Clinical performance of nanopore targeted sequencing for diagnosing endophthalmitis

Abstract

Purpose: Rapid identification of pathogenic bacteria in the vitreous and/or aqueous humor of patients with acute clinical diagnosis of endophthalmitis via nanopore sequencing technology.

Methods: We recruited a total of 12 patients (12 eyes) who were diagnosed with endophthalmitis at an ophthalmic outpatient clinic of Peking University Third Hospital from January 2022 to October 2022. Clinical evaluation is conducted in the order of consultation, symptom evaluation, physical sign evaluation, and ophthalmic special examination, all of which are completed by the same experienced clinical physician. Finally, 19 aqueous humor and/or vitreous samples were obtained via anterior chamber wash, vitreous tap and vitrectomy. The samples were separated for cultivation, biochemical drug sensitivity identification, and targeted nanopore sequencing (NTS), and the results of nanopore sequencing were validated via Sanger sequencing.

Results: In patients with endophthalmitis, NTS can identify infected pathogens within 8-12 h. Six samples (31.6%) were subjected to culture-based diagnosis, while NTS revealed the presence of pathogenic microorganisms in 19 samples (100%), of which bacteria and fungi were detected in three samples. A total of 19 samples were subjected to Sanger sequencing, of which 16 (84.2%) tested positive, including 6 culture-positive samples and 10 culture-negative samples, of which 5 (26.3%) were positive for two bacterial genera. In culture-positive cases, there is a high-quality match between culture and targeted nanopore sequencing.

Conclusions: NTS can quickly detect pathogenic bacteria in samples from patients with endophthalmitis. Moreover, the use of vitreous and/or aqueous humor for the NTS has potential. NTS is a promising diagnostic platform for endophthalmitis, especially for mixed infections and culture-negative cases.

Keywords: 16S rRNA; Endophthalmitis; Infectious pathogens; Microbiome; Nanopore sequencing.

Fig. 1

Stacked box plot displaying the relative abundances of the bacterial taxa (≥ 5%) obtained via NTS. A Culture-positive endophthalmitis. B Culture-negative endophthalmitis. C Stacked box plot displaying the relative abundances of the fungal taxa (≥5%)

Fig. 2

Heatmap of genus-level bacterial abundances in samples from patients with presumed infectious, culture-positive and culture-negative endophthalmitis. The color bar on the right side indicates the average relative abundances of these genera in each patient

Fig. 3

Venn diagram of consistency between dominant bacterial genera at the level of bacterial culture, Sanger sequencing, and NTS