Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing

Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):280-288. doi: 10.1167/iovs.17-21617.


Purpose: We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis.

Methods: This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool.

Results: Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge.

Conclusions: Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Corneal Ulcer* / microbiology
  • Corneal Ulcer* / parasitology
  • Corneal Ulcer* / virology
  • Eye Infections / diagnosis*
  • Eye Infections, Bacterial / diagnosis
  • Eye Infections, Fungal / diagnosis
  • Eye Infections, Parasitic / diagnosis
  • Eye Infections, Viral / diagnosis
  • Female
  • Fixatives
  • Formaldehyde
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Male
  • Middle Aged
  • Real-Time Polymerase Chain Reaction
  • Retrospective Studies
  • Sequence Analysis, DNA
  • Software
  • Tissue Fixation


  • Fixatives
  • Formaldehyde