1. Clinical characteristics of the enrolled neonates
A total of 66 full-term neonates were enrolled in this study, including 40 neonates with sepsis (10 EOS and 30 LOS) and 26 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 C reactive protein (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). Detailed information of the clinical characteristics was shown in Table 1.
Table 1 The clinical characteristics of the enrolled neonates
Variables
|
Neonate sepsis (n=40)
|
Control (n=26)
|
p value
|
Age, days
|
12(3.25-19.75)
|
10(7-16.25)
|
0.672
|
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
|
/
|
Early of sepsis, n (%)
|
30(75)
|
/
|
/
|
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 Figure 1, 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). 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). Interestingly, the levels of serum CXCL5, IL-2 and IL-4 in female were higher than those in male (Table S1).
Subsequently, we further analyzed the differences in these cytokines and chemokines between the EOS and LOS groups. There were 10 EOS and 30 LOS. The ages of EOS and LOS were 2 days (IQR,1day-3days) and 17days (IQR,11.75days-21.25days), respectively (p<0.001). The body temperature was 37.15℃ (IQR, 36.63℃-37.5℃) and 38.45℃ (IQR, 38℃-38.73℃) in EOS and LOS, respectively (p=0.0006). All cases who were blood culture positive were LOS (Table 2). As shown in Figure 2, the levels of serum CCL20 and IL-17 were higher in LOS than those in EOS (both p<0.05). Conversely, serum IL-16 was lower in LOS than that in EOS (p<0.05).
Table 2 The clinical characteristics of EOS and LOS
Variables
|
EOS (n=10)
|
LOS (n=30)
|
p value
|
Age, days
|
2(1-3)
|
17(11.75-21.25)
|
<0.001
|
Male gender, n (%)
|
4(40)
|
21(70)
|
0.135
|
Temperature (℃)
|
37.15(36.63-37.5)
|
38.45(38-38.73)
|
0.0006
|
Blood culture, n (%)
|
0
|
13(43.3)
|
<0.001
|
Note: EOS, early of sepsis; LOS, late of sepsis.
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, CCL8, and CCL23 in neonates with positive blood culture were significantly higher than those with negative blood culture (all p<0.05) (Table S2). There was no difference in the levels of other cytokines and chemokines between the two groups (data not shown).
3. The association between the levels of serum cytokines/chemokines and age in neonatal sepsis
We further analyzed the association between the levels of cytokines/chemokines and age among neonatal sepsis. As shown in Table S3, the levels of CCL21, CCL27, CCL22, CCL19, CXCL16, CCL17, IL-10, and IL-16 were negative correlation with their ages, and IL-17 was positively correlated with ages (all p<0.05).
Of 15 neonatal sepsis (3 EOS and 12 LOS) were treated with antibacterial therapy and were effective. The sera of the15 neonates with sepsis were collected after treatment. The point-in-time of collection was 5.5days (IQR, 2days-7.25days) after treatment. A total of 41 cytokines and chemokines were measured. As shown in Figure 3, IL-6, IL-8, TNF-α, IL-1β, CXCL13, CXCL16, CCL27, CCL3, CCL23, and CX3CL1 were significantly decreased after treatment (all p<0.005).