Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad, Telangana, India.
Smt. Kannuri Santhamma Centre for vitreoretinal diseases, L V Prasad Eye Institute, Hyderabad, India.
Sci Rep. 2019 Jan 29;9(1):844. doi: 10.1038/s41598-018-37502-w.
The proof-of-concept, study to investigate the presence of microorganisms in presumed infectious endophthalmitis using Next generation sequencing (NGS) was carried out in vitreous biopsies from 34 patients with endophthalmitis, and thirty patients undergoing surgery for non-infectious retinal disorders as controls. Following DNA extraction using the Qiagen mini kit and PCR amplification of the V3-V4 regions of the bacterial 16S rRNA and ITS 2 region of fungus, they samples were sequenced on an Illumina HiSeq 2500 Machine. Paired reads were curated, taxonomically labeled, and filtered. Culture based diagnosis was achieved in 15/34 (44%) patients while NGS diagnosed the presence of microbes in 30/34 (88%) patients (bacteria in 26/30, fungi in 2/30, mixed infections in 2/30 cases). All 30 controls were negative for bacteria or fungus by NGS. There was good agreement between culture and NGS for culture-positive cases. Among culture negative cases, DNA of common culturable bacteria were identified like Streptococcus sp., Staphylococcus sp., Pseudomonas sp., Gemella sp., Haemophilus sp., Acinetobacter sp. The specificity of NGS with culture and clinical diagnosis was found to be 20% and 100% respectively and sensitivity of NGS with culture and clinical diagnosis was found to be 87.5% and 88% respectively. NGS appears to be promising diagnostic platform for the diagnosis of infectious culture negative endophthalmitis.
本研究旨在通过下一代测序（NGS）检测疑似感染性眼内炎中微生物的存在，对 34 例眼内炎患者和 30 例因非感染性视网膜疾病接受手术的患者进行了玻璃体活检。使用 Qiagen mini 试剂盒提取 DNA 后，对细菌 16S rRNA 的 V3-V4 区和真菌的 ITS 2 区进行 PCR 扩增，然后在 Illumina HiSeq 2500 机器上对样本进行测序。对配对读取进行了整理、分类标记和过滤。在 34 例患者中，有 15 例（44%）通过培养诊断，30 例（88%）通过 NGS 诊断（26 例为细菌，2 例为真菌，2 例为混合感染）。所有 30 例对照均通过 NGS 检测出无细菌或真菌。培养阳性病例中培养和 NGS 之间具有良好的一致性。在培养阴性病例中，鉴定出了常见可培养细菌的 DNA，如链球菌、葡萄球菌、假单胞菌、孪生球菌、嗜血杆菌、不动杆菌。NGS 与培养和临床诊断的特异性分别为 20%和 100%，敏感性分别为 87.5%和 88%。NGS 似乎是一种很有前途的诊断平台，可用于诊断培养阴性的感染性眼内炎。
