1. Clinical characteristics of the enrolled neonates
Fifty-nine full-term neonates were enrolled in this study, including 40 neonates with sepsis (30 EOS and 10 LOS) and 19 uninfected neonates as controls. The body temperature was 38.1℃ (IQR 37.13℃-38.65℃) and 36.8℃ (IQR 36.5℃-37.2℃) in neonates with sepsis and those in the control group, respectively (p<0.0001). In 13 neonates with positive blood culture, the following pathogens were isolated: Escherichia coli (seven cases), Streptococcus lactis (four cases), Klebsiella (one case), and Enterococcus faecium (one case). The levels of serum CRP and serum amyloid protein A (SAA) in neonates with sepsis were 9.11 mg/mL (IQR 6.54 mg/mL-10.13 mg/mL) and 29.38 mg/mL (IQR 4.83 mg/mL-36.24 mg/mL), respectively, which were significantly higher than those in the control group (both p<0.0001). The neutrophil counts of the neonates with sepsis and those in the control group were 9.06x109/L (IQR 5.34 x109/L-14.84 x109/L) and 6.14x109/L (IQR 4.29 x109/L-7.76 x109/L) (p=0.018), respectively. There were no significant differences in the monocyte, lymphocyte, and platelet counts between the two groups (all p>0.05). The duration of disease was 4.5h (IQR 1h-12h) and 24h (IQR 5.75h-30h) in EOS and LOS, respectively (p=0.0026). Detailed information on the clinical characteristics is shown in Table 1.
Table 1 The clinical characteristics of the enrolled neonates
Variable
|
Neonate sepsis (n=40)
|
Control (n=19)
|
p value
|
Age
|
12(3.25-19.75)
|
10(7-16.25)
|
0.672
|
(Median, IQR), days
|
Male gender, n (%)
|
25(62.5)
|
13(68.4)
|
0.775
|
Temperature (℃)
|
38.1(37.13-38.65)
|
36.8(36.5-37.2)
|
<0.0001
|
Blood culture, n (%)
|
13(32.5)
|
nd
|
nd
|
Early of sepsis, n (%)
|
30(75)
|
0
|
nd
|
WBC(x109/L)
|
17(11.68-21.13)
|
12.1(9.9-14.7)
|
0.005
|
Neutrophils(x109/L)
|
9.06(5.34-14.84)
|
6.14(4.29-7.76)
|
0.018
|
Monocytes(x109/L)
|
1.49(0.90-2.05)
|
1.26(0.96-51.46)
|
0.189
|
lymphocytes(x109/L)
|
4.07(3.14-5.53)
|
4.22(3.43-5.02)
|
0.942
|
Platelets(x109/L)
|
316.5(249-436)
|
262(227-345)
|
0.076
|
CRP (mg/ml)
|
9.11(6.54-10.13)
|
0.35(0.18-0.69)
|
<0.0001
|
SAA (mg/ml)
|
29.38(4.83-36.24)
|
0.38(0.04-3.86)
|
<0.0001
|
Note: nd, not done; WBC, White Blood Cell; CRP, C reactive protein; SAA, serum amyloid protein A. Values are given as median (interquartile range).
2. Levels of serum cytokines and chemokines in neonatal sepsis
We compared serum cytokine and chemokine levels between neonates with sepsis and those in the control group. Since the level of CCL15 was not in the quantitative range of the assay and exceeded the highest calculated concentration, we did not analyze it further. As shown in Table 2, the levels of the pro-inflammatory cytokines, IL-6, IL-8, TNF-α, and IL-1β, were significantly higher in neonates with sepsis than those in the control group (all p<0.05). There was no difference in the levels of IL-4, IFN-γ, IL-17, IL-2, IL-10, GM-CSF, and IL-16 between the two groups (all p>0.05). Serum MIF level was remarkably increased in neonates with sepsis, and was 6,270 pg/mL (IQR 4,615 pg/mL-11,820 pg/mL) and 4,947 pg/mL (IQR 3,204 pg/mL-6,767 pg/mL) in the sepsis and control groups, respectively (p=0.0223). As shown in Table 3, serum CXCL13, CXCL1, CXCL2, CXCL5, CXCL6, CXCL16, CCL27, CCL2, CCL8, CCL3, CCL20, CCL23, CCL27, and CX3CL1 levels were significantly increased in neonates with sepsis compared to those in the control group (all p<0.05).
Table 2 The serum levels of cytokines in control group and neonatal sepsis group.
Cytokine
|
Control group
|
Sepsis group
|
p value
|
GM-CSF (pg/ml)
|
125.2(87.8-234.3)
|
149.4(96.62-228.9)
|
0.4533
|
IFN-γ(pg/ml)
|
105.5(78.75-167.2)
|
107.8(76.35-160.6)
|
0.6977
|
IL-1β (pg/ml)
|
10.93(5.955-11.22)
|
12.02(10.68-18.19)
|
0.0071
|
IL-2 (pg/ml)
|
30.52(14.01-46.2)
|
36.81(24.28-45.02)
|
0.2794
|
IL-4 (pg/ml)
|
38.02(25.45-42.63)
|
36.1(30.99-43.43)
|
0.418
|
IL-6 (pg/ml)
|
24.25(12.9-39.01)
|
58.57(37.03-236.6)
|
< 0.0001
|
IL-8 (pg/ml)
|
38.5(17.45-64.08)
|
262.1(130.1-630)
|
< 0.0001
|
IL-10 (pg/ml)
|
89.27(42.78-134.7)
|
95.2(67.34-137.3)
|
0.2871
|
IL-16 (pg/ml)
|
1176(617.8-1832)
|
1005(727.2-1331)
|
0.3417
|
MIF (pg/ml)
|
4947(3204-6767)
|
6270(4615-11820)
|
0.0223
|
TNF-α (pg/ml)
|
45.72(26.27-55.92)
|
53.14(44.26-79.38)
|
0.0213
|
IL-17 (pg/ml)
|
1.13(0-2.525)
|
1.792(0.3479-5.423)
|
0.1723
|
Table 3 The chemokine levels of serum in control group and neonatal sepsis group.
Chemokine
|
Control group
(n=19)
|
Sepsis group
(n=40)
|
p value
|
CCL21(pg/ml)
|
7380(4948-15970)
|
6356(3958-9688)
|
0.1434
|
CXCL13(pg/ml)
|
53.38(41.35-72.76)
|
86.9(51.3-142.8)
|
0.003
|
CCL27(pg/ml)
|
1565(945-2621)
|
2041(1385-2895)
|
0.0361
|
CXCL5(pg/ml)
|
969.7(550.4-1366)
|
1187(929.4-1947)
|
0.0321
|
CCL11(pg/ml)
|
63.4(50.19-89.31)
|
77(58.22-89.35)
|
0.4947
|
CCL24(pg/ml)
|
263(122.4-485.8)
|
231.1(124.8-356.8)
|
0.4493
|
CCL26(pg/ml)
|
70.31(46.26-102.6)
|
77.38(58.56-98.76)
|
0.3283
|
CX3CL1(pg/ml)
|
365.9(223.1-584.3)
|
609.7(529.9-857.1)
|
< 0.0001
|
CXCL6(pg/ml)
|
48.4(30.76-95.48)
|
88.18(54.28-108.4)
|
0.005
|
CXCL1(pg/ml)
|
277.9(244.3-332.5)
|
436.8(359.2-763)
|
< 0.0001
|
CXCL2(pg/ml)
|
143.3(54.57-315.1)
|
331.3(186.3-700.9)
|
0.0027
|
CCL1(pg/ml)
|
112.6(96.16-145.3)
|
116.7(88.1-153.7)
|
0.7378
|
CXCL10(pg/ml)
|
171.4(87.76-262.7)
|
210(97.66-593.5)
|
0.185
|
CXCL11(pg/ml)
|
23.95(7.658-36.5)
|
20.28(13.38-53.64)
|
0.8251
|
CCL2(pg/ml)
|
77.22(28.18-166.8)
|
168(89.07-363.4)
|
0.0011
|
CCL8(pg/ml)
|
32.67(21.28-39.7)
|
61.6(40.28-101.8)
|
< 0.0001
|
CCL7(pg/ml)
|
204(145.8-377.9)
|
238.5(151.5-370.6)
|
0.5557
|
CCL13(pg/ml)
|
53.52(12.8-87.72)
|
47.06(22.71-91.9)
|
0.7478
|
CCL22(pg/ml)
|
1694(1061-3421)
|
1234(774.3-1615)
|
0.0829
|
CXCL9(pg/ml)
|
658.3(251.5-960.4)
|
604.7(394.3-1069)
|
0.4413
|
CCL3(pg/ml)
|
16.28(9.845-30.3)
|
58.16(32.58-136.5)
|
< 0.0001
|
CCL20(pg/ml)
|
25.24(13.16-42.4)
|
50.93(20.63-97.86)
|
0.0074
|
CCL19(pg/ml)
|
902.2(274.9-1714)
|
772.5(466.5-1381)
|
0.8356
|
CCL23(pg/ml)
|
273.6(107.9-609.3)
|
1092(554.5-2366)
|
< 0.0001
|
CXCL16(pg/ml)
|
965.4(774.3-1152)
|
1218(1105-1364)
|
0.0004
|
CXCL12(pg/ml)
|
1559(423-1790)
|
1215(410.4-1607)
|
0.279
|
CCL17(pg/ml)
|
269.7(100.5-999.8)
|
417.5(122.4-1024)
|
0.705
|
CCL25(pg/ml)
|
1156(698.6-1498)
|
1285(895.3-1792)
|
0.3123
|
Subsequently, we further analyzed the differences in these cytokines and chemokines between the EOS and LOS groups. As shown in Table 4, the levels of serum CXCL6, CXCL1, IL-6, CXCL10, CXCL11, CCL20, and IL-17 were higher in LOS than those in EOS (all p<0.05). Conversely, the levels of serum IL-16, CXCL16, and CCL22 were lower in LOS than those in EOS (all p<0.05).
Table 4 The levels of serum cytokines and chemokines between EOS and LOS.
Cytokine/
Chemokine
|
EOS
(n=10)
|
LOS
(n=30)
|
p value
|
CXCL6(pg/ml)
|
66.55(37.7-97.69)
|
93.27(61.68-121.5)
|
0.0299
|
CXCL1(pg/ml)
|
370.1(266-449.8)
|
528.6(393.9-957.2)
|
0.0408
|
IL-6(pg/ml)
|
44.18(32.08-57.81)
|
87.68(44.9-354.5)
|
0.0479
|
IL-16(pg/ml)
|
1449(1099-1948)
|
885(607.4-1211)
|
0.0015
|
CXCL10(pg/ml)
|
110.9(62.27-268.6)
|
253.3(136.2-705.1)
|
0.0415
|
CXCL11(pg/ml)
|
11.81(5.853-20.38)
|
22.48(16.25-62.85)
|
0.0152
|
CCL22(pg/ml)
|
1485(1179-3912)
|
1164(619.3-1440)
|
0.0276
|
CCL20(pg/ml)
|
22.86(14.3-40.77)
|
62.76(32.03-124.9)
|
0.0093
|
CXCL16(pg/ml)
|
1403(1169-1532)
|
1207(1007-1334)
|
0.0351
|
IL-17(pg/ml)
|
0.43(0-0.97)
|
2.89(1.16-6.47)
|
0.003
|
Among the 40 neonates with sepsis, there were 13 neonates with positive blood culture and 27 neonates with negative blood culture. We compared the levels of serum cytokines and chemokines between neonates with positive and negative blood cultures. The levels of CXCL6, TNF-α, CCL8, and CCL23 in neonates with positive blood culture were significantly higher than those with negative blood culture (all p<0.05) (Table 5). There was no difference in the levels of other cytokines and chemokines between the two groups (data not shown).
Table 5 The chemokine and cytokine levels of serum in blood culture positive group and blood culture negative group.
Cytokine/
Chemokine
|
Positive
(n=13)
|
Negative
(n=27)
|
p value
|
CXCL6(pg/ml)
|
99.88(65.67-157.9)
|
82.05(51.1-102.2)
|
0.0444
|
CCL8 (pg/ml)
|
92.06(49.62-375.7)
|
43.81(38.66-76.02)
|
0.0373
|
CCL23(pg/ml)
|
1954(528-3292)
|
746.2(366.1-1223)
|
0.0311
|
3. Levels of cytokines and chemokines after treatment
The sera of 15 neonates with sepsis were collected before and after treatment. A total of 41 cytokines and chemokines were measured. As shown in Supplemental Digital Content (Figure 1), IL-6, IL-8, TNF-α, IL-1β, CXCL13, CXCL16, CCL27, CCL3, CCL23, and CX3CL1 were significantly decreased after treatment (all p<0.005).
4. The association between the levels of cytokines and chemokines and the severity of organ function
We further analyzed the association between the levels of cytokines and chemokines and the SOFA score. As shown in the Supplemental Digital Content (Figure 2), the levels of CX3CL1, CXCL2, CCL8, and TNF-α were all positively correlated with SOFA scores (all p<0.05).