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Potential biomarkers for diagnosing neonatal sepsis
Journal Pre-proof Potential biomarkers for diagnosing neonatal sepsis Ankita Sharma, Anup Thakur, Chitra Bhardwaj, Neelam Kler, Pankaj Garg, Manvender Singh, Sangeeta Choudhury PII:
S2352-0817(20)30001-5
DOI:
https://doi.org/10.1016/j.cmrp.2019.12.004
Reference:
CMRP 460
To appear in:
Current Medicine Research and Practice
Received Date: 21 September 2019 Revised Date:
25 November 2019
Accepted Date: 31 December 2019
Please cite this article as: Sharma A, Thakur A, Bhardwaj C, Kler N, Garg P, Singh M, Choudhury S, Potential biomarkers for diagnosing neonatal sepsis, Current Medicine Research and Practice, https:// doi.org/10.1016/j.cmrp.2019.12.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Sir Ganga Ram Hospital.
Title Page
Title: Potential biomarkers for diagnosing neonatal sepsis
Authors Ankita Sharmaa,c# a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
c
Department of Biotechnology, University Institute of Engineering & Technology, Maharshi
Dayanand University, Rohtak, India Anup Thakurb# b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Chitra Bhardwaja a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
Neelam Klerb b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Pankaj Gargb b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Manvender Singhc c
Department of Biotechnology, University Institute of Engineering & Technology, Maharshi
Dayanand University, Rohtak, India Sangeeta Choudhurya* a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
# Equal Contribution * Corresponding author: Email Id: [email protected] Address: Department of Research, Room no. 1258, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi-110060, INDIA
TITLE PAGE
Article Category: Review
Title: Potential biomarkers for diagnosing neonatal sepsis
Running Title: Biomarkers for neonatal sepsis
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Abstract Vulnerable population of neonates has suboptimal immune function that results towards their susceptibility to infection and sepsis related mortality. Till date, neonatal innate immunity has not been completely understood, yet molecular characterization of this complex system has paved a path for identifying biomarkers that hold the potential to aid clinicians in diagnosing sepsis. The last decade has seen emergence of multiple putative biomarkers for early detection of neonatal sepsis. These include surface markers, acute phase reactants, cytokines and chemokines. Analysis of these components has led us to the conclusion that biological factors/molecules have the potential to identify the sepsis progression in neonates. Early onset sepsis can be detected by C-reactive protein (CRP), Procalcitonin (PCT) and soluble triggering receptor expressed on myeloid cells-1 (sTREM1). On the other hand, specific neutrophil and monocyte markers (Cluster of Differentiation 64 and Toll like receptors) along with the soluble urokinase-type plasminogen activator receptor can effectively detect late onset sepsis. These markers need to be validated by conducting multicenter clinical trials with target of enrolling more number of neonates so that statistically effective biomarkers could be identified.
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Keywords Biomarkers Cytokines Inflammation Neonates Sepsis
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Introduction Sepsis is a life threatening condition in neonates accounting for high rates of morbidity and mortality. Infants admitted in neonatal intensive care unit (NICU) are vulnerable to develop sepsis and its complications due to compromised host defense, poor mucosal barrier and exposure to invasive procedures such as central catheter insertion and mechanical ventilation.1 Blood culture is the gold standard to diagnose sepsis but there is usually a delay of 24-48 hours in availability of reports.2 Many conventional hematological tests such as total leukocyte count (TLC), absolute neutrophil count (ANC), immature/total neutrophil ratio(I:T), micro-erythrocyte sedimentation rate (ESR), platelet count, Procalcitonin (PCT) and C-reactive protein (CRP) have been used for early diagnosis of sepsis; however the positive predictive value (PPV) of these putative markers remains low.3 The current practice of starting empirical antibiotic therapy in all neonates showing sepsis like symptoms results in exposure to adverse drug effects, hospital acquired complications and emergence of resistant strains. On the other hand, delayed start of antimicrobial treatment in case of a fulminant sepsis may lead to major sequelae such as systemic inflammatory responses (SIRs) and multiple organ dysfunctions (MODs) that ultimately lead to death.3An early marker to identify and differentiate infants who need treatment from those who do not is essential. This review attempts to elucidate the efficacy of sepsis biomarkers to decipher the risk stratification in neonates. These have been categorized into two subdivisions namely “Biomarkers in practice” and “Potential biomarkers”.
• BIOMARKERS IN PRACTICE
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In this section described herein are the meta-analysis including randomized controlled trials which have shown the effectiveness of mentioned biomarkers in clinical practice. C-reactive protein C-reactive protein (CRP) is synthesized in liver in response to inflammatory cytokines. Its level may increase up to a thousand-fold during infection. It has a short half-life of approximately 19 hours and falls rapidly after elimination of the microbial source. 4 In recent years, meta-analysis has been performed in large cohorts to evaluate the sensitivity and specificity of CRP in neonatal sepsis. In 2019, a meta-analysisby Brown et al, analyzed 20 studies enrolling 1615 infants to determine diagnostic accuracy of CRP in detecting late onset infection. The data showed the cut-off range of CRP was between 5mg/L to 10mg/L; for which median specificity was 0.74 and sensitivity was 0.62 (95% CI 0.50 - 0.73). 5 Liu et al (2019) in their meta-analysis of 1819 neonates corroborate a similar data with respect to accuracy of CRP test. Positive likelihood ratio (PLR), sensitivity, negative likelihood ratio (NLR), specificity, Diagnostic Odds-ratio (DOR) and AUC were 5.63 (95% CI=2.86 to 11.09), 0.70 (95% CI=0.66 to 0.75), 0.36 (95% CI=0.21 to 0.60), 0.89 (95% CI=0.87 to 0.91), 17.99 (95% CI=6.50 to 49.83), and 0.9 respectively. 6 Patel et al (2018) in their systemic review and meta-analysis suggested that CRPbased algorithms decrease the duration of antibiotic treatment in neonates by 1.45 days (95% CI −2.61 to –0.28) in two randomized controlled trials (RCTs), and by 1.15 days (95% CI −2.06 to –0.24) in two cohort studies, with no differences in mortality or infection relapse. 7 Procalcitonin Procalcitonin (PCT) is a precursor of the hormone calcitonin and is produced by para-follicular cells of thyroid and neuroendocrine cells of lungs and intestine. The levels of PCT rise in response to an inflammatory stimulus, especially of bacterial origin. PCT levels rise within 4
5
hours of exposure to bacterial toxins with a half-life of 25-30 hours.
8
A meta-analysis was
carried out to determine the diagnostic utility of PCT in early onset sepsis (EOS) and late onset sepsis (LOS). Data showed PCT to be 70-77% sensitive in EOS and 82-90% sensitive in LOS. Area under curve (AUC) for PCT was 0.87, pooled sensitivity (95% CI) and specificity were 81% (74-87%) and 79% (69-87%), respectively. 9 Based on the literature as mentioned above, PCT is a better marker than CRP. However, both the markers have low negative predictive value (NPV), hence compromising their diagnostic utility. This calls a search for new biomarkers for diagnosing neonatal sepsis with better diagnostic accuracy. POTENTIAL BIOMARKERS This section reviews newer biomarkers for sepsis. These have been sub-grouped into three categories; acute phase reactants (APR), cytokines and cell surface markers. • Acute phase reactants are inflammation markers whose levels either increase or decrease in patient’s sera during any acute tissue injury or inflammation. • Cell surface markers are proteins/carbohydrates attached to the plasma membrane of cell and expressed uniquely by each cell type. They play a major role in inter-cellular communication, immune regulation and recognition. • Cytokines are signaling proteins, usually less than 80 kDa in size, known to regulate a wide range of biological functions including hematopoiesis, innate and acquired immunity, inflammation and repair, division and proliferation through extracellular signaling. These are secreted by a diverse range of immune cells like macrophages, B lymphocytes, T lymphocytes, NK cells, and mast cells. • ACUTE PHASE REACTANTS
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Serum amyloid A Serum amyloid A (SAA) is an apolipoprotein predominately produced by the liver. SAA is also secreted by various other cells such as endothelial cells, monocytes, and smooth muscle cells. It is known to be regulated by cytokines IL1, IL6 and TNFα.
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SAA is released as a response to
infection or injury 11 and is altered by hepatic function and host nutritional status. 12Arnon et al, compared the diagnostic accuracy of SAA with CRP in early onset septic term neonates and found SAA to be a reliable screening marker in first 24 hours of onset of infection wherein its levels was inversely proportionate to neonatal mortality. SAA had better diagnostic accuracy than CRP at 0 hours in terms of sensitivity (96% vs 30%), specificity (95% vs 98%), PPV (85% vs 78%), NPV (99% vs 83%), positive likelihood ratio (PLR; 19 vs 12), and negative likelihood ratio (NLR; 0.05 vs 0.71) respewctively.13
Lipopolysaccharide-binding protein Lipopolysaccharide-binding protein (LPB) is primarily produced by hepatocytes, epithelial and muscle cells. It is a soluble pattern-recognition molecule crucial for interaction with endotoxin of gram-negative bacteria. LPB recognizes microbial-associated molecular patterns of bacteria to transport endotoxins to CD14 immune effector cells in response to infections.14 Binding to lipopolysaccharide (LPS) component of bacteria, it forms a complex that links to the host macrophage to initiate a response against infection. Level of LPB peaks early, within 6–8 hours, after an acute infection (LPS-induced) resulting in higher sensitivity and NPV for diagnosing EOS in comparison to CRP and PCT. Study by Pavcnik-Arnol et al showed that serum LBP levels were higher in infants with SIRS/sepsis as compared to infants with no infection. Serum LBP, IL-6 and PCT levels were estimated serially for 2 consecutive days. AUC for LBP was 0.89 on day 1.15 Similar trend was observed in another study by Pavcnik-Arnol et al, where LPB
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had the best AUC (0.97) for infants less than 48hours of life on day 1 as compared to lipopolysaccharide( AUC= 0.77) and soluble CD14 (AUC=0.74). Authors found LPB to be the best indicator of sepsis in newborn infants.
15
Presently there exists an ambiguity on usefulness
of LBP since studies report that its levels are based on LPS induced infection and on the other hand less influenced by obstetrical events.16 •
CELL SURFACE BIOMARKERS
Cluster of Differentiation 64 Cluster of Differentiation 64 (CD64) is an Fc-receptor located in membrane glycoprotein that binds monomeric IgG-type antibodieswith high affinity.17 It is more commonly known as Fcgamma receptor 1 (FcγRI). Structurally CD64 is composed of a signal peptide that allows its transport to the surface membrane, three extracellular immunoglobulin domains of the C2-type used to bind antibody, a hydrophobic trans-membrane domain and a short cytoplasmic tail. 18 CD64 is expressed by macrophages, monocytes and weakly by naive neutrophils. Stimulation with cytokines such as IFNγ and Granulocyte colony stimulating factor (G-CSF) can induce higher expression CD64 on these cells. Neutrophil CD64 (nCD64) expression appear to be promising marker for diagnosing early and late onset sepsis. 19 Meta-analysis by Shi et al, shows that nCD64 has an overall pooled sensitivity, specificity, PLR, NLR and diagnostic odds ratio (DOR) of 0.77 (95 % CI: 0.74–0.79), 0.74 (95 % CI: 0.72–0.75), 3.58 (95 % CI: 2.85–4.49), 0.29 (95 % CI: 0.22–0.37) and 15.18 (95 % CI: 9.75–23.62), respectively with an AUC of 0.86.20 Ng et al, in their study assessed two neutrophil surface markers - CD11b, CD64 and two lymphocyte surface markers - CD25, CD45RO. The authors showed that CD64 had highest sensitivity (97%), specificity (90%), and NPV (99%) to diagnose early onset neonatal infection both at the onset and 24 hours later. The study also suggested that
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combining CD64 with IL6 or CRP may improve the sensitivity and NPV which could enhance the ability to diagnose localized infection. 21 The limitation of the study was its small sample size and low PPV (50%) in diagnosing neonatal sepsis. Cluster of Differentiation 11b Cluster of differentiation 11b (CD11b) is a subunit of the b2 integrin adhesion molecule, weakly expressed by non-activated neutrophils.
22
A study by Qui et al, (2019) was conducted to
compare neutrophil CD11b expression between neonates and adults culture positive sepsis. The data showed a 2-4 fold increase in expression of nCD11b in both neonates as well as adults. Thus establishing its functional role in modulating inflammatory response. This systemic review analyzed nine studies, accounting for 843 neonates, showed that overall pooled sensitivity, specificity, PLR, NLR, DOR, post-test positive probability and post-test negative probability and AUC of CD11b were 0.82 (95% CI 0.71-0.90), 0.93 (95% CI 0.62-0.99), 11.51 (95% CI 1.55- 85.62), 0.19 (95% CI 0.10- 0.36), 59.50 (95% CI 4.65- 761.58), 74%, 5% and 0.90 respectively. These studies suggest that nCD11b is a promising biomarker to distinguish sepsis and infection. 23 Human Leukocyte Antigen- DR Isotype Human Leukocyte Antigen- DR Isotype (HLA-DR)is an MHC class II cell surface receptor, constituting a ligand for the T-cell receptor of T-helper cells. Monocytes strongly express HLADR and it is up-regulated in response to inflammation. Genel et al, observed a decreased cellsurface expression of HLA-DR on circulating monocytes from neonates with sepsis.4 This study showed a significant decrease in monocyte HLA-DR expression between non-survivor (n = 8) and survivor (n = 32) preterm and term (median gestation age 36 weeks) neonates with confirmed (n = 18) and clinical (n = 22) LOS (mean postnatal age 16.3 days). Infants with a
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monocyte HLA-DR expression <30% had 30-fold higher risk of mortality.
24
Studies have
reported that significant decrease in monocyte HLA-DR expression in neonates correlates to confirmed or clinical sepsis.25 Toll like receptors Toll like receptors (TLRs) are vital trans-membrane receptors that initiate the innate immune response. TLRs are involved in native immunity recognition and the stimulation of antimicrobial mechanisms. The typical initial response to bacterial infection is recognition of pathogenassociated molecular patterns (PAMPs) via cell surface receptors. Altered neonatal TLR function contributes to increased susceptibility towards infection and sustained inflammatory condition in term and preterm neonates.
26
TLR 1, 2, 4, 5, and 6 chiefly recognize bacterial
products, whereas TLR-3, 7 and 8 are specific for viral detection. TLR 9 seems to be involved in both microbial and viral recognition. TLR-2 and TLR-4 mainly recognize components of Grampositive and Gram-negative bacteria, respectively. Studies have shown that Toll-like receptors, especially TLRs 2 and 4 to be associated with necrotizing enterocolitis, periventricular leukomalacia and sepsis.
27
A prospective cross-sectional study by Yunanto et al showed
significant increase in the levels of neutrophils (p= 0.021, 0.00) as well as neutrophil expression of TLR2 (p= 0.011, 0.003) and TLR4 (p= 0.044, 0.00) in saliva and blood respectively from newborns with sepsis risk factors compared to those of healthy newborns. 28 Soluble form of Cluster of differentiation 14 Cluster of differentiation 14 (CD14) is the receptor of lipopolysaccharide-lipopolysaccharide binding protein (LPS-LBP) complexes. CD14 has two forms: membrane-bound CD14 (mCD14) and soluble CD14 (sCD14). mCD14 has high affinity to LPS, and is mainly expressed on the cell surface of monocytes/macrophages and neutrophils; whereas sCD14 is found in plasma and
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is also called as presepsin. It is produced by mCD14 fall-off or cell secretion. 29 sCD14 plays an important role in mediating the immune responses to LPS of CD14-negative cells such as endothelial cells and epithelial cells. The level of sCD14 increases significantly in neonates with sepsis, and this change is significantly related to the severity and prognosis of the disease. 30 According to a meta-analysis by Bellos et al, analyzing eleven studies and 783 enrolled neonates; the pooled sensitivity and specificity of serum sCD14 for prediction of neonatal sepsis was 0.91 (95% CI 0.87-0.93) and 0.91 (95% CI 0.88-0.94) respectively. The diagnostic odds ratio was 170.28 (95% CI 51.13-567.11) and AUC was 0.97 (SE 0.0117). 31 Studies report AUC for sCD14 to be 0.97 whereas the AUC of CRP and PCT is 0.86 and 0.78 respectively. sCD14 presents itself as a possible biomarker for sepsis. It can be measured via immunoassay and appears to possess better diagnostic capacity for sepsis than PCT.High levels of sCD14 correlates to increased sepsis related mortality rates, and its low false-negative rate indicates patient safety. The data herein shows sCD14 could be a potential biomarker of sepsis. 32 Soluble triggering receptor expressed on myeloid cells-1 Triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily and is expressed on the outer surface of neutrophils and monocytes (myeloid lineage) instrumental in perpetuating an early immune response.
33
Soluble triggering
receptor on myeloid cells -1 (sTREM-1) is a comparatively new biomarker explored for neonatal sepsis. It is a glycoprotein regulated by neutrophils released by phagocytes. Involved in the innate inflammatory response and sepsis, sTREM-1 may be useful as a biomarker of early onset sepsis. Its diagnostic ability to identify patients at greater risk of developing sepsisis significant. sTREM levels correlate significantly with white cell counts and I:T ratios in case of acute infections.
34
A study by Adly et. al, shows that at a cut-off of 310 pg/ml, sensitivity and
11
specificity were 100%, suggesting its role in early diagnosis of neonatal sepsis.
35
A meta-
analysis by Wu et al including eight studies with a total number of 667 neonates showed the estimated sensitivity and specificity of sTREM to be 0.95 [95% CI (0.81-0.99)] and 0.87 [95% CI (0.56-0.97)] respectively. The diagnostic odds ratio was 132.49 [95% CI (6.85-2560.70)]. The study also mentioned about the discrepancies in the cut-off used to calculate the sensitivity and specificity in the studies analyzed. 36 More studies are needed to determine the optimal cutoff value that may discriminate normal levels from those suggestive of neonatal sepsis Soluble urokinase-type plasminogen activator receptor The urokinase-type plasminogen activator receptor, is a 3-domain glycosylated protein (D1–D3) that binds to GPI anchor on cell surface to release the soluble form i.e. soluble urokinase-type plasminogen activator receptor (sUPAR). sUPAR has chemotactic properties and is expressed mostly on immunologically active cells and found in most of the biological fluids such as serum, plasma, cerebrospinal fluid and urine.
37
Systemic levels of sUPAR have been suggested as a
novel diagnostic marker of inflammation in term neonates. Saihanidou et al, reported that increased suPAR levels were significantly higher in the septic infants as compared to healthy controls, suggesting its role in diagnosing sepsis.38 The study also found a correlation between circulating sUPAR levels and systemic inflammation suggesting its association with immune activation. More studies are required to validate the role of sUPAR in sepsis. •
CHEMOKINES & CYTOKINES
Chemokines are cytokines that have ability to direct white blood cell migration towards the site of infection. All cytokines are cellular signaling proteins that play a crucial role in modulating the host immunological response to infection. 39 Leukocyte immune regulation and trafficking into areas of tissue infection and injury is primarily controlled by chemokines and cytokines.
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Cytokines are classified into the subsets of pro-inflammatory (Th1-type, stimulatory), antiinflammatory (Th2-type, inhibitory) or multifunction depending on the final balance of their effects on the immune system (Table 1). Pro-inflammatory cytokines include interferon-gamma (IFNγ), Interleukin (IL)-2, IL-6, IL-8, IL-12, and IL-17. Multiple functional inflammatory cytokines include IL-1β, IL-3, monocyte chemoattractant protein (MCP-1), and growth factors, IL-3, G-CSF. Anti-inflammatory cytokines are IL-4, IL-10, tumor necrosis factor soluble receptor, IL-1 receptor alpha, and transforming growth factor beta 2 (TGF- β2). The evolution of disease and clinical symptoms during neonatal sepsis is dependent upon a complex and delicate balance between the pro-inflammatory, anti-inflammatory, and multiple function cytokines based on their final effect on the immune system. 21,40 Among the group of pro-inflammatory cytokines, IL-6 has been widely studied for its potential use as a biomarker of early neonatal sepsis.41,42,43 During the onset of an infection, B and T lymphocytes are stimulated to produce IL-6 cytokine, which in turn induces hepatocyte production of acute phase reactants such as CRP.15,43 Although IL-6 has a higher sensitivity(90%) as compared to CRP as an early phase biomarker with a negative predictive value of 91%
44,45,46
but its limitation is its short half-life. Usefulness of IL-6 as a sensitive
biomarker was studied by Hu et al in a meta-analysis of 33 studies that included 3135 neonates diagnosed with sepsis. Their study showed a sensitivity of 0.79 (95% CI 0.76-0.81), specificity 0.83 (95% CI 0.81-0.85), AUC was 0.89 and Q (*) index was 0.83 suggesting IL-6 can be used as a diagnostic biomarker. 47 IL-8, another pro-inflammatory cytokine is a probable biomarker for neonatal sepsis with a reported sensitivity of 90% and specificity between 75%–100%. IL-8 regulates leukocyte migration and activation; and has been extensively investigated in neonatal infections.
48
Eight
13
studies (548 neonates) analyzed in a meta-analysis by Zhou et. al, showed IL-8 had a moderate accuracy (AUC=0.89) for the diagnosis of neonatal sepsis. The pooled sensitivity and specificity were 0.78 and 0.84, respectively. The pooled DOR and AUC was 21.64 and 0.89 (Q*=0.8215), respectively.
49
In a study by Ng et al, serum IL-8 concentrations increased within 2–4 hours of
an infection, and decline in 4 hours. The study demonstrated that the use of IL-8 in conjunction with CRP as a biomarker of sepsis enhanced its diagnostic utility.50 The inflammatory process is highly regulated by anti-inflammatory mediators such as IL-10 and TGF-β. These cytokines prevent a severe pro-inflammatory response in reaction to pathogen invasion.
51
In premature infants, the ability to mount an aggressive anti-inflammatory response
is limited, leading to increased susceptibility to target organ injury with excessive SIRS. Thus, serum levels of anti-inflammatory cytokines is an important aspect that needs to be explored as markers of prognostication and survival.50,51 Boskabadi et al., recently reported that concentration above 14 pg/mL of IL-10 showed a higher sensitivity/ NPV (77.7% / 73.6%), with a moderate specificity/PPV (87.8% / 90%).52 He et al (2017) compared multiple cytokines to assess the predictive plasma biomarkers of EOS. Amongst IL-6, IL-8, IL-27, TNF-α, HSP 70, MMP-8, granzyme B, MIP-1α, MIP-1β, and CRP; the authors found IL-27 and PCT to be independent predictors of EOS.53 Other cytokines like RANTES, IL-17A, IL12, IL23p40 play a role in protective host immunity. Though the levels of these cytokines are observed to be inversely proportionate to severity of sepsis in adults but in pre-term neonates, the levels are under the detectable limits. 54 Implementation of anti- and pro-inflammatory ratio analysis emphasizes the importance of host immune balance in disease development and progression, providing an avenue to guide therapy
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in prediction of morbidity and mortality. Combining the cytokines to study their diagnostic utility may be fruitful area of exploration in the early identification of neonatal sepsis. Limitations Having a surrogate biomarker for early and accurate diagnosis of neonatal sepsis would be an important addition to the conventional clinical practice. Yet the studies so far show variable results with respect to diagnostic accuracy (Table 2). The biomarkers reviewed in this article have different sensitivities and specificities for both EOS and LOS. Some of the mentioned biomarkers have a short half-life, so the time point at which the sample should be analyzed is very critical. Non-specificity of some of the markers for sepsis has been identified in conditions like inflammation, infection and stress. Studies in the literature reports small sample size, therefore these upcoming newer biomarkers should be validated in clinical trials with larger cohorts. Conclusion The conventional hematological and microbiological techniques that are routinely used to diagnose neonatal sepsis remain variable due to associated limitations i.e low accuracy and NPV and more time consuming. There is need for an ideal biomarker/s that provides early, specific, and reliable identification of the neonates at higher risk of infection. Potential use of technological developments and ever evolving understanding of biomarker’s strengths and limitations has led to conclusive interpretations on their usage in the clinical systems. In addition, advanced understanding of neonatal immune system and its response to infection has led to identification of potential biomarkers that may translate to early diagnosis and better prognosis of neonatal sepsis in the future.
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Despite many promising biomarker candidates, to date no single biomarker, combination of biomarkers, or score system can exclusively be considered to accurately diagnose early neonatal sepsis. Further studies are needed to explore, test, analyze and validate the reviewed biomarkers for their diagnostic and prognostic value in neonatal sepsis. Identifying biomarkers that are specific and sensitive will aid in enhancing diagnostic accuracy and help in early identification of critical cases. These biomarkers if used as tests for neonatal sepsis can assist the clinicians in better understanding of neonatal host immune responses leading to quick clinical decisions and appropriate antibiotic usage. The authors herein suggest that there is a current need for biomarker or a set of biomarkers that correlates with neonatal sepsis at its various stage of progression. This review brings out the effectiveness of specific biomarkers as indicators of sepsis progression. For identifying EOS; CRP, PCT and sTREM1 are good biomarkers. CD64, sUPAR and TLRs are sensitive markers for LOS. Biomarkers such as sCD14, SAA and HLA-DR are good predictors of mortality. The review also concludes that cytokines such as IL-2/4/6/8/10, TNFα and TGFβ have not been able to show any clinical significance in terms of diagnostic or prognostic utility. Thus, a good biomarker should not only risk stratify in predicting outcome but also be classified based on its detection ability for disease severity and mortality.
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Conflict of Interest The authors have no conflict of interest relevant for this review article.
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Acknowledgement The authors acknowledge the grant provided by Research and Development programme (RDP) by Sir Ganga Ram Hospital (SGRH). SA is a recipient of DST-INSPIRE fellowship (IF140078).
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25. Ng PC, Li G, Chui KM, Chu WCW, Li K, Wong RPO, et al. Quantitative Measurement of Monocyte HLA-DR Expression in the Identification of Early-Onset Neonatal Infection. Biol Neonate. 2006;89:75-81. 26. Watson R, O'Hare FM, William, Molloy EJ. Toll-like receptors in neonatal sepsis. ActaPaediatr. 2013;102:572-8 27. Glaser K, Speer CP. Toll-like receptor signaling in neonatal sepsis and inflammation: a matter of orchestration and conditioning. Expert Rev ClinImmunol. 2013;9:1239-52 28. Yunanto A, Endharti A, Widodo A. Neutrophil, TLR2, and TLR4 expression in newborns at risk of sepsis. Paed Indo. 2013;53:132 29. Mussap M, Puxeddu E, Burrai P, Noto A and Cibecchini F. Soluble CD14 subtype (sCD14-ST) presepsin in critically ill preterm newborns: preliminary reference ranges. J Matern Fetal Neonatal Med. 2012; 25:51-3. 30. Masson S, Caironi P, Spanuth E, Thomae R, Panigada M and Sangiorgi G. Presepsin (soluble CD14 subtype) and procalcitonin levels for mortality prediction in sepsis: data from the Albumin Italian Outcome Sepsis trial. Crit Care. 2014;18:R6 31. Ulla M, Pizzolato E and Lucchiari M. Diagnostic and prognostic value of Presepsin in the management of sepsis in the emergency department: a multicentre prospective study. Critical Care. 2013;17:R168 32. Bellos I, Fitrou G, Pergialiotis V, Thomakos N, Perrea DN, Daskalakis G. The diagnostic accuracy of presepsin in neonatal sepsis: a meta- analysis. Eur J Pediatr. 2018;177:625–32 33. Ford JW, McVicar DW. TREM and TREM-like receptors in inflammation and disease. Curr Opin Immunol. 2009;21:38–46.
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34. Bellos I, Fitrou G, Daskalakis G, Thomakos N, Papantoniou N, Pergialiotis V. Soluble TREM-1 as a predictive factor of neonatal sepsis: a meta-analysis. Inflamm. Res. 2018;67:571. 35. Adly AA, Ismail EA, Andrawes NG, El-Saadany MA. Circulating soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as diagnostic and prognostic marker in neonatal sepsis. Cytokine. 2014;65:184–91 36. Wu Y, Wang F, Fan X, Bao R, Bo L, Li J, et al. Accuracy of plasma sTREM-1 for sepsis diagnosis in systemic inflammatory patients: a systematic review and meta-analysis. Crit Care. 2012;16:R229. 37. Eugen-Olsen J. suPAR – a future risk marker in bacteremia. J Intern Med. 2011; 270:29– 31. 38. Siahanidou T, Margeli A, Tsirogianni C, Charoni S, Giannaki M, Vavourakis E, et al. Clinical value of plasma soluble urokinase-type plasminogen activator receptor levels in term neonates with infection or sepsis: a prospective study. Mediators Inflamm. 2014;2014:375702.doi:10.1155/2014/375702 39. Commins SP, Borish L, Steinke JW. Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy ClinImmunol. 2010;125:S53–72. 40. Polinski C. The value of the white blood cell count and differential count in the prediction of neonatal sepsis. Neonatal Netw. 1996;15:13-23. 41. Silveira RC, Procianoy RS. Evaluation of interleukin-6, tumour necrosis factor-alpha and interleukin-1beta for early diagnosis of neonatal sepsis. ActaPaediatr. 1999;88:647-50. 42. Hodge G, Hodge S, Han P, Haslam R. Multiple leucocyte activation markers to detect neonatal infection. ClinExpImmunol. 2004;135:125–9.
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43. Kishimoto T. The biology of interleukin-6. Blood. 1989;74:1–10. 44. Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed. 2004;89:F229–35. 45. Turunen R, Andersson S, Nupponen I, Kautiainen H, Siitonen S, Repo H. Increased CD11b-density on circulating phagocytes as an early sign of late-onset sepsis in extremely low-birth-weight infants. Pediatr Res. 2005;57:270–5. 46. Küster H, Weiss M, Willeitner AE, Detlefsen S, Jeremias I, Zbojan J, et al. Interleukin-1 receptor antagonist and interleukin-6 for early diagnosis of neonatal sepsis 2 days before clinical manifestation. Lancet. 1998;352:1271–7. 47. HU J, DU PF, Bei DD. Diagnostic value of interleukin 6 for neonatal sepsis: A metaanalysis. CJCP. 2015;17:1176-82. 48. Orlikowsky TW, Neunhoeffer F, Goelz R, Eichner M, Henkel C, Zwirner M, et al. Evaluation of IL-8- concentrations in plasma and lysed EDTA-blood in healthy neonates and those with suspected early onset bacterial infection. Pediatr Res. 2004;56:804–9. 49. Zhou M, Cheng S, Yu J, Lu Q. Interleukin-8 for diagnosis of neonatal sepsis: a metaanalysis. PLoS One.2015;10:e0127170. doi: 10.1371/journal.pone.0127170 50. Ng PC, Li K, Wong RP, Chui K, Wong E, Li G, et al. Proinflammatory and antiinflammatory cytokine responses in preterm infants with systemic infections. Arch Dis Child Fetal Neonatal Ed. 2003;88:F209–13. 51. Schultz C, Temming P, Bucsky P, Göpel W, Strunk T, Härtel C. Immature antiinflammatory response in neonates. ClinExpImmunol 2004;135:130–6
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52. Boskabadi H, Maamouri GA, Ghayour-Mobarhan M, TavakkolAfshari J, Shakeri MT, Ferns GAA. Early diagnosis of late neonatal sepsis by measuring interleukin 10: a case control study. J Neonatol. 2011;25:82-6. 53. He Y, Du W, Jiang H, Ai Q, Feng J, Liu Z, Yu J. Multiplex Cytokine Profiling Identifies Interleukin-27 as a Novel Biomarker For Neonatal Early Onset Sepsis. SHOCK. 2017; 47:140–7. 54. Kasztelewicz B, Piotrowska E, Tołłoczko J, Borszewska-Kornacka M, Gregorek H, Dzierżanowska-Fangrat K. Assessment of interleukin-17A, C5a and RANTES for early diagnosis of neonatal sepsis – a preliminary study. Cent Eur J Immunol. 2016;41:376-82.
25
Table 1: Cytokine classification Pro-Inflammatory
Anti-Inflammatory
Multi-functional
Th1 Type, Stimulatory
Th2 Type, Inhibitory
IFNγ
IL-4, IL-10, IL-5, IL-6, IL-9,IL-13, IL-3, IL1α/β IL-25
TNFα
IL-1RA, IL-33, IL-31
MCP1, TNF α,β
IL-2, IL-6, IL-8,IL-10, IL-27
TNFsr
Growth factors: G-CSF
IL-12, IL-17, IL-18, IL-27
TGFβ2
IL-8
(Abbreviations:G-CSF: Granulocyte-colony stimulating factor,IFNᵧ: Interferon gamma, IL: Interleukin, MCP1: Monocyte chemo-attractant protein 1, TGFβ2: Transforming growth factor beta 2, TNF: Tumor necrosis factor)
26
Table 2: Promising biomarkers for neonatal sepsis Biomarker
n
Cut-off
AUC
Sensitivity Specificity PPV
NPV
(%)
(%)
(%)
(%)
Ref
CRP
1615 4-10 mg/l
0.68
95.7
88.9
78
98
[5]
CD11b
843
0.63
75
100
100
86
[22]
CD64
3478 2.19-3.62
0.73-0.83
75-78
59-77
29-54
81-96
[19]
IL-6
3135 10-150 pg/ml
0.78-0.85
75-87
50-82
92
52
[46]
IL-8
548
0.6-0.72
90
75-100
78
88
[48]
PCT
1915 0.5-5.75 ng/ml 0.8-0.9
93.1
53
79.4
80
[8]
SAA
84
>6.8mg/dl
0.71-0.88
44.7-76.4
100
100
38-58
[12]
sCD14
783
1483 pg/ml
0.97
91
91
91
91
[31]
sTREM-1
667
310 pg/ml
0.8
97
87
68
85
[35]
sUPAR
60
5-11ng/ml
0.6-0.72
96
60
79
90
[37]
TLR 2
60
0.81
0.68
63
80
80
60
[27]
TLR4
60
0.68
0.72
75
82
86
75
[27]
290
60-300 pg/ml
(Abbreviations: CRP: C-reactive protein, CD11b: Cluster of differentiation 11b, CD64: Cluster of differentiation 64, IL-6: Interleukin-6, IL-8: Interleukin-8, PCT: Procalcitonin, SAA: Serum amyloid A, sCD14: soluble Cluster of differentiation 14, sTREM-1: Soluble triggering receptor
27
expressed on myeloid cells-1, sUPAR: Solubleurokinase-type plasminogen activator receptor, TLR2: Toll like receptor 2, TLR4: Toll like receptor 4)
28
Table 1: Cytokine classification Pro-Inflammatory
Anti-Inflammatory
Th1 Type, Stimulatory
Th2 Type, Inhibitory
IFNγ
Multi-functional
IL-4, IL-10, IL-5, IL-6, IL-9,IL-13, IL-3, IL1α/β IL-25
TNFα
IL-1RA, IL-33, IL-31
IL-2, IL-6, IL-8,IL-10, IL- TNFsr
MCP1, TNF α,β Growth factors: G-CSF
27 IL-12, IL-17, IL-18, IL-27
TGFβ2
IL-8
(Abbreviations: G-CSF: Granulocyte-colony stimulating factor, IFNᵧ: Interferon gamma, IL: Interleukin, MCP1: Monocyte chemo-attractant protein 1, TGFβ2: Transforming growth factor beta 2, TNF: Tumor necrosis factor)
1
Table 2: Promising biomarkers for neonatal sepsis Biomarker
Cut-off
AUC
Sensitivity
Specificity
PPV
NPV
(%)
(%)
(%)
(%)
[Ref] CRP [6]
4-10 mg/l
0.68
95.7
88.9
78
98
CD11b [29]
290
0.63
75
100
100
86
CD64 [26]
2.19-3.62
0.73-0.83
75-78
59-77
29-54
81-96
HLA-DR [31]
70
0.6-0.72
65-72
85
85
60-78
IL-6 [54]
10-150 pg/ml
0.78-0.85
75-87
50-82
92
52
IL-8 [56]
60-300 pg/ml
0.6-0.72
90
75-100
78
88
PCT [10]
0.5-5.75 ng/ml
0.8-0.9
93.1
53
79.4
80
SAA [17]
>6.8mg/dl
0.71-0.88
44.7-76.4
100
100
38-58
sCD14 [38]
1483 pg/ml
0.97
91
91
91
91
sTREM-1[40]
310 pg/ml
0.8
97
87
68
85
sUPAR [44]
5-11ng/ml
0.6-0.72
96
60
79
90
TLR 2 [32]
0.81
0.68
63
80
80
60
TLR4 [32]
0.68
0.72
75
82
86
75
TNFα [47]
12-60 pg/ml
0.58-0.62
52-68
82
85
60-70
(Abbreviations: CRP: C-reactive protein, CD11b: Cluster of differentiation 11b, CD64: Cluster of differentiation 64, HLA-DR: Human Leukocyte Antigen- DR Isotype, IL-6: Interleukin-6, IL8: Interleukin-8, PCT: Procalcitonin, SAA: Serum amyloid A, sCD14: soluble Cluster of differentiation 14, sTREM-1: Soluble triggering receptor expressed on myeloid cells-1, sUPAR:
2
Soluble urokinase-type plasminogen activator receptor, TLR2: Toll like receptor 2, TLR4: Toll like receptor 4, TNFα : Tumor necrosis factor α)
3
S2352-0817(20)30001-5
DOI:
https://doi.org/10.1016/j.cmrp.2019.12.004
Reference:
CMRP 460
To appear in:
Current Medicine Research and Practice
Received Date: 21 September 2019 Revised Date:
25 November 2019
Accepted Date: 31 December 2019
Please cite this article as: Sharma A, Thakur A, Bhardwaj C, Kler N, Garg P, Singh M, Choudhury S, Potential biomarkers for diagnosing neonatal sepsis, Current Medicine Research and Practice, https:// doi.org/10.1016/j.cmrp.2019.12.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier, a division of RELX India, Pvt. Ltd on behalf of Sir Ganga Ram Hospital.
Title Page
Title: Potential biomarkers for diagnosing neonatal sepsis
Authors Ankita Sharmaa,c# a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
c
Department of Biotechnology, University Institute of Engineering & Technology, Maharshi
Dayanand University, Rohtak, India Anup Thakurb# b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Chitra Bhardwaja a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
Neelam Klerb b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Pankaj Gargb b
Department of Neonatology, Sir Ganga Ram Hospital, New Delhi, India
Manvender Singhc c
Department of Biotechnology, University Institute of Engineering & Technology, Maharshi
Dayanand University, Rohtak, India Sangeeta Choudhurya* a
Department of Research, Sir Ganga Ram Hospital, New Delhi, India
# Equal Contribution * Corresponding author: Email Id: [email protected] Address: Department of Research, Room no. 1258, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi-110060, INDIA
TITLE PAGE
Article Category: Review
Title: Potential biomarkers for diagnosing neonatal sepsis
Running Title: Biomarkers for neonatal sepsis
1
Abstract Vulnerable population of neonates has suboptimal immune function that results towards their susceptibility to infection and sepsis related mortality. Till date, neonatal innate immunity has not been completely understood, yet molecular characterization of this complex system has paved a path for identifying biomarkers that hold the potential to aid clinicians in diagnosing sepsis. The last decade has seen emergence of multiple putative biomarkers for early detection of neonatal sepsis. These include surface markers, acute phase reactants, cytokines and chemokines. Analysis of these components has led us to the conclusion that biological factors/molecules have the potential to identify the sepsis progression in neonates. Early onset sepsis can be detected by C-reactive protein (CRP), Procalcitonin (PCT) and soluble triggering receptor expressed on myeloid cells-1 (sTREM1). On the other hand, specific neutrophil and monocyte markers (Cluster of Differentiation 64 and Toll like receptors) along with the soluble urokinase-type plasminogen activator receptor can effectively detect late onset sepsis. These markers need to be validated by conducting multicenter clinical trials with target of enrolling more number of neonates so that statistically effective biomarkers could be identified.
2
Keywords Biomarkers Cytokines Inflammation Neonates Sepsis
3
Introduction Sepsis is a life threatening condition in neonates accounting for high rates of morbidity and mortality. Infants admitted in neonatal intensive care unit (NICU) are vulnerable to develop sepsis and its complications due to compromised host defense, poor mucosal barrier and exposure to invasive procedures such as central catheter insertion and mechanical ventilation.1 Blood culture is the gold standard to diagnose sepsis but there is usually a delay of 24-48 hours in availability of reports.2 Many conventional hematological tests such as total leukocyte count (TLC), absolute neutrophil count (ANC), immature/total neutrophil ratio(I:T), micro-erythrocyte sedimentation rate (ESR), platelet count, Procalcitonin (PCT) and C-reactive protein (CRP) have been used for early diagnosis of sepsis; however the positive predictive value (PPV) of these putative markers remains low.3 The current practice of starting empirical antibiotic therapy in all neonates showing sepsis like symptoms results in exposure to adverse drug effects, hospital acquired complications and emergence of resistant strains. On the other hand, delayed start of antimicrobial treatment in case of a fulminant sepsis may lead to major sequelae such as systemic inflammatory responses (SIRs) and multiple organ dysfunctions (MODs) that ultimately lead to death.3An early marker to identify and differentiate infants who need treatment from those who do not is essential. This review attempts to elucidate the efficacy of sepsis biomarkers to decipher the risk stratification in neonates. These have been categorized into two subdivisions namely “Biomarkers in practice” and “Potential biomarkers”.
• BIOMARKERS IN PRACTICE
4
In this section described herein are the meta-analysis including randomized controlled trials which have shown the effectiveness of mentioned biomarkers in clinical practice. C-reactive protein C-reactive protein (CRP) is synthesized in liver in response to inflammatory cytokines. Its level may increase up to a thousand-fold during infection. It has a short half-life of approximately 19 hours and falls rapidly after elimination of the microbial source. 4 In recent years, meta-analysis has been performed in large cohorts to evaluate the sensitivity and specificity of CRP in neonatal sepsis. In 2019, a meta-analysisby Brown et al, analyzed 20 studies enrolling 1615 infants to determine diagnostic accuracy of CRP in detecting late onset infection. The data showed the cut-off range of CRP was between 5mg/L to 10mg/L; for which median specificity was 0.74 and sensitivity was 0.62 (95% CI 0.50 - 0.73). 5 Liu et al (2019) in their meta-analysis of 1819 neonates corroborate a similar data with respect to accuracy of CRP test. Positive likelihood ratio (PLR), sensitivity, negative likelihood ratio (NLR), specificity, Diagnostic Odds-ratio (DOR) and AUC were 5.63 (95% CI=2.86 to 11.09), 0.70 (95% CI=0.66 to 0.75), 0.36 (95% CI=0.21 to 0.60), 0.89 (95% CI=0.87 to 0.91), 17.99 (95% CI=6.50 to 49.83), and 0.9 respectively. 6 Patel et al (2018) in their systemic review and meta-analysis suggested that CRPbased algorithms decrease the duration of antibiotic treatment in neonates by 1.45 days (95% CI −2.61 to –0.28) in two randomized controlled trials (RCTs), and by 1.15 days (95% CI −2.06 to –0.24) in two cohort studies, with no differences in mortality or infection relapse. 7 Procalcitonin Procalcitonin (PCT) is a precursor of the hormone calcitonin and is produced by para-follicular cells of thyroid and neuroendocrine cells of lungs and intestine. The levels of PCT rise in response to an inflammatory stimulus, especially of bacterial origin. PCT levels rise within 4
5
hours of exposure to bacterial toxins with a half-life of 25-30 hours.
8
A meta-analysis was
carried out to determine the diagnostic utility of PCT in early onset sepsis (EOS) and late onset sepsis (LOS). Data showed PCT to be 70-77% sensitive in EOS and 82-90% sensitive in LOS. Area under curve (AUC) for PCT was 0.87, pooled sensitivity (95% CI) and specificity were 81% (74-87%) and 79% (69-87%), respectively. 9 Based on the literature as mentioned above, PCT is a better marker than CRP. However, both the markers have low negative predictive value (NPV), hence compromising their diagnostic utility. This calls a search for new biomarkers for diagnosing neonatal sepsis with better diagnostic accuracy. POTENTIAL BIOMARKERS This section reviews newer biomarkers for sepsis. These have been sub-grouped into three categories; acute phase reactants (APR), cytokines and cell surface markers. • Acute phase reactants are inflammation markers whose levels either increase or decrease in patient’s sera during any acute tissue injury or inflammation. • Cell surface markers are proteins/carbohydrates attached to the plasma membrane of cell and expressed uniquely by each cell type. They play a major role in inter-cellular communication, immune regulation and recognition. • Cytokines are signaling proteins, usually less than 80 kDa in size, known to regulate a wide range of biological functions including hematopoiesis, innate and acquired immunity, inflammation and repair, division and proliferation through extracellular signaling. These are secreted by a diverse range of immune cells like macrophages, B lymphocytes, T lymphocytes, NK cells, and mast cells. • ACUTE PHASE REACTANTS
6
Serum amyloid A Serum amyloid A (SAA) is an apolipoprotein predominately produced by the liver. SAA is also secreted by various other cells such as endothelial cells, monocytes, and smooth muscle cells. It is known to be regulated by cytokines IL1, IL6 and TNFα.
10
SAA is released as a response to
infection or injury 11 and is altered by hepatic function and host nutritional status. 12Arnon et al, compared the diagnostic accuracy of SAA with CRP in early onset septic term neonates and found SAA to be a reliable screening marker in first 24 hours of onset of infection wherein its levels was inversely proportionate to neonatal mortality. SAA had better diagnostic accuracy than CRP at 0 hours in terms of sensitivity (96% vs 30%), specificity (95% vs 98%), PPV (85% vs 78%), NPV (99% vs 83%), positive likelihood ratio (PLR; 19 vs 12), and negative likelihood ratio (NLR; 0.05 vs 0.71) respewctively.13
Lipopolysaccharide-binding protein Lipopolysaccharide-binding protein (LPB) is primarily produced by hepatocytes, epithelial and muscle cells. It is a soluble pattern-recognition molecule crucial for interaction with endotoxin of gram-negative bacteria. LPB recognizes microbial-associated molecular patterns of bacteria to transport endotoxins to CD14 immune effector cells in response to infections.14 Binding to lipopolysaccharide (LPS) component of bacteria, it forms a complex that links to the host macrophage to initiate a response against infection. Level of LPB peaks early, within 6–8 hours, after an acute infection (LPS-induced) resulting in higher sensitivity and NPV for diagnosing EOS in comparison to CRP and PCT. Study by Pavcnik-Arnol et al showed that serum LBP levels were higher in infants with SIRS/sepsis as compared to infants with no infection. Serum LBP, IL-6 and PCT levels were estimated serially for 2 consecutive days. AUC for LBP was 0.89 on day 1.15 Similar trend was observed in another study by Pavcnik-Arnol et al, where LPB
7
had the best AUC (0.97) for infants less than 48hours of life on day 1 as compared to lipopolysaccharide( AUC= 0.77) and soluble CD14 (AUC=0.74). Authors found LPB to be the best indicator of sepsis in newborn infants.
15
Presently there exists an ambiguity on usefulness
of LBP since studies report that its levels are based on LPS induced infection and on the other hand less influenced by obstetrical events.16 •
CELL SURFACE BIOMARKERS
Cluster of Differentiation 64 Cluster of Differentiation 64 (CD64) is an Fc-receptor located in membrane glycoprotein that binds monomeric IgG-type antibodieswith high affinity.17 It is more commonly known as Fcgamma receptor 1 (FcγRI). Structurally CD64 is composed of a signal peptide that allows its transport to the surface membrane, three extracellular immunoglobulin domains of the C2-type used to bind antibody, a hydrophobic trans-membrane domain and a short cytoplasmic tail. 18 CD64 is expressed by macrophages, monocytes and weakly by naive neutrophils. Stimulation with cytokines such as IFNγ and Granulocyte colony stimulating factor (G-CSF) can induce higher expression CD64 on these cells. Neutrophil CD64 (nCD64) expression appear to be promising marker for diagnosing early and late onset sepsis. 19 Meta-analysis by Shi et al, shows that nCD64 has an overall pooled sensitivity, specificity, PLR, NLR and diagnostic odds ratio (DOR) of 0.77 (95 % CI: 0.74–0.79), 0.74 (95 % CI: 0.72–0.75), 3.58 (95 % CI: 2.85–4.49), 0.29 (95 % CI: 0.22–0.37) and 15.18 (95 % CI: 9.75–23.62), respectively with an AUC of 0.86.20 Ng et al, in their study assessed two neutrophil surface markers - CD11b, CD64 and two lymphocyte surface markers - CD25, CD45RO. The authors showed that CD64 had highest sensitivity (97%), specificity (90%), and NPV (99%) to diagnose early onset neonatal infection both at the onset and 24 hours later. The study also suggested that
8
combining CD64 with IL6 or CRP may improve the sensitivity and NPV which could enhance the ability to diagnose localized infection. 21 The limitation of the study was its small sample size and low PPV (50%) in diagnosing neonatal sepsis. Cluster of Differentiation 11b Cluster of differentiation 11b (CD11b) is a subunit of the b2 integrin adhesion molecule, weakly expressed by non-activated neutrophils.
22
A study by Qui et al, (2019) was conducted to
compare neutrophil CD11b expression between neonates and adults culture positive sepsis. The data showed a 2-4 fold increase in expression of nCD11b in both neonates as well as adults. Thus establishing its functional role in modulating inflammatory response. This systemic review analyzed nine studies, accounting for 843 neonates, showed that overall pooled sensitivity, specificity, PLR, NLR, DOR, post-test positive probability and post-test negative probability and AUC of CD11b were 0.82 (95% CI 0.71-0.90), 0.93 (95% CI 0.62-0.99), 11.51 (95% CI 1.55- 85.62), 0.19 (95% CI 0.10- 0.36), 59.50 (95% CI 4.65- 761.58), 74%, 5% and 0.90 respectively. These studies suggest that nCD11b is a promising biomarker to distinguish sepsis and infection. 23 Human Leukocyte Antigen- DR Isotype Human Leukocyte Antigen- DR Isotype (HLA-DR)is an MHC class II cell surface receptor, constituting a ligand for the T-cell receptor of T-helper cells. Monocytes strongly express HLADR and it is up-regulated in response to inflammation. Genel et al, observed a decreased cellsurface expression of HLA-DR on circulating monocytes from neonates with sepsis.4 This study showed a significant decrease in monocyte HLA-DR expression between non-survivor (n = 8) and survivor (n = 32) preterm and term (median gestation age 36 weeks) neonates with confirmed (n = 18) and clinical (n = 22) LOS (mean postnatal age 16.3 days). Infants with a
9
monocyte HLA-DR expression <30% had 30-fold higher risk of mortality.
24
Studies have
reported that significant decrease in monocyte HLA-DR expression in neonates correlates to confirmed or clinical sepsis.25 Toll like receptors Toll like receptors (TLRs) are vital trans-membrane receptors that initiate the innate immune response. TLRs are involved in native immunity recognition and the stimulation of antimicrobial mechanisms. The typical initial response to bacterial infection is recognition of pathogenassociated molecular patterns (PAMPs) via cell surface receptors. Altered neonatal TLR function contributes to increased susceptibility towards infection and sustained inflammatory condition in term and preterm neonates.
26
TLR 1, 2, 4, 5, and 6 chiefly recognize bacterial
products, whereas TLR-3, 7 and 8 are specific for viral detection. TLR 9 seems to be involved in both microbial and viral recognition. TLR-2 and TLR-4 mainly recognize components of Grampositive and Gram-negative bacteria, respectively. Studies have shown that Toll-like receptors, especially TLRs 2 and 4 to be associated with necrotizing enterocolitis, periventricular leukomalacia and sepsis.
27
A prospective cross-sectional study by Yunanto et al showed
significant increase in the levels of neutrophils (p= 0.021, 0.00) as well as neutrophil expression of TLR2 (p= 0.011, 0.003) and TLR4 (p= 0.044, 0.00) in saliva and blood respectively from newborns with sepsis risk factors compared to those of healthy newborns. 28 Soluble form of Cluster of differentiation 14 Cluster of differentiation 14 (CD14) is the receptor of lipopolysaccharide-lipopolysaccharide binding protein (LPS-LBP) complexes. CD14 has two forms: membrane-bound CD14 (mCD14) and soluble CD14 (sCD14). mCD14 has high affinity to LPS, and is mainly expressed on the cell surface of monocytes/macrophages and neutrophils; whereas sCD14 is found in plasma and
10
is also called as presepsin. It is produced by mCD14 fall-off or cell secretion. 29 sCD14 plays an important role in mediating the immune responses to LPS of CD14-negative cells such as endothelial cells and epithelial cells. The level of sCD14 increases significantly in neonates with sepsis, and this change is significantly related to the severity and prognosis of the disease. 30 According to a meta-analysis by Bellos et al, analyzing eleven studies and 783 enrolled neonates; the pooled sensitivity and specificity of serum sCD14 for prediction of neonatal sepsis was 0.91 (95% CI 0.87-0.93) and 0.91 (95% CI 0.88-0.94) respectively. The diagnostic odds ratio was 170.28 (95% CI 51.13-567.11) and AUC was 0.97 (SE 0.0117). 31 Studies report AUC for sCD14 to be 0.97 whereas the AUC of CRP and PCT is 0.86 and 0.78 respectively. sCD14 presents itself as a possible biomarker for sepsis. It can be measured via immunoassay and appears to possess better diagnostic capacity for sepsis than PCT.High levels of sCD14 correlates to increased sepsis related mortality rates, and its low false-negative rate indicates patient safety. The data herein shows sCD14 could be a potential biomarker of sepsis. 32 Soluble triggering receptor expressed on myeloid cells-1 Triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily and is expressed on the outer surface of neutrophils and monocytes (myeloid lineage) instrumental in perpetuating an early immune response.
33
Soluble triggering
receptor on myeloid cells -1 (sTREM-1) is a comparatively new biomarker explored for neonatal sepsis. It is a glycoprotein regulated by neutrophils released by phagocytes. Involved in the innate inflammatory response and sepsis, sTREM-1 may be useful as a biomarker of early onset sepsis. Its diagnostic ability to identify patients at greater risk of developing sepsisis significant. sTREM levels correlate significantly with white cell counts and I:T ratios in case of acute infections.
34
A study by Adly et. al, shows that at a cut-off of 310 pg/ml, sensitivity and
11
specificity were 100%, suggesting its role in early diagnosis of neonatal sepsis.
35
A meta-
analysis by Wu et al including eight studies with a total number of 667 neonates showed the estimated sensitivity and specificity of sTREM to be 0.95 [95% CI (0.81-0.99)] and 0.87 [95% CI (0.56-0.97)] respectively. The diagnostic odds ratio was 132.49 [95% CI (6.85-2560.70)]. The study also mentioned about the discrepancies in the cut-off used to calculate the sensitivity and specificity in the studies analyzed. 36 More studies are needed to determine the optimal cutoff value that may discriminate normal levels from those suggestive of neonatal sepsis Soluble urokinase-type plasminogen activator receptor The urokinase-type plasminogen activator receptor, is a 3-domain glycosylated protein (D1–D3) that binds to GPI anchor on cell surface to release the soluble form i.e. soluble urokinase-type plasminogen activator receptor (sUPAR). sUPAR has chemotactic properties and is expressed mostly on immunologically active cells and found in most of the biological fluids such as serum, plasma, cerebrospinal fluid and urine.
37
Systemic levels of sUPAR have been suggested as a
novel diagnostic marker of inflammation in term neonates. Saihanidou et al, reported that increased suPAR levels were significantly higher in the septic infants as compared to healthy controls, suggesting its role in diagnosing sepsis.38 The study also found a correlation between circulating sUPAR levels and systemic inflammation suggesting its association with immune activation. More studies are required to validate the role of sUPAR in sepsis. •
CHEMOKINES & CYTOKINES
Chemokines are cytokines that have ability to direct white blood cell migration towards the site of infection. All cytokines are cellular signaling proteins that play a crucial role in modulating the host immunological response to infection. 39 Leukocyte immune regulation and trafficking into areas of tissue infection and injury is primarily controlled by chemokines and cytokines.
12
Cytokines are classified into the subsets of pro-inflammatory (Th1-type, stimulatory), antiinflammatory (Th2-type, inhibitory) or multifunction depending on the final balance of their effects on the immune system (Table 1). Pro-inflammatory cytokines include interferon-gamma (IFNγ), Interleukin (IL)-2, IL-6, IL-8, IL-12, and IL-17. Multiple functional inflammatory cytokines include IL-1β, IL-3, monocyte chemoattractant protein (MCP-1), and growth factors, IL-3, G-CSF. Anti-inflammatory cytokines are IL-4, IL-10, tumor necrosis factor soluble receptor, IL-1 receptor alpha, and transforming growth factor beta 2 (TGF- β2). The evolution of disease and clinical symptoms during neonatal sepsis is dependent upon a complex and delicate balance between the pro-inflammatory, anti-inflammatory, and multiple function cytokines based on their final effect on the immune system. 21,40 Among the group of pro-inflammatory cytokines, IL-6 has been widely studied for its potential use as a biomarker of early neonatal sepsis.41,42,43 During the onset of an infection, B and T lymphocytes are stimulated to produce IL-6 cytokine, which in turn induces hepatocyte production of acute phase reactants such as CRP.15,43 Although IL-6 has a higher sensitivity(90%) as compared to CRP as an early phase biomarker with a negative predictive value of 91%
44,45,46
but its limitation is its short half-life. Usefulness of IL-6 as a sensitive
biomarker was studied by Hu et al in a meta-analysis of 33 studies that included 3135 neonates diagnosed with sepsis. Their study showed a sensitivity of 0.79 (95% CI 0.76-0.81), specificity 0.83 (95% CI 0.81-0.85), AUC was 0.89 and Q (*) index was 0.83 suggesting IL-6 can be used as a diagnostic biomarker. 47 IL-8, another pro-inflammatory cytokine is a probable biomarker for neonatal sepsis with a reported sensitivity of 90% and specificity between 75%–100%. IL-8 regulates leukocyte migration and activation; and has been extensively investigated in neonatal infections.
48
Eight
13
studies (548 neonates) analyzed in a meta-analysis by Zhou et. al, showed IL-8 had a moderate accuracy (AUC=0.89) for the diagnosis of neonatal sepsis. The pooled sensitivity and specificity were 0.78 and 0.84, respectively. The pooled DOR and AUC was 21.64 and 0.89 (Q*=0.8215), respectively.
49
In a study by Ng et al, serum IL-8 concentrations increased within 2–4 hours of
an infection, and decline in 4 hours. The study demonstrated that the use of IL-8 in conjunction with CRP as a biomarker of sepsis enhanced its diagnostic utility.50 The inflammatory process is highly regulated by anti-inflammatory mediators such as IL-10 and TGF-β. These cytokines prevent a severe pro-inflammatory response in reaction to pathogen invasion.
51
In premature infants, the ability to mount an aggressive anti-inflammatory response
is limited, leading to increased susceptibility to target organ injury with excessive SIRS. Thus, serum levels of anti-inflammatory cytokines is an important aspect that needs to be explored as markers of prognostication and survival.50,51 Boskabadi et al., recently reported that concentration above 14 pg/mL of IL-10 showed a higher sensitivity/ NPV (77.7% / 73.6%), with a moderate specificity/PPV (87.8% / 90%).52 He et al (2017) compared multiple cytokines to assess the predictive plasma biomarkers of EOS. Amongst IL-6, IL-8, IL-27, TNF-α, HSP 70, MMP-8, granzyme B, MIP-1α, MIP-1β, and CRP; the authors found IL-27 and PCT to be independent predictors of EOS.53 Other cytokines like RANTES, IL-17A, IL12, IL23p40 play a role in protective host immunity. Though the levels of these cytokines are observed to be inversely proportionate to severity of sepsis in adults but in pre-term neonates, the levels are under the detectable limits. 54 Implementation of anti- and pro-inflammatory ratio analysis emphasizes the importance of host immune balance in disease development and progression, providing an avenue to guide therapy
14
in prediction of morbidity and mortality. Combining the cytokines to study their diagnostic utility may be fruitful area of exploration in the early identification of neonatal sepsis. Limitations Having a surrogate biomarker for early and accurate diagnosis of neonatal sepsis would be an important addition to the conventional clinical practice. Yet the studies so far show variable results with respect to diagnostic accuracy (Table 2). The biomarkers reviewed in this article have different sensitivities and specificities for both EOS and LOS. Some of the mentioned biomarkers have a short half-life, so the time point at which the sample should be analyzed is very critical. Non-specificity of some of the markers for sepsis has been identified in conditions like inflammation, infection and stress. Studies in the literature reports small sample size, therefore these upcoming newer biomarkers should be validated in clinical trials with larger cohorts. Conclusion The conventional hematological and microbiological techniques that are routinely used to diagnose neonatal sepsis remain variable due to associated limitations i.e low accuracy and NPV and more time consuming. There is need for an ideal biomarker/s that provides early, specific, and reliable identification of the neonates at higher risk of infection. Potential use of technological developments and ever evolving understanding of biomarker’s strengths and limitations has led to conclusive interpretations on their usage in the clinical systems. In addition, advanced understanding of neonatal immune system and its response to infection has led to identification of potential biomarkers that may translate to early diagnosis and better prognosis of neonatal sepsis in the future.
15
Despite many promising biomarker candidates, to date no single biomarker, combination of biomarkers, or score system can exclusively be considered to accurately diagnose early neonatal sepsis. Further studies are needed to explore, test, analyze and validate the reviewed biomarkers for their diagnostic and prognostic value in neonatal sepsis. Identifying biomarkers that are specific and sensitive will aid in enhancing diagnostic accuracy and help in early identification of critical cases. These biomarkers if used as tests for neonatal sepsis can assist the clinicians in better understanding of neonatal host immune responses leading to quick clinical decisions and appropriate antibiotic usage. The authors herein suggest that there is a current need for biomarker or a set of biomarkers that correlates with neonatal sepsis at its various stage of progression. This review brings out the effectiveness of specific biomarkers as indicators of sepsis progression. For identifying EOS; CRP, PCT and sTREM1 are good biomarkers. CD64, sUPAR and TLRs are sensitive markers for LOS. Biomarkers such as sCD14, SAA and HLA-DR are good predictors of mortality. The review also concludes that cytokines such as IL-2/4/6/8/10, TNFα and TGFβ have not been able to show any clinical significance in terms of diagnostic or prognostic utility. Thus, a good biomarker should not only risk stratify in predicting outcome but also be classified based on its detection ability for disease severity and mortality.
16
Conflict of Interest The authors have no conflict of interest relevant for this review article.
17
Acknowledgement The authors acknowledge the grant provided by Research and Development programme (RDP) by Sir Ganga Ram Hospital (SGRH). SA is a recipient of DST-INSPIRE fellowship (IF140078).
18
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25
Table 1: Cytokine classification Pro-Inflammatory
Anti-Inflammatory
Multi-functional
Th1 Type, Stimulatory
Th2 Type, Inhibitory
IFNγ
IL-4, IL-10, IL-5, IL-6, IL-9,IL-13, IL-3, IL1α/β IL-25
TNFα
IL-1RA, IL-33, IL-31
MCP1, TNF α,β
IL-2, IL-6, IL-8,IL-10, IL-27
TNFsr
Growth factors: G-CSF
IL-12, IL-17, IL-18, IL-27
TGFβ2
IL-8
(Abbreviations:G-CSF: Granulocyte-colony stimulating factor,IFNᵧ: Interferon gamma, IL: Interleukin, MCP1: Monocyte chemo-attractant protein 1, TGFβ2: Transforming growth factor beta 2, TNF: Tumor necrosis factor)
26
Table 2: Promising biomarkers for neonatal sepsis Biomarker
n
Cut-off
AUC
Sensitivity Specificity PPV
NPV
(%)
(%)
(%)
(%)
Ref
CRP
1615 4-10 mg/l
0.68
95.7
88.9
78
98
[5]
CD11b
843
0.63
75
100
100
86
[22]
CD64
3478 2.19-3.62
0.73-0.83
75-78
59-77
29-54
81-96
[19]
IL-6
3135 10-150 pg/ml
0.78-0.85
75-87
50-82
92
52
[46]
IL-8
548
0.6-0.72
90
75-100
78
88
[48]
PCT
1915 0.5-5.75 ng/ml 0.8-0.9
93.1
53
79.4
80
[8]
SAA
84
>6.8mg/dl
0.71-0.88
44.7-76.4
100
100
38-58
[12]
sCD14
783
1483 pg/ml
0.97
91
91
91
91
[31]
sTREM-1
667
310 pg/ml
0.8
97
87
68
85
[35]
sUPAR
60
5-11ng/ml
0.6-0.72
96
60
79
90
[37]
TLR 2
60
0.81
0.68
63
80
80
60
[27]
TLR4
60
0.68
0.72
75
82
86
75
[27]
290
60-300 pg/ml
(Abbreviations: CRP: C-reactive protein, CD11b: Cluster of differentiation 11b, CD64: Cluster of differentiation 64, IL-6: Interleukin-6, IL-8: Interleukin-8, PCT: Procalcitonin, SAA: Serum amyloid A, sCD14: soluble Cluster of differentiation 14, sTREM-1: Soluble triggering receptor
27
expressed on myeloid cells-1, sUPAR: Solubleurokinase-type plasminogen activator receptor, TLR2: Toll like receptor 2, TLR4: Toll like receptor 4)
28
Table 1: Cytokine classification Pro-Inflammatory
Anti-Inflammatory
Th1 Type, Stimulatory
Th2 Type, Inhibitory
IFNγ
Multi-functional
IL-4, IL-10, IL-5, IL-6, IL-9,IL-13, IL-3, IL1α/β IL-25
TNFα
IL-1RA, IL-33, IL-31
IL-2, IL-6, IL-8,IL-10, IL- TNFsr
MCP1, TNF α,β Growth factors: G-CSF
27 IL-12, IL-17, IL-18, IL-27
TGFβ2
IL-8
(Abbreviations: G-CSF: Granulocyte-colony stimulating factor, IFNᵧ: Interferon gamma, IL: Interleukin, MCP1: Monocyte chemo-attractant protein 1, TGFβ2: Transforming growth factor beta 2, TNF: Tumor necrosis factor)
1
Table 2: Promising biomarkers for neonatal sepsis Biomarker
Cut-off
AUC
Sensitivity
Specificity
PPV
NPV
(%)
(%)
(%)
(%)
[Ref] CRP [6]
4-10 mg/l
0.68
95.7
88.9
78
98
CD11b [29]
290
0.63
75
100
100
86
CD64 [26]
2.19-3.62
0.73-0.83
75-78
59-77
29-54
81-96
HLA-DR [31]
70
0.6-0.72
65-72
85
85
60-78
IL-6 [54]
10-150 pg/ml
0.78-0.85
75-87
50-82
92
52
IL-8 [56]
60-300 pg/ml
0.6-0.72
90
75-100
78
88
PCT [10]
0.5-5.75 ng/ml
0.8-0.9
93.1
53
79.4
80
SAA [17]
>6.8mg/dl
0.71-0.88
44.7-76.4
100
100
38-58
sCD14 [38]
1483 pg/ml
0.97
91
91
91
91
sTREM-1[40]
310 pg/ml
0.8
97
87
68
85
sUPAR [44]
5-11ng/ml
0.6-0.72
96
60
79
90
TLR 2 [32]
0.81
0.68
63
80
80
60
TLR4 [32]
0.68
0.72
75
82
86
75
TNFα [47]
12-60 pg/ml
0.58-0.62
52-68
82
85
60-70
(Abbreviations: CRP: C-reactive protein, CD11b: Cluster of differentiation 11b, CD64: Cluster of differentiation 64, HLA-DR: Human Leukocyte Antigen- DR Isotype, IL-6: Interleukin-6, IL8: Interleukin-8, PCT: Procalcitonin, SAA: Serum amyloid A, sCD14: soluble Cluster of differentiation 14, sTREM-1: Soluble triggering receptor expressed on myeloid cells-1, sUPAR:
2
Soluble urokinase-type plasminogen activator receptor, TLR2: Toll like receptor 2, TLR4: Toll like receptor 4, TNFα : Tumor necrosis factor α)
3