Combined Molecular Gram Typing and High-Resolution Melting Analysis for Rapid Identification of a Syndromic Panel of Bacteria Responsible for Sepsis-Associated Bloodstream Infection

Effective diagnosis and treatment of bloodstream infections are often hampered by a lack of time-critical information from blood cultures. Molecular techniques aimed at the detection of circulating pathogen DNA have the potential to dramatically improve the timeliness of infection diagnosis. Our aim in this study was to establish a rapid, low-cost PCR approach using high-resolution melting analysis to identify a syndromic panel of 21 pathogens responsible for most bloodstream bacterial infections encountered in critical care environments. A broad-range, real-time PCR technique that combines primers for molecular Gram classification and high-resolution melting analysis in a single run was established. The differentiation of bacterial species was achieved using a multiparameter, decision-tree approach that was based on Gram type, grouping according to melting temperature, and sequential comparisons of melting profiles against multiple reference organisms. A preliminary validation study was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. Fifty isolates contained organisms that were present in the panel, and 96% of these were identified correctly at the genus or species level. A correct Gram classification was reported for all 53 isolates. This technique shows promise as a cost-effective tool for the timely identification of bloodstream pathogens, allowing clinicians to make informed decisions on appropriate antibiotic therapies at an earlier stage.