Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India
Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India
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DOI：
10.3390/microorganisms7100411
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发表时间：
2019-10-01
期刊：
影响因子：
4.5
通讯作者：
Joseph, Joveeta
中科院分区：
文献类型：
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作者：
Gandhi, Jaishree;Jayasudha, Rajagopalaboopathi;Joseph, Joveeta
为了评价基于高通量测序（HTS）方法分析临床诊断为眼内炎患者玻璃体液中细菌和真菌基因组的临床实用性，我们对临床假定感染性眼内炎患者的75份玻璃体液进行了高通量测序（Illumina HiSeq 2500）进行DNA提取和扩增后的16 S rRNA用于检测细菌，ITS 2区域用于检测真菌病原体。作为对照，我们纳入了70例诊断为其他非感染性视网膜疾病的患者的玻璃体活检。在构建策划的微生物基因组数据库和过滤步骤以减少来自环境的模糊性/污染物之后，分析配对读数。我们的HTS读数在几乎所有情况下都显示了通过常规微生物检查在培养物中生长的相同生物体（细菌-14/15，真菌3/3）。HTS还诊断出42/57例（73.7%）常规阴性患者中存在微生物（真菌病原体36/57例，细菌病原体11/57例，包括5例显示细菌和真菌生物体均存在的病例）。曲霉属，镰刀菌属，突脐孢属，和念珠菌属是培养阴性的眼内炎病例中最主要的属。基于热图的微生物聚类分析显示，这些生物体在分类学上与通过常规培养方法鉴定的物种相似。有趣的是，4/70的对照样品也显示存在细菌读数，尽管其临床意义尚不确定。HTS可用于检测无法进行常规诊断的眼内炎病例中的病原体，并可提供与管理相关的可操作信息，特别是在高度怀疑真菌性眼内炎的情况下，特别是在热带国家。结果分析和临床试验解决的HTS的成功和节省费用的诊断眼内炎的建议。
To evaluate the clinical utility of high-throughput sequencing (HTS) approach-based analysis of the bacterial and fungal genome in vitreous fluids from patients clinically diagnosed as endophthalmitis, we subjected 75 vitreous fluids from clinically presumed infectious endophthalmitis patients to high-throughput sequencing (Illumina HiSeq 2500) after DNA extraction and amplification of the 16S rRNA for the detection of bacteria, and ITS 2 region for detection of fungal pathogens. As controls, we included vitreous biopsies from 70 patients diagnosed with other non-infectious retinal disorders. Following the construction of the curated microbial genome database and filtering steps to reduce ambiguousness/contaminants from the environment, the paired reads were analyzed. Our HTS reads revealed in almost all cases the same organism that was grown in culture (bacterial-14/15, fungal 3/3) by conventional microbiological workup. HTS additionally diagnosed the presence of microbes in 42/57 (73.7%) patients who were conventionally negative (fungal pathogens in 36/57, bacterial pathogens in 11/57, including five cases that showed the presence of both bacterial and fungal organisms). Aspergillus sp., Fusarium sp., Exserohilum sp., and Candida sp. were the most predominant genera in our cohort of culture-negative endophthalmitis cases. Heat map based microbial clustering analysis revealed that these organisms were taxonomically similar to the species identified by conventional culture methods. Interestingly, 4/70 control samples also showed the presence of bacterial reads, although their clinical significance is uncertain. HTS is useful in detecting pathogens in endophthalmitis cases that elude conventional attempts at diagnosis and can provide actionable information relevant to management, especially where there is a high index of suspicion of fungal endophthalmitis, particularly in tropical countries. Outcome analyses and clinical trials addressing the success and cost savings of HTS for the diagnosis of endophthalmitis are recommended.