Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis

Abstract

To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one culture positive endophthalmitis patients, admitted to a tertiary care hospital were processed for targeted metagenomics. NGS of 7 variable regions of 16S rRNA gene was done using Ion Torrent Personal Genome Machine (PGM). Sequence data were analyzed using Ion Reporter software using QIIME and BLSATN tools and Greengenes and NCBI–Genbank databases. Bacterial genome sequences were detected in 15 culture negative and culture positive vitreous specimens. The sequence reads varied between 25,245–540,916 with read length between 142bp–228bp and coverage depth was 41.0X and 81.2X. Operational taxonomic unit (OTUs) of multiple bacterial genera and species were detected in 13 culture negative vitreous specimens and OTUs of a single bacterial species were detected in 2 culture negative and 1 culture positive specimens; one negative specimen had no bacterial DNA. Maximum numbers of OTUs detected by NGS for a bacterial species from any vitreous specimen was the one which was detected and identified by Sanger's sequencing in broad range PCR. All the bacteria were belonging to clinically relevant species. Broad range PCR with sequencing failed to identify bacteria from 5 of the 16 (31.25%) culture negative vitreous specimens. Metagenomics could detect and identify bacterial pathogens in 15 of the 16 culture negative vitreous specimen's up to species level. With rapidly decreasing cost, metagenomics has a potential to be used widely in endophthalmitis diagnosis, in which culture negativity is usually high.

Nanopore techniques as a potent tool in the diagnosis and treatment of endophthalmitis: a literature review

“…Metagenomic methods can provide taxonomic analysis of the microbiome, assess its potential function, and quantify it. The clinical applications include identifying microbial species, metabolic pathways, and metabolites related to the development and treatment of human diseases, and further promoting the discovery of microbiome-targeted drugs, and improving human health management efforts [34,59] . By simultaneously conducting NGS and culture on infected endophthalmitis and normal vitreous specimens, Deshmukh et al [44] concluded that the specificity of culture and clinical diagnoses of NGS was 20% and 100%, and the sensitivity of culture and clinical diagnoses of NGS was 87.5% and 88%, respectively.…”

Update on Current Microbiological Techniques for Pathogen Identification in Infectious Endophthalmitis

“…In a similar fashion, Mishra et al took 16 culture-negative vitreous samples, purified DNA using commercial kits, and targeted seven regions of the 16S rRNA gene for amplification and metagenomic analysis against the genomic bank database [ 42 ]. The HTS/NGS method was able to identify pathogens for every sample of culture-negative endophthalmitis.…”

“…These shortcomings are further compounded by the insufficient and suboptimal results attained from standard microbiological methods that are currently the “gold standard”. Gram stains of intraocular samples are negative in 50–60% of cases, and negative cultures rates are up to 60% from aqueous tap biopsies, and up to 55% in vitreous tap biopsies [ 1 , 42 , 43 , 44 ]. The reported rates for positive cultures that result from samples obtained from surgical vitrectomy range widely, from 44.6 to 90% of cases [ 1 ].…”

Diagnostic and Therapeutic Challenges

“…Specifically, 16S or 28S sequencing can identify bacterial or fungal organisms from vitreous aspirate. 1,2 Finally, there are enzyme immunoassays for invasive fungal disease such as (1–3)-β-d-glucan (BDG) and galactomannan (GM), both of which detect fungal cell wall components—BDG more broadly and galactomannan specifically for Aspergillus. 3…”

“…Specifically, 16S or 28S sequencing can identify bacterial or fungal organisms from vitreous aspirate. 1,2 Finally, there are enzyme immunoassays for invasive fungal disease such as (1-3)b-d-glucan (BDG) and galactomannan (GM), both of which detect fungal cell wall components-BDG more broadly and galactomannan specifically for Aspergillus. 3 A similarly rigorous evaluation for malignancy is required with a detailed history and physical as well as imaging including magnetic resonance imaging of brain and orbits, liver ultrasound, and computed tomography of chest, abdomen, and pelvis if there a reasonably high index of suspicion; consultation with an ocular oncologist is recommended.…”