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Customised 96-ocular TaqMan (iCAM) microarray PCR card for rapid diagnosis of microbial keratitis
Yunfei Yang1, Ahmed Roble2, Rashmi Deshmukh1
1Department of Ophthalmology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.
Aim:
To validate the diagnostic performance of a custom 96-micro-organism TaqMan PCR card (iCAM) for microbial keratitis (MK) from a single corneal epithelial sample.
Methods:
Patients over the age of 18 referred to Cambridge University Hospital with MK were recruited in this single-site prospective cohort study between September 2021 and January 2023. An ocular-specific, customised microarray card (iCAM) was constructed according to primer and probe nucleotide sequences developed in our department to detect bacteria, viruses, Acanthamoeba and fungi commonly implicated in MK using a single corneal epithelial sample. Part of the corneal epithelial sample was taken for conventional cultures per local protocol, followed by iCAM array. Microbial detection rate and positive predictive value (PPV) were evaluated.
Results:
38 corneal epithelial samples from 32 patients with MK and 4 control samples from healthy participants were obtained from 36 consecutive patients. A causative microbe was isolated in 15/34 samples (44%) using the iCAM test, compared with 15 by conventional methods (44%). iCAM test processing time varied between 6 and 24 hours compared with up to 7 days for conventional tests. Combined, the microbial detection rate was 65%, with the correlation between methods at 62%. The iCAM test could detect all major micro-organism groups with 56% sensitivity and 60% PPV.
Conclusions:
The iCAM test can detect bacterial, fungal, viral and protozoan organisms using one corneal epithelial sample. The limitations include small patient cohort size and reduced volume of available corneal epithelial sample when shared between the iCAM PCR test and conventional culture methods utilised in the study. A multicentre trial is being planned to validate the clinical impact of using iCAM test on accuracy of diagnosis, early institution of appropriate antimicrobials and clinical outcomes.
Trial Registration Number:
ISRCTN17422545.
Insights
A new TaqMan PCR card (iCAM) accurately detects microbes causing microbial keratitis (MK) from a single corneal sample, offering faster results than traditional methods. This diagnostic tool shows promise for improving MK diagnosis and treatment.
Area of Science:
- Ophthalmology
- Microbiology
- Molecular Diagnostics
Background:
- Microbial keratitis (MK) is a significant cause of vision impairment.
- Accurate and rapid diagnosis of the causative microbe is crucial for effective treatment.
- Current diagnostic methods, including cultures, can be time-consuming.
Purpose of the Study:
- To validate the diagnostic performance of a custom 96-micro-organism TaqMan PCR card (iCAM) for detecting microbes in corneal epithelial samples.
- To compare the iCAM test's performance against conventional microbiological methods.
Main Methods:
- A prospective cohort study involving patients with MK was conducted.
- Corneal epithelial samples were analyzed using both the iCAM PCR test and conventional culture methods.
- Microbial detection rates and positive predictive values (PPV) were evaluated.
Main Results:
- The iCAM test identified a causative microbe in 44% of samples, comparable to conventional methods (44%).
- iCAM test processing time ranged from 6 to 24 hours, significantly faster than conventional tests (up to 7 days).
- The overall microbial detection rate was 65%, with a 62% correlation between methods; sensitivity was 56% and PPV was 60%.
Conclusions:
- The iCAM test effectively detects a broad range of microbial pathogens (bacteria, fungi, viruses, protozoa) from a single corneal sample.
- Limitations include a small patient cohort and sample volume constraints when split for multiple tests.
- A multicenter trial is planned to further validate the clinical impact of iCAM on diagnostic accuracy and patient outcomes.
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