Sepsis is a serious clinical condition with a considerable morbidity and mortality 1. Clinicians are in need of good diagnostic and prognostic markers to identify infected patients who could benefit from prompt empirical antibiotic therapy and other supportive therapy as early as possible. An increased knowledge of the immunopathogenesis of sepsis could have the potential of generating new diagnostic and treatment modalities for this serious condition.

High-mobility group-box 1 protein (HMGB1) is a nuclear chromosomal protein 23. A new role for HMGB1 has been explored in recent years. HMGB1 has been suggested to have an important role as a 'late-onset' proinflammatory cytokine 45. HMGB1 was rediscovered in this role when cultures of macrophages were exposed to endotoxin 4. Animal models confirmed these observations, and there has been considerable attention on this protein especially in relation to sepsis and rheumatoid arthritis 4. Lipopolysaccharide-binding protein (LBP) is an acute-phase protein with an important role in the innate immune system 67. For the past 15 years attention has been pointed at the inflammatory marker procalcitonin (PCT) 89, which has been associated with severe bacterial infections among adults and children 9.

The present study purpose was to examine levels of HMGB1, LBP and PCT in patients with sepsis of different severity, in bacteraemic patients and in relation to the outcome of the patients. Another purpose was to examine the diagnostic test abilities of HMGB1, LBP and PCT to predict bacteraemia. Finally, correlations between the examined markers were explored.

HMGB1 has been known for many years as a chromosomal protein. In recent years there has been interest in HMGB1's role as a proinflammatory cytokine 45. Animal models have shown that HMGB1 has an important role in immunopathogenesis in sepsis 4. Administration of exogenous HMGB1 to septic animals increased mortality, and administration of antibodies against HMGB1 ameliorated the clinical outcome of septic animals 4. HMGB1 has been characterised as a 'late-onset' proinflammatory cytokine involved in the late phases of the septic process, after the early induction of 'early-onset' proinflammatory cytokines such as TNFα and IL-1 45. Disappointing results in trials trying to suppress early proinflammatory pathways in sepsis have made HMGB1 an interesting target molecule in sepsis 4516.

HMGB1 levels have been measured in several clinical sepsis cohorts 41417181920. Three of these studies used blotting methods 41720 and three of the studies used ELISA techniques 141819. In the study by Wang and colleagues, patients with fatal sepsis had median HMGB1 levels of 84 ng/ml and surviving sepsis patients had median HMGB1 levels of 25 ng/ml 4. In the study by Sunden-Cullberg and colleagues, the HMGB1 levels in critically ill patients remained elevated for up to 1 week, with mean levels of HMGB1 over 340 ng/ml after a 144-hour observation period 17. In a study of community-acquired pneumonia by Angus and colleagues, median HMGB1 levels of 190 ng/ml were observed 20. Much lower levels were seen in the three studies using HMGB1 ELISA techniques 141819. In the study by Hatada and colleagues, infected patients had median HMGB1 levels of 4.54 ng/ml 18; Yasuda and colleagues, studying infected patients with severe acute pancreatitis, observed mean HMGB1 levels of 7.8 ng/ml 19; and, finally, in a study performed by our group, the median HMGB1 level in mild sepsis was 2.14 ng/ml 14.

In the present study the HMGB1 levels were comparable with the latter three aforementioned studies using ELISA for HMGB1 measurements 141819. HMGB1 levels in the present study were higher in bacteraemic patients compared with those in nonbacteraemic patients and HMGB1 correlated to several proinflammatory markers (LBP, CRP, white blood cells and neutrophils). These correlations seem to confirm a proinflammatory role for HMGB1 in human sepsis. HMGB1 did not perform well in a ROC analysis examining its ability to identify bacteraemic patients, with an AUC of only 0.62. As mentioned earlier, levels of HMGB1 were much lower than levels reported in studies using blotting techniques. The reason for this is not clear. One possibility is that our patients who were recruited from an ordinary department of internal medicine were less ill compared with studies conducted on intensive care units. Another possibility is that we sampled patients in the early phase of disease (within 24 hours after admission), which perhaps could explain the low levels of a 'late-onset' proinflammatory cytokine such as HMGB1. Finally, the chosen laboratory technique might explain the low levels. The presence of interfering inhibitory factors/autoantibodies to HMGB1 in human serum could affect results of HMGB1 measurements with ELISA techniques 21. It is still unknown whether the currently used assays detect biologically active HMGB1. This is an important issue for future studies focusing on HMGB1 levels and disease activity.

LBP is a protein with a central role in the innate immune response in both Gram-negative and Gram-positive infection when the host is challenged by an invading pathogen 67. In Gram-negative infection, LBP carries the endotoxin lipopolysaccharide to the CD14 receptors on the monocyte-macrophage cell lineage 2223. CD14 receptors then interact with the Toll-like receptor 4, initiating cytokine production 2223. The lipotheichoic acid from pneumococci and staphylococci activates a cellular response through Toll-like receptor 2 24. This response can be enhanced by LBP and CD14 7.

Several clinical studies have examined the levels of LBP in infected patients 2526272829, in which the median levels of LBP were between 21.1 μg/ml and 59.7 μg/ml. Only one previous study has examined LBP's diagnostic test abilities in diagnosing Gram-negative bacteraemia 29. The authors found a sensitivity of 100% and a specificity of 92% with a high cut-off level (46.3 μg/ml) for LBP. The study only included four patients with Gram-negative bacteraemia 29. In the present study, the median levels of LBP were high compared with the previous studies. LBP levels in the present study were higher in bacteraemic patients compared with nonbacteraemic patients, and LBP correlated to several proinflammatory markers (HMGB1, PCT and CRP). LBP correlated to the severity of infection. LBP did not perform well in a ROC analysis examining its ability to identify bacteraemic patients, with an AUC of 0.74.

PCT is a protein involved in the immunopathogenesis of sepsis. Many different parenchymal cells are able to produce PCT when the host is challenged by a pathogen 30. Animal models have shown that administration of exogenous PCT to septic animals increased mortality and administration of antibodies against PCT to septic animals protected against fatal outcome 3132. Elevated levels of PCT have been associated with several conditions, such as toxic shock syndrome, bacterial sepsis, postoperative infectious complications, meningitis, cholangitis, pancreatitis with infection, malaria and fungemia 33. PCT has been shown to be a marker associated with the severity of sepsis 3435363738. Several previous studies have examined PCT's diagnostic test abilities in diagnosing bacteraemia 394041424344. These studies found AUCs between 0.71 and 0.85 394041424344. In the present study the PCT levels increased with increasing severity of infection, with the highest levels in severe sepsis (median 4.4 ng/ml) and in septic shock (median 46.1 ng/ml). These data confirm findings from earlier studies showing that PCT is a severity marker in sepsis.

Our study data showed more than 20-fold higher PCT levels in bacteraemic patients compared with nonbacteraemic patients. The AUC of PCT in diagnosing bacteraemia was 0.79. This result regarding PCT's diagnostic test abilities in diagnosing bacteraemia confirms the abovementioned findings in previous studies. Diagnosis of bacteraemia at the present time is a relatively slow process, requiring up to several days of culturing/processing in the laboratory of microbiology. It is possible that a good biochemical marker for the presence of bacteraemia could have a role in stratifying patients to faster microbiological diagnosis with molecular diagnostic techniques. A broad-range PCR could perhaps be a possible strategy speeding up the species diagnosis in bacteraemia. PCT may have a role in identifying patients that could benefit from fast molecular diagnostics.

The strengths of the present study are its prospective design, the relatively large sample size, well-characterised patients and fast blood sampling after admission to the hospital. The study focused on patients admitted to a general department of internal medicine. Many previous studies examining immunological, prognostic and diagnostic markers in sepsis have focused upon critically ill patients on intensive care units. Most patients with infections and sepsis are, however, at the milder end of the sepsis spectrum and will be treated on general departments of internal medicine or surgery. The risk of work-up bias was reduced by classifying the infectious status of the patients without access to the biochemical laboratory results. The laboratory technicians were blinded from the clinical data. The risk of spectrum bias was reduced by using relatively broad inclusion criteria, including all age groups over 18 years, all kinds of infectious foci, different aetiology, different severity and comorbidity.

A drawback of the study design was the risk of an imperfect gold standard bias. Before inclusion of patients was begun, the criteria for infection and sepsis severity were established by the study group. These criteria were followed rigorously to minimise the risk of an imperfect gold standard bias. Patients with uncertain diagnosis were excluded for the same reason. Drawbacks of the present study, as in many other clinical sepsis studies, were the heterogeneity among included patients, a heavy burden of comorbidity, variable severity of disease, variation in infectious focus, variation in microbiological aetiology and different lengths of disease prior to hospitalisation.

This is the largest prospective study that has been conducted regarding HMGB1 measurements in infections and sepsis. Elevated levels of HMGB1, LBP and PCT were associated with the presence of infection and with the presence of bacteraemia in patients with community-acquired infections. None of the examined inflammatory markers had prognostic abilities in identifying patients with fatal outcome. PCT had better diagnostic test abilities in diagnosing the presence of bacteraemia compared with HMGB1 and LBP. PCT could have a future role in identifying patients who would benefit from new faster molecular diagnostic techniques for diagnosing bacteraemia.

• HMGB1 is a proinflammatory cytokine in severe infections and bacteraemia.

• LBP and PCT are severity markers in severe infections and bacteraemia.

• PCT is a better diagnostic test marker for bacteraemia compared with HMGB1 and LBP.

AUC = area under the curve; CRP = C-reactive protein; ELISA = enzyme-linked immunosorbent assay; FiO₂ = fraction of inspired oxygen; HMGB1 = high-mobility group box-1 protein; IL = interleukin; LBP = lipopolysaccharide-binding protein; PaO₂ = partial pressure of arterial oxygen; PCR = polymerase chain reaction; PCT = procalcitonin; ROC = receiver–operator characteristic; SIRS = systemic inflammatory response syndrome; TNF = tumour necrosis factor.

The authors declare that they have no competing interests.

SG planned the study, wrote the protocol, collected and analysed the data, and wrote the report. OGK was responsible for the PCT, LBP and IL-6 analyses. HJM was responsible for the HMGB1 analyses. CP and SSP were involved in planning the study, in revising the manuscript and in practical clinical aspects. All authors read and approved the final manuscript.