Identification of pathogens in the vitreous of patients with infectious uveitis by metagenomic sequencing

Abstract

Objective: To investigate the role of metagenomic sequencing in the diagnosis of infectious uveitis. Methods: Cross-sectional study. A total of 19 vitreous specimens of patients with suspected infectious uveitis from March 2016 to July 2018 in Beijing Tongren Hospital were collected, including 8 males and 11 females, 19 to 68 years old. There were 10 cases in the right eye, 8 cases in the left eye and 1 case in both eyes. Acute retinal necrosis was clinically diagnosed in 8 patients (9 eyes), and the diagnosis was unknown in 11 patients (11 eyes). About 1 ml of the vitreous fluid was reserved for each specimen, 800 μl for metagenomic sequencing and 200 μl for real-time fluorescence quantitative PCR verification. The TIANamp Micro DNA Kit was used to extract the sample DNA for metagenomic sequencing, and the ultrasonic fragment was broken to 200-300 bp. The BGISEQ-500 platform was used for sequencing. The data with low quality and length less than 35 bp were cleared from the sequencing data to obtain high-quality data. Through biological authentication software, the reference human genome sequence and low complexity were removed from high-quality data. The data obtained were compared with a special microorganism database regarding the percentage of microbial sequences, the number of unique sequences, coverage and sequencing depth, so as to determine positive sequencing parameters, which were classified into bacteria, viruses, fungi and parasites. Real-time fluorescence quantitative PCR was performed to validate the accuracy. Results: A variety of microorganisms were detected by metagenomic sequencing in 19 specimens, including 3 cases of varicella zoster virus, 2 cases of Candida albicans, 1 case of Propionibacterium acnes and 1 case of Haemophilus parainfluenzae. The percentage of microbial sequences was 77.93% （1 794/2 302）, 99.98% （12 843/12 845） and 98.88%（5 733/5 798）, and the number of unique sequences was 1 794, 12 843 and 57 33 in varicella zoster virus cases, respectively. The verification of varicella zoster virus by PCR was consistent with that by metagenomic sequencing. Conclusion: Metagenomic sequencing can be used as an alternative method for laboratory diagnosis of infectious uveitis. (Chin J Ophthalmol, 2020, 56: 519-523).