Decrease of HC and Hypoproteinemia: Possible Predictors of Complication of Neonatal Sepsis in a Developing Country?

Background: The main purpose of our study was to determine the predictors of the complications of neonatal sepsis. Methods: This was a retrospective cohort study conducted in neonatal intensive care unit between June 2016 to February 2020. Neonates with late-onset sepsis (Age > 3 days old) were enrolled in this study. According to whether merged complication after sepsis, children were divided into complication group and non-complication group. The demographic data, perinatal conditions, blood cell count analysis, blood culture, hypoproteinemia within 1 week after the onset and treatment measures were compared. Results: A total of 87 infants with late-onset sepsis were enrolled in this study. (1) Signicant differences were observed between the complication and non-complication group with regard to hemoglobin concentration (HC), decrease of HC, hypoproteinemia and red blood cell transfusion (P < 0.05). (2) Further comparison found that children with sepsis who had moderate and more severe anemia at the time of onset were more likely to have complications than those with mild or no anemia. (3) The results of binomial stepwise logistic regression suggested that signicant decrease of HC (OR = 1.113, P = 0.000 < 0.05) and hypoproteinemia (OR = 3.953, P = 0.012 < 0.05) were independent risk factors for complication in infants with sepsis. (4) A receiver operating characteristic analysis showed that the AUC was 0.807 for decrease of HC. Conclusions: Signicant decrease of HC and hypoproteinemia were independent risk factors for complication and may predict the occurrence of complication in infants with late-onset sepsis in the early stage.


Background
Neonatal sepsis is one of the most common infectious diseases in newborns with high morbidity and mortality, and remains as the third major cause of neonatal death, especially in developing countries [1][2] .
Sepsis can cause many diseases, such as septic shock, neonatal necrotizing enterocolitis, meningitis, osteomyelitis, etc [3] . The occurrence of these diseases not only increases the mortality of newborns, and also cause short-term and long-term sequelae, such as intestinal stenosis, short bowel syndrome, neurological and skeletal system sequelae, etc [4][5][6] , Seriously affect the quality of newborns's life.
Therefore, exploring the predictors of complications of neonatal sepsis will help to take targeted prevention measures to reduce the incidence of complications and mortality. Up to now, there are many studies on the severity and prognosis of sepsis all over the world, but very few studies on the predictors of complications of neonatal sepsis, especially quantitative indicators. Hence, we hope that through this study, we can obtain quantitative indicators which are easy to obtain in clinical practice to predict the occurrence of complications, with a view to reducing the incidence of complications, and provide new clues for optimizing the treatment strategy in the early clinical stage.

Study subjects
This was a retrospective cohort study conducted in neonatal intensive care unit (NICU). Neonates with late-onset sepsis (age > 3d) diagnosed by a neonatal specialist at the Southwest Hospital of the First Hospital A liated to Army Medical University, China between June 2016 to February 2020. According to whether merged complications after sepsis, infants were divided into complication and noncomplications group. Exclude: (1) Intrauterine infection leads to early-onset sepsis (Age ≤ 3d), meconium aspiration syndrome; (2) Those with genetic metabolic diseases and congenital deformity; (3) Anemia caused by intracranial hemorrhage, gastrointestinal hemorrhage, pulmonary hemorrhage, etc; (4) Incomplete case data.

Clinical de nitions
The diagnostic criteria for sepsis refer to the Guidelines for the Diagnosis and Treatment of Neonatal Sepsis (2003) revised by the Chinese Medical Association [7] . Diagnostic criteria for sepsis: infants have fever or low body temperature, less crying, poor response and feeding and other clinical manifestations.

Data collection
Demographic data were obtained from the electronic medical records and included mother's prenatal condition, such as whether there was premature rupture of membranes, amniotic uid meconium pollution, maternal hypertension and diabetes, etc. At the same time, the information of children, such as gender, gestational age, birth weight, age of onset, comorbidities, blood cell count analysis, blood culture, albumin within 1 week after the onset and treatment measures (after the occurrence of sepsis and before the occurrence of complications) was also recorded. When patients rst showed the symptoms of sepsis (such as fever, poor response and feeding), the blood samples collected for the rst time would be examined. Laboratory parameters such as WBC, PLT, HC, the decrease of HC, blood culture and albumin were measured. The decrease of HC was the result of comparing the most recent HC before sepsis with HC at the time of sepsis.

Statistical analysis
SPSS 20.0 was used for statistical analysis. The measurement data was expressed as mean ± standard deviation (x ± s), Categorical variables were presented as absolute number and percentage, and were compared using Chi-square test and Fisher's Exact test. Multivariate logistic regression analysis was used to assess the risk factors of complication in neonatal sepsis. The sensitivity and specifcity were compared by analyzing the area under the ROC curve. P < 0.05 was considered signi cant. In the upper right corner of P value: ' §' is the representative of Fisher's Exact test.

Baseline characteristics
Total 87 children with sepsis were included, 56 in the non-complication group and 31 in the complication group, including 14 cases of NEC, 6 of septic shock, 8 of meningitis, 2 of osteomyelitis, and 1 of DIC.
Demographic characteristics of the two groups were presented in Table 1, including their gender, gestational age, birth weight, age of onset, perinatal situation, comorbidities, et al. The table showed that there were no signifcant differences were observed regarding these data among the complication group and non-complication group P > 0.05).

signi cant differences in HC, decrease of HC, hypoproteinemia and red blood cell transfusion
Signi cant differences were observed between the complication and non-complication group with regard to HC, decrease of HC, hypoproteinemia within 1 week after the onset and red blood cell transfusion (P < 0.05). No signi cant difference in WBC, PLT, HC, blood culture and mechanical ventilation was observed among the two groups (P > 0.05, Table 2). Further analysis of the impact of different degrees of anemia on the incidence of complication suggested that children with moderate or more severe anemia were more likely to had complications than those with mild or no anemia (P < 0.05, Table 3).

Signi cant decrease of HC and hypoproteinemia were independent risk factors for complication in infants with sepsis
Binomial stepwise logistic regression was used with whether complication occurred as the dependent variable, and the above statistically signi cant indicators, including HC, decrease of HC, hypoproteinemia and red blood cell transfusion were used as independent variables to test whether each factor had a signi cant effect on complication. The results suggested that signi cant decrease of HC (OR = 1.113, P = 0.000 < 0.05) and hypoproteinemia (OR = 3.953, P = 0.012 < 0.05) were independent risk factors for complication in infants with sepsis. (Table 4).

The high predictive value of decrease of HC for complication in infants with sepsis
The receiver operating characteristic (ROC) analysis of sepsis showed that area under the curve (AUC) was 0.807, suggesting that decrease of HC may predict the occurrence of complication in infants with sepsis (Table 5, Figure 1).

Discussion
The neonatal immune system is immature, highly susceptible to many microorganisms and prone to sepsis, especially premature infants and low birth weight infants. Long-term use of invasive procedures and intravenous nutrition increase the incidence of sepsis. Due to the low immunity and weak resistance of the newborn, sepsis often develops rapidly from subclinical symptoms to severe systemic infection symptoms, causing damage to systemic organs and the occurrence of diseases, such as NEC, meningitis, septic shock, DIC, etc, making sepsis become an important cause of neonatal death [10] . Therefore, it is great signi cance to analyze the speci c risk factors of complications and death of sepsis, taking active control measures to reduce the incidence of complications and mortality. Our study found that there was signi cant difference in HC, decrease of HC, hypoproteinemia within 1 week after the onset and red blood cell transfusion between the complication group and non-complication group. Further logistic regression showed that signi cant decrease of HC and hypoproteinemia were independent risk factors for complication in infants with sepsis.

The effect of anemia and decrease of HC on the complications and prognosis of children with sepsis
Sepsis is often accompanied by anemia or a decrease in hemoglobin concentration. Many causes can lead anemia, such as iatrogenic blood loss, reduced serum iron levels, shortened red blood cell life and increased destruction, etc. In addition, vascular endothelial glycocalyx shedding and intravenous uid administration lead to blood thinning [11][12][13] , which is also manifested by a decrease in HC, causing thinning 'anemia'. But in the early stage of sepsis, the decrease of HC is mostly caused by increased destruction of red blood cells and damage to the glycocalyx layer. In severe infection, a large number of in ammatory factors are released, on the one hand, directly destroying red blood cells, causing a decrease in HC. On the other hand, a large number of in ammatory factors destroy the glycocalyx layer of the vascular endothelium, causing dilution anemia. Therefore, we speculate that the decrease of HC may re ect the level of in ammation in the body, the more obvious the decrease in HC, the more severe the in ammation. After the occurrence of sepsis, the body may have microcirculation disturbances, leading to tissue ischemia and hypoxia. When severe infection occurs, a large amount of in ammatory factors are secreted in the body, which leads to true anemia or dilute 'anemia' through various mechanisms. The signi cantly reduced HC further causes ischemia and hypoxia, and redistribution of blood in the body, causing or aggravating the microcirculation disorder. Eventually, sepsis and signi cantly reduced HC complement each other and cause adverse effects on tissue organs. Studies have shown that anemia is a high-risk factor of NEC [14][15][16][17][18] , and the incidence of NEC in children with moderate or above anemia is higher than that without anemia or mild anemia [19] . Jung SM [20] found that low hemoglobin levels ( < 9.0 g/dL) were observed in approximately 20% of patients with septic shock, and the severity of decrease in these levels correlated with mortality, the lower the HC, the higher the mortality rate. Loftus TJ [21] found that reducing anemia may improve the prognosis of sepsis patients. This study found that the incidence of complications in children with sepsis was closely related to the severity of anemia and the degree of HC reduction at the onset. Moderate or above anemia and signi cantly reduced HC are more likely to have complications. At the same time, this study found that after the occurrence of sepsis and before the occurrence of complications, the transfusion of red blood cells may increase the incidence of complications. Many studies have also shown that the transfusion of red blood cells can lead to the occurrence of NEC [22][23] , and increase the chance of surgery in children with NEC and reduce the survival rate [24] . A foreign study on blood transfusion after sepsis in children showed that after sepsis-related anemia, compared to the restrictive strategy group, there had a signi cantly higher incidence of acute respiratory distress syndrome and acute lung injury in the liberal transfusion group. Moreover, mortality was signi cantly higher, and liberal transfusion might be associated with a worse outcome [25] . In addition, other studies found that the transfusion of red blood cells may increase the mortality of critically ill patients [26][27] . It should be pointed out that the anemia associated with sepsis is not all true anemia, and sometimes it may be caused by blood dilution related to uid load [28][29] . Therefore, after the occurrence of sepsis, even if the HC is reduced, it is necessary to strictly grasp the blood transfusion pointer and carefully infuse red blood cells.
Further logistic regression showed that decrease of HC was an independent predictors for complication in infants with sepsis. By comparing the ROC curves of the decrease of HC the results showed that the AUC was 0.807 for the decrease of HC, which suggests that decrease of HC (cut-off value: 14.5) may predicted the occurrence of complication in infants with sepsis.

The effect of hypoproteinemia on the complications and prognosis of children with sepsis
This study found that the incidence of hypoproteinemia in the complication group was signi cantly higher than that in the non-complication group. Logistic regression showed that hypoproteinemia was an independent risk factor for complication in infants with sepsis. Hypoproteinemia is a common complication of sepsis and albumin can be reduced by about 10-15 g/L within 1 week after sepsis. After infection, a large number of in ammatory mediators such as interleukin -1 (IL -1), IL -6 and tumor necrosis factor -α (TNF -α) are released, which can inhibit albumin synthesis. In addition, when the children have sepsis, the body will have fever, stress and in ammation. These symptoms will accelerate metabolism, a large amount of albumin in the body quickly synthesizes acute-phase protein, causing the albumin level to drop [30] . At the same time, increased permeability and destruction of vascular endothelial integrity can also lead to hypoalbuminemia [9] . Hypoproteinemia can cause the plasma colloid osmotic pressure to drop and a large amount of uid remains in the tissue gap, which reduces the effective blood volume of the body and causes damage to multiple organ functions. In addition, albumin can clear free radicals in the body and inhibit the production of oxygen free radicals by multinuclear cells. But when sepsis occurs, a large number of in ammatory factors are secreted, leading to proin ammatory and anti-in ammatory imbalances in the body, the activity of free radicals is out of control, and a large number of free radicals are produced. The occurrence of hypoalbuminemia leads to a weakening of the body's ability to scavenge free radicals and promote the development of sepsis. Therefore, it is necessary to pay attention to children with hypoalbuminemia after the occurrence of infection. For those with sepsis complicated with hypoalbuminemia, the hypoalbuminemia should be actively corrected to reduce the incidence of complications.
This study has some limitations. This study investigated the relationship between complications among infants with late-onset sepsis and decrease of HC and hypoalbuminemia. The incidence of complications in infants with sepsis in our hospital is not high, so the sample size of the complication group is small, conclusions must be further validated with larger sample sizes. In addition, the retrospective nature of the study is a limitation, as only blood cell count analysis and blood culture at the initial stage of the disease were counted, and the results of other onset times were not recorded. Finally, we only report on the situation in a developing country. Our results may not correlate with those of other countries on the grounds of racial differences, as well as different healthcare systems and medical technologies.

Conclusion
A careful analysis of the decrease of HC and hypoproteinemia within 1 week after sepsis, which is easily accessible and affordable, represents a valuable tool to predict the occurrence of complication in infants with late-onset sepsis in the early stage.

Declarations Ethical statement
This study was approved by the Ethics Committee of the First Hospital A liated of Army Medical University (Chongqing, China. KY2020065). All methods were performed in accordance with the relevant guidelines and regulations.

Consent for publication
The informed consent for participation in the study was obtained from legal guardians.

Availability of data and materials
The data analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no con icts of interest to declare.

Funding
No funding has been received.    Figure 1 The ROC curve of decrease of HC for predicting complication in infants with sepsis.