Performance evaluation of InfectID-BSI: A rapid quantitative PCR assay for detecting sepsis-associated organisms directly from whole blood.

BACKGROUND: Bloodstream infections (BSIs) (presence of pathogenic organism in blood) that progress to sepsis (life-threatening organ dysfunction caused by the body's dysregulated response to an infection) is a major healthcare issue globally with close to 50 million cases annually and 11 million sepsis-related deaths, representing about 20% of all global deaths. A rapid diagnostic assay with accurate pathogen identification has the potential to improve antibiotic stewardship and clinical outcomes. METHODS: The InfectID-Bloodstream Infection (InfectID-BSI) test is a real-time quantitative PCR assay, which detects 26 of the most prevalent BSI-causing pathogens (bacteria and yeast) directly from blood (without need for pre-culture). InfectID-BSI identifies pathogens using highly discriminatory single nucleotide polymorphisms located in conserved regions of bacterial and fungal genomes. This report details the findings of a patient study which compared InfectID-BSI with conventional blood culture at two public hospitals in Queensland, Australia, using 375 whole blood samples (from multiple anatomical sites, eg. left arm, right arm, etc.) from 203 patients that have been clinically assessed to have signs and symptoms of suspected BSI, sepsis and septic shock. FINDINGS: InfectID-BSI was a more sensitive method for microorganism detection compared with blood culture (BacT/ALERT, bioMerieux) for positivity rate (102 vs 54 detections), detection of fastidious organisms (Streptococcus pneumoniae and Aerococcus viridans) (25 vs 0), detection of low bioburden infections (measured as genome copies/0.35 mL of blood), time to result (<3 h including DNA extraction for InfectID-BSI vs 16 h-48 h for blood culture), and volume of blood required for testing (0.5 mL vs 40-60 mL). InfectID-BSI is an excellent 'rule out' test for BSI, with a negative predictive value of 99.7%. InfectID-BSI's ability to detect 'difficult to culture' microorganisms re-defines the four most prevalent BSI-associated pathogens as E. coli (28.4%), S. pneumoniae (17.6%), S. aureus (13.7%), and S. epidermidis (13.7%). INTERPRETATION: InfectID-BSI has the potential to alter the clinical treatment pathway for patients with BSIs that are at risk of progressing to sepsis.

Evaluation of a closed loop-blood sampling system in intensive care: A pilot randomised controlled trial. The ENCLOSE trial.

OBJECTIVE: To test the feasibility of conducting a randomised controlled trial to evaluate the impact of a closed-loop blood sampling system and blood conservation bundle. METHODS: Single site, parallel group, pilot randomised control trial comparing open system sampling to closed system sampling and conservation bundle aligned with national guidelines. Randomisation sequence was generated by an independent statistician and allocation concealment maintained via sealed opaque envelopes. The study setting was the general intensive care unit of a major metropolitan public hospital in Queensland, Australia. Participants were ≥ 18 years who had an arterial catheter inserted in intensive care. Main outcome measures included trial feasibility, blood sample loss, haematocrit (HCT) change, and packed red blood cell transfusion use. RESULTS: Eighty patients were randomised (n = 39 open group, n = 41 closed group). Characteristics in each group were equal at baseline with overall median age 60 years (IQR 48.6-70.4), 58 % male, and median APACHE II score 16 (IQR 11-22). The proportion of patients eligible was 29 % and missed eligible was 65 %. Otherwise, feasibility criteria were met with proportion of eligible patients agreeing to enrolment 99 %, 100 % of patients receiving allocated treatment; only 1 % of data missing. Analysis demonstrated a significant reduction in mean daily blood sample losses (open 32.7 (SD 1.58) mL vs closed 15.5 (SD 5.79) mL, t = -8.454, df = 78, p < 0.001). CONCLUSIONS: A large, multi-site trial is feasible with enhanced eligibility criteria, increased recruitment support. The intervention reduced daily blood sample volumes and transfusion use. Further trials are required to provide both effectiveness and implementation outcomes.

Core and Accessory Genome Comparison of Australian and International Strains of O157 Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australian STEC epidemiology differs from many other countries, as severe outbreaks and HUS cases appear to be more often associated with non-O157 serogroups. It is not known why Australian strains of O157 STEC might differ in virulence to international strains. Here we investigate the reduced virulence of Australian strains. Multiple genetic analyses were performed, including SNP-typing, to compare the core genomes of the Australian to the international isolates, and accessory genome analysis to determine any significant differences in gene presence/absence that could be associated with their phenotypic differences in virulence. The most distinct difference between the isolates was the absence of the stx2a gene in all Australian isolates, with few other notable differences observed in the core and accessory genomes of the O157 STEC isolates analyzed in this study. The presence of stx1a in most Australian isolates was another notable observation. Acquisition of stx2a seems to coincide with the emergence of highly pathogenic STEC. Due to the lack of other notable genotypic differences observed between Australian and international isolates characterized as highly pathogenic, this may be further evidence that the absence of stx2a in Australian O157 STEC could be a significant characteristic defining its mild virulence. Further work investigating the driving force(s) behind Stx prophage loss and acquisition is needed to determine if this potential exists in Australian O157 isolates.

Molecular Prediction of the O157:H-Negative Phenotype Prevalent in Australian Shiga Toxin-Producing Escherichia coli Cases Improves Concordance of In Silico Serotyping with Phenotypic Motility.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australia is unique in its STEC epidemiology, as severe cases are typically associated with non-O157 serogroups, and locally acquired O157 isolates are H-negative/nonmotile. The H-negative phenotype and reduced severity of disease compared to that associated with H7/motile strains are distinct features of Australian O157 strains, but the molecular mechanism behind this phenotype has not been reported. Accurate characterization of the H-negative phenotype is important in epidemiological surveillance of STEC. Serotyping is moving away from phenotype-based methods, as next generation sequencing allows rapid extrapolation of serotype through in silico detection of the O-antigen processing genes, wzx, wzy, wzm, and wzt, and the H-antigen gene, fliC The detection and genotyping of fliC alone is unable to determine the motility of the strain. Typically, most Australian O157:H-negative strains carry an H7 genotype yet phenotypically are nonmotile; thus, many are mischaracterized as H7 strains by in silico serotyping tools. Comparative genomic analysis of flagellar genes between Australian and international isolates was performed and an insertion at nucleotide (nt) 125 in the flgF gene was identified in H-negative isolates. Chi-square results showed that this insertion was significantly associated with the H-negative phenotype (P < 0.0001). Phylogenetic analysis was also completed and showed that the Australian H-negative isolates with the insertion in flgF represent a clade within the O157 serogroup, distinct from O157:H7 serotypes. This study provides a genetic target for inferring the nonmotile phenotype of Australian O157 STEC, which increases the predictive value of in silico serotyping.