Procalcitonin, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in community-acquired infections and sepsis: a prospective study

Patients were included in a prospective manner in the period January–May 2003. The patients were referred by a general practitioner or were admitted from the Emergency Room. Odense University Hospital is a 1,200 bed health care facility serving a local population of approximately 185,000 inhabitants. The study setting was a Department of Internal Medicine covering the specialties of infectious diseases, rheumatology, pulmonary medicine and general internal medicine. Inclusion criteria for study were suspected diagnosis of infection as judged by the referring physician and blood cultures drawn at the time of admission. The exclusion criteria were age <18 years, earlier participation in the study or prior hospitalization within seven days before admission. Plasma for later analyses of PCT, LBP and IL-6 were drawn immediately after admission. The samples were processed and frozen at -80°C within 1.5 hours. Sampling was performed before any antibiotic treatment was started at the hospital. The patients received a standard of care according to the departmental guidelines. The project protocol was approved by the Ethics Committee of Fyns and Vejle Counties. Informed consent was obtained from all patients or their close relatives.

Baseline characteristics, demographic data, biochemical parameters, SIRS criteria and severity score were obtained at the time of inclusion. Severity was assessed with the Sepsis-related Organ Failure Assessment score [18]. Comorbidity was assessed with the Charlson Index [19]. Patients were classified at the time of admission according to the SIRS criteria [3]. Severe sepsis was defined as the presence of sepsis and one or several of the following indices of organ dysfunction: Glasgow coma scale ≤14, PaO₂ ≤9.75 kPa, oxygen saturation ≤92%, PaO₂/FiO₂ ≤250, systolic blood pressure ≤90 mmHg, systolic blood pressure fall ≥40 mmHg from baseline, pH ≤7.3, lactate ≥2.5 mmol/l, creatinine ≥177 μmol/l, 100% increase of creatinine in patients with known kidney disease, oliguria ≤30 ml/hour in >3 hours or ≤0.7 l/24 hours, prothrombin time ≤0.6 (reference: 0.70–1.30), platelets ≤100 × 10⁹/l, bilirubin ≥43 μmol/l, and paralytic ileus. Septic shock was defined as hypotension persisting despite adequate fluid resuscitation for at least 1 hour. If a patient had any comorbidity that could more probably explain one or more of the criteria for organ dysfunction stated earlier, the patient could not be categorized as having severe sepsis.

Infection was categorized according to the following definitions: culture/microscopy of a pathogen from a clinical focus; positive urine dip test in the presence of dysuria symptoms; chest X-ray-verified pneumonia with no identified pathogen; infection documented with another imaging technique with no identified pathogen; obvious clinical infection (for instance, erysipelas, wound infection); and identification of a pathogen by serology or PCR. The classification of the status of infection was made by a single physician who was blinded to all biochemical laboratory results. The patients were divided into the following groups for the subsequent statistical analyses: noninfected patients without SIRS, noninfected patients with SIRS, infected patients without SIRS, patients with sepsis, and patients with severe sepsis/septic shock. Patients who could not be classified were excluded from the analyses.

PCT was measured with a time-resolved amplified cryptate emission technology assay (Kryptor PCT^®; Brahms, Hennigsdorf, Germany). The functional assay sensitivity was 0.06 ng/ml. LBP and IL-6 were measured with a chemiluminescent immunometric assay (Immulite-1000^®; DPC, Los Angeles, CA, USA). The detection limit of LBP was 0.2 μg/ml. The detection limit of IL-6 was 2 pg/ml. CRP was measured with an immunoturbidometric principle (Modular P^®; Hitachi, Tokyo, Japan). White blood cells and neutrophils were counted on a Sysmex SE 9000^® (TOA^®, Kobe, Japan). PCT, LBP and IL-6 measurements were carried out in duplicate and the mean values were used for analyses.

Data are presented as medians, interquartile ranges and means ± standard deviation. Significance testing was carried out using the Kruskal–Wallis test. A two-tailed P value < 0.05 was considered statistically significant. Receiver-operator characteristic (ROC) curves and the area under the curve (AUC) were determined for PCT, LBP, IL-6, CRP, white blood cells and neutrophils. AUC values are reported with the 95% confidence interval (95% CI). The method described by DeLong and colleagues was used as the significance test for ROC and AUC comparison [20]. Sensitivities, specificities, positive predictive values and negative predictive values were calculated from cross-tabulations. The positive likelihood ratio and negative likelihood ratio were also reported.

Prior to the study we chose the following cut off levels for reporting sensitivities, specificities, positive predictive values, negative predictive values, positive likelihood ratios and negative likelihood ratios: PCT, 0.1 ng/ml, 0.25 ng/ml and 0.5 ng/ml; LBP, 20 μg/ml and 40 μg/ml; CRP, 50 mg/l and 100 mg/l; and IL-6, 25 pg/ml and 50 pg/ml. We also planned to report cut off levels, specificities, positive predictive values, negative predictive values, positive likelihood ratios and negative likelihood ratios with sensitivities of approximately 80%. We intended to compare the test performance by comparing the AUCs and by comparing the specificities when the sensitivity was approximately 80%. The Spearman rank correlation test was used to determine correlations. At the time of the study the Department of Clinical Biochemistry did not report levels of CRP below 10 mg/l; CRP measurements below 10 mg/l were therefore assigned a value of 10 mg/l for calculations. The detection limit of our method for IL-6 measurements was 2 pg/ml; IL-6 measurements below 2 pg/ml were therefore assigned a value of 2 pg/ml for calculations. Statistical calculations were performed in STATA 8 (STATA Corporation^®, College Station, TX, USA).

One hundred and ninety-four adult patients were included in our study. The patients were divided according to our definitions into the following groups: 48 noninfected patients without SIRS, 19 noninfected patients with SIRS, 32 infected patients without SIRS, 47 patients with sepsis, and 27 patients with severe sepsis or septic shock. Only one patient had septic shock. This patient was included in the severe sepsis group. Twenty-one patients could not be classified and were excluded from analyses. Fifteen (22.4%) of the noninfected patients were treated with prednisolone and one treated with methotrexate at the time of admission. Fifteen (14.2%) of the infected patients were treated with prednisolone at the time of admission. The baseline characteristics, the outcome, and the microbiology and focus of infection are presented in Tables 1, 2, 3. The final diagnoses of the noninfected patients are described in Table 4.

The levels of PCT, LBP, IL-6 and CRP were statistically significantly higher among all infected patients compared with noninfected patients (P < 0.001) (Table 5). There was a small increase in PCT levels from the group of noninfected patients to the group of infected patients without SIRS and to the group of sepsis patients. Patients with severe sepsis had almost 10-fold higher levels of PCT compared with patients with sepsis. Levels of LBP, IL-6 and CRP increased progressively with increasing severity of infection/sepsis.

Diagnostic performance of PCT, LBP, IL-6, CRP, white blood cell count and neutrophils in diagnosing infection, sepsis and severe sepsis

In a ROC analysis to distinguish between noninfected patients and infected patients, CRP and IL-6 had the highest AUC values of 0.83 (95% CI 0.76–0.89) and 0.82 (95% CI 0.75–0.88) (Figure 1). PCT performed with an AUC of 0.77 (95% CI 0.69–0.84) and LBP with an AUC of 0.78 (95% CI 0.71–0.85) (Figure 1). Using a cut off level of 30 mg/l, CRP had a sensitivity of 80.2% and a specificity of 62.7% in diagnosing infection (Table 6). Using a cut off level of 16.3 pg/ml, IL-6 had a sensitivity of 79.2% and a specificity of 64.2% in diagnosing infection (Table 6).

In a ROC analysis to distinguish between patients with noninfectious SIRS and patients with sepsis/severe sepsis, IL-6, LBP and CRP had an AUC of 0.87 (95% CI 0.78–0.96), 0.86 (95% CI 0.77–0.95) and 0.84 (95% CI 0.75–0.92), respectively (Figure 2). PCT had an AUC of 0.75 (95% CI 0.63–0.87) (Figure 2). Using a cut off level of 25 pg/ml, IL-6 had a sensitivity of 81.1% and a specificity of 78.9% in diagnosing sepsis/severe sepsis (Table 7). Using a cut-off level of 20 μg/ml, LBP had a sensitivity of 81.0% and a specificity of 68.4% in diagnosing sepsis/severe sepsis (Table 7). Using a cut off level of 38 mg/l, CRP had a sensitivity of 79.7% and a specificity of 57.9% in diagnosing sepsis/severe sepsis (Table 7).

In a ROC analysis to distinguish between patients with sepsis and patients with severe sepsis, PCT performed best with an AUC of 0.74 (95% CI 0.61–0.87) (Figure 3).

A strong correlation was found between LBP and CRP (r = 0.842, P < 0.0001) and a weaker correlation was found between LBP and IL-6 (r = 0.568, P < 0.0001). Weak correlations were found between PCT, CRP and IL-6.