This case-control study was performed in the 10-bed surgical intensive care unit (ICU) and the 20-bed trauma ICU of Wonju Severance Christian Hospital, between March 2011 and October 2014. The protocol was approved by the Institutional Review Board (IRB) of Wonju Severance Christian Hospital (IRB no. CR-314041). We did not obtained written informed consent from each patient. Before data analysis, the records of patients were anonymized and de-identified. The approaches are following ordinances concerning informed consent and enrollment expounded in guidelines.

When patients had a fever above 38°C or hypothermia below 35°C in the ward or ICUs, microbiological tests were performed on blood samples, urine specimens, sputum (endotracheal suction), and body fluid from other regions. Complete blood count (CBC) and DNI were measured daily in these patients. All consecutive ICU patients who were clinically diagnosed as AB pneumonia were included. The electronic medical charts of these patients were reviewed retrospectively, and the following parameters were recorded and analyzed: age, sex, underlying diseases, cause of admission, operation type, Acute Physiology and Chronic Health Evaluation (APACHE) II score, duration of mechanical ventilation, duration of ICU admission, overall mortality, and infectious disease mortality. To assess the patient’s condition at the time of bacterial culture, we obtained information on the following: mechanical ventilation, occurrence of shock, presence of a thoracostomy tube, acute renal failure, WBC, platelet, CRP, and DNI laboratory findings. Microbiologic data, such as the presence of MDR and concurrent isolation of other bacteria, were also collected. Cause of admission was categorized as trauma, non-traumatic emergency operation, elective operation and conservative management.

AB pneumonia was defined by all of the following conditions: (1) isolation of AB in the sputum culture (sputum group 4 or 5 in patient without endotracheal tube); (2) new development of clinical symptoms and signs including fever (=38°C), leukopenia (<4,000 WBC/mm3) or leukocytosis (≥12,000 WBC/mm3) and a large amount of sputum production; and (3) new or progressive lung infiltration on chest x-ray. MDR was defined as resistance to three or more classes of drugs, including quinolones, cephalosporins, and carbapenems. Antibiotic sensitivity was determined using the disc diffusion methods as described by the Clinical and Laboratory Standard Institute [12]. Empirical antibiotics were started for patients who were diagnosed as pneumonia, the regimes were adjusted as microbiologic report of sputum culture after 3∼7 days. Respiratory disease was defined as chronic obstructive pulmonary disease, history of pulmonary tuberculosis, asthma, pneumoconiosis, or adult respiratory distress syndrome. Renal disease was defined as chronic renal failure or end-stage renal disease. Cardiac diseases included unstable angina, myocardial infarction, congestive heart failure, and atrial fibrillation. Hepatobiliary disease was defined as hepatitis or liver cirrhosis. Shock was defined as an arterial systolic pressure less than 90 mmHg despite adequate fluid resuscitation or use of a vasopressor. Acute renal failure at the time of bacterial culture was defined as a serum creatinine elevation of more than 50% compared to a baseline or a urine output of less than 0.5 mg/kg/h over 6 hours. With regard to mortality, death within 7 days and death within 30 days from the diagnosis of pneumonia were evaluated.

Blood samples for the analysis of the DNI and other laboratory were drawn from an arterial catheter or venipuncture. A specific type of automatic cell analyzer (ADVIA 120/212; Siemens, Tarrytown, NY, USA) was used for the calculation of the DNI. This flow cytometry-based hematologic analyzer used two independent WBC methods, which are a MPO channel and a lobularity/nuclear density channel. DNI values were calculated using the following formula: DNI=(leukocyte subfraction assayed using the MPO channel of a cytochemical reaction)-(leukocyte subfraction assayed using the nuclear lobularity channel based on reflected light beam measurements) [8]. In Wonju Severance Christian Hospital, the DNI count was provided with the CBC at no additional cost.

All statistical analyses were performed using PASW Statistics ver. 18.0 (IBM Co., Armonk, NY, USA). Continuous variables are presented as the mean±standard deviation or median values and ranges, and comparative analysis was conducted using an independent sample t-test. A chi-square test and Fisher’s exact test were used for the comparative analysis of categorical variables. To identify the independent risk factors, a multivariate analysis was performed using logistic regression. A receiver-operating characteristics (ROC) curve was constructed and the Youden Index method was used to determine the optimal cut-off values for the DNI, CRP, and WBC to predict infectious disease mortality. Statistical significance was accepted for p-values <0.05.

A total of 104 patients were enrolled, of which 65 patients (62.5%) were men. The mean patient age was 60.8±18.8 year, and the mean APACHE II score was 15.8±5.3. Diabetes mellitus was the most common underlying disease (14 cases, 13.5%), followed by respiratory disease (10 cases, 9.6%) and renal disease (7 cases, 6.7%). The cause of admission was trauma, emergency operation, and elective operation for 40 (38.5%), 46 (44.2%) and 15 patients (14.4%), respectively. Three patients (2.9%) were managed conservatively. When the sputum cultures were performed, 57 (54.8%) were supported by mechanical ventilation, 16 (15.4%) had a thoracostomy tube, 16 (15.4%) were in shock, and 16 patients (15.4%) had acute renal failure. The mean WBC and platelet values were 14,007±7,017/mm3 and 207±167×103/mm3, respectively. The median serum DNI was 2.7% (0∼39.4%). When pneumonia was diagnosed, cefoperazone-sulbactam (37.5%) was most commonly used, followed by 3rd generation cephalosporin (29.8%), vancomycin (17.3%), and tazoabctam-piperacillin (11.5%). The median length of ICU stay was 16 days (0∼143 days), and overall ICU mortality rate was 25.0% (26 patients). Twenty-four patients died due to the infection and two died of hemorrhage shock and brain ischemia respectively (Table 1). All 24 deaths due to infection were exacerbations of sepsis caused by pneumonia. Of 24 deaths due to infection, 21 died within 30 days from the day of pneumonia diagnosis, and 8 died within 7 days.

When the survivor and non-survivor groups were compared, age was significantly higher in the non-survivor group (58.5±19.5 vs. 68.3±13.9, p=0.008). There were also significant differences in the occurrence of shock at the time of the sputum culture between the survivor and non-survivor groups (p<0.001). Non-survivor group had more thoracostomy tube due to pleural effusion at the time of the sputum culture than survivor group (1.3% vs. 20.8%, p=0.003). Non-survivor patients had significantly lower serum platelet counts and higher serum DNI values than the survivor patients (125±84 vs. 232±177×103/mm3, p<0.001 and 2.85±2.23% vs. 8.31±11.47%, p=0.029, respectively) (Fig. 1). There were more renal failure at the time of culture in non-survival group (33.3% vs. 10.0%, p=0.005). Carbapenem was used as the empirical antibiotic more frequently in the non-survivor group than in the survivor group (29.2% vs. 12.5%, p=0.053) (Table 1). There was no significant difference in the DNI according to the isolation of MDR AB (p=0.403).

Logistic regression model constructed by age, DNI, renal failure, shock, and platelet count at the time of bacterial culture showed that the following were independently correlated with infectious disease mortality: renal failure (odds ratio [OR], 3.811; p=0.046), decreased platelet level (OR, 0.994; p=0.046) and elevated serum DNI (OR, 1.136; p=0.047) at the time of bacterial culture (Table 2).

When the ROC curves of the DNI, CRP and WBC were conducted to predict mortality in 24 patients with infectious mortalities, area under curve (AUC) of the DNI, CRP, and WBC were 0.633 (95% confidence interval [CI], 0.496∼0.770), 0.465 (95% CI, 0.314∼0.616), and 0.371 (95% CI, 0.222∼0.520), respectively. Eight patients died from infectious causes within seven days of the diagnosis of AB pneumonia. When the ROC curves of the DNI, CRP, and WBC were conducted to predict infectious disease mortality in these patients, the DNI was the best indicator of infectious disease mortality, with an AUC of 0.839 (95% CI, 0.694∼0.985). The optimal cut-off point for the serum DNI was 6.85% (sensitivity, 75.0%; specificity, 94.8%) (Fig. 2). When the difference between the first DNI and the follow-up DNI within 24 hours was denominated as the DNI delta value (DNI delta=follow-up DNI-First DNI; median value: -0.05; range: -3.8∼39.5), it had an AUC of 0.974 (95% CI, 0.940∼1.000) and its optimal cut-off value was 1.65% (sensitivity, 100%; specificity, 87.6%) (Table 3, Fig. 3).