JMIR Publications

ABSTRACT

Background:

Blood culture (BC) is the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48h and 10-40% false-negative results. Rapid diagnosis would facilitate earlier treatment and/or earlier switch to narrow-spectrum antibiotics. The aim of this study is to develop and implement a multiplex droplet digital PCR (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3h of incubation.

Objective:

The study consists of three phases: 1) Design of primers-probe (PP) pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms in a multiplex reaction. 2) Determination of the analytical sensitivity and specificity of the multiplex ddPCR assay. 3) Clinical study in sepsis-patients using the assay.

Methods:

The QX200 ddPCR System (Bio-rad) will be used for the detection of species-specific genes, i.e. coa (staphylocoagulase) in S. aureus, cpsA (capsular polysaccharide) in S. pneumoniae, uidA (beta-D-glucuronidase) in E. coli, oprL (peptidoglycan-associated lipoprotein) in P. aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria in blood from sepsis patients. All data will be analyzed using QuantaSoft™ Analysis Pro Software (Bio-rad).

Results:

In Phase 1, for the optimal annealing temperature for the designed PP pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In Phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In Phase 3, the clinical sensitivity and specificity compared to the blood culture technique will be determined using 301 clinical samples.

Conclusions:

Successful design of PP pairs in the first phase, and subsequent optimization and determination of limit of detection will allow progression to phase three to compare the method with existing blood culture methods.