Introduction

Sepsis is common in small animals and is associated with substantial morbidity and a high risk of death, with reported mortality rates in dogs of 20–68%.^1-3 Sepsis is a clinical syndrome that, in veterinary medicine, is presently defined as the systemic inflammatory response to infection, and diagnosed by documenting the hallmarks of systemic inflammation (fever, tachycardia, tachypnea, and leukocytosis) and a concurrent infection. Commonly used SIRS criteria are sensitive for sepsis diagnosis,² but lack specificity and have limited prognostic utility⁴. Illness severity in sepsis can be assessed using scoring systems such as SPI2,⁵ APPLE,^6,7 and the SOFA score,⁸ but they are insensitive to the underlying immune dysfunction that characterizes sepsis. In humans, biomarkers have been widely evaluated in sepsis in an attempt to provide accurate diagnosis and prognostication and to enable clinicians to individualize patient care.^9,10 The ideal biomarker provides biologic insight into the disease process and improves our understanding of pathogenesis. Identification of a biomarker that can be rapidly measured patient-side and that correlates with illness severity and prognosis in small animals with sepsis would advance the pursuit of individualized medicine. Acute phase reactants, such as serum amyloid A (SAA) and C-reactive protein (CRP), are increased in cats and dogs with sepsis but are not prognostic.^11,12 Increased procalcitonin (PCT) concentrations are prognostic in humans with septic shock,^13,14 and PCT may be superior to CRP in this regard. Initial efforts to measure PCT in dogs were unsuccessful,¹⁵ but there is now a validated assay that will enable future studies of PCT in veterinary patients¹⁶. Since sepsis is a syndrome defined by the presence of immune dysregulation and systemic inflammation, analyses of cytokines have been the subject of considerable investigation.^17-24 There is also potential that biochemicals released following formation of neutrophil extracellular traps (NETs) and biomarkers that act as endogenous damage-associated molecular patterns might prove useful as clinical biomarkers.^25-27 Technological developments in high-throughput screening tools mean evaluating the transcriptome, proteome and metabolome are now within reach.^28-32

Biomarkers

Acute Phase Proteins

C-reactive protein (CRP) is a highly conserved plasma protein that binds specific damage- and pathogen-associated molecular patterns and functions as a soluble pattern recognition receptor.³³ As such, CRP is an innate immune response component, that is synthesized rapidly following tissue injury or infection. It is this rapid synthesis and correlation with the degree of inflammation that has made CRP an attractive biomarker in critical illness. Serum concentrations of CRP increase rapidly in dogs with babesiosis, leishmaniosis, leptospirosis, parvoviral enteritis and sepsis.³⁴ CRP is very sensitive and offers biologic insight, but it cannot differentiate infectious from non-infectious inflammation and although it parallels disease severity, it is typically not prognostic. Alpha-1 acid glycoprotein is a recognized biomarker of inflammation in cats, with increased concentrations reported in FIP,³⁵ and neoplasia,³⁶ although it too is not prognostic. Serum amyloid A may also be a useful marker in the cat because it is rapidly responsive to a variety of inflammatory and infectious conditions.^37,38 CRP is not a useful biomarker in the cat and although haptoglobin is increased during the feline acute phase response, additional work will be needed to determine its potential diagnostic utility in feline sepsis specifically.^39-43 Plasma fibrinogen concentration is sensitive to inflammation in dogs, but the use of this biomarker in the evaluation of inflammation in canine sepsis is complicated by the role of fibrinogen in the hemostatic system. Fibrinogen is a positive acute phase protein but since it is the substrate for the formation of fibrin by thrombin, it may be reduced as a result of consumption despite ongoing augmented production in inflammation.

Cytokines

Recently the importance of sepsis-induced immunoparalysis in the morbidity and mortality of sepsis has been recognized.⁴⁴ In human medicine, there is huge potential for precision medicine through immunophenotyping to provide targeted interventions specific to the patient's underlying pathology. For instance, those patients with a hyperactive immune response may benefit from anti-inflammatory therapies, while those with suppressed and inadequate immune responses may benefit from immunostimulatory therapies. Achieving a better understanding of the nature of the inflammatory and immune responses to sepsis is a crucial first step in this endeavor in veterinary medicine. Accordingly, profiling cytokines and chemokines may provide a snapshot of the current immunological state that could be used to tailor treatment.^45,46 Cytokines have the potential to be ideal biomarkers for inflammatory and infectious diseases by providing sensitivity, specificity, and biological insight. Dogs with parvoviral enteritis have increased concentrations of TNF-α.⁴⁷ Proinflammatory cytokines have also been documented in other inflammatory disease states including pyometra and non-septic SIRS.⁴⁸ In a study of dogs with IMHA, cytokines associated with macrophage activity and recruitment were prognostic and appear to provide insight into the nature of this disease process.⁴⁹ Immunophenotyping data characterizing sepsis are now available for both dogs,^19,20 and cats^23,50. This may be particularly valuable given the challenges recognizing and characterizing sepsis in cats.^51,52

Procalcitonin

In humans, procalcitonin (PCT) is a valuable biomarker for sepsis diagnosis and is also used as a therapeutic guide. In sepsis, PCT originates from stimulated mononuclear leukocytes,⁵³ and its release occurs within hours of an endotoxin challenge before returning to baseline within 48 hours⁵⁴. Although the exact role of PCT in patients with sepsis is still unknown, it has been shown to increase inducible nitric oxide synthase (iNOS)-mediated nitric oxide release and therefore may play a role in amplification of the inflammation.⁵⁵ Studies in humans suggest PCT may be able to differentiate bacterial sepsis from non-infectious SIRS and may be used as a guide to initiate antimicrobial therapy.⁵⁶ In some studies, PCT levels correlate with disease severity, and may have prognostic value in humans with sepsis and septic shock.^14,57 There is evidence of procalcitonin gene expression in both thyroidal and extra-thyroidal tissues in dogs with SIRS; while in contrast, normal dogs only express procalcitonin in the thyroid.⁵⁸ Although early attempts to measure PCT were unsuccessful,¹⁵ several more recent studies have demonstrated that PCT is increased in dogs with endotoxemia,⁵⁴ and sepsis,^16,18 and that PCT is prognostic in canine sepsis⁵⁹.

Cell-Free DNA and Nucleosomes

Neutrophil extracellular trap formation or NETosis has now been described in a number of diseases and syndromes in critically ill humans including those with sepsis, acute respiratory distress syndrome, burns, cancer and trauma.^60-64 This cfDNA may originate from apoptotic cells, necrotic tissue or from neutrophil extracellular traps (NETs).^65,66 Increased cell-free DNA (cfDNA) has been shown to have prognostic significance in human patients with severe sepsis, and those with bacteremia.^67,68 As such, cfDNA has the potential to provide biologic insight into the extent and nature of the innate immune response to infection and inflammation. In addition to being an indicator of nature of the disease process and its severity, it has been demonstrated that NET formation may directly contribute to pathogenesis in sepsis through the phenomenon of immunothrombosis,⁶⁹ while cfDNA can inhibit plasmin-mediated fibrinolysis⁷⁰. Increased cfDNA concentrations have also been detected in dogs with sepsis,^18,71 and these concentrations may have prognostic utility⁷².

Nucleosomes are complexes formed by DNA and histone proteins that are released into circulation during cell death and cellular damage such as apoptosis and necrosis.⁷³ They can also be due to the process of NET formation.⁷⁴ As such, cfDNA and nucleosomes share potential origins, but are distinct entities,⁷⁵ with differential potential for immune cell activation through pattern recognition receptors⁷³. Measuring both biomarkers may provide better insights into the disease process than either alone.⁷⁵ Humans with septic shock have significantly higher nucleosome concentrations than patients with sepsis, non-infectious SIRS, or fever.⁷³ In humans, plasma nucleosome concentrations distinguish septic from non-septic critically ill patients and correlate with the severity of immunosuppression and with organ dysfunction.⁷⁶ Nucleosome concentrations are increased in dogs with sepsis,⁷⁷ and may have prognostic utility in some conditions⁷⁸.

-Omics

The evaluation of host gene expression is known as transcriptomics and involves measurement of the patient’s messenger RNAs (mRNAs) in blood or circulating leucocytes.⁷⁹ This approach has been widely studied in human medicine, but there are only a handful of publications in veterinary medicine to date.^58,80,81 Transcriptomics offers the potential for rapid diagnosis of sepsis and for patient stratification for prognostic or therapeutic purposes. From a diagnostic perspective, transcriptomics offers the tantalizing possibility of differentiation of severe infections from noninfectious inflammation.^82,83 Several studies have also investigated both proteomics and metabolomics to discover new diagnostic and prognostic biomarkers for sepsis in humans.^30,84 Proteomics evaluates the array of translated proteins and offers a more distinct picture than the underlying transcriptome profile because of differences in the timing and extent of translation, the influence of posttranslational modifications, and the half-lives of proteins relative to those of mRNA. For veterinary medicine, proteomics also offers advantages over traditional protein assays such as ELISAs, where the applicability and diagnostic accuracy of the assay is dependent upon immunologic reagent availability and efficacy. Several studies have recently been published using proteomics to study veterinary patients with sepsis.^28,29 Metabolomics is the most recent of all these high-throughput screening technologies. This process involves measuring endogenous and exogenous compounds in the blood such as nucleic acids, amino acids, carbohydrates, and lipids in addition to microbial components, and panels of metabolites can differentiate patients with sepsis from healthy subjects,⁸⁵ and predict adverse events and mortality in sepsis³¹.

Individualized Therapy

Many clinical trials in sepsis have tested various interventions to modulate the immune system, but have not resulted in the integration of new drugs into clinical practice. This suggests novel strategies to treat sepsis should be considered. Our current understanding of the pathophysiology of sepsis may be inadequate to design such interventions, particularly when faced with changing patient populations, the effects of therapeutic interventions and the ever-changing dynamic interaction between host and pathogen. The heterogeneity of sepsis and of the patients we manage is also part of the hurdle any novel therapy must overcome. Given the complexity of the host response to sepsis, it may be impossible for a single drug to benefit all patients with sepsis, but biomarkers may help us to overcome some of these issues. Indeed, a recent reanalysis of a trial evaluating an IL-1R antagonist identified benefit in a subgroup of patients with signs of a macrophage activation syndrome,⁸⁶ despite the overall trial being negative. A good biomarker provides biologic insight as well as sensitivity, specificity, diagnosis and prognostication. Sets of biomarkers that provide insight into biologic activity and pathophysiology can be used to target specific patients for intervention, to stratify risk and to monitor response to therapy. Veterinary medicine is not that far behind and personalized medicine for our patients may yet be within reach.⁸⁷

Despite therapeutic advances over the last decade, published survival rates for sepsis have not significantly improved, and range from 30–64%.^88,89 This high mortality rate is attributed to the dysregulated host response to the infection that manifests as cardiovascular instability, hypotension, and organ dysfunction.^90,91 Early recognition and aggressive management comprising cardiovascular stabilization, administration of broad-spectrum antimicrobials and surgical source control is essential to maximizing survival.^92,93 Dogs with bacterial pneumonia or intra-abdominal infections are typically prescribed antimicrobials for 3–6 weeks, to limit potential complications such as recurrence, novel infection development and death.⁹⁴ Studies in humans with intra-abdominal infections show similar outcomes when treated for four days compared to eight days.⁹⁵ Similar findings are described in patients with ventilator-associated pneumonia with no change in outcome when treated for 8 or 15 days.⁹⁶ Thus, human guidelines for treatment of intra-abdominal infection following surgery and for ventilator-associated pneumonia, now recommend shorter courses of antibiotics.⁹⁷ Despite this, the average duration of antimicrobials in humans remains longer than recommended,⁹⁸ likely due to concerns of increased complications for individual patients with premature antimicrobial discontinuation. A recent study in dogs suggested that for urinary tract infections, a 3-day, high-dose course of antimicrobials was non-inferior to a standard 14-day course.⁹⁹ This suggests that, as in humans, dogs may not require extended antimicrobial courses for some disease processes.

Inflammatory biomarkers are now widely used in humans to guide antimicrobial administration in sepsis,¹⁰⁰ and to support decisions to discontinue antimicrobial therapy while minimizing risk^101-104. Various inflammatory biomarkers have been evaluated to aid identification of the appropriate time to safely discontinue antimicrobials. Procalcitonin is a hormokine associated with the calcitonin gene family whose expression is upregulated in response to endotoxins or cytokines. Retrospective and prospective studies using absolute or relative changes in PCT concentrations have shown that patients in whom antimicrobials were discontinued based on PCT values received shorter courses of treatment with no difference in outcome.¹⁰³ The acute phase reactant C-reactive protein has also been evaluated as part of a decision-making algorithm for antimicrobial therapy in patients suffering from sepsis.¹⁰⁵ CRP-guided antimicrobial therapy in humans is associated with shorter antimicrobial administration without changes in patient outcome,¹⁰⁶ while a pilot study in canine pyometra suggests that novel post-operative increases in CRP may help identify post-operative complications such as surgical wound infections¹⁰⁷. The optimal duration of antimicrobial in dogs with sepsis is unknown, but data from humans suggest we may be able to safely reduce the duration of antimicrobials for those diseases. Veterinarians typically rely on clinical, clinicopathologic and radiographic criteria to discontinue therapy. For instance, current recommendations for canine pneumonia are to continue antimicrobials for a least 1 week post-radiographic resolution. Biomarkers of the inflammatory response might provide superior guidance.¹⁰⁸

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