Since 2010, consecutive patients over age 18 with LRTIs requiring hospital admission were asked to provide additional blood samples for investigation. The study protocol was approved by the local ethical committee, and all of the participants provided written informed consent for the collection and storage of blood upon admission. Plasma was obtained by centrifugation and stored frozen at -80ºC. For the present study, 85 plasma samples were randomly selected.

LRTI was defined by the presence of at least one respiratory symptom (e.g., cough, sputum production, dyspnea, tachypnea, pleuritic pain) plus at least one finding during auscultation (i.e., rales) or one sign of infection (temperature >38.0°C, shivering, leukocyte count >10 or <4 x10⁹ cells) regardless of antibiotic use. For CAP, a new infiltrate on the chest radiograph was also required. COPD was defined by post-bronchodilator spirometric criteria according to the Global initiative for Chronic Obstructive Lung Disease (GOLD)-guidelines as a FEV1/FVC ratio < 70%. Acute bronchitis was defined as a LRTI in the absence of an underlying lung disease or focal chest signs and infiltrates on chest X-rays [18, 19]. A panel of three specialists had been previously reviewed chest X-rays from the patient population. Panel consensus in classifying LRTI was required for study inclusion.

The exclusion criteria were: 1) severe immunosuppression (HIV infection or severe haematological diseases); 2) immunosuppressive therapy (prednisone or an equivalent dose of 20 mg daily for 2 weeks or any immunosuppressive regimen (azathioprine, cyclosporine, cyclophosphamide and/or other immunosuppressant drugs); 3) leukopenia (<1,000 leukocytes per mm³) or neutropenia (< 500 neutrophils per mm³) and/or chemotherapy in the previous year; 4) pulmonary abscess (radiological cavitation), aspiration pneumonia and obstructive pneumonia; 5) possible nosocomial origin (30 days from hospital discharge); and 6) known active neoplasm.

The patients’ clinical parameters were included in an electronic database. Comorbidities were documented and defined as the presence of one or more of the following: congestive heart failure, chronic obstructive pulmonary disease, chronic renal disease, chronic liver disease, cerebrovascular disease, malignancy, or diabetes mellitus. The microbiological work-up in the patients with pneumonia included sputum and/or bronchoalveolar lavage fluid samples for the Gram stain and culture, a urine sample for detecting Legionella pneumophila and Streptococcus pneumoniae antigens, and serological testing for Mycoplasma pneumoniae and Chlamydophila pneumoniae. The detection of respiratory pathogens was performed according to standard methods. In patients with severe exacerbations of COPD, a sputum sample was collected for the Gram stain and sputum culture. No microbiological tests were performed in the patients with acute bronchitis.

The severity assessment was made with the CURB-65 score and performed upon admission to the emergency department. The CURB-65 score consists of five variables: confusion, urea, respiratory rate ≥ 30 breaths/min, blood pressure (systolic) < 90 mm Hg or diastolic ≤ 60 mmHg, and age ≥ 65 years [20]. One point is given for each parameter present, which results in CURB-65 scores of 0-5. The criteria for ICU admission were based on the American Thoracic Society (ATS) guidelines [21]. In brief, ICU admission was considered in patients with severe CAP, which was defined as the presence of one of two major criteria (the need for mechanical ventilation or septic shock), the presence of two of three minor criteria (systolic blood pressure <90 mmHg, multilobar disease, or PaO2/FIO2ratio< 250) or more than two CURB points. For COPD patients, the ICU criteria included severe acidosis or respiratory failure (pH <7.25, pO2 < 40 mmHg), no response to initial treatment in the emergency department or worsening mental status (confusion, coma) despite adequate therapy [19].

Both MR-proADM and MR-proANP were measured in duplicated in stored EDTA plasma samples by the TRACE® technology, as previously described [22, 23]. The procedure was fully automated in a KRYPTOR Compact analyzer (B.R.A.H.M.S. AG, Hennigsdorf, Germany). The other biochemical determinations were routinely performed as part of the admission study using conventional laboratory methods.

The categorical variables were expressed as counts (percentages), and the continuous variables were expressed as medians and interquartile ranges (IQR), as appropriate. The χ2 and Fisher’s exact tests were used, as appropriate, to compare the qualitative variables. In the case of quantitative variables, the Mann-Whitney U-test was used.

To assess the accuracy of the biomarkers for predicting the presence of pneumonia, we generated receiver operating characteristic curves and determined the area under the curve (AUC) and its 95% confidence interval (95% CI). For each ROC curve, a cut-off point was determined as the value of the biomarker that maximised the sum of the sensitivity and specificity for diagnosing pneumonia. Pairwise comparisons of the AUC were conducted, following the Hanley and McNeil procedure. To adjust for confounders, a logistic regression model estimated the simultaneous impact of CRP and MR-proADM, along with significant variables in the univariate analysis predicting pneumonia. Associations were considered to be statistically significant if the p-value was <0.05. The statistical software packages used were SPSS 15.0 (SPSS Inc., Chicago, IL, USA) and MedCalc version 12.3.0 (MedCalc, Mariakerke, Belgium).

A total of 85 patients who were hospitalized for LRTIs were recruited; 56 were diagnosed with pneumonia and 29 with other LRTIs (13 with acute exacerbation of chronic obstructive pulmonary disease and 16 with acute bronchitis). The mean patient age was 71.0 years (range 20-96); 69.4% were males, and 62.4% had comorbidities. Of the participants, 8.2% arrived from nursing home facilities. The patients with pneumonia had a median CURB65 score of 2.5. During the follow-up, 3 (3.5%) patients were transferred to the ICU, and 7 (8.2%) died (6 with pneumonia and 1 with other LRTIs). The mean in-patient stay was 8.4 days (range 2-41).

Microbiological work-up were performed in patients with pneumonia or with severe exacerbations of COPD. A final etiological diagnosis was established in 29 (42.0%) patients. The microorganisms identified were: Streptococcus pneumoniae (19), Chlamydophila pneumoniae (6), Legionella pneumophila, Staphylococcus aureus, Escherichia coli, and Aspergillus fumigatus (1 each).

A comparison of the clinical, analytical and follow-up data between the groups is shown in Table 1. In a univariate analysis, 5 variables were different between the patients with pneumonia and those with other LRTIs: acute onset of symptoms (48% vs 14%), heart rate (100 vs 89 beats/min), the presence of rales (64% vs 14%), CRP (222 vs 102 mg/L) and MR-proADM (1.46 vs 0.88 nmol/L). However, in the multivariate analysis, the presence of rales, CRP and MR-proADM maintained significant differences only.

Fig. (1) shows MR-proANP levels in the study population. The patients with pneumonia had similar MR-proANP levels (median 233 pmol/L [IQR 25-75, 115-398 pmol/L]) compared with those with other LRTIs (median 259 pmol/L [110-l/L]) (p=0.88). Conversely, the patients with pneumonia had increased MR-proADM levels (median 1.46 nmol/L [IQR 25-75, 0.82-2.02 nmo/mL]) compared with the patients with other LRTIs (median 0.88 nmol/L [0.71-1.39 nmol/L]) (p=0.04) (Fig. 2).

Fig. (1). Box plots of the MR-proANP plasma levels in the study population. The patients with pneumonia had similar MR-proANP levels (median 233 pmol/L [IQR 25-75, 115-398 pmol/L]) compared with those with other LRTIs (median 259 pmol/L [110-399 pmol/L]) (p=0.88). The Box plots represent the 25th and 75th percentiles, with the internal horizontal lines showing the median. T-shaped bars represent the 10th and 90th percentiles. IQR, interquartile range; MR-proANP, mid-regional proatrial natriuretic peptide.

Fig. (2). Box plots of the MR-proADM plasma levels in the study population. The patients with pneumonia had increased MR-proADM levels (median 1.46 nmol/L [IQR 25-75, 0.82-2.02 nmol/mL]) compared with those with other LRTIs (median 0.88 nmol/L [0.71-1.39 nmol/L]) (p=0.04). The MR-proADM units are expressed as natural logarithm. The box plots represent the 25th and 75th percentiles, with the internal horizontal lines showing the median. T-shaped bars represent the 10th and 90th percentiles. IQR, interquartile range; MR-proADM, mid-regional proadrenomedullin.

The AUC for MR-proADM to discriminate between the patients with pneumonia and other LRTIs was 0.65 (95% CI, 0.53-0.76), which was not inferior to the result obtained for CRP (0.78 [95% CI, 0.67-0,86], p=0.11). The optimal threshold (minimal false negative and false positive results) of MR-proADM to predict pneumonia was 1.5 nmol/L, which yielded a sensitivity of 51.7% (95% CI, 38.0-65.3), a 79.3% specificity (95% CI, 60.3-92.0). As Table 2 shows, combining MR-proADM with CRP in an “and” rule increased the specificity to 96% (95% CI, 82-99) and the likelihood ratio positive to 9.2 (95% CI, 1.3-65.1) for detecting pneumonia.