To investigate postnatal lipopolysaccharide-binding protein (LBP) kinetics in term neonates and to test its diagnostic accuracy for early-onset bacterial infection (EOBI).
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.
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.
LBP kinetics were age-dependent. LBP was not sufficiently sensitive in the prediction of EOBI.
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
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%.
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).
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.
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).
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).
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.
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.
Ng PC . Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed 2004; 89: F229–F235.
Mathers NJ, Pohlandt F . Diagnostic audit of C-reactive protein in neonatal infection. Eur J Pediatr 1987; 146: 147–151.
Hack CE, Hart M, van Schijndel RJ, Eerenberg AJ, Nuijens JH, Thijs LG et al. Interleukin-8 in sepsis: relation to shock and inflammatory mediators. Infect Immun 1992; 60: 2835–2842.
Orlikowsky TW, Neunhoeffer F, Goelz R, Eichner M, Henkel C, Zwirner M et al. Evaluation of IL-8-Concentrations in Plasma and Lysed EDTA-Blood in Healthy Neonates and Those with suspected Early Onset Bacterial Infection. Pediatr Res 2004; 56: 804–809.
Schumann RR, Kirschning CJ, Unbehaun A, Aberle HP, Knope HP, Lamping N et al. Lipopolysaccharide binding protein: its role and therapeutical potential in inflammation and sepsis. Biochem Soc Trans 1994; 22: 80–82.
Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC . CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249: 1431–1433.
Dammann O, Leviton A . Maternal intrauterine infection, cytokines, and brain damage in the preterm newborn. Pediatr Res 1997; 42: 1–8.
Ng PC, Li K, Wong RP, Chui K, Wong E, Li G et al. Proinflammatory and anti-inflammatory cytokine responses in preterm infants with systemic infections. Arch Dis Child Fetal Neonatal Ed 2003; 88: F209–F213.
Froon AH, Dentener MA, Greve JW, Ramsay G, Buurman WA . Lipopolysaccharide toxicity-regulating proteins in bacteremia. J Infect Dis 1995; 171: 1250–1257.
Rietschel ET, Brade H, Holst O, Brade L, Muller-Loennies S, Mamat U et al. Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification. Curr Top Microbiol Immunol 1996; 216: 39–81.
Grunfeld C, Marshall M, Shigenaga JK, Moser AH, Tobias P, Feingold KR . Lipoproteins inhibit macrophage activation by lipoteichoic acid. J Lipid Res 1999; 40: 245–252.
Berner R, Furll B, Stelter F, Drose J, Muller HP, Schutt C . Elevated levels of lipopolysaccharide-binding protein and soluble CD14 in plasma in neonatal early-onset sepsis. Clin Diagn Lab Immunol 2002; 9: 440–445.
Pavcnik-Arnol M, Hojker S, Derganc M . Lipopolysaccharide-binding protein in critically ill neonates and children with suspected infection: comparison with procalcitonin, interleukin-6, and C-reactive protein. Intensive Care Med 2004; 30: 1454–1460.
Berendt D, Dembinski J, Heep A, Bartmann P . Lipopolysaccharide binding protein in preterm infants. Arch Child Fetal Neonatal Ed 2004; 89: F551–554.
Franz AR, Kron M, Pohlandt F, Steinbach G . Comparison of procalcitonin with interleukin 8, C-reactive protein and differential white blood cell count for the early diagnosis of bacterial infections in newborn infants. Pediatr Infect Dis J 1999; 18: 666–671.
Franz AR, Bauer K, Schalk A, Garland SM, Bowman ED, Rex K et al. Measurement of interleukin 8 in combination with C-reactive protein reduced unnecessary antibiotic therapy in newborn infants: a multicenter, randomized, controlled trial. Pediatrics 2004; 114: 1–8.
Chiesa C, Panero A, Osborn JF, Simonetti AF, Pacifico L . Diagnosis of neonatal sepsis: a clinical and laboratory challenge. Clin Chem 2004; 50: 279–287.
Schroedl W, Jaekel L, Krueger M . C-reactive protein and antibacterial activity in blood plasma of colostrum-fed calves and the effect of lactulose. J Dairy Sci 2003; 86: 3313–3320.
Turner MA, Power S, Emmerson AJ . Gestational age and the C reactive protein response. Arch Dis Child Fetal Neonatal Ed 2004; 89: F272–F273.
Vreugdenhil AC, Dentener MA, Snoek AM, Greve JW, Buurman WA . Lipopolysaccharide binding protein and serum amyloid A secretion by human intestinal epithelial cells during the acute phase response. J Immunol 1999; 163: 2792–2798.
Dentener MA, Vreugdenhil AC, Hoet PH, Vernooy JH, Nieman FH, Heumann D et al. Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins. Am J Respir Cell Mol Biol 2000; 23: 146–153.
Hsu KK, Pelton SI, Shapiro DS . Detection of group B streptococcal bacteremia in simulated intrapartum antimicrobial prophylaxis. Diagn Microbiol Infect Dis 2003; 45: 23–27.
Buttery JP . Blood cultures in newborns and children: optimising an everyday test. Arch Dis Child Fetal Neonatal Ed 2002; 87: F25–F28.
Ottolini MC, Lundgren K, Mirkinson LJ, Cason S, Ottolini MG . Utility of complete blood count and blood culture screening to diagnose neonatal sepsis in the asymptomatic at risk newborn. Pediatr Infect Dis J 2003; 22: 430–434.
Luck S, Torny M, d’Agapeyeff K, Pitt A, Heath P, Breathnach A et al. Estimated early-onset group B streptococcal neonatal disease. Lancet 2003; 361: 1953–1954.
Hengst JM . The role of C-reactive protein in the evaluation and management of infants with suspected sepsis. Adv Neonatal Care 2003; 3: 3–13.
Pourcyrous M, Bada HS, Korones SB, Baselski V, Wong SP . Significance of serial C-reactive protein responses in neonatal infection and other disorders. Pediatrics 1993; 92: 431–435.
Messer J, Eyer D, Donato L, Gallati H, Matis J, Simeonai U . Evaluation of interleukin-6 and soluble receptors of tumor necrosis factor for early diagnosis of neonatal infection. J Pediatr 1996; 129: 574–580.
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Cite this article
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
- lipopolysaccharide-binding protein
- C-reactive protein
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