Early detection to enable timely therapeutic intervention is crucial for improved outcome in patients with sepsis, but diagnosis is difficult, as the clinical signs associated with the condition commonly occur in patients with systemic inflammatory response syndrome (including sterile SIRS). This article discusses the current and emerging PCR-based technologies for the diagnosis of sepsis.

by Dr Satyanarayana Maddi, Dr Paul Dark and Dr Geoffrey Warhurst

Sepsis and issues for its early diagnosis
Sepsis is the clinical syndrome resulting from the host’s response to infection and represents a major international healthcare problem being a major cause of mortality and morbidity as well as a massive burden on resources [1]. The clinical signs associated with sepsis, such as changes in respiration, pulse, temperature and circulating immune cell counts, are non-specific and commonly seen in patients with a systemic inflammatory response syndrome (or SIRS) as well as in other insults such as tissue injury, where there is no infective cause. Early identification of sepsis and the ability to differentiate it from sterile SIRS is an important diagnostic goal in international medical practice. Evidence suggests that giving the most appropriate antimicrobial therapy at the earliest opportunity to patients with severe forms of sepsis saves more lives than any other medical intervention [1]. The Surviving Sepsis Campaign, which promotes early goal-directed management of sepsis, recommends initiation of antimicrobial therapy within one hour of clinical suspicion of sepsis [1]. Ideally, this requires rapid confirmation that infection is present and identification of the organism(s) involved. The guidelines advocate taking a whole blood sample and, where possible, other supporting clinical samples for microbiological culture prior to antibiotic administration. The problem facing clinicians is that blood cultures routinely take two to three days to confirm the presence of pathogens in the bloodstream (‘pathogenaemia’) and up to five days to either rule it out or to obtain a complete profile of the pathogen including its antibiotic susceptibility/resistance pattern. Also, since viable organisms are needed for culture, the tests can be compromised if the patient has received antimicrobial therapy prior to sampling, which is common in this clinical field.

In the face of this lack of time-critical information on the infection status of the patient coupled with the knowledge that delaying antimicrobial therapy will impair the survival chances of those patients that have infection, current opinion favours the early use of broad-spectrum and high potency antibiotics with focussing to specific organisms when microbiological evidence becomes available [1, 2]. This ‘safety first’ approach is currently the best available but does have negative consequences, particularly in terms of the overuse of antibiotics. The widespread use of broad-spectrum antibiotics is implicated in the emergence of antibiotic resistant pathogens and increasing rates of infection with Clostridium difficile and fungi. In addition many patients who will subsequently be shown to have had no infection are exposed to unnecessary treatment with powerful and potentially toxic drugs.

Application of PCR to diagnosis of pathogenaemia in suspected sepsis
While microbiological culture is likely to remain the gold standard for infection diagnosis, there is growing interest in the potential of PCR technology to provide early, time critical information based on the detection and recognition of bacterial or fungal pathogen DNA in blood [1, 2]. Platforms based on real-time PCR have proved to be the most effective in this field allowing continuous monitoring of amplicon production with either fluorescent dyes that bind non-specifically to double stranded DNA or fluorescently labelled probes that bind to specific sequences. In real-time PCR, the whole process of amplification, product detection and analysis is achieved in a single reaction vessel. Furthermore, several sequence-specific probes with different fluorescent reporters can be added to the reaction, allowing simultaneous determination of multiple products. This process is therefore ideally suited to sepsis diagnosis in which a variety of pathogen species could be involved. In terms of its application to infection diagnosis in blood (and other clinical samples), PCR offers a number of potential advantages; results are available in a matter of hours rather than days, the extreme sensitivity facilitates detection of even minute amounts of pathogen DNA in clinical samples and the test is not significantly affected by prior administration of antibiotics.

Two basic approaches to assay design have been used, either using specific primers to detect a particular organism or, more commonly, universal primers that bind to conserved sequences in bacterial but not human DNA and can detect a broad range of organisms [1]. The latter approach is ideally suited to sepsis diagnosis which can be caused by a variety of pathogen species. For bacteria, the most favourable targets are sequences in the 16S and 23S rRNA genes which are ubiquitous in bacteria and therefore ideally suited for universal detection of bacterial pathogens. More recently, the gene sequence between the 16S and 23S regions, the so-called internally transcribed region (ITS), has been targeted because it contains additional hypervariable regions that allow even better discrimination between bacterial species. Fungal pathogens can be detected by targeting analogous regions in the fungal genome [1].

Following PCR of these regions, pathogen species present can be identified by (a) specific binding of fluorescent hybridisation probes to the amplified target (b) sequencing of the amplified DNA (c) hybridisation to microarrays (d) melting temperature profiling of the amplified products.

Commercial PCR platforms for bloodstream infection diagnosis
Based on these approaches, a number of commercial systems are now available for detection of bacterial/fungal DNA in blood. Lightcycler SeptiFast, the first real-time PCR system to receive a European CE-mark (2006) for use in diagnosis of bloodstream infection, is manufactured by Roche Diagnostics (Basel, Switzerland) [Figure 1]. SeptiFast is a multiplex assay for detection and identification of a defined panel of 25 bacterial and fungal pathogens known to cause the majority of bloodstream infections in critical care. The assay can be completed in 6-8 hours and has a reported sensitivity of between 3 and 30 colony forming units (CFU) per mL of blood. SeptiFast is currently the most studied commercial PCR-based test for sepsis-associated blood-stream infection with numerous clinical validity studies published to date. At the time of writing, the author’s laboratory is hosting the first independent multicentre systematic validity study comparing SeptiFast with culture for the diagnosis of suspected healthcare-associated bloodstream infection [2]. Based on the results of this study, independent recommendations will be made to the UK’s Department of Health as to whether this real-time PCR technology has sufficient clinical diagnostic accuracy to move forward to efficacy testing during the provision of routine clinical care.

SepsiTest (Molzym GmbH & Co. KG, Bremen, Germany) [Figure 2], which was awarded a CE mark in 2008, uses universal primers to detect bacterial or fungal DNA in blood and other clinical samples but relies on post-test sequencing of the products for subsequent species identification [1, 3]. Studies evaluating the use of SepsiTest in a clinical setting are beginning to appear in the literature [3]. A third CE marked commercial platform, VYOO PCR identification test from SIRS-Lab GmbH, Germany is a semi-automated method combining broad range PCR with multiplex detection plus microarray hybridisation. In addition to detecting 34 bacterial and six fungal species covering 99% of sepsis-associated pathogens, it also detects five resistance markers i.e. mecA for Methicillin Resistance Staphylococcus aureus, vanA and vanB for vancomycin resistance in enterococci and blaCTX-M15 and blaSHV for extended spectrum β-lactamases in gram negative bacilli. To date no published clinical validity studies of this product are available.

Future/emerging approaches and technologies
High resolution melting analysis (HRMA) is a post PCR amplification method of analysing DNA that does not require multiple expensive fluorescent probes, and is solely dependent on intercalating dye chemistry for its results. Using universal primers, the target regions are amplified and then melting curve analysis is performed in high resolution (high resolution with HRM analysis) after the PCR. Thanks to the advances in instrumentation which can delineate minute shifts in the melting temperatures, the species are identified using shifts in the melting profile of the amplicons [4]. HRM analysis is quick and cost effective. The HRMA is still in developmental stage but the future looks encouraging.

Other approaches under various stages of developments for diagnosis of sepsis are FilmArrays (Idaho Technology Inc. USA) [5] [Figure 3], and ‘‘Lab-on-a-Chip’’ using microfluidic-technology i.e. taqMan Low-density array (TLDA), which overcomes limitations in multiplex PCR assays, namely the narrow range of probe regions needed for multiplexing and the inability of the PCR instrument to detect more than six fluorophores simultaneously [6].

Conclusions and future
Widespread technology adoption of these PCR systems will not occur in healthcare until clinical effectiveness has been proven. No adequately powered systematic clinical effectiveness studies have been performed to date in the field of sepsis, resulting in the absence of data that would support optimal pricing of the available technologies alongside health service adoption. There is clearly an unmet need in the field of sepsis diagnostics, but a more coordinated approach to health technology assessment and adoption in this field is urgently required to help patients benefit from the elegant technologies currently available and from those under development.

References
1. Dark PM, Dean P, Warhurst G. Bench-to-bedside review: the promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection. Crit Care 2009;13(4):217.
2. Dark P, Dunn G, Chadwick P, Young D, Bentley A, Carlson G et al. The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology. BMJ Open 2011 Jan 1;1(1):e000181.
3. Kühn C, Disqué C, Mühl H, Orszag P, Stiesch M, and Haverich A. Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis. J Clin Microbiol. 2011 August; 49(8): 2919–2923.
4. Ozbak H, Dark P, Maddi S, Chadwick P, Warhurst G. Combined molecular gram typing and high-resolution melting analysis for rapid identification of a syndromic panel of bacteria responsible for sepsis-associated bloodstream infection. J Mol Diagn 2012 Mar;14(2):176-184.
5. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011; 52 (4): S326-30
6. Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ et al. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011 Jun;49(6):2175-2182.

The authors
Satyanarayana Maddi, Paul Dark, Geoffrey Warhurst
Infection Inflammation Injury Research Group (3IRG)
Salford Royal NHS Foundation Trust, UK. School of Translational Medicine
The University of Manchester, UK.