Bench-to-bedside review: The promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection

Abstract

Early infection diagnosis as the cause of a patient's systemic inflammatory syndrome is an important facet of sepsis care bundles aimed at saving lives. Microbiological culture provides the main route for infection diagnosis but by its nature cannot provide time-critical results that can impact on early management. Consequently, broad-spectrum and high-potency antibiotics are essential during the immediate management of suspected sepsis in critical care but are associated with the development of drug-resistant organisms and superinfections. Established molecular laboratory techniques based on polymerase chain reaction (PCR) technology can detect pathogen DNA rapidly and have been developed for translation into a clinical diagnostic setting. In the setting of sepsis in critical care, emerging commercial systems are now available for the analysis of whole blood within hours, with the presumed aim of adoption into the current care bundles. In this review, we consider the importance of early infection diagnosis in sepsis, how this is limited by culture approaches and how the emerging PCR methods are showing promise in early clinical observational studies. The strengths and weaknesses of culture and PCR pathogen detection in whole-blood samples will be highlighted and recommendations made for urgent appropriately powered diagnostic validation studies in advance of clinical effectiveness trials before these emerging PCR pathogen detection techniques can be considered for adoption in clinical practice.

Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis

“…Laboratory validation studies have focused on two approaches using PCR for genomic amplification with either (a) broad range detection of bacterial or fungal DNA with universal primers, followed by species identification using a post-PCR technique such as gene sequencing or electrospray mass spectrometry or (b) using species-specific hybridisation probes that provide direct confirmation of the species present [10]. While the laboratory analytical accuracy of these techniques for the detection of pathogen DNA in blood has been evaluated, there is a lack of reported clinical trial data to define the utility of such tests in patients [2,10]. This has been due in part to the lack of standardised technology platforms that meet accepted regulatory standards for clinical diagnosis.…”

Molecular typing of bacteria for epidemiological surveillance and outbreak investigation / Molekulare Typisierung von Bakterien für die epidemiologische Überwachung und Ausbruchsabklärung

“…For selected pathogens, specific PCR detection assays, including real-time PCR methods, have been developed: they are very powerful, simple, and effective tools for fast detection and identification, even directly from clinical or environmental specimens (Maheux et al, 2013). To bypass the main disadvantage of PCR-based identification technologies, that is, the one primer pair for each pathogen principle, diverse broad range and multiplex PCR protocols have been published to allow detection of the 35 main important pathogens in a single and closed-tube reaction format, considerably shortening the time to result and improving the outcome of patients (Dark et al, 2009;Mancini et al, 2010;Lucignano et al, 2011). In our laboratory, a high-resolution melting (HRM) PCR assay was developed for rapid and accurate differentiation of highly pathogenic Yersinia pestis strains from Yersinia pseudotuberculosis and highly pathogenic Bacillus anthracis strains from Bacillus cereus strains that allowed specific, rapid, and simple identification of these highly pathogenic bacterial species .…”

Detection of microbial DNAemia: does it matter for sepsis management?

“…While broad spectrum empirical antimicrobial therapy allows to initially treat a majority of likely pathogens, every effort should be made to rapidly identify the causative pathogens and narrow the spectrum of therapy to minimize the development of antibiotic resistance. Currently, microbiological testing in sepsis still relies on blood cultures, microscopic examination and culture of specimens from the suspected focus of infection [5]. By nature, microbiological culture is a slow process.…”

“…Multiplex analysis of nucleic acid sequences of microbial pathogens allows rapid detection and identification of pathogenic agents in clinical samples such as blood [5] or sputum [7]. Target genes include those of 16S or 23S ribosomal RNA, or the intervening spacer region, which allow reliable discrimination between bacterial species [5].…”

“…Target genes include those of 16S or 23S ribosomal RNA, or the intervening spacer region, which allow reliable discrimination between bacterial species [5]. Two technologies have been developed: (1) real-time PCR, in which amplified segments of DNA are being monitored quantitatively by fluorescent dyes or labeled hybridization probes and (2) DNA microarrays, in which labeled ribosomal RNA or genomic DNA is detected by hybridization with specific DNA probes spotted on a solid phase [7,8].…”