http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.916683
ORIGINAL ARTICLE
Diagnostic value of elevated CXCR4 and CXCL12 in neonatal sepsis Turan Tunc1, Ferhat Cekmez1, Merih Cetinkaya2, Tugce Kalayci2, Kursat Fidanci3, Mehmet Saldir3, Oguzhan Babacan3, Erkan Sari3, Galip Erdem3, Tuncer Cayci4, Mustafa Kul5, and Sultan Kavuncuoglu2
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1
Division of Neonatology, Department of Pediatrics, Gulhane Military School of Medicine, Ankara, Turkey, 2Division of Neonatology, Department of Pediatrics, Istanbul Kanuni Sultan Su¨leyman Teaching Hospital, Istanbul, Turkey, 3Department of Pediatrics, Gulhane Military School of Medicine, Ankara, Turkey, 4Department of Clinical Biochemistry, Gulhane Military School of Medicine, Ankara, Turkey, and 5Department of Pediatrics, Gulhane Haydarpasa Military Hospital, Istanbul, Turkey Abstract
Keywords
Objective: Neonatal sepsis remains a major cause of morbidity and mortality in newborns. The chemokine CXCL12 and its receptor CXCR4 are now known to play an important role in inflammatory states. However, it is unclear how chemokines respond to late-onset neonatal sepsis. Methods: Patients were classified into the groups of septic and non-septic ones. Samples of venous blood were obtained from all septic and non-septic newborns at the beginning and within 48–72 h after initiation of treatment. Serum levels of CXCR4 and CXCL12 were measured. Results: Concentrations of IL-6, CXCR4 and CXCL12 at the time of diagnosis were significantly higher in the septic neonates compared with the non-septic ones. Additionally, there were statistically significant differences in septic neonates between the first and the second levels of IL-6, CXCR4, CXCL12 and I/T ratio. ROC curve analyses revealed that IL-6, CXCR4, CXCL12 and I/T ratio resulted in significant AUC with respect to early identification of septic neonates. Univariate logistic regression analysis showed that increased IL-6, CXCR4 and CXCL12 were strong predictors of neonatal LOS. Conclusions: Serum CXCR4 and CXCL12 levels increase in septic neonates and that both chemokines decrease within 48–72 h of treatment. Serum concentrations of both chemokines represent promising novel biomarkers for neonatal sepsis.
Biomarkers, chemokine, newborn, sepsis
Introduction Neonatal sepsis remains a major cause of mortality and morbidity in the first month of life [1]. Despite the considerable advances in modern treatments, it is still a leading cause of death in critically ill babies, with a mortality rate ranging from 1.5% in term to 40% in very-low-birth weight infants [2]. The signs and symptoms of neonatal sepsis may not be easily distinguished from a variety of noninfectious conditions, such as respiratory distress syndrome and metabolic disturbances [3]. The high mortality and severe outcome of neonatal sepsis are mainly related to the combination of the derangements in the innate immune system and the complex interactions between the microorganism and the host defense [4]. In addition, neonatal sepsis is considered as early-onset (EOS) if it is diagnosed in the first 72 h of life and late-onset (LOS) if it is diagnosed after this period [5].
Address for correspondence: Turan Tunc, MD, Associate Professor of Pediatrics, Department of Pediatrics, Division of Neonatology, Gulhane Military Medical Academy, 06018 Ankara, Turkey. Tel: +90 312 304 1893. Fax: +90 312 304 4381. E-mail: [email protected]
History Received 16 February 2014 Revised 8 April 2014 Accepted 16 April 2014 Published online 22 May 2014
Sepsis usually originates from a rapid development of infections in the first days of life. When the microorganisms gain access to the blood stream, they may cause different clinical circumstances such as septicemia, pneumonia or meningitis [6]. Sepsis can be considered an abrupt evolution of infections supported by a cytokine-mediated condition consisting of immune, inflammatory and coagulation homeostasis impairment. The clinical course of neonatal sepsis may abruptly progress towards shock, disseminated intravascular coagulation (DIC) and death within very few hours from the onset of disease [7]. There are a variety of tests that are helpful for screening newborns with sepsis. The widely used are white blood cell (WBC) count, differential count, an absolute neutrophil count, a ratio of immature neutrophils, platelet count and micro-erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) [8–10]. Chemokines are chemotactic cytokines that give directional guidance for leukocyte migration. They are classified as some subgroups by the position of the first two-conserved cysteine residues near the amino terminus [11]. Although cytokines, leukotriens, proteases, integrins and bacterial products have been implicated in bone marrow neutrophil release, attention has centered on CXC chemokine and
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Toll-like receptor signaling during neutrophil release [12,13]. CXCR4 is expressed by both native and memory T lymphocytes although often in intracellular stores [14]. CXCL12 (stromal cell-derived factor-1) is found ubiquitously throughout the body although its precise role is unknown [15]. Previous studies have suggested that CXCL12 and its receptor CXCR4 play important role in HIV infection, neurodegenerative diseases, cancer development and progression [16–18]. The roles of CXCL12 and CXCR4 in sepsis have not previously been identified, although recently, Ding et al. [14] demonstrated that CXCR4 expression on the surface of circulating blood lymphocytes was up-regulated during sepsis or after lipopolysaccharide (LPS) stimulation. The aim of this study was to determine serum CXCR4 and CXCL12 levels in septic neonates and to assess their possible value in early diagnosis of LOS against interleukin (IL)-6, another commonly used sepsis biomarker with high diagnostic value in detecting bacterial infections in neonates.
Patients and methods Study subjects This prospective study was conducted in the neonatal intensive care units (NICUs) of Kanuni Sultan Su¨leyman Teaching Hospital and Gu¨lhane Military School of Medicine, Turkey. The study protocol was approved by the local Ethical Committee of the Teaching Hospital and written informed consent was obtained from the parents before enrollment. The study population included late preterm (gestational age of 434 weeks) and term neonates evaluated for LOS during the follow-up period. Between January 2012 and December 2012, we studied 30 consecutive infants who were admitted to our neonatal units because of suspected sepsis 472 h after birth. Inclusion criteria were postnatal age 72 h, presence of nonspecific signs of sepsis (temperature instability, apneic spells, need for supplemented oxygen, need for ventilation, tachycardia/bradycardia, hypotension, feeding intolerance, abdominal distension and necrotizing enterocolitis), and clinical deterioration considered to be due to sepsis [19]. Babies born to mothers with clinical chorioamnionitis and babies diagnosed with sepsis in 72 h of life or those who used antibiotic treatment for early-onset sepsis, and who had congenital infections and anomalies were excluded. Diagnosis of infection Patients were classified according to the criteria reported by Gitto et al. [19]. They were classified into the groups of septic and non-septic ones. Neonates with proven infection (positive blood or cerebrospinal fluid (CSF) cultures for microorganisms) and probable infection (negative cultures but clinical and laboratory evidence of sepsis; CRP45 mg/dl, 3 sepsis-related clinical signs) were grouped as sepsis. All patients in the septic group were treated with antibiotics according to standard protocols. Patients with negative cultures and those having no clinical and laboratory evidence of infection were classified as non-septic group. The likelihood of infection was assessed at admission and laboratory investigations included complete blood count and differential, white blood cell (WBC) and platelet counts, absolute
J Matern Fetal Neonatal Med, Early Online: 1–6
neutrophil count (ANC), immature/total neutrophil (I/T) ratio, CRP, arterial blood gases, blood, urine and CSF cultures. Laboratory evidence of infection was accepted as positive blood or CSF cultures, metabolic acidosis, leukopenia/ leukocytosis, thrombocytopenia, I/T neutrophil ratio 0.2, and CRP 45 mg/dl. Gestational age, birth weight, gender, survival, microorganisms isolated from blood and CSF cultures and Apgar scores at 1 and 5 min for all newborns were included in the two groups (septic newborns and nonseptic newborns). Blood sampling Samples of venous blood were obtained from a peripheral vein of all septic and non-septic newborns at the time of initial laboratory evaluation before any treatment and within 48–72 h after initiation of treatment. Samples were centrifuged directly after sampling and collected into sterile tubes. Thereafter the serum obtained was kept at 70 C until analysis. Serum levels of CXCR4 and CXCL12 were measured using a human CXCR4 and CXCL12 Quantikine Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Statistical analysis SPSS for Windows, version 15.0 (SPSS, Chicago, IL) was used for statistical analysis. Descriptive statistics were presented as mean, standard deviation, median (Interquartile range), frequency and percent. The distribution characteristics of continuous variables were evaluated with Kolmogorov– Smirnov test. Student’s t-test was used as parametric test and Mann–Whitney U-test was used as non-parametric test where appropriate. Paired t-test or Wilcoxon signed rank tests were used to compare first and second measures in the study group as appropriate. Pearson’s Chi-square test or Fisher’s exact test were used to compare categorical variables as appropriate. Spearman’s rho correlation test was used to determine the linear association between variables. Receiver operating characteristic (ROC) curve analysis was used to determine the best cut-off points for IL-6, CXCR4, CXCL12 and I/T ratio. And then sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios were calculated with respect to determined cut-off values. Univariate logistic regression analysis was used to calculate odds ratio (OR) values. A p value of50.05 was accepted as statistically significant.
Results The septic group consisted of 30 infected neonates (five with proven and 25 with probable sepsis). The non-septic group consisted of 20 neonates who were admitted to NICU for conditions other than sepsis. Demographic characteristics of septic and non-septic patients are shown in Table 1. The two groups of newborns did not differ from each other with respect to gestational age or birth weight (p40.05). Also there were no differences between the septic and nonseptic infants with respect to other demographic characteristics such as gender, mode of delivery, duration of ruptured membranes, evidence of maternal fever or number of assisted vaginal delivery (p40.05) (Table 1). Five patients developed
CXCR4 and CXCL12 in neonatal sepsis
DOI: 10.3109/14767058.2014.916683
positive blood cultures; two of Gram-positive and three of Gram-negative microorganisms. Urine and CSF cultures were negative in all infants. No infant died in both groups. Concentrations of IL-6, CXCR4 and CXCL12 at the time of diagnosis were significantly higher in the septic neonates compared with the non-septic ones [median ¼ 21.0, interquartile range (IQR) ¼ 15.5–34.8 pg/ml versus median ¼ 1.0,
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Table 1. Demographic characteristics of septic versus non-septic newborns.
Gestational age (weeks) Birth weight (g) Male sex Maternal fever Premature rupture of membranes Intrapartum antibiotics Cesarean section Apgar at min 1 Apgar at min 5 Umbilical artery pH Age at study entry (h)
Non-septic (N ¼ 20)
Septic (N ¼ 30)
p value
38.5 ± 1.4 3285 ± 342 11 (55%) 3 (15%) 3 (15%) 4 (20%) 8 (40%) 8.2 ± 0.8 9.6 ± 0.5 7.20 ± 0.07 93.8 ± 17.9
37.5 ± 1.5 3227 ± 357 12 (40%) 8 (26.7%) 5 (16.7%) 7 (23.3%) 14 (46.7%) 7.8 ± 0.8 9.4 ± 0.6 7.18 ± 0.07 98.9 ± 22.2
0.33 0.57 0.29 0.49 1.00 1.00 0.64 0.08 0.34 0.19 0.39
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IQR ¼ 0.0–3.8 pg/ml, p50.001; median ¼ 236.0, IQR ¼ 216.8–265.5 pg/ml versus median ¼ 163.5, IQR ¼ 129.5– 174.5 pg/ml, p50.001; median ¼ 620.5, IQR ¼ 351.0– 1733.8 pg/ml versus median ¼ 73.0, IQR ¼ 46.0–139.0 pg/ ml, p50.001, respectively] (Figure 1). Also there was a significant difference between the septic and non-septic groups with respect to I/T ratio [median ¼ 0.29, IQR ¼ 0.21–0.39 versus median ¼ 0.12, IQR ¼ 0.10–0.16, p50.001] at the initial evaluation. Additionally, there were statistically significant differences in septic neonates between the first and the second levels of IL-6, CXCR4, CXCL12 and I/T ratio [median ¼ 21.0, IQR ¼ 15.5–34.8 pg/ml versus median ¼ 5, IQR ¼ 2.0– 6.5 pg/ml, p50.001; median ¼ 236.0, IQR ¼ 216.8–265.5 pg/ ml versus median ¼ 202.3, IQR ¼ 199.9–210.7 pg/ml, p50.001; median ¼ 620.5, IQR ¼ 351.0–1733.8 pg/ml versus median ¼ 268.5, IQR ¼ 120.3–431.9 pg/ml, p50.001; median ¼ 0.29, IQR ¼ 0.21–0.39 versus median ¼ 0.12, IQR ¼ 0.10–0.16, p50.001, respectively] (Figure 1). Although there was a significant difference between the septic and non-septic groups with respect to the second values of IL-6, CXCR4 and CXCL12, the difference disappeared for the second value of I/T ratio (data not shown).
Figure 1. Serum levels of (a) IL-6, (b) CXCR4, (c) CXCL12 and (d) I/T ratio in control group and septic neonates at the time of diagnosis [Sepsis 1] and 48–72 h after initiation of treatment [Sepsis 2].
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Table 2. Diagnostic accuracy of serum IL-6, CXCR4, CXCL12, and I/T ratio in septic neonates.
Diagnostic parameters
Cut-off point
Biomarkers IL-6 (pg/ml) CXCR4 (pg/ml) CXCL12 (pg/ml) I/T ratio
7 185 200 0.19
ROC AUC (95%-CI) 0.97 0.95 0.94 0.91
(0.918–0.998) (0.885–0.998) (0.877–0.998) (0.833–0.991)
p value
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
PLR
NLR
50.001 50.001 50.001 50.001
96.7 86.7 83.3 80.0
95 95 95 90
96.7 96.3 96.2 92.3
95.0 82.6 79.2 75.0
19.3 17.3 16.7 8.0
0.04 0.14 0.18 0.22
ROC AUC: receiver-operating characteristic area under the curve, PPV: positive predictive value, NPV: negative predictive value, PLR: positive likelihood ratio, NLR: negative likelihood ratio, IL-6: interleukin-6, I/T ratio: immature by total ratio.
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Table 3. Univariate logistic regression analysis of diagnostic markers as an indicator of sepsis. Diagnostic markers
p value
Odds ratio (95% CI)
IL-6 CXCR4 CXCL12 I/T ratio
50.001 50.001 50.001 50.001
551.0 58.5 95.0 36.0
(32.5–93151.3) (9.7–354.1) (10.2–882.0) (6.5–199.7)
Receiver operating characteristic (ROC) curve analyses revealed that IL-6, CXCR4, CXCL12 and I/T ratio resulted in significant areas under the curve (AUC) with respect to early identification of septic neonates. Overall, IL-6 performed the best to distinguish septic neonates from non-septic ones. The AUC was 0.97 (95% CI ¼ 0.918–0.998, p50.001) for IL-6, 0.95 for CXCR4 (95% CI ¼ 0.885–0.998, p50.001), 0.94 for CXCL12 (95% CI ¼ 0.877–0.998, p50.001) and 0.91 for I/T ratio (95% CI ¼ 0.833–0.991, p50.001). According to the ROC analysis, the optimal cut-off point, sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ratio for each marker are displayed and compared in Table 2. Univariate logistic regression analysis showed that increased IL-6, CXCR4 and CXCL12 were strong predictors of neonatal LOS. The strongest association with neonatal LOS [Odds ratio (OR) ¼ 551.0 (95% CI ¼ 32.5–9351.3), p50.001] belonged to IL-6, followed by CXCR4 [OR ¼ 58.5 (95% CI ¼ 9.7–354.1), p50.001] and CXCL12 [OR ¼ 95.0 (95% CI ¼ 10.2–882.0), p50.001]. Odds ratio for I/T ratio were found as 36.0 (95% CI ¼ 6.5–199.7, p50.001) (Table 3).
Discussion In this prospective study on term and late preterm infants with LOS, we aimed to define predictive values and the kinetics of CXCR4 and CXCL12 concentrations, and to correlate these markers with the other biomarkers commonly used to assess neonatal sepsis. The results of this study revealed that the levels of CXCR4, CXCL12 and IL-6 increased significantly in the sera of neonates with LOS. Our findings suggest that CXCR4 and CXCL12 may have significant diagnostic performance to detect LOS, but they are not better than that of IL-6. Further, our results showed that both of these markers might be useful in monitoring the response to therapy in neonates with LOS. To the best of our knowledge, this is the first study investigating these
two markers both in diagnosis and monitoring of LOS in neonates. Early and accurate diagnosis of sepsis in neonates has always been a major challenge in NICUs. It is a complex disorder that is still incompletely understood, despite the advances in neonatal intensive care management and novel therapeutics. Identifying mediators that modulate innate immunity would be invaluable in the neonatal period due to the complexity of the host response to sepsis [20]. Although serum levels of some cytokines such as IL-6 and IL-8 were suggested as early markers of neonatal sepsis, cytokine analyses are far from being diagnostic for bacterial sepsis in neonates [21,22]. This study showed that CXCR4 and CXCL12 may be helpful as diagnostic markers in newborn infants with late-onset neonatal sepsis. Most of the studies investigating the relationship between sepsis and chemokines were conducted in adults. Ding et al. [23] analyzed CXCR4 expression on lymphocytes and chemotaxis – in response to CXCL12 – in critically ill patients in a tertiary care ICU. They demonstrated that CXCR4 expression on the surface of circulating blood lymphocytes was up-regulated during sepsis or after LPS stimulation, suggesting that CXCR4 may be important in lymphocyte infiltration into tissues. In another experimental study, Delano et al. [24] investigated the mechanisms involved in bone marrow mobilization of neutrophils in response to a polymicrobial sepsis challenge. They showed that neutrophil efflux from the bone marrow during acute infection depends on CXCL12/CXCR4 signaling. They also identified CXCL12 as an important survival factor in polymicrobial sepsis necessary for both neutrophil mobilizations from the bone marrow and neutrophil recruitment to peripheral sites of infection. In the present study, we found that CXCR4 and CXCL12 serum levels significantly increased in septic neonates compared to the non-septic ones, similar to previous studies. Therefore, we imply that increased chemokine levels may be due to both increased expression on the surface of circulating lymphocytes and efflux of neutrophils from bone marrow to peripheral blood during septic inflammation. Recently, cell surface antigens (CD64 and CD11b), adhesion molecules (soluble intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and cytokines (IL-1b, IL-1ra, IL-2, sIL-2R, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-a) have been intensively studied in septic patients [25–30]. However, none of them is able to be the only parameter in the diagnosis of neonatal sepsis. So far, the
CXCR4 and CXCL12 in neonatal sepsis
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DOI: 10.3109/14767058.2014.916683
combination of CRP and IL-6 remains to be the diagnostic resource of choice in detection of late-onset neonatal sepsis [25]. There are few studies examining the relationship between chemokines and neonatal sepsis in the literature [25]; however, none of them investigated CXCR4 and CXCL12 chemokines in neonatal sepsis. Our data support the elevation of these molecules in the serum of septic neonates. According to our results, the AUC values of both chemokines were slightly lower than those of IL-6. Therefore, we propose that both chemokines may be useful in predicting neonates with LOS. The AUC value of CRP was not performed because it was taken into consideration for the differential diagnosis of neonatal sepsis. Although I/T ratio was also statistically significant but it was not considered clinically significant compared with IL-6 and chemokines as far as the values of positive and negative likelihood ratio (PLR and NLR) are concerned (Table 2). An important point is that we found significant decrease in repeat measurements of CXCR4 and CXCL12 concentrations compared to the initial values in our patient population. We believe that this decline may be due to the effectiveness of antibiotic therapy. Therefore, we also speculate that a progressive decline in levels of CXCR4 and CXCL12 may indicate a favorable progress in neonates with LOS. Hence, we suggest that these two chemokines may be used in the follow-up of neonatal late-onset sepsis. A limitation of this study is the low incidence of cultureproven septic neonates. Neonates with probable sepsis were assumed similar to culture-proven sepsis. Besides, neonatal sepsis cannot be ruled out solely on the basis of a negative blood culture result. Although blood culture remains the ‘‘gold standard’’ in the diagnosis of neonatal sepsis, obtaining sufficient quantity of blood from an ill neonate is difficult and therefore false negative results are common. Furthermore, exposure of neonates to intrapartum antibiotic therapy during labor and delivery increases the rate of false negative blood cultures. In conclusion, we report that serum CXCR4 and CXCL12 levels increase in septic neonates and that both chemokines decrease within 48–72 h of treatment. However, the diagnostic accuracy of circulating CXCR4 and CXCL12 are not as high as that of IL-6. Serum concentrations of CXCR4 and CXCL12 represent promising novel biomarkers for neonatal sepsis. To determine the diagnostic importance of CXCR4 and CXCL12 measurements in late-onset neonatal sepsis, additional trials in larger number of septic newborns should be conducted.
Acknowledgements We thank our colleagues and FMF Arthritis Vasculitis and Orphan disease Research in Paediatric Rheumatology (FAVOR) for their helpful contributions and suggestions.
Declaration of interest The authors report no declarations of interest. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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References 1. Weston EJ, Pondo T, Lewis MM, et al. The burden of invasive early-onset neonatal sepsis in the United States, 2005–2008. Pediatr Infect Dis J 2011;30:937–41. 2. Fanaroff AA, Stoll BJ, Wright LL, et al. Trends in neonatal morbidity and mortality for very low birthweight infants. Am J Obstet Gynecol 2007;196:147.e1–8. 3. Murray BE. Can antibiotic resistance be controlled? N Engl J Med 1994;28:330:1229–30. 4. Mussap M. Laboratory medicine in neonatal sepsis and inflammation. J Matern Fetal Neonatal Med 2012;25:32–4. 5. Vouloumanou EK, Plessa E, Karageorgopoulos DE, et al. Serum procalcitonin as a diagnostic marker for neonatal sepsis: a systematic review and meta-analysis. Intensive Care Med 2011; 37:747–62. 6. Russell JA. Management of sepsis. N Engl J Med 2006;355: 1699–713. 7. Ng PC, Li K, Leung TF, et al. Early prediction of sepsis-induced disseminated intravascular coagulation with interleukin-10, interleukin-6, and RANTES in preterm infants. Clini Chem 2006;52: 1181–9. 8. Chirico G, Loda C. Laboratory aid to the diagnosis and therapy of infection in the neonate. Pediatr Rep 2011;24:3:e1. 9. Kul M, Saldır M, Gu¨lgu¨n M, et al. Retrospective evaluation of low birth weight newborns followed up with the diagnosis of respiratory distress syndrome in the newborn intensive care unit. Gulhane Med J 2005;47:290–3. 10. Kilic S, Tezcan S, Tascilar ME, et al. Diagnosis and treatment characteristics of the in-patients who were treated at the Gu¨lhane Military Medical Academy, Department of Pediatrics in 2001. Gulhane Med J 2003;45:169–74. 11. Frangogiannis NG, Mendoza LH, Smith CW, et al. Induction of the synthesis of the C-X-C chemokine interferon-gamma-inducible protein-10 in experimental canine endotoxemia. Cell Tissue Res 2000;302:365–76. 12. Metcalf D. Control of granulocytes and macrophages: molecular, cellular, and clinical aspects. Science 1991;254:529–33. 13. Christopher MJ, Link DC. Regulation of neutrophil homeostasis. Curr Opin Hematol 2007;14:3–8. 14. Ding Z, Xiong K, Issekutz TB. Regulation of chemokine-induced transendothelial migration of T lymphocytes by endothelial activation: differential effects on naive and memory T cells. J Leukoc Biol 2000;67:825–33. 15. Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat Immunol 2001;2:123–8. 16. Ma Q, Jones D, Borghesani PR, et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4and SDF-1-deficient mice. Proc Natl Acad Sci U S A 1998;95: 9448–53. 17. Lee HJ, Jo DY. The role of the CXCR4/CXCL12 axis and its clinical implications in gastric cancer. Histol Histopathol 2012;27: 1155–61. 18. Li M, Hale JS, Rich JN, et al. Chemokine CXCL12 in neurodegenerative diseases: an SOS signal for stem cell-based repair. Trends Neurosci 2012;35:619–28. 19. Gitto E, Karbownik M, Reiter RJ, et al. Effects of melatonin treatment in septic newborns. Pediatr Res 2001;50: 756–60. 20. Cuenca AG, Wynn JL, Kelly-Scumpia KM, et al. Critical role for CXC ligand 10/CXC receptor 3 signaling in the murine neonatal response to sepsis. Infect Immun 2011;79:2746–54. 21. 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; 18:666–71. 22. Buck C, Bundschu J, Gallati H, et al. Interleukin-6: a sensitive parameter for the early diagnosis of neonatal bacterial infection. Pediatrics 1994;93:54–8. 23. Ding Z, Jia SH, Marshall JC, et al. Up-regulation of functional CXCR4 expression on human lymphocytes in sepsis. Crit Care Med 2006;34:3011–17. 24. Delano MJ, Kelly-Scumpia KM, Thayer TC, et al. Neutrophil mobilization from the bone marrow during
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polymicrobial sepsis is dependent on CXCL12 signaling. J Immunol 2011;187:911–18. 25. Umlauf VN, Dreschers S, Orlikowsky TW. Flow cytometry in the detection of neonatal sepsis. Int J Pediatr 2013;2013: 763191. 26. Streimish I, Bizzarro M, Northrup V, et al. Neutrophil CD64 as a diagnostic marker in neonatal sepsis. Pediatr Infect Dis J 2012;31: 777–81. 27. Nupponen I, Andersson S, Jarvenpaa AL, et al. Neutrophil CD11b expression and circulating interleukin-8 as diagnostic markers for early-onset neonatal sepsis. Pediatrics 2001; 108:E12.
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28. Raynor LL, Saucerman JJ, Akinola MO, et al. Cytokine screening identifies NICU patients with Gram-negative bacteremia. Pediatr Res 2012;71:261–6. 29. Figueras-Aloy J, Gomez-Lopez L, Rodriguez-Miguelez JM, et al. Serum soluble ICAM-1, VCAM-1, L-selectin, and P-selectin levels as markers of infection and their relation to clinical severity in neonatal sepsis. Am J Perinatol 2007;24:331–8. 30. Edgar JD, Gabriel V, Gallimore JR, et al. A prospective study of the sensitivity, specificity and diagnostic performance of soluble intercellular adhesion molecule 1, highly sensitive C-reactive protein, soluble E-selectin and serum amyloid A in the diagnosis of neonatal infection. BMC Pediatr 2010;10:22.
ORIGINAL ARTICLE
Diagnostic value of elevated CXCR4 and CXCL12 in neonatal sepsis Turan Tunc1, Ferhat Cekmez1, Merih Cetinkaya2, Tugce Kalayci2, Kursat Fidanci3, Mehmet Saldir3, Oguzhan Babacan3, Erkan Sari3, Galip Erdem3, Tuncer Cayci4, Mustafa Kul5, and Sultan Kavuncuoglu2
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1
Division of Neonatology, Department of Pediatrics, Gulhane Military School of Medicine, Ankara, Turkey, 2Division of Neonatology, Department of Pediatrics, Istanbul Kanuni Sultan Su¨leyman Teaching Hospital, Istanbul, Turkey, 3Department of Pediatrics, Gulhane Military School of Medicine, Ankara, Turkey, 4Department of Clinical Biochemistry, Gulhane Military School of Medicine, Ankara, Turkey, and 5Department of Pediatrics, Gulhane Haydarpasa Military Hospital, Istanbul, Turkey Abstract
Keywords
Objective: Neonatal sepsis remains a major cause of morbidity and mortality in newborns. The chemokine CXCL12 and its receptor CXCR4 are now known to play an important role in inflammatory states. However, it is unclear how chemokines respond to late-onset neonatal sepsis. Methods: Patients were classified into the groups of septic and non-septic ones. Samples of venous blood were obtained from all septic and non-septic newborns at the beginning and within 48–72 h after initiation of treatment. Serum levels of CXCR4 and CXCL12 were measured. Results: Concentrations of IL-6, CXCR4 and CXCL12 at the time of diagnosis were significantly higher in the septic neonates compared with the non-septic ones. Additionally, there were statistically significant differences in septic neonates between the first and the second levels of IL-6, CXCR4, CXCL12 and I/T ratio. ROC curve analyses revealed that IL-6, CXCR4, CXCL12 and I/T ratio resulted in significant AUC with respect to early identification of septic neonates. Univariate logistic regression analysis showed that increased IL-6, CXCR4 and CXCL12 were strong predictors of neonatal LOS. Conclusions: Serum CXCR4 and CXCL12 levels increase in septic neonates and that both chemokines decrease within 48–72 h of treatment. Serum concentrations of both chemokines represent promising novel biomarkers for neonatal sepsis.
Biomarkers, chemokine, newborn, sepsis
Introduction Neonatal sepsis remains a major cause of mortality and morbidity in the first month of life [1]. Despite the considerable advances in modern treatments, it is still a leading cause of death in critically ill babies, with a mortality rate ranging from 1.5% in term to 40% in very-low-birth weight infants [2]. The signs and symptoms of neonatal sepsis may not be easily distinguished from a variety of noninfectious conditions, such as respiratory distress syndrome and metabolic disturbances [3]. The high mortality and severe outcome of neonatal sepsis are mainly related to the combination of the derangements in the innate immune system and the complex interactions between the microorganism and the host defense [4]. In addition, neonatal sepsis is considered as early-onset (EOS) if it is diagnosed in the first 72 h of life and late-onset (LOS) if it is diagnosed after this period [5].
Address for correspondence: Turan Tunc, MD, Associate Professor of Pediatrics, Department of Pediatrics, Division of Neonatology, Gulhane Military Medical Academy, 06018 Ankara, Turkey. Tel: +90 312 304 1893. Fax: +90 312 304 4381. E-mail: [email protected]
History Received 16 February 2014 Revised 8 April 2014 Accepted 16 April 2014 Published online 22 May 2014
Sepsis usually originates from a rapid development of infections in the first days of life. When the microorganisms gain access to the blood stream, they may cause different clinical circumstances such as septicemia, pneumonia or meningitis [6]. Sepsis can be considered an abrupt evolution of infections supported by a cytokine-mediated condition consisting of immune, inflammatory and coagulation homeostasis impairment. The clinical course of neonatal sepsis may abruptly progress towards shock, disseminated intravascular coagulation (DIC) and death within very few hours from the onset of disease [7]. There are a variety of tests that are helpful for screening newborns with sepsis. The widely used are white blood cell (WBC) count, differential count, an absolute neutrophil count, a ratio of immature neutrophils, platelet count and micro-erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) [8–10]. Chemokines are chemotactic cytokines that give directional guidance for leukocyte migration. They are classified as some subgroups by the position of the first two-conserved cysteine residues near the amino terminus [11]. Although cytokines, leukotriens, proteases, integrins and bacterial products have been implicated in bone marrow neutrophil release, attention has centered on CXC chemokine and
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Toll-like receptor signaling during neutrophil release [12,13]. CXCR4 is expressed by both native and memory T lymphocytes although often in intracellular stores [14]. CXCL12 (stromal cell-derived factor-1) is found ubiquitously throughout the body although its precise role is unknown [15]. Previous studies have suggested that CXCL12 and its receptor CXCR4 play important role in HIV infection, neurodegenerative diseases, cancer development and progression [16–18]. The roles of CXCL12 and CXCR4 in sepsis have not previously been identified, although recently, Ding et al. [14] demonstrated that CXCR4 expression on the surface of circulating blood lymphocytes was up-regulated during sepsis or after lipopolysaccharide (LPS) stimulation. The aim of this study was to determine serum CXCR4 and CXCL12 levels in septic neonates and to assess their possible value in early diagnosis of LOS against interleukin (IL)-6, another commonly used sepsis biomarker with high diagnostic value in detecting bacterial infections in neonates.
Patients and methods Study subjects This prospective study was conducted in the neonatal intensive care units (NICUs) of Kanuni Sultan Su¨leyman Teaching Hospital and Gu¨lhane Military School of Medicine, Turkey. The study protocol was approved by the local Ethical Committee of the Teaching Hospital and written informed consent was obtained from the parents before enrollment. The study population included late preterm (gestational age of 434 weeks) and term neonates evaluated for LOS during the follow-up period. Between January 2012 and December 2012, we studied 30 consecutive infants who were admitted to our neonatal units because of suspected sepsis 472 h after birth. Inclusion criteria were postnatal age 72 h, presence of nonspecific signs of sepsis (temperature instability, apneic spells, need for supplemented oxygen, need for ventilation, tachycardia/bradycardia, hypotension, feeding intolerance, abdominal distension and necrotizing enterocolitis), and clinical deterioration considered to be due to sepsis [19]. Babies born to mothers with clinical chorioamnionitis and babies diagnosed with sepsis in 72 h of life or those who used antibiotic treatment for early-onset sepsis, and who had congenital infections and anomalies were excluded. Diagnosis of infection Patients were classified according to the criteria reported by Gitto et al. [19]. They were classified into the groups of septic and non-septic ones. Neonates with proven infection (positive blood or cerebrospinal fluid (CSF) cultures for microorganisms) and probable infection (negative cultures but clinical and laboratory evidence of sepsis; CRP45 mg/dl, 3 sepsis-related clinical signs) were grouped as sepsis. All patients in the septic group were treated with antibiotics according to standard protocols. Patients with negative cultures and those having no clinical and laboratory evidence of infection were classified as non-septic group. The likelihood of infection was assessed at admission and laboratory investigations included complete blood count and differential, white blood cell (WBC) and platelet counts, absolute
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neutrophil count (ANC), immature/total neutrophil (I/T) ratio, CRP, arterial blood gases, blood, urine and CSF cultures. Laboratory evidence of infection was accepted as positive blood or CSF cultures, metabolic acidosis, leukopenia/ leukocytosis, thrombocytopenia, I/T neutrophil ratio 0.2, and CRP 45 mg/dl. Gestational age, birth weight, gender, survival, microorganisms isolated from blood and CSF cultures and Apgar scores at 1 and 5 min for all newborns were included in the two groups (septic newborns and nonseptic newborns). Blood sampling Samples of venous blood were obtained from a peripheral vein of all septic and non-septic newborns at the time of initial laboratory evaluation before any treatment and within 48–72 h after initiation of treatment. Samples were centrifuged directly after sampling and collected into sterile tubes. Thereafter the serum obtained was kept at 70 C until analysis. Serum levels of CXCR4 and CXCL12 were measured using a human CXCR4 and CXCL12 Quantikine Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Statistical analysis SPSS for Windows, version 15.0 (SPSS, Chicago, IL) was used for statistical analysis. Descriptive statistics were presented as mean, standard deviation, median (Interquartile range), frequency and percent. The distribution characteristics of continuous variables were evaluated with Kolmogorov– Smirnov test. Student’s t-test was used as parametric test and Mann–Whitney U-test was used as non-parametric test where appropriate. Paired t-test or Wilcoxon signed rank tests were used to compare first and second measures in the study group as appropriate. Pearson’s Chi-square test or Fisher’s exact test were used to compare categorical variables as appropriate. Spearman’s rho correlation test was used to determine the linear association between variables. Receiver operating characteristic (ROC) curve analysis was used to determine the best cut-off points for IL-6, CXCR4, CXCL12 and I/T ratio. And then sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios were calculated with respect to determined cut-off values. Univariate logistic regression analysis was used to calculate odds ratio (OR) values. A p value of50.05 was accepted as statistically significant.
Results The septic group consisted of 30 infected neonates (five with proven and 25 with probable sepsis). The non-septic group consisted of 20 neonates who were admitted to NICU for conditions other than sepsis. Demographic characteristics of septic and non-septic patients are shown in Table 1. The two groups of newborns did not differ from each other with respect to gestational age or birth weight (p40.05). Also there were no differences between the septic and nonseptic infants with respect to other demographic characteristics such as gender, mode of delivery, duration of ruptured membranes, evidence of maternal fever or number of assisted vaginal delivery (p40.05) (Table 1). Five patients developed
CXCR4 and CXCL12 in neonatal sepsis
DOI: 10.3109/14767058.2014.916683
positive blood cultures; two of Gram-positive and three of Gram-negative microorganisms. Urine and CSF cultures were negative in all infants. No infant died in both groups. Concentrations of IL-6, CXCR4 and CXCL12 at the time of diagnosis were significantly higher in the septic neonates compared with the non-septic ones [median ¼ 21.0, interquartile range (IQR) ¼ 15.5–34.8 pg/ml versus median ¼ 1.0,
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Table 1. Demographic characteristics of septic versus non-septic newborns.
Gestational age (weeks) Birth weight (g) Male sex Maternal fever Premature rupture of membranes Intrapartum antibiotics Cesarean section Apgar at min 1 Apgar at min 5 Umbilical artery pH Age at study entry (h)
Non-septic (N ¼ 20)
Septic (N ¼ 30)
p value
38.5 ± 1.4 3285 ± 342 11 (55%) 3 (15%) 3 (15%) 4 (20%) 8 (40%) 8.2 ± 0.8 9.6 ± 0.5 7.20 ± 0.07 93.8 ± 17.9
37.5 ± 1.5 3227 ± 357 12 (40%) 8 (26.7%) 5 (16.7%) 7 (23.3%) 14 (46.7%) 7.8 ± 0.8 9.4 ± 0.6 7.18 ± 0.07 98.9 ± 22.2
0.33 0.57 0.29 0.49 1.00 1.00 0.64 0.08 0.34 0.19 0.39
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IQR ¼ 0.0–3.8 pg/ml, p50.001; median ¼ 236.0, IQR ¼ 216.8–265.5 pg/ml versus median ¼ 163.5, IQR ¼ 129.5– 174.5 pg/ml, p50.001; median ¼ 620.5, IQR ¼ 351.0– 1733.8 pg/ml versus median ¼ 73.0, IQR ¼ 46.0–139.0 pg/ ml, p50.001, respectively] (Figure 1). Also there was a significant difference between the septic and non-septic groups with respect to I/T ratio [median ¼ 0.29, IQR ¼ 0.21–0.39 versus median ¼ 0.12, IQR ¼ 0.10–0.16, p50.001] at the initial evaluation. Additionally, there were statistically significant differences in septic neonates between the first and the second levels of IL-6, CXCR4, CXCL12 and I/T ratio [median ¼ 21.0, IQR ¼ 15.5–34.8 pg/ml versus median ¼ 5, IQR ¼ 2.0– 6.5 pg/ml, p50.001; median ¼ 236.0, IQR ¼ 216.8–265.5 pg/ ml versus median ¼ 202.3, IQR ¼ 199.9–210.7 pg/ml, p50.001; median ¼ 620.5, IQR ¼ 351.0–1733.8 pg/ml versus median ¼ 268.5, IQR ¼ 120.3–431.9 pg/ml, p50.001; median ¼ 0.29, IQR ¼ 0.21–0.39 versus median ¼ 0.12, IQR ¼ 0.10–0.16, p50.001, respectively] (Figure 1). Although there was a significant difference between the septic and non-septic groups with respect to the second values of IL-6, CXCR4 and CXCL12, the difference disappeared for the second value of I/T ratio (data not shown).
Figure 1. Serum levels of (a) IL-6, (b) CXCR4, (c) CXCL12 and (d) I/T ratio in control group and septic neonates at the time of diagnosis [Sepsis 1] and 48–72 h after initiation of treatment [Sepsis 2].
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Table 2. Diagnostic accuracy of serum IL-6, CXCR4, CXCL12, and I/T ratio in septic neonates.
Diagnostic parameters
Cut-off point
Biomarkers IL-6 (pg/ml) CXCR4 (pg/ml) CXCL12 (pg/ml) I/T ratio
7 185 200 0.19
ROC AUC (95%-CI) 0.97 0.95 0.94 0.91
(0.918–0.998) (0.885–0.998) (0.877–0.998) (0.833–0.991)
p value
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
PLR
NLR
50.001 50.001 50.001 50.001
96.7 86.7 83.3 80.0
95 95 95 90
96.7 96.3 96.2 92.3
95.0 82.6 79.2 75.0
19.3 17.3 16.7 8.0
0.04 0.14 0.18 0.22
ROC AUC: receiver-operating characteristic area under the curve, PPV: positive predictive value, NPV: negative predictive value, PLR: positive likelihood ratio, NLR: negative likelihood ratio, IL-6: interleukin-6, I/T ratio: immature by total ratio.
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Table 3. Univariate logistic regression analysis of diagnostic markers as an indicator of sepsis. Diagnostic markers
p value
Odds ratio (95% CI)
IL-6 CXCR4 CXCL12 I/T ratio
50.001 50.001 50.001 50.001
551.0 58.5 95.0 36.0
(32.5–93151.3) (9.7–354.1) (10.2–882.0) (6.5–199.7)
Receiver operating characteristic (ROC) curve analyses revealed that IL-6, CXCR4, CXCL12 and I/T ratio resulted in significant areas under the curve (AUC) with respect to early identification of septic neonates. Overall, IL-6 performed the best to distinguish septic neonates from non-septic ones. The AUC was 0.97 (95% CI ¼ 0.918–0.998, p50.001) for IL-6, 0.95 for CXCR4 (95% CI ¼ 0.885–0.998, p50.001), 0.94 for CXCL12 (95% CI ¼ 0.877–0.998, p50.001) and 0.91 for I/T ratio (95% CI ¼ 0.833–0.991, p50.001). According to the ROC analysis, the optimal cut-off point, sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ratio for each marker are displayed and compared in Table 2. Univariate logistic regression analysis showed that increased IL-6, CXCR4 and CXCL12 were strong predictors of neonatal LOS. The strongest association with neonatal LOS [Odds ratio (OR) ¼ 551.0 (95% CI ¼ 32.5–9351.3), p50.001] belonged to IL-6, followed by CXCR4 [OR ¼ 58.5 (95% CI ¼ 9.7–354.1), p50.001] and CXCL12 [OR ¼ 95.0 (95% CI ¼ 10.2–882.0), p50.001]. Odds ratio for I/T ratio were found as 36.0 (95% CI ¼ 6.5–199.7, p50.001) (Table 3).
Discussion In this prospective study on term and late preterm infants with LOS, we aimed to define predictive values and the kinetics of CXCR4 and CXCL12 concentrations, and to correlate these markers with the other biomarkers commonly used to assess neonatal sepsis. The results of this study revealed that the levels of CXCR4, CXCL12 and IL-6 increased significantly in the sera of neonates with LOS. Our findings suggest that CXCR4 and CXCL12 may have significant diagnostic performance to detect LOS, but they are not better than that of IL-6. Further, our results showed that both of these markers might be useful in monitoring the response to therapy in neonates with LOS. To the best of our knowledge, this is the first study investigating these
two markers both in diagnosis and monitoring of LOS in neonates. Early and accurate diagnosis of sepsis in neonates has always been a major challenge in NICUs. It is a complex disorder that is still incompletely understood, despite the advances in neonatal intensive care management and novel therapeutics. Identifying mediators that modulate innate immunity would be invaluable in the neonatal period due to the complexity of the host response to sepsis [20]. Although serum levels of some cytokines such as IL-6 and IL-8 were suggested as early markers of neonatal sepsis, cytokine analyses are far from being diagnostic for bacterial sepsis in neonates [21,22]. This study showed that CXCR4 and CXCL12 may be helpful as diagnostic markers in newborn infants with late-onset neonatal sepsis. Most of the studies investigating the relationship between sepsis and chemokines were conducted in adults. Ding et al. [23] analyzed CXCR4 expression on lymphocytes and chemotaxis – in response to CXCL12 – in critically ill patients in a tertiary care ICU. They demonstrated that CXCR4 expression on the surface of circulating blood lymphocytes was up-regulated during sepsis or after LPS stimulation, suggesting that CXCR4 may be important in lymphocyte infiltration into tissues. In another experimental study, Delano et al. [24] investigated the mechanisms involved in bone marrow mobilization of neutrophils in response to a polymicrobial sepsis challenge. They showed that neutrophil efflux from the bone marrow during acute infection depends on CXCL12/CXCR4 signaling. They also identified CXCL12 as an important survival factor in polymicrobial sepsis necessary for both neutrophil mobilizations from the bone marrow and neutrophil recruitment to peripheral sites of infection. In the present study, we found that CXCR4 and CXCL12 serum levels significantly increased in septic neonates compared to the non-septic ones, similar to previous studies. Therefore, we imply that increased chemokine levels may be due to both increased expression on the surface of circulating lymphocytes and efflux of neutrophils from bone marrow to peripheral blood during septic inflammation. Recently, cell surface antigens (CD64 and CD11b), adhesion molecules (soluble intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and cytokines (IL-1b, IL-1ra, IL-2, sIL-2R, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-a) have been intensively studied in septic patients [25–30]. However, none of them is able to be the only parameter in the diagnosis of neonatal sepsis. So far, the
CXCR4 and CXCL12 in neonatal sepsis
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DOI: 10.3109/14767058.2014.916683
combination of CRP and IL-6 remains to be the diagnostic resource of choice in detection of late-onset neonatal sepsis [25]. There are few studies examining the relationship between chemokines and neonatal sepsis in the literature [25]; however, none of them investigated CXCR4 and CXCL12 chemokines in neonatal sepsis. Our data support the elevation of these molecules in the serum of septic neonates. According to our results, the AUC values of both chemokines were slightly lower than those of IL-6. Therefore, we propose that both chemokines may be useful in predicting neonates with LOS. The AUC value of CRP was not performed because it was taken into consideration for the differential diagnosis of neonatal sepsis. Although I/T ratio was also statistically significant but it was not considered clinically significant compared with IL-6 and chemokines as far as the values of positive and negative likelihood ratio (PLR and NLR) are concerned (Table 2). An important point is that we found significant decrease in repeat measurements of CXCR4 and CXCL12 concentrations compared to the initial values in our patient population. We believe that this decline may be due to the effectiveness of antibiotic therapy. Therefore, we also speculate that a progressive decline in levels of CXCR4 and CXCL12 may indicate a favorable progress in neonates with LOS. Hence, we suggest that these two chemokines may be used in the follow-up of neonatal late-onset sepsis. A limitation of this study is the low incidence of cultureproven septic neonates. Neonates with probable sepsis were assumed similar to culture-proven sepsis. Besides, neonatal sepsis cannot be ruled out solely on the basis of a negative blood culture result. Although blood culture remains the ‘‘gold standard’’ in the diagnosis of neonatal sepsis, obtaining sufficient quantity of blood from an ill neonate is difficult and therefore false negative results are common. Furthermore, exposure of neonates to intrapartum antibiotic therapy during labor and delivery increases the rate of false negative blood cultures. In conclusion, we report that serum CXCR4 and CXCL12 levels increase in septic neonates and that both chemokines decrease within 48–72 h of treatment. However, the diagnostic accuracy of circulating CXCR4 and CXCL12 are not as high as that of IL-6. Serum concentrations of CXCR4 and CXCL12 represent promising novel biomarkers for neonatal sepsis. To determine the diagnostic importance of CXCR4 and CXCL12 measurements in late-onset neonatal sepsis, additional trials in larger number of septic newborns should be conducted.
Acknowledgements We thank our colleagues and FMF Arthritis Vasculitis and Orphan disease Research in Paediatric Rheumatology (FAVOR) for their helpful contributions and suggestions.
Declaration of interest The authors report no declarations of interest. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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