The study was conducted in the clinical/surgical 26-bed intensive care unit (ICU) of the Hospital de Clínicas de Porto Alegre (HCPA), a tertiary-care–teaching institution with 744 hospital beds.
All patients consecutively admitted to the ICU suspected of VAP were eligible for this prospective observational cohort study. Patients at least 18 years old were recruited. Exclusion criteria were a previous diagnosis of AIDS or neutropenia below 500 cells/ml. Pneumonia was considered ventilator-associated when it occurred after 48 hours of mechanical ventilation and was judged to not have been incubating before starting mechanical ventilation. VAP was considered early-onset when it occurred during the first four days of mechanical ventilation and late-onset when it developed five or more days after the initiation of mechanical ventilation [23]. APACHE II was calculated during the first 24 hours of admission to ICU. Patients were considered immunosuppressed when they had received chemotherapy within the preceding 45 days, or had neutropenia of less than 1,000/mm3.
Diagnosis of pneumonia was suspected when a patient developed a new and persistent radiographic infiltrate plus two of the following: (1) body temperature more than 38°C or less than 36°C; (2) white blood cells more than 11,000 or less than 4,000/mm3 (3) macroscopically purulent tracheal aspirate [24]. Purulent endotracheal aspirate was defined on inspection by the assistant team. The axillary temperature used was the highest in the previous 24 hours before the inclusion on the study.
Chest X-ray, arterial blood gases, complete blood count, creatinine, total bilirubin, albumin, PCT and CRP were obtained by the that time VAP was suspected (D0) and repeated on the fourth day of treatment (D4). Quantitative endotracheal aspirate (QEA) was obtained on D0, repeated on the third day after the diagnosis (D3) and then weekly. Sterile endotracheal aspirates were obtained with a suction catheter adapted to a mucus collector without saline instillation, and two samples of hemocultures were collected from different veins with a 15-minute interval before starting antimicrobial treatment.
CPIS [25], modified as described by Singh and colleagues [20], was calculated on the basis of data on D0 and D3. Patients were assumed to have VAP when the CPIS was 7 points or more. CPIS was calculated with data from D0, adding points for microbiological results and progression of pulmonary infiltrate on a new chest X-ray on D3. To calculate CPIS on D3, data from D3 were used to study the kinetics of the modified CPIS.
For a diagnosis of VAP, there should be no evidence of another medical condition to which the presenting symptoms, signs or radiological findings could be attributed. A SOFA score was calculated on D0 and D4. QEA was considered positive when values were at least 105 colony-forming units/ml.
All patients with a clinical suspicion of VAP, later confirmed by a CPIS of at least 7 and fulfilling inclusion criteria, were included and received empirical antimicrobial therapy on D0. The choice of antibiotics and changes rested solely with the critical care team or primary service caring for the patient. Modifications to empirical therapy were based on the results of QEA and hemocultures. Mechanical ventilation, physiotherapy and airway management were performed in accordance with a standard protocol in all patients.
Patients' progress was followed until the 28th day (D28) after the diagnosis of VAP, when they were considered survivors. Patients who died before D28 were non-survivors. Patients discharged from the ICU before D28 were also considered survivors. All patients with VAP were reviewed by one of the investigators to confirm the diagnosis on the basis of predetermined criteria.
Seventy-five patients were enrolled from October 2003 to August 2005 and constituted the study population. The research protocol was reviewed and approved by the Human Research Committee from the HCPA, and informed written consent was obtained from patients' representatives before enrollment. The study protocol conforms to the ethical guidelines of the Declaration of Helsinki.
Trained investigators collected data on D0, D3, D4, and weekly until D28. Recorded data included age, sex, cause of ICU admission, arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2), APACHE II score, SOFA score, CPIS, co-morbidities including chronic obstructive pulmonary disease, whether active smoker, history of congestive heart failure, history of malignancy, immunosuppression, albumin, use of histamine type-2 receptor (H2) antagonist, use of proton pump inhibitor, use of corticosteroids, dialysis, central vein catheterization, urinary tract catheterization, duration of mechanical ventilation, duration of stay in ICU before VAP, cardiopulmonary resuscitation, intubation (orotracheal versus nasotracheal), and tracheotomy.
Adequacy of the empirical antimicrobial treatment was recorded on the basis of microbiological results. Adequate antibiotic therapy was defined as coverage of all the pathogens isolated (from QEA culture or from blood), by at least one antimicrobial administered by the onset of VAP, determined by the sensitivity pattern in the antibiogram [26]. Treatment was considered adequate when cultures were negative.
PCT was determined with the commercially available immunoluminometric assay (Brahms PCT LIA; Brahms Diagnostika, Berlin, Germany) with an analytical sensitivity of 0.1 ng/ml and analyzed with a Lumat LB 9507 Luminometer (Berthold, Bad Wildbad, Germany). Blood was drawn when a diagnosis of VAP was clinically suspected, before empirical antibiotic treatment was started. Samples of serum were prepared and frozen immediately after blood was drawn, then stored at -80°C in the HCPA research laboratory. Assays were performed in batches at the end of the study period.
CRP was measured by nephelometry (Bade Behring, Marburg, Germany), routinely determined at the HCPA laboratory.
Kinetics definitions
Dichotomized Δ was calculated by the formula Δ = D4 - D0. Therefore ΔPCT = PCTD4 - PCTD0, ΔCRP = CRPD4 - CRPD0, and ΔSOFA = SOFAD4 - SOFAD0.
CPIS was calculated on D0 and D3. Consequently, ΔCPIS = CPISD3 - CPISD0.
Δ > 0 means increasing values and Δ ≤ 0 means decreasing values.
Microbiological processing
Endotracheal samples were initially analyzed with Gram stain. They were rejected if there were more than ten squamous epithelial cells per low-power field (magnification × 100), requiring a new sample [27]. Samples considered acceptable were mixed in a 1:1 proportion with N-acetylcysteine, mechanically liquefied and homogenized with a vortex mixer for two minutes. After incubation for two hours at room temperature, samples were again vortex-mixed for 30 s and serially diluted in sterile 0.9% saline solution to obtain final concentrations of 1:100 and 1:10,000. Aliquots of 0.1 ml were plated on chocolate agar. Depending on the Gram stain results, samples also were plated on sheep blood agar, azide blood agar or MacConkey agar. All plates were incubated at 35°C overnight in a 5% carbon dioxide incubator, except for those in MacConkey agar, which were incubated in normal atmosphere without carbon dioxide. Isolates were assessed within 24 and 48 hours and were characterized by colony morphology and Gram stain. Microorganisms were identified by standardized laboratory methods. For plates inoculated with 1:100 dilution, the presence of 5 colonies was considered to show 104, 50 colonies 105, and 500 colonies 106 colony-forming units/ml. In plates inoculated with 1:10,000 dilution, the presence of 5 colonies was considered to show 106 colony-forming units/ml.
Statistical analysis
Continuous baseline data are expressed as means ± SD. Categorical variables were compared with the χ2 test. Continuous kinetics data are expressed as medians (range). ΔPCT, ΔCRP and ΔSOFA were categorized as increasing or unchanged/decreasing. The Kruskal–Wallis test was used to compare groups for continuous variables. For these analyses, two-tailed tests and p ≤ 0.05 were considered statistically significant. Logistic regression analysis was used to determine the relation of risk factors to clinical outcome. In the multivariable model we considered significant variables with biological importance. Variables with p < 0.20 in univariable logistic regression were entered into the multivariable model. In the multivariable model we considered as significant those variables with p < 0.05. SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.