Development and evaluation of high-resolution melting assays for direct and simultaneous pathogen identification in bloodstream infections in pediatric oncology patients

Blood culture, the gold-standard method for identifying pathogens in bloodstream infections, is time-consuming and demonstrates low sensitivity. These drawbacks are related to high mortality, especially among pediatric oncology patients presenting febrile neutropenia episodes. Here we describe two novel High-Resolution Melting assays designed for pathogen detection in bloodstream infections. The assays were initially evaluated using five sepsis-associated pathogens. Both assays demonstrated 100 % specificity, detected as low as 100 fg of bacterial DNA, and exhibited reproducibility. Clinical isolates from blood cultures were 100 % identified by both assays. Moreover, blind and direct identification of blood samples from pediatric cancer patients demonstrated sensitivities of 61.5 % and 69.2 % for "Primer Set 1" and "Primer Set 2", respectively. Our study highlights the potential of HRM-based assays as a rapid and efficient diagnostic approach for sepsis-related microorganisms. Further advancements could enhance their clinical utility for better management of febrile neutropenia episodes, especially in pediatric oncology patients.