Development Of A Ultrasensitive Crispr-Based Diagnsotic Platform For Point-Of-Care Diagnosis Of Fungal Keratitis

Despite being a disease of near epidemic proportions in developing tropical countries, diagnostic difficulties have ensured that fungal keratitis requires tertiary care facilities and patients are generally referred to cornea specialists by primary care physicians and general ophthalmologists for establishing diagnosis and initiating treatment. The diagnosis of fungal keratitis remains the bottleneck which precludes its management at the primary heath care level, in this project, we propose to develop a rapid and accurate molecular detection system for detection of fungal keratitis that combines iso-thermal amplification, CRISPR/Cas12a enzymology and the lateral flow detection method. 

For validation of the assay, thirty patients with presumed fungal corneal ulcers presenting to the outpatient department and specialty service of our tertiary eye care facility were included in this prospective study.

 

The genome sequences from a comprehensive list of 49 fungal species was aligned to identify the conserved regions and for designing the Recombinase Polymerase Amplification (RPA) primers targeting the 18S rRNA region. LbCas12a protein was preassembled with identified crRNAs and ss-DNA fluroscent reporter. The sensitivity and specificity of the assay was first established using clinical isolates and reference strains of 9 different fungi as positive controls and 4 bacteria as negative controls.

 

Of the 30 presumed cases of mycotic keratitis, CRISPR assay showed positivity in 93%, culture in 43%, hyphae in KOH in 80%, and Gram’s staining in 45%. The sensitivities of PCR, KOH, and Gram’s were 93%, 81%, and 60% and specificities 42%, 53%, and 61%, respectively. 

This work establishes a CRISPR based rapid point-of-care clinical diagnostic for the diagnosis of fungal keratitis.