Objectives

We investigated whether nanopore amplicon sequencing of aqueous humor was capable of rapid pathogen identification in infectious endophthalmitis.

Methods

5 cases of culture-positive bacterial endophthalmitis and 3 cases of fungal endophthalmitis (1 culture-positive and 2 presumed) were included. DNA was extracted from the aqueous humor and vitreous specimen, and PCR of bacterial rDNA (16S) and fungal rDNA (ITS1 and D1/2/3) was performed. Then, nanopore amplicon sequencing was performed for 2 h. The results of amplicon sequencing were compared to those of conventional culture studies.

Results

In all cases, pathogens were identified by amplicon sequencing of aqueous humor specimens. In 3 cases of bacterial endophthalmitis, the identified microbes were confirmed by culture studies of both aqueous humor and vitreous specimens. In 2 cases of bacterial and 1 case of fungal endophthalmitis, the identified pathogens were confirmed only by culture studies of vitreous specimens. In all cases, amplicon sequencing identified pathogen in a shorter turnaround time than culture studies. In 2 cases with negative culture results, amplicon sequencing of aqueous humor identified fungal pathogens.

Conclusions

Our data demonstrates the potential of amplicon nanopore sequencing using aqueous humor to enable rapid, sensitive and less invasive microbial diagnosis of endophthalmitis.

中文翻译：

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纳米孔扩增子测序技术对眼内炎的微生物诊断

目标

我们调查了房水的纳米孔扩增子测序是否能够在传染性眼内炎中快速鉴定病原体。

方法

包括培养阳性细菌性眼内炎5例和真菌性眼内炎3例（培养阳性1例，推测2例）。从房水和玻璃体标本中提取DNA，并进行细菌rDNA（16S）和真菌rDNA（ITS1和D1 / 2/3）的PCR。然后，进行纳米孔扩增子测序2小时。将扩增子测序的结果与常规培养研究的结果进行比较。

结果

在所有情况下，通过房水标本的扩增子测序来鉴定病原体。在3例细菌性眼内炎病例中，通过房水和玻璃体标本的培养研究证实了所鉴定的微生物。在2例细菌和1例真菌性眼内炎中，仅通过对玻璃体标本进行培养研究才能确定已鉴定的病原体。在所有情况下，与培养研究相比，扩增子测序都能在更短的周转时间内识别出病原体。在2例培养结果阴性的病例中，房水的扩增子测序鉴定出了真菌病原体。

结论

我们的数据证明了使用房水进行扩增子纳米孔测序的潜力，以实现眼内炎的快速，敏感和侵入性较小的微生物诊断。