hemorrhagic fever with renal syndrome ( hfrs ) is a rodent - borne disease that is caused by hantavirus ( hv ) , with major clinical characteristics of fever , hemorrhage , hypotension , and renal damage [ 1 , 2 ] . the typical disease progresses through five phases : febrile , hypotensive , oliguric , diuretic , and convalescent [ 3 , 4 ] . china is the most severe endemic area of hfrs in the world , with a high incidence rate in the last ten years . xi'an city is the center of shaanxi province and one of the most severely affected regions in china . the pathogen that causes hfrs having been discovered in the city is hantaan virus , the major serotype of hv carried by the rodents apodemus agrarius . furthermore , the hfrs patients in this district display more severe manifestations , usually accompanied with refractory shock , acute respiratory distress syndrome ( ards ) , encephalopathy , disseminated intravascular coagulation ( dic ) , and multiple organ dysfunction syndrome ( mods ) , which leads to a greatly increased fatality rate . there is no specific antiviral therapy for hfrs , and the identification of a single laboratory parameter that is routinely tested clinically to evaluate severity and predict the outcome remains challenging because of the nonspecific , complicated clinical courses and the pathophysiology of the disease . nevertheless , an accurate early determination of disease severity and the investigation of new biomarker are still very important to timely and systematic treatment . in this study , we detected the concentrations of the high mobility group box protein-1 ( hmgb-1 ) in hfrs patients and explored its predictive value on the disease severity and prognosis . the perspective study was approved by the institutional review board of tangdu hospital . before inclusion , the patients were informed about the objectives of this study ; they or their direct relatives agreed and signed the informed consent form so that blood samples and medical records could be obtained . one hundred and five patients with hfrs that were treated at our center between october 2011 and december 2012 were randomly enrolled in this study . patients who had other kidney diseases , diabetes , cardiovascular disease , hematological disease , autoimmune disease , viral hepatitis , and other liver diseases were excluded . the diagnosis of hfrs was made based on the positive enzyme linked immunosorbent assay ( elisa ) result for specific igm and igg antibodies against hantaan virus in acute phase serum . the assay was performed using igg / igm capture elisa kits and was analyzed via a multifunctional autoanalyzer ( biorad-680 , united states ) . according to the hfrs criteria of clinical classification , the severity of hfrs was classified into four types : ( 1 ) mild , defined as patients who had kidney injury without oliguria and hypotension ; ( 2 ) moderate , defined as patients who had uremia , effusion ( bulbar conjunctiva ) , hypotension , hemorrhage ( skin and mucous membranes ) , and aki with typical oliguria ; ( 3 ) severe , defined as patients who had severe uremia , effusion ( bulbar conjunctiva and either peritoneum or pleura ) , hemorrhage ( skin and mucous membranes ) , hypotension , and aki with oliguria ( urine output of 50500  ml / day ) for 5 days or anuria ( urine output of < 100  ml / day ) for 2 days ; and ( 4 ) critical , defined as patients who usually had one or more of the following complications compared with the severe patients : refractory shock ( 2 days ) , visceral hemorrhage , heart failure , pulmonary edema , brain edema , severe secondary infection , and severe aki with oliguria ( urine output of 50500  ml / day ) for > 5 days or anuria ( urine output of < 100  ml / day ) for > 2 days . considering the clinical conditions that a majority of the survival patients had been discharged before the convalescent phase and the degree of acute kidney injury ( aki ) that was still severe during the early stage of the diuretic phase , the acute stage was defined as the period that included the febrile , hypotensive , and oliguric phases and the early three days of the diuretic phase in this study , and the convalescent stage was defined as the diuretic and convalescent phase except the early three days of the diuretic phase . furthermore , the patients were followed up until 28 days after discharge , and the prognosis ( death ) in this study was defined as patient death during hospitalization or within the 28 days following discharge . ninety - three venous blood samples were drawn randomly from the patients during the acute stage , and 78 samples were drawn randomly during the convalescent stage . all of the samples were stored in edta tubes and were centrifuged at 2,000  rpm for 10  min at 4c within 2 hours after drawing . the plasma supernatant was pipetted carefully and transferred to polypropylene tubes and then stored at 80c prior to hmgb-1 analysis . 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 twice and the sensitivity of the minimum concentration of hmgb-1 was below 0.3  ng / ml . seven laboratory parameters including white blood cells ( wbc ) , platelets ( plt ) , hematocrit ( hct ) , albumin ( alb ) , blood urea nitrogen ( bun ) , serum creatinine ( scr ) , and uric acid ( ua ) were routinely tested using autoanalyzers ( sysmex xt-4000i , japan ; hitachi 7600 - 100 , japan ) . all the laboratory parameters mentioned above and hmgb-1 were measured in the same time frame . tables were created using excel 2003 ( microsoft ) , and figures were created using graphpad prism 5 ( graphpad software , san diego , ca ) . continuous variables are presented as the mean  sd and were analyzed by kolmogorov - smirnov 's test for normal distribution and by levene 's test for the homogeneity of variance . the variables among the four types were compared by snk test for normally distributed variables . the nonnormally distributed variables are presented as medians with interquartile ranges and were compared by the nonparametric kruskal - wallis h test . the frequencies and percentages are given for qualitative variables , and the differences among the four types were tested using pearson 's chi - square test . pearson 's correlation coefficient was used to determine the relationship between hmgb-1 and the laboratory parameters as mentioned above . the predictor values of hmgb-1 for disease prognosis were tested using receiver operating characteristic ( roc ) curves and quantified by calculating the area under the roc curve ( auc ) and the 95% confidence interval ( ci ) . of the enrolled patients , 19 cases were classified as mild , 25 cases were classified as moderate , 27 cases were classified as severe , and 34 cases were classified as critical according to the hfrs criteria of clinical classification . twelve critical individuals died during the acute stage with a hospital mortality rate of 11.42% . there was no significant difference in the sex or age distribution among the groups ( p > 0.05 ) ( table 1 ) . the duration from disease onset to sample collection among the groups was not significantly different ( p > 0.05 ) ( table 2 ) . the levels of hmgb-1 in the patients from the acute stage were significantly higher than control ( p < 0.001 ) and were increased with the severity of the disease . the levels of hmgb-1 in the patients in the convalescent stage were higher than the control group , except for the mild - type group ( p < 0.05 ) ; the hmgb-1 levels of the critical - type group were higher compared with the mild- and moderate - type groups ( p < 0.05 ) ( table 3 , figure 1 ) . pearson correlation analysis revealed that hmgb-1 was positively correlated with wbc and bun and was negatively correlated with plt , alb , and ua ( p < 0.001 ) ( table 4 , figure 2 ) . roc analysis revealed that hmgb-1 showed statistical significance for predicting prognosis with the area under the curve ( auc ) equal to 0.800 ( 95% ci : 0.6450.955 , p < 0.001 ) . the sensitivity and specificity of the hmgb-1 , wbc , plt , and alb in combination for predicting outcome were better than with individual analysis ( auc = 0.892 , p < 0.001 ) ( table 5 , figure 3 ) . this is the first study to explore the predictive capacity of hmgb-1 for the severity and prognosis of hfrs . it can be released by activated monocytes , macrophages , neutrophils , and platelets and , in turn , mediates inflammation and enhanced cell motility . it has been proven that the lipopolysaccharide ( lps ) that is released by gram - negative bacteria and proinflammatory factors such as tnf- , il-1 , and ifn- can positively stimulate the secretion of hmgb-1 ; extracellular hmgb-1 can be secreted passively by necrotic rhagiocrine cell . extracellular hmgb-1 can be considered an effective mediator that further induces and promotes the inflammatory response [ 10 , 11 ] . as a key late - phase proinflammatory cytokine , hmgb-1 participates in the physiopathological course of sepsis   and has become an important target for the prevention and treatment of sepsis . in the past ten years , only a minority of research focused on observing the predictive role of hmgb-1 on disease severity and prognosis in patients with sepsis , including different findings . found increased hmgb-1 serum levels in patients with sepsis or septic shock in a prospective study with 33 septic patients . they did not find a significant difference between the septic and septic shock patients , and hmgb-1 was not found to be correlated with the prognosis of sepsis . in another study that was recently published , gibot et al . reported that the septic shock nonsurvivors had higher hmgb-1 concentrations than survivors on day 3 but not at the time of hospital admission . they also found that the hmgb-1 concentrations on day 3 could be beneficial to predicting the prognosis of the septic shock patients , and hmgb-1 was closely correlated with the degree of multiple organ dysfunctions . in one study , angus et al .   found that the circulating blood of patients with pneumonia and with pneumonia combined with serious sepsis demonstrated greatly elevated levels of hmgb-1 , while there was no significant difference between the two groups . they also found that the hmgb-1 concentrations in nonsurvivors were lower compared with the survivors . considering that the role of a single laboratory parameter that is routinely tested clinically for severity evaluation and outcome prediction is still challenging , we observed levels of high mobility group box protein-1 ( hmgb-1 ) in different clinical phases of hfrs and explored its predictive value on disease severity and prognosis . unlike the results from the research in sepsis patients as mentioned above , this study demonstrated that hmgb-1 levels increased with the severity of hfrs ( table 3 , figure 1 ) ; dynamic monitoring of hmgb-1 could benefit the early prediction of prognosis ( table 5 , figure 3 ) , which indicates that hmgb-1 plays an essential role in the pathogenesis of hfrs as a proinflammatory cytokine . hmgb-1 can be considered a novel biomarker to evaluate disease severity and outcome in hfrs patients , with potential for applications in clinical practice . it is believed that hfrs has the basic clinical characteristics of sepsis , but it also has a unique pathophysiologic feature . the hypotensive phase of hfrs ( e.g. , low blood pressure and circulation collapse ) usually occurs between day 3 and day 7 of the clinical course , and grave hfrs patients can manifest more severe leukemoid reaction , plasma leakage , and coagulation disorders [ 18 , 19 ] compared with septic patients , which would lead to massive bleeding , profound shock , severe tissue hypoperfusion , and severe hypoxia , potentially rendering renal , cardiac , cerebellar , and hepatic injury [ 4 , 19 , 20 ] . this is similar to the data shown in this study , except that hmgb-1 , wbc , plt , and alb also demonstrated statistical significance for predicting prognosis , which can reflect the degree of inflammatory reaction , destruction , or dysfunction of plt   and the loss of vascular integrity with increasing degree of vascular permeability [ 3 , 22 , 23 ] . in this study , pearson 's correlation analysis revealed that hmgb-1 was closely correlated with wbc , plt , and alb ( table 4 , figure 2 ) , and the sensitivity and specificity of the combined hmgb-1 , wbc , plt , and alb for predicting prognosis were better than those of single analysis ( table 5 , figure 3 ) ; this further indicates the effective role of hmgb-1 for evaluating the severity and outcome , and the use of hmgb-1 , wbc , plt , and alb in combination to predict the outcome in patients with hfrs exhibited an acceptable diagnostic capability . as a perspective study , we evaluated the predictive role of hmgb-1 on the severity and prognosis of hfrs , while there were still some limitations . the length of time form collection of the blood samples from the patients was not unified or precise , considering the different clinical conditions and phases on admission , and we can only define two periods , the acute and convalescent stages . although there was no significant difference in the median collection time of the samples , the dynamic change of the levels of hmgb-1 can also be influenced by this variation . there were only 12 patients who died , which would influence the result of the roc curve analysis . third , the clinical outcomes and classifications of the hfrs patients might be biased due to the lack of a more standardized protocol for the management of patients with hfrs until now .
objective . to examine the predictive capacity of the high mobility group box protein-1 ( hmgb-1 ) for disease severity and prognosis of hemorrhagic fever with renal syndrome ( hfrs ) 
 . methods . 
 one hundred and five hfrs patients and 28 controls were studied . 
 the concentrations of hmgb-1 in the blood were measured with a commercially available elisa . 
 the levels of white blood cells ( wbc ) , platelets ( plt ) , hematocrit ( hct ) , albumin ( alb ) , blood urea nitrogen ( bun ) , serum creatinine ( scr ) , and uric acid ( ua ) were routinely tested in the same time frame . 
 results . 
 the levels of hmgb-1 increased with the severity of the disease ( p < 0.001 ) . 
 hmgb-1 was positively correlated with wbc and bun and negatively correlated with plt , alb , and ua ( p < 0.001 ) . 
 hmgb-1 showed statistical significance for predicting prognosis ( auc = 0.800 , p < 0.001 ) . 
 the sensitivity and specificity of hmgb-1 , wbc , plt , and alb used in combination for predicting outcome were better than those of single analyses ( auc = 0.892 , p < 0.001 ) . 
 conclusions . 
 hmgb-1 can be considered a novel biomarker for severity and outcome in patients with hfrs . 
 the use of hmgb-1 , wbc , plt , and alb in combination to predict the outcome in patients with hfrs exhibited an acceptable level of diagnostic capability .