Rapid and sensitive identification of Candida in blood based on M1 beads enrichment combined with multiple recombinase-aided PCR: a culture-independent approach

Abstract

Introduction: Clinically, timely diagnosis and effective treatment of Candida bloodstream infections rely on rapid and sensitive detection methods. However, the long turn-around time and low detection rate of blood culture (the gold standard) make rapid diagnosis of Candida challenging. This study develops a novel molecular assay (M1-mRAP) designed for the rapid and sensitive detection of three Candida species in blood samples: Candida albicans(CA), Candida tropicalis(CT), and Candida glabrata(CG).

Methods: We used the M1-mRAP method aimed at detecting Candida DNA in blood samples, in which we developed a novel multiplex recombinase-aided PCR (mRAP) assay for sensitive amplification of Candida DNA and used a self-developed recombinant human mannan-binding lectin beads (M1 beads)method for enrichment of Candida in blood. The analytical sensitivity of mRAP was evaluated using Candida recombinant plasmids. The analytical sensitivity of the M1-mRAP method for blood sample detection was assessed using quantitative Candida simulated blood samples. The clinical performance of the mRAP and M1-mRAP methods was evaluated in 120 non-blood samples and 9 blood samples and compared with conventional qPCR methods.

Results: The limit of detection(LOD) for CA, CT, and CG by the mRAP method were 4, 4, and 3 copies/μL, respectively. The LOD for CA, CT, and CG simulated blood samples by the M1-mRAP were 2, 2, and 1 CFU/mL, and the overall detection time was about 3.5 h. Clinical assays of mRAP and M1-mRAP showed that these two methods were consistent with qPCR (P<0.05), but had better clinical detection ability than qPCR. Specifically, the mRAP method identified 5 (4.2%) qPCR-negative samples, while M1-mRAP detected 1 (11.1%) classified as the qPCR grey zone sample.

Conclusion: The M1-mRAP method provides rapid and sensitive detection of low concentrations of CA, CT, and CG blood samples and has the potential to emerge as an important tool for the early detection of Candida bloodstream infections in clinical settings.

Keywords: Candida; bloodstream infection; multiple detection; recombinant human mannan-binding lectin protein beads (M1 beads); recombinase-aided PCR assay(RAP).

Figure 1

Schematic diagram of the M1-mRAP method. The process of M1 beads enrichment of blood samples includes the preparation of M1 beads and the enrichment of blood samples (A); the two-system mRAP method and the single-system mRAP method possess different reaction systems but the same detection principle, RAP (B). M1 protein, recombinant human mannan-binding lectin protein; M1 beads, Magnetic beads coated with M1 protein; RAP, recombinase-aided PCR. This figure was created by Biorender.

Figure 2

The sensitivity of single-system mRAP, two-system mRAP, and qPCR were analyzed using recombinant plasmids of 10⁵-10⁰ copies/μL. The CA (A), CT (B), and CG (C) recombinant plasmids were amplified by single-system mRAP; The CA (D), CT (E), and CG (F) recombinant plasmids were amplified by two-system mRAP method; The CA (G), CT (H), and CG (I) recombinant plasmids were amplified by PCR. NC, negative control; CA, Candida albicans; CT, Candida tropicalis; CG, Candida glabrata..

Figure 3

qPCR and single-system mRAP LODs assay for high-concentration simulated blood samples (20-1000 CFU/mL). The CA, CT, and CG mRAP assays (A–C). The CA, CT, and CG qPCR assays (D–F). CFU, colony forming unit; CA, Candida albicans; CT, Candida tropicalis; CG, Candida glabrata..

Figure 4

qPCR and single-system mRAP LODs assay based on M1-enriched low-concentration simulated blood samples (<10 CFU/mL). The CA, CT, and CG single-system mRAP assays (A–C). The CA, CT, and CG qPCR assays (D–F). CFU, colony forming unit; CA, Candida albicans; CT, Candida tropicalis; CG, Candida glabrata..