Combined serum biomarkers in the noninvasive diagnosis of complicated parapneumonic effusions and empyema

Globally, pneumonia remains a major cause of morbidity and mortality in lung disease. Parapneumonic effusion (PPE) refers to the filling of the pleural cavity with exudative pleural fluids, and this condition develops secondary to pneumonia [1]. The presence of pleural effusion can be defined with a chest radiograph and/or ultrasound-guided thoracentesis [2]. Identifying the cause of PPE is relatively difficult because patients have the same clinical symptoms, such as cough, chest pain and fever [3]. Traditionally, PPEs are divided into uncomplicated PPE (UPPE), complicated PPE (CPPE), and empyema based on pleural fluid biochemical parameters. Including pH, glucose, and LDH [4, 5]. In addition to routine antibiotic treatments for PPE, patients with severe infection may need an invasive procedure, such as surgery, for resolution [3]. Many reports have indicated that routine and optional pleural fluid tests, including biochemical parameters, proinflammatory cytokines, or enzymes, can be used to guide the clinician toward identifying infectious versus non-infectious pleural effusion and to further stage the PPE [6‐8].

Serum biomarkers are feasibly used as a complementary strategy to imaging and histopathological techniques, and the aim of their use is to provide noninvasive and differential information. The acute-phase protein (C-reactive protein [CRP]) and many inflammatory cytokines (IL-6, IL-8, IL-1α, IL-1β and TNF-α) have been studied in both the sera and pleural effusions of pneumonia patients [9‐13]. CRP is an acute-phase protein, and the CRP level is widely used as a marker of infectious disease and sepsis [14, 15]. Bielsa et al reported that serum CRP (at the optimum cutoff value of 200 mg/l) had a sensitivity and specificity for identifying CPPE of 58 and 81%, respectively [12]. The pleural levels of various inflammatory cytokines are useful for identifying PPE and CPPE, but the serum levels of these markers are only useful for identifying PPE from other exudates [10, 11, 13]. Additionally, none of the useful markers can predict which PPE patient may need invasive treatment. We previously found that four neutrophil-associated proteins (neutrophil gelatinase-associated lipocalin [NGAL], calprotectin, bactericidal permeability-increasing [BPI] and AZU1) in pleural effusions were significantly elevated in PPE and served as useful markers for identifying CPPE and empyema [16]. The serum levels of these proteins in PPE and noninfectious pleural effusions have not been studied. In this study, we investigate whether the serum levels of these four proteins had diagnostic value and to determine the best combination of serum biomarkers for a rapid and accurate diagnosis of CPPE and empyema.

The clinical symptoms and chief complaints of UPPE and CPPE are similar and one of the key diagnostic challenges is how to rapidly identify CPPE and empyema patients who require an invasive intervention. In this study, we demonstrated that patients with PPE expressed significantly higher serum levels of NGAL, calprotectin, and BPI than those in transudates, noninfectious exudates, and healthy controls. Furthermore, serum levels of NGAL and calprotectin were significantly elevated in CPPE and empyema compared to UPPE. The combination of serum calprotectin and CRP constitutes a more highly sensitive and specific assay for identifying CPPE and empyema.

Elevated serum NGAL levels have been reported in kidney disease, sepsis, heart disease, metabolic diseases and cancers [17‐20]. Additionally, serum NGAL increases with community-acquired pneumonia (CAP) and is an independent predicator of hospital mortality in CAP patients with a cutoff value 457 ng/ml [21]. Gumus et al reported that both serum and pleural NGAL levels are highly effective in differentiating PPE from non-PPE [22]. In sepsis patients, Otto et al analyzed 15 patients with severe sepsis, and the mean concentration of plasma NGAL was approximately 381.7 ng/ml [23]. However, these previous reports did not subgroup the PPE patients into CPPE, empyema, or UPPE categories. In our study, the serum NGAL levels of healthy individuals, transudates, and noninfectious exudates were approximately 47.8 to 53.4 ng/ml. However, the serum levels in UPPE and CPPE/empyema were increased by approximately 3-fold (165.0 ng/ml) and 7-fold (322 to 361 ng/ml), respectively. These results may indicate that circulating neutrophils produce higher NGAL levels under the severe inflammation that is typical of pneumonia patients with CPPE.

Many pathological conditions, such as infection and inflammation, are associated with altered calprotectin levels in body fluids [24‐26]. A study by van Zoelen et al. showed that serum calprotectin was elevated in sepsis caused by pneumonia and that the mean concentration of serum calprotectin was 4 μg/ml in 29 pneumonia patients; however, there was no apparent correlation between serum calprotectin and the severity of disease [27]. The calprotectin levels in pleural effusion are significantly lower in malignant PE than in benign effusions and have a high accuracy for predicting malignancy in patients with exudative PE [28]. The diagnostic accuracy of serum calprotectin for predicting CPPE/empyema in patients has not been addressed previously. Our results directly demonstrate that serum calprotectin is elevated in patients with PPE compared to healthy controls, transudates, and noninfectious exudates. Furthermore, serum calprotectin is higher in CPPE and empyema (8.6 to 9.5 μg/ml) than in UPPE (4.6 μg/ml).

C-reactive protein (CRP) is a well-known marker for sepsis and/or infection [14, 15]. Increased serum CRP levels are associated with invasive infections and various diseases [15, 29]. In our study, the AUC value of CRP only in identifying PPE from transudates and noninfectious exudates were 0.861 and 0.831, respectively. The diagnostic value of CRP in identifying PPE from other types of effusion, particularly CPPE/empyema was not higher than those three serum biomarkers (NGAL, calprotectin, BPI). The individual protein of these three serum biomarkers was also not good enough for clinical. When we set the cutoff value of serum CRP was 32.6 mg/dl, the sensitivity was 100% for identifying CPPE/empyema, which approach can identify all severe patients. Patients with serum CRP lower than 32.6 mg/dl may perform second serum test and the combination of neutrophil-associated proteins (calprotectin or NGAL) and CRP could provide useful information for CPPE and empyema diagnosis.

The pleural to serum ratios of the NGAL and calprotectin proteins showed significant differences in CPPE/empyema and UPPE. For NGAL, the pleural/serum ratios were 9.30 and 3.46 in CPPE/empyema and UPPE, respectively. For calprotectin, the pleural/serum ratios were 22.60 and 10.23 in CPPE/empyema and UPPE, respectively. These results indicated that the proteins were most elevated in the local inflammation site compared to the systemic circulation. Furthermore, the pleural/serum ratios of NGAL and calprotectin were higher in CPPE/empyema than in UPPE (p = 0.015 for NGAL, p <  0.001 for calprotectin). The accuracy of serum calprotectin alone (AUC = 0.765) and the pleural/serum ratio of calprotectin (AUC = 0.786) were similar in confirmed CPPE and empyema.

The main limitation of our study was the small sample size and patients were recruited from only one hospital. Thus, further large-scale studies that enroll various causes of pleural effusion will enable further evaluation of the diagnostic value of these serological markers. The other limitation was the sensitivity and specificity values of serological markers were not high enough and not superiority than pleural markers we had identified. This phenomenon may due to serum is a heterogeneous body fluid sample contained various proteins and may be affected by different physiological conditions. Third, only one time-point specimen was obtained from patients, and we could not evaluate the serial NGAL or calprotectin levels of multiple hospital days. The kinetics of serological markers may be helpful for PPE diagnosis and for monitoring disease severity or therapeutic response. In summary, we concluded that patients present with pneumonia and pleural effusion signs on the chest x-ray and the combination of the serum CRP with the serum calprotectin constitutes a more highly sensitive and specific assay for the differential diagnosis of UPPE and CPPE/empyema. These findings can be helpful in early clinical decision-making for the treatment of these patients as these markers may lead to a better prognosis and the avoidance of potential adverse consequences.