Applying Metagenomic Analysis Using Nanopore Sequencer (MinION) for Precision Medicine in Bacterial Keratoconjunctivitis: Comprehensive Validation of Molecular Biological and Conventional Examinations

Abstract

Smear microscopic examination and culture of the corneal scrapings are the gold standards for the diagnosis of bacterial keratoconjunctivitis. High-sensitivity molecular biological examinations of the ocular surface specimens are used clinically. However, the results require careful interpretation to avoid the unintentional detection of indigenous bacteria. Results of conventional and state-of-the-art examinations require clinical verification for specificity and sensitivity. In this study, smear microscopic examination, culture, and nanopore sequencing using the MinION of ocular surface specimens from eight clinically diagnosed bacterial keratoconjunctivitis cases were performed and compared. Seven of the eight cases (87.5%) were smear positive and five (62.5%) were culture positive. The former showed the same genus in >60% of the classified reads as one specific bacterium inferred from the smear microscopy when sequenced by the MinION. In two of the three culture-negative cases, the smear-positive images were highly reminiscent of the species comprising most of the MinION sequences. Four of the five culture-positive cases were consistent with the most prevalent bacteria in the sequencing results. Probable contamination among specimens processed on the same day were observed. In conclusion, the microscopic examination of the corneal scraping specimens may be more sensitive and specific than the culture examination. Additionally, although metagenomic analysis using the MinION contributes to more precise medication for bacterial keratoconjunctivitis, contamination can affect the results.

Keywords: bacterial keratoconjunctivitis; corneal scraping; culture; nanopore sequencer (MinION); smear microscopic examination.

Figure 1

(a) Anterior segment photograph. The entire graft is opaque due to infection after corneal transplantation. (b) Smear microscopy. Many large Gram-negative rods were detected. (c) Nanopore sequencing results (relative abundance) of Case 1. Moraxella lacunata was detected as the dominant species by MinION.

Figure 2

(a) Anterior segment photograph. A paracentral corneal abscess is present. (b) Smear microscopy. Several large Gram-negative rods were detected. (c) Nanopore sequencing results (relative abundance) of Case 2. Moraxella lacunata was detected as the dominant species by MinION.

Figure 3

(a) Anterior segment photograph. The entire cornea has pale opacity and hypopyon is obvious. (b) Smear microscopy. Elongated Gram-negative rods were detected. (c) Nanopore sequencing results (relative abundance) of Case 3. (c) An overwhelming majority of Pseudomonas aeruginosa was detected by MinION.

Figure 4

(a) Anterior segment photograph. The lower 3/4 of the flap created by former LASIK forms a circular abscess. (b) Smear microscopy. Gram-positive chain cocci were detected. (c) Nanopore sequencing results (relative abundance) of Case 4. An overwhelming majority of Streptococcus dysgalactoae was detected by MinION.

Figure 5

(a) Anterior segment photograph. The entire corneal graft was opaque, with abscess and infiltration, and temporal corneal graft showed melting. (b) Smear microscopy. Diplococci that appear Gram-negative were determined primarily due to heterogeneous staining but were found to be encapsulated Gram-positive. (c) Nanopore sequencing results (relative abundance) of Case 5. An overwhelming majority of Streptococcus pneumoniae was detected by MinION.

Figure 6

(a) Anterior segment photograph. Corneal infiltration and abscess were found in nasal cornea, suggestive of infectious keratitis. (b) Smear microscopy. Few Gram-positive rods are detected (c) Nanopore sequencing results (relative abundance) of Case 6. No bacterial species with a relative abundance greater than 50% was detected by MinION.

Figure 7

(a) Anterior segment photograph. Corneal infiltration and corneal abscess were found, suggestive of infectious keratitis. (b) Smear microscopy. No organisms were detected by Gram staining. (c) Nanopore sequencing results (relative abundance) of Case 7. An overwhelming majority of Staphylococcus aureus was detected by MinION.

Figure 8

(a) Anterior segment photograph. Severe muco-purulent eye discharge and conjunctival hyperemia were found. The immune response caused corneal infiltration. These findings are mostly suggestive of gonococcal keratoconjunctivitis. (b) Smear microscopy. Gram staining of ocular surface aspirates containing eye discharge detected abundant Gram-negative diplococci. (c) Nanopore sequencing results (relative abundance) of Case 8. An overwhelming majority of Neisseria gonorrhoeae was detected by MinION.