Dear Editor, Acanthamoeba keratitis (AK) is a rare but sight-threatening corneal infection, accounting for approximately 2–3% of microbial keratitis cases globally, with rising incidence among contact lens users and immunocompromised individuals1. The pathophysiology involves trophozoite invasion of the corneal epithelium and stroma, triggering severe inflammatory responses, ring-shaped stromal infiltrates, and progressive tissue necrosis. Symptoms include ocular pain, photophobia, tearing, and blurred vision, but clinical overlap with viral, fungal, or herpetic keratitis contributes to misdiagnosis in more than 50% of cases2. Conventional diagnostic methods such as culture, confocal microscopy, and PCR remain limited, with sensitivity ranging from 40 to 60% and specificity from 65 to 75%, often requiring more than 48 hours for results3. Current treatment modalities include topical biguanides (polyhexamethylene biguanide), diamidines (propamidine isethionate), and keratoplasty in refractory cases, yet delayed diagnosis remains a major determinant of poor visual outcomes4. CRISPR-Cas12a technology, a DNA-guided DNA-targeting system, has redefined molecular diagnostics by offering attomolar sensitivity and collateral cleavage activity. Unlike PCR, Cas12a enables real-time detection of pathogen-specific DNA fragments without thermal cycling5. Platforms integrating recombinase polymerase amplification (RPA) with Cas12a have demonstrated diagnostic accuracy exceeding 92% for infectious agents in under 45 minutes6. This approach has been successfully applied to tuberculosis, SARS-CoV-2, and human papillomavirus, improving prognosis through rapid detection7, 8. For AK, Cas12a assays targeting conserved genomic regions such as 18S rRNA or mitochondrial DNA fragments could identify species-specific signatures of Acanthamoeba castellanii, A. polyphaga, and A. hatchetti9. The potential to detect DNA levels as low as 10–100 copies/µl surpasses qPCR by nearly two orders of magnitude, offering unprecedented accuracy for ophthalmic infections. Beyond AK, CRISPR-based diagnostics have demonstrated utility in malaria, Zika virus, and influenza, underscoring their broad applicability in infectious disease surveillance10. Recent literature supports this translational potential. In one study, Xiao et al demonstrated that CRISPR-Cas12a integrated with RPA achieved rapid detection of Mycobacterium tuberculosis with sensitivity above 90%, highlighting its applicability in resource-limited settings11. In another investigation, Broughton et al validated Cas12a-based assays for SARS-CoV-2, achieving results in under 45 minutes with high specificity7. Together, these findings illustrate complementary strengths: tuberculosis-focused research highlights diagnostic accuracy in low-resource environments, while viral disease studies confirm broader applicability of CRISPR-Cas12a in point-of-care diagnostics. Translating these successes to AK could significantly improve early detection, reduce misdiagnosis, and prevent vision-threatening complications. Despite promise, several hurdles limit clinical adoption. Variability in sample preparation and DNA extraction from corneal swabs may affect assay reproducibility. Most CRISPR-based diagnostic studies remain preclinical, with limited validation in ophthalmic infections. Cost barriers and infrastructure requirements may hinder implementation in low-resource settings, while ethical concerns regarding data privacy and regulatory approval processes for CRISPR-based diagnostics remain unresolved. Furthermore, integration into clinical workflows requires standardized protocols and clinician training. Literature emphasizes that while predictive accuracy is high, prospective multicenter validation and long-term safety assessments are still lacking3, 7. In conclusion, CRISPR-Cas12a diagnostics targeting Acanthamoeba DNA represent a novel frontier in ophthalmic infection detection. By merging molecular microbiology, CRISPR technology, and portable diagnostics, this approach offers early, non-invasive, and cost-effective identification of AK. Future strategies should prioritize multicenter trials, cost-effectiveness analyses, and awareness campaigns to ensure safe adoption. Integrating Cas12a assays into lateral flow or microfluidic point-of-care platforms could reduce turnaround time to under 45 minutes and cost to under US10 per test, vastly improving accessibility. Clinical drives to evaluate efficacy and side effects will be essential to establish trust and optimize outcomes. This letter adheres to the Transparency in the Reporting of Artificial Intelligence (TITAN) guidelines 2025. 12
Habib et al. (Thu,) studied this question.