Rapid Detection of Pathogens in Sepsis- Patients using Multiplex Droplet Digital PCR

Background: Sepsis is a major public health concern, with more than 11 million deaths annually. Every hour of delay in antibiotics increases mortality by 7.6%. Blood culture (BC) is the cornerstone of diagnosis, with a detection time up to 72h and 10-40% false-negative results (based on quantitative PCR (qPCR)). Rapid detection would facilitate earlier treatment and/or earlier switch to narrow-spectrum antibiotics. Reduced broad-spectrum therapy reduces antibiotic resistance and other complications e.g. Clostridium difficile infection.

Methods: We are developing a multiplex droplet digital PCR (ddPCR) assay for rapid diagnosis of sepsis using the QX200 System (Bio-Rad). The study has three phases: 1) Design of primers and probes for accurate and reliable quantification of common sepsis-causing microorganisms. 2) Determination of sensitivity and specificity of the multiplex ddPCR assay. 3) Clinical study in critically care sepsis-patients using the assay. In the first phase, primer-probe (PP) pairs for the targets of coa (S. aureus), cpsA (S. pneumoniae), uidA (E. coli), and oprL (P. aeruginosa) were designed using AllelID 7.85 software (Premier Biosoft) for the detection of bacterial genomic DNA. The designed PP pairs were tested using the following ATCC strains: 29213 (S. aureus), 49619 (S. pneumoniae), 25922 (E. coli), and 27853 (P. aeruginosa).

Results: After modification of the DNA extraction protocol for gram-positive bacteria, the designed PP pairs for cpsA and uidA detected the bacterial DNA with high amplitude (3-fold increase for positive droplets compared to negative droplets) and with no rain droplets (droplets that fall between the positive and negative ‘clouds’). In comparison, PP pairs for oprL showed extreme rain droplets, while the PP pairs for coa detected no signal. A systematic bioinformatics search demonstrated a mismatch between the reverse primer for oprL and ATCC 27853 and a mismatch between the forward primer and probe for coa and ATCC 29213, explaining both results. Therefore, the PP sequences for both targets (oprL and coa) were redesigned, and it was ensured that both designs match 100% the ATCC strains of both microorganisms, respectively.

Conclusions: The redesigned sequences will be re-tested using the ddPCR. Completion of this first phase of the study will allow progression to stages 2) and 3).