Performance evaluation and further development of the PCR and microarray-based Prove-it™ Sepsis assay

Introduction

The Prove-it™ Sepsis assay is a rapid, broad-range PCR and microarray-based assay designed to identify the majority of sepsis-causing bacteria from positive blood cultures. The pathogen panel covers 50 Gram-negative and Gram-positive bacterial species (Table 1). It also reports methicillin resistance by detecting the mecA gene. The assay time is 3 hours. Our objective was to conduct a performance evaluation study for Prove-it™ Sepsis according to the EN 13612-standard (Performance evaluation of in vitro diagnostic medical devices) and to compare obtained results with those of current culture-based diagnostics. We evaluated the sensitivity, specificity and time to result of Prove-it™ Sepsis in two major teaching hospitals in Helsinki and London.

Table 1 Bacteria and an antibacterial resistance marker identified by the Prove-it™ Sepsis assay

Materials

A total of 3,318 blood samples from patients with suspected sepsis were collected. Blood culture bottles of BacT/ALERT 3D (bioMérieux) and BACTEC 924 (Becton Dickinson) were incubated for a total of 6 days or until flagged as positive.

Methods

DNA was extracted from blood cultures using the automated DNA extraction instrument easyMAG (bioMérieux) prior to the Prove-it™ Sepsis assay. Conventional blood culture was conducted in parallel and results were only revealed for comparison at the statistical analysis stage. Discordant results were studied by DNA sequencing and case-by-case review of original microbiology laboratory data.

Results

Of the analyzed 3,318 blood cultures, 2,107 yielded a pathogen by conventional techniques. Of these, 302 samples contained microbes not covered by Prove-it™ Sepsis, and an additional 137 cultures contained more than one organism. Sensitivity and specificity for Prove-it™ Sepsis were 94.7% and 98.7%, respectively. Of particular significance was the assay's faultless ability to differentiate MRSA from MSSA and from CNS. Furthermore, it provided results on average 1 day earlier than reference methods.

Conclusion

Prove-it™ Sepsis was considered to be a fast, robust, and high-performance diagnostic platform, which is easily implemented into everyday laboratory workflow. Both study sites identified cases where timely information provided by Prove-it™ Sepsis would have significantly improved patient management. Examples include more rational management and antibiotic choice subsequent to earlier differentiation of Gram-positive cocci in clumps into MRSA, MSSA, or CNS, and earlier speciation of Gram-negative organisms. Prove-it™ Sepsis is further configured for detection of Candida spp. and new bacterial targets. The assay now identifies 60 out of the 302 samples not covered during the evaluation, increasing the pathogen coverage from 86% to 89%. The earlier speciation provided by Prove-it™ Sepsis could contribute to faster, more evidence-based patient management and, thus, positive outcomes.