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Journal of Neonatal-Perinatal Medicine 6 (2013) 153–163 DOI 10.3233/NPM-1364112 IOS Press

Original Research

Cytokines patterns in newborn infants with late onset sepsis S. Lusyatia,b,∗ , C.V. Hulzebosb , J. Zandvoortb , H. Sukandarc and P.J.J. Sauerb a Department

of Pediatrics, Harapan Kita Women and Children’s Hospital, Jakarta, Indonesia of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, The Netherlands c Department of Epidemiology and Biostatistics, University of Padjadjaran, Bandung, Indonesia b Department

Received 27 July 2012 Revised 5 February 2013 Accepted 2 April 2013

Abstract. BACKGROUND: Cytokines might be helpful to diagnose late onset sepsis (LOS) in newborn infants. Many studies on cytokines did not discriminate culture-proven from clinically-suspected sepsis; however, such differentiation is clinically useful. OBJECTIVES: To evaluate the feasibility to differentiate among culture-proven LOS, clinical LOS and controls using a battery of cytokines. STUDY DESIGN: This prospective study was conducted at the NICU of Harapan-Kita Women and Children’s Hospital, JakartaIndonesia. Three groups of infants with postnatal age >72 hours of age were enrolled in the study: culture-proven sepsis group (PS) (n = 18), clinical sepsis group (CS) (n = 25) and control group (n = 34). A battery of 25 cytokines was measured in each infant five times: at enrollment, after 4 hrs, 12 hrs, 24 hrs, and 48 hrs using Invitrogen-immunoassays-LuminexTM 100. RESULTS: There were no significant differences in gestational age or mode of delivery among the three groups. IL-1␤, IL-2r, IL-6, IL-8, IL-10 and MIP-1a were significantly higher at all measurement points in group PS compared to controls. IL-13 was lower at all measurement moments in group CS compared to controls, IL-12 was lower and IP-10 higher between 0 and 24 hrs. IL-1Ra, IL-6, IL-8, IL-13, IL-15, TNF␣, MIP-1a and MIP-1b were higher at all the measurement moments in group PS compared to group CS. The ROC curves show that IL-6, IL-8, IL-15, MIP-1a, MIP-1b and TNF␣ have a sensitivity and specificity between 80 and 85% during the first 24–48 hours after the onset of infection. IL-6, IL-15, MIP-1a, MIP-1b and TNF␣ showed the best likelihood ratios. CONCLUSIONS: IL-6, IL8, IL 15, MIP-1a, MIP-1b and TNF␣ are potentially good markers for detecting a proven LOS. In case these cytokines are not elevated in sick infants, other causes than an infection have to be identified. Keywords: Neonates, late onset sepsis, cytokines, chemokines

1. Introduction

∗ Corresponding

author: Dr. Setyadewi Lusyati, Neonatology Working Group-Harapan Kita Women and Children’s Hospital, S.Parman Kav 87 Slipi-West Jakarta, Jakarta, Indonesia. Fax: +62 21 5657547; E-mail: [email protected].

Infections by newborn infants are important contributors to morbidity and mortality in both developed as well as in developing countries [1]. In Indonesia, the neonatal mortality is at present 33.9 per 1000 live births. This is comparable to other developing

1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved

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countries [2–4]. Gram-negative infections are the most important causes of mortality [1, 3–5]. Antibiotics are very frequently used in case of a suspected neonatal infection [4–6]. At the moment it is very difficult, if not impossible, to differentiate between a deterioration in the clinical condition of a newborn infant due to a bacterial infection or due to other causes. Clinical symptoms and laboratory investigations such as the white-cell count, I/T-ratio, temperature instability, grey skin and capillary refill are not conclusive [7, 8]. C-reactive protein (CRP) has been proven to have an inadequate sensitivity and specificity and elevated levels may not be detected in the first 24 hours after the first symptoms of a possible infection are revealed [9, 10]. Procalcitonin (PCT) has a sensitivity of 60–88% and a specificity of 54–80%, depending on the cut-off level [11]. Recently was hepcidin suggested as marker for a late onset sepsis, but further studies are needed [12]. Since the results of a blood culture are only available 48 to 72 hours after the onset of symptoms, it is often impossible to wait with antibiotic treatment until the blood culture results are available. This implies that antibiotics are given to all infants who are suspected to have an infection. In developing countries antibiotics are not only used to treat, but also to prevent infections [4, 5]. However, extensive use of antibiotics will induce resistance. Methods in order to reduce the use of antibiotics are therefore important. The incorporation of cytokines in the diagnostic work-up with infants with the suspicion of LOS could reduce the use of antibiotics. Different studies have shown that IL-6, IL-8, IL-10, TNF␣ and IP-10 are elevated in infants with late-onset sepsis compared to the controls [13–29]. Despite these reports, these interleukins are still not being used in clinical practice to diagnose a neonatal infection. There might be a number of reasons as to why the results of these studies have not been implemented in clinical practice yet. Most of these studies were not able to differentiate between infants with a proven sepsis and infants with a clinical sepsis without a positive blood culture. In many of the studies infants with suspected sepsis, regardless whether the result of the blood culture was positive or not, were compared to infants without signs of an infection. A useful test should, however, differentiate between infants with clinical signs of an infection and a positive blood culture versus those with the same signs but with a negative culture. This is important not only to assess the need for antibiotics, but also to know

whether or not other causes than an infection should be considered. Finally it has been discovered that IL-6, IL8 and TNF␣ decrease rapidly after the onset of a neonatal sepsis, possibly due to the administration of antibiotics. Most studies evaluated levels of cytokines at the moment of infection and 24 hours later [26–28]. Therefore the moment of the highest value of cytokines/chemokines might have been missed. The aim of the present study was to determine levels of 25 cytokines/chemokines in the blood of newborn infants with a suspected infection and comparing the levels of these cytokines in infants with or without a positive blood culture. Secondly, the levels were compared to results obtained in infants without an infection. The diagnostic capacity of these cytokines to distinguish between these groups of infants was evaluated. Finally, we aimed to detect the most optimal time point for the measurement of these cytokines/chemokines.

2. Patients and methods This is part of a large prospective study on cytokine/chemokine levels in newborn infants conducted at the NICU of Harapan-Kita Women and Children’s Hospital, Jakarta, Indonesia. All infants admitted to our unit from October-2007 until October2009 with the suspicion of a sepsis, both early and late onset sepsis, and without a major congenital abnormality were included. Infants were suspected of an infection when they showed at least two clinical signs, i.e: increased frequency of apnea accompanied by either desaturation or persistent bradycardia (HR 160/min), respiratory dysfunction with requirement of respiratory support and need for more oxygen, poor capillary perfusion or hypotension with requirement of circulatory support, hyperirritability/lethargy, seizures, temperature instability (37.2◦ C on two occasions within 24 hours), feeding intolerance, abdominal signs of Necrotizing-Enterocolitis (NEC), hyperglycemia (>10 mmol/L) and metabolic acidosis (BE 72 hours of age and showed clinical signs of a sepsis were selected. Infants with a positive culture were identified and included in group Proven Sepsis (PS). In this group we also included two infants with meningitis and two infants with NEC stage 3. Infants with at least two signs of an infection but a negative culture were included in the group Clinical Sepsis (CS). Infants were included as control if they were clinically stable, >72 hrs of age, had no signs compatible with an infection, except mild respiratory problems for which they had received CPAP in the first three days after birth, and had a negative blood culture. In control infants, samples were taken daily from day 3 to 7 after birth. As we did not observe a trend in any of the cytokines during this period, we used the median of these daily measurements as control values.

3. Cytokine measurements When in a patient clinical signs compatible with an infection were observed and 4, 12, 24 and 48 hours later, 0.3 ml blood was taken for cytokine levels together with blood taking for routine clinical purposes. The blood sample was centrifuged; 50 uL serum was stored at minus 80◦ C within 15–30 min after blood collection. Frozen serum was shipped on dry ice to the laboratory of the University Medical Center Groningen, Netherlands where they were analyzed. Sera were analyzed using Invitrogen’s Multiplex Bead Immunoassay. In a 96 well plate samples were prepared by adding beads of defined spectral properties, which were conjugated to protein-specific capture antibodies, incubation buffer to bind cytokines to the protein-specific capture antibodies and biotinylated detector antibodies. Finally, streptavidin conjugated to the light-sensitive fluorescent protein R-Phycoerythrin was added and cytokine concentrations were analyzed with the Luminex detection system (Luminex Corp.,

155

Austin, Texas) using the program StarStation2.3. By monitoring the spectral properties of the beads and the amount of associated R-Phycoerythrin (RPE) fluorescence, the concentration of proteins was determined. The following interleukins were measured: IL-1b, IL1Ra, IL-2, IL-2r, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, TNF␣, IFNa, IFNg, IP-10, MIP-1a, MIP-1b, Eotaxin, Rantes, GM-CSF, MIG and MCP-1.

4. Statistical analysis We compared serum cytokine levels at all time points (0, 4, 12, 24 and 48 hours) between the three groups. A non-parametric test (Mann-Whitney U) from a computerized database (SPSS-11 for windows, SPSS Inc, 2001) was used to compare cytokine/chemokine values between PS vs. control, CS vs. Control and PS vs. CS. Because of the multiple comparisons, significance was set at p < 0.01, 95% of confidence intervals were calculated. Based on the receiver-operating characteristic (ROC), sensitivity, specificity and positive and negative likelihood ratios were calculated. We aimed at including 20 infants in both the proven and clinical sepsis groups. We chose this number, as we were not aiming to find a significant difference between groups but a clinically relevant test. If a test could not distinguish between groups of 20 infants, we did believe it was not clinically useful. Based on the experience of the past years, we expected to include that number of infants in less than one and a half year. However, due to more strict hygienic control, the incidence of proven sepsis was lower than expected, so we extended the inclusion period to two years.

5. Results Forty-three neonates of more than 72 hours of age, who showed minimally two clinical signs compatible with an infection, were included in the study. Eighteen of them were included in the group PS and twenty-five of them in the group CS. In the group PS, 2 infants had gram-positive bacteria, 10 infants had gram-negative, 2 infants had candida, 2 infants with meningitis and 2 infants had NEC stage 3. Thirty-four infants were included into the control group. Clinical characteristics of the three groups are presented in Table 1. There were no significant differences

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in gestational age, gender and Apgar score of less than 5 at 5 minutes between the groups. Birth weight was significantly lower in the group PS compared to the other groups. Temperature instability, clinical seizures and cardio-respiratory insufficiency were more frequent in group PS compared to CS. CRP was higher and abnormal platelet counts more frequent in group PS than CS. In the group PS 3 infants died after the sampling period, but before day seven. Cytokines/chemokines were detectable in all patients at almost all the measurement moments. RANTES, IL-2, IL-12, Eotaxin, MIG and GM-CSF did not show different levels between groups. Compared to the control group, significantly higher values in the group PS were found for IL-1b, IL-2r, IL6, IL-10 and MIP-1a at all the measurement moments. At t = 0, IL-1Ra and IL-15 were significantly higher, but decreased later on. TNF␣ was significantly higher at 24 and 48 hours, other cytokines were higher only at one of the measurement moments. IL-8 was higher in group PS at all the measurement moments with pvalues from 0.04 at t = 0 to 0.01 at t = 48 hours. When comparing CS to controls, IL-13 was lower at all the measurement moments compared to controls. IP-10 was higher and IL-12 lower in the first 24 hours. When comparing the group PS to CS, higher values in the group PS for IL1-Ra, IL-6, IL-8, IL-13, IL-15 and TNF␣, MIP-1a and MIP-1b at all the measurement moments were found. Results for six cytokines, IL1Ra, IL-6, IL-8, IL-15, MIP1-a and TNF␣ are shown in Fig 1. Almost all cytokines, both in group PS and CS, did not show a trend during the 48 hours observation period, except IL-1Ra, IL-6, TNF␣ (Fig. 1). In group PS, IL-1Ra was higher at 0 and 4 hours compared to later measurement moments, IL-6 showed a decreasing trend over time but all these levels remained higher than the control values. TNF␣ started to increase at 4 hours. Sensitivity, specificity, positive and negative likelihood calculated from the ROC curves to distinguish between proven sepsis, clinical sepsis and control groups are shown in Tables 2–4. IL-6, IL-15, TNF␣ and MIP-1a and MIP-1b showed a sensitivity of 75 to 100 % and a specificity of 60–95% when comparing the groups with proven and clinical sepsis during the 48hour period. IL-8 showed a good sensitivity, but a low specificity during the first 24 hours (Table 2). Almost the same sensitivity and specificity was found when comparing proven sepsis and controls, but both IL-8 and TNF␣ showed a low sensitivity and high speci-

ficity (Table 3). According to our finding that almost all cytokine levels were not different between groups clinical sepsis and controls, were sensitivity and specificity low in the comparison between both groups (Table 4).

6. Discussion In this study we show that the levels of 8 out of the 25 tested interleukins are higher during the 48 hours after clinical signs of an infection in infants with a proven late onset sepsis compared to non-infected patients. In infants with clinical symptoms of an infection but a negative culture, IP-10 was significantly increased while IL-12 and IL-13 were lower. Eight cytokines, IL-1Ra, IL-2r, IL-6, IL-8, IL-15, MIP1-a, MIP1-b and TNF␣ were higher in the infants with a proven sepsis compared to a clinical sepsis. According to the ROC curves IL-6, IL-15, MIP-1a MIP-1b and TNF␣ at 12 and 24 hours are potentially good markers to differentiate between a proven and clinical sepsis. A number of studies compared levels of cytokines/chemokine’s between infants with a proven or clinical LOS versus non-infected infants [13–29]. Most studies measured levels of IL-6, IL-8, IL-10, TNF␣ and IP-10. De Bont et al. [15] were one of the first to show elevated levels of IL-6, IL-1b and TNF␣ in infants with LOS. They compared infants with proven, mainly gram-positive infections, or the combination of proven and suspected cases to controls. Kuster et al. [24] found significantly elevated levels of IL-1Ra and IL-6 in infants with a proven sepsis compared to controls. These levels became elevated one or more days before the onset of the infection. The group of infants with signs of an infection but without a positive culture showed intermediate results for both cytokines, and no significant differences with either the proven sepsis or control group were observed. Ng et al. [17] compared levels of IL-1b, IL-6, IL-8, IL-10, IL-12, IP-10, MIG, MIP-1 and TNF␣ between infants with a proven sepsis and controls. They observed significantly higher levels of IL-6, IL-8, IL-10, IP-10, TNF␣, MIG and MIP-1 at the onset of disease. IL-6, IL-10, IP-10 and MIG were still elevated at the moment of the second blood sampling, 24 hours after the first sample. In another study by Ng et al. [18], higher concentrations of IL-2, IL-4, IL-6, IL-10, IFNg and TNF␣ were found at the onset of symptoms in infected infants than in those not infected. For most cytokines, however, there was

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Table 1 Characteristics of the study groups Characteristic GA (wks), median (min-max) BW (gram), mean (range) C-Section Gender: Male AS less than 5 at 5 minutes Day first symptoms (days) Clinical Signs: -Temperature Instability -Apnoea,bradycardia,desaturations -Increasing of need oxygen -Respiratory support IMV CPAP -Clinical seizure -Cardiorespiratory. insufficiency -GI tract problems Leucocyte30.000/mm3 Thrombocyte600.000/mm3 CRP median (min-max) Antibiotics days median (min-max) Mortality within 7 days Length of admission NICU days mean (range)

Control (n = 34) Clinical Sepsis (CS) (n = 25) Proven Sepsis /PS (n = 18) CS vs. PS p value 34 (32–34) 1958 (1480–2035) 20 11 1 0

34 (30–39) 2000 (1057–3352) 18 13 3 8

32 (25–34) 1700 (1134–2350) 10 8 5 7

n.s n.s n.s n.s n.s n.s

0 0 0

8 7 8

15 8 9

33∗ >61∗ >47∗ >203∗

94.44 85.71 94.12 41.18

72.6–99.1 57.2–97.8 71.2–99.0 18.5–67.0

55.56 70.00 48.57 100.00

35.3–74.5 45.7–88.0 31.4–66.0 89.0–100.0

2.12 2.86 1.83

0.10 0.20 0.12 0.59

>3∗ >3∗ >3∗ >2∗

80.00 78.57 80.00 86.67

51.9–95.4 49.2–95.1 51.9–95.4 59.5–98.0

76.00 70.00 81.82 66.67

54.9–90.6 45.7–88.0 64.5–93.0 47.2–82.7

3.33 2.62 4.40 2.60

0.26 0.31 0.24 0.20

>80∗ >137∗ >80∗ >49∗

84.62 58.33 84.62 92.31

54.5–97.6 27.8–84.7 54.5–97.6 63.9–98.7

75.00 100.00 75.76 66.67

53.3–90.2 83.0–100.0 57.7–88.9 47.2–82.7

3.38 3.30 3.49 2.77

0.21 0.42 0.20 0.12

>30∗ >67∗ >42∗ >49∗

85.71 66.67 84.62 76.92

57.2–97.8 34.9–89.9 54.5–97.6 46.2–94.7

76.00 100.00 81.82 80.00

54.9–90.6 83.0–100.0 64.5–93.0 61.4–92.2

3.57 3.40 4.65 3.85

0.19 0.33 0.19 0.29

>1786∗ >4018∗ >883∗ >1778∗

86.67 64.29 78.57 66.67

59.5–98.0 35.2–87.1 49.2–95.1 38.4–88.1

84.00 90.00 75.76 80.00

63.9–95.4 68.3–98.5 57.7–88.9 61.4–92.2

5.42 6.43 3.24 3.33

0.16 0.40 0.28 0.42

with infection vs. controls, while levels in infants with clinical signs of infection and negative culture were not increased. In another study by the same group, IL-10 levels were higher in suspected infants vs. those not infected at 0 and 24 hours, but their levels were significantly lower compared to septic infants [22]. Zeitoun et al. showed a high sensitivity and specificity of IL-10, but in that study both infants with a positive and negative culture were included in the sepsis group [25]. Our results are in line with most of these results, increased levels at t = 0 and t = 24 hours in infants with a proven sepsis, but not in infants with a clinical sepsis. Due to the overlap in results, IL-10 is not a specific and sensitive marker for bacterial infection in newborn infants. If IP-10 can detect infants with proven sepsis was, so far, only studied by Ng et al. [17], who found IP-10 to be a good marker for LOS. In our study the IP-10 levels were elevated in the group CS at all the measurement moments except t = 48 hours, while it was only elevated in the group PS at t = 12 hours. The increase of IP-10 therefore might be due to more and other fac-

tors than a bacterial infection. This is in line with the study of Ng et al. [17] who found that infants with sepsis complicated by intravascular coagulation (DIC) showed elevated levels, while levels in infants with sepsis without DIC were less elevated. TNF␣, a pro-inflammatory cytokine, is secreted predominantly by monocytes in response to inflammatory stimuli [32]. A study in adults showed that TNF␣ was increased in all patients with septic shock and not increased in severely ill patients with trauma [29]. Girardin et al. [31] showed that neonates can produce TNF␣ as well as adults, in response to endotoxaemia. They showed higher levels of TNF␣ in neonates with bacterial sepsis than in neonates with only bacterial colonization. De Bont et al. [15] showed that TNF␣ levels may be influenced by antibiotic treatment, the level was decreased significantly 8 hours after treatment. The levels in our study were higher in the group PS than in the group CS at all the measurement moments. TNF␣ might be a good marker to distinguish between proven and clinical sepsis.

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Table 3 Sensitivity, specificity, positive and negative likelihood ratio of IL-6, IL-8, IL-15, MIP-1a, MIP-1b and TNF␣ between proven sepsis (PS) and controls Interleukins IL-6 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 8 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. TNF␣ T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. MIP-1a T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 15 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 1Ra T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr.

Criterion

Sensitivity

95% CI

Specificity

95% CI

+LR

−LR

2.60 5.00 4.12

0.38 0.00 0.22 0.35

>93∗ >25∗ >40∗ >88∗

72.22 100.00 82.35 64.71

46.5–90.3 76.8–100.0 56.6–96.2 38.3–85.8

72.22 80.00 80.00 100.00

46.5–90.3 56.3–94.3 56.3–94.3 84.6–100.0

>396∗ >210∗ >104∗ >33∗

38.89 50.00 64.71 94.12

17.3–64.3 23.0–77.0 38.3–85.8 71.3–99.9

100.00 95.00 80.00 45.45

81.5–100.0 75.1–99.9 56.3–94.3 24.4–67.8

>6∗ >10∗ >8∗ >7∗

40.00 42.86 40.00 60.00

16.3–67.7 17.7–71.1 16.3–67.7 32.3–83.7

100.00 100.00 100.00 100.00

69.2–100.0 75.3–100.0 73.5–100.0 76.8–100.0

>96∗ >31∗ >80∗ >53∗

84.62 91.67 84.62 92.31

54.5–97.6 61.5–98.6 54.5–97.6 63.9–98.7

80.00 61.54 75.00 78.57

44.4–96.9 31.6–86.0 42.8–94.2 49.2–95.1

4.23 2.38 3.38 4.31

0.19 0.14 0.21 0.10

>69∗ >56∗ >57∗ >59∗

71.43 75.00 76.92 76.92

41.9–91.4 42.8–94.2 46.2–94.7 46.2–94.7

100.00 92.31 83.33 85.71

69.0–100.0 63.9–98.7 51.6–97.4 57.2–97.8

9.75 4.62 5.38

0.29 0.27 0.28 0.27

>2726∗ >4482∗ >2726∗ >1716∗

86.67 64.29 57.14 66.67

59.5–98.0 35.2–87.1 28.9–82.2 38.4–88.1

90.00 100.00 100.00 92.86

55.5–98.3 75.1–100.0 73.4–100.0 66.1–98.8

IL-4 and IL-5 are both anti-inflammatory cytokines. Ng et al. [18] found a higher level of IL-4 at onset of infection in infants with proven infection compared to controls and no difference in levels of IL-5. Our results are in line with this study. We found higher levels of both cytokines in the group PS compared to CS, IL-4 at t = 12 and 24 hours and IL-5 at 24–48 hours. IL-4 showed good sensitivity and specificity at t = 24 hours, but had an overlap between PS and the clinical sepsis group. IL-5 showed poor diagnostic performances. Therefore IL-4 and IL-5 are not feasible to identify newborn infants with proven LOS. MIP-1a and MIP-1b are pro-inflammatory cytokines, defined as B-chemokine’s, involved in the initiation and propagation of the inflammatory response. MIP-1a and MIP-1b activate IL-1, IL-6 and TNF␣ production [33, 34]. Fotopoulos et al. [35] showed higher levels of MIP-1a in infants with a nosocomial infection compared to infants with perinatal asphyxia. In our study, MIP-1a had higher levels at all the measurement moments in the group

10.00 3.24 1.73

0.61 0.53 0.44 0.13 0.60 0.57 0.60 0.40

8.67

9.33

0.15 0.36 0.43 0.36

PS compared to the clinical sepsis group. MIP-1a taken at 0, 12, 24 and 48 hours can be a good infection marker for the following reasons; First, the levels in proven sepsis were not only elevated at the time when infection was suspected, but still elevated at 24 and 48 hours. Secondly levels of MIP-1a in proven sepsis infants have a good sensitivity and specificity. Finally, MIP-1a is the first cytokine to increase in response to bacterial infections and is not dependent on other cytokines. MIP-1b showed the same results, but with lowers sensitivity and specificity. IL-15 is considered to be able to stimulate natural killer cells and their killing activity. Natural killer cells are part of the innate immune system and play an important role in infections and autoimmune diseases [36, 37]. IL-15 showed higher levels in the group PS compared to CS at 0, 12 and 24 hours. The elevated level of IL-15 in the proven septic infants might indicate a good and active performance of NK-cells, which could be seen as a response to the infection. A second conclusion is that neonates can produce IL-15 as well

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Table 4 Sensitivity, specificity, positive and negative likelihood ratio of IL-6, IL-8, IL-15, MIP-1a, MIP-1b and TNF␣ between clinical sepsis (CS) and controls Interleukins IL-6 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 8 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. TNF␣ T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. MIP-1a T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 15 T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr. IL 1Ra T = 0 hr. T = 12 hr. T = 24 hr. T = 48 hr.

Criterion

Sensitivity

95% CI

≤28∗ >10∗ ≤13∗ >3∗

81.48 70.00 57.14 100.00

61.9–93.6 45.7–88.0 39.4–73.7 89.0–100.0

≤33∗ ≤55∗ ≤53∗ >51∗

55.56 60.00 60.00 62.50

≤2∗ ≤1∗ ≤0∗ ≤1∗

95% CI

+LR

-LR

61.11 60.00 70.00 31.82

35.8–82.6 36.1–80.8 45.7–88.0 13.9–54.9

2.10 1.75 1.90 1.47

0.30 0.50 0.61 0.00

35.3–74.5 36.1–80.8 42.1–76.1 43.7–78.9

88.89 75.00 65.00 54.55

65.2–98.3 50.9–91.2 40.8–84.5 32.2–75.6

5.00 2.40 1.71 1.37

0.50 0.53 0.62 0.69

68.00 50.00 51.52 53.33

46.5–85.0 27.2–72.8 33.6–69.2 34.3–71.6

80.00 92.31 100.00 92.86

44.4–96.9 63.9–98.7 73.4–100.0 66.1–98.8

3.40 6.50 7.47

0.40 0.54 0.48 0.50

≤26∗ ≤137∗ ≤26∗ ≤26∗

45.83 100.00 45.45 53.33

25.6–67.2 83.0–100.0 28.1–63.6 34.3–71.6

90.00 23.08 91.67 78.57

55.5–98.3 5.3–53.8 61.5–98.6 49.2–95.1

4.58 1.30 5.45 2.49

0.60 0.00 0.60 0.59

≤22∗ ≤7∗ ≤16∗ ≤10∗

68.00 50.00 63.64 60.00

46.5–85.0 27.2–72.8 45.1–79.6 40.6–77.3

90.00 100.00 100.00 100.00

55.5–98.3 75.1–100.0 73.4–100.0 76.7–100.0

6.80

0.36 0.50 0.36 0.40

≤503∗ ≤317∗ ≤567∗ ≤79∗

56.00 40.00 60.61 20.00

34.9–75.6 19.2–63.9 42.1–77.1 7.8–38.6

70.00 100.00 83.33 100.00

34.8–93.0 75.1–100.0 51.6–97.4 76.7–100.0

1.87

0.63 0.60 0.47 0.80

as adults. As far as we know, no other study analyzed this cytokine in LOS infants. To confirm our findings further studies regarding the use of IL-15 as indicator of LOS are necessary. A number of studies evaluated serially cytokines, at the onset of infection and 24 hours later. Kuster et al. [23] showed that IL-6 was elevated at the onset of sepsis and returned to control levels at 24 hours. Ng et al showed a lower level of IP-10, MIG, IL-6 and IL-10 in culture proven infants at 24 compared to 0 hours, although levels at that moment were still higher than the controls. We found that IL-6 was lower at 24 and 48 hours in the group PS compared to t = 0 hours. IL10 showed a clear decrease in levels in the group PS from t = 0 to t = 48 hours. Our results indicate that the diagnostic performance of cytokines will not improve with more sampling in the first 24 hours after the onset of symptoms. Sampling at 24 hours can be used to confirm the results of t = 0. When IL-6 is not higher than control values at 24 hours in not severely sick

Specificity

3.64

infants, it can be used as indicator of a negative culture, together with the absence of an increase in MIP-1a. Our study however does have limitations. We have conducted our studies in a developing country, where the use of antibiotics is high; therefore gram-negative bacteria were the main cause of LOS. The control group included in this study also received antibiotics, as this is routine practice in our unit. The duration of the use of antibiotics in this group however was significantly shorter than in both other groups. As these infants showed no clinical signs of an infection, except for very mild respiratory problems, and had negative blood cultures, we are convinced that these infants can serve as controls. For this study we separated infants in the groups proven and clinical sepsis based on the presence or absence of a positive blood culture. It has been suggested that a blood culture might be a false negative, because sometimes not enough blood was taken or the mother received antibiotics intra-partum. We believe that the present method to culture a blood sample, the

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Bactec method, which we used, is a very sensitive testing method in order to detect a bacterial infection [38]. A study comparing 548 blood samples of infants with suspected sepsis found 25 positive cultures and 27 positive specimens using PCR. We might have missed one infant with a positive sample; this however would not have influenced our results. We were very careful in assuring, that in all samples for a blood culture at least 1 ml blood was taken. We included two patients with meningitis and two patients with NEC stage 3 in the group proven sepsis, although no positive cultures were present. From the patients with NEC no blood culture was taken, our method to culture cerebrospinal fluid might not be optimal. Finally, the birth weight of infants in group PS was lower than in the other groups. If cytokines that were not higher in group PS compared to CS might show other results in more mature infants needs further study. In conclusion, in this study we show that eight out of twenty-five tested cytokines are higher during the 48 hour period after the start of symptoms in newborn infants with a culture proven LOS compared to infants with clinical signs of a sepsis but with a negative culture. IL-6, IL-8, IL-15, MIP-1a MIP-1b and TNF␣ measured at 12 and 24 hours after the onset of symptoms are potentially good markers to differentiate between sick infants with or without a bacterial infection. In sick infants without an increase in these cytokines, other causes for the deterioration must be considered. Determination of cytokines at the onset of symptoms and four hours later however does not improve the predictive value.

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Acknowledgments We would gratefully like to acknowledge the Dutch Organization for International Cooperation in Higher Education (NUFFIC) for funding this study. We would like to gratefully acknowledge the following people: Paul van den Broek, MD, for the cytokines analysis; Hani Handayani for the data base compilations; all the neonatologists and nurses at the NICU, Harapan-Kita Hospital and the NICU, UMCG, the Netherlands, for supporting this study. Without their help this study would not have been possible. Financial disclosure statement The authors declare no conflict of interest.

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Cytokines patterns in newborn infants with late onset sepsis.

Cytokines might be helpful to diagnose late onset sepsis (LOS) in newborn infants. Many studies on cytokines did not discriminate culture-proven from ...
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