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LITHUANIAN UNIVERISTY OF HEALTH SCIENCES MEDICAL ACADEMY
FACULTY OF MEDICINE DEPARTMENT OF NEONATOLOGY
Modern Biomarkers those are effective in diagnosing neonatal
sepsis (Early and Late)
Author: Christina Joseph Supervisors: Asist. Kristina Štuikienė Co-supervisor: Prof. Dr. Rasa Tameliene
Kaunas 2020
2 Table of Contents: 1. SUMMARY ...3 2. ACKNOWLEGEMENTS ...4 3. CONFLICTS OF INTEREST...4 4. ABBREVIATIONS ...5 5. INTRODUCTION...6
6. AIM AND OBJECTIVES ...8
7. LITERATURE REVIEW...9
7.1 Incidence of neonatal sepsis: ...9
7.1 Aetiology ... 10 7.2 Clinical features ... 12 7.3 Current diagnostics ... 12 8 METHODOLOGY ... 14 9 RESULTS ... 16 10 DISSCUSIONS ... 19 10.1 C - reactive protein (CRP): ... 19 10.2 Procalcitonin (PCT): ... 20
10.3 Serum Amyloid A (SAA): ... 22
10.4 Chemokines and Cytokines ... 22
10.41 Interleukin-6 (IL-6): ... 22
10.42 Interleukin-10 (IL-10): ... 23
10.43 Cluster of differentiation (CD64): ... 24
10.44 Hepcidin: ... 25
10.45 Presepsin (Soluble CD14 Subtype): ... 26
10.5 Acute phase reactants vs Chemokines and Cytokines: ... 27
10.6 Molecular Biomarkers:... 28 10.7 Novel Biomarkers: ... 29 11 STUDY LIMITATIONS ... 31 12 CONCLUSION ... 32 13 PRACTICLE RECOMMENDATIONS ... 33 14 REFERENCES ... 34
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1. SUMMARY
Author: Christina Joseph
Title: Modern Biomarkers those are effective in diagnosing neonatal sepsis (Early and Late): A Review of
Literature.
Aim: The aim of this literature review is to identify biomarkers for diagnosing neonatal sepsis based on
available literature
Objectives: The goal of this literature review was to:
1. To review the aetiology, clinical features and current diagnosis of neonatal sepsis.
2. To gather a list of papers that look into neonatal biomarkers with potential value now and for the future. 3. To compare acute phase reactants with chemokines /cytokines and summarise the collected information on biomarkers of neonatal sepsis for future consideration.
Methods: Literature was identified by searching PubMed with combination of the following terms:
“Neonatal sepsis” OR “Early onset neonatal sepsis” OR “Late onset neonatal sepsis” OR “Sepsis in Neonates” AND “biomarkers” OR “markers” OR “interleukins” OR “CRP” OR “chemokines”. Articles that were in English and within ten years of the search date were manually sorted according to inclusion and exclusion criteria.
Results: Initial search returned n=573. After activating filters, n=333 were identified of which n=40 were
included for the literature review. The markers that were investigated were, C-reactive protein (CRP), Procalcitonin (PCT), Serum Amyloid A (SAA), Interleukin (IL-6), Interleukin (IL-10), Cluster of differentiation (CD64), Presepsin (P-SEP), Hepcidin, Molecular biomarkers and Novel biomarkers. Information on these biomarkers were summarised in a table, along with a graphical comparison between acute phase reactants and chemokines/cytokines.
Conclusion:
1. The research showed that the etiologic agent of neonatal sepsis can vary depending on whether it is early- onset neonatal sepsis or late-onset neonatal sepsis.
2. In terms of the main clinical features of neonatal sepsis, it seems that respiratory distress, neonatal jaundice along with feeding intolerance seems to predominate.
3. Blood culture along with CBC and CRP being the gold standard, but with errors such as low volume of blood and false negatives making it somewhat unreliable.
4. Out of the three acute phase reactants serum amyloid A shows the most variation in values, having the highest specificity and lowest sensitivity. Whereas out of the chemokines/cytokines P-SEP had the greatest specificity and highest PPV.
5. Because of the complexity of the sepsis response, it is very difficult to determine a single effective biomarker that can be used in clinical practice. Each biomarker has a different cut-off value along with sensitivity and specificity, due to varying sample sizes, detection methods used and the type of analysis. Therefore a combination of several sepsis biomarkers may be more effective.
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1. ACKNOWLEGEMENTS
I want to extend my sincere gratitude to my supervisor Asist. Kristina Štuikienė for her support.
2. CONFLICTS OF INTEREST
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3. ABBREVIATIONS
1H-NMR: Proton nuclear magnetic resonance LBP:Lipopolysaccharide binding protein
Aa: Amino Acid Lf: Lactoferrin
A-SAA: Acute-phase serum amyloid A LOS: Late onset sepsis
AUC: Area under the curve LPS: Lipopolysaccharide
BC: Blood culture nCD64: Neutrophil CD64
B-cells: B lymphocytes NICU: Neonatal intensive care unit
CALC-1 gene : Calcitonin-1 gene NK-cells: Natural killer cells
CBC: Complete blood count NPV: Negative predictive value
CD14:Cluster of differentiation 14 NS: Neonatal sepsis
CD64:Cluster of differentiation 64 PCA:Principal component analysis
CONS: Coagulase-negative staphylococci PCDHB-gene
CRP : C-reactive protein PCR: Polymerase chain reaction
C-SAA: Constitutive serum amyloid A PCT: Procalcitonin
CSF: Cerebrospinal fluid PN: Parenteral nutrition
DNA: Deoxyribonucleic Acid PPV: Positive predictive value
ELBW: Extremely low birth weight P-SEP: Presepsin
EOI: Early onset infection rRNA: Ribosomal Ribonucleic Acid
EOS: Early onset sepsis SAA: Serum amyloid A
FDR: False discovery rate sCD14-ST: Presepsin
GBS: Group B streptococcus Th-cells: T-helper cells
GC-MS: Gas-chromatography-mass spectrometry THBA: 2,3,4-trihydroxybutyric acid
gDNA: Genomic DNA TLRs: Toll-like receptors
hsCRP: High sensitivity C-reactive protein TNF: Tumour necrotic factor
IL-1:Interleukin-1 VLBW: Very low birth weight
IL-10: Interleukin-10 WBC: White blood count
IL-12: Interleukin-12 WHO: World health organisation
IL-35: Interleukin-35
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4. INTRODUCTION
The term neonatal sepsis is used to designate a systemic condition of bacterial, viral, or fungal (yeast) origin that is associated with haemodynamic changes and other clinical manifestations and results in substantial morbidity and mortality [7] . World Health Organisation (WHO) has recently recommended that sepsis should be defined as “life threatening organ dysfunction caused by a dysregulated host response to infection” and septic shock as “a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone” [1].
Analysing the onset, we can distinguish "early onset sepsis" when microbiological cultures positive for external pathogens come from new-borns during the first 3 days of life. "Late onset sepsis" when microbiological cultures positive for external pathogens come from new-borns after the first 3 days from delivery (postnatal acquisition) [2]. Additionally a more precise definition of early-onset sepsis is variable from less than or equal to 3 days (American Academy of Paediatrics definition) to less than or equal to 7 days (Centres for Disease Control definition based on epidemiology studies) [94].Subsequently the causative agents and risk factors vary according to the type of sepsis; along with the detection of the type of biomarkers in diagnosis.
According to epidemiological data; in the year 2010, of 7·6 million deaths in children younger than 5 years, 64·0% (4·879 million) were attributable to infectious causes and 40·3% (3·072 million) occurred in neonates. Sepsis or meningitis (5·2%), along with intrapartum complication and preterm birth, were the leading causes of neonatal death [5]. Despite major advances in neonatal care and increasing research, in developed countries, four of every ten infants with sepsis die or experience major disability [6].
Neonatal sepsis remains one of the leading causes of neonatal morbidity and mortality (mainly among low birth weight neonates) [3]; although measures such as intrapartum antibiotic prophylaxis have been put in place, the incidence still remains high [4]. In a report by WHO, listed neonatal sepsis as a key health-care priority for the coming decade [1] [2]. The report by WHO recognised neonatal sepsis as a global issue with a rapid rise in incidence; predicted to affect both high and low income countries. Therefore early detection and management is of importance in reduction of neonatal mortality [1].
7 For over a decade the gold standard for diagnosing neonatal sepsis includes CBC, culture and clinical features, along with CRP [9]. However, these remain unreliable or insensitive for reasons such as, intrapartum antimicrobial administration and low blood sample volume [3]. Accurate diagnosis of neonatal sepsis is challenging, at this moment, no single marker has a significant advantage over the others in diagnosing neonatal sepsis. Certain diagnostics utilities such as sensitivity, specificity, positive predictive value and negative predictive value, determine the usefulness of a clinical test [33]. For a biomarker to be ideal, it has to fit certain characteristics. An ideal biomarker should rise rapidly and should have a good diagnostic window (table 1) [94]. This review aims to provide an overview of old and novel diagnostic biomarkers in neonatal sepsis and explore their usefulness in diagnosing neonatal sepsis.
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5. AIM AND OBJECTIVES
The aim of this literature review is to identify biomarkers for diagnosing neonatal sepsis based on available literature.
Objectives:
1. To review the aetiology, clinical features and current diagnosis of neonatal sepsis.
2. To gather a list of papers that look into neonatal biomarkers with potential value now and for the future.
3. To compare acute phase reactants with chemokines /cytokines and summarise the collected information on biomarkersof neonatal sepsis for future consideration.
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6. LITERATURE REVIEW 7.1 Incidence of neonatal sepsis:
Neonatal sepsis remains a global issue, despite advances in perinatal care. Early onset neonatal sepsis (EOS) is defined as an infection that occurs in the first 72hrs after life and mainly involves vertical transmission; whereas Late onset neonatal sepsis (LOS), occurs after 72hrs after life and usually involves horizontal transmission [98]. Although data on exact estimate of global burden is not available, published data suggests that 3 million new-borns and 1.2 million children suffer from sepsis [8]; with an estimated toll of over 400 000 annual deaths worldwide [9]. Early neonatal sepsis causes about 8% of all neonatal deaths, but the proportion of late neonatal deaths due to sepsis is four times higher [11]. The incidence of neonatal sepsis varies in different geographic regions also, which shows a differences in resources, maternal and infant risk factors, and prevention strategies, but sepsis remains one of the most common neonatal diseases even in high-income countries [7]. Most recently the WHO called for action in reducing the global burden of neonatal sepsis by the year 2030; at the forefront of this is in considering the importance of effective prevention, identification, and management of maternal and neonatal sepsis in reducing maternal and new-born deaths [10]. Therefore, research into detection of neonatal sepsis at this time is one of critical importance and as part of this many researches have being carried out for the ideal biomarker. As previously discussed there are certain aspects of a biomarker, which determines its usefulness. A review conducted by Mally et al concluded that an ideal biomarker should rise rapidly and should have a good diagnostic window. This review explored sensitivity, specificity, predictive value and more [94].
Table 1: Characteristics of an ideal biomarker for neonatal sepsis Adapted from Sharma D et al [94].
Discriminate aetiology of sepsis
Able to identify causes of sepsis such as viral, bacterial, or fungal
High sensitivity The ability to detect sepsis in infants where sepsis is present (approaching 100%)
High specificity The ability to rule out sepsis in infants where sepsis is absent (85%)
High predictive value
Likelihood that the test accurately predicts presence or absence of sepsis (approaching 100%)
Rapid timely results Necessary in early sepsis diagnosis generally in ,60 minutes
Reliable and precise Informs in early diagnosis, guides treatment decisions or prognosis
Readily available standardized method
Technology can be available from commonly obtainable small volume sample and expanded routinely among care institutions
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7.1 Aetiology
Neonatal sepsis is associated with infection with either bacterial, viral or fungal agents. The etiologic agent can also vary depending on whether it is early- onset neonatal sepsis or late-onset neonatal sepsis. A study conducted on EOS (containing 18,778 neonates) in Lithuania by prof.Rasa Tameliene et al found that “in 4 neonates, microbiologically confirmed sepsis was caused by Group B streptococcus (GBS), and in 2 neonates by L.monocytogenes, while E. coli, coagulase-negative Staphylococcus, S.
epidermidis, and Corynebacterium spp. caused EOS in 1 neonate each” [96]. Additionally, an earlier
study undertaken in the Lithuanian university of health sciences hospital, performed by prof.Rasa Tameliene et al, looked into the prevalence of E.coli in women and children, a total of 193 women(out of 970) had vaginal E. coli colonization; the prevalence of colonization was 19.9%. E. coli colonization was documented in 119 (14.4%) of the 827 neonates [97]; showing E.coli as one of the major players in the cause of EOS.
This was similar to findings in others studied, Group B streptococcus (GBS) was the most common etiologic agent, while Escherichia coli was the most common cause of mortality. These organisms are commonly colonizers of the maternal genitourinary tract, which leads to contamination of the amniotic fluid, placenta, cervix, or vaginal canal [12].A study done in USA by Barbara J. Stoll MD. Among 396 586 LBs (live births) 389 of these neonates developed EOS. GBS (43%) and E coli (29%) were most frequently isolated. Most infants with GBS were term (73%); 81% with E coli were preterm [13]. Suggesting term neonates are more likely to be infected with GBS and preterm with E coli.
Figure 1: Shows the causative agents of early-onset neonatal sepsis by prof.Rasa Tameliene et al (Lithuania) [96].
11 Risk factors for LOS include immaturity, intravascular catheters, mechanical ventilation, and prolonged parenteral nutrition (PN) [14]. The etiologic pathogen in LOS vary compared to EOS,
Coagulase-negative staphylococci (CONS) have emerged as the predominant pathogens of LOS, accounting for
53.2%–77.9% of LOS in industrialised countries and 35.5%–47.4% in some developing regions have been reported [ 15-18]. A study by Lim et al, reported the vast majority of infections (60.7%) were caused by Gram-positive organisms [G (+)], and overall Coagulase-negative staphylococci (CoNS) (52.5%) out of 158 episodes of sepsis [20]. Specifically Staphylococcus epidermidis, was a predominating pathogen for LOS in very low birth weight (VLBW) infants [19]. Recent advances in molecular microbiology show that S. epidermidis is a bacterial species equipped with remarkable genetic flexibility, and can employ a variety of evasion mechanisms to become adapted to the changing environment [19].
Figure 2: Shows the incidence of pathogens, causing EOS by Barbara J. Stoll et al [13].
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7.2 Clinical features
Clinical symptoms can vary in new-borns according to gestational age as well as the type of onset (EOS or LOS). A study carried out by Xu et al, on 341 culture positive neonates out of which 161 was EOS cases and 180 was LOS cases. This study showed that, respiratory distress (39.13%), neonatal jaundice (70.19%), hypoglycaemia (12.42%), pulmonary hypertension (14.19%) and neonatal asphyxia (22.98%) were the most common clinical manifestations of EOS. In contrast, fever (40.56%), feeding intolerance (49.44%) and abdominal distension (20.56%) occurred most commonly in LOS group [21]. In EOS preterm neonates it was found that apnea, and/or bradycardia, and/or cyanosis were the most common presenting features of sepsis, followed by poor activity and increased respiratory effort [20]. In EOS term neonates the most infants presents with respiratory distress [23].
7.3 Current diagnostics
Diagnosing neonatal sepsis is either culture dependent or independent. When talking about culture dependent gold standard diagnostics; it means confirmation by isolating the causative agent from a normally sterile body site; which includes blood, CSF, urine, and pleural, joint, and peritoneal fluids [29]. Although this is a common practise, a study done by Connell TG et al, concluded that a negative blood culture result is almost inevitable for a large proportion of blood cultures because of the submission of an inadequate volume of blood [30], making use of culture for routine diagnostics, highly unreliable. There are 2 types of errors that can occur, type 1 error, is distinguishing true pathogen from contaminant. Type 2 error refers to when an insufficient volume of blood is obtained for culture, the blood culture may be falsely negative [27]. Even though a culture is considered as a gold standard for diagnosing neonatal sepsis, culture negative sepsis can be a reality; this is to say a culture negative sepsis can be present even in the presence of shock [28]
For this reason currently diagnosis of neonatal sepsis relies on biomarkers, which include complete blood count with WBC differentials, as well as acute phase reactants. Although CBC may aid diagnosis, there are many draw backs. For example there is evidence that in ELBW neonates, low neutrophil counts was at a rate five times that reported in the general NICU population [31]. Many other factors can affect the WBC and differential counts including maternal hypertension, the method of delivery, the infant’s sex, age in hours, and the method of blood sampling [24]. A study conducted by Hornik et al. on 166,092 neonates with suspected EOS with cultures admitted to 293 neonatal intensive care units; concluded that Low white blood cell count, absolute neutrophil count and high immature-to-total neutrophil ratio were
13 associated with increasing odds of infection, but no complete blood cell count-derived index possesses the sensitivity to rule out reliably EOS in neonates [25]In addition to this a further study of LOS and CBC was carried out by Hornik et al, which found no complete blood cell count index possessed adequate sensitivity to reliably rule out late-onset sepsis [26].
A more widely researched and used biomarker and acute phase reactant is CRP, along with CBC. CRP is an acute phase protein and is a component of first line of innate host defence [32]. Although its sensitivity has come into question, mainly due to the fact that its value is affected by non-infectious maternal and perinatal factors, and is hampered by a physiological 3-day increase, resulting in a low sensitivity to detect sepsis at an early stage [33]. Despite this it is very much the forefront of diagnosing neonatal sepsis.
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8 METHODOLOGY Search strategy:
Literature was identified by searching PubMed with combination of the following terms: “Neonatal sepsis” OR “Early onset neonatal sepsis” OR “Late onset neonatal sepsis” OR “Sepsis in Neonates” AND “Biomarkers” OR “Markers” OR “Interleukins” OR “CRP” OR “Chemokines” (Table 2).
After this further, filters were applied (using the advanced filter section on PubMed): Date range: Within ten years (until 23rd of January, 2020), Species: Human, Language: English, Population type: Neonates (Table 3).
Then, the resulting papers were included or excluded (inclusion/exclusion criteria) as shown in Table 4 by manually reading the title and abstract. This is to say that articles which were free texts, with a study population, with no other concomitant diseases, had bacterial and viral aetiology sepsis, infants who were under 28 days old and clinical studies on biomarkers, were included as part of this literature review. Additionally, any articles that studied the adult population, which were literature reviews, met analysis, systematic reviews and abstracts only with no external links were excluded and were not used as part of the literature review.
- Neonatal sepsis - Sepsis in neonates
- Early onset neonatal sepsis - Late onset neonatal sepsis
AND - Markers - CRP - Biomarkers - Chemokines - Interleukins - Molecular markers - Proteomics - Metabolics
These terms where used alone or in combination with the below in the initial search.
15 Table 2: Filters that where applied after initial search
Date range: Within ten years (until 23rd of January, 2020)
Species: Human Language: English Population type: Neonates
.
Inclusion Exclusion
Free text Adults
No concomitant diseases Literature reviews Bacterial and viral aetiology sepsis Met analysis Child under 28 days old Systematic reviews
Clinical studies on biomarkers Abstracts only with no external link Table 3: Criteria for inclusion and exclusion
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9 RESULTS
The search was done by combining following terms: “Neonatal sepsis”, “Sepsis in neonates”, “Early onset neonatal sepsis”, “Late onset neonatal sepsis” AND “Markers”, “CRP”, “Biomarkers”, “Chemokines”, “Interleukins”, “Molecular markers”, “Proteomics”, “ Metabolic”. This returned 573 records which were narrowed to 333 after activating the filters, Date range: Within ten years (until 23rd of January, 2020), Species: Human, Language: English, Population type: Neonates on PubMed.
Afterwards, the title and abstracts were manually sorted and matched according to the inclusion and exclusion criteria (Table 4), resulting in 40 articles; which means 293 articles did not fit the inclusion and exclusion criteria.
Information on sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) from each article on the biomarkers where extracted to form a table of overview. The table of values where formed by calculating the Mean for each biomarker according to the collected number of literature for each potential biomarker.
Finally two bar charts (one for acute phase reactants and one for chemokines and cytokines) were formulated, using the calculated Mean values, to draw comparison between the two groups of biomarkers.
Identification
Screening
Eligibility
Inclusion
Articles found after initial database search
n=573
The articles that where screened after application of filter. n=333 Records excluded: . Adults . Literature reviews . Met analysis . Systematic reviews . Abstracts only with
no external link n= 293 Studies that where included into
analysis n= 40
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Table 4: The Mean values for the biomarkers of neonatal sepsis
Biomarker Sensitivity (%) Specificity (%) Negative predictive value (NPV) (%) Positive predictive value (PPV) (%)
Acute phase reactants
CRP 75.28 78.1 75.18 48.30
Procalcitonin 77.92 79.20 74.63 63.97
Serum Amyloid A 23.5 92.8 66.5 66.6 Cytokines and Chemokines
IL-6 83.73 89.73 98.7 42.1 IL-10 89.75 72.88 86 80 CD64 85.02 85.03 91.58 71.55 Hepcidin 90 91 97 75 Soluble CD14 subtype (presepsin) 88.52 94.16 89.25 92.35 Molecular Biomarkers
Multiplex PCR Sensitivity, Specificity, NPV and PPV where not identifiable for PC. However PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%).
DNA ELISA(DNA Methylation)
Highly significant difference in percentage of gDNA methylated was found between the cases and controls (Cases: 2.4 ± 0.39; Controls: 2.07 ± 0.35; P < 0.0001). Concluding that although the global DNA methylation was not a highly sensitive diagnostic method, this study showed that this has a potential for the future in terms of use as a neonatal sepsis biomarker
16S rRNA Gene
Sequencing(PCR form)
The number of positive cultures and positive PCR results were 95 (13.5%) and 123 (17.4%), respectively. Compared with blood culture, the diagnosis of bacterial sepsis by PCR revealed a 100.0% sensitivity, 95.4% specificity, 77.2% positive predictive value, and 100.0% negative predictive value.
Novel Biomarkers
Urine metabolomics Sixteen septic neonates and 16 controls were studied, the research showed that analysis of the spectral data by multivariate analysis showed that, even by unsupervised Principal Component Analysis (PCA), septic infants (confirmed and possible) could be discriminated from non-septic ones (controls) at the time point of sepsis suspicion.
IL-35 The cut-off concentration for IL-35 was 31.7 pg/ml, with a sensitivity of 78.48%, a specificity of 66.67%, PPV of 70.46% and NPV of 75.36%
Melatonin Melatonin concentration was significantly increased in sepsis group when compared to control group (27.2 ± 3.3 versus 11.4 ± 3.2, p = 0.001) and had a sensitivity of 93.5% and specificity 85.6%
Lactoferrin A total of 15 infants where involved in this study, the AUC of Lf was 0.90. Sensibility and specificity were of 100 and 81.0%, respectively, with optimal cut-off <1.2 μg/ml for lactoferrin
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Figure 3: Summary Bar chart of acute phase reactants
Figure 4: Summary Bar chart of Chemokines and Cytokines.
0 10 20 30 40 50 60 70 80 90 100
CRP Procalcitonin Serum Amyloid A
Perc en ta ge (% ) Biomarkers
Acute phase reactants
Sensitivity(%) Specificity(%)
Negative predictive value (NPV)(%) Positive predictive value(PPV)(%)
0 20 40 60 80 100 120
IL-6 IL-10 CD64 Hepcidin Soluble CD14
subtype (presepsin) Perc en ta ge (% ) Biomarkers
Chemokines and Cytokines
Sensitivity (%) Specificity (%)
Negative predictive value (NPV) (%) Positive predictive value (PPV) (%)
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10 DISSCUSIONS
10.1 C - reactive protein (CRP):
CRP has been a widely researched biomarker for neonatal sepsis. A total of six studies where identified involving CRP [43, 44, 47, 49-51]. CRP is an acute phase reactant, a protein which is made by hepatocytes under the control of cytokine IL-6 and released, according to tissue injury, inflammation or infection (peaks at 48hr) [86]. A met analysis performed on CRP by Brown et al, suggested that rapid and accurate diagnosis of late-onset infection in new-born infants could inform treatment decisions and avoid unnecessary administration of antibiotics [87]. Subsequently many studies have been undertaken in order to prove the usefulness of CRP in early diagnosis of neonatal sepsis.
Studies have indicated varying differences in specificity and sensitivity of CRP and the useful nature of CRP in early diagnosis of Late onset sepsis (LOS) and early onset sepsis (EOS). A 5-year retrospective cohort study, conducted by Beltempo et al, on 416 VLBW infants born at less than 1500 g, there were 590 separate late-onset sepsis evaluations. CRP in this case was measured at T0 (CRP was drawn at the time of initial blood culture), T24 (at 16-24 h) and T48 (40-48 h). It was found that at T0, combining the CBC and the CRP had the highest sensitivity of 66% and at T24, CRP’s sensitivity was 84%. This study revealed how CRP in combination with CBC at T0, gave the greatest sensitivity at 88% with 93% negative predictive value, mainly for LOS [50]. Another study, which compared CRP with PCT; blood samples were collected from neonates with clinical signs of sepsis, before antibiotics treatment. The sensitivity of both CRP and PCT was 88.90% each. The specificity for CRP was higher at 89.40% [43].
Moreover there are other research indicating C-reactive protein (CRP) as an effective biomarker for neonatal sepsis. Result observed significant difference in CRP and IL-6 in early onset sepsis (EOS) when compared with late onset sepsis (LOS) neonates. In terms of biomarkers accuracy, the result showed that CRP had the best diagnostic accuracy with cut-off value of 3.6 ng/ml (sensitivity 78% and specificity of 70%). The best combination is shown with CRP and IL-6 in which sensitivity increased to 89% and specificity to 79% [44]. In addition to this, CRP in combination WBC have been studied in developing countries such as Ethiopia. This cross sectional study undertaken by Sorsa et al, further backups the effectiveness of CRP and reiterates the need for useful markers in developing areas of the world. The sensitivity, specificity, PPV and NPV of CRP were 78.5, 66, 43 and 90.4%, respectively. This high sensitivity in developing could come from the fact that better tests are limited and diseases undergo an aggressive nature [49]
20 A more new and more sensitive CRP measurement comes from hsCRP, although not yet routinely available, studies have shown its effectiveness in EOS. A hospital based cross sectional study of 168 neonates, with both conventional CRP and hsCRP measurement; showed a higher serum levels of CRP among late onset versus early onset sepsis group with significantly higher serum levels of hsCRP among early onset compared with the late onset sepsis group (p < 0.05 for all) [51].Concluding to the fact that, CRP maybe more useful in early diagnosis of LOS in comparison with hsCRP, which seems more effective in early diagnosis of EOS. In addition to this, hsCRP was analysed in a German study by Bartolovic et al, which studied 31 neonates with suspected sepsis and 31 control neonates. This study revealed that Neonates with bacterial infection had significantly higher values of PCT (p <0.001), WBC (p <0.001) and CRP (p <0.05) compared to healthy babies [47]. Further supporting the use of CRP as an effective marker for neonatal sepsis.
All in all, studies on CRP seem to conclude that (CRP) is generally considered a helpful marker for diagnosis of sepsis used in addition to blood culture [51]; with a high sensitivity. Although, the analysis of these clinical studies, show that CRP is more useful for diagnosing LOS, in comparison to hsCRP which seem to be effective for EOS.
10.2 Procalcitonin (PCT):
In addition to CRP, Procalcitonin has been also been studied extensively in the last 10 years. Procalcitonin (PCT) is a 116 amino acid peptide and belongs to the calcitonin (CT) superfamily of peptides [87]. Procalcitonin expression occurs in a tissue-specific manner and in the C cells of the thyroid gland where expression of CALC-1 gene produces PCT (a precursor of CT in healthy and non-infected individuals) [88]. In the presence of microbial infection, non-neuroendocrine tissues also express the CALC-1 gene to produce PCT. So in case of an infection the increase in gene expression in all parenchymal cells leads to production of PCT [88].
In this review a total of 7 studies where identified, which researched the use PCT as a diagnostic biomarker for neonatal sepsis, but more mainly EOS. A prospective cross sectional study conducted in a hospital in India, showed that along with CRP, PCT was an effective biomarker. The study was carried out for 5 months and blood samples were taken from neonates with clinical signs of sepsis. This research yielded a sensitivity of 88.90% for both CRP and PCT and specificity of 89.40% for CRP and 80.30% for PCT [42-43] showing adequate effectiveness in sepsis diagnosis. According to many studies, determination of Procalcitonin in the umbilical vein blood could be a parameter of sufficient specificity and sensitivity for diagnosis of neonatal infection. For example a study done by Steinberger E et al in
21 Austria, on cord blood Procalcitonin, revealed that the optimal cut-off value for PCT was 0.235 μg/L (sensitivity 78.6%, specificity 86.3%), although the highest diagnostic accuracy came from combining IL-6 in clinical EOS [45]. Additionally, another study of intensive care neonates where carried out, similarly cord blood was taken from the mothers. In this study again there showed a higher median number of Procalcitonin at 0.188 (ng/mL) in sick borns compared to 0.121 (ng/mL) in healthy new-borns [47], therefore it was concluded that PCT could be of value in early diagnosis of neonatal sepsis.
Furthermore, a study carried out by H.Altunhan et al [46] showed that PCT measurement during birth and at 24hrs could be useful for early diagnosis of EOS, rather than just one single measurement of PCT. In this prospective study 2 groups where indicated, one patient group and one control group. At 24 h of age, PCT and CRP levels were significantly higher in the patient group than in the control group (p < 0.001). The cut-off levels of PCT with optimum diagnostic efficiency derived from the ROC curves were ≥0.59 ng/ml at birth (sensitivity 48.7%, specificity 68.6%) and ≥5.38 ng/ml at 24 h of life (sensitivity 83.3%, specificity 88.6%)[46]. This clearly showed a great effectiveness in measuring PCT at 24hrs of age as compared to at birth ad reinforcing the idea that PCT is a useful biomarker of neonatal sepsis. Moreover, another study that also concluded with the positive effectiveness of PCT as diagnostic biomarker for neonatal sepsis is a study done on 320 neonates by M. A. Fattah et al, concluded that PCT had a specificity of 72% and sensitivity of 70% [44].
One study conducted by Nagwan I.Rashwan et al, showed a very high sensitivity and specificity. Cut-off value of PCT >389 pg/ml showed sensitivity of 97.00% and specificity of 100.00%. However the best diagnostic accuracy came from combining hs-CRP and PCT together with any IL-6 or presepsin (especially in EOS) which means that the combination, rather than one biomarker alone as better predictor for neonatal sepsis [51]. This was in conjunction with a similar study carried out by Ali Kh Al-Zahrani et al, which also states that the combination of markers (hs-CRP, PCT, and IL-6) is better than single markers to diagnose sepsis; even though PCT had a greater diagnostic value [90]. Finally, one final study available on PCT show a low sensitivity (65%) and low specificity (60%), compared to other studies carried out on the same biomarker. However this could possibly be attributed to the fact that the method of testing was different for all the other studies and this particular study used the ELISA methodology [48] . Despite this, PCT could be an effective biomarker for neonatal sepsis, especially EOS.
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10.3 Serum Amyloid A (SAA):
A total of 2 studies where identified that included Serum amyloid A (SAA), as filters applied did not reveal extensive research carried on this particular biomarker since 2010. Although research prior to this have indicated the usefulness of SAA, as a biomarker for neonatal sepsis, for example a study by A Lannergård et al showed that the sensitivity values for SAA and hsCRP were 0.47 and 0.75, respectively [91]. SAA is also an acute phase reactant and just like CRP is synthesises in the liver hepatocyte [92]. There is also evidence of extrahepatic expression in humans mainly in the lymphocytes and plasma cells of many normal tissues and in lymphoid follicles [92]. There is some reported evidence to suggest that the normal physiological levels of SAA is serum is around 1-2 µg/ml [93]. During the acute phase response, the production of A-SAA (Acute-phase serum amyloid A), but not of C-SAA (Constitutive serum amyloid A), in the liver and in extrahepatic tissues is stimulated by exogenous TLR ligands (e.g. LPS) and endogenous cytokines (IL-1, IL-6 and TNF-α) [92].
One such steady that looked in SAA, carried out by J David M Edgar [76], showed that in EOS SAA has a sensitivity of 58.3% and specificity 67.1%; and in LOS the sensitivity was 28.6% and specificity of 91.4% in infected neonates. The results seem to show that SAA is useful for neonatal sepsis, even though the sensitivity and specificity is lower than other acute phase reactants. Additionally, a study conducted by Daniela Bartolovic et al [47] states that SAA has its disadvantages as it’s neither sufficiently specific nor sufficiently sensitive, because its values increase only with longer duration of infection. So this biomarker is questionable in its effectiveness as a biomarker for neonatal sepsis; as it does not fit the usual characteristics of an ideal biomarker, with a low sensitivity and specificity.
10.4 Chemokines and Cytokines
10.41 Interleukin-6 (IL-6):
Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by a variety of cells in response to infection and tissue injury [95]. Many proinflammatory cytokines are involved in the pathogenesis of sepsis including IL-6. It has come to light that microorganisms interact with a family of toll-like receptors (TLRs) belonging to the innate immune system, which trigger intracellular activation of nuclear factor kappa B and various kinases, leads to the production and release of cytokines [22]. Additionally, IL-6 has been reported to be produced in the early phase of infectious inflammation by monocytes and macrophages immediately after the stimulation of TLRs [89]. This acute IL-6 expression plays a central role in the body’s defence against infection or injury by stimulating various cell populations [66].
23 A total of 2 studied where collected which fit all of the inclusion criteria [45, 52]. One of these studies was conducted by Qing Ye et al, the study group included 420 patients with neonatal sepsis with a male to female ratio of 1.32:1. The results showed that IL-6 and IL-6/IL-10 were more effective than CRP in the diagnosis of neonatal sepsis. IL-6 had the highest diagnostic sensitivity, whereas IL-6/IL-10 had the best diagnostic specificity. Serum IL-6 level of >12.5 pg/ml had a sensitivity of 93.75% and a specificity of 94.12% in the differential diagnosis of neonatal sepsis [52].Moreover another study looked into the predictive value of cord blood IL-6, this study analysed IL-6 for early-onset sepsis (EOS) in the preterm infants. Thirty of 218 preterm infants (13.8%) were diagnosed as having EOS. The optimal cut-off value for IL-6 15.85 ng/L (sensitivity 73.7%, specificity 84.2%) and the positive and negative predictive values were 42.1% (26.3–59.2%) and 98.7% (92.8–99.8%) respectively [45]. Overall IL-6 seems to fit most of the characteristics of an ideal biomarker, making it a prime contender as an effective biomarker [94].
10.42 Interleukin-10 (IL-10):
The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo [63].
2 studies which involved IL-10 was discovered, fitting the inclusion criteria [48, 52]. Much like IL-6 the IL-10 was researched by Qing Ye et al. This study also analysed IL-10 as a biomarker for neonatal sepsis, a serum IL-10 level of >3.8 pg/ml had a sensitivity of 87.50% and a specificity of 61.76% [52]. Although this is not as high as IL-6, IL-10 is still showing some potential. A further study conducted showed higher sensitivity and specificity, IL-10 had sensitivity of 92% and specificity of 84% at a cut-off level of ≥17.3 pg/ml. As well as this IL-10 has a positive predictive value (PPV) of 80% and negative predictive value (NPV) of 86% [48]. This shows IL-10 has the possibility to be an effective biomarker for neonatal sepsis.
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10.43 Cluster of differentiation (CD64):
CD64 as a biomarker for neonatal sepsis has been extensively been researched during the last couple of years. Around 8 articles where discovered that fit well with the inclusion criteria [48, 58-62, 72, 74]. CD64, is a high affinity Fc receptor, which is normally expressed by monocytes and only weakly on resting neutrophils [98]. Upregulation of CD64 on neutrophils (nCD64) is thought to be a very early step of host’s immune response to bacterial infection, increasing approximately one hour after invasion [65].
CD64 is usually measured on flow cytometry. One prospective study which was conducted in a neonatal intensive care unit, looked into a total of 158 preterm neonates, 88 of whom were with suspected infection. This study found that CD64 had sensitivity and specificity of 81.82% and 70% respectively; additionally with a PPV (77.40) and NPV (75.4) [58]. Thus fitting some of the main criteria for an ideal biomarker. Similarly a study conducted by Zongsheng Tang et al concluded that CD64 index is unique in its capacity to diagnose neonatal infections early, this found that the levels of the CD64 index both in the non-sepsis group and in the sepsis group were significantly higher than that in the control group, but there were no differences between the non-sepsis and sepsis groups (P = 0.0012, P = 0.0005 and P > 0.9999 respectively [59]. Furthermore, studies have not only been carried out on preterm neonates but VLBW neonates’ as well. A study on VLBW neonates was performed by Motta M et al specifically for early onset neonatal sepsis. The finding of this study was as follows; Neutrophil CD64 expression (CD64 index) was assessed in 129 VLBW neonates within 72 h after birth. The average sensitivity, specificity, NPV and PPV value came to 70.6%, 80%, 98.3% and 12.6% respectively over a 72 h period [62].
One particular study on diagnostic utility of CD64 revealed very high sensitivity and specificity. This prospective study was carried out on 60 neonates admitted to the Neonatal Intensive Care Unit (NICU) with clinical and laboratory signs of sepsis. Inclusion criteria were: gestational age > 30 weeks, birth weight > 1.2 kg. The ROC curve analysis revealed that nCD64 cut-off value > 34.1% was able to discriminate patients with NS from healthy controls is with 96.67% sensitivity, 100% specificity, 92.6% negative predictive value (NPV) and 100% positive predictive value (PPV). Likewise a study conducted on 75 neonates by Sanaa Elawady et al, presented results of high sensitivity and specificity for CD64. CD64 had a sensitivity of 96%, a specificity of 100%, a PPV of 96.2%, and an NPV of 100% with a cut-off value of 45.8% and 46.0%in the confirmed and the clinical sepsis groups, respectively [61]. Besides this in a research conducted in India, blood from 100 neonates suspected of sepsis and 29 healthy controls was collected on clinical suspicion of sepsis, and the expression of nCD64, was evaluated by Flow Cytometry; the cut‐off for nCD64 was calculated to be 126.10. The sensitivity and specificity of nCD64 at day 1 was 73.01% and 89.18%, respectively [72].Finally a study on forty-nine infants with
25 sepsis and 49 healthy between July 2008 and March 2009 included. They were retrospectively allocated into a ‘sepsis’ group and a ‘no sepsis’ group before nCD64 analyses were done. This study was undertaken by Alaa A.H. Zeitoun et al, CD64 showed sensitivity of 92% and specificity of 71% at a cut-off value 2.6% [48]. All in all CD64 was capable of differentiating infected from non-infected infants.
There are advantages of using nCD64 as a diagnostic marker, as the flow cytometric analysis can be performed with a minimal blood volume (50–100 μl of whole blood); the result is available within a few hours after the specimen reaches the laboratory; the measurement is ‘quantitative’ and thus enables the comparison of results across different centres; rendering nCD64 as one of the best infection markers for the identification of both early-onset and late-onset neonatal sepsis [48].
10.44 Hepcidin:
The hormone Hepcidin, a 25-amino-acid (aa) peptide, is the principal regulator of iron absorption and its distribution to tissues. Hepcidin is synthesized predominantly in hepatocytes, but its low levels of expression in other cells and tissues, including macrophages, adipocytes and brain, may also be important for the autocrine and paracrine control of iron fluxes at the local level [73]. Hepcidin is also an acute-phase reactant expressed on neutrophils and macrophages, stimulated by lipopolysaccharides and IL-6 in response to microorganisms [77]. In human hepatocyte cultures, Hepcidin expression was induced after direct exposure to LPS or medium from LPS-activated human monocytes, and this response could be ablated by the addition of anti-interleukin-6 (anti-IL-6) antibodies [74]
Of a total of 2 studies on Hepcidin, one analysed Hepcidin as a biomarker for early onset sepsis; a study conducted by Mehmet Nevzat Cizmeci et al. This study looked at cord blood Hepcidin, it revealed some promising results as to the use of cord blood Hepcidin as a biomarker for EOS. They showed that all infants with sepsis in their study had very high levels of cord blood Hepcidin (min–max: 118.1–8400 ng/mL), which may indicate the intrauterine initiation of the inflammatory process [76]. On the other hand a more promising study was conducted for the use of Hepcidin as a biomarker for LOS. A total of 44 VLBW and 21 term infants were enrolled in this study between October 2008 and September 2011. Hepcidin values > 92.2 ng/mL was set as the cut-off point, which correctly classified 91% of all infants. When analysis was restricted to infants with positive blood cultures, the PPV, NPV, specificity, and sensitivity of Hepcidin were 75%, 97%, 91%, and 90% respectively [77]. In addition to blood culture and other markers of infection, Hepcidin concentration may be a useful adjunct test in the evaluation of late-onset sepsis in VLBW infants, as well as EOS in term and preterm infants. However much more research would be required in order to adopt this into daily clinical practice.
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10.45 Presepsin (Soluble CD14 Subtype):
CD14 is a multifunctional cell surface glycoprotein [55]. Presepsin, a soluble subtype of CD14 or sCD14-ST, is a complex product of CD14 cleavage that is released in the general circulation after bacterial antigen binding. Presepsin is stable in the general circulation and an automated rapid quantification is currently available [56].CD14 is constitutively expressed on the surface of various cells, including monocytes, macrophages, neutrophils, chondrocytes, B cells, dendritic cells, gingival fibroblasts, keratinocytes, and human intestinal epithelial cell lines[55]. Most importantly, CD14 is involved in the recognition of a wide variety of bacterial products, including peptidoglycans. Upon binding of the LBP complex, CD14 activates the toll-like receptor 4 (TLR4)-specific proinflammatory signalling cascade, thereby starting the inflammatory reaction of the host against infectious agents [55].
A total of 5 studies where identified [57, 64, 67-68, 70], which looked into Presepsin (P-SEP) as a biomarker for neonatal sepsis. This single-centre study was carried out between January 2013 and March 2016 and involved 60 preterm neonates. The study uncovered that P-SEP achieved the best accuracy for prediction of sepsis at the cut-off of 788 ng/ with 93% sensitivity and 100% specificity. The PPV and NPV were 100% and 94% [67]. This study found that P-SEP was significantly higher in preterm with EOS, compared to non-infected neonates. Although the sample size in this study was small, the research shows potential for future development; allowing this data to support the use of P-SEP as a promising biomarker for sepsis. Additionally a study conducted by Sevilay Topcuoglu et al, looked into the role of P-SEP in LOS. Preterm infants who were smaller than 32 weeks’ gestational age and between 4 and 30 d’ postnatal age were included in the study and a total of 82 infants where included in the study. A Presepsin value of 800.5 pg/mL was established as a cut-off value with 67% sensitivity and 100% specificity. The positive and negative predictive values were 100% and 74% respectively [68]. The main limitation of this study was that, there was no comparison to other biomarkers, nevertheless this study showed that P-SEP could be a useful biomarker for LOS.
A larger study with a sample size of 784 patients, P‐SEP 795 pg/mL was established as the cut‐off, with 85% sensitivity and 89% specificity. The PPV and NPV were 85% and 89%, respectively [70]. Although this study had a larger sample, it was still not big enough, however this study shows that P-SEP can better is valuable in discriminating infectious and non-infectious inflammatory conditions.
27 One study compared Presepsin to Procalcitonin and CRP. In this study a total of 41 neonates where involved, the study identified that the area under the curve (AUC) for Presepsin was 0.9 with sensitivity and specificity, 97.6%, 95.1%, respectively at a cut-off value of 1800 μg/l [64]. This study concluded that Presepsin had the best sensitivity and NPV, therefore giving it potential value for use as a biomarker for neonatal sepsis. Moreover another research compared P-SEP to CD64. In this research it found that Presepsin level was significantly higher in sepsis group than control group (P<0.05). AUC for Presepsin was higher (0.95). The cut-off value at 767 pg/mL showed a sensitivity of 100% and specificity of 86.7%. The PPV and NPV were 84.4% and 100%, respectively; which was significantly higher than CD64 [57]. All in all here again it’s possible to say P-SEP could be a potential effective biomarker for neonatal sepsis.
10.5 Acute phase reactants vs Chemokines and Cytokines :( Fig3 and Fig 4)
The information in the bar chart allows us to see that acute phase reactants and chemokines/cytokines, vary to a certain degree in their sensitivity, specificity, NPV and PPV. Out of the three acute phase reactants serum amyloid A shows the most variation in values, having the highest specificity (92.8%) and lowest sensitivity (23.5%) and highest positive predictive value (PPV) (66.6%), CRP on the other hand had the highest negative predictive value (NPV) (75.18%). On the other had chemokines and cytokines biomarkers did not seem to show a wide contrast in its values; in saying this however P-SEP had the greatest specificity (94.16%) and highest PPV (92.35%), whereas Hepcidin had the highest sensitivity(90%), IL-6 had the highest NPV(98.7%) and lowest PPV(42.1%). Comparison of both types of biomarkers is a challenging task as each biomarker will have a different cut-off value along with sensitivity and specificity, due to varying sample sizes, detection methods used and the type of analysis.
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10.6 Molecular Biomarkers:
Advances in molecular microbiology have provided culture-independent molecular assays for rapid diagnosis of the causative agent of infection. Specific detection of pathogens in blood using PCR- assay has been described as early as 1993 [69]. The development of broad-spectrum PCR-assays facilitates more universal detection of microorganisms [71]. A study looked at The SeptiFast®-test, which uses real-time multiplex PCR to detect more than 20 different pathogens in blood. This study revealed that out of the performed prospective, multi-centre study that included 133 VLBW with 214 episodes of suspected late onset sepsis (LOS); PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%) [37].This means that, Multiplex-PCR results had a moderate impact on clinical management. The main advantage of multiplex-PCR was the rapid detection of pathogens from micro-volume blood samples. Similarly a study investigated the use of Septifast PCR system for LOS. The study demonstrated that, in comparison to routinely used blood culture techniques, the Roche SeptiFast MGRADE multiplex PCR system applying a modified DNA extraction protocol showed 90.2% sensitivity for the diagnosis of blood-culture positive sepsis episodes, a moderately high number of positive test results in episodes without infection potentially due to contamination during blood drawing and/or processing, and an increased pathogen detection rate in patients with clinical sepsis [35].
Molecular genetic studies have become increasingly popular over the past couple of years; one such study which looks into DNA methylation for the diagnosis of neonatal sepsis was conducted by Dhas BB et al. Fifty one new-borns as cases and thirty seven new-borns as controls were enrolled in the study, Highly significant difference in percentage of gDNA methylated was found between the cases and controls (Cases: 2.4 ± 0.39; Controls: 2.07 ± 0.35; P < 0.0001). Concluding that although the global DNA methylation was not a highly sensitive diagnostic method, this study showed that this has a potential for the future in terms of use as a neonatal sepsis biomarker [39]. Furthermore another study has also reported the use of DNA methylation, it compared the DNA methylation pattern in septic and non-septic borns. Functional annotation revealed that a group of PCDHB genes was hyper methylated in new-borns with sepsis [40]. Another study applied gene expression from arterial umbilical cord blood of preterm infants. Using a rank-based statistics, comparison of gene expression of infants with and without EOI revealed 292 differentially regulated genes (FDR ≤ 0.1). Of these, 219 genes had significantly higher gene expression levels in infants with EOI, while 73 genes had significantly lower expression levels [36].
29 16S rRNA Gene Sequencing/PCR is another molecular method that has been investigated. On study compared this to conventional blood culture for diagnosing bacterial neonatal bacterial sepsis. Sepsis was suspected in 706 infants. The number of positive cultures and positive PCR results were 95 (13.5%) and 123 (17.4%), respectively. Compared with blood culture, the diagnosis of bacterial sepsis by PCR revealed a 100.0% sensitivity, 95.4% specificity, 77.2% positive predictive value, and 100.0% negative predictive value [41]. Finally one study also researched 16S rRNA Gene. In this study despite the equal results of PCR and BC in, the detection of bacteraemia by BC was (26%), while by using a molecular method, namely broad range 16S rRNA PCR, the rate was improved to (35.4%) [38]. This means that the sensitivity was 62.5%, specificity 86.9%, PPV 62.5 % and NPV 86.9% [38]. Therefore in comparison with BC, the 16S rRNA PCR increased the sensitivity in detecting bacterial DNA in new-borns, with a high PPV and NPV.
10.7 Novel Biomarkers:
Due to the ever increasing need for an effective biomarker, there is always new and fresh research introduced in order to find an answer to early and fast diagnosis of neonatal sepsis. Therefore one such research is in the proteomics and metabolomics field. Urine metabolomics in late onset neonatal sepsis was studied. 16 septic neonates (9 with confirmed and 7 with possible LOS) and 16 controls were studied (Urine metabolic profiles were assessed using non-targeted nuclear magnetic resonance spectroscopy), the research showed that analysis of the spectral data by multivariate analysis showed that, even by unsupervised Principal Component Analysis (PCA), septic infants (confirmed and possible) could be discriminated from non-septic ones (controls) at the time point of sepsis suspicion (D0) [78]. This means to say that, there where varying difference in septic and non- septic neonates. One such difference which came to light was the data showed that several metabolic pathways are influenced in neonatal sepsis. One particular elevations in the amounts of urinary taurine and hypotaurine, the presence of which had a significant statistical impact on the differentiation of septic neonates from those without the disease [78]. Similarly one study combined the use of both nuclear magnetic resonance (1H-NMR) and gas-chromatography-mass spectrometry (GC-MS) techniques, the purpose of the study was to evaluate the capability of the urine metabolomics approach to identify a potential metabolic profile related to the neonatal septic condition. 25 neonates where included in this study. In this study, metabolomics led to identifying the molecules responsible for the differences in the metabolic profiles, among which glucose, lactate and acetate, the urine content of which had increased in septic neonates compared to controls, while THBA, ribitol, ribonic acid and citrate had decreased [79]. There is still a need for highly sensitive and specific biomarkers, therefore making metabolomics a method of great diagnostic potential.
30 Interleukins such as, IL-6 and IL-10 have been widely researched in the past; however due to the ongoing need for highly sensitive biomarkers for the efficient diagnosis of neonatal sepsis, new interleukins (cytokine) have been researched. One such marker is IL-35, which is in the family of IL-12 and plays a major role in host immunity [81]. Studies have shown that IL-35 is an important anti-inflammatory cytokine and can suppress T-helper (Th) 1, Th2, and Th17 cell responses [54].157 patients were included in this study, the cut-off concentration for IL-35 was 31.7 pg/ml, with a sensitivity of 78.48%, a specificity of 66.67%, PPV of 70.46% and NPV of 75.36% [1]. IL-35 was compared with PCT and CRP in this same study. The comparison showed that IL-35 had a higher sensitivity than CRP and a higher specificity than PCT. This goes to say with wider research on IL-35, it has the potential to be an effective biomarker for neonatal sepsis.
One other novel marker that has been investigated is melatonin as a biomarker for neonatal sepsis. Melatonin is s an indolamine endogenously produced by pineal body and has many functions. It has involvement in neuroendocrine, cerebrovascular and sleep systems [83]. Melatonin also has important role as an anti-oxidant, anti-inflammatory and anti-apoptotic agent [53]. This prospective study included 40 neonates, 20 with clinical LOS and 20 as control. Melatonin concentration was significantly increased in sepsis group when compared to control group (27.2 ± 3.3 versus 11.4 ± 3.2, p = 0.001) and had a sensitivity of 93.5% and specificity 85.6% [84]. However an interesting finding in this study was that sensitivity and specificity were increased by combining melatonin concentration with HsCRP to 97.3% and 93.3%, respectively [84]. This means melatonin can be potential effective biomarker for neonatal sepsis, with the best results achieved when combined with CRP.
Finally another biomarker lactoferrin has been researched as a potential biomarker for neonatal sepsis. Lactoferrin (Lf) is a non-heme iron-binding glycoprotein with a molecular weight of about 80 kDa that belongs to the transferrin family [80]. It is also a cell-secreted mediator that bridges the innate and adaptive immune responses [8]. A total of 15 infants where involved in this study, the AUC of Lf was 0.90. Sensibility and specificity were of 100 and 81.0%, respectively, with optimal cut-off <1.2 μg/ml for lactoferrin [80]. Showing potential for its use as a biomarker for neonatal sepsis.
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11 STUDY LIMITATIONS
The data collection during this thesis was conducted by one person, this could impact the type and number of articles that were selected. Moreover, the data and articles were searched for in one database in order to form an overview; however this could mean other numerous articles which were relevant on other respected databases could have been missed. Finally the use of an English language filter, could have limited other potentially appropriate articles.
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12 CONCLUSION
1. The research showed that the etiologic agent of neonatal sepsis can vary depending on whether it is early- onset neonatal sepsis or late-onset neonatal sepsis. In EOS, Group B streptococcus (GBS) is the most common etiologic agent, while the etiologic pathogen in LOS vary compared to EOS, coagulase-negative staphylococci (CONS) have emerged as the predominant pathogens of LOS.
2. In terms of the main clinical features of neonatal sepsis it seems that the clinical symptoms can vary in new-borns according to gestational age as well as the type of onset, respiratory distress, neonatal jaundice along with feeding intolerance seems to predominate.
3. The current diagnostic method of neonatal sepsis is either culture dependent or independent; blood culture along with CBC and CRP being the gold standard, but with errors such as low volume of blood and false negatives making it somewhat unreliable.
4. Out of the three acute phase reactants serum amyloid A shows the most variation in values, having the highest specificity and lowest sensitivity. Whereas out of the chemokines/cytokines P-SEP had the greatest specificity and highest PPV.
5. A varied amount of research has been conducted over last 10 years on biomarkers of neonatal sepsis, for early diagnostics with some useful results. Because of the complexity of the sepsis response, it is very difficult to determine a single effective biomarker that can be used in clinical practice. Each biomarker has a different cut-off value along with sensitivity and specificity, due to varying sample sizes, detection methods used and the type of analysis. In terms of molecular and novel biomarkers, there is a need for an abundance of studies to be able consider their effectives; therefore a combination of several sepsis biomarkers may be more effective.
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13 PRACTICLE RECOMMENDATIONS
One way could be to combine biomarkers and compare them, with a large sample size and the clinical features of neonatal sepsis. This could give way to higher sensitivity and specificity in biomarkers. In this Presepsin shows the most promise, however the question remains how available it can be in clinical practice, as cost effectiveness and accessibility has to be taken into consideration and researched further.
With regards to an optimal cut-off value for different markers, more studies are needed to be undertaken and needs to provide more accurate results and associate it with patients or within the context of the clinical situation, and whether the aim is for the early diagnosis of EOS or LOS
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