Expert Review of Molecular Diagnostics

ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: https://www.tandfonline.com/loi/iero20

Molecular and biomarker-based diagnostics

in early sepsis: current challenges and future
perspectives

Judith Schenz, Markus A. Weigand & Florian Uhle

To cite this article: Judith Schenz, Markus A. Weigand & Florian Uhle (2019): Molecular and
biomarker-based diagnostics in early sepsis: current challenges and future perspectives, Expert
Review of Molecular Diagnostics, DOI: 10.1080/14737159.2020.1680285

To link to this article: https://doi.org/10.1080/14737159.2020.1680285

Accepted author version posted online: 12

Oct 2019.

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Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group

Journal: Expert Review of Molecular Diagnostics

DOI: 10.1080/14737159.2020.1680285
Molecular and biomarker-based diagnostics in early sepsis: current
challenges and future perspectives

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Judith Schenz1, Markus A. Weigand1 and Florian Uhle1*

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Department of Anesthesiology, Heidelberg University Hospital, 69120 Heidelberg, Germany
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*Corresponding author:
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Florian Uhle
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Dept. of Anesthesiology, Heidelberg University Hospital, Im Neuenheimer Feld

110 69120 Heidelberg,
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Phone: +49 (0) 6221 – 56 35 140

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Fax: +49 (0) 6221 – 56 33 726

email: florian.uhle@med.uni-heidelberg.de
Molecular and biomarker-based diagnostics in early sepsis: current
challenges and future perspectives

Judith Schenz, M.Sc.a, Markus A. Weigand, MDa and Florian Uhle, Ph.D.a*

Affiliations

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Department of Anesthesiology, Heidelberg University Hospital, 69120 Heidelberg, Germany

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* Corresponding author

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Dr. Florian Uhle

Dept. of Anesthesiology

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Heidelberg University Hospital
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Im Neuenheimer Feld 110
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69120 Heidelberg

Phone: +49 (0) 6221 – 56 35 140

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Fax: +49 (0) 6221 – 56 33 726

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Mail: florian.uhle@med.uni-heidelberg.de
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Molecular and biomarker-based diagnostics in early sepsis: current
challenges and future perspectives

Abstract

Introduction: Sepsis, defined as a life-threatening organ dysfunction resulting from

dysregulated host response to infection, is still a major challenge for healthcare systems.
Early diagnosis is highly needed, yet challenging, due to the non-specificity of clinical

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symptoms. Rapid and targeted application of therapy strategies is crucial for patient’s

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outcome.

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Areas covered: Faster and better diagnostics with high accuracy is promised by novel
host response biomarkers and a wide variety of direct pathogen identification

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technologies, which have emerged over the last years. This review will cover both - host
response-guided diagnostics and methods for direct pathogen detection. Some of the
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markers and technologies are already market-ready, others are more likely aspirants.
We will discuss them in terms of their performance and benefit for use in clinical
diagnostics.
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Expert opinion: Latest technological advances enable the development of promising

diagnostic tests, detecting the host response as well as identifying pathogens without the
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need of cultivation. However, the syndrome’s heterogeneity makes it difficult to

develop a universal test suitable for routine use. Moreover, the robustness of the
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biomarkers and technologies still has to be verified. Combining these technologies and
clinical routine parameters with bioinformatic methods (e.g., machine-learning
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algorithms) may revolutionize sepsis diagnostics.

Keywords:
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bacteremia, biomarker, bloodstream infection, diagnosis, diagnostics, fungemia, host

response, molecular, pathogen detection, sepsis

Article highlights:

• Rapid diagnosis of sepsis is urgently needed but still challenging. Novel host response

biomarkers and a plethora of technologies for direct pathogen detection promise faster

and better diagnostics.

 • Each single biomarker has its own advantages and limitations resulting from its

biology.

• Promising novel diagnostic approaches based on biomarkers, biomechanical properties

or host gene expression are under development.

• Direct pathogen detection might overcome the limitations of blood culture-based

detection approaches.

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• Amplification-based systems detect an a priori determined panel of pathogens. The

sensitivity is influenced by the concentration of pathogen-derived nucleic acids as well

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as the presence of PCR inhibitors and the sample volume.

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• Most amplification free technologies do not require purification, extraction, or

amplification steps. Furthermore, next-generation sequencing might enable unbiased

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testing.
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1. Introduction
Sepsis is defined as a life-threatening organ dysfunction resulting from dysregulated host
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response to infection [1]. It is still a major challenge for healthcare systems worldwide and

has an incidence of more than 400 per 100,000 person-years in high-income countries [2].
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Mortality ranges between around 20% and over 40% depending on the severity of syndrome
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and patients’ predisposition [3]. It is controversially debated whether the incidence has risen

or remained stable in recent years [4]. Nevertheless, despite a higher proportion of severe
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cases, the overall mortality falls [5]. Underlying reasons are both the adherence to

standardized treatment bundles [6] and an improvement in general intensive care treatment

[3]. Rapid and accurate diagnostics permit a fast and targeted use of available therapy

strategies, a prerequisite for a further decrease of morbidity and mortality [7]. Estimating the

sepsis-induced healthcare costs is difficult, since there is high heterogeneity in the applied
calculation methods [8]. However, early diagnosis is also relevant from the economic view,

since the earlier the diagnosis is made, the lower the costs are [9].

A major challenge in early diagnosis of sepsis is the non-specificity of clinical

symptoms such as fever, tachycardia, tachypnoea, and leukocytosis/leukopenia. Many of

these symptoms can also occur in a variety of other clinical conditions, e.g., heart failure [10]

or pancreatitis [11]. The extent of a patient’s organ dysfunction can be assessed clinically

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using the sequential organ failure assessment score (SOFA), which has been postulated more

than 20 years ago as a metascore for following the patient’s clinical trajectory [12]. If a

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patient presents with a de novo increase of at least two points in combination with a clinical or

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microbiological suspicion or proof of infection, the diagnostic criteria of sepsis according to

the latest Sepsis-3 definition are fulfilled [1]. The new definition was extensively debated, as
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it was statistically modeled for prognosis, resulting in an enrichment of more severe ill

patients with infection, but not for specific diagnosis. In addition, a sterile, non-infectious
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systemic inflammatory response syndrome (SIRS) can also induce life-threatening organ
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dysfunction [13]. Ideally, a comprehensive diagnostic test for sepsis not only detects the

dysregulated host response but also discriminates between a sterile and an infectious cause
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within short turnaround time. Going beyond, providing insights about the causative pathogen

(e.g., bacteria vs. viruses in respiratory disease) and its resistance pattern are desirable, since
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diagnostics serves the choice of adequate therapy strategies. Ease of use, low demands

concerning preanalytical handling and cost-effectiveness are vital for a diagnostic test, not
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only in economically highly developed regions but especially in low- and middle-income

countries. It is also important to note, that, in comparison, the causative pathogen spectrum

responsible for the majority of cases differs largely among different regions in the world [14].

Over the last years, novel host response biomarkers as well as a wide variety of

technologies for the identification of pathogens have emerged, promising a better diagnostic

accuracy. In this review, we will deal with those markers and technologies in the context of
their performance and benefit for use in early clinical diagnostics. With regard to the current

consensus definition for sepsis diagnosis [1], we will point out both the diagnostic

possibilities detecting the dysregulated host response (Table 1 & 2) as well as the triggering

infection (Table 3). Our aim is to provide a comprehensive overview of the current state of

development and evidence for diagnostic in adult patients. Nevertheless, diagnostic can rarely

be separated from prognosis and therapy decision, meaning that these areas at least partially

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will be touched despite the diagnostic focus.

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2. Host response-guided diagnostic

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The Sepsis-3 definition underlines the dysregulated host response to infection as a

pathophysiological core element of the syndrome. Although a direct assessment of this

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dysregulation would be favorable, the heterogeneity of the syndrome’s manifestation makes it

a sophisticated approach.
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2.1 Classical biomarkers

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Serum lactate is included in the Sepsis-3 definition as diagnostic criteria of septic shock.

Serum lactate levels greater than 2 mmol/l despite an adequate fluid resuscitation together
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with the continuous dependency on vasopressors defines patients with septic shock [15].
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Blood lactate is a widely available parameter but on the other hand, an extremely unspecific

marker of cellular stress or microcirculatory disturbances [16], hampering its use for early
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sepsis diagnosis. Nevertheless, the assessment of blood lactate levels has been incorporated

into the recent guidelines of the Surviving Sepsis Campaign to guide hemodynamic therapy

monitoring and patient’s risk stratification [17]. Lactate clearance, which means the decline of

plasma lactate level towards reference values, is commonly used as a surrogate parameter to

judge the therapy success of fluid resuscitation, hinting towards a reestablishment of tissue

perfusion [18]. However, very recently a head-to-head comparison of lactate versus capillary
refill time for therapy guidance found no difference between these concepts [19].

Interleukin-6 (IL-6) is a pro-inflammatory cytokine produced early during an

immune response and involved in lymphocyte activation, fever, and induction of acute phase

response. In a meta-analysis including six case-control studies, IL-6 identified adult patients

with sepsis with a sensitivity of 85% and a specificity of 62% [20]. This poor performance as

a diagnostic biomarker is hardly surprising since IL-6 and related cytokines are produced

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from innate immune cells (e.g., monocytes and neutrophils) during various inflammatory

reactions and not limited to infections. The concentration of C-reactive protein (CRP) is a

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routinely available diagnostic laboratory parameter. CRP is an acute-phase reactant, produced

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and released by the liver in response to rising levels of pro-inflammatory cytokines (mainly

IL-6). Therefore, CRP, too, is not exclusively produced in response to an infection. Sterile
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inflammatory reactions with a high cytokine production secondarily lead to increased CRP

levels, too. In a meta-analysis by Simon et al. which included in total 905 patients, CRP was
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found to distinguish bacterial from non-infectious inflammation only with a sensitivity of

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75% and a specificity of 67%. However, it was shown to be slightly better suited to

differentiate between bacterial and viral infections (sensitivity of 86% and specificity of 70%)
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[21]. In this regard, it is routinely used in the context of lower respiratory infections [22].

An intensively evaluated marker is procalcitonin (PCT). PCT is the precursor of the

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peptide hormone calcitonin essential for calcium homeostasis: In response to hypercalcemia,

PCT is secreted by the C cells of the thyroid and neuroendocrine cells in the lung and bowel.
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Typically, serum PCT levels are below the limit of detection of common assays. During

systemic inflammation, blood PCT levels rise, in particular in those caused by Gram-negative

bacterial infections, but less pronounced also in patients with Gram-positive bacteremia and

candidemia [23]. PCT measurement is superior to CRP measurement not only to distinguish

between bacterial and viral infections but also between a bacterial or a non-infectious cause of

systemic inflammation [21]. In a meta-analysis including 30 studies with overall 3,244 critical
ill patients, PCT was shown to distinguish between sepsis and sterile SIRS with a pooled

sensitivity of 77% and a pooled specificity of 79%. In surgical patients, it has a slightly higher

diagnostic accuracy than in medical patients [24]. A very recent, but significantly smaller

analysis, reported comparable results [25]. However, other analyses revealed lower accuracies

with respect to diagnosis [26]. Further, a recent meta-analysis showed that currently there is

not enough evidence for PCT alone being sufficient to distinguish bacteremia and candidemia.

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Notwithstanding, in combination with other biomarkers like β-glucan, it may improve

diagnostic accuracy [27]. The actual strength of PCT lies not in the one-time measurement for

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diagnosis, but as a prognostic marker for disease progression and mortality when measured

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sequentially [28,29] as well as for antibiotic therapy guidance [30]. In this context, it has been

incorporated into the Surviving Sepsis Campaign recommendations [18]. A point-of-care

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device (B·R·A·H·M·S PCT direct; Thermo Fisher Scientific, Hennigsdorf, Germany) is

available allowing rapid, bedside detection without being dependent on opening hours of a lab
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service.
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2.2 Novel biomarkers

Presepsin (also sCD14-ST) is a proteolytically truncated subtype (ST) of soluble cluster of

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differentiation 14 (sCD14), consisting of 64 amino acids. CD14 is a co-receptor of the toll-

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like receptor 4 (TLR4) for the binding of lipopolysaccharide (LPS)-lipopolysaccharide-

binding protein (LBP) complex, subsequently facilitating the LPS-induced TLR4 activation.
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By metalloproteinase shedding from the cell surface, the CD14-LPS-LBP complex is released

into the blood where proteases can further cleave it, resulting in presepsin formation. Due to

its origin in bacterial pattern recognition, the level of free presepsin in the blood is considered

a diagnostic biomarker in sepsis. A meta-analysis of eleven studies with heterogenous

inclusion criteria revealed an overall sensitivity of 83% and a specificity of 78%. The

introduction of presepsin as a diagnostic criterion improved sepsis probability from 56% (pre-
test probability among all subjects) to 81% (post-test probability for a positive result).

Therefore, it is considered an adjunct biomarker [31]. A more recent meta-analysis, including

19 studies, reported comparable results with a sensitivity of 84% and a specificity of 73%.

Moreover, the authors reported a similar diagnostic accuracy for presepsin (area under the

receiver operating characteristic curve (AUC 0.87) as for PCT (AUC 0.84) in critically ill

adults [32]. Wu et al. performed a subgroup analysis for the diagnostic accuracy of presepsin

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and PCT in the emergency department (ED) (AUC 0.90 vs. 0.88) and intensive care unit

(ICU) (AUC 0.87 vs. 0.82). In contrast to others, they could not find a difference between

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presepsin and CRP (AUC 0.85 and 0.85) although this would have been expected since PCT

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is considered to have better accuracy than CRP [33]. As for PCT, there is a commercially

available point-of-care assay for presepsin (PATHFAST Presepsin, Mitsubishi Chemical

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Europe, Düsseldorf, Germany).

Such soluble forms of proteins released into the circulation during immune reactions
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are a direct outflow of immune cell activation and promise to have a higher specificity for
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sepsis-related immune activation compared to “indirect” marker as CRP or PCT. Triggering

receptor expressed on myeloid cells-1 (TREM-1) is expressed on monocytes and neutrophils

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infiltrating tissues after bacterial infection. The soluble form (sTREM-1) is released when

these phagocytes are activated. A meta-analysis published in 2012 examined the ability of
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sTREM-1 in plasma to differentiate between sepsis caused by a bacterial infection and sterile,

non-infectious SIRS. Incorporating 11 studies, the authors reported a moderate diagnostic

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accuracy (AUC 0.87) for the diagnosis of sepsis with a pooled sensitivity of 79% and a pooled

specificity of 80% [34]. Based on these results, sTREM-1 is not superior to the biomarkers

presented so far. sTREM-1 is not only released into the blood, but also found in other body

fluids such as cerebrospinal fluid [35], urine [36] or fluids obtained during diagnostic

procedures like bronchoalveolar lavage fluids (BALF). With an AUC of 0.91, sTREM-1 in
BALF is a good diagnostic marker for bacterial lung infections in ICU patients [37],

indicating that sTREM-1 might be a useful biomarker for diagnosis in such specimens.

Detecting the expression level of specific immune cell surface receptors is another

approach to identify suitable biomarkers. Neutrophil CD64 expression has been evaluated

and classified as a useful marker for improving early diagnostic accuracy. Wang et al.

revealed a pooled sensitivity and specificity of 76% and 85%, respectively, considering 8

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studies in their meta-analysis [38]. Cellular markers can be assessed by flow cytometry,

however implicating a higher personnel and apparatus expenditure, rendering implementation

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in fast routine diagnostic difficult to accomplish outside of specialized centers.

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Mid-regional pro-adrenomedullin (MR-proADM) is explored as a biomarker for the

detection of current and future organ failure instead of the dysregulated immune response.
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The mid-regional fragment of the pro-adrenomedullin is more stable than the biologically

active adrenomedullin (ADM), which is expressed in a wide variety of tissues, but mainly by
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endothelial and vascular smooth muscle cells. ADM is biologically involved in the regulation
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of vasodilation and vascular integrity and MR-proADM is found in high levels in septic

patients [39]. It was discovered to be predictive at an early stage for disease progression in
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patients with suspected infections in the ED [40] and to be superior to established markers,

such as PCT as a marker, for treatment response [41].

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Based on current evidence, none of these markers is sufficient for correct

discrimination of all patients with sepsis from patients with systemic inflammation from non-
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infectious etiology in real life. Parlato et al. conducted a multicenter cohort study enrolling

279 ICU patients with the presence of SIRS and evaluated 53 biomarkers for diagnostic

performance for infection diagnosis. Surprisingly, CRP proved to be the best biomarker

available, while others like, e.g., PCT provided no information above chance [42]. This study

underlines that the quest for suitable biomarker combinations has not ended and requires a

great effort. Importantly, markers must ultimately work in the intended environment (ED or
ICU), raising the need to consider respective pre-test probabilities and influencing factors

during discovery. It is unclear if it will be possible to find a single marker able to detect all

cases with sufficient accuracy. However, combinatorial strategies might be a strategy to

overcome this challenge.

2.3 Sophisticated technologies based on …

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2.3.1 … biomarkers

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Celeras Dx (part of Cube Dx GmbH, St. Valentin, Austria) a commercial test under

development pursues this strategy. Using the principle of microarrays, the company

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developed a multiplex technology called hybcell. Similar to classic competitive

immunoassays, the targeted molecules from the sample are competing with fluorescence-
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labelled antigens for the bond to immobilized specific antibodies. The so called xA panel
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comprises a set of 9 different biomarkers indicating an inflammatory reaction (CRP, PCT,

serum amyloid A, Substance P), the failure of individual organs (Cystatin C, Myoglobin,
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NGAL), and coagulation disorders (plasminogen, D-dimer). Unfortunately, no information is

currently available indicating the diagnostic accuracy of this panel in a real-life clinical
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setting. However, the individual markers are highly characterized and only with an

algorithmic approach to combine the results into a binary decision support for the physician,
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the advantage of this concept might be incremental.

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2.3.2 … biomechanical properties

Using biomechanical properties of immune cells is an entirely different way to approach the

diagnostic challenge. A commercial test still under development is based on altered cell

deformability (Cytovale, San Francisco CA, USA). Using a combination of microfluidics and

single cell imaging, thousands of cells per second are analyzed. A pilot study revealed higher

deformability of granulocytes from patients with sepsis compared to healthy controls [43]. For
lymphocytes, no difference was found. There are two major limitations: The authors included

only 25 patients with sepsis and they have chosen healthy volunteers as a comparator.

Including critically ill patients or patients with sterile SIRS instead would have been clinically

more appropriate.

2.3.3 … host gene expression

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A third fundamentally different approach is the use of gene expression-based methods,

primarily from whole blood. This rests on the fact that the host response to the infection leads

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to a specific change in the patient’s blood transcriptome. Such a comprehensive host response

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analysis postulates to provide information for accurate differentiation of sepsis and sterile

inflammation, estimation of infection severity, risk stratification and prognosis. Moreover,

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different classes of causative pathogens are thought to induce different changes in the host’s

transcriptome pattern.
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Currently, two commercial tests based on this concept are under development.

SeptiCyte LAB (Immunexpress Inc., Seattle WA, USA) is a host response-targeted, PCR-
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based test detecting the expression level of four genes (CEACAM4, LAMP1, PLAC8,

PLA2G7) in whole blood [44]. These levels are offset against each other leading to the
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calculation of a score called SeptiScore. The test is FDA-cleared for clinical evaluation of its
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usefulness in critically ill patients on the first day after ICU admission to differentiate

between sepsis and SIRS. A validation study conducted by the developers, including 447
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critical care patients, yielded a sensitivity of 92% and a specificity of 65% [45]. Meanwhile,

several independent studies have been published with less optimistic results concerning the

clinical performance. These are reviewed extensively elsewhere [46]. The assay’s current

turnaround time is 6 hours, but a faster point-of-care format is under development.

Information about the pathogen and potential resistances are lacking at the moment. However,

Sampson et al. published another gene set (ISH15, IL16, OASL, ADGRE5), able to identify a
viral infection causing the systemic inflammation [47]. The authors claim that combining both

gene signatures may close the currently existing gap.

The second test is called HostDX Sepsis (Inflammatix, Burlingame CA, USA). It is

based on the expression level of eleven host genes (CEACAM1, ZDHHC19, 210

C9orf95/NMRK1, GNA15, BATF, C3AR1, KIAA1370, TGFBI, MTCH1, RPGRIP1, HLA-

DPB1) [48]. The mathematically calculated score was named Sepsis MetaScore. The test

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distinguished with a sensitivity of 95% and a specificity of 60% between neonates with sepsis

and healthy ones in a validation study conducted by the developers [49]. Similar to the

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developers of the SeptiCyte LAB test, further research is undertaken to improve the test

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towards the causative pathogen: Sweeney et al. published a group of seven genes (IFI27, JUP,

LAX1, HK3, TNIP1, GPAA1, CTSB) sufficient to classify between bacterial and viral
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infections [50].

Recently, the diagnostic accuracies of both tests were independently validated using
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gene expression data prospectively collected in the context of a randomized controlled trial in
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an ICU. This study revealed an AUC of 0.80 using the Sepsis MetaScore and an AUC of 0.68

for the SeptiScore. In less heterogeneous subpopulations, the diagnostic performance raised in
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both cases [51].

The various commercial concepts are promising, but based on the current technology
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readiness, none provides sufficient diagnostic accuracy to empower decision-making in

complex clinical settings. Above all, time is a critical dimension and tests aiming to answer
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the necessity for antimicrobial therapy must yield information within minutes to project into

clinical benefit.

3. Pathogen identification

The most recent international consensus definition emphasize that sepsis, in contrast to sterile,

non-infectious SIRS, is caused by a dysregulated immune response to infection [1]. While a

suspicion of infection can be based on clinical symptoms, the rapid confirmation by the

identification of the causative pathogens is favorable, not only for diagnostic purposes.

Effective source control and initiation of antimicrobial therapy are decisive to eliminate the

causative trigger of the syndrome. Each hour delay in the beginning of antimicrobial therapy

leads to an increased mortality rate [52]. In best case scenario, a rapid pathogen identification

can inform a targeted antibiotic therapy. As discussed above, host-derived biomarkers might

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hint towards the class of triggering pathogens thus supporting the choice for antimicrobial

therapy. However, microbial diagnostic specifies the causative pathogen and its individual

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resistance properties.

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3.1 Current gold standard: blood culture-based approaches
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Conventional blood culture is still the gold standard in this regard. The grown and identified

pathogens serve as basis for subsequent, direct testing of antimicrobial susceptibility. Over the
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last years, the introduction of different new post-culture technologies improved and shortened

the pathogen identification in positive cultures [53]. However, the time between blood
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draw/inoculation and positivity of blood culture remains to be the weak spot. Depending on

the initial pathogen load and the growth rate, it takes several hours to days. Thus, these
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essential diagnostic tools remain inadequate to support early diagnosis and treatment
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decisions. This current diagnostic practice leads to empiric therapy choices using broad-

spectrum combination therapies, bearing the risk of an inappropriate antimicrobial therapy at

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the beginning, which is associated with a fivefold reduced survival [54]. Depending on the

study population and disease severity, between 30% [55] and 70% [56] of the patients are

blood culture-negative. Reasons for this may be the choice of appropriate culture media, non-

cultivable bacteria [57] or viral etiology, inappropriate pre-analytic handling [58], prior

antibiotic treatment [59] or a low or no pathogen load in the bloodstream. Such failings limit

the utility to clinicians.

3.2 Direct pathogen detection

Direct pathogen-detection from whole blood, other body fluids (e.g., urine, sputum) or fluids

from diagnostic procedures (e.g., BALF, smear) would overcome these limitations. Several

innovative technologies addressing this challenge have emerged recently. Many of those

direct detection methods are based on the amplification of microbial nucleic acids. However,

the concentration of these nucleic acids in the blood is low and host nucleic acids may hamper

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the accuracy. Polymicrobial samples may additionally complicate the determination.

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Sophisticated test developments tackle these issues by combining several technologies.

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3.2.1 Amplification-based systems

As early as 2006, the multiplex PCR-based SeptiFast test (Roche Diagnostics, Rotkreuz,
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Switzerland) was introduced. It is CE-marked and commercially available in the European

Union. Within a turnaround time of 6 hours, the test can detect 25 different bacteria and fungi
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responsible for a large proportion of sepsis cases in Europe. Compared to conventional blood
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culture diagnostics, the SeptiFast test reaches a sensitivity level of 68% and a specificity of

86% in a meta-analysis including 41 clinical diagnostic accuracy studies with a total of 7,727
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patients [60]. Despite this rather low sensitivity, indicating that the test misses pathogens

being detected by blood culture, Mongelli et al. reported that most pathogens detected via
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SeptiFast, but not via blood culture, could be found in other sample specimen taken from
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other sites of the same patients, indicating to be associated with the sepsis-triggering infection

[61]. The manufacturer states that down to 1.5ml blood are sufficient for a reliable result. This

small quantity may be advantageous in many circumstances. At the same time, it should be

considered, that this is achieved at the expense of a higher sensitivity, due to the low levels of

circulating pathogen-derived nucleic acids as well as the presence of PCR inhibitors such as

iron or immunoglobulins [62]. Currently, the test cannot replace, but rather reasonably

complement conventional blood culture diagnostics, especially improving a rapid start of

targeted therapy and providing added value for culture-negative patients.

SepsiTest (Molzym, Bremen, Germany) combines 16S and 18S ribosomal RNA gene

PCR with Sanger sequencing technology and currently is able to detect 1,359 bacteria and

fungi [63] from a broad range of body fluids and other specimens derived from medical

interventions. This broad detection range overcomes one of the major drawbacks of the panel-

based SeptiFast test. The test is commercially available in Europa. The turnaround time for

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the whole test is around 8 – 10 hours. However, the combined approach leads to an interim

result within <4 hours providing information about the presence of bacteremia or fungaemia

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without further information about the exact pathogen. Currently, both tests based on nucleic

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acid testing cannot replace culture-based diagnostic in terms of the availability of information

on antibiotic resistances. For the SeptiFast test, a mecA test is available at least. In
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comparison with the diagnostic tests based on host-derived factors, another severe limitation

of both tests is that they are not available in a point-of-care format, but require specialized
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staff and accredited labs to perform. Based on the same principle, Molzym has developed an
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automated test (Micro-Dx), too, but currently it detects less species compared to the

SepsiTest. This is indicating that molecular diagnostic approaches are under ongoing
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development and it is very likely that currently existing limitations can be overcome in the

foreseeable future. On the downside, promising and broadly developed systems may be pulled
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back from the market. This has happened to the IRIDICA system (Abbott, Lake Bluff IL,

USA) of late. The system comprised a variety of detectable microbes, comparable with the
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SepsiTest [64]. Despite a commercial availability in Europa since 2014 and an only recently

completed trial for FDA clearance, the production was terminated, not mainly for

performance issues but due to logistical and financial deliberations [65]. This process

emphasizes that ingenious technologies and clinical impact alone are not sufficient to

revolutionize diagnostics in the long term. Nevertheless, a randomized crossover trial with the

intention to shed light on the cost-benefit calculation revealed a weak dominance of direct
pathogen detection using SeptiFast compared to conventional diagnostic procedures for

patients with severe sepsis [66]. A meta-analysis conducted by Stevenson et al. assessed both

clinical power and cost-effectiveness of all three tests. They found higher specificity than

sensitivity levels for all three tests, but none was statistically significant superior to the current

gold standard. Because they did not find enough evidence, the authors draw no definitive

conclusion concerning the cost-effectiveness of direct pathogen tests [67]. The BioFire

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FilmArray System (BioMerieux, Marcy-l’Étoile, France) and Unyvero (Curetis, Holzgerlingen,

Germany) ameliorate the problem of a limited number of simultaneously detectable species

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by providing focus-specific panels, e.g., for pneumonia, meningitis, or abdominal infections.

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Both systems use tailored single-use cartridges, which detect in a culture-free, PCR-based

manner those species commonly causing these infections. In case of a known or suspected
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focus this is feasible.
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3.2.2 Magnetic resonance technology

The T2MR technology (T2 biosystems, Lexington MA, USA) differs from the previously
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discussed nucleic acid amplification-based systems. It is a miniaturized magnetic resonance

technology, meaning in this case that the behavior of water molecules within a magnetic field
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is detected. So far, the T2Bacteria panel and the T2Candida panel are the only systems for
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pathogen detection directly from blood, which are FDA-cleared and CE-marked. The method

does not require any purification, extraction or amplifications step leading to a faster
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turnaround time of 3 – 5 hours as compared to the nucleic acid amplification-based methods.

Moreover, the T2Bacteria panel displayed not only specificity but also sensitivity levels well

above 90% in a prospective multicenter study analyzing samples from 1,427 patients [68].

The test is limited to six species (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P.

aeruginosa, E. coli). Even though this covers a large fraction of the common bacterial sepsis

pathogens, the restricted number is a comparatively large limitation not taking into account
many other bacterial pathogens triggering sepsis. The T2Candida panel also detects only five

Candida spp., but with high overall sensitivity (91.1%) and specificity (99.4%), too [69]. The

technology is also able to detect growth-inhibited Candida cells. Especially after prior

antifungal treatment, this is an important superiority over conventional culture-based

diagnostics [70]. Important to note in dealing with positive T2Candida results is the overall

prevalence of invasive Candida infections. In relation to the start of antifungal treatment, such

t
ip
a result should be evaluated differently with a patient in ICU in comparison to a patient in ED

[71]. In addition to the high diagnostic accuracy, the panels currently under development are

cr
of particular interest in near future. A resistance panel detecting 13 resistance genes as well as

us
a panel detecting Candida auris address the clinical need about potential resistances and

emerging pathogens.
an
3.2.3 Further amplification free technologies
M

There are also some other promising systems for pathogen detection that do not require

amplification of nucleic acids, shortening the delay between sample collection and assay
ed

result. The cylindric microarray called hybcell (Cube Dx, St. Valentin, Austria) is used as a

platform for direct pathogen detection. Pathogen derived DNA fragments in the sample bind
pt

to immobilized DNA oligonucleotides and the interaction is visualized via fluorescence.

ce

Unfortunately, the performance of this technology has only been presented at conferences so

far. Next-generation sequencing of cell-free plasma together with bioinformatic analysis

Ac

enables unbiased testing and therefore is more comprehensive (including bacteria [72], fungi

[73], and viruses [74]) than the fixed panels. This advantage is purchased at the cost of a

higher turnaround time of currently 24 hours. Analysis and interpretation of the sequencing

data are of particular importance – the lower the pathogenic load still being detectable, the

more crucial is to distinguish between infectious agents and commensals [75,76]. Various

companies such as Karius (Redwood City CA, USA) and Noscendo (Duisburg, Germany)
are working on commercially available solutions to make this method widely available.

Almost none of the technologies presented have already been tested to such an extent

that they can be used as the sole microbiological test. Large multicenter trials are needed to

test the efficacy, added benefit, and practicability in clinical routine. The technologies need to

be tested not only in ICUs, but in particular also in ED settings. This means a higher effort

concerning study design and patient inclusion and holds also true for the host response-guided

t
ip
tests. A major problem is the choice of an appropriate reference value. As stated earlier, the

current diagnostic gold standard blood culture is not an analytically robust method. Judging

cr
the new technologies on the basis of these results could disguise their actual performance.

us
Blood culture-positivity typically requires bacterial cell numbers around 10,000 to 1,000,000

CFUs/ml. Most of the new technologies already detect more than one thousandth lower
an
concentrations. This raises new demands in regard to the necessity to distinguish true

infections from colonization or even contamination.

M

4. Conclusion
ed

Latest technological developments in different areas allowed the development of novel rapid

and accurate diagnostic tools for patients with suspected sepsis. Some of these technologies
pt

are more likely to have candidate status while others are already market-ready. All have the
ce

potential to revolutionize diagnostics and to lead away from empiric treatment to rapid and

objectively driven decision-making. Nevertheless, robustness of these biomarkers and

Ac

technologies still has to be verified in large, heterogenous cohorts. This is particularly

necessary for use in the ED, being the main gateway for most patients with sepsis. In addition,

most tools lack reliable cut-off values allowing easy result interpretation in routine settings.

Prospectively, the further development towards screening tools for patients at risk predicting

sepsis before the onset of the first symptom or at least the progression to organ dysfunction

would enormously improve treatment decisions. This is most likely to succeed with multi-
marker panels, including both host response assessment and direct pathogen detection.

5. Expert Opinion
On the one hand, there is an urgent need for rapid and, above all, accurate tests that support

early diagnosis of sepsis. At the same time, even the most matured commercial technologies

have not yet entered clinical routine, although considerable progress has been made in recent

t
years. This apparent contradiction results from the disease itself. Sepsis is a complex

ip
syndrome with a high degree of heterogeneity of both the underlying pathogens and the

cr
clinical appearance of patients. Almost all marked-ready developed systems cover only a part

of the sepsis cases and therefore, merely support the diagnosis of a specific patient subgroup.

us
These test procedures are therefore less suitable for unselected and routine use, e.g., in ED
an
settings. An improved diagnostic option, only aiding diagnosis of a subgroup, does not pay

off in real-life, considering the high economically effort, staff training and not lastly, patient
M
outcome. The routine use of these test systems is more likely to work in specialised ICUs with

more homogeneous group of patients. Apart from study settings, broad implementation
ed

currently fails in particular due to a lack of simple, robust cut-off diagnostic values. However,

this situation can also change very quickly, as soon as some of the diversified tests, which are
pt

still in the early stages of development, are ready for the market. The often erratic,
ce

unpredictable technical development as well as information on the exact current development

status of individual tests being not published for company strategic reasons makes it difficult
Ac

to make a serious statement about when this will be the case. However, it can be mentioned

with considerable certainty that there will be no single, universally applicable test accurately

diagnosing all cases of sepsis. Rather, it can be assumed that different tests will take root in

different divisions (e.g., ICU, ED), based on the predominant patient populations.

Combinations of host-response guided tests with tests that capture the triggering pathogens

and their possible resistances are not only conceivable, but likely and promising as they can
be adapted to local patient populations.

Besides all this, the influence of the human factor must not be underestimated. The quickest

and most accurate diagnostic test will not improve patient’s outcome, when a diagnostic

sample is not further processed immediately or, even worse, is not taken at all, despite

suspicion of infection.

Apart from technical improvements and biomarker research, bioinformatics has also

t
ip
developed dramatically [77]. Combining intelligent bioinformatic tools as e.g., machine-

learning algorithms, with results from clinical examination and laboratory parameters has the

cr
potential to revolutionize diagnosis. This approach increases not only diagnostic accuracy, but

us
can also open up completely new possibilities for therapy control. Patients with sepsis may be

identified in different phases of the disease, even before the onset of symptoms using a neural
an
network analysis of several biomarkers [78,79]. Such approaches are especially helpful for

high-risk patients e.g., postoperative patients or patients staying long on ICU [80] and may
M

also predict the further progression of severe sepsis [81]. It has already been shown that
ed

artificial intelligence is able to diagnose sepsis in different settings such as ICU, ED or

general ward, using routine data [82,83].

pt

The future diagnostics lies in the convergence of clinical examination, real-time

recorded vital functions and laboratory-based technologies using bioinformatic methods. This
ce

connection of classical and novel diagnostic strategies will lead to a high predicative and

diagnostic accuracy, improved therapy guidance and finally to better patient outcome.
Ac

Conflicts of Interest and Source of Funding

No funding has been received for this review. The authors have no conflicts of interest to

declare.
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Abbreviations

t
ip
AUC: area under the receiver operating characteristic curve

cr
BALF: bronchoalveolar lavage fluids

us
CD14: Cluster of differentiation 14

CE: Conformité Européenne (French for European Conformity)

an
CRP: C-reactive protein
M

DNA: deoxyribonucleic acid

ed

e.g.: exempli gratia

pt

ED: emergency department

ce

FDA: U.S. Food and Drug Administration

ICU: intensive care unit

Ac

IL-6: interleukin-6

LBP: lipopolysaccharide-binding protein

LPS: lipopolysaccharide

MR-proADM: mid-regional pro-adrenomedullin

PCR: polymerase chain reaction

PCT: procalcitonin

RNA: ribosomal ribonucleic acid

sCD14: soluble Cluster of differentiation 14

t
SIRS: systemic inflammatory response syndrome

ip
cr
SOFA: sequential organ failure assessment score

us
ST: subtype

sTREM-1: soluble triggering receptor expressed on myeloid cells-1

an
TLR4: toll-like receptor 4
M

TREM-1: triggering receptor expressed on myeloid cells-1

ed
pt
ce
Ac
Table 1. Biomarkers in sepsis diagnostics

Biomarker Sensitivity Specificity Reference

Serum lactate marker of cellular stress or [15]

microcirculatory disturbances

IL-6 pro-inflammatory cytokine 85% 62% [20]

t
ip
CRP acute-phase reactant 75% 67% [21]

cr
PCT precursor of the peptide hormone 77% 79% [24,25]

us
calcitonin

Presepsin truncated subtype of soluble

an 83 - 84% 73 - 78% [31,32]

cluster of differentiation 14
M

sTREM-1 soluble form of a receptor 79% 80% [34]

expressed on monocytes and

ed

neutrophils infiltrating tissues

pt

after bacterial infection

ce

Neutrophil surface receptor on neutrophils 76% 85% [38]

CD64
Ac

MR-proADM expressed mainly by endothelial [39]

and vascular smooth muscle cells

involved in vasodilation and

vascular integrity regulation

Abbreviations: CRP: C-reactive protein, IL-6: interleukin-6, MR-proADM : mid-regional pro-

adrenomedullin , PCT: procalcitonin, sTREM-1: soluble triggering receptor expressed on
myeloid cells-1
Ac
ce
pt
ed
M
an
us
cr
ip
t
Table 2. Host response-guided diagnostic assays

Assay Manufacturer Technique Panel

Celeras Dx Cube Dx GmbH competitive 9 biomarkers

(St. Valentin, immunoassay (CRP, PCT, serum

amyloid A, Substance P,
Austria)
Cystatin C, Myoglobin,

t
ip
NGAL, plasminogen, D-

dimer)

cr
Cytovale Cytovale microfluidic and granulocyte

us
(San Francisco CA, single cell imaging deformability

USA)
an
SeptiCyte LAB Immunexpress Inc host gene expression 4 genes
M

(Seattle WA, USA) (CEACAM4, LAMP1,

PLAC8, PLA2G7)
ed

HostDX Sepsis Inflammatix host gene expression 11 genes

(Burlingame CA, (CEACAM1, ZDHHC19,

pt

210 C9orf95/NMRK1,
USA)
ce

GNA15, BATF, C3AR1,

KIAA1370, TGFBI,
Ac

MTCH1, RPGRIP1,

HLA-DPB1)
Table 3. Direct pathogen detection assays

Assay Manufacturer Technique Turnaround Panel

time

SeptiFast Roche Diagnostics PCR-based detection of 6 hours 25 bacteria

(Rotkreuz, microbial DNA and fungi

t
Switzerland)

ip
SepsiTest Molzym 16S and 18S rRNA PCR 8 - 10 hours 1,359 bacteria

cr
(Bremen, Germany) and sanger sequencing and fungi

us
BioFire BioMerieux DNA purification and
an 1 hour Tailored

FilmArray (Marcy-l’Étoile, multiplex PCR cartridges (up

France) to 33 bacterial,
M
viral, or

parasitic
ed

targets,

including
pt

resistance
ce

genes)

Unyvero Curetis DNA purification and 4 – 5 hours Tailored

Ac

(Holzgerlingen, multiplex PCR cartridges

Germany) (up to 105

pathogens, 3

toxins, 22

resistance
 genes)

T2MR T2 biosystems magnetic resonance 3 – 5 hours T2bacteria: 6

(Lexington MA, technology species

USA) T2Candida: 5

species

t
ip
hybcell Cube Dx microarray

(St. Valentin,

cr
Austria)

us
Karius (Redwood City CA, an unbiased

USA) testing
next generation around 24
(including
Noscendo (Duisburg, sequencing hours
M
bacteria, virus,
Germany)
fungi)
ed

Abbreviations: DNA: deoxyribonucleic acid, PCR: polymerase chain reaction, rRNA: ribosomal
ribonucleic acid
pt
ce
Ac