Development and evaluation of real-time recombinase polymerase amplification assay for fast identification of Rhizopus arrhizus

Abstract

Purpose

Timely diagnosis of mucormycosis is challenging as the disease is rare and confusing to laboratory physicians without experience or expertise. Molecular tools like real-time PCR have been developed to diagnose mucormycosis and can circumvent these issues. However, their use requires an expensive thermocycler. Hence, there is a need for an alternative rapid, sensitive, and specific low-cost molecular test. Here, we developed and evaluated a real-time recombinase polymerase amplification (RPA) based test for detection of R. arrhizus, the principal cause of mucormycosis in patient samples with and without COVID-19 associated mucormycosis.

Methods

Primers and probes targeting Rhizopus arrhizus for RPA-based assay were designed using PrimedRPA and screened per the manufacturer's guidelines. DNA from 40 clinically relevant bacteria and molds were used to determine the analytical specificity of the assay, and probit regression analysis using plasmid DNA standards were used to determine the analytical sensitivity of the assay. The developed assay was evaluated on 110 tissue samples from patients with suspected mucormycosis.

Results

The developed assay was able to detect 9 mucorales viz. R. arrhizus, R. microsporus, R. stolonifer, R. homothallicus, S. racemosum, M. indicus, M. circinelloides, A. variabilis and Cunninghamella spp. and did not cross-react with the remaining 31 molds or bacteria. Its limit of detection at 95 % probability was 18.58 copies. The test demonstrated a sensitivity of 96 % (95 % CI: 86.3 %–99.5 %) and specificity of 95 % (95 % CI: 86.1 %–98.9 %).

Conclusion

The developed RPA assay for R. arrhizus demonstrates high diagnostic sensitivity(96 %), specificity(95 %), and low detection limit(18.58 copies). While initial testing using stored unfixed tissue showed promise, comprehensive clinical validation studies are needed to establish the assay's diagnostic utility across diverse clinical settings and specimen types.