Infectious Diseases
ISSN: 2374-4235 (Print) 2374-4243 (Online) Journal homepage: http://www.tandfonline.com/loi/infd20
Diagnostic utility of biomarkers for neonatal sepsis – a systematic review Sofie Sommer Hedegaard, Kirsten Wisborg & Anne-Mette Hvas To cite this article: Sofie Sommer Hedegaard, Kirsten Wisborg & Anne-Mette Hvas (2015) Diagnostic utility of biomarkers for neonatal sepsis – a systematic review, Infectious Diseases, 47:3, 117-124, DOI: 10.3109/00365548.2014.971053 To link to this article: http://dx.doi.org/10.3109/00365548.2014.971053
Published online: 18 Dec 2014.
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Date: 13 July 2017, At: 11:43
Infectious Diseases, 2015; 47: 117–124
REVIEW ARTICLE
Diagnostic utility of biomarkers for neonatal sepsis – a systematic review
SOFIE SOMMER HEDEGAARD1, KIRSTEN WISBORG2 & ANNE-METTE HVAS3 From the 1Department of Pediatrics, Aarhus University Hospital, 2Head & Neuro Centre, Aarhus University Hospital, and 3Department of Clinical Biochemistry, Aarhus University Hospital and Aarhus University, Aarhus, Denmark
Abstract Neonatal sepsis is a major cause of morbidity and mortality. Early diagnosis and treatment of the neonate with suspected sepsis are essential to prevent life-threatening complications. Diagnosis of neonatal sepsis is a challenge due to non-specific clinical signs and the fact that infection markers are difficult to interpret in the first and critical phase of neonatal sepsis. The objective of the present study was to systematically evaluate existing evidence of the diagnostic utility of biomarkers for prediction of sepsis in neonates. We conducted a systematic literature search performed in PubMed and Embase. The study population was neonates with gestation age ⬎ 24 weeks in their first 28 days of life with suspected sepsis. The included manuscripts were rated due to criteria from a modified rating scale developed by Douglas Altman. Of 292 potentially relevant manuscripts, 77 fulfilled the inclusion and exclusion criteria; 16 (21%) were rated as high-quality studies. C-reactive protein (CRP) was the most extensively studied biomarker evaluated. The high-quality studies indicated that the acute phase protein serum amyloid A had high sensitivity, both at onset of symptoms and 2 days after. The studies evaluating serum amyloid A presented a variable positive predictive value (PPV, 0.67 and 0.92) with a high negative predictive value (NPV, 0.97 and 1.00). The existing evidence of the diagnostic value of serum amyloid A for neonatal sepsis showed promising results, and should be further investigated in clinical settings.
Keywords: Newborn, procalcitonin, C-reactive protein, CRP, serum amyloid A
Introduction Worldwide, infection is the single largest cause of neonatal death [1]. In developed countries the incidence of neonatal bacterial sepsis varies from 1 to 4 cases per 1000 live births [2]. Neonatal septicemia is defined as a clinical syndrome of systemic illness accompanied by bacterium in the first 28 days of life [3]. Neonatal sepsis is classified in two major categories: early-onset sepsis, which usually presents with respiratory distress within 72 h of age; and late-onset sepsis, which usually presents with septicemia after 72 h of age [4]. Early diagnosis and treatment of the neonate with suspected sepsis are essential to prevent severe and life-threatening complications. Diagnosis of neonatal sepsis is a challenge due to variable and non-specific clinical signs; moreover, infection markers are difficult to evaluate in the first and critical phase [5]. To avoid serious complications such as meningitis,
cerebral abscess, and neonatal death, treatment is initiated in many newborns with clinical signs of sepsis without proven infection [1]. The utility of infection markers in the diagnosis of neonatal sepsis has been evaluated in many studies with different results. The biomarkers potentially used for diagnosis of neonatal sepsis can be divided into four groups: acute phase proteins, cell surface antigens, cytokines and chemokines, and soluble adhesion molecules. The acute phase proteins investigated in multiple studies concerning neonatal sepsis are C-reactive protein (CRP), procalcitonin, serum amyloid A, and hepcidin. CRP is commonly used as an indicator for bacterial sepsis in neonates and children. CRP has several drawbacks; it is not useful as an early phase infection marker because it can only be detected 12 h after manifestation of clinical signs, it reaches a plateau after 20–72 h [6], and it lacks specificity [7].
Correspondence: Sofie Sommer Hedegaard MD, Department of Pediatrics, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark. Tel: ⫹ 45 28747699. E-mail: [email protected] (Received 16 July 2014 ; accepted 22 September 2014 ) ISSN 2374-4235 print/ISSN 2374-4243 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.971053
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A previous study indicated that procalcitonin had a higher predictive value than CRP in detecting sepsis in patients [8]. Procalcitonin is released into the circulation within 3 h after endotoxin injection, it reaches a plateau at 6 h, and remains elevated for 24 h. This makes procalcitonin a promising marker for early-phase infection and sensitive identification of infected infants [9]. However, in neonates its value in the first day of life is limited by a marked physiological increase after birth [10]. Serum amyloid A has shown as high as a 1000-fold increase during infection and increases 8–24 h after the onset of sepsis [11]. This marker has been reported to be useful in various acute conditions and in the diagnosis of sepsis in neonates [12]. Hepcidin, a highly conserved antimicrobial peptide, plays a critical role in inflammation and iron homeostasis [13], and is one of the later markers for diagnosis of neonatal sepsis. In the group of cell surface antigens, CD64 and CD11b are the primary biomarkers investigated in studies on neonatal sepsis. CD64 is a high-affinity receptor normally expressed by monocytes and macrophages, and only to a small extent on neutrophils [5]. During bacterial infection, neutrophilic expression of CD64 (nCD64) is markedly increased; nCD64 density thus has the potential to differentiate between infected and non-infected neonates at a very early stage [5]. In the group of cytokines and chemokines, interleukin (IL) IL-1, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and soluble TNF receptor (sTNFR) have been evaluated for diagnosis of neonatal sepsis. The biological function of cytokines and chemokines is among other things to induce fever and attract and activate neutrophilocytes [14]. IL-6, TNF-α, and IL-1 all play a critical role in the induction of acutephase proteins such as CRP synthesis in the liver; thus, these cytokines can be detected in the blood earlier than CRP [15]. In the group of soluble adhesion markers, E-selectin and intercellular adhesion markers (ICAM) are the primary biomarkers evaluated for diagnosis of neonatal sepsis. The expression of E-selectin and ICAM is stimulated by cytokines IL-1 and TNF. The function of these markers is adhesion and recruitment of leukocytes to endothelial cells and platelet aggregation [12]. The objective of the present study was to systematically evaluate existing evidence from 1980 to January 2014 of the diagnostic value of biomarkers for predicting sepsis in neonates. Literature search The literature search was performed in the databases PubMed and Embase using the following keywords:
‘infant newborn,’ ‘neonates,’ ‘sepsis,’ ‘infection,’ ‘sensitivity and specificity,’ ‘predictors,’ ‘early diagnosis,’ and ‘biological markers’ with the limitations ‘humans’ and ‘English language.’ All studies from 1980 until January 2014 were included in the present review. Duplicate publications were identified and removed. Relevant publications were identified on the basis of titles and abstracts using the following inclusion and exclusion criteria. Inclusion criteria: (1) the study population was neonates with gestational age ⬎ 24 weeks in their first 28 days of life with suspected sepsis, (2) the study population had cultureproven sepsis or suspected infection based on clinical algorithm, (3) the manuscript evaluated biomarkers for diagnosing the risk of neonatal sepsis, (4) the specific biomarker was measured in serum or plasma, (5) the publication was an original paper including short communications and technical notes. The following publications were excluded: reviews, metaanalyses, editorials, letters to the editor, case reports, and guidelines. We also excluded publications with the biomarkers measured in umbilical cord, urine, and cerebrospinal fluid or in the mother before childbirth. After selection on the basis of the title and abstract, each manuscript was read to further evaluate whether inclusion or exclusion criteria were met. Two of the authors evaluated the validity of the manuscripts independently concerning patient sample, follow-up, outcome, prognostic variable, and analysis, as described in Table I. If consensus was not found between the two authors, the manuscript was rated and discussed among all three authors until consensus was reached. The rating criteria were slightly modified on the basis of the rating scale by Altman [16]. The modification was performed to obtain the most relevant rating of manuscripts evaluating biomarkers. The modification only implied deletion of the criteria of ‘treatment’ as this was not included in the evaluation of the biomarker. Scoring of the manuscript was either 1 (yes – fulfilled) or 0 (not fulfilled or data not available) points. The highest possible score was 11. We categorized the manuscripts in accordance with Khan et al. [17], with a slight adjustment. We established three categories: (1) high (ⱖ 10 points), (2) moderate (6–9 points), and (3) low-quality studies (⬍ 6 points). The reported sensitivity and specificity of the test, integrated in a receiver operating characteristic (ROC) curve, were used to evaluate the diagnostic value of the biomarkers. We considered an area under the curve (AUC) of ⬎ 0.75 as indicating a moderate biomarker, an AUC ⬎ 0.90 was considered good, and an AUC of 1.0 was an ideal biomarker. Furthermore,
Diagnostic utility of biomarkers for neonatal sepsis Table I. Rating criteria for included manuscripts [16]. A 1 2 B 1
C 1 2
3
D 1
2 3 E 1
2
Sample of patients Sample selection explained (inclusion and exclusion criteria defined). Clinical and demographic description of study population. Follow-up of patient Sufficiently long (baseline blood sample collected at time 0 at suspicion of sepsis and at least one more blood sample minimum after 8 h. (If blood samples are only collected once it should be explained.) Outcome (neonatal sepsis) Unbiased (assessment blinded to prognostic information). Fully defined (reference from WHO criteria or international guidelines) or adequate description of diagnostic criteria for neonatal sepsis. Known for all or high proportion of patients (if the study included both EOS/LOS it has to be shown how many in each group). Prognostic variable Fully defined (in background it has to be described why the biomarker is interesting as a marker of predicting neonatal sepsis). Information on measurement methods (principle of analysis/kit). Precisely measured – description of the coefficient of variation of the biomarker. Analysis Assessment of specificity and sensitivity and/or positive predictive value/negative predictive value and/or ROC curves and other correlation analysis and/or regression analysis. If a study includes neonates with both EOS and LOS they have to adjust for this by statistical analysis. If a study includes only one group, they will get 1 point.
Each item was given the score ‘0’ (not fulfilled) or ‘1’ (fulfilled). EOS, early-onset sepsis; LOS, late-onset sepsis; ROC, receiver operating characteristic; WHO, World Health Organization.
we used the positive and negative predictive values (PPV, NPV) when evaluating the diagnostic value of the biomarkers.
119
292 Rejected on the basis of title and abstract according to the criteria of inclusion and exclusion n = 169.
123
Rejected after reading full manuscripts, did not fulfil criteria of inclusion n = 46. 77
Figure 1. Flow chart of the literature search showing included and excluded manuscripts on biomarkers of neonatal sepsis.
were primarily immunoturbidimetric and nephelometric assays. In the highly rated studies CRP showed a great variability in sensitivity (0.30–0.80) but a higher specificity (0.83–1.00) at symptom onset. There was a tendency towards an increasing sensitivity and specificity after 24 and 48 h, respectively. Both PPV (0.77–1.00) and NPV (0.73–0.98) showed variable results. IL-6 was investigated in 21 studies, of which 8 had a high rating. The detection method for IL-6 was primarily enzyme-linked immunoassays (ELISA). In high-rated studies, IL-6 showed variable sensitivity (0.61–0.89) and specificity (0.65–0.96) at symptom onset, while it was lower after 24 and 48 h. PPV and NPV also showed better results at symptom onset than after 24 h. Procalcitonin was investigated in 18 studies, of which 2 manuscripts had a high rate and 6 had a score of 9. The study populations had early-onset sepsis in both studies, and different cut-off values depending on time after birth were used. The method used for measurement of procalcitonin was an immunoluminometric assay. The sensitivity (0.72–0.79) and specificity (0.72–0.90) at symptom onset were moderate and almost constant after 24 and 48 h, respectively. Only one of the studies presented
Results 20 Number of manuscripts
Of 292 potentially relevant manuscripts, 77 were selected for final review (Figure 1). Among these, 16 (21%) manuscripts [12,13,19–32] had a high rating score of 10 or 11 [18–31], 49 manuscripts had a moderate score of 6–99, while 12 publications had a low score below 6 (Figure 2). Among the moderately rated manuscripts, a considerable number (36%) had a score of 9 (Figures 1–3). Figure 3 shows the numbers and distribution of the rated manuscripts (high/moderate/low). Table II summarizes the diagnostic information of the highrated studies and is briefly commented on below. CRP was the most extensively studied biomarker evaluated in 37 original manuscripts, of which 10 had a high rating. The cut-off points of CRP were between 4 and 15 mg/L and the detection methods
15
10
5
0 1
2
3
4
5 6 7 Rating score
8
9
10 11
Figure 2. Distribution of rating score (0–11) in 77 manuscripts validating biomarkers used for prediction of neonatal sepsis.
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Number of manuscripts
40
30 Score 10-11 Score 6-9 Score < 6
20
10
C
R P IL -6 PC C T D 64 SA TN A Fα IL -8 Se IC -s AM el ec ti sT I n N l-1 F b sT Rp N 75 FR p I 55 H L-1 ep ra ic i C d in D 11 B
0
Biomarkers Figure 3. Evaluated biomarkers for prediction of neonatal sepsis and number of manuscripts investigating each biomarker.
predicted values reporting poor PPV (0.51 and 0.61) and moderate NPV (0.88 and 0.91). CD64 expression was investigated in 12 studies, of which 3 had a high rating. The detection method for CD64 was flow cytometry. In the latter three studies the study population had late-onset sepsis. CD64 showed sensitivity between 0.75 and 0.95 and specificity between 0.77 and 0.88 at symptom onset; both sensitivity and specificity were highest 24 h after symptom onset. Serum amyloid A was investigated in six studies, of which two studies had a high rating. In both these studies sensitivity and specificity were high (above 0.93) at symptom onset and after 24 h. The two highly rated studies presented a variable PPV (0.67 and 0.92), while both studies showed a high NPV (0.97 and 1.00). Seven studies evaluated TNF-α, and three of these had a high rating. Sensitivity (0.54–0.82) and specificity (0.60–0.92) were variable both at symptom onset and 24 h after. Also PPV (0.52–0.90) was variable while the NPV moderately improved (0.73–0.86). Of the 16 high-rated studies, 10 included neonates after their gestational age. Among those eight studies included both preterm and full-term neonates, one study included term neonates, and one study only included preterm infants (⬍ 37 weeks). The last six studies included the neonates on the basis of birth weight, thus one study included neonates with extremely low birth weight and five studies included neonates with very low birth weight. As shown in Table II, four studies included both early- and late-onset sepsis, while five studies included only neonates with early-onset sepsis and seven studies included only neonates with late-onset sepsis.
Of the included 77 publications only 16 were highquality studies. This systematic review provides an indication of which biomarkers are well investigated and which show promising results. Due to the serious complications of neonatal sepsis, the ideal biomarker should have both a high sensitivity and a high specificity. From a clinical point of view, a high sensitivity is the most important to avoid overlooking sepsis in neonates. CRP was the most frequently investigated biomarker for predicting neonatal sepsis. Based on results from the studies included in this review it was clear that CRP lacked sensitivity at symptom onset, whereas specificity was higher at this point. Thus, CRP had limited value as a diagnostic marker of early neonatal sepsis. The results of the present systematic review indicate that the acute phase protein serum amyloid A had a high sensitivity at both symptom onset and 2 days after. As this biomarker also showed high sensitivity as well as high NPV after 2 days, it might also be valuable for monitoring the treatment. However, the clinical usefulness of serum amyloid A is limited by the fact that so far, it is only possible to measure this marker with the both expensive and time-consuming method ELISA. It is worth mentioning that the two high-rated studies evaluating serum amyloid A were published by the same research group. Results on procalcitonin were reported in 18 of the included manuscripts but only 11% of these studies were rated as high-quality manuscripts. However, 6 of the 12 manuscripts with a moderate rating had a score of 9. Sensitivity ranged between 0.72 and 0.79 for procalcitonin; this biomarker will miss up to 28% of truly infected cases. It is important to use age-specific cut-off points for procalcitonin in the first 48 h of life because its value in the first day of life is limited by a marked physiological increase after birth. Chiesa et al. [18] proposed an age-related 95th centile nomogram for term infants while Turner et al. [10] made a similar proposal for preterm infants. The need for age-specific cut-off points of procalcitonin greatly limits its clinical use. The cytokine IL-6 showed a high sensitivity at 0 h; however, it was low after 24 h due to a short half-life. This implies that IL-6 might only be a valuable marker very early in the course of neonatal sepsis. CD64 expression showed promising results for detecting late-onset sepsis. This is in line with a very recent study that investigated CD64 expression for diagnosis of early-onset sepsis in neonates with very low birth weight [33].
Diagnostic utility of biomarkers for neonatal sepsis
121
Table II. Overview of manuscripts with a high score (10 or 11) for evaluation of biomarkers for predicting neonatal sepsis (n ⫽ 16).
Biomarker
Ref. no.
Sample size: EOS/LOS
Reference interval
Cut-off value
Sensitivity
Specificity
PPV
NPV
12
104 EOS
NR
7.0 mg/L
0 h: 0.30
0 h: 0.98
0 h: 0.78
0 h: 0.83
144 EOS; 2 LOS Immunoturbidimetric assay 90 EOS Immunoturbidimetric assay 68 LOS Immunoturbidimetric assay
5.0–37.0
5.0 mg/L
24 h: 0.91 0.7
24 h: 0.98 0.91
NR
5.0 mg/L
0.69
0.86
0.84
0.73
NR
12.0 mg/L
0 h: 0.6
0 h: 1.0
0 h: 1.0
0 h: 0.75
12 EOS; 8 LOS; Immunoturbidimetric 60 neonates assay
0 h: 0.0–1.1
15.2 mg/L
24 h: 0.82 0 h: 0.80
24 h:0.96 0 h: 0.92
0 h: 3.1–3.5 24 h: 4.1–5.4 48 h: 4.0–5.1 NR 0 h: 0.0–4.2 8 h: 3.0–7.3 24 h: 1.3–6.3 0 h: 0.3–3.0
0 h: 4.0 mg/L 24 h:10 mg/L 48 h:10 mg/L 15.0 mg/L
10.0 mg/dl
24 h: NR 0 h: 0.74 24 h: 0.89 48 h: 0.89 0.5 0 h: 0.32 8 h: 0.53 24 h: 0.84 0 h: 0.69
24 h: NR 0 h: 0.83 24 h: 0.87 48 h: 0.84 0.93 0 h: 0.97 8 h:0.92 24 h:0.90 0 h: 0.96
NR NR NR 0 h: 0.86 0 h: 0.74 8 h: 0.77 8 h: 0.80 24 h: 0.80 24 h: 0.92 0 h: 0.93 0 h: 0.80
12.0 mg/L
24 h: 0.78 0 h: 0.65
24 h: 0.94 0 h: 0.99
0 h: 0.96
0 h: 1.0 ng/ml
24 h: 0.72 48 h: 0.65 0 h: 0.79
24 h: 1.00 48 h:1.00 0 h: 0.95
24 h: 1.00 24 h: 0.90 48 h: 1.00 48 h: 0.87 NR NR
24 h: 0.89 48 h: 0.89 0 h: 0.72
24 h: 0.87 48 h: 0.84 0 h: 0.72
0 h: 0.51
24 h: 0.74 48 h: 0.78
24 h: 0.81 48 h: 0.79
24 h: 0.61 24 h: 0.88 48 h: 0.56 48 h: 0.91
0 h: 0.93 8 h: 0.85 24 h: 0.77 0 h: 0.95
0 h: 0.87 0 h: 0.97 8 h: 0.76 8 h: 1.00 24 h: 0.67 24 h: 0.98 0 h: 0.85 0 h: 0.99
Method
Acute-phase proteins CRP
20 21 25
28
NR
Procalcitonin
Immunoturbidimetric assay
27
134 EOS
Nephelometric assay
22 19
101 LOS 116 LOS
Nephelometric assay ELISA
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent ELISA
26
80 LOS
Immunoturbidimetric assay
27
24
134 EOS
317 EOS
Immunoluminometric assay
24 h: 0.4–3.0 0 h: 1.0–3.0
0 h: 0.18–.27
10.0 mg/L
24 h: 4.28–7.3 24 h: 100ng/ml 48 h: 1.69–2.70 48 h: 50 ng/ml Immunoluminometric 0 h: 0.23–0.64 0 h: 0.55 ng/ml assay 24 h: 0.68–3.78 24 h: 4.7 ng/ml 48 h: 0.45–1.48 48 h: 1.7 ng/ml
24 h: 0.91 4 h: 0.98 AUC ⫽ 0.86
24 h: 0.95 24 h: 0.87 AUC ⫽ 0.75
NR
0 h: 0.87
0 h: 0.88
Serum amyloid A
IL-1ra Hepcidin Cell surface antigens
19
116 LOS
ELISA
0 h: 2.0–8.0 8 h: 2.0–7.0 24 h: 1.0–9.0 NR
12
104 EOS
Photometric immunoassay
22 13
101 LOS 44 LOS
26
80 LOS
10.0 mg/L
0 h: 8.0 mg/L
0 h: 0.95 8 h: 1.00 24 h: 0.97 0 h: 0.96
ELISA Capture ELISA
0–28 240 5.3–89.8 ng/ml
24 h: 10.0 mg/L 12 000 pg/ml 92.2 ng/ml
24 h: 0.96 1 0.76
24 h: 0.98 0.92 1
24 h: 0.92 24 h: 0.99 NR NR 1 0.87
Flow cytometry
0 h: 1116–2890 4000 molecules/ cell
0 h: 0.95
0 h: 0.88
0 h: 0.80
CD64
CD64 index
29
24 h: 0.97 24 h: 0.90 24 h: 0.80 24 h: 0.99 48 h: 0.87 48 h: 0.86 48 h: 0.80 48 h: 0.93 EOS: 1.00; EOS: 0.68; NR NR LOS: 0.75 LOS: 0.77 0.88 1 AUC ⫽ 0.95
3 EOS; 47 LOS; 749 neonates 16 LOS; 16 controls
Flow cytometry
NR
Automated Leuko64 Assay kit
0.5–2.8
EOS: 2.38; LOS: 3.62 2.85
30 31
30 LOS 106 EOS
Flow cytometry Flow cytometry
0 h: 45.0–146.0 NR
139.0 RFU 60 FU
0 h: 1.00 0.96
0 h: 0.56 1
0.5 0.99
30
30 LOS
Flow cytometry
0 h: 43.0–125.0
225.0 RFU
0 h: 0.86
0 h: 0.94
0.86
32
0 h: 0.97
CD11b neutrophils 1 1
CD11b monocytes 0.94 (Continued)
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S. S. Hedegaard et al.
Table II. (Continued)
Biomarker
Ref. no.
Sample size: EOS/LOS
Method
Reference interval
19
116 LOS
ELISA
0 h: 4.0–22.0
Cut-off value
Sensitivity
Specificity
PPV
NPV
Cut–off: 31.0 pg/ml
0 h: 0.78
0 h: 0.89
0 h: 0.64
0 h: 0.88
8 h:1.00 24 h:0.97 0 h: 0.89 24 h: 0.71 48 h: 0.70 0.65 0.83 0 h: 0.96 24 h: 0.89 0 h: 0.92 24 h: 0.93 48 h: 0.91 0 h. 0.73
8 h: 1.00 8 h: 0.80 24 h: 0.98 24 h: 0.69 NR NR
Cytokines/ chemokines Interleukin-6
8 h: 7.0–28.0 24 h: 2.0–14.0 0 h: 16.1–26.6 0 h: 200.0 ng/L 24 h: 8.8–15.0 24 h: 30.0 ng/L 48 h: 7.3–11.7 48 h: 20.0 ng/L 10.0–3028 20 pg/ml 0.0–1253 25.0 pg/ml NR 31.0 pg/ml
27
134 EOS
ELISA
20 22 25
144 EOS; 22 LOS 101 LOS 68 LOS
Colorimetric assay ELISA ELISA
26
80 LOS
ELISA
0 h: 1.0–6.0
31.0 pg/ml
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
0 h: 6.0–22.0
18.0 pg/ml
8 h: 0.47 24 h: 0.19 0 h: 0.74 24 h: 0.63 48 h: 0.53 0.65 0.89 0 h: 0.89 24 h: 0.67 0 h: 0.78 24 h: 0.44 48 h: 0.46 0 h: 0.76
28
12 EOS; 8 LOS; 60 neonates
Chemiluminescent immunometric assay
24 h: 5.0–18.0 0 h: 1.0–654.0
30.0 pg/ml
24 h: 0.63 0 h: 0.61
24 h:0.76 0 h: 0.80
NR
NR
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
0 h: 20.0–134.0
100.0 pg/ml
0 h: 0.75
0 h: 0.66
0 h: 0.60
0 h: 0.80
28
12 EOS; 8 LOS; 60 neonates
Chemiluminescent immunometric assay
24 h: 16.0–71.0 0 h: 17.6–140.0
63.0 pg/ml
24 h: 0.49 0 h: 0.62
24 h: 0.79 0 h: 0.96
NR
NR
24 h: NR
24 h: NR
0 h: 0.82 24 h: 0.58 0 h: 0.60
0 h: 0.68 24 h: 0.84 0 h: 0.87
24 h: NA
24 h: NR
0 h: 0.66
0 h: 0.60
0 h: 0.52
24 h: 0.49 0 h: 0.82 24 h: 0.82 0 h: 0.54
24 h: 0.72 0 h: 0.86 24 h: 0.93 0 h: 0.92
0 h: 0.82 0 h: 0.85 24 h: 0.90 24 h: 0.86 NR NR
24 h: NR
24 h: NR
0.78 0.82
0.9 0.64
0 h: 0.64 24 h: 0.51
0 h: 0.89 24 h: 0.93
NR NR NR NR 0 h: 0.95 0 h: 0.91 24 h: 0.84 24 h: 0.77 0 h: 0.81 0 h: 0.91 24 h: 0.72 24 h: 0.81 48 h: 0.68 48 h: 0.80 0 h: 0.67 0 h: 0.81
Interleukin-8
Interleukin-1b 25
68 LOS
ELISA
NR
1.0 pg/ml
28
12 EOS; 8 LOS; 60 neonates
ELISA
0 h: 0.2–100
24.5 pg/ml
0 h: 0.68 0 h: 0.82 24 h: 0.75 24 h: 0.71 NR NR
TNF-α 23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
25
68 LOS
28
12 EOS; 8 LOS; 60 neonates
0 h: 6.0–16.0
12.0 pg/ml
ELISA
24 h: 5.0–15.0 NR
17.0 pg/ml
ELISA
0 h: 1.0–32.1
3.5 pg/ml
0 h:0.73
Soluble adhesion markers ICAM 20 21 22
144 EOS; 22 LOS 90 EOS 101 LOS
ELISA ELISA ELISA
95.2–500.0 NR 68.0–1304.0
NR 300.0 ng/ml 425.0 ng/ml
20 25
144 EOS; 22 LOS 68 LOS
ELISA ELISA
2.0–217.8 NR
NR 174 ng/ml
AUC ⫽ 0.72 0.9 NR
0.8 NR
Soluble E-selectin AUC ⫽ 0.72 0 h: 0.83 0 h: 0.75 24 h: 0.85 24 h: 0.70
AUC, area under the curve; CRP, C-reactive protein; EOS, early-onset sepsis; ICAM, intercellular adhesion molecule; IL, interleukin; LOS, late-onset sepsis; nCD64, neutrophilic CD64; NPV, negative predicted value; NR, not reported; PPV, positive predicted value; RFU, relative fluorescence unit; SAA, serum amyloid A; Sensitivity 0 h, sensitivity at symptom onset; Sensitivity 24 h, sensitivity after 24 h of symptoms; TNFα, tumor necrosis factor-α.
Diagnostic utility of biomarkers for neonatal sepsis The cell surface antigen CD11b showed high sensitivity and specificity for both early and late diagnosis. Also the acute phase proteins hepcidin and IL-1ra showed promising results. These results were based on a high-quality manuscript, but as evidence was based on only one manuscript the results should be interpreted with caution. As shown in Figure 2, around one-third of the manuscripts had a rating score of 9. We decided beforehand that a high-rated study should comply with almost every criterion indicated by a score of 10 or 11; only a limited part of the studies fulfilled this requirement. One of the strengths of the present review was the compliance with defined stringent criteria for performing a systematic review. The literature search was performed systemically, the basis for the rating of the manuscripts was strict and based on a score developed beforehand, and strict assessment of the quality of the studies was performed independently by at least two authors. Some potential limitations of our review should be considered. During the ratings, all rating issues were assessed as equally important. This might be a problem, especially considering the importance of mentioning the coefficient of variation and blinding of the technicians. More often a coefficient of variation of a non-automated ELISA analysis will appear in the manuscripts compared with an automated high volume test. The same difference exists for information about a laboratory technician being blinded to the results; this was probably the case for most of the studies but it was only mentioned in a few. Therefore, the studies investigating biomarkers measured by non-automated methods had a tendency to be rated higher than studies evaluating markers analyzed by well-known automated methods. An evaluation of biomarkers of infection requires assessment of the PPVs and NPVs; however, in six (38%) of the studies with a high rate [20,22,27– 29,32] these data were not available. In 50% of the studies the population included both preterm and term neonates, even though the clinical picture of infection in preterm and term infants is quite different. Also the kinetics of the biological processes associated with inflammatory responses and biomarkers used for assessment of infection may differ markedly in correlation with gestational age. We differentiated between early- and late-onset sepsis, although the limit between early and late sepsis varies throughout publications. Most studies use 72 h to mark a limit for early- or lateonset sepsis; however, in clinical practice the clinician often use the same biomarker and the same reference interval for diagnosis of neonatal sepsis regardless of debut of the sepsis.
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During the literature search we used the term: ‘biological markers’ and chose not to include studies investigating 16s ribosomal RNA. This might, however, be a promising future marker for predicting neonatal sepsis. There was a great variability of the definition of sepsis between the studies. The majority of the highrated studies used clinical conditions to make the diagnosis [12,19–27], whereas others used a positive blood culture as the gold standard [28–30]. Four of the 16 high-rated studies included in the present review [19,24,25,30] compared the biomarker in the sepsis group with a group of healthy neonates. However, in clinical practice it is often a challenge to differentiate septic neonates from neonates that are ill due to other diseases, for example, respiratory distress syndrome, necrotizing enterocolitis, asphyxia, or hemodynamic failure. In the light of multidrug resistance, it is mandatory to avoid unnecessary use of antibiotics. In addition, the intravenous administration of antibiotics demands hospitalization of the neonate and thereby separation from the mother during these first important days of life. An ideal biomarker should thus identify the neonate at risk and at the same time be useful to distinguish the infected neonate from the neonate that is ill due to other diseases. In conclusion, the present review showed that only a few high-quality clinical studies have evaluated biomarkers for neonatal sepsis. As yet, there is no single ideal biomarker for neonatal sepsis and each marker has strengths and weaknesses. We suggest that serum amyloid A should be investigated further in clinical settings.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Sankar MJ, Agarwal R, Deorari AK, Paul VK. Sepsis in the newborn. Indian J Pediatr 2008;75:261–6. [2] Kliegman R. In: Kliegman R, Stanton B, St Geme J, Schor N, Behrman R, editors. Nelson textbook of pediatrics, 19th edn. Elsevier Saunders; 2013 Chapter 109, p 794. [3] Goldstein B, Giroir B, Randolph A. International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med 2005;6:2–8. [4] Gotoft SP. Infection of the neonatal infant. WB Saunders Co. 2000; pp 538–49. [5] Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed 2004;89:229–235. [6] Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999; 340:448–54.
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[7] Ng PC, Lam HS. Diagnostic markers for neonatal sepsis. Curr Opin Pediatr 2006;18:125–31. [8] Arkader R, Troster EJ, Lopes MR, Junior RR, Carcillo JA, Leone C, et al. Procalcitonin does discriminate between sepsis and systemic inflammatory response syndrome. Arch Dis Child 2006;91:117–20. [9] Fendler WM, Piotrowski AJ. Procalcitonin in the early diagnosis of nosocomial sepsis in preterm neonates. J Paediatr Child Health 2008;44:114–18. [10] Turner D, Hammerman C, Rudensky B, Schlesinger Y, Goia C, Schimmel MS. Procalcitonin in preterm infants during the first few days of life: introducing an age related nomogram. Arch Dis Child Fetal Neonatal Ed 2006;91:283–6. [11] Pizzini C, Mussap M, Plebani M, Fanos V. C-reactive protein and serum amyloid A protein in neonatal infections. Scand J Infect Dis 2000;32:229–35. [12] Arnon S, Litmanovitz I, Regev RH, Bauer S, Shainkin-Kestenbaum R, Dolfin T. Serum amyloid A: an early and accurate marker of neonatal early-onset sepsis. J Perinatol 2007;27:297–302. [13] Wu TW, Tabangin M, Kusano R, Ma Y, Ridsdale R, Akinbi H. The utility of serum hepcidin as a biomarker for late-onset neonatal sepsis. J Pediatr 2013;162:67–71. [14] Calder PC, Ahluwalia N, Albers R, Bosco N, Bourdet-Sicard R, Haller D. A consideration of biomarkers to be used for evaluation of inflammation in human nutritional studies. Br J Nutr 2013;109(Suppl 1):S1–34. [15] Noor MK, Shahidullah M, Mutanabbi M, Barua C, Mannan Ma, Afroza S. Comparison between CRP and IL-6 as early markers of neonatal sepsis. Mymensingh Med J 2008;17:72–76. [16] Altman DG. Systematic reviews of evaluations of prognostic variables. BMJ 2001;28;323:224–8. [17] Khan K. Systematic reviews to support evidence-based medicine: how to review and apply findings of healthcare research, 2nd edn. Hodder Arnold; 2011. [18] Chiesa C, Panero A, Rossi N, Stegagno M, De Giusti M, Osborn JF, et al. Reliability of procalcitonin concentrations for the diagnosis of sepsis in critically ill neonates. Clin Infect Dis 1998;26:664–72. [19] Arnon S, Litmanovitz I, Regev R, Bauer S, Lis M, ShainkinKestenbaum R, et al. Serum amyloid A protein is a useful inflammatory marker during late-onset sepsis in preterm infants. Biol Neonate 2005;87:105–10. [20] Dollner H, Vatten L, Austgulen R. Early diagnostic markers for neonatal sepsis: comparing C-reactive protein, interleukin-6, soluble tumour necrosis factor receptors and soluble adhesion molecules. J Clin Epidemiol 2001;54:1251–7. [21] Hansen AB, Verder H, Staun-Olsen P. Soluble intercellular adhesion molecule and C-reactive protein as early markers of infection in newborns. J Perinat Med 2000;28:97–103.
[22] Kuster 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;17;352:1271–7. [23] Laborada G, Rego M, Jain A, Guliano M, Stavola J, Ballabh P et al. Diagnostic value of cytokines and C-reactive protein in the first 24 hours of neonatal sepsis. Am J Perinatol 2003;20:491–501. [24] Lopez Sastre JB, Solis DP, Serradilla VR, Colomer BF, Cotallo GD; Grupo de Hospitales Castrillo. Evaluation of procalcitonin for diagnosis of neonatal sepsis of vertical transmission. BMC Pediatr 2007;7:9 [25] Ng PC, Cheng SH, Chui KM, Fok TF, Wong MY, Wong W, et al. Diagnosis of late onset neonatal sepsis with cytokines, adhesion molecule, and C-reactive protein in preterm very low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1997;77:F221–F227. [26] Ng PC, Li K, Wong RP, Chiu KM, Wong E, Fok TF. Neutrophil CD64 expression: a sensitive diagnostic marker for late-onset nosocomial infection in very low birthweight infants. Pediatr Res 2002;51:296–303. [27] Chiesa C, Pellegrini G, Panero A, Osborn JF, Signore F, Assumma M, et al. C-reactive protein, interleukin-6, and procalcitonin in the immediate postnatal period: influence of illness severity, risk status, antenatal and perinatal complications, and infection. Clin Chem 2003;49:60–8. [28] Santana RC, Garcia-Munoz F, Reyes D, Gonzales G, Dominguez C, Domenech E. Role of cytokines (interleukin1beta, 6, 8, tumour necrosis factor-alpha, and soluble receptor of interleukin-2) and C-reactive protein in the diagnosis of neonatal sepsis. Acta Paediatr 2003;92:221–7. [29] Streimish I, Bizzarro M, Northrup V, Wan C, Renna S, Koval N, et al. Neutrophil CD64 as a diagnostic marker in neonatal sepsis. Pediatr Infect Dis J 2012;31:777–81. [30] 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. [31] Weirich E, Rabin RL, Maldonado Y, Benitz W, Modler S, Herzenberg LA, et al. Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection. J Pediatr 1998;132:445–51. [32] Mazzucchelli I, Garofoli F, Ciardelli L, Borghesi A, Tzialla C, Di CA, et al. Diagnostic performance of triggering receptor expressed on myeloid cells-1 and CD64 index as markers of sepsis in preterm newborns. Pediatr Crit Care Med 2013;14:178–82. [33] Motta M, Zini A, Regazzoli A, Garzoli E, Chirico G, Caimi L, et al. Diagnostic accuracy and prognostic value of the CD64 index in very low birth weight neonates as a marker of early-onset sepsis. Scand J Infect Dis 2014;46:433–9.
ISSN: 2374-4235 (Print) 2374-4243 (Online) Journal homepage: http://www.tandfonline.com/loi/infd20
Diagnostic utility of biomarkers for neonatal sepsis – a systematic review Sofie Sommer Hedegaard, Kirsten Wisborg & Anne-Mette Hvas To cite this article: Sofie Sommer Hedegaard, Kirsten Wisborg & Anne-Mette Hvas (2015) Diagnostic utility of biomarkers for neonatal sepsis – a systematic review, Infectious Diseases, 47:3, 117-124, DOI: 10.3109/00365548.2014.971053 To link to this article: http://dx.doi.org/10.3109/00365548.2014.971053
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Date: 13 July 2017, At: 11:43
Infectious Diseases, 2015; 47: 117–124
REVIEW ARTICLE
Diagnostic utility of biomarkers for neonatal sepsis – a systematic review
SOFIE SOMMER HEDEGAARD1, KIRSTEN WISBORG2 & ANNE-METTE HVAS3 From the 1Department of Pediatrics, Aarhus University Hospital, 2Head & Neuro Centre, Aarhus University Hospital, and 3Department of Clinical Biochemistry, Aarhus University Hospital and Aarhus University, Aarhus, Denmark
Abstract Neonatal sepsis is a major cause of morbidity and mortality. Early diagnosis and treatment of the neonate with suspected sepsis are essential to prevent life-threatening complications. Diagnosis of neonatal sepsis is a challenge due to non-specific clinical signs and the fact that infection markers are difficult to interpret in the first and critical phase of neonatal sepsis. The objective of the present study was to systematically evaluate existing evidence of the diagnostic utility of biomarkers for prediction of sepsis in neonates. We conducted a systematic literature search performed in PubMed and Embase. The study population was neonates with gestation age ⬎ 24 weeks in their first 28 days of life with suspected sepsis. The included manuscripts were rated due to criteria from a modified rating scale developed by Douglas Altman. Of 292 potentially relevant manuscripts, 77 fulfilled the inclusion and exclusion criteria; 16 (21%) were rated as high-quality studies. C-reactive protein (CRP) was the most extensively studied biomarker evaluated. The high-quality studies indicated that the acute phase protein serum amyloid A had high sensitivity, both at onset of symptoms and 2 days after. The studies evaluating serum amyloid A presented a variable positive predictive value (PPV, 0.67 and 0.92) with a high negative predictive value (NPV, 0.97 and 1.00). The existing evidence of the diagnostic value of serum amyloid A for neonatal sepsis showed promising results, and should be further investigated in clinical settings.
Keywords: Newborn, procalcitonin, C-reactive protein, CRP, serum amyloid A
Introduction Worldwide, infection is the single largest cause of neonatal death [1]. In developed countries the incidence of neonatal bacterial sepsis varies from 1 to 4 cases per 1000 live births [2]. Neonatal septicemia is defined as a clinical syndrome of systemic illness accompanied by bacterium in the first 28 days of life [3]. Neonatal sepsis is classified in two major categories: early-onset sepsis, which usually presents with respiratory distress within 72 h of age; and late-onset sepsis, which usually presents with septicemia after 72 h of age [4]. Early diagnosis and treatment of the neonate with suspected sepsis are essential to prevent severe and life-threatening complications. Diagnosis of neonatal sepsis is a challenge due to variable and non-specific clinical signs; moreover, infection markers are difficult to evaluate in the first and critical phase [5]. To avoid serious complications such as meningitis,
cerebral abscess, and neonatal death, treatment is initiated in many newborns with clinical signs of sepsis without proven infection [1]. The utility of infection markers in the diagnosis of neonatal sepsis has been evaluated in many studies with different results. The biomarkers potentially used for diagnosis of neonatal sepsis can be divided into four groups: acute phase proteins, cell surface antigens, cytokines and chemokines, and soluble adhesion molecules. The acute phase proteins investigated in multiple studies concerning neonatal sepsis are C-reactive protein (CRP), procalcitonin, serum amyloid A, and hepcidin. CRP is commonly used as an indicator for bacterial sepsis in neonates and children. CRP has several drawbacks; it is not useful as an early phase infection marker because it can only be detected 12 h after manifestation of clinical signs, it reaches a plateau after 20–72 h [6], and it lacks specificity [7].
Correspondence: Sofie Sommer Hedegaard MD, Department of Pediatrics, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark. Tel: ⫹ 45 28747699. E-mail: [email protected] (Received 16 July 2014 ; accepted 22 September 2014 ) ISSN 2374-4235 print/ISSN 2374-4243 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.971053
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A previous study indicated that procalcitonin had a higher predictive value than CRP in detecting sepsis in patients [8]. Procalcitonin is released into the circulation within 3 h after endotoxin injection, it reaches a plateau at 6 h, and remains elevated for 24 h. This makes procalcitonin a promising marker for early-phase infection and sensitive identification of infected infants [9]. However, in neonates its value in the first day of life is limited by a marked physiological increase after birth [10]. Serum amyloid A has shown as high as a 1000-fold increase during infection and increases 8–24 h after the onset of sepsis [11]. This marker has been reported to be useful in various acute conditions and in the diagnosis of sepsis in neonates [12]. Hepcidin, a highly conserved antimicrobial peptide, plays a critical role in inflammation and iron homeostasis [13], and is one of the later markers for diagnosis of neonatal sepsis. In the group of cell surface antigens, CD64 and CD11b are the primary biomarkers investigated in studies on neonatal sepsis. CD64 is a high-affinity receptor normally expressed by monocytes and macrophages, and only to a small extent on neutrophils [5]. During bacterial infection, neutrophilic expression of CD64 (nCD64) is markedly increased; nCD64 density thus has the potential to differentiate between infected and non-infected neonates at a very early stage [5]. In the group of cytokines and chemokines, interleukin (IL) IL-1, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and soluble TNF receptor (sTNFR) have been evaluated for diagnosis of neonatal sepsis. The biological function of cytokines and chemokines is among other things to induce fever and attract and activate neutrophilocytes [14]. IL-6, TNF-α, and IL-1 all play a critical role in the induction of acutephase proteins such as CRP synthesis in the liver; thus, these cytokines can be detected in the blood earlier than CRP [15]. In the group of soluble adhesion markers, E-selectin and intercellular adhesion markers (ICAM) are the primary biomarkers evaluated for diagnosis of neonatal sepsis. The expression of E-selectin and ICAM is stimulated by cytokines IL-1 and TNF. The function of these markers is adhesion and recruitment of leukocytes to endothelial cells and platelet aggregation [12]. The objective of the present study was to systematically evaluate existing evidence from 1980 to January 2014 of the diagnostic value of biomarkers for predicting sepsis in neonates. Literature search The literature search was performed in the databases PubMed and Embase using the following keywords:
‘infant newborn,’ ‘neonates,’ ‘sepsis,’ ‘infection,’ ‘sensitivity and specificity,’ ‘predictors,’ ‘early diagnosis,’ and ‘biological markers’ with the limitations ‘humans’ and ‘English language.’ All studies from 1980 until January 2014 were included in the present review. Duplicate publications were identified and removed. Relevant publications were identified on the basis of titles and abstracts using the following inclusion and exclusion criteria. Inclusion criteria: (1) the study population was neonates with gestational age ⬎ 24 weeks in their first 28 days of life with suspected sepsis, (2) the study population had cultureproven sepsis or suspected infection based on clinical algorithm, (3) the manuscript evaluated biomarkers for diagnosing the risk of neonatal sepsis, (4) the specific biomarker was measured in serum or plasma, (5) the publication was an original paper including short communications and technical notes. The following publications were excluded: reviews, metaanalyses, editorials, letters to the editor, case reports, and guidelines. We also excluded publications with the biomarkers measured in umbilical cord, urine, and cerebrospinal fluid or in the mother before childbirth. After selection on the basis of the title and abstract, each manuscript was read to further evaluate whether inclusion or exclusion criteria were met. Two of the authors evaluated the validity of the manuscripts independently concerning patient sample, follow-up, outcome, prognostic variable, and analysis, as described in Table I. If consensus was not found between the two authors, the manuscript was rated and discussed among all three authors until consensus was reached. The rating criteria were slightly modified on the basis of the rating scale by Altman [16]. The modification was performed to obtain the most relevant rating of manuscripts evaluating biomarkers. The modification only implied deletion of the criteria of ‘treatment’ as this was not included in the evaluation of the biomarker. Scoring of the manuscript was either 1 (yes – fulfilled) or 0 (not fulfilled or data not available) points. The highest possible score was 11. We categorized the manuscripts in accordance with Khan et al. [17], with a slight adjustment. We established three categories: (1) high (ⱖ 10 points), (2) moderate (6–9 points), and (3) low-quality studies (⬍ 6 points). The reported sensitivity and specificity of the test, integrated in a receiver operating characteristic (ROC) curve, were used to evaluate the diagnostic value of the biomarkers. We considered an area under the curve (AUC) of ⬎ 0.75 as indicating a moderate biomarker, an AUC ⬎ 0.90 was considered good, and an AUC of 1.0 was an ideal biomarker. Furthermore,
Diagnostic utility of biomarkers for neonatal sepsis Table I. Rating criteria for included manuscripts [16]. A 1 2 B 1
C 1 2
3
D 1
2 3 E 1
2
Sample of patients Sample selection explained (inclusion and exclusion criteria defined). Clinical and demographic description of study population. Follow-up of patient Sufficiently long (baseline blood sample collected at time 0 at suspicion of sepsis and at least one more blood sample minimum after 8 h. (If blood samples are only collected once it should be explained.) Outcome (neonatal sepsis) Unbiased (assessment blinded to prognostic information). Fully defined (reference from WHO criteria or international guidelines) or adequate description of diagnostic criteria for neonatal sepsis. Known for all or high proportion of patients (if the study included both EOS/LOS it has to be shown how many in each group). Prognostic variable Fully defined (in background it has to be described why the biomarker is interesting as a marker of predicting neonatal sepsis). Information on measurement methods (principle of analysis/kit). Precisely measured – description of the coefficient of variation of the biomarker. Analysis Assessment of specificity and sensitivity and/or positive predictive value/negative predictive value and/or ROC curves and other correlation analysis and/or regression analysis. If a study includes neonates with both EOS and LOS they have to adjust for this by statistical analysis. If a study includes only one group, they will get 1 point.
Each item was given the score ‘0’ (not fulfilled) or ‘1’ (fulfilled). EOS, early-onset sepsis; LOS, late-onset sepsis; ROC, receiver operating characteristic; WHO, World Health Organization.
we used the positive and negative predictive values (PPV, NPV) when evaluating the diagnostic value of the biomarkers.
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292 Rejected on the basis of title and abstract according to the criteria of inclusion and exclusion n = 169.
123
Rejected after reading full manuscripts, did not fulfil criteria of inclusion n = 46. 77
Figure 1. Flow chart of the literature search showing included and excluded manuscripts on biomarkers of neonatal sepsis.
were primarily immunoturbidimetric and nephelometric assays. In the highly rated studies CRP showed a great variability in sensitivity (0.30–0.80) but a higher specificity (0.83–1.00) at symptom onset. There was a tendency towards an increasing sensitivity and specificity after 24 and 48 h, respectively. Both PPV (0.77–1.00) and NPV (0.73–0.98) showed variable results. IL-6 was investigated in 21 studies, of which 8 had a high rating. The detection method for IL-6 was primarily enzyme-linked immunoassays (ELISA). In high-rated studies, IL-6 showed variable sensitivity (0.61–0.89) and specificity (0.65–0.96) at symptom onset, while it was lower after 24 and 48 h. PPV and NPV also showed better results at symptom onset than after 24 h. Procalcitonin was investigated in 18 studies, of which 2 manuscripts had a high rate and 6 had a score of 9. The study populations had early-onset sepsis in both studies, and different cut-off values depending on time after birth were used. The method used for measurement of procalcitonin was an immunoluminometric assay. The sensitivity (0.72–0.79) and specificity (0.72–0.90) at symptom onset were moderate and almost constant after 24 and 48 h, respectively. Only one of the studies presented
Results 20 Number of manuscripts
Of 292 potentially relevant manuscripts, 77 were selected for final review (Figure 1). Among these, 16 (21%) manuscripts [12,13,19–32] had a high rating score of 10 or 11 [18–31], 49 manuscripts had a moderate score of 6–99, while 12 publications had a low score below 6 (Figure 2). Among the moderately rated manuscripts, a considerable number (36%) had a score of 9 (Figures 1–3). Figure 3 shows the numbers and distribution of the rated manuscripts (high/moderate/low). Table II summarizes the diagnostic information of the highrated studies and is briefly commented on below. CRP was the most extensively studied biomarker evaluated in 37 original manuscripts, of which 10 had a high rating. The cut-off points of CRP were between 4 and 15 mg/L and the detection methods
15
10
5
0 1
2
3
4
5 6 7 Rating score
8
9
10 11
Figure 2. Distribution of rating score (0–11) in 77 manuscripts validating biomarkers used for prediction of neonatal sepsis.
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Number of manuscripts
40
30 Score 10-11 Score 6-9 Score < 6
20
10
C
R P IL -6 PC C T D 64 SA TN A Fα IL -8 Se IC -s AM el ec ti sT I n N l-1 F b sT Rp N 75 FR p I 55 H L-1 ep ra ic i C d in D 11 B
0
Biomarkers Figure 3. Evaluated biomarkers for prediction of neonatal sepsis and number of manuscripts investigating each biomarker.
predicted values reporting poor PPV (0.51 and 0.61) and moderate NPV (0.88 and 0.91). CD64 expression was investigated in 12 studies, of which 3 had a high rating. The detection method for CD64 was flow cytometry. In the latter three studies the study population had late-onset sepsis. CD64 showed sensitivity between 0.75 and 0.95 and specificity between 0.77 and 0.88 at symptom onset; both sensitivity and specificity were highest 24 h after symptom onset. Serum amyloid A was investigated in six studies, of which two studies had a high rating. In both these studies sensitivity and specificity were high (above 0.93) at symptom onset and after 24 h. The two highly rated studies presented a variable PPV (0.67 and 0.92), while both studies showed a high NPV (0.97 and 1.00). Seven studies evaluated TNF-α, and three of these had a high rating. Sensitivity (0.54–0.82) and specificity (0.60–0.92) were variable both at symptom onset and 24 h after. Also PPV (0.52–0.90) was variable while the NPV moderately improved (0.73–0.86). Of the 16 high-rated studies, 10 included neonates after their gestational age. Among those eight studies included both preterm and full-term neonates, one study included term neonates, and one study only included preterm infants (⬍ 37 weeks). The last six studies included the neonates on the basis of birth weight, thus one study included neonates with extremely low birth weight and five studies included neonates with very low birth weight. As shown in Table II, four studies included both early- and late-onset sepsis, while five studies included only neonates with early-onset sepsis and seven studies included only neonates with late-onset sepsis.
Of the included 77 publications only 16 were highquality studies. This systematic review provides an indication of which biomarkers are well investigated and which show promising results. Due to the serious complications of neonatal sepsis, the ideal biomarker should have both a high sensitivity and a high specificity. From a clinical point of view, a high sensitivity is the most important to avoid overlooking sepsis in neonates. CRP was the most frequently investigated biomarker for predicting neonatal sepsis. Based on results from the studies included in this review it was clear that CRP lacked sensitivity at symptom onset, whereas specificity was higher at this point. Thus, CRP had limited value as a diagnostic marker of early neonatal sepsis. The results of the present systematic review indicate that the acute phase protein serum amyloid A had a high sensitivity at both symptom onset and 2 days after. As this biomarker also showed high sensitivity as well as high NPV after 2 days, it might also be valuable for monitoring the treatment. However, the clinical usefulness of serum amyloid A is limited by the fact that so far, it is only possible to measure this marker with the both expensive and time-consuming method ELISA. It is worth mentioning that the two high-rated studies evaluating serum amyloid A were published by the same research group. Results on procalcitonin were reported in 18 of the included manuscripts but only 11% of these studies were rated as high-quality manuscripts. However, 6 of the 12 manuscripts with a moderate rating had a score of 9. Sensitivity ranged between 0.72 and 0.79 for procalcitonin; this biomarker will miss up to 28% of truly infected cases. It is important to use age-specific cut-off points for procalcitonin in the first 48 h of life because its value in the first day of life is limited by a marked physiological increase after birth. Chiesa et al. [18] proposed an age-related 95th centile nomogram for term infants while Turner et al. [10] made a similar proposal for preterm infants. The need for age-specific cut-off points of procalcitonin greatly limits its clinical use. The cytokine IL-6 showed a high sensitivity at 0 h; however, it was low after 24 h due to a short half-life. This implies that IL-6 might only be a valuable marker very early in the course of neonatal sepsis. CD64 expression showed promising results for detecting late-onset sepsis. This is in line with a very recent study that investigated CD64 expression for diagnosis of early-onset sepsis in neonates with very low birth weight [33].
Diagnostic utility of biomarkers for neonatal sepsis
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Table II. Overview of manuscripts with a high score (10 or 11) for evaluation of biomarkers for predicting neonatal sepsis (n ⫽ 16).
Biomarker
Ref. no.
Sample size: EOS/LOS
Reference interval
Cut-off value
Sensitivity
Specificity
PPV
NPV
12
104 EOS
NR
7.0 mg/L
0 h: 0.30
0 h: 0.98
0 h: 0.78
0 h: 0.83
144 EOS; 2 LOS Immunoturbidimetric assay 90 EOS Immunoturbidimetric assay 68 LOS Immunoturbidimetric assay
5.0–37.0
5.0 mg/L
24 h: 0.91 0.7
24 h: 0.98 0.91
NR
5.0 mg/L
0.69
0.86
0.84
0.73
NR
12.0 mg/L
0 h: 0.6
0 h: 1.0
0 h: 1.0
0 h: 0.75
12 EOS; 8 LOS; Immunoturbidimetric 60 neonates assay
0 h: 0.0–1.1
15.2 mg/L
24 h: 0.82 0 h: 0.80
24 h:0.96 0 h: 0.92
0 h: 3.1–3.5 24 h: 4.1–5.4 48 h: 4.0–5.1 NR 0 h: 0.0–4.2 8 h: 3.0–7.3 24 h: 1.3–6.3 0 h: 0.3–3.0
0 h: 4.0 mg/L 24 h:10 mg/L 48 h:10 mg/L 15.0 mg/L
10.0 mg/dl
24 h: NR 0 h: 0.74 24 h: 0.89 48 h: 0.89 0.5 0 h: 0.32 8 h: 0.53 24 h: 0.84 0 h: 0.69
24 h: NR 0 h: 0.83 24 h: 0.87 48 h: 0.84 0.93 0 h: 0.97 8 h:0.92 24 h:0.90 0 h: 0.96
NR NR NR 0 h: 0.86 0 h: 0.74 8 h: 0.77 8 h: 0.80 24 h: 0.80 24 h: 0.92 0 h: 0.93 0 h: 0.80
12.0 mg/L
24 h: 0.78 0 h: 0.65
24 h: 0.94 0 h: 0.99
0 h: 0.96
0 h: 1.0 ng/ml
24 h: 0.72 48 h: 0.65 0 h: 0.79
24 h: 1.00 48 h:1.00 0 h: 0.95
24 h: 1.00 24 h: 0.90 48 h: 1.00 48 h: 0.87 NR NR
24 h: 0.89 48 h: 0.89 0 h: 0.72
24 h: 0.87 48 h: 0.84 0 h: 0.72
0 h: 0.51
24 h: 0.74 48 h: 0.78
24 h: 0.81 48 h: 0.79
24 h: 0.61 24 h: 0.88 48 h: 0.56 48 h: 0.91
0 h: 0.93 8 h: 0.85 24 h: 0.77 0 h: 0.95
0 h: 0.87 0 h: 0.97 8 h: 0.76 8 h: 1.00 24 h: 0.67 24 h: 0.98 0 h: 0.85 0 h: 0.99
Method
Acute-phase proteins CRP
20 21 25
28
NR
Procalcitonin
Immunoturbidimetric assay
27
134 EOS
Nephelometric assay
22 19
101 LOS 116 LOS
Nephelometric assay ELISA
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent ELISA
26
80 LOS
Immunoturbidimetric assay
27
24
134 EOS
317 EOS
Immunoluminometric assay
24 h: 0.4–3.0 0 h: 1.0–3.0
0 h: 0.18–.27
10.0 mg/L
24 h: 4.28–7.3 24 h: 100ng/ml 48 h: 1.69–2.70 48 h: 50 ng/ml Immunoluminometric 0 h: 0.23–0.64 0 h: 0.55 ng/ml assay 24 h: 0.68–3.78 24 h: 4.7 ng/ml 48 h: 0.45–1.48 48 h: 1.7 ng/ml
24 h: 0.91 4 h: 0.98 AUC ⫽ 0.86
24 h: 0.95 24 h: 0.87 AUC ⫽ 0.75
NR
0 h: 0.87
0 h: 0.88
Serum amyloid A
IL-1ra Hepcidin Cell surface antigens
19
116 LOS
ELISA
0 h: 2.0–8.0 8 h: 2.0–7.0 24 h: 1.0–9.0 NR
12
104 EOS
Photometric immunoassay
22 13
101 LOS 44 LOS
26
80 LOS
10.0 mg/L
0 h: 8.0 mg/L
0 h: 0.95 8 h: 1.00 24 h: 0.97 0 h: 0.96
ELISA Capture ELISA
0–28 240 5.3–89.8 ng/ml
24 h: 10.0 mg/L 12 000 pg/ml 92.2 ng/ml
24 h: 0.96 1 0.76
24 h: 0.98 0.92 1
24 h: 0.92 24 h: 0.99 NR NR 1 0.87
Flow cytometry
0 h: 1116–2890 4000 molecules/ cell
0 h: 0.95
0 h: 0.88
0 h: 0.80
CD64
CD64 index
29
24 h: 0.97 24 h: 0.90 24 h: 0.80 24 h: 0.99 48 h: 0.87 48 h: 0.86 48 h: 0.80 48 h: 0.93 EOS: 1.00; EOS: 0.68; NR NR LOS: 0.75 LOS: 0.77 0.88 1 AUC ⫽ 0.95
3 EOS; 47 LOS; 749 neonates 16 LOS; 16 controls
Flow cytometry
NR
Automated Leuko64 Assay kit
0.5–2.8
EOS: 2.38; LOS: 3.62 2.85
30 31
30 LOS 106 EOS
Flow cytometry Flow cytometry
0 h: 45.0–146.0 NR
139.0 RFU 60 FU
0 h: 1.00 0.96
0 h: 0.56 1
0.5 0.99
30
30 LOS
Flow cytometry
0 h: 43.0–125.0
225.0 RFU
0 h: 0.86
0 h: 0.94
0.86
32
0 h: 0.97
CD11b neutrophils 1 1
CD11b monocytes 0.94 (Continued)
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Table II. (Continued)
Biomarker
Ref. no.
Sample size: EOS/LOS
Method
Reference interval
19
116 LOS
ELISA
0 h: 4.0–22.0
Cut-off value
Sensitivity
Specificity
PPV
NPV
Cut–off: 31.0 pg/ml
0 h: 0.78
0 h: 0.89
0 h: 0.64
0 h: 0.88
8 h:1.00 24 h:0.97 0 h: 0.89 24 h: 0.71 48 h: 0.70 0.65 0.83 0 h: 0.96 24 h: 0.89 0 h: 0.92 24 h: 0.93 48 h: 0.91 0 h. 0.73
8 h: 1.00 8 h: 0.80 24 h: 0.98 24 h: 0.69 NR NR
Cytokines/ chemokines Interleukin-6
8 h: 7.0–28.0 24 h: 2.0–14.0 0 h: 16.1–26.6 0 h: 200.0 ng/L 24 h: 8.8–15.0 24 h: 30.0 ng/L 48 h: 7.3–11.7 48 h: 20.0 ng/L 10.0–3028 20 pg/ml 0.0–1253 25.0 pg/ml NR 31.0 pg/ml
27
134 EOS
ELISA
20 22 25
144 EOS; 22 LOS 101 LOS 68 LOS
Colorimetric assay ELISA ELISA
26
80 LOS
ELISA
0 h: 1.0–6.0
31.0 pg/ml
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
0 h: 6.0–22.0
18.0 pg/ml
8 h: 0.47 24 h: 0.19 0 h: 0.74 24 h: 0.63 48 h: 0.53 0.65 0.89 0 h: 0.89 24 h: 0.67 0 h: 0.78 24 h: 0.44 48 h: 0.46 0 h: 0.76
28
12 EOS; 8 LOS; 60 neonates
Chemiluminescent immunometric assay
24 h: 5.0–18.0 0 h: 1.0–654.0
30.0 pg/ml
24 h: 0.63 0 h: 0.61
24 h:0.76 0 h: 0.80
NR
NR
23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
0 h: 20.0–134.0
100.0 pg/ml
0 h: 0.75
0 h: 0.66
0 h: 0.60
0 h: 0.80
28
12 EOS; 8 LOS; 60 neonates
Chemiluminescent immunometric assay
24 h: 16.0–71.0 0 h: 17.6–140.0
63.0 pg/ml
24 h: 0.49 0 h: 0.62
24 h: 0.79 0 h: 0.96
NR
NR
24 h: NR
24 h: NR
0 h: 0.82 24 h: 0.58 0 h: 0.60
0 h: 0.68 24 h: 0.84 0 h: 0.87
24 h: NA
24 h: NR
0 h: 0.66
0 h: 0.60
0 h: 0.52
24 h: 0.49 0 h: 0.82 24 h: 0.82 0 h: 0.54
24 h: 0.72 0 h: 0.86 24 h: 0.93 0 h: 0.92
0 h: 0.82 0 h: 0.85 24 h: 0.90 24 h: 0.86 NR NR
24 h: NR
24 h: NR
0.78 0.82
0.9 0.64
0 h: 0.64 24 h: 0.51
0 h: 0.89 24 h: 0.93
NR NR NR NR 0 h: 0.95 0 h: 0.91 24 h: 0.84 24 h: 0.77 0 h: 0.81 0 h: 0.91 24 h: 0.72 24 h: 0.81 48 h: 0.68 48 h: 0.80 0 h: 0.67 0 h: 0.81
Interleukin-8
Interleukin-1b 25
68 LOS
ELISA
NR
1.0 pg/ml
28
12 EOS; 8 LOS; 60 neonates
ELISA
0 h: 0.2–100
24.5 pg/ml
0 h: 0.68 0 h: 0.82 24 h: 0.75 24 h: 0.71 NR NR
TNF-α 23
22 EOS; 28 LOS; 57 non-septic
Chemiluminescent immunometric assay
25
68 LOS
28
12 EOS; 8 LOS; 60 neonates
0 h: 6.0–16.0
12.0 pg/ml
ELISA
24 h: 5.0–15.0 NR
17.0 pg/ml
ELISA
0 h: 1.0–32.1
3.5 pg/ml
0 h:0.73
Soluble adhesion markers ICAM 20 21 22
144 EOS; 22 LOS 90 EOS 101 LOS
ELISA ELISA ELISA
95.2–500.0 NR 68.0–1304.0
NR 300.0 ng/ml 425.0 ng/ml
20 25
144 EOS; 22 LOS 68 LOS
ELISA ELISA
2.0–217.8 NR
NR 174 ng/ml
AUC ⫽ 0.72 0.9 NR
0.8 NR
Soluble E-selectin AUC ⫽ 0.72 0 h: 0.83 0 h: 0.75 24 h: 0.85 24 h: 0.70
AUC, area under the curve; CRP, C-reactive protein; EOS, early-onset sepsis; ICAM, intercellular adhesion molecule; IL, interleukin; LOS, late-onset sepsis; nCD64, neutrophilic CD64; NPV, negative predicted value; NR, not reported; PPV, positive predicted value; RFU, relative fluorescence unit; SAA, serum amyloid A; Sensitivity 0 h, sensitivity at symptom onset; Sensitivity 24 h, sensitivity after 24 h of symptoms; TNFα, tumor necrosis factor-α.
Diagnostic utility of biomarkers for neonatal sepsis The cell surface antigen CD11b showed high sensitivity and specificity for both early and late diagnosis. Also the acute phase proteins hepcidin and IL-1ra showed promising results. These results were based on a high-quality manuscript, but as evidence was based on only one manuscript the results should be interpreted with caution. As shown in Figure 2, around one-third of the manuscripts had a rating score of 9. We decided beforehand that a high-rated study should comply with almost every criterion indicated by a score of 10 or 11; only a limited part of the studies fulfilled this requirement. One of the strengths of the present review was the compliance with defined stringent criteria for performing a systematic review. The literature search was performed systemically, the basis for the rating of the manuscripts was strict and based on a score developed beforehand, and strict assessment of the quality of the studies was performed independently by at least two authors. Some potential limitations of our review should be considered. During the ratings, all rating issues were assessed as equally important. This might be a problem, especially considering the importance of mentioning the coefficient of variation and blinding of the technicians. More often a coefficient of variation of a non-automated ELISA analysis will appear in the manuscripts compared with an automated high volume test. The same difference exists for information about a laboratory technician being blinded to the results; this was probably the case for most of the studies but it was only mentioned in a few. Therefore, the studies investigating biomarkers measured by non-automated methods had a tendency to be rated higher than studies evaluating markers analyzed by well-known automated methods. An evaluation of biomarkers of infection requires assessment of the PPVs and NPVs; however, in six (38%) of the studies with a high rate [20,22,27– 29,32] these data were not available. In 50% of the studies the population included both preterm and term neonates, even though the clinical picture of infection in preterm and term infants is quite different. Also the kinetics of the biological processes associated with inflammatory responses and biomarkers used for assessment of infection may differ markedly in correlation with gestational age. We differentiated between early- and late-onset sepsis, although the limit between early and late sepsis varies throughout publications. Most studies use 72 h to mark a limit for early- or lateonset sepsis; however, in clinical practice the clinician often use the same biomarker and the same reference interval for diagnosis of neonatal sepsis regardless of debut of the sepsis.
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During the literature search we used the term: ‘biological markers’ and chose not to include studies investigating 16s ribosomal RNA. This might, however, be a promising future marker for predicting neonatal sepsis. There was a great variability of the definition of sepsis between the studies. The majority of the highrated studies used clinical conditions to make the diagnosis [12,19–27], whereas others used a positive blood culture as the gold standard [28–30]. Four of the 16 high-rated studies included in the present review [19,24,25,30] compared the biomarker in the sepsis group with a group of healthy neonates. However, in clinical practice it is often a challenge to differentiate septic neonates from neonates that are ill due to other diseases, for example, respiratory distress syndrome, necrotizing enterocolitis, asphyxia, or hemodynamic failure. In the light of multidrug resistance, it is mandatory to avoid unnecessary use of antibiotics. In addition, the intravenous administration of antibiotics demands hospitalization of the neonate and thereby separation from the mother during these first important days of life. An ideal biomarker should thus identify the neonate at risk and at the same time be useful to distinguish the infected neonate from the neonate that is ill due to other diseases. In conclusion, the present review showed that only a few high-quality clinical studies have evaluated biomarkers for neonatal sepsis. As yet, there is no single ideal biomarker for neonatal sepsis and each marker has strengths and weaknesses. We suggest that serum amyloid A should be investigated further in clinical settings.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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