Abstract
Sepsis is associated with high morbidity and mortality rates worldwide. Rapid and reliable diagnostic methods are needed for efficient and evidence-based treatment of septic patients. Recently, new molecular tools have emerged to complement the conventional culture-based diagnostic methods. In this study, we used spiked whole blood samples to evaluate together two ready-to-use molecular solutions for the detection of sepsis-causing bacteria. We spiked whole blood with bacterial species relevant in sepsis and extracted bacterial DNA with the NorDiag Arrow device, using the SelectNA Blood pathogen DNA isolation kit. DNA extracts were analyzed by the polymerase chain reaction (PCR)- and microarray-based Prove-it™ Bone and Joint assay, resulting in correctly identified bacterial species with detection limits of 11–600 colony-forming unit/mL (CFU/mL). To understand the recovery losses of bacterial DNA during the sample preparation step and the capability of the PCR- and microarray-based platform to respond to the sensitivity requirements, we also determined the analytical sensitivity of the PCR and microarray platform to be 1–21 genome equivalents for the tested bacterial species. In addition, the inclusivity of the Prove-it™ Bone and Joint assay was demonstrated with methicillin-resistant Staphylococcus aureus (MRSA) clones carrying SCCmec types I, II, IV, or V and a nontypable SCCmec type. The proof-of-concept for accurate multiplex pathogen and antibacterial resistance marker detection from spiked whole blood samples was demonstrated by the selective bacterial DNA extraction method combined with the high-throughput PCR- and microarray-based platform. Further investigations are needed to study the promising potential of the concept for sensitive, semi-automated identification of sepsis-causing pathogens directly from whole blood.
Highlights
Sepsis is defined as the presence of systemic inflammatory response syndrome (SIRS) in addition to a confirmed or presumed infection
We studied the capacity of Prove-itTM Bone and Joint assay to meet the sensitivity requirements for whole blood sepsis diagnostics
DNA concentrations representing approximtely 103, 102, and 101 GEs for K. pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), S. agalactiae, E. faecalis, and L. monocytogenes and approximately 103, 102, 101, and 1 GEs for E. coli and S. aureus were analyzed as duplicates using the Prove-itTM Bone and Joint assay
Summary
Sepsis is defined as the presence of systemic inflammatory response syndrome (SIRS) in addition to a confirmed or presumed infection. Blood culture is the gold standard for the determination of sepsis-causing bacteria. Identification of causative pathogens and determination of antibiotic sensitivity profiles require typically 2–5 days. Novel nucleic acid (NA)–based amplification methods have been developed to speed up diagnosis of sepsis. Some of these new concepts are aimed at detecting causative agents directly from whole blood without any culture periods (Klouche and Schro€der 2008; La Scola and Raoult 2009; Mancini et al 2010; Paolucci et al 2010). Identification of bacteria from whole blood eliminates time-consuming culture a 2013 The Authors.
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