capriCORN - Comprehensive Analysis of Pathogens, Resistomes, and Inflammatory-markers in the Cornea

Undiagnosed corneal infection leads to blindness. Worldwide, vision loss from
corneal ulcers is a major public health problem. Identification of causative
organisms is key in the treatment of any infectious disease, yet many corneal
infections go undiagnosed. Empirical treatment of infectious keratitis for a
presumed organism is the rule not the exception. Only a minority of corneal
infections are actually cultured. More accurate identification of the cause of
infectious ulcers can help lower the burden of vision loss from corneal ulcers.

Global surveillance of corneal infections is needed. The cause of corneal
ulcers varies widely by location. Climatic, geographic, season, cultural, and
economic differences all contribute to this variability. Awareness of the most
common organisms in a region can guide initial therapy
while lab tests are pending, or where labs are not available. As the prior
information, knowledge of the likely spectrum of organisms in a region can even
improve the interpretation of indeterminate lab results.

Metagenomic deep sequencing (MDS) is transforming infectious disease
diagnostics. Traditional corneal ulcer smear and culture have limited
sensitivity and cannot identify a number of etiologies. Targeted nucleic acid
amplification tests can be sensitive but requires clinical suspicion of a
specific etiology. MDS allows for the identification of pathogen, whether
anticipated or not. In fact, the sequence can add to our knowledge about the
pathogen. For example, microsporidia was previously classified as an
intracellular parasite but now is identified as a fungus. MDS also allows for
identification of pathogens at the subspecies level. Subspecies variability has
been associated with clinical outcome in many diseases, including
Fusarium keratitis.

Antimicrobial resistance (AMR) is increasing in keratitis. Decreased
susceptibility to an antimicrobial predicts worse visual outcomes in both
bacterial and fungal keratitis. The same MDS analysis that reveals the organism
also provides known AMR determinants for the identified organism. Cataloging
AMR in infectious keratitis over different regions can facilitate appropriate
initial treatment. Global surveillance of AMR patterns may prevent vision loss.

Characterization of host response transcription signatures may aid in pathogen
identification and clarify disease outcome. If surface samples of the cornea
fail to reveal a deeper stromal infection, deep sequencing the host immune
response transcriptome may reveal a fingerprint characteristic of bacterial,
fungal, parasitic, or viral infection. The transcriptional profile may also
predict which cases are more likely to perforate.

In this proposal, we will collect samples from both US and international
corneal ulcers, identify etiology, AMR, and local host immune response, and
correlate with presentation and outcome.

Specific Aim 1. Worldwide surveillance of organisms responsible for infectious
keratitis.
SA 1A: To identify pathogens causing infectious keratitis in an unbiased manner
with MDS.
SA 1B: To determine the optimal diagnostic technique (stains, cultures, MDS)
for corneal ulcers.
SA 1C: To determine the effect of seasonality on pathogen profile and visual
outcomes.

We hypothesize the spectrum of etiology will vary significantly by geographic
location, and novel organisms not typically associated with infectious
keratitis will be identified with MDS. In the absence of a diagnostic gold
standard, we hypothesize the use of latent class analysis (LCA) will predict
MDS affords the highest sensitivity compared to other tests. Furthermore, we
hypothesize that geographic location and seasonality will affect pathogen
profile and disease outcomes.

Specific Aim 2. Worldwide surveillance of antimicrobial resistance (AMR) in
corneal ulcer pathogens.
SA 2A: To determine the frequency and richness of AMR in pathogens causing
keratitis.
SA 2B: To determine AMR genotype and phenotype correlation with clinical
outcomes.

We hypothesize that AMR will differ by geographic location and that genotypic
AMR profiles will predict clinical outcomes.

Specific Aim 3. To define host immune response transcriptional profiles in
keratitis.
SA 3A: To determine the host transcriptional signatures for pathogen types.
SA 3B: To determine the host transcriptional signatures for clinical outcomes.

We hypothesize that host transcriptome signatures can differentiate between
bacterial, fungal, viral, and parasitic infections and predict clinical
outcomes.

Rapid and accurate identification of etiology can inform vision-saving therapy.
The results of this proposed study will provide a new paradigm for diagnostics
and treatment approaches to improve clinical outcomes.

This study is designed to apply new technologies to increase the diagnosis rate
and track AMR for infectious corneal ulcers. Genotypic data including sequences
of microbial RNA transcripts, antimicrobial resistance determinants, and human
RNA transcripts derived from each patient*sdonated specimen. Phenotypic data
includes, but is not limited to a patient*s age, diagnosis, sex, race, or
ethnicity.
We will obtain corneal ulcer and conjunctival samples from patients with signs
and symptoms of infectious corneal ulcers from sites around the world. Samples
will be shipped to UCSF for all molecular analysis. We will also collect
clinical photography on eligible patients.

- We will ask the patient if we can collect 2 conjunctival swabs and a corneal
swab to be analyzed using high throughput sequencing.
- We will also collect 2 clinical photos (of the eyes) using a mobile device.

There are minimal risks (<1:100) to the participant during corneal and
conjunctiva swabbing, the risks are comparable to the risks when material is
taken as part of standard diagnostics. Trained staff will collect the samples
from the patient. Participants may experience some temporary discomfort, but
the swabbing involves minimal risk.

Adverse effects that may occur as a minimum are:
- Enlargement of the epithelium of the infection, increasing the damage.
However, this chance is also present if material is taken for standard
diagnostics to discover which pathogen is causing the corneal infection.
- Irritation/discomfort of the cornea or conjunctiva

Clinical photography offers minimal risk as well. Participants may experience
temporary discomfort from the flash of the camera.
For the study, it is necessary that the patient visits the hospital one more
time after sample collection for a check-up visit. This appointment may be part
of a scheduled visit as part of standard treatment or scheduled as a specific
study visit. A visit takes about 10 minutes.