Biomarkers of Infection and

Sepsis
a, b
Steven M. Opal, MD *, Xavier Wittebole, MD

KEYWORDS
 Rapid  Culture-independent  Microbial diagnostic techniques
 Host-derived biomarkers for early diagnosis of sepsis and septic shock
 Machine-learning systems to identify susceptible patients with sepsis
 Rapid genotypic and phenotypic methods to measure antimicrobial
susceptibility patterns

KEY POINTS
 Promising molecular diagnostic techniques are under development, but not yet displaced
the traditional rather insensitive, and slow microbial diagnostic measures in use today.
 Identifying a pathogen and determining its susceptibility to antimicrobial agents generally
takes more than 24 hours, forcing clinicians to give empiric, broad-spectrum, antimicro-
bial agents to cover the likely pathogen(s).
 Clear evidence exists that correct administration of antimicrobial agents can be life-saving
in patients with sepsis.
 Two major strategies are in development to assist treating physicians to make expeditious
decisions on appropriate antibiotic choices that specifically targets the invading patho-
gens in the septic patient.
 This article reviews the current status of rapid molecular markers to optimally define which
patients need urgent treatment.

INTRODUCTION

Intensive care unit (ICU) practitioners and emergency department clinicians are in
urgent need of a major upgrade in their access to accurate and fast microbial diag-
nosis (Table 1). The diagnostic techniques for bloodstream infection available to
most ICU clinicians today do not fundamentally differ from Robert Koch’s laboratory
methods circa 1887.1,2 That was the year when 2 co-workers in Koch’s laboratory,
Fanny Hess and Richard Petri, first suggested using semisolid agar and 2 glass plates
of slightly different size for isolation of bacteria in pure culture.

a
Infectious Disease Division, Alpert Medical School of Brown University, Ocean State Clinical
Coordinating Center at Rhode Island Hospital, 1 Virginia Avenue Suite 105, Providence, RI
02905, USA; b Critical Care Department, (Pr Laterre), Cliniques Universitaires Saint-Luc, Avenue
Hippocrate 10, 1200 Brussels, Belgium
* Corresponding author.
E-mail address: Steven_Opal@brown.edu

Crit Care Clin - (2019) -–-

https://doi.org/10.1016/j.ccc.2019.08.002 criticalcare.theclinics.com
0749-0704/19/ª 2019 Elsevier Inc. All rights reserved.
2 Opal & Wittebole

Table 1
Rapid detection methods and molecular biomarkers to improve the diagnosis of sepsis

Analytical Technique Potential Uses References

29,36–39
Microfluidics Rapid antibiotic susceptibility method;
neutrophil nuclear membrane
deformability as an early diagnostic
method
40–56
Proteomics and plasma protein Identify new patterns of plasma protein
biomarkers interactions and responses; predict
outcomes and possibly direct new
therapies by using plasma biomarkers
53,57–59
Genomics Full genomic or exon-expressed SNPs as
indicators for host susceptibility to
infection and sepsis; rate of LPS removal
in LDLRs by PCSK9 levels
60–72
Transcriptomics Method to define DEGs to assess the acute
and more chronic host–response to sepsis
stimuli, determine the dominant host
response from proinflammatory to
immunosuppression to coagulopathic
phenotypes
73,74
metabolomics Measure metabolic perturbations in
confined spaces such as pleural fluid or
cerebrospinal fluid indicative of tissue
stress, cell injury or immune–metabolic
alterations for diagnosis and prognosis
75–79
Large data set machine learning Identify pathophysiologic patterns of host
response phenotypes and endotypes

Abbreviations: DEGs, differentially expressed genes; LDLR, low-density lipoprotein receptor; LPS,
lipopolysaccharide; PCSK9, proprotein convertase subtilisin kexin type 9; SNP, single nucleotide
polymorphism.

Most clinical microbiology laboratories still use the same basic method of single
colony isolation before conducting antimicrobial susceptibility testing. The drug sensi-
tivity results are usually not available for 24 to 72 hours. However, it should be noted
that current treatment guidelines for treating sepsis promulgate initiation of effective
antibiotics within 1 to 3 hours!3–5 Although clinicians at large medical centers might
now have access to early microbial identification via innovations in mass spectrometry
(MS) and nuclear magnetic resonance methods, rarely do they have antimicrobial sus-
ceptibility results at the critical moment of ordering the antibiotic treatment regimen.
Antibiotic therapy is therefore empiric by nature and purposely given as broad-
spectrum treatment to avoid missing the drug susceptibility profile of the causative
pathogen.
Compelling but not uniformly accepted evidence exists in support of
urgent administration of empiric antibiotics in patients with severe sepsis of septic
shock.5–9 Ideally, antibiotic intervention should be completed within 1 hour of
initial diagnosis of sepsis or septic shock.6–8 Living up to this 1-hour imperative
is hampered by the fact that there is no single, rapid, and accurate diagnostic
test for the syndrome of sepsis or septic shock. The diagnosis rests on a
diagnosis of severe infection accompanied by a constellation of signs and
symptoms, blood studies, and pathophysiologic findings clinically recognized as
sepsis.10
 Biomarkers of Infection and Sepsis 3

Yet, choosing an effective, empiric, antimicrobial drug regimen, before knowing

the sensitivity profile of the causative pathogen, is becoming a real challenge.
Progressive emergence of antimicrobial resistance among bacterial and fungal
pathogens is an omnipresent threat.10–13 Widespread use of broad-spectrum anti-
biotics likely contributes to the spread of antibiotic resistance genes, further exac-
erbating the emergence of multidrug-resistant microbial pathogens and secondary
fungal infections.14,15 Antibiotic stewardship programs are focused on curbing
excessive use of empiric antibiotics, placing the ICU clinician in a therapeutic
conundrum.15,16 To make matters worse, empiric antibiotics are often administered
to patients before arriving in the hospital and before exhibiting progressive signs of
sepsis, thereby precluding an accurate microbial diagnosis by standard culture
methods.
What is needed now are a set of molecular tools that can provide (1) rapid diagnostic
methods for invasive infection without the need for compulsory delays with laboratory
cultivation, (2) the capacity for urgent determination of antimicrobial susceptibility
results from the causative pathogen and (3), a new generation of biomarkers that
can clearly distinguish between infection-mediated organ dysfunction and sepsis
mimics with non–infection-mediated systemic inflammation. Progress is being made
to advance the laboratory diagnosis of severe infection and early recognition of sepsis
and septic shock.16–18 We provide a brief review of the subject of biomarkers in sepsis
and their status in clinical development. Hopefully, some of these innovations will soon
be available as bedside tools for the diagnosis and treatment of the enigmatic
syndrome called sepsis.

RAPID MICROBIAL DIAGNOSTIC METHODS

Advances in molecular diagnosis without the need for traditional, time-consuming cul-
ture methods is finally reaching the stage for actual clinical usage. This advance could
revolutionize the clinical care and treatment decisions of critically ill, patients with
sepsis. Knowing the molecular identity of the causative organism, and perhaps
even the susceptibility data, in a few hours rather than 24 to 72 hours could allow
the ICU physician to make more informed decisions on antibiotic choices.16–23 Most
of these rapid diagnostic methodologies are based on the polymerase chain reaction
(PCR) to amplify the microbial DNA signal. This is followed by various analytical ap-
proaches to determine the genomic sequence identity by MS16–23 or nuclear magnetic
resonance.14,24 The most common system currently used in clinical microbiology lab-
oratory at present is matrix assisted laser desorption/ionization time of flight mass
spectroscopy. Many studies report reduced mortality rates in comparative studies
with traditional microbiologic diagnostic measurements.16,19,21,22 Fast detection of
specific pathogens such as Staphylococcus aureus versus coagulase-negative staph-
ylococci by peptide–nucleic acid fluorescence in situ hybridization can inform the clini-
cian with a rapid determination if the infection is a highly virulent or low virulence
microorganism.25–27 Linking rapid diagnostics results with antimicrobial stewardship
programs with the hospital is particularly useful in realizing the maximum benefits of
rapid microbial diagnostic testing16,19,22
Screening for the presence of antibiotic resistance genes is also certainly feasible as
a part of rapid genomic testing, but there are hundreds of known antibiotic resistance
genes and the list of new resistance genes continues to grow. Moreover, the
resistance gene sequence might be present in the genomic DNA of the pathogen
without being fully expressed.16 Clinical correlation between genotype and pheno-
typic expression in vivo during infection needs further investigation.
4 Opal & Wittebole

The potential clinical usefulness of a rapid, non–culture-based microbial diagnosis

was well-demonstrated in a recent large, multicenter, European observational trial.
They simultaneously compared standard culture method s versus a PCR electrospray
ionization mass spectroscopy–based diagnostic system in 616 bloodstream infec-
tions.23 Routine blood cultures were positive in only 11% of patients with sepsis,
whereas the PCR-based system from the same samples was positive for the pathogen
in 37%. This large difference was attributed to the widespread use of empiric antibac-
terial therapy in these critical ill patients. Antibiotics limit the diagnostic validity of
blood cultures but have little effect on the PCR-based samples with the ability to iden-
tify circulating bacterial DNA despite antibiotic exposure. If the PCR electrospray ioni-
zation mass spectroscopy data had been available to the investigators in real time,
treatment would have been altered in up to 57% of these patients.

BIOMARKERS FOR RAPID DIAGNOSIS OF ANTIMICROBIAL SUSCEPTIBILITY TESTING

Another unmet medical need in managing critically ill patients with severe infection is
the lack of ability to provide rapid and directed antibiotic therapy guided by specific,
antimicrobial susceptibility testing.21,22 In this era of progressive loss of antimicrobial
activity from bacterial evolution of antibiotic resistance genes, rapid susceptibility
testing is urgently needed but rarely available. There are exceptions of course. Strep-
tococcus pyogenes (group A strep) remains uniformly susceptible to penicillin,28 but
for almost everything else direct antibiotic sensitivity testing to guide therapy is desir-
able if possible. Unfortunately, laboratory testing usually entails time-consuming incu-
bation times to fully express and define the susceptibility pattern for each identified
pathogen.
Progress is being made in the provision of novel methodologies to bring rapid sus-
ceptibility testing into clinical practice. Microfluidic systems with single cell imaging
assays,29 laser light scatter rapid imaging technologies,30 and time-lapse microscopic
imaging analyses31 are only some of the antimicrobial sensitivity testing systems un-
derway to fill the current void in rapid and clinically accessible susceptibility testing.
These rapid diagnostic systems are best applied in normally sterile fluids like blood,
cerebrospinal fluid, pleural fluid etc. and are not particularly useful at present for
microbiologic diagnosis from tissues with an endogenous microbiome (sputum, urine,
and gastrointestinal or genitourinary tract pathogens).32–35

BIOMARKERS FOR INFECTION VERSUS NONINFECTIOUS SYSTEMIC INFLAMMATION

Microfluidics
Advances in microfluidics and microchip assembly offer the potential to be a rapid
diagnostic technique for distinguishing infection versus inflammation in the emergency
department.36 The internal flexibility and deformability of the nucleus found inside
single cells of circulating neutrophils can be analytically measured within minutes in
a microfluidics apparatus.37 Activated neutrophils responding to systemic infection
can be distinguished from resting neutrophils in the circulation by this technique.
Deformability indices of thousands of neutrophils can be calculated over a few
minutes. This technique could rapidly assess the state of neutrophil activation in a
patient presenting acutely in the emergency department to aid in initial triage and
management.37–39
Plasma Biomarkers, Proteomics, and Soluble Pattern Recognition Receptors
More than 100 proteins, soluble receptors, cytokines, and chemokines have proposed
in the literature to serve as biomarkers to distinguish between infection and
 Biomarkers of Infection and Sepsis 5

inflammation.40,41 The most widely studied biomarkers as an indicator of sepsis are

procalcitonin (PCT) and C-reactive protein (CRP). PCT is a prohormone synthesized
and rapidly released by many cell types during period s of generalized inflammation.
Plasma levels are usually highest during episodes of severe bacterial infection, but
noninfectious injury such as major surgery, severe trauma, and some virus infections
can also elevate PCT levels.42–44 Serial measurement of PCT levels might prove to be
useful in antibiotic stewardship programs to encourage clinicians to withdraw empiric
antibiotics if PCT levels were never elevated or were low and drop precipitously. The
clinical usefulness of applying PCT levels as a guide to discontinue antibiotics, if no
objective evidence of bacterial infection is found, remains the subject of considerable
debate.40–44
CRP is a hepatically synthesized, acute phase protein whose blood levels dramati-
cally increase with tissue injury, infection, and acute severe inflammatory states.45
The pentameric plasma protein functions as a soluble pattern recognition molecule
that binds to C-polysaccharide of the pneumococcus. CRP also binds to dead or dying
bacteria in the blood and promotes complement-mediated bacterial clearance. The
binding site for CRP on bacteria and injured host cells is lysophosphatidal choline.
The protein synthesis and secretion of CRP is IL-6 driven.46 CRP levels in the blood
can increase in sepsis and septic shock to greater than 10,000-fold greater resting
levels. CRP has been used as a nonspecific but reliable measure of systemic inflamma-
tion of any type. CRP has been used by clinicians as a biomarker of inflammation for de-
cades. It is highly sensitive for sepsis, but lacks specificity when compared with PCT.47
Several other protein biomarkers of recent interest include a neutrophil activation
marker known as heparin-binding protein,48,49 a mesenteric ischemia marker known
as pancreatic stone protein,50,51 and a detection assay for measuring circulating
endothelial cells.52 Elevated plasma levels of heparin-binding protein (>30 ng/mL) is
an accurate indicator of neutrophil activation with evidence of neutrophil chemo-
taxis.48 If left untended, progressive rise in heparin-binding protein levels portend
endothelial damage, diffuse capillary leakage, sepsis, and shock. In a recent multi-
center clinical trial, heparin-binding protein levels detected in the blood in 7 emer-
gency departments compared favorably with other standard measures (CRP, blood
lactate, and PCT) as a predictor of adverse outcomes.49
Another potentially promising biomarker is pancreatic stone protein. This protein is
released during periods of splanchnic hypoperfusion and seems to be a good
biomarker for mesenteric perfusion in patients with sepsis. Elevated levels can be
readily detected with a rapid diagnostic assay, and pancreatic stone protein measure-
ment might be able to outperform other biomarkers for sepsis like PCT and CRP.50,51
Another interesting detection assay in development for the prediction of sepsis is
direct measurement of detached endothelial cells in the blood.52 Sepsis-induced
disruption of the endothelial barrier throughout the circulatory system is the hallmark
pathophysiologic finding in septic shock.53 A specific assay for endothelial cell dam-
age might prove to be a useful tool to make an early diagnosis and management guide
for novel sepsis therapeutics.
Measuring a biomarker that is the target of a new therapeutic intervention to prevent
sepsis is a logical approach in using biomarkers. Measuring circulating endotoxin
before starting an anti-endotoxin therapy54 or measuring soluble triggering receptor
expressed on myeloid cells before beginning a clinical trial targeting triggering recep-
tor expressed on myeloid cells with a new experimental inhibitor.55 Moreover, a holis-
tic approach to proteomics where the entire plasma proteome is analyzed for critical
interactions between signaling peptides in sepsis and septic shock could provide new
insights on when and where to intervene.56
6 Opal & Wittebole

Genomics
Advances in DNA sequencing technologies now make it feasible to sequence the exon
segments of the human genome and search for single nucleotide polymorphisms.
Myriads of single nucleotide polymorphisms are already identified in the human
genome that affect susceptibility to infection and alter the expression of the host
response during sepsis.53 We are just beginning to decipher the magnitude of interact-
ing genomic elements induced by sepsis, and the computational biology, which will be
essential to fully understanding the complexity of host susceptibility and resistance to
infection.
At present we can only scratch the surface of the host–pathogen interactions that
participate in the daily standoff between our genomes and an array of potential
microbial pathogens.53 Single nucleotide polymorphisms have already been identi-
fied in complement factors, immunoglobulin structure, intracellular signaling net-
works, and clearance capacity to eliminate lipopolysaccharides and other toxic
biolipids.57–59 Much basic research and applied science research is needed to
bring advances in functional genomics to the bedside in support of sepsis
management.

Transcriptomics
The major methodology now in use to analyze components of gene activation or
inhibition of the host response in sepsis is with upregulation or downregulation of
differentially expressed genes from a given specific baseline determination. Thou-
sands of genes are differentially expressed in response to a powerful and patho-
logic pattern recognition molecule like bacterial endotoxin. Complicating matters
further is that transcription gene profiles between individuals even with single site
of infection (eg, pneumonia or blood stream infections) are markedly heteroge-
neous.60 In response, investigators have endeavored to use unsupervised searches
for common transcriptional clusters to detect recognizable and reproducible
signatures of gene activation. This process requires a careful analysis for primary
driver transcripts from different individuals with the same or similar acute disease
process.61–63
Initial unsupervised clusters of transcripts are then linked in together from patients
with similar disease features to determine recognizable, organ-specific, tissue-spe-
cific, and circulating blood transcript patterns. Meta-clustering and data pooling
methods have improved the reproducibility of detection of common gene clusters
associated with specific diseases. Research teams have now shown it is possible
to distinguish infection-related systemic inflammatory states from noninfectious
inflammatory states by analyzing differences in the transcription profiles.64–67
McHugh and colleagues64 have been able to reduce the number of transcribed
genes necessary to predict the presence or absence of infection in critically ill patients
with only 4 targeted enzymes. The 4 gene products are LAMP1 (lysosomal-associated
membrane protein), PLAG7 (phospholipase A2, group VII), PLAC8 (placenta-specific
gene 8), and CEACAM 4 (carcinoembryonic antigen-related cell adhesion molecule
4). This work is of extreme importance in antibiotic stewardship practices in the ICU
by avoiding the use of antibiotics if no infection is present.67–69
Efforts are underway to streamline this process and develop diagnostic tests by nar-
rowing down the gene search to a small number of critical transcriptional determinants
of infection versus noninfection. This promising approach is ongoing in many labora-
tories around the globe.70,71 Although still in the developmental phase, these analyses
are beginning to decipher patterns of host response genes that might help guide
 Biomarkers of Infection and Sepsis 7

treatment strategies and improve patient selection in future experimental intervention

trials. Sweeney and colleagues71 have uncovered consistent patterns in transcriptome
analysis that differentiate into 3 major clusters designated as inflammopathic, adap-
tive, and coagulopathic. Each of these groups are identifiable in numerous publicly
available transcriptome databases and have significant outcomes. This work could
begin to direct therapies in patients with sepsis with reduced heterogeneity in the pa-
tient population. Heterogeneity across different studies has stymied attempts to
reproduce the results of interventional trials in sepsis. Hopefully, cluster analysis of
transcriptomics-based classification will solve the problem of reproducibility of trial
results by defining broadly relevant endotypes.72

Metabolomics
Another potentially rapid diagnostic methodology is the assessment of the metabolic
state of tissues within the host by state-of-the-art 1H nuclear magnetic resonance and
gas chromatographic MS metabolomics.73 Using principal components analysis for
pattern recognition of unsupervised clustering behaviors of patient samples, it is
possible to generate a 2-dimensional principal components analysis to rapidly recog-
nize the presence of infection versus sterile inflammation and predict the risk of mor-
tality in acutely ill patients. Both host-derived and pathogen-derived molecules can be
measured by this technique and provide early distinction between bacterial and viral
pathogens.74

Machine Learning Techniques and Early Sepsis Recognition Methods

A final novel biomarker strategy in development at present is instruction of ICU-
focused machine-derived risk prediction and early recognition of sepsis.75–79 Using
large databases with thousands if not millions of physiologic and pathophysiologic
data points from previous patients with sepsis, it is possible to train computers to
recognize early patterns of sepsis development in real time. These pattern recogni-
tion elements can be improved on by adding more and more new data from ICU pa-
tients. Machine learning in the form of integrating input data points for early detection
toward sepsis-specific patterns might serve as a monitoring assist device in ICU pa-
tients or ward patients who are developing sepsis. A number of groups are working
on machine learning in the ICU and it is likely that this investigation will continue to
evolve.
A recent study by Seymour and colleagues75 in a machine learning in sepsis trial
design is particularly intriguing. These investigators have used several large database
assets from electronic medical records and focused on patients who developed
sepsis. Unstructured clustering of patient physiologic elements repeatedly detected
4 specific patient phenotypes. Group 1 was the most common and were less ill with
a lower need for vasopressors (33% of study population). Group 2 were older with
lots of comorbid illnesses and acute kidney injury. Group 3 had more inflammatory
markers and lung injury. Group 4 was least common (13%) with more septic shock
and more liver dysfunction. Using statistics and machine learning, they then ran Monte
Carlo simulations using different groupings of sepsis phenotypes at baseline against
data available from several recent, phase 3 sepsis trials. Even though the clinical trial
patients all met the standard sepsis definitions, changing the proportion of the 4 pa-
tient groups at baseline could alter the results. Some phenotype groups would drive
outcomes toward harm and other phenotype ratios toward benefit. This information
needs to be validated in future sepsis trials. If confirmed, this could serve as the basis
for better patient phenotype selection into future trials. Most important, these patient
phenotypes could be used to exclude patients at possible risk of harm.
8 Opal & Wittebole

SUMMARY

The role of biomarkers for the detection of sepsis has come a long way in the field of
sepsis management and enrollment of patients into clinical trials. Molecular bio-
markers are taking front stage at present but machine learning and other computa-
tional measures using big datasets is promising as well. Clinical research in sepsis
is hampered but lack of specificity of the diagnosis itself as sepsis is a syndrome
with no uniformly agreed definition.
One consequence of the lack of diagnostic precision in sepsis is who or what is the
gold standard for the final diagnosis of sepsis. Currently the final conclusion is expert
opinion, which is not bad but not perfect.80,81 Perhaps machine learning (or some
highly specific biomarker) will displace expert opinion as the final and most accurate
definition for sepsis.

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