 |
Ocular
Microbiology and Immunology Group
Back
to OMIG Main Page
< Previous | 2021
Agenda and Abstracts | Next >
2021 OMIG Abstract
Novel Molecular Barcoding for Rapid Pathogen Detection in Infectious Keratitis
Lawson Ung1,2, Nicole L. Belanger1, James Chodosh1, Michael S. Gilmore1, and Paulo J.M. Bispo*1
1Department of Ophthalmology, Infectious Disease Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston Massachusetts; 2Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
Purpose: This proof-of-principle study applies novel molecular barcoding on the NanoString platform for rapid and comprehensive pathogen detection in infectious keratitis.
Methods: We recruited adult study participants presenting with infectious keratitis, and who were determined according to our emergency department algorithm – the Assess, Culture, and Treat (1-2-3-ACT) Rule – to have an immediately sight-threatening lesion requiring corneal cultures. Following routine swab collection for microscopy and culture, an additional sample of the infected lesion underwent nucleic acid purification, with DNA quality assessed by performing dual internal control real-time PCR assays targeting human ß-globin, and variable regions 3 and 4 of bacterial 16S ribosomal RNA. Following multiplexed targeted enrichment, patient specimens were processed using a custom-designed NanoString assay for common ocular infections, covering 48 genomic targets each 150-300bp in length. Paired 50mer oligonucleotide probes were designed per target, consisting of a biotin-bound capture probe, and a reporter probe bound to a fluorescent barcode unique to each pathogen sequence. Patient specimens were then hybridized to these 50mer probes, with barcoded complexes digitally enumerated to obtain a relative hybrid count for each target. A threshold of >100 mean sequence-specific hybrids defined positive results.
Results: 17 culture-positive specimens that were PCR-positive for both ß-globin and 16S rDNA, were included. All cases were bacterial in etiology, with 6 Gram-positive, 9 Gram-negative, and 2 polymicrobial cases. Mean NanoString probe counts normalized for hybridization efficiency ranged from 129.54 (±30.40) to 91297.27 (±8598.04), transformed to a log10 count of 2.11 to 4.96. Captured sequences included genus-level targets, including staphylococci (28S rDNA) and streptococci (16S rDNA), and species-specific targets including Staphylococcus aureus (spa), Streptococcus agalactiae (cfb), Streptococcus pneumoniae (lytA), Pseudomonas aeruginosa (proA), Serratia marcescens (gyrB) and Haemophilus influenzae (pstA). There was complete agreement between culture and our multiplex panel for monomicrobial cases, and partial agreement for 2 polymicrobial infections included. Compared to a median time to growth of 3 days (range, 1-5 days), all samples underwent NanoString analysis within 12 hours.
Conclusions: Molecular barcoding may add to our growing diagnostic arsenal to provide cultureless identification of corneal pathogens. Its clinical application may inform appropriate treatment strategies at earlier stages of disease, thereby salvaging vision.
Disclosure: S: Tej Kohli Foundation; NIH/NEI (MSG, JC), Research to Prevent Blindness (JC); and C: US FDA (JC).
< Previous | 2021
Agenda and Abstracts | Next >
|
|
