Endometriosis is a common estrogen-dependent disease affecting approximately 176 million women worldwide. Presently, a blood test for endometriosis remains elusive with laparoscopic surgery followed by histopathological confirmation of lesions remaining the gold standard for diagnosis. Women with endometriosis experience long delays between the onset of symptoms and a definitive diagnosis. The search for single or even panels of markers in the blood for the diagnosis of endometriosis has long been underway and typically met with disappointing results. Recently, plasma concentrations of brain-derived neurotrophic factor (BDNF) have been shown to have potential as a diagnostic marker of endometriosis and a novel test method was developed to enable its detection. Herein we summarize the literature suggesting a role for BDNF in the pathophysiology of endometriosis, explain why it is a promising clinical marker for this vexing condition, and introduce a point-of-care device for diagnosis.

by Dr W. G. Foster, Dr J. M. Wessles and Dr L. Soleymani

Introduction
Endometriosis is a common disease in reproductive-aged women characterized by the growth of endometrial cells anywhere in the body outside of the uterus. Epidemiological studies suggest that 6–11% of women are affected by endometriosis [1] reaching an estimated 176 million women globally. Frequent sites of endometrial disease implants include the fallopian tubes, surface of the ovaries and the space between the vagina and rectum; although implants can be found throughout the body. The cause of endometriosis remains to be elucidated; however, in some women, it is thought that during menstruation, some cells from the uterus migrate through the fallopian tubes into the pelvic cavity where they adhere to surrounding structures attach, establish a new blood supply, and grow under the influence of estrogens from the ovaries. Other potential explanations include intravasation of endometrial cells during menstruation, neonatal uterine bleeding, celomic metaplasia, immune dysfunction, and environmental factors [2].

Non-menstrual pelvic pain and infertility are common features of endometriosis that bring women with this disease to seek medical attention. Unfortunately, diagnosis has been reported to be delayed by between 6 and 12 years with an average time-to-diagnosis of 9 years from the onset of symptoms to receipt of a definitive diagnosis [3]. Hence, identification of a clinical tool for the diagnosis of endometriosis has become a high-priority research objective. Techniques in phenomics, genomics, proteomics, metabolomics and biochemistry have been employed to identify single or even panels of markers that could be exploited in the diagnosis of endometriosis. A brief overview of clinical markers is summarized below along with discussion of the data implicating brain-derived neurotrophic factor (BDNF) in the pathophysiology of endometriosis and pelvic pain leading to the suggestion of its use as a clinical marker of endometriosis.

Diagnosis of endometriosis
Healthcare providers and patients face a number of challenges in arriving at a diagnosis of endometriosis including early age at onset of symptoms, normalization of pain by primary care providers, and suppression of symptoms through intermittent use of oral contraceptive pills. Endometriosis is presumptively diagnosed through assessment of patient history, signs and symptoms, and imaging studies. However, the gold standard for diagnosis remains visualization of endometriotic lesions typically by laparoscopy followed by histopathological confirmation of disease. Unfortunately, a biochemical test for the diagnosis of endometriosis remains elusive. Multiple mechanistic pathways including dysregulation of cell adhesion, tissue remodelling, apoptosis, cell proliferation, immune function, and angiogenesis have all been explored in women with endometriosis. A plethora of biochemical differences in the peripheral circulation, peritoneal fluid, and endometrial tissues of women with endometriosis versus healthy controls have been documented [4] and explored as markers of endometriosis. For example, endometriosis induces a chronic inflammatory reaction that is characterized by alterations in interleukin-1, 6, 8, tumour necrosis factor-alpha, RANTES, and interferon gamma concentrations. However, no protein marker of endometriosis has been found to have suitable sensitivity or specificity for the diagnosis of endometriosis whether used alone or in a panel of clinical markers [4]. Consequently, the hunt for a clinical marker of endometriosis continues.

Emerging markers of interest
Emerging markers of interest for the diagnosis of endometriosis include nerve fibre density, microRNA (miRNA), and the neurotrophins. Recent studies report that nerve fibre density in the functional layer of the eutopic endometrium is greater in women with endometriosis compared to controls [5], although this conclusion was recently challenged. The measurement of nerve fibre density was suggested as a diagnostic tool for minimal to mild endometriosis (stage I and II disease). Unfortunately, measurement of nerve fibre density requires an invasive endometrial biopsy and thus is more technically demanding, painful, time consuming, and resource intensive than a simple blood test, and is therefore potentially less appealing to patients.

Recent studies have documented aberrant expression of different miRNAs in the endometrium and ectopic lesions of women with endometriosis [6]. miRNAs are short non-coding RNAs that negatively regulate mRNA translation by repressing the protein translational machinery or degrading their target transcripts. Greater than 2000 mature human miRNA sequences have been identified and are thought to regulate approximately 50% of all protein coding genes. Although widely studied in cancer, the role of miRNAs in regulation of proteins important in the pathophysiology of endometriosis is relatively unexplored. While encouraging results have been reported, replication of miRNA findings, with the exception of miR-451a, has not been demonstrated. In contrast, we suggest that complementary findings from different studies using different techniques and study populations, suggests that the neurotrophins are potentially useful clinical markers of endometriosis.

Neurotrophins and endometriosis
Neurotrophins of the nerve growth factor (NGF) family are soluble polypeptides that are best known for their role in neurite survival and differentiation. Neurotrophins include but are not limited to the following: NGF, BDNF, neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT4/5). Although the neurotrophin family shares a common low affinity receptor, the tumour necrosis factor family neurotrophin growth factor receptor (NGFR), they also signal via high affinity neurotrophin receptors. Specifically, NGF preferentially activates neurotrophic tyrosine kinase receptor 1 (Ntrk1) whereas BDNF and NT4/5 activate Ntrk2, and NT-3 preferentially signals via Ntrk3. The neurotrophin receptors and their ligands are widely expressed in non-neuronal tissues [7] including endocrine glands [7], granulosa cells, and oocytes of fetal and adult mammalian ovaries. Furthermore, we have shown that BDNF and its receptor Ntrk2 are present in endometrial epithelial cells [8] and are expressed in the eutopic endometrium of healthy as well as the eutopic endometrium and ectopic lesions of women with endometriosis (Fig. 1). Both BDNF and Ntrk2 are localized in vascular smooth muscle and endothelial cells as well as activated macrophages and endometrial epithelium. Moreover, we have shown that BDNF can be localized to endometrial cells of ectopic lesions in women with endometriosis (Fig. 2). Hence, we suggest that BDNF and its receptor family are expressed in the endometrium and endometriotic lesions.

Previous studies have established that BDNF is synthesized as a large precursor protein (pro-BDNF) that is cleaved internally by pro-protein convertases in the trans-Golgi network and secretory granules or is cleaved extracellularly by plasmin or matrix metalloproteinases to mature BDNF (mBDNF). While pro-BDNF may be released constitutively, mBDNF is packaged in vesicles and secreted via the regulated pathway facilitated by the sorting receptor, Sortilin-I. Recently, it has been suggested that BDNF regulates divergent pathways including apoptosis, as well as differentiation and survival of discrete nerve cell populations in a receptor dependent manner. A recent proteomic study further demonstrated that BDNF and NT4/5 are both expressed in the endometrium at higher concentrations in women with endometriosis versus disease free controls [9], results that further support a role for neurotrophins as potential clinical markers of endometriosis. Thus, we suggest that the neurotrophins are potentially important in the pathophysiology of endometriosis. Specifically, similar to their roles in the central nervous system, we believe that pro-BDNF dimerizes with Sortilin-I and NGFR to promote apoptosis and inhibit macrophage infiltration whereas mBDNF binds with Ntrk2 and NGFR to facilitate resistance to apoptosis and promote cell survival, differentiation, and nerve outgrowth.

BDNF role in endometriosis and diagnosis
BDNF and the receptors Ntrk2, Sortilin-I and p75NTR are expressed in the endometrium of women (Fig. 1.) and different  mammalian species [10]. A proteomic analysis of the endometrium from women with and without endometriosis revealed that BDNF protein is expressed at greater levels in the endometrium of women with endometriosis than healthy controls [9]. Using immunohistochemistry we localized BDNF to epithelial cells and blood vessels of women with endometriosis (Fig. 2). Circulating concentrations of BDNF were 2-fold greater in women with endometriosis compared to healthy fertile controls [11]. Moreover, plasma concentrations of BDNF returned to baseline levels 3 months after laparoscopic surgery to remove endometriotic lesions. We suggest that greater circulating concentrations of BDNF prior to laparoscopy followed by a decline to concentrations indistinguishable from the control population strongly implicates BDNF in the pathophysiology of endometriosis and the endometriotic lesions as a potential source of circulating BDNF. In our subsequent study [12], plasma BDNF concentrations were 1.5-fold greater in women with endometriosis compared to symptomatic controls, values that were greater in women with stage I and II disease compared to stages III and IV, suggesting a potential value of this marker in earlier stages of disease. Sensitivity and specificity of BDNF as a clinical marker of endometriosis in our laboratory has varied from 68.3–91.7% and 69.4–80.8%, respectively, depending on the population studied and the BDNF cut-off value used. Recently, although BDNF quantified in the serum was not found to be of particular value for the diagnosis of endometriosis, there was a significant correlation between serum BDNF and pelvic pain [13]. We believe this discrepancy between plasma and serum results can be explained by BDNF storage in platelets, which are lysed during blood clotting for serum collection, and might thus confound the relationship. Furthermore, since BDNF expression is estrogen regulated, stage of menstrual cycle may be important in characterizing the utility of this clinical marker in the diagnosis of endometriosis. Finally, BDNF expression in endometrial stromal cells has recently been shown to be regulated by interleukin-1β (IL-1β), an effect that is mediated through the c-Jun and NF-κB [14]. Taken together, these data suggest that the neurotrophin BDNF is expressed in the endometrium and epithelial cells of endometriotic lesions where it may contribute to neurogenesis and pain of endometriosis. Moreover, the findings of increased concentrations of BDNF in the plasma of women with endometriosis suggests potential value as a clinical marker of endometriosis.

Point-of-care diagnostic tool
Given the fact that we and others are able to detect a 1.5–2-fold difference in plasma BDNF concentrations between women with and without endometriosis, we sought to develop a clinically useful device for the diagnosis of endometriosis. Biosensors are devices that combine biorecognition with signal transduction to analyse biologically-relevant targets [15]. The glucose monitor is an example of a handheld, easy-to-use biosensor with electrochemical signal transduction used for disease management. Inspired by the widespread clinical adoption of the glucose monitor, we developed an electrochemical biosensor for diagnosing endometriosis. BDNF concentration in plasma is higher in women with endometriosis in comparison with reference populations [11, 12]. The newly developed BDNF biosensor was created using nanoporous and wrinkled electrodes [16]. These nano/microstructured electrodes enhance the sensitivity of biosensors by increasing the surface area of the transducer [17] and increasing the accessibility of the target to the biorecognition elements [18]. The specificity of the BDNF biosensor is achieved by functionalizing the nanoporous and wrinkled electrodes with anti-BDNF antibodies [19]. Signal is transduced by using an electrochemical reporter [20]. Protein, in our case BDNF, is captured at the electrode surface, and sterically hinders the access of the reporter to the electrode surface, reducing the recorded electrochemical current. As the concentration of the BDNF protein increases, the electrochemical current decreases. The decrease in electrochemical current is correlated with the concentration of BDNF measured in plasma using enzyme-linked immunosorbent assay (ELISA), the gold standard for protein analysis.

Summary
In summary, identifying a clinical marker for the diagnosis of endometriosis has been a difficult challenge. Recent evidence implicates the neurotrophin BDNF in the pathophysiology of endometriosis that is correlated with pelvic pain and potential for the diagnosis. A novel electrochemical polymer chip-based technology has been developed that can detect BDNF in human plasma and discriminate between women with and without endometriosis [16]. Combining BDNF, a novel biomarker for diagnosing endometriosis, with a sensitive electrochemical biosensor for analysing protein targets is paving the way for a diagnostic blood test for endometriosis.

Acknowledgements
The authors gratefully acknowledge the contributions of the women who have participated in our studies by providing blood and tissue samples without which our projects would not have been possible. We also gratefully acknowledge the staff of the Endo@Mac program and the clinical teams of Drs Nick Leyland, Sanjay Agarwal, Dustin Costescu and Sarah Scattolon who have enabled the tissue collection for the experiments described in this report. Annette Bullen has made our work possible through study participant recruitment and unwavering support for our efforts. The authors also are grateful for the contributions of our student Marina Bockaj without whom the endochip would not have been possible. The support of our funding partner the Canadian Institutes of Health Research (MOP142230 to WGF) is also greatly appreciated. Leyla Soleymani in the Canada Research Chair (Tier II) in Miniaturized Biomedical Devices and is supported by the Canada Research Chair program.

References
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The authors
Warren G. Foster*^1,2 PhD, Jocelyn M. Wessles¹ PhD and Leyla Soleymani^2,3 PhD
¹Department of Obstetrics & Gynecology, McMaster University. Hamilton, Ontario, Canada
²School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada
³Department of Engineering Physics, McMaster University, Hamilton, Ontario, Canada

*Corresponding author
E-mail: fosterw@mcmaster.ca

Sepsis is a medical emergency that needs rapid identification and treatment to create the best possible outcomes. However, in the early stages it can be very difficult to distinguish sepsis from uncomplicated infection. This article summarizes recent developments in sepsis nomenclature and definitions as well as providing an insight into the role that biomarkers might play in diagnosis and prognosis.

Background
Sepsis is a life-threatening condition associated with high morbidity and mortality, with the risk of death ranging from 30% to 80% depending on the severity of the disease. The World Health Organization estimates that more than 30 million people are affected by sepsis worldwide every year [1], although for reasons discussed by Candel et al., the actual epidemiology of sepsis is difficult to ascertain [2]. In the UK and USA it is thought that sepsis is the cause of around 37 000 and nearly 270 000 deaths per year, respectively [3, 4]. Outcomes of sepsis are better if it is detected and treated early, but despite the large numbers of people affected by it, public awareness of it is still low. In recent years, awareness campaigns have been launched and this year several popular TV and radio programmes in the UK have featured sepsis storylines (Call the Midwife, Coronation Street and The Archers).
Definitions
The difficulties experienced in studying the epidemiology of sepsis are likely to reflect the problems of characterization and diagnosis of the disease, which is in turn a reflection of the complex nature of the condition. Original definitions of sepsis date back to 1991, with the idea that sepsis was caused by systemic inflammatory response syndrome (SIRS) in resulting from infection. In 2001 the definitions were re-examined but left largely unchanged. In 2016, a task force re-evaluated and updated definitions of sepsis and septic shock (Box 1), taking into account improved understanding of the pathobiology of sepsis, which is now recognized to involve early activation of both pro- and anti-inflammatory responses, along with major modifications in non-immunologic pathways such as cardiovascular, neuronal, autonomic, hormonal, bioenergetic, metabolic, and coagulation [5]. A lay definition of sepsis published in 2011 [6] was also accepted by the 2016 task force (Box 1). The definitions created in 1991, 2001 and 2016 have been designated Sepsis-1, Sepsis-2 and Sepsis-3, respectively, to indicate the need for ongoing refinement.

Diagnosis of sepsis
Early diagnosis and treatment of sepsis is associated with improved outcomes, but the difficulty lies in distinguishing sepsis from uncomplicated infection. Identification of patients with sepsis is largely achieved through the use of the Sequential (or Sepsis-Related) Organ Failure Assessment (SOFA) score (Table 1) in the hospital setting or the quick SOFA (qSOFA) score (See Figure 1 “Operationalization of Clinical Criteria Identifying Patients With Sepsis and Septic Shock” in Singer et al. [5]). Commencement of treatment should occur within the first hour of admission and should not be delayed by waiting for results from the lab, as the SOFA score can be applied retrospectively. Management of sepsis also requires (amongst other things) that blood samples are taken before broad spectrum antibiotics are administered and that once the pathogen has been identified antibiotic usage can be refined to aid antimicrobial stewardship (See the Surviving Sepsis Campaign [7] and NICE guidelines [8] for full details of early sepsis management). Sepsis is most commonly caused by bacterial infection, but can also be due to fungal, viral or parasitic infection. However, identification of the pathogen and its antibiotic susceptibility and/or resistance by classic culture techniques is slow and molecular- and proteomic-based approaches, such as matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) spectroscopy, may improve turnaround times [9].

Biomarkers
The difficulty of distinguishing sepsis from uncomplicated infection has long driven the search for suitable biomarkers to aid sepsis diagnosis. An ideal biomarker would be able to distinguish sepsis from non-infectious causes of critical illness, having a fast and specific increase in sepsis and a rapid decrease after effective therapy. A number of potential biomarkers have been identified, although none are specific enough to be used alone.
Procalcitonin and C-reactive protein
The most-studied biomarkers are procalcitonin and C-reactive protein (CRP). CRP is an acute-phase protein that is secreted from the liver in the response to inflammatory processes and is therefore sensitive but not specific for sepsis. Procalcitonin, again is produced in response to inflammation and infection, and is so far the only biomarker to be used clinically, as it differentiates better than CRP between infectious and non-infectious causes of critical illness. A meta-analysis found that procalcitonin had a mean sensitivity and specificity of around 70% and an area under receiver operator characteristic curve of less than 0.80 [10]. However as levels of procalcitonin are known to be raised after surgery, trauma and viral infection, the Surviving Sepsis Campaign concluded that procalcitonin levels are not adequate to distinguish sepsis from other causes of inflammation [11], although it may be useful for indicating when treatment with antibiotics can end [12].

Interleukin 6 (IL-6)
IL-6 was initially a biomarker of interest for rapid sepsis diagnosis as it has a fast kinetic profile – the concentration increases within 2 hours of onset of sepsis and decreases within 6 hours. However, the results from studies have been mixed, with some suggesting that it was able to discriminate between sepsis and non-infectious illness, whereas others found that procalcitonin was better, hence it has not been added to current guidelines [11].

Promising biomarkers
A number of other biomarkers have been identified that show promise include soluble urokinase-type plasminogen activator receptor, presepsin and proadrenomedullin [2, 13]. Additionally, recently, reduced serum levels of fetuin-A (a major hepatokine) were found to be independently associated with predicting progression to septic shock and higher rates of mortality [14].

Biomarker panels
Even today, no single biomarker has the diagnostic strength to identify patients suffering from sepsis and it is likely that assessing panels of biomarkers will increase the sensitivity and accuracy of diagnosis of sepsis, compared to any individual biomarker (for example, see the study by Kofoed et al. [15]). More recently, the power of mass spectrometry and “-omics studies” is being investigated with some promise, although still suffering from limitations [13].

References
1. Sepsis. World Health Organization 2018; http://www.who.int/news-room/fact-sheets/detail/sepsis.
2. Candel FJ, et al. Current aspects in sepsis approach. Turning things around. Rev Esp Quimioter 2018; 31(4): 298–315.
3. Improving outcomes for patients with sepsis: a cross-system action plan. NHS England 2015; https://www.england.nhs.uk/wp-content/uploads/2015/08/Sepsis-Action-Plan-23.12.15-v1.pdf.
4. Sepsis. Centers for Disease Control and Prevention 2018; https://www.cdc.gov/sepsis/datareports/index.html.
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6. Czura CJ. Merinoff symposium 2010: Sepsis – speaking with one voice. Mol Med 2011; 17(1-2): 2–3.
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9. Ward KM, Harris R. Sepsis: earlier organism identification using MALDI-TOF. Clin Lab Int 2015; Nov: 14–18.
10. Wacker C, et al. Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. Lancet Infect Dis 2013; 13: 426–435.
11. Dellinger RP, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013; 41(2): 580–637.
12. Sager R, et al. Procalcitonin-guided diagnosis and antibiotic stewardship revisited. BMC Med 2017; 15: 15.
13. Ludwig KR, Hummon AB. Mass spectrometry for the discovery of biomarkers of sepsis. Mol Biosyst 2017; 13(4): 648–664.
14. Karampela. Karampela I, Kandri E, Antonakos G, Vogiatzakis E, Christodoulatos GS, Nikolaidou A, Dimopoulos G, Armaganidis A, Dalamaga M. Kinetics of circulating fetuin-A may predict mortality independently from adiponectin, high molecular weight adiponectin and prognostic factors in critically ill patients with sepsis: A prospective study. J Crit Care 2017; 41: 78–85.
15. Kofoed K, et al. Use of plasma C-reactive protein, procalcitonin, neutrophils, macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble triggering receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study. Crit Care 2007; 11(2): R38. 

Extracellular vesicles as markers and mediators in sepsis
^{Raeven P, Zipperle J, Drechsler S. Theranostics 2018; 8(12):3348–3365}

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It remains a highly lethal condition in which current tools for early diagnosis and therapeutic decision-making are far from ideal. Extracellular vesicles (EVs), 30 nm to several micrometers in size, are released from cells upon activation and apoptosis and express membrane epitopes specific for their parental cells. Since their discovery two decades ago, their role as biomarkers and mediators in various diseases has been intensively studied. However, their potential importance in the sepsis syndrome has gained attention only recently. Sepsis and EVs are both complex fields in which standardization has long been overdue. In this review, several topics are discussed. First, we review current studies on EVs in septic patients with emphasis on their variable quality and clinical utility. Second, we discuss the diagnostic and therapeutic potential of EVs as well as their role as facilitators of cell communication via micro RNA and the relevance of microorganism-derived EVs. Third, we give an overview over the potential beneficial but also detrimental roles of EVs in sepsis. Finally, we focus on the role of EVs in selected intensive care scenarios such as coagulopathy, mechanical ventilation and blood transfusion. Overall, the prospect for EV use in septic patients is bright, ranging from rapid and precise (point-of-care) diagnostics, prevention of harmful iatrogenic interventions, to using EVs as guides of individualized therapy. Before the above is achieved, however, the EV research field requires reliable standardization of the current methods and development of new analytical procedures that can close the existing technological gaps.

Diagnostic accuracy of lipopolysaccharide-binding protein for sepsis in patients with suspected infection in the Emergency Department
^{García de Guadiana Romualdo L, Albaladejo Otón MD, Rebollo Acebes S, Esteban Torrella P, Hernando Holgado A, et al. Ann Clin Biochem 2018; 55(1): 143–148}

BACKGROUND: Biomarkers can facilitate the diagnosis of sepsis, enabling early management and improving outcomes. Lipopolysaccharide-binding protein (LBP) has been reported as a biomarker for the detection of infection, but its diagnostic value is controversial. In this study, we assessed the diagnostic accuracy of LBP for sepsis in the Emergency Department (ED) patients, comparing it with more established biomarkers of sepsis, including procalcitonin (PCT) and C-reactive protein (CRP).

METHODS: LBP and other sepsis biomarkers, including PCT and CRP, were measured on admission in 102 adult patients presenting with suspected infection. Classification of patients was performed using the recently updated definition for sepsis (Sepsis-3). The diagnostic accuracy of LBP, CRP and PCT for sepsis was evaluated by using receiver operating characteristic curve (ROC) analysis.

RESULTS: A total of 49 patients were classified as having sepsis. In these patients, median (interquartile range) LBP (41.8 [41.1] µg/dL vs 26.2 [25] µg/dL), CRP (240 [205] mg/L vs 160 [148] mg/dL) and PCT (5.19 [13.68] µg/L vs 0.39 [1.09] µg/L) were significantly higher than in patients classified as not having sepsis (P<0.001 for all three biomarkers). ROC curve analysis and area under curve (AUC) revealed a value of 0.701 for LBP, similar to CRP (0.707) and lower than that for PCT (0.844) (P=0.012).

CONCLUSION: In adult ED patients with suspected infection, the diagnostic accuracy for sepsis of LBP is similar to that of CRP but lower than that of PCT.

An innovative approach for the integration of proteomics and metabolomics data in severe septic shock patients stratified for mortality
^{Cambiaghi A, Díaz R, Martinez JB, Odena A, Brunelli L, et al. Sci Rep 2018; 8(1):6681}

In this work, we examined plasma metabolome, proteome and clinical features in patients with severe septic shock enrolled in the multicentre ALBIOS study. The objective was to identify changes in the levels of metabolites involved in septic shock progression and to integrate this information with the variation occurring in proteins and clinical data. Mass spectrometry-based targeted metabolomics and untargeted proteomics allowed us to quantify absolute metabolite concentration and relative protein abundance. We computed the ratio D7/D1 to take into account their variation from day 1 (D1) to day 7 (D7) after shock diagnosis. Patients were divided into two groups according to 28-day mortality. Three different elastic net logistic regression models were built: one on metabolites only, one on metabolites and proteins and one to integrate metabolomics and proteomics data with clinical parameters. Linear discriminant analysis and partial least squares discriminant analysis were also implemented. All the obtained models correctly classified the observations in the testing set. By looking at the variable importance (VIP) and the selected features, the integration of metabolomics with proteomics data showed the importance of circulating lipids and coagulation cascade in septic shock progression, thus capturing a further layer of biological information complementary to metabolomics information.

Validity of biomarkers in screening for neonatal sepsis – a single-center hospital-based study
^{Rashwan NI, Hassan MH, Mohey El-Deen ZM, Ahmed AE. Pediatr Neonatol 2018; doi: 10.1016/j.pedneo.2018.05.001 [Epub ahead of print]}

BACKGROUND: The diagnosis of neonatal sepsis still considered to be a challenge for both clinicians and the laboratory owing to the non-specific clinical presentations. The present study aimed to compare and assess the diagnostic and prognostic values of C-reactive protein (CRP), high sensitivity CRP (hsCRP), presepsin, interleukin-6 (IL-6) and procalcitonin (PCT) in neonatal sepsis separately and in combination.

METHODS: This hospital-based cross-sectional study has been conducted on 168 neonates recruited from the Neonatal Intensive Care Unit of Qena University Hospitals, Upper Egypt. Measurements of CRP using the latex agglutination test, hsCRP, presepsin, IL6 and PCT assays using commercially available ELISA assay kits were done to all included neonates.

RESULTS: There were significantly higher serum levels of CRP among late onset versus early onset sepsis group with significantly higher serum levels of hsCRP and presepsin among early onset compared with the late onset sepsis group (P<0.05 for all). There were significantly higher hsCRP, presepsin and PCT serum levels in proven versus probable sepsis group (P<0.05 for all). Significantly higher serum levels of presepsin and PCT were noted among survivors versus non-survivors sepsis group (P<0.05 for all). The cut-off value of the serum level of CRP >6 mg/dL showed lower sensitivity and specificity than that of hsCRP at cut-off >140 ng/mL in diagnosing neonatal sepsis. The cut-off value of presepsin >200 ng/mL showed equal sensitivity and specificity to IL-6 at cut-off >22 pg/mL. The cut-off value of PCT at >389 pg/mL showed sensitivity and specificity approximate to that of hsCRP.

CONCLUSIONS: CRP could be a helpful prognostic marker in late onset neonatal sepsis. hsCRP and PCT have higher diagnostic accuracy in neonatal sepsis in comparison to other studied markers. Both IL-6 and presepsin have equal diagnostic utility in neonatal sepsis, but presepsin could be a helpful diagnostic marker in early onset neonatal sepsis.

Plasma miRNA-223 correlates with risk, inflammatory markers as well as prognosis in sepsis patients
^{Wu X, Yang J, Yu L, Long D. Medicine 2018; 97(27):e11352}

The purpose was to evaluate the role of plasma microRNA-223 (miRNA-223) in risk and prognosis in sepsis patients, and its correlation with inflammatory markers. In this study, 187 sepsis patients from July 2015 to December 2016 were consecutively enrolled. Blood samples from septic patients and healthy controls (HCs) were collected, and plasma was separated for miRNA-223 expression detected by quantitative real-time PCR (qPCR). Enzyme-linked immune sorbent assay (ELISA) was performed to detect inflammatory markers. The results were as follows: miRNA-223 was highly expressed in sepsis patients compared to HCs (P<0.001). Receiver operating characteristic (ROC) curve revealed miRNA-223 disclosed a good diagnostic value of sepsis with area under curve (AUC) of 0.754, 95 % CI: 0.706–0.803. Sensitivity and specificity were 56.6 % and 86.6 % at the best cut-off point, respectively. Multivariate logistic analysis indicated that miRNA-223 could predict sepsis risk independently. Spearman’s correlation disclosed that miRNA-223 relative expression positively correlated with APCHE II score (r=0.459, P<0.001), CRP (r=0.326, P<0.001), TNFα (r=0.325, P<0.001), IL-1β (r=0.165, P=0.024), IL-6 (r=0.229, P=0.002) and IL-8 (r=0.154, P=0.035), while it negatively correlated with IL-10 (r=−0.289, P<0.001). miRNA-223 expression in non-survivors was higher than that in survivors (P<0.001). ROC curve revealed miRNA-223 could distinguish sepsis non-survivors from survivors with AUC of 0.600, 95 % CI: 0.505–0.695. Sensitivity and specificity were 83.5 % and 38.9 % respectively at the best cut-off point. In conclusion, plasma miRNA-223 correlates with disease severity and inflammatory markers levels, and it might serve as a novel diagnostic and prognostic biomarker in sepsis patients.

Biomarkers of endothelial dysfunction predict sepsis mortality in young infants: a matched case-control study
^{Wright JK, Hayford K, Tran V, Al Kibria GM, Baqui A, et al. BMC Pediatr 2018; 18(1):118}

BACKGROUND: Reducing death due to neonatal sepsis is a global health priority, although there are limited tools to facilitate early recognition and treatment. We hypothesized that measuring circulating biomarkers of endothelial function and integrity (i.e. Angiopoietin-Tie2 axis) would identify young infants with sepsis and predict their clinical outcome.

METHODS: We conducted a matched case-control (1:3) study of 98 young infants aged 0–59 days of life presenting to a referral hospital in Bangladesh with suspected sepsis. Plasma levels of Ang-1, Ang-2, sICAM-1, and sVCAM-1 concentrations were measured at admission. The primary outcome was mortality (n=18); the secondary outcome was bacteremia (n=10).

RESULTS: Ang-2 concentrations at presentation were higher among infants who subsequently died of sepsis compared to survivors (aOR 2.50, P=0.024). Compared to surviving control infants, the Ang-2:Ang-1 ratio was higher among infants who died (aOR 2.29, P=0.016) and in infants with bacteremia (aOR 5.72, P=0.041), and there was an increased odds of death across Ang-2:Ang-1 ratio tertiles (aOR 4.82, P=0.013).

CONCLUSIONS: This study provides new evidence linking the Angiopoietin-Tie2 pathway with mortality and bacteremia in young infants with suspected sepsis. If validated in additional studies, markers of the angiopoietin-Tie2 axis may have clinical utility in risk stratification of infants with suspected sepsis.

Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis
^{van de Groep K, Bos MP, Savelkoul PHM, Rubenjan A, Gazenbeek C, et al. Eur J Clin Microbiol Infect Dis 2018; 37(7):1333–1344}
Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100 %. Sensitivity was 26 % for the Gram-positive PCR, 32 % for the Gram-negative PCR, and ranged 0 to 7 % for yeast PCRs. Yeast detection was improved to 40 % in a smaller set-up. There was no overall association between BSI-PCR sensitivity and time-to-positivity of BC (which was highly variable), yet Ct-values were lower for true-positive versus false-positive PCR results. False-positive results were observed in 84 (4 %) of the 2200 species-specific PCRs in 200 culture-negative samples, and ranged from 0 to 6 % for generic PCRs. Sensitivity of BSI-PCR was promising for individual bacterial pathogens, but still insufficient for yeasts and generic PCRs. Further development of BSI-PCR will focus on improving sensitivity by increasing input volumes and on subsequent implementation as a
bedside test.

From traditional biochemical signals to molecular markers for detection of sepsis after burn injuries
^{Muñoz B, Suárez-Sánchez R, Hernández-Hernández O, Franco-Cendejas R, Cortés H, Magaña JJ. Burns 2018; doi: 10.1016/j.burns.2018.04.016 [Epub ahead of print]}

Sepsis is a life-threatening organ-dysfunction condition caused by a dysregulated response to an infectious condition that can cause complications in patients with major trauma. Burns are one of the most destructive forms of trauma; despite the improvements in medical care, infections remain an important cause of burn injury-related mortality and morbidity, and complicated sepsis predisposes patients to diverse complications such as organ failure, lengthening of hospital stays, and increased costs. Accurate diagnosis and early treatment of sepsis may have a beneficial impact on clinical outcome of burn-injured patients. In this review, we offer a comprehensive description of the current and traditional markers used as indicative of sepsis in burned patients. However, although these are markers of the inflammatory post-burn response, they usually fail to predict sepsis in severely burned patients because they do not reflect the severity of the infection. Identification and measurement of biomarkers in early stages of infection is important in order to provide a timely response and the effective treatment of burned patients. Therefore, we compiled important experimental evidence, demonstrating novel biomarkers, including molecular markers such as genomic DNA variations, alterations of transcriptome profiling (mRNA, miRNAs, lncRNAs and circRNAs), epigenetic markers, and advances in proteomics and metabolomics. Finally, this review summarizes next-generation technologies for the identification of markers for detection of sepsis after burn injuries.