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.

Standard drug testing is regularly carried out using urine, blood or oral fluid. However, fingerprints present a good alternative, as the sample collection is non-invasive, rapid and safe. Herein, we describe the application of two different testing methods for the detection of cocaine in fingerprint samples.

by Dr Catia Costa, Dr Mahado Ismail and Dr Melanie J. Bailey

Drug abuse in the United Kingdom is on the rise and it is a cause for concern, with widespread financial and social implications [1, 2]. The ever-growing drug and alcohol culture in the UK has led to the implementation of workplace drug testing in many industries, especially those in high-risk operational environments. Consequently, there has been a surge in the demand for drug-screening suppliers to develop faster and more reliable testing. This demand is set to increase the market value of drug and alcohol testing in the UK from £167 million to £231 million by 2019 [3].

Conventionally, drug testing is carried out using biological matrices such as blood, urine and, more recently, oral fluid. These matrices and methods of analysis, although established, present a few problems relating to sample collection and transportation. The collection of blood requires medically trained personnel and sample collection is considered invasive, whereas urine carries privacy concerns. Oral fluid is an alternative matrix used for non-invasive drug testing, although sample collection can be time-consuming. All these three matrices are also biohazardous, making sample storage and transportation a potential issue. The potential use of fingerprints for drug testing has become the subject of many recent publications. Fingerprint samples present a good alternative for drug testing as collection is non-invasive and rapid, and there are no known biohazards associated with the sample. Additionally, the fingerprint pattern can be used for donor identification.

Chemical analysis of fingerprints
The chemical information embedded in a fingerprint sample has been reviewed by many, and several publications have explored the detection of substances such as cocaine [4–6], heroin [7], methadone [8], lorazepam [9], methamphetamines [10], caffeine [11] and cough medicine [12] in fingerprints after administration of the substances. These reports are predominantly based on liquid chromatography-mass spectrometry (LC-MS), which is very well established in the field of toxicology for its quantitative potential as well as its sensitivity and reliability. New advances in the field of mass spectrometry saw the rise of ambient ionization mass spectrometry techniques that allow the sample to be analysed in its native state, under ambient conditions. Examples include desorption electrospray ionization (DESI), liquid extraction surface analysis (LESA) and paper spray, which have been applied to the detection of cocaine and metabolites in fingerprint samples [4–6].

Most of these reports in the literature have looked at fingerprint samples collected after administration of the substances. However, no research has investigated the significance of the detection of these substances compared to a large background population of non-drug users. This is of particular importance as a positive test result may be the outcome of contamination by contact with contaminated surfaces or handling the parent drug rather than ingestion. This directly highlights the need for a sampling strategy that removes any contact residue while providing enough fingerprint material for the analysis.

Detection of cocaine in fingerprints
The detection of cocaine in fingerprints has been studied and reported by Ismail et al. [7]. This study looked at fingerprints collected from the background population (i.e. non-drug users) and from patients at a drug rehabilitation clinic. Both sets of samples were collected as presented and after handwashing, followed by wearing nitrile gloves for 10 minutes. Fingerprint results were supported by oral fluid analysis and patient testimony. Analysis of samples collected from patients (n=13) at the rehabilitation clinic yielded a 100% detection rate for cocaine for samples collected as presented and after handwashing. However, the detection of the cocaine metabolite, benzoylecgonine (BZE), decreased from 94% from samples collected as presented, to 87% for samples collected after handwashing. To evaluate the significance of the results above, fingerprint samples collected from the background population were analysed to investigate the prevalence of these substances in non-drug users. Samples collected as presented (n=99 samples) returned a 13% and 5% detection rate for cocaine and BZE, respectively. After handwashing, cocaine was only detected in 1% of the samples analysed (n=100) and no BZE was present. These findings suggest that cocaine can be detected in the background population owing to environmental exposure (e.g. contact with bank notes). However, after using a handwashing procedure, cocaine and benzoylecgonine were not prevalent. Collection of fingerprint samples after a hand-cleaning procedure is therefore advantageous to reduce potential false-positive rates that can be observed from environmental exposure.

As previously mentioned, the use of chromatographic methods is well established in the field of toxicology. However, such methods often rely on extensive sample preparation and analysis. To overcome this issue we have developed paper spray-mass spectrometry (PS-MS) for the detection of cocaine in under 4 minutes from fingerprints collected from patients seeking treatment at a rehabilitation centre [5]. For this method fingerprints are collected on a triangular piece of paper, which is in turn placed on the paper spray source for analysis. An internal standard, solvent and voltage are applied to the paper, resulting in the extraction and ionization of the fingerprint residues before detection on the mass spectrometer (Fig. 1). The method was evaluated with 239 fingerprint samples collected from drug users at the National Health Service (NHS)  rehabilitation clinics and from the background population. A positive result was based on the detection of cocaine or one of its two main metabolites, BZE and ecgonine methyl ester (EME). A 99% true-positive rate was achieved on the samples collected from patients at drug rehabilitation centres, which was supported by standard saliva drug testing and patient testimony. Analysis of samples collected from the general population yielded a 2.5% false-positive rate. This follows from the work by Ismail et al. [7] described above, where in the absence of a hand-cleaning procedure cocaine was detected in the background population. Both studies highlight the need for a well-defined sample collection procedure to eliminate false-positive results while maintaining true-positives.

This method has since its publication been shortened to 30 seconds and it has also been applied to the detection of heroin, morphine, codeine, 6-AM and explosive materials. This highlights the potential for the technique to be on a par with current testing methods that target a wide range of substances.

Fingerprint visualization
Another advantage of using fingerprints for drug testing is the possibility to integrate a fingerprint visualization step for donor identification. This would be of particular benefit for preventing cheating and also in cases of disputed results where one would be able to prove that the results were derived from the correct person. Silver nitrate was used to visualize fingerprint samples collected from drug users by treating the substrate before sample collection. Upon collection, samples were exposed to ultraviolet light to bring out the fingerprint pattern (Fig. 2). Analysis of fingerprint samples collected from drug users after silver nitrate development yielded a 100% detection rate for cocaine, showing great potential for this development step to be included in the fingerprint testing routine.

The future: treatment adherence monitoring
Treatment non-adherence is a well-known problem in the NHS and it is estimated that it can cost over £500 million each year [13]. Thus, the establishment of an adherence monitoring tool could result in substantial savings for the NHS. Fingerprint testing offers the opportunity for remote testing where the samples can be collected by the patient at home and sent to the laboratory for analysis. In cases of non-adherence, medical professionals may intervene and ensure the patient is receiving adequate treatment. This is of particular interest for conditions known to have poor adherence rates such as diabetes, cardiovascular diseases and mental health disorders [14] or for highly infectious diseases such as tuberculosis.

References
1. Barber S, Harker R, Pratt A. Human and financial costs of drug addiction. House of Commons Library 2017.
2. Health matters: preventing drug misuse deaths (GOV.CO.UK2017). Public Health England 2017 (https: //www.gov.uk/government/publications/health-matters-preventing-drug-misuse-deaths/health-matters-preventing-drug-misuse-deaths).
3. Eurofins Workplace Drug Testing launches new holistic ‘wrap around service’ to assist UK plc. Eurofins 2018 (https: //www.eurofins.co.uk/forensic-services/press-releases/uk-growing-drug-culture/).
4. Bailey MJ, Bradshaw R, Francese S, Salter TL, Costa C, Ismail M, Webb RP, Bosman I, Wolff K, de Puit M. Rapid detection of cocaine, benzoylecgonine and methylecgonine in fingerprints using surface mass spectrometry. Analyst 2015; 140(18): 6254–629.
5. Costa C, Webb R, Palitsin V, Ismail M, de Puit M, Atkinson S, Bailey MJ. Rapid, secure drug testing using fingerprint development and paper spray mass spectrometry. Clin Chem 2017; 63(11): 1745–17525.
6. Bailey MJ, Randall EC, Costa C, Salter TL, Race AM, de Puit M, Koeberg M, Baumert M, Bunch J. Analysis of urine, oral fluid and fingerprints by liquid extraction surface analysis coupled to high resolution MS and MS/MS – opportunities for forensic and biomedical science. Anal Methods 2016; 8(16): 3373–3382.
7. Ismail M, Stevenson D, Costa C, Webb R, de Puit M, Bailey M. Noninvasive detection of cocaine and heroin use with single fingerprints: determination of an environmental cutoff. Clin Chem 2018; 64(6): 909–917.
8. Jacob S, Jickells S, Wolff K, Smith N. Drug testing by chemical analysis of fingerprint deposits from methadone-maintained opioid dependent patients using UPLC-MS/MS. Drug Metab Lett 2008; 2(4): 245–247.
9. Goucher E, Kicman A, Smith N, Jickells S. The detection and quantification of lorazepam and its 3-O-glucuronide in fingerprint deposits by LC-MS/MS. J Sep Sci 2009; 32(13): 2266–2272.
10. Zhang T, Chen X, Yang R, Xu Y. Detection of methamphetamine and its main metabolite in fingermarks by liquid chromatography-mass spectrometry. Forensic Sci Int 2015; 248: 10–14.
11. Kuwayama K, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H. Time-course measurements of caffeine and its metabolites extracted from fingertips after coffee intake: a preliminary study for the detection of drugs from fingerprints. Anal Bioanal Chem 2013; 405(12): 3945–3952.
12. Kuwayama K, Yamamuro T, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H. Time-course measurements of drugs and metabolites transferred from fingertips after drug administration: usefulness of fingerprints for drug testing. Forensic Toxicol 2014: 32(2): 235–242.
13. Trueman P, Taylor D, Lowson K, Bligh A, Meszaros A, Wright D, Glanville J, Newbould J, Bury M, et al. Evaluation of the scale, causes and costs of waste medicines. York Health Economics Consortium/School of Pharmacy, University of London 2010.
14. Cutler RL, Fernandez-Llimos F, Frommer M, Benrimoj C, Garcia-Cardenas V. Economic impact of medication non-adherence by disease groups: a systematic review. BMJ Open 2018; 8(1): e016982.

The authors
Catia Costa*¹ PhD, Mahado Ismail² PhD and Melanie J. Bailey² PhD
¹Ion Beam Centre, University of Surrey, Surrey, GU2 7XH, UK
²Department of Chemistry, University of Surrey, Surrey, GU2 7XH, UK

*Corresponding author
E-mail: c.d.costa@surrey.ac.uk