Novel DNA microarray in sepsis diagnostics

Sepsis is defined as a documented infection with systemic inflammatory response syndrome (SIRS). When pathogens have been detected by blood culturing method, the condition is classified as a bloodstream infection (BSI). The frequency of severe sepsis is approximately 90.4 cases per 100 000 population in Europe and circa 751 000 cases annually in United States. Sepsis is associated with high mortality rates ranging up to 50 % in most severe cases. The presence of immunocompromising conditions, chronic diseases, prosthetic devices such as intravenous lines or urinary catheters and higher age are factors which typically increase the infection risk. Currently, common causative bacteria such as Staphylococcus aureus, other staphylococci, Escherichia coli and Klebsiella pneumoniae are detected using blood culturing method. It is time-consuming, especially in case of fastidious and slow growing bacteria and thus initial empirical therapy typically contains broad-spectrum antimicrobial(s). Rapid methods for sepsis/BSI diagnostics are needed to improve patient outcomes, decrease length of stay in hospital and related costs. When causative pathogens are identified earlier, also appropriate antimicrobials can be administered earlier. The aim of this study was to develop a polymerase chain reaction (PCR) and microarray-based assay for the detection of main causative pathogens and methicillin resistance marker from patients with suspected sepsis/BSI. The assay, which utilized the Prove-itTM TubeArray platform, was first developed for detection of 12 bacterial species, coagulase negative Staphylococcus group and methicillin resistance marker. The performance of this assay was evaluated with blood culture samples. The bacterial panel was further improved for the detection of over 50 causative pathogens in sepsis/BSI. This optimized assay was clinically validated with over 3300 blood culture samples collected from HUSLAB, Finland and UCLH, United Kingdom. The developed assay, named Prove-itTM Sepsis, demonstrated 94.7 % sensitivity and 98.8 % specificity. Based on this validation study, the assay was CE-marked for in vitro diagnostics in Europe. This diagnostics assay with the improved target panel was also successfully transferred and optimized to the Prove-itTM StripArray platform, whose capacity of 1-96 simultaneous analyses responds to the need of hospital laboratories dealing with larger sample amounts. Another aim of this study was to evaluate the PCR and microarray assay’s suitability for identification of pathogens directly from whole blood samples without a culturing step. The assay was combined with a selective bacterial deoxyribonucleic acid (DNA) isolation method and the performance of this combination was evaluated with spiked blood samples. Detection limit of 11-600 colony forming units per mL was obtained depending on the target organism. In addition, analytical sensitivity of 1-21 genome equivalents for the PCR and microarray assay was demonstrated. These results showed proof-of-concept for the combination assay and feasibility of the PCR and microarray assay to be used for more sensitive applications after an extensive optimization phase. Molecular assays have opened a new era in microbiological laboratories and brought a broadened perspective parallel to the conventional culturing and phenotype-based method. Also in this study, genotype-based characterization was utilized to offer more accurate identification than conventional culturing. In future, understanding the clinical relevance of DNAemia may open new strategies to the management of septic patients using nucleic acids-based assays.