Detection assay of Cryptococcus neoformans bloodstream infection based on recombined MBL- magnetic beads enrichment

ObjectiveTo establish an assay based on recombined MBL protein-magnetic beads enrichment technique for the detection of Cryptococcus neoformans directly from whole blood, and provide guidance for clinical diagnosis of C. neoformans infection.

MethodsWestern blot was used to detect the direct binding of recombined MBL protein (M1) to C. neoformans. M1-protein A beads were used to enrich C. neoformans with different concentration gradients in phosphate buffer saline (PBS)-simulated and rabbit blood-simulated samples, and the enrichment efficiencies were obtained. Three different assays of detecting C. neoformans in rabbit blood-simulated samples were compared., i.e. assay 1, M1-protein A enrichment followed by qPCR identification, assay 2, M1-protein A enrichment followed by culture and MS identification, assay 3, blood culture followed by MS identification (standard gold method).

ResultsM1 protein could bind with standard and clinical strains of C. neoformans. The enrichment efficiency of M1-protein A magnetic beads in PBS-simulated samples with pathogens of 20 CFU/ml, 10 CFU/ml, 5 CFU/ml, and 1 CFU/ml was 71.65%, 72.40%, 62.70%, and 83.00% respectively, and in rabbit blood-simulated samples was 35.76%, 18.00%, 21.04%, and 23.30% respectively. Among the three assays of detecting C. neoformans, assay 1 needed the shortest time (4.25 h), and sensitivity was 10 CFU/ml. The sensitivity of assay 2 could reach ≤ 1 CFU/ml and needed a total time of 57 h. Although the sensitivity of assay 3 could also reach ≤ 1 CFU/ml, it lasted for 120 h.

ConclusionCompared with standard blood culture, the detection assays with qPCR or MALDI-TOF MS based on M1-protein A beads enrichment need much less time, but still have high sensitivity and specificity.