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

Abstract

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.

Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR

“…Indeed, we observed 99% detection across a panel of 10 species commonly causative of sepsis at the level of 2 genome copies per reaction (n = 120). The dye based approach used here also has potential to give limited speciation data if multi-step high resolution melt curve analysis of the product is employed [ 25 ].…”

Rapid Identification of Candida Species in Candidemia Directly from Blood Samples Using Imperfect Match Probes

“…The IM Q-probe method does not use melting curve shapes, instead relying only on Tm values. For this reason, although melting curve shapes are affected by various DNA concentrations 15 , Tm values are not. In fact, fungal concentrations in candidemia vary among individual patients.…”

Epidemiology of bloodstream infection in Saudi Arabia

“…HRMA, when combined with real time PCR, can be used for characterizing PCR amplicons based on differences in melting profile (curve shape) and therefore identifying a range of clinically important organisms from clinical isolates and positive culture (Cheng et al, 2006;Yang et al, 2009). Ozbak and co-authors in 2012 reported that the HRMA combined with PCR is a low-cost molecular approach that has ability for identification of the 21 organisms responsible for bloodstream infection from pathogens isolated from positive blood culture (Ozbak et al, 2012). In 2018, this PCR-HRMA approach was tested further for its ability for detection and identification of antibiotic resistant genes (Ozbak, 2018).…”