Predictive value of pentraxin-3 on disease severity and prognosis in patients with hemorrhagic fever with renal syndrome

Pentraxin-3 is an acute-phase protein involved in the processes of inammatory and infection. This study aimed to analyze the changes of plasma pentraxin-3 prospectively and evaluate its predictive value on disease severity and prognosis in patients with hemorrhagic fever with renal syndrome (HFRS). Methods 105 patients with admitted by from to 2014 were randomly enrolled in this study. All patients were divided into a mild-type group (17 cases), a moderate-type group (27 cases), a severe-type group (26 cases) and a critical-type group (35 cases) according to the HFRS criteria for clinical classication. 96 venous blood samples of acute phase and 65 of convalescent phase were collected from patients during hospitalization, while venous blood samples of 27 healthy volunteers were taken as controls. The levels of plasma pentraxin-3 were detected using the enzyme linked immunosorbent assay (ELISA), and which were compared among the acute and convalescent phases in different types of patients, as well as the control group. Spearman correlation analysis was used to evaluate the correlation between pentraxin-3 and conventional laboratory parameters. The predictive effectiveness for prognosis of pentraxin-3 was evaluated by receiver operating characteristic (ROC) curve analysis.


Results
There was no signi cant difference in gender and age distribution between all types of patients and control group (P > 0.05). In all types of patients, the levels of pentraxin-3 in acute phase were signi cantly higher than that of control group and convalescent phase of the same type (P < 0.05). The levels of pentraxin-3 increased with the aggravation of the disease, and showed the highest expression in criticaltype patients (P < 0.05). Pentraxin-3 was positively correlated with WBC, AST and APTT, and negatively correlated with PLT, ALB and Fib (|r s |>0.500, P < 0.001). Pentraxin-3 showed signi cant predictive value for the prognosis of patients with HFRS, with the area under ROC curve (AUC) of 0.753 (95%CI: 0.593 ~ 0.914, P = 0.003).

Conclusions
The detection of plasma pentraxin-3 can be bene cial to the evaluation of disease severity and prognosis in patients with HFRS.
Background Page 3/15 Hemorrhagic fever with renal syndrome (HFRS) is a kind of natural focus disease caused by Hantavirus infection, which is characterized by fever, hemorrhage and renal impairment [1]. Hantavirus infection could induce the destruction of vascular endothelial cells [2], the diffuse damage of systemic microvessels, the increase of capillary permeability, and the decrease of platelets [3]. Patients with intemperate immunoreaction may further develop "capillary leakage syndrome", which could result in secondary edema, hypovolemic shock, acute kidney injury (AKI), coagulation disorder, and even multiple organ dysfunction syndrome (MODS) [4]. It has been accepted that HFRS has immunopathological features of systemic in ammatory response syndrome (SIRS) [5]. The early diagnosis and disease severity assessment may help the clinicians to choose the best therapeutic schedule for patients and nally improve the therapeutic effect of patients with HFRS [6]. Given the limited predictive value of conventional laboratory parameters, it is necessary to explore novel biomarkers for disease severity and prognosis in patients with HFRS.
As the rst long-pentraxin discovered by human, pentraxin-3 is mainly produced by monocyte macrophages and myeloid dendritic cells stimulated by pro-in ammatory signals such as IL-1β, TNF-α and Toll-like receptor activation, as well as the secretion of neutrophils, lymphocytes, and endothelial cells [7]. Pentraxin-3 plays an important role in innate humoral immune response, in ammatory response, antiinfection, as well as tissue damage and repair [8]. Many previous studies have shown that the level of plasma pentraxin-3 is positively related to the severity of sepsis, acute pancreatitis, acute myocardial injury and other diseases, which can be severed as a new biomarker for in ammation, infection and tissue damage [9][10][11][12][13]. Additionally, the over-expressed pentraxin-3 of neutrophils may be associated with the overproduction of reactive oxygen species (ROS) and vascular endothelial dysfunction, and which may represent an emerging biomarker for the progression of vascular injury in patients with hemodialysis [14]. Laine et al [15] showed that plasma pentraxin-3 was highly correlated with PLT, Fib, APTT and other coagulation indicators in patients with acute Puumala virus infection, and which got a favorable predictive value for the severity of epidemic nephropathy patients. However, the role of pentraxin-3 in HFRS caused by Hantaan virus infection has not been reported. Given the above research background, we prospectively analyzed the changes of plasma pentraxin-3 in patients with HFRS, and investigated its predictive value for disease severity and prognosis (death) of HFRS.

Study Population
105 patients with HFRS admitted by the Second A liated Hospital of Air Force Medical University from October 2012 to December 2014 were randomly enrolled in this study. All patients were con rmed by serological examination with the positive results of speci c IgM and IgG antibodies against Hantaan virus in the acute phase. The assay was performed using IgM/IgG capture ELISA kits and was analyzed via a multifunctional autoanalyzer (BIORAD-680, United States). Patients with chronic kidney diseases, diabetes, cardiovascular diseases, hematological diseases, autoimmune diseases, viral hepatitis and other liver diseases were excluded. Data of demographic, laboratory and clinical outcomes were reviewed and extracted by two physicians who had been treating patients with HFRS from the electronic medical records, and which con rmed independently by at least two researchers. In addition, we recruited 27 healthy volunteers as controls. The study was approved by the ethics committee of the Second A liated Hospital of Air Force Medical University. Before inclusion, the patients and healthy volunteers were informed about the objectives of this study, and they or their guardians agreed and signed the informed consent form.

Procedures and De nitions
According to the clinical classi cation criteria of HFRS [16], all enrolled patients were divided into the following four groups: (1) mild-type: patients with mild renal impairment without oliguria and hypotension; (2) moderate-type: patients with obvious symptoms of effusion (bulbar conjunctiva), hypotension, hemorrhage (skin and mucous membranes), and AKI with a typical oliguria stage; (3) severe-type: patients with severe uremia, effusion (bulbar conjunctiva and either peritoneum or pleura), hemorrhage (skin and mucous membranes), hypotension, and AKI with oliguria (urine output 100 ~ 500 mL/day) ≤ 5 days or anuria (urine output < 100 mL/day) ≤ 2 days; (4) critical-type: patients with one or more of the following complications compared with the severe-type patients: refractory shock (≥ 2 days), visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe AKI with either oliguria (urine output 100 ~ 500 mL/day) > 5 days or anuria (urine output < Statistical analysis Continuous variables of normal distribution and non-normal distribution were presented as mean ± standard deviation and median (interquartile range) respectively, and were compared by one-way analysis of variance (one-way ANOVA) and Kruskal-Wallis H test, respectively. The categorical variables were presented as numbers (percentage) and compared by chi-square test. The levels of pentraxin-3 in acute phase and convalescent phase were compared by Wilcoxon matched-pairs signed-ranks test. Spearman correlation analysis was used to evaluate the correlation between pentraxin-3 and conventional laboratory parameters. The predictive e cacy of pentraxin-3 for the prognosis (death) of patients with HFRS was evaluated by the receiver operating characteristic (ROC) curve analysis and quanti ed by the area under the ROC curve (AUC). A two-sided P < 0.05 was considered statistically signi cant. All statistical analyses were performed using SPSS software (IBM SPSS Statistics, version 23.0).

Clinical typing and demographic characteristics of the enrolled patients
There were a total of 105 patients with a mean age of 41.85 ± 15.35 years enrolled in this study, including 21 (20.0%) females and 84 (80.0%) males. According to the grouping criteria mentioned above, 17 cases were classi ed as mild-type, 27 cases were classi ed as moderate-type, 26 cases were classi ed as severe-type, and 35 cases were classi ed as critical-type. Of all the enrolled patients, 14 (13.3%) critical patients were died and the rest were recovered and discharged. There was no signi cant difference in gender and age distribution between all types of patients and control group (P > 0.05) ( Table 1).

Levels of plasma pentraxin-3 in patients with HFRS
In all types of patients, the levels of pentraxin-3 in acute phase were signi cantly higher than that of control group and convalescent phase of the same type (P < 0.05). The levels of pentraxin-3 had an increasing tendency with the aggravation of the disease, and showed the highest expression in criticaltype patients (P < 0.05). The comparison of pentraxin-3 during the convalescent phase among the four types demonstrated no signi cant difference (P < 0.05) ( Table 2 and Fig. 1).

The correlation between pentraxin-3 and conventional laboratory parameters
The results of spearman correlation analysis showed that pentraxin-3 was positively correlated with WBC, AST and APTT, and negatively correlated with PLT, ALB and Fib (|r s |>0.500, P < 0.001) ( Table 3 and Fig. 2).

Predictive e cacy of pentraxin-3 for the prognosis (death) in patients with HFRS
The results of ROC curve analysis demonstrated obvious predictive value of pentraxin-3 for the prognosis (death) of patients with HFRS, and which with the AUC of 0.753 (95%CI: 0.593 ~ 0.914, P = 0.003). The sensitivity and speci city of pentraxin-3 for predicting the prognosis (death) was 71.4% and 80.5%, respectively (Table 4 and Fig. 3).

Discussion
After infection with Hantavirus, the human body could develop a strong and rapid immune response characterized by the hyperactivity of immune cells and the ooding of cytokines, so as to the damage of vascular endothelium and visceral organs [1][2][3][4][5][6]. As one of the most important immune cells in innate immunity, neutrophils play a crucial part in resisting bacterial infection. Nevertheless, the neutrophil counts are also elevated in most patients with HFRS triggered by the Hantavirus infection, and which are also positively correlated with the disease severity of HFRS [16,18]. Therefore, neutrophils may also play an important role in the immunopathological injury of HFRS. Pentraxin-3 synthesized by neutrophils is mainly stored in neutrophil granules, which can interact with a variety of bacteria, fungi and viruses after release and then propel the phagocytosis and clearance of pathogenic microorganisms [19]. As an important component of innate humoral immunity, pentraxin-3 has the ability to bind complement component C1q and then activate the classical pathway of complement [20,21]. Recently, many studies have showed that pentraxin-3 could modulate in ammatory cells, interact with P-selectin, reduce the nitric oxide (NO) synthesis of endothelial cells, inhibit endothelial cells proliferation and alter their functions, and nally promote vascular in ammatory response and endothelial dysfunction [22,23]. Therefore, the release of pentraxin-3 by neutrophil degranulation may be an important link in the immunopathological injury of HFRS, and the level of plasma pentraxin-3 may indirectly re ect the severity of vascular endothelial injury in patients with HFRS.
In present study, the results have showed that the levels of pentraxin-3 in acute phase were signi cantly higher than that of control group and convalescent phase of the same type, and had an increasing tendency with the aggravation of HFRS. In addition, the level of plasma pentraxin-3 was highly correlated with WBC, PLT, AST, ALB, APTT, Fib and other conventional laboratory parameters, which is consistent with the ndings of pentraxin-3 in epidemic nephropathy [15]. The above ndings indicate that pentraxin-3 can serve as an early predictor for the disease severity of HFRS. The results of ROC curves analysis demonstrated the signi cant predictive value of pentraxin-3 for the prognosis (death) of patients with HFRS, which was comparable with the predictive value of conventional laboratory parameters such as PLT. Therefore, pentraxin-3 could serve as a novel and e cient biomarker for predicting the disease severity and prognosis of patients with HFRS. The detection of plasma pentraxin-3 may help clinicians quickly identify the severe patients at an early stage and timely take optimal therapeutic schedule for them, so as to improve the therapeutic effect of patients with HFRS.
As an observational prospective study, although we got a meaningful conclusion that the detection of plasma pentraxin-3 might be bene cial to evaluating the disease severity and prognosis of the HFRS patients, while there were still some limitations as following: First, this study was conducted in a single center for infectious diseases. The results might be limited by the relatively small sample size because of the gradually declining incidence of HFRS in Xi'an city. Because some patients had tided over the acute phase on admission and only convalescence samples available, and the dead patients had died before entering the convalescence phase, so in fact, only 96 venous blood specimens in acute phase and 65 in convalescence phase were collected from patients during hospitalization. All these adverse factors may affect the accuracy of the research results. Second, the de nition of blood sample collection time of HFRS patients was too broad in this study. Given to the individual differences of patients' condition and the clinical process on admission, we could only randomly collect venous blood specimens according to the acute phase and convalescence phase de ned in the study. Although there was no signi cant statistical difference in the median collection time of all samples in the acute phase, the level of plasma pentraxin-3 was also in uenced by the different time-points and the variability of pathological injury during the acute phase of Hantaan virus infection. Third, it is essential to conduct a prospective, large sample, multicenter cohort study to further con rm the predictive e cacy and clinical application value of plasma pentraxin-3 in acute phase. Last but not least, the research limitations caused by the experimental measurement errors and outdated clinical typing criteria should not be overlooked.

Conclusions
The detection of plasma pentraxin-3 might be bene cial to the evaluation of disease severity and prognosis in patients with HFRS, which could help clinicians quickly identify the severe patients at an early stage and timely take optimal therapeutic schedule for them, so as to improve the therapeutic effect of patients with HFRS. University and was performed in accordance with the Helsinki Declaration. Before inclusion, the patients and healthy volunteers were informed about the objectives of this study, and they or their guardians agreed and signed the informed consent form.

Consent for publication
Written informed consents were obtained from all patients or their guardians and the healthy volunteers.

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