Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04030403 |
| Other study ID # |
5680 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
September 10, 2019 |
| Est. completion date |
December 1, 2022 |
Study information
| Verified date |
June 2022 |
| Source |
Liverpool University Hospitals NHS Foundation Trust |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Design: Prospective observational diagnostic study and methods-comparison.
Aims:
The overall aim of the project is to better define the pathogenic microorganisms in patients
with microbial keratitis (MK) through a better understanding of the corneal and ocular
surface microbiome in health and disease.
This will be achieved through the following objectives:
1. Using NGS, analyse the corneal microbiome of the affected and unaffected eye of patients
with and without MK and compare with simultaneous results from CDC and MTPCR.
2. Determine the microbiological spectrum of the cornea, ocular surface and contiguous
structures, in patients with MK, healthy controls, contact lens wearers and eye drop
users.
Outcome measures:
1. A comparison of isolation rates and identified bacteria obtained from CDC, MTPCR and NGS
processing of MK corneal samples will be made.
2. Microorganisms identified in the eyes with MK will be compared to the fellow eye and
other control groups and subtractive bioinformatics methodology applied to identify the
most likely pathogenic organisms compared to those seen in the healthy corneal and
ocular surface microbiome.
3. Comparisons of the relative abundance of microorganisms obtained from MK corneal samples
over the participant's follow-up visits will be used to evaluate longitudinal changes in
the corneal and ocular surface microbiome during treatment and resolution of MK.
4. A direct comparison between the relative abundance of microorganisms isolated from
participants cornea, conjunctiva, eyelids and nose (contiguous structures) will be made
to identify any possible endogenous sources of infection for MK.
Population Eligibility:
- All patients aged 18 years and over presenting with unilateral clinically suspected MK
to St. Paul's Eye Unit, The Royal Liverpool University Hospital.
- Patients with keratoconus undergoing cross-linking, subjects with no history of MK,
subjects with no history of MK who are contact lens wearers and subjects with no history
of MK but who are on eye drop treatment for glaucoma.
Duration: Three years.
Description:
Microbial keratitis (MK) is an ophthalmological emergency that can lead to sight threatening
complications such as corneal scarring, perforation, endophthalmitis and ultimately
blindness. The patient requires aggressive topical antimicrobial therapy and close treatment
response monitoring, often including hospitalisation followed by frequent outpatient visits.
Improving outcomes depends on rapidly identifying the causative microorganism. Currently the
likely causative microorganism is only isolated in around 30 to 40% of cases using
traditional scraping methods and standard conventional diagnostic culture (CDC), with results
typically taking up to 4 days to become available to the clinician. More sensitive methods
such as microorganism targeted polymerase chain reaction (MTPCR) and metagenomic analysis
increase the potential to detect microorganisms but may increase the likelihood of picking up
commensal microorganisms, making it difficult for the treating clinician to interpret which
of the isolated organisms is likely to be causative in MK.
One of the barriers to identifying the organisms has been the difficulty in collecting
samples from the cornea. In 2015, a group led by Professor Kaye at the University of
Liverpool developed a non- invasive corneal sampling methodology using a corneal impression
membrane (CIM) made from polytetrafluoroethylene. This was shown to have a significantly
higher overall isolation rate compared to conventional scraping methods. As it is minimally
invasive, the CIM sampling method offers a unique opportunity to sample both the affected
cornea and unaffected cornea of patients presenting with MK as well as the eyes of patients
unaffected by MK. This will enable a much better understanding of the clinical significance
of isolated organisms in MK.
Metagenomic next-generation sequencing (NGS) techniques enable the genomic analysis of all
the microbes in a sample, giving a wealth of information regarding the presence and
interaction of microorganisms as well as the presence or absence of antimicrobial resistance
(AMR) genes. Previous studies attempting to characterise the ocular surface microbiome in
health using metagenomics have demonstrated a diverse resident homeostatic microbiome,
however, have been limited to swabs taken from the lid, conjunctiva and tears rather than the
cornea. Variations in the microbiome have been associated with conditions such as dry eye
syndrome, contact lens wear and infectious pathologies, including a pathological microbiome
dominated by pseudomonas spp. in a small number of patients with bacterial keratitis. Despite
the cornea being the surface affected by MK, there is no data regarding the healthy corneal
microbiome because traditional sampling methods have been invasive.
Despite NGS generating personalised results in a matter of hours, its sensitivity to detect
all organisms present without an understanding of their clinical significance is a barrier to
its introduction into clinical practice. In this project, the CIM sampling method and NGS
processing will be used to improve understanding of the causative organisms in MK.
Furthermore, through sampling both the affected and unaffected eyes of MK patients and from
those without MK, a diagnostic rule will be developed to identify and discount commensal
organisms. The inclusion of different control groups will also provide information on the
influence of contact lenses and eye drops on the microbiome, which have a role both on the
development and treatment of MK. This will greatly facilitate current interpretation of
corneal sample results and facilitate potential future use of NGS in routine ophthalmic
clinical practice.
Aims and objectives
The overall aim of the project is to better define the pathogenic microorganisms in patients
with MK through a better understanding of the corneal microbiome in health and disease.
This will be achieved through the following objectives.
1. Using NGS, analyse the corneal microbiome of the affected and unaffected eye of patients
with and without MK and compare with simultaneous results from CDC and MTPCR.
2. Determine the microbiological spectrum of the cornea, ocular surface and contiguous
structures, in patients with MK, healthy controls, contact lens wearers and eye drop
users.
Study Design:
Design: Prospective observational diagnostic study and methods-comparison.
Study Duration: Three years.
Number and type of subjects:
151 patients with MK will be recruited. This is based on the following sample size
calculation: Current standard diagnostic reference method (CDM) has a diagnostic accuracy of
40%. For clinical implementation of NGS, diagnostic accuracy will be required to increase to
at least 80%. NGS can only increase the proportion of detected micro-organisms. To estimate a
diagnostic rate of 80% with a 95% confidence interval (of width 15%) 137 participants are
required. This calculation assumes that the smallest diagnostic group has a prevalence of
20%. To adjust for a 10% rate of possible loss of follow-up, we will recruit 151 patients.
We will also recruit 90 participants to four control groups:
1. 20 patients with no history of MK who use no eye drop medication
2. 20 patients with no history of MK who are contact-lens wearers
3. 20 patients who have no history of MK but are on eye drop treatment for glaucoma. This
group has been included to assess for changes in the corneal microbiome that could be
secondary to drop treatment.
4. 30 patients with keratoconus who are undergoing cross-linking will be recruited. These
participants as part of the routine cross-linking procedure will have their corneal
epithelium removed. This removed epithelium from an otherwise healthy corneal surface
will allow for a direct comparison between the corneal microbiome characterised from the
CIM and that characterised directly from the epithelium.
Clinical data collection
At presentation, patient demographics, risk factors (ocular surface disease, contact lens
wear, previous MK), and treatment received in the past or using at presentation will be
obtained from interview, together with ulcer characteristics (major and minor axes of the
corneal ulcer measured using a continuous scale). A standardised data collection form will be
utilised.
Best corrected visual acuity (BCVA), size, location and depth of the ulcer will be recorded
using a slit lamp biomicroscope. Ulcer size (minor and major axes) and location (minimum
distance from the limbus) will be measured. Ulcer depth will be measured on a nominal scale
of 1-4 based on percentage of remaining corneal thickness underneath the ulcer.
Specimen Collection
For each participant, the following samples will be collected:
- Three CIMs from the affected and unaffected eyes of participants with MK, or from one
eye of control participants.
- Swabs from conjunctiva of the participant's affected and unaffected eyes, upper eyelids
and nose.
Three CIMs will be applied (PTFE Millipore culture insert, pore size 0.40micrometres) to the
surface of the cornea for 5 seconds and one CIM to the participants inferior fornix
conjunctiva for 5 seconds using sterile gloves. A topical anaesthetic (one drop of 0.5%
proxymethacaine) will be instilled into the lower conjunctival fornix prior to application of
the CIMs. The first CIM sample obtained from the participants cornea will be placed and
transported in a bottle containing 0.5mL of BHI broth for culture. The CIMs obtained from the
participants cornea and the inferior conjunctiva will be kept dry in a sterile tube. In
addition to the collection of samples described, the corneal epithelium removed from patients
undergoing corneal cross-linking will be placed in a sterile eppendorf and stored at -80 C.
Swabs will be taken from the participant's conjunctiva, upper eyelids and anterior nares in
order to characterize the surrounding microbiome and endogenous sources.
MK participant follow up
Participants presenting with clinically suspected MK will be followed up at 3 days, 7 days
and 1 month following presentation, as per normal procedure for follow-up of MK. At each
follow up appointment BCVA, size and depth of the ulcer will be recorded. All samples will be
repeated at each follow up appointment from the participants affected eye only. These will
include three corneal CIMS, conjunctival, upper eyelid and nasal swabs.
Processing of samples and identification of microorganisms:
1. CDC (established diagnostic standard): Blood, chocolate, and Sabouraud's dextrose agar
plates and a 24-hour subculture of the BHI broth inoculated with the bacterial swabs and
membranes will be examined for evidence of bacterial growth after 24 and 48 h
incubation.
2. MTPCR:DNA will be extracted and quantified from the CIM and virology conjunctival swab.
A multiplex microorganism targeted PCR (MTPCR) master mix, including primers for HSV-1,
HSV2, acanthamoeba, 16S and 18S ribosomal DNA will be prepared and PCR performed using a
real-time PCR instrument
3. NGS: Metagenomic testing will be performed using high-throughput platforms, such as the
Illumina HiSeq 4000. Sequencing depth will be determined empirically. Genome alignments,
filtering and pathogen calling will be performed using established pipelines. An
analysis approach based on read classification will be applied for taxonomic
classification. A subtractive analysis method based on removal of host DNA and healthy
control eye metagenome will be applied.
Statistical analysis:
A comparison of isolation rates and identified bacteria obtained from CDC, MTPCR and NGS
processing of MK corneal samples will be made. This will be visualised using a Venn Diagram.
For NGS data, we will work with the Centre of Genomic Research (CGR) to assign taxonomy
labels and calculate relative abundances in each sample. For detection of AMR, software will
be used to directly map reads to those in a comprehensive AMR database and report the AMR
genes present.
Microorganisms identified in the eyes with MK will then be compared to the control fellow eye
and other control groups and subtractive bioinformatics methodology applied to identify the
most likely pathogenic organisms compared to those seen in the healthy corneal microbiome.
Comparisons of the relative abundance of microorganisms obtained from MK corneal samples over
the participant follow up visits will be used to evaluate longitudinal changes in the corneal
microbiome during treatment and resolution of MK.
A direct comparison between the relative abundance of microorganisms isolated from
participants cornea, conjunctiva, eyelids and nose will be made to identify any possible
endogenous sources of infection for MK. Overlaps of microorganisms between the sample sites
will be visualised using a Venn diagram and non-parametric tests used to assess for
differences between the sample sites.
