Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Bloodstream infections (BSIs) are a significant cause of morbidity and mortality. Successful patient outcomes are diminished by a failure to rapidly diagnose these infections and initiate appropriate therapy. A rapid and reliable diagnostic platform of high sensitivity is needed for the management of patients with BSIs. The combination of an RNA-dependent nucleic acid sequence-based amplification and molecular beacon (NASBA-MB) detection system in multiplex format was developed to rapidly detect medically important BSI organisms. Probes and primers representing pan-gram-negative, pan-gram-positive, pan-fungal, pan-Candida, and pan-Aspergillus organisms were established utilizing 16S and 28S rRNA targets for bacteria and fungi, respectively. Two multiplex panels were developed to rapidly discriminate bacterial or fungal infections at the subkingdom/genus level with a sensitivity of 1 to 50 genomes. A clinical study was performed to evaluate the accuracy of this platform by evaluating 570 clinical samples from a tertiary-care hospital group using blood bottle samples. The sensitivity, specificity, and Youden's index values for pan-gram-positive detection and pan-gram-negative detection were 99.7%, 100%, 0.997 and 98.6%, 95.9%, 0.945, respectively. The positive predictive values (PPV) and the negative predictive values (NPV) for these two probes were 100, 90.7, and 99.4, 99.4, respectively. Pan-fungal and pan-Candida probes showed 100% sensitivity, specificity, PPV, and NPV, and the pan-Aspergillus probe showed 100% NPV. Robust signals were observed for all probes in the multiplex panels, with signal detection in <15 min. The multiplex real-time NASBA-MB assay provides a valuable platform for the rapid and specific diagnosis of bloodstream pathogens, and reliable pathogen identification and characterization can be obtained in under 3 h.