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Lipopolysaccharide-binding protein in noninfected neonates and those with suspected early-onset bacterial infection

Abstract

Objectives:

To investigate postnatal lipopolysaccharide-binding protein (LBP) kinetics in term neonates and to test its diagnostic accuracy for early-onset bacterial infection (EOBI).

Study design:

A total of 99 neonates with clinical and serological signs of EOBI comprised the study group; 198 neonates with risk factors, but without EOBI, served as controls. LBP, C-reactive protein (CRP) and interleukin-8 (IL-8) were determined.

Results:

LBP in the noninfected group increased until 24 h after birth (P<0.05 vs 6 h). LBP and CRP correlated strongly in neonates with suspected EOBI (r=0.63). Although LBP reached a higher sensitivity than CRP 6 and 12 h after clinical suspicion (45 (24–68) and 79% (54–94) vs 9 (0–24) and 39% (17–64); P<0.05)), EOBI was most reliably detected by IL-8.

Conclusion:

LBP kinetics were age-dependent. LBP was not sufficiently sensitive in the prediction of EOBI.

Introduction

The laboratory diagnosis of early-onset bacterial infection (EOBI) remains a challenge (rev. in 1), since C-reactive protein (CRP) has a limited sensitivity in the early phase1, 2 and proinflammatory plasma cytokines generally decrease to normal values within hours,3 potentially leaving a diagnostic gap between cytokine decrease and CRP increase.4

Lipopolysaccharide-binding protein (LBP) is an acute-phase protein produced mainly by hepatocytes.5 It transforms lipopolysaccharide (LPS) and facilitates interaction with monocyte membrane CD14.6 LBP initiates a cascade of proinflammatory cytokines which are key mediators for a systemic inflammatory response syndrome (SIRS), the main form of EOBI.7

Since LBP is constitutively synthesized and secreted, its blood concentrations range between 5 and 15 μg/ml in adults.8 Its induction occurs at 12 h9 after stimulation with both Gram-negative, for example, Escherichia coli,10 and Gram-positive bacteria, for example, Streptococcus agalactiae,11 the two most important inducers of EOBI. Elevated LBP concentrations were found in cord blood of culture-proven EOBI,12 septic neonates within 48 h,13 and preterm neonates 12 h after bacterial infection.14

Since LBP in the early postnatal phase has not been investigated, we were interested in its kinetics in noninfected term neonates and those with suspected EOBI, and tested the hypothesis that LBP may serve as an accurate parameter in the prediction of EOBI.

Patients, materials and methods

Patients

Neonates consecutively admitted and meeting inclusion criteria were enrolled with institutional ethics committee approval and parental consent. In all, 32 neonates in the noninfected group and 12 in the suspected EOBI group also participated in an investigation on lysed whole blood interleukin-8 (IL-8).4 Blood was collected from neonates who underwent blood testing because of risk factors for EOBI, but had no clinical signs or subsequent laboratory changes (noninfected group). To exclude EOBI, blood was drawn up to three times per patient at 6–12, 12–24 and 24–72 h post partum in addition to close observation by experienced neonatologists at least three times per day. In addition, blood was collected from neonates with suspected EOBI within 0–6, 12–24 and 48–72 h after clinical suspicion as defined below (suspected EOBI group). LBP samples were obtained prior to and after antibiotic treatment. A prerequisite was a nonhemolytic sample by venipuncture, processed within 2 h.

Definition of bacterial infection

A diagnosis of EOBI/suspected EOBI was based upon the presence of at least two of the following criteria within the first 72 h of life:15 one or more clinical signs compatible with EOBI, plus a consecutive elevation of CRP >10 mg/l within 24 h after first suspicion, or positive blood culture results. Clinical signs were fever (37.8°C rectal), hypothermia (36.5°C), temperature instability (1.5°C), pallor, grayish skin colour, poor perfusion (capillary refill >2 s), tachypnea (>60 respirations per minute at rest), dyspnea (grunting, nasal flaring, retractions), respiratory insufficiency, apnea, rising FiO2 in previously stable neonates, arterial hypotension (mean arterial blood pressure <37 mmHg), muscular hypotonia, irritability, hyperexcitability, neck stiffness, and lethargy.4, 15 The CRP cutoff of 10 mg/l has been used in our institution for many years according to previous investigations.4, 15, 16

Workup program for suspected EOBI

Indications for close clinical observation and our blood screening program were one or a combination of the following criteria: history of amniotic infection, maternal leukocytosis (>12 000 leukocytes/mm3), and/or maternal CRP elevation to >10 mg/l after exclusion of infectious foci unrelated to the fetus (gastrointestinal or urinary tract infections), fetal tachycardia (>160 beats/min), prolonged rupture of membranes (12 h) in the absence of labour, maternal fever (rectal temperature 38.0°C), and foul smelling amniotic fluid. Growth of group B streptococci in vaginal smear was routinely screened in case of prolonged rupture of membrane.

Sample processing and detection of LBP

LBP concentrations were detected in plasma. A volume of 10 μl was mixed with 1000 μl sample diluent (DPC-Biermann, Bad Nauheim, Germany) and incubated in LBP test units for 10 min at room temperature. Each test unit containd beads, coated with monoclonal murine anti-LBP antibody. Samples were assayed by the fully automated enzyme immunoassay Immulite LBP (test code 109, DPC Biermann, Bad Nauheim, Germany). The detection limit was 0.2 μg/ml (standardized in accordance with the National Institute for Biological Standards and Controls Reference Preparation (89/520)), and was calibrated to 200 μg/ml. Inter- and intra-assay variations were <5% at 20 pg/ml.

Biochemical and hematologic determinations

IL-8 was detected in plasma (DPC Biermann, Bad Nauheim, Germany) with a detection limit of 2 pg/ml and was calibrated to 7500 pg/ml. Inter- and intra-assay variations were <5% at 100 pg/ml. According to an analysis using receiver operator characteristics (ROC) curves, the threshold for IL-8 was set to 60 pg/ml. CRP was measured by enzyme sandwich immunoassay (Vitros 250, Ortho Diagnostics, Rochester, NY, USA). Intra- and interassay variations were <5%.

Statistical analysis

For LBP, IL-8 and CRP, specificity, sensitivity, positive and negative predictive value as well as the appropriate 95% confidence intervals were calculated. ROC curves were constructed to describe the relationship between the sensitivity and the false-positive rate (1−specificity) for different parameters. LBP, CRP and IL-8 concentrations are shown as Box–Whisker plots. Data were grouped; results obtained between 0 and 6 h are depicted as 6 h; results obtained between 6 and 12 h as 12 h; those obtained between 12 and 24 h as 24 h, etc. For the comparison of healthy neonates and those with suspected EOBI within the same interval, the Student's t-test was applied, using the decadic logarithm of the measurements. Comparisons between means of the transformed LBP concentrations across time were performed using univariate ANOVA. Dunnett's test was used for pairwise post-hoc analysis using 6 h as reference period. The correlations between CRP, LBP, apgar score and cord blood pH were analyzed by Spearman coefficient. Comparisons between the sensitivity of LBP and CRP were performed by a two-tailed Fisher's exact test. A probability of P<0.05 was defined as statistically significant. All charts were created with the help of software (SigmaPlot 2000; SPSS, Chicago, IL, USA).

Results

Patients

Serological and clinical follow-up data were complete for 297 term neonates with pre-, peri- or postnatal risk factors and/or symptoms of EOBI. In all, 19 additional neonates had to be excluded because of incomplete records or simultaneous presence of other diseases, potentially causing elevated LBP or CRP levels, such as chromosomal abnormalities, measurements after surgery, meconium aspiration, or hypoxic injury. The groups comprised 198 noninfected neonates and 99 with suspected EOBI. No patient in our noninfected group was readmitted with late-onset infection. In the suspected EOBI group, a blood culture was obtained in 99 patients; no positive result was found. Patient characteristics are presented in Table 1. We found 65% males in the suspected EOBI group, compared to 53% in our noninfected group (P<0.05). All patients in the suspected EOBI group received antibiotics (median 6 days) compared to 8% (16/198) in the noninfected group. The mean postnatal age at clinical suspicion was 14.5 h (s.d. 9.3). Since the suspicion of EOBI did not hold (CRP<10 mg/l, negative blood culture), treatment was discontinued after 3.6 days (s.d. 1.8 days) in the latter group; these newborns were regarded as noninfected.

Table 1 Patient data

LBP concentrations in noninfected neonates

In the first 6 h of life, the median LBP plasma concentration was 3.7 μg/ml (range: 1.0–17.0) and increased to 9.5 μg/ml (2.5–24.0) after 12, and 12.6 μg/ml (range: 4.3–27.4) after 24 h, indicating a threefold increase (both P<0.05 vs 6 h; Figure 1). In the remaining study period, LBP concentrations remained stable, with 12.5 μg/ml (range: 2.6–27.0) after 48 and 11.1 μg/ml (4.1–24.8) after 72 h. We found no influence of gender (male 3.8 μg/ml (range: 1.0–17.0); female 3.8 μg/ml (range: 1.1–16.8), P>0.05) or mode of delivery (spontaneous delivery 4.0 μg/ml (range: 1.0–17.0); elective caeserian section 4.1 μg/ml (range: 1.6–11.7); vacuum extraction 4.3 μg/ml (range: 2.6–10.6), all P>0.05) on LBP concentrations 6 h postnatally, and poor correlations to 5 min Apgar score (r=0.2), or cord blood pH (r=0.32).

Figure 1
figure1

Age-dependent LBP kinetics in 198 noninfected neonates in the first 72 h.

LBP, CRP and IL-8 kinetics in neonates with suspected EOBI

LBP concentrations in the suspected EOBI group were elevated in the first 6 h (9.5 μg/ml; range: 1.6–49.0), 12 h (16.9 μg/ml; range: 6.2–42.0), 24 h (24.3 μg/ml; range: 10.1–46.5) and 48 h (20.3 μg/ml; range: 4.4–55.0) after clinical suspicion (all P<0.05 vs noninfected group) and declined to 13.2 μg/ml (1.6–30.6) after 72 h (Figure 2a; NS). CRP concentrations increased 12–24 h after clinical suspicion, reached their maximum at 48 h and remained elevated for another 24 h (P<0.05 vs the noninfected group; Figure 2b). IL-8 concentrations were elevated 6 h (228.1 pg/ml; range: 44–2165) after clinical suspicion (noninfected group: 29 pg/ml; range: 5–73; P<0.05), but declined after 24 h (54.9 pg/ml; range: 10–114; NS; Figure 2c). There was a good correlation between CRP and LBP values in the suspected EOBI group (r=0.63).

Figure 2
figure2

LBP (a), CRP (b), and IL-8 plasma concentrations (c) in 99 neonates with suspected early-onset bacterial infection. Numbers represent the numbers of blood samples analyzed in each time interval, lines the cutoff of 10 mg/l for CRP (b) and 60 pg/ml for IL-8 (c), *P<0.05 vs the noninfected group measured at the same time, using a two-sample t-test for the logarithm of the values.

The sensitivity, specificity, positive and negative predictive values of LBP, IL-8 and CRP are shown in Table 2. At 6 and 12 h after clinical suspicion, the sensitivity of LBP was higher than that of CRP (P<0.05). Of the parameters studied, IL-8 showed the highest negative predictive value (74%; prevalence 0.245; Table 2). ROC analysis revealed age-dependent LBP cutoff values with a maximum sensitivity of 79% at 24 h (12 μg/ml; Figure 3a, b). A combination of LBP and IL-8 reached sensitivities of 73, 79 and 85% at 6, 12 and 24 h after clinical suspicion.

Table 2 Diagnostic accuracies of LBP, IL-8 and CRP within 6, 12 and 24 h after the first suspicion of early-onset bacterial infection
Figure 3
figure3

ROC curves for LBP, CRP, and IL-8, 6 (a) and 24 h (b) after clinical suspicion of early-onset bacterial infection. Sensitivity is plotted against 1−specificity for LBP level thresholds between 1.1 and 49.0 μg/ml, IL-8 level thresholds between 5.0 and 2165 pg/ml, and CRP level thresholds between 11.0 and 73.0 mg/l for patients with suspected EOBI.

Discussion

LBP concentrations in noninfected neonates revealed an age-dependent threefold increase in the first 24 h and remained stable within the study interval (Figure 1). LBP concentrations initially, however, were lower than described,12, 13 but comparable to adult levels 24 h after birth.8 In those studies,16, 17 LPB either had not been detected with a commercially available assay and/or values were pooled over the first 24 h. As in a study on preterm infants,14 we did not find an influence of gender and mode of delivery, nor correlations to the 5 min APGAR score, or cord blood pH. Other perinatal confounders that might affect LBP in analogy to CRP17 cannot be ruled out. Our noninfected group, however, certainly does not represent an ideal population, since neonates were not necessarily free of a history of maternal and intrapartum complications. Similar to CRP, the ‘physiological’ postnatal LBP increase may be attributed to intestinal colonization, contact with bacterial components, colostrum, lactulose,18 or postnatal maturation of hepatic function.19

Neonates with suspected EOBI showed elevated IL-8 and LBP concentrations within 6 and 12 h, which was in contrast to CRP (Table 2, Figure 2, Figure 3a). At the expense of a lower specificity, LPB had a higher sensitivity than CRP (Table 2, Figure 3a). Although the liver is the central organ for IL-6-induced LBP production, recent evidence suggests additional synthesis by cytokines in the intestinal mucosa and lung,20, 21 partly explaining the faster LBP increase compared to CRP in suspected EOBI.

Our investigation again reveals the problem of defining EOBI. Although the clinical course was compatible, there were no positive blood culture results, which is still the ‘gold standard’ in diagnosis. This may be due to consequent implementation of peripartal maternal antibiotic treatment,22 the use of small amounts of blood (<0.5 ml) in only one culture23 and the generally low sensitivity of blood cultures (0–15%5, 24). In all, 33% of our neonates with suspected EOBI were positive for group B Streptococci. Their impact on EOBI, as indicated by culture-proven cases, may be underestimated, since the latter can be falsely negative in at least 50%.25

Although the clinical work-up was mostly performed by experienced neonatologists, we are aware of the intrinsic restrictions, related to heterogeneity of patients, limited comparability to other studies or dependence on physicians’ experience. The CRP cutoff applied in our study (10 mg/l) is arbitrary, but has been used by other investigators.2, 15, 26 As with CRP, the clinical use of LBP may be limited because of its lack of specificity in differentiating infection from inflammation.27

Of the parameters tested, IL-8 showed the highest sensitivity and specificity (Table 2, Figures 2, 3a), as described by others.15, 16 Since, however, IL-8 plasma half-life comprises only 1–3 h, its determination may lead to false-negative results when sampling is performed later in the course of disease.3, 4, 28 Our hopes to predict EOBI and to close the diagnostic gap with LBP did not hold true, since its diagnostic value was remarkably lower than that of IL-8 in plasma (Table 2) or lysed whole blood.4

In conclusion, LBP is not sufficiently sensitive to predict EOBI. IL-8 in plasma and lysed whole blood4 are ‘early sensitive’ markers, whereas CRP is a ‘late specific’ diagnostic test. Since in this pattern LBP is not superior to IL-8 or CRP, it is probably of no additional value for the early diagnosis of EOBI in term neonates.

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Orlikowsky, T., Trüg, C., Neunhoeffer, F. et al. Lipopolysaccharide-binding protein in noninfected neonates and those with suspected early-onset bacterial infection. J Perinatol 26, 115–119 (2006). https://doi.org/10.1038/sj.jp.7211422

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Keywords

  • newborn
  • infection
  • lipopolysaccharide-binding protein
  • interleukin-8
  • C-reactive protein

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