Molecular Methodologies for Improved Polymicrobial Sepsis Diagnosis

Abstract

Polymicrobial sepsis is associated with worse patient outcomes than monomicrobial sepsis. Routinely used culture-dependent microbiological diagnostic techniques have low sensitivity, often leading to missed identification of all causative organisms. To overcome these limitations, culture-independent methods incorporating advanced molecular technologies have recently been explored. However, contamination, assay inhibition and interference from host DNA are issues that must be addressed before these methods can be relied on for routine clinical use. While the host component of the complex sepsis host–pathogen interplay is well described, less is known about the pathogen’s role, including pathogen–pathogen interactions in polymicrobial sepsis. This review highlights the clinical significance of polymicrobial sepsis and addresses how promising alternative molecular microbiology methods can be improved to detect polymicrobial infections. It also discusses how the application of shotgun metagenomics can be used to uncover pathogen/pathogen interactions in polymicrobial sepsis cases and their potential role in the clinical course of this condition.

Polymicrobial Infections and Biofilms: Clinical Significance and Eradication Strategies

“…The current standard approach for polymicrobial detection remains culture-dependent [ 119 ]. However, culture-dependent approaches are prone to disadvantages, such as low turnaround and false negatives for slow-growing or low-titer pathogens.…”

Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures

“…The blood was spiked with 40 CFU/mL from an overnight plate culture of the clinical strain. The lower CFU was used as an inoculum because, in the case of sepsis, the CFU/mL ranges typically from 10-100 (Doualeh et al, 2022). E. coli was grown overnight on a BHI agar plate and then suspended in saline to measure the OD.…”

“…12-14 h). When growing bacteria from clinical samples in blood cultures, however, a far lower inoculum is experienced, i.e., <100 CFU or 0.5 McFarland units (MFU), and growth must be pursued for a minimum of 24 h up to several days to detect potential pathogens (Doualeh et al, 2022;Hardy et al, 1992;Waldeisen et al, 2011). The lengthy growth of fastidious bacteria in blood cultures to reach detectable titers is a significant bottleneck in the workflow of testing pathogens for AMR (Menchinelli et al, 2019;Somily et al, 2018;Verroken et al, 2015).…”

Visible label-free detection of bacterial DNA using flocculation of sterically stabilised cationic latexes

“…These methods include loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR), the latter of which is currently the most used and researched method for amplification of nucleic acids. [15][16][17][18] A Nucleic Acid Amplification Test (NAAT) such as PCR involves the extraction, amplification and consequent detection of DNA using a variety of methods in order to determine the presence or absence of an infection.…”