对棘阿米巴的检测的适用性。从角膜刮片中提取200个DNA并通过棘阿米巴筛选使用内部16S-18S NGS测定法分析特异性实时PCR。其中,有24例通过实时荧光定量PCR阳性,其中21例通过NGS分析呈阳性。与实时PCR相比;NGS检测的特异性和敏感性分别为100%和88%。通过NGS分析鉴定的基因型包括T4(n = 19)和T6(n = 2)。在31份棘阿米巴阴性样本中鉴定出具有潜在临床相关性的真菌和细菌种类,例如铜绿假单胞菌n = 11),莫拉氏菌。(n = 6),金黄色葡萄球菌n = 2),镰刀菌spp。(n = 4)和白色念珠菌n = 1)。总之,在检测角膜刮屑中的棘阿米巴特异性DNA时,16S-18S测定的灵敏度比实时PCR稍差。通过NGS分析提供了可靠的基因型信息,在16%的棘阿米巴阴性样品中鉴定出了具有潜在临床相关性的其他病原体。基于NGS的角膜刮片中核糖体基因的检测可能是一种有效的筛选方法,用于检测IK的非病毒原因,包括棘阿米巴
更新日期:2021-01-21
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