Clinical characteristics, risk factors, prognosis and immune status of secondary infection of sepsis: a retrospective observational study

Background: Secondary infection has a higher incidence in septic patients and affects clinical outcomes. This study aims to investigate the clinical characteristics, risk factors, prognosis and immune status of secondary infection of sepsis. Methods: A four-year retrospective study was carried out in Zhongshan Hospital, Fudan University, enrolling septic patients admitted between January, 2014 and January, 2018. Clinical data were acquired from medical records. CD14+ monocyte human leukocyte antigen-D related (HLA-DR) expression and serum cytokines levels were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA) respectively. Results: A total of 297 septic patients were enrolled, 92 of whom developed 150 cases of secondary infections. Respiratory tract was the most common site of secondary infection (n = 84, 56%) and Acinetobacter baumanii the most commonly isolated pathogen (n = 40, 31%). Urinary and deep venous catheterization increased the risk of secondary infection development. Lower HLA-DR expression and elevated IL-10 level were found in secondary infection group. The expected prolonged in-hospital stay owing to secondary infection was 4.63 days. Secondary infection was also associated with higher in-hospital, 30-day and 90-day mortality. Kaplan-Meier survival curves and Log-rank test revealed secondary infection group had a worse survival between day 15 and day 90. Conclusions: Urinary and deep venous catheter indwelling increased the risk of developing secondary infection. Secondary infection influenced outcomes of septic patients and prolonged in-hospital length of stay. Underlying immunosuppression led to a higher tendency to developing secondary infection.


Background
Sepsis accounts for a considerable number of hospital and intensive care unit (ICU) admission and adds to the overall in-hospital mortality [1,2]. Lack of consensus and knowledge in its pathological mechanism has posed a threat to patient management. After proper treatment, conditions of many septic patients became stable. However, some other patients developed secondary infection which led to the aggravation of disease and multiple organ dysfunction syndrome (MODS).
Previous studies have provided some findings on the risk factors of developing secondary infection, such as age, severity of disease on admission, length of stay (LOS) in ICU and invasive procedures [3,4]. Some studies also focused on the association between secondary infection and the prognosis of septic patients but the results were inconsistent in how secondary infection influenced the prognosis and whether it was the major cause of death [5,6].
It has also been widely studied that the underlying immune dysfunction of sepsis could lead to secondary infection. The early phase of sepsis features hyper inflammation caused by the systemic release of pro-inflammatory cytokines called "cytokine storm" [7,8].
Immunosuppression is then observed at later phase of sepsis as a result of the imbalance of pro-and anti-inflammatory activities [9]. Sepsis could lead to a variety of mechanisms such as the apoptosis and autophagy of immune cells, endotoxin tolerance and relevant center nervous system regulation, which is presented as immunosuppression consequently [8,10,11]. CD14 + monocyte human leukocyte antigen-D related (HLA-DR) expression is an effective biomarker of immune status, which reflects net sum of pro-and antiinflammatory process during sepsis [12][13][14]. Low HLA-DR expression is associated with immunosuppression and higher risk of secondary infection, especially during early phase of sepsis [15][16][17][18][19]. Serum cytokine levels are also commonly used by clinicians to monitor immune status. A higher release of anti-inflammatory cytokines such as IL-10, together with acute pro-inflammatory activities were found in the patients prone to secondary infection [20][21][22][23][24].
Because of the illuminating but inconsistent findings of previous studies, the clinical characteristics, risk factors and the prognosis of secondary infection of sepsis were further investigated. Additionally, the association between immune status and secondary infection of sepsis based on data of HLA-DR expression and serum cytokines levels were also explored in the current study.

Study setting and population
A retrospective study was carried out in emergency intensive care unit (EICU) of Zhongshan Hospital, Fudan University, Shanghai, China. Patients diagnosed with sepsis on admission between January, 2014 and January, 2018 were enrolled in this study. The diagnosis of sepsis referred to The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) , namely suspected infection with Sequential Organ Failure Assessment (SOFA) score ≥ 2 [2]. Information of infection and SOFA score were acquired form Electronic Medical Record System (EMRS). Patients were excluded if they had one of the following conditions: ① under the age of 18; ② suffering chronic heart failure (New York heart function assessment -IV), advanced malignancy, end-stage liver (Child-Pugh C) or kidney diseases (CKD-5); ③ having received in-hospital treatment in other hospitals prior to being admitted to this hospital; ④ in-hospital LOS less than 48 hours. Antiinfection treatments of all included patients were applied by experienced physicians based on either etiological evidence or empirical therapy plan. The study was approved by the Ethics Committee Study Board of Zhongshan Hospital, Fudan University (record number: 2006-23).

Data collection
EMRS and Computerized Physician Order Entry (CPOE) were screened for available data.
The following data of each patient were collected: ① baseline characteristics: age, gender, comorbidity and smoking history; ② site of primary infection; ③ index of severity of the disease at the time of admission: Acute Physiology and Chronic Health Evaluation II (APACHE II) score, SOFA score and hemodynamic status; ④ interventions such as the use of glucocorticoids, anticoagulation therapy, mechanical ventilation, urinary catheterization, deep venous catheterization, continuous renal replacement therapy and blood transfusion (whether those interventions were applied before or after the onset of secondary infection was noticed); ⑤ occurrence time, site and pathogen of secondary infection; ⑥ LOS in hospital and ICU, the outcome of hospital stay.

Measurement of monocyte HLA-DR expression and serum levels of cytokines
In order to explore the underlying immune mechanism of secondary infection, we acquired the data from Database of Clinical Sample and Information for Sepsis of Zhongshan Hospital, an database founded in 2008 intended for the collection and perseveration of clinical samples of sepsis. According to the guideline of database, the peripheral blood samples were collected in the BD Vacutainer® tubes at day 1, 3 and 7 after admission. In some patients, sample at day 3 and 7 were not collected. As the nature of an retrospective study, data of only a part of the included patients were available. To explore The level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8 and IL-10 were measured by ELISA method (R&D System, MN, USA) according to manufacturer's instructions. The experiment of flow cytometry and ELISA were conducted right after the samples were collected and the results were recorded in the database. In this retrospective study, the results were directly acquired from the database.

Statistical analysis
The Kolmogorov-Smirnov test was used to verify the normality of all data. Normally distributed data were expressed as means and standard deviation (SD). Abnormally distributed continuous data were expressed as median and the 25th and 75th quartiles.
Categorical data were expressed as frequency and percentage.
The risk factors of secondary infection of septic patients were explored by a two-step method. Firstly, univariate analysis were conducted. Covariates included age, gender, comorbidities, smoking history, site of primary infection, hemodynamic status and severity of disease on admission, HLA-DR expression and serum cytokines levels and clinical interventions before onset of secondary infection. Student's t test was used to compare normally distributed data and Mann-Whitney U test was utilized to compare abnormally distributed data. Categorical data were compared by Pearson's chi-square test or Fisher's exact test when appropriate. Secondly, covariates with statistical significance in univariate analysis were tested in multivariate binary logistic regression analysis to identify the independent risk factors by means of Backward: Conditional method. Because the data missing of HLA-DR expression and serum cytokine levels, they were not brought into multivariate analysis.
In our study, we treated in-hospital LOS as an outcome of secondary infection, rather than a potential risk factor. A multistate model with 4 states (state 0: admission, state 1: development of secondary infection, state 2: being discharged alive, state 3: in-hospital death) was performed using "etm" package in R in order to explore the influence of secondary infection on in-hospital LOS, where the data of patients with an in-hospital LOS longer than 100 days were omitted to eliminate the impact of extreme cases (see Additional file 1: Figure S1) [3]. Survival analysis was conducted by Kaplan-Meier method and Log-rank test was used to compare survival curves and it was conducted in every division once two curves had intersections. The two-step method was also used to explore the risk factors of mortality. Univariate analysis was conducted first and followed by multivariate binary logistic regression analysis. Secondary infection was among covariates, together with age, gender, comorbidities, smoking history, site of primary infection, hemodynamic status and severity of disease on admission, clinical interventions and ICU and in-hospital LOS.
All statistical analyses were two-sided, and the significance level was set to P < 0.05. We checked the model assumptions before using each statistical methods. Statistical analysis was conducted on SPSS 25.0 (SPSS Inc., IL, USA) and R 3.5.1 (R Development Core Team).

Characteristics of septic patients
From January, 2014 to January, 2018, a total of 297 patients were enrolled in this study. A flowchart to illustrate the recruitment of study samples was shown in  Table 1. Staphylococcus aureus (n = 9, 6%) were common identified pathogens. In 23 cases, pathogens were not identified. The characteristics of secondary infections were shown in Table 2 and distribution of pathogens, time of onset and diagnostic criteria of each infection were shown in Table S1 (see Additional file 2) .

Risk factors of secondary infection in septic patients
No statistical significance existed between septic patients with and without secondary infection concerning age, gender, comorbidity and site of primary infection. In univariate analysis, statistical significance was found in severity of illness on admission (APACHE II score: P < 0.001; SOFA score: P = 0.007) and some interventions before the onset of secondary infection such as the use of mechanical ventilation ( Table 3).

The association between HLA-DR expression, cytokine levels and secondary infection of sepsis
Data of a part of patients were available for HLA-DR expression and cytokines. The exact numbers were shown in Table 1. In the univariate analysis of the risk factors of secondary infection, statistical significance was found in HLA-DR expression at day 3 (P = 0.048) , IL-0.035). The results were shown in Table 1, Figure 2 and Additional file 3: Table S2.
Although statistical significance was not found at every time point, a trend of decrease of HLA-DR expression and increase of IL-10 level in secondary infection group was observed, which is indicative of immunosuppression (Figure 2A & B). Interestingly, a reverse trend of dynamic change was found between two pro-inflammatory cytokines IL-6 and IL-8 in both secondary infection and non-secondary infection groups ( Figure 2C & D).
Representative flow cytometry profiles for HLA-DR expression were shown in Figure 3.

The association between secondary infection and the outcomes of sepsis
Secondary infection group had longer LOS in hospital and ICU than non-secondary infection group (in-hospital LOS: P < 0.001; ICU LOS: P < 0.001) (  Table S3). Multivariate binary logistic regression analysis also found out that secondary infection was an independent risk factor of in-hospital mortality (OR 3.476, 95% CI 1.599 to 8.219, P = 0.003) (see Additional file 5: Table S4). Kaplan-Meier survival curves and Log-rank test revealed no difference between two groups before day 15 (P = 0.426) (see Additional file 6: Figure S2). But non-secondary infection group had a better survival between day 15 and day 90 (P < 0.001) ( Figure 5) and subgroup analysis showed that the difference remained significant in patients with and without septic shock (P = 0.04 and P < 0.001) (see Additional file 7: Figure S3).

Discussion
Our study confirmed a high incidence of secondary infection in septic patients (31.0%). We found higher APACHE II and SOFA scores on admission in patients with secondary infection, which were similar to previous studies [3,4,31]. Although illness severity was not found to be an independent risk factor of secondary infection. It could be explained that the more severely ill patients died mostly at the very early period of disease before developing secondary infections, which might impact the true association between the risk and illness severity.

Moreover
It's widely acknowledged that catheter indwelling was a major cause of nosocomial infection [32-34]. We found that urinary catheterization was an independent risk factor of secondary infection. A study revealed that catheter-associated urinary tract infection was not only affected by duration of urinary catheterization, but also the presence of another site of nosocomial infection, which was confirmed by our study that many cases of secondary infections in urinary tract were subsequent to secondary infections at other sites [35]. Deep venous catheterization was also common in ICU setting. Our finding was consistent with the study by van Vught et al. that it was also an independent risk factor of secondary infection [4]. Also, mechanical ventilation and blood transfusion were found to be risk factors in univariate analysis. The need for mechanical ventilation of critical ill patients incurred high prevalence of ventilator-associated pneumonia, which accounted for nearly half of nosocomial infections and was found to be an independent risk factor of secondary infection in some studies [3,4,36]. But the result of multivariate analysis of our study was not consistent with previous findings. As we did not tell apart invasive and noninvasive ventilation due to limited medical record and the duration of ventilation was not recorded, the result should be interpreted cautiously. The association between blood transfusion and secondary infection of sepsis could be induced by transfusion-related immune modulation (TRIM) caused by the altered immune function [37][38][39][40].
Immune status of septic patients and its underlying mechanism have been widely studied.
Innate immune function was altered as a dysfunction of neutrophils, monocytes, dendritic cells and myeloid-derived suppressor cells (MDSCs) which causes altered first-line of defense, inhibition of T cell proliferation, altered inflammatory response and incomplete activation of T cells [8]. Adaptive immune function was also altered as sepsis affects the effector functions and phenotypes of T cells, B cells and innate-type lymphocytes [8]. HLA-DR and cytokines were chosen to reflect the immune status in this study. HLA-DR was an marker reflecting both innate and adaptive immune function and lower expression indicates immunosuppression [8]. IL-10 was an anti-inflammatory cytokine and elevated level reflected the down-regulation of inflammation process. It might generate MDSCs and enhances the immunosuppression during sepsis [20,41]. In secondary infection group of this study, HLA-DR expression was lower and IL-10 level showed a trend of increase, which was a sign of immunosuppression. A more severe pro-inflammatory response in secondary infection group presented as a higher IL-6 and IL-8 level, was also observed, which were both pro-inflammatory cytokines. This confirmed that higher pro-and anti-inflammatory process might exist at the same time in septic patients with secondary infection [21,23]. A reverse trend of dynamic change between IL-6 and IL-8 was noticed, though they were both pro-inflammatory cytokines. This might be explained by that the increase of IL-6 demonstrated the progress of inflammation, as the blood sample collected at day 3 and 7 were more often from severely ill patients. IL-8, as we hypothesized, might be involved in early phase inflammatory process rather than later phase and thus showed a trend of decrease. As the dynamic changes were only statistical significant in some timepoints, studies with a larger sample size are needed to further the study. Those results enlightened us that the identification and risk stratification of immunosuppression and the therapies that boost immunity could be beneficial to the prevention of secondary infection [13,42,43]. When it comes to mortality, secondary infection was an independent risk factor of inhospital death in this study. Survival analysis demonstrated that patients with secondary infection had worse outcome after first 15 days. In the first 15 days, secondary infection group even had better survival. That could be explained by that patients who were severely sick died earlier before they developed secondary infections. This was consistent with the previous concept that the mortality of patients who survived that early period was more likely affected by secondary infection [13]. A re-increased microbiological burden revealed by positive blood cultures at later phase of sepsis (> 15 days) was

Ethics approval and consent to participate
The clinical study protocol was approved by the Ethics Committee Study Board of Zhongshan Hospital, Fudan University, Shanghai, China (record number 2006-23). Written informed consent was obtained from subjects or their legal surrogates before enrollment.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there are no conflicts of interest.

Acknowledgments
We would like to thank Prof. Weibing Wang for his help in statistical methods and Prof.
Yiqun Chen for his help in English language editing. leukocyte antigen-DR is independently associated with nosocomial infections after

Additional Files
Additional file 1: Figure S1. Illustration of multistate model to explore the expected ength of stay. (JPEG) Additional file 2: Table S1.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.