Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection

Background: Rapid and reliable diagnostic methods for Aspergillus fumigatus infec-tion are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus.Methods: The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplifica-tion (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical iso-lates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay.Results: No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus ter-reus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation exper-iments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus sec-tion, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003).

Automated summary

A highly sensitive and specific method for the detection of A. fumigatus using the CRISPR/Cas13a system has been developed. The method shows a promising potential for clinical diagnosis with high specificity and sensitivity.