Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India.

Jaishree Gandhi,Joveeta Joseph,Savitri Sharma,Vivek Pravin Dave,Poonam Naik,Rajagopalaboopathi Jayasudha

doi:10.3390/microorganisms7100411

Jaishree Gandhi, Joveeta Joseph + Show 4 more

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https://doi.org/10.3390/microorganisms7100411

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Journal: Microorganisms	Publication Date: Oct 1, 2019
Citations: 29	License type: CC BY 4.0

Affiliation: L V Prasad Eye Institute

Abstract

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.

Highlights

Endophthalmitis is a purulent vision-threatening infection of the intraocular fluids and is reported to vary by geographic location in incidence and cause, following ocular surgery or injury and rarely by hematogenous spread [1]
While direct sequencing of the 16S rRNA/ITS region can be applied on clinical specimens to detect the presence of microorganisms [3,4], in low pathogen loads or polymicrobial infections it is usually challenging to sort out ambiguous signals from mixed chromatograms of samples, and sequences often remain unidentified/misidentified
Targeted High-throughput sequencing (HTS) refers to the selective capture or amplification of specific genomic regions of interest prior to massive parallel sequencing, compared to unbiased random amplification which results in the sequencing of all the nucleic acids, including host and pathogen nucleic acids, and millions of reads need to be analyzed to identify the pathogen(s) of interest

Summary

Introduction

Endophthalmitis is a purulent vision-threatening infection of the intraocular fluids and is reported to vary by geographic location in incidence and cause, following ocular surgery or injury and rarely by hematogenous spread [1]. High-throughput sequencing (HTS) termed as next-generation sequencing (NGS), is a novel platform with the potential to simultaneously detect and independently sequence virtually all the DNA sequences of the infectious agents present in a sample [5,6,7]. This unbiased amplification results in a large number of reads of both host and pathogen DNA, and the complexities in detecting and interpreting millions of sequences to identify the microbe(s) of interest is considerable and challenging. Targeted HTS, on the other hand, provides better sensitivity and specificity along with ease of downstream analysis and a lower cost by allowing more samples to be tested in one run [8]

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