Study Population
A total of 105 patients with HFRS admitted by the Second Affiliated Hospital of Air Force Medical University from October 2012 to December 2014 were randomly enrolled in this study. All patients were confirmed by serological examination with the positive results of specific 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 HFRS patients from the electronic medical records, and which confirmed 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 Affiliated 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 Definitions
According to the clinical classification criteria of HFRS [18], 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 <100 mL/day) >2 days. The classification of all patients was confirmed by their attending physicians respectively. Based on the classically defined five stages of HFRS [19], the clinical course was divided into the acute phase (including the febrile, hypotensive, and oliguric stages) and the convalescent phase (including the diuretic and convalescent stages). The patients received a follow-up visit until 28 days after discharge, and the death in this study was defined as patient died during hospitalization or the follow-up period.
Blood samples and the detection of pentraxin-3
Under sterile conditions, 96 venous blood samples in acute phase and 65 in convalescent phase from the enrolled patients, and 27 from healthy volunteers were collected by ethylene diamine tetraacetic acid (EDTA) tubes. All specimens were collected on an empty stomach in the morning, and were centrifuged at 3000 rpm for 10 min at 4 °C within 2 hours after drawing. The plasma supernatant was pipetted carefully and transferred to polypropylene tubes, and then stored at −80 °C prior to analysis. To avoid the experimental errors caused by repeated freeze-thaw of plasma, all specimens were tested together after collection.
Pentraxin-3 levels were measured with commercially available ELISA kits (Quantikine, XiTang Inc., Shanghai, China) and were tested using a multifunctional autoanalyzer (BIORAD-680, United States) according to the manufacturer’s instructions. Each sample was detected in duplicate. The sensitivity of the minimum concentration of pentraxin-3 was between 0.008ng/mL and 0.116ng/mL. According to the difference of clinical typing, some samples of the patients were further taken multiple dilutions according to 1:2, 1:4, 1:16, 1:32 and 1:64, and finally selected the reasonable detection results.
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 indexes. The predictive efficacy of pentraxin-3 for the prognosis (death) of patients with HFRS was evaluated by the receiver operating characteristic (ROC) curve analysis and quantified by the area under ROC curve (AUC). A two-sided P<0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (IBM SPSS Statistics, version 23.0).