The study was conducted in the clinical/surgical 26-bed intensive care unit (ICU) of the Hospital de Clínicas de Porto Alegre, 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. The exclusion criteria were a previous diagnosis of AIDS or neutropenia <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 4 days of mechanical ventilation and was considered late-onset when it developed 5 days or more after the initiation of mechanical ventilation [9]. The Acute Physiology and Chronic Health Evaluation (APACHE) II score was calculated during the first 24 hours of admission to the ICU [10]. Patients were considered immunosuppressed when they had received chemotherapy within the preceding 45 days, or had neutropenia 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 signs/symptoms: body temperature >38°C or <36°C; white blood cells >11,000 or <4,000/mm3; and macroscopically purulent tracheal aspirate [11]. Purulent endotracheal aspirate was defined on inspection by the assistant team. The axillary temperature used was the highest in the previous 24 hours before inclusion into the study.
A chest X-ray scan, arterial blood gases, complete blood count, creatinine, total bilirubin, and albumin were obtained by the time VAP was suspected (D0) and were 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 obtained 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.
The Clinical Pulmonary Infection Score (CPIS) [12], modified as described by Singh and colleagues [13], 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. The CPIS was calculated with data from D0, adding points for microbiological results and progression of pulmonary infiltrate on a new chest X-ray scan on D3. To calculate the CPIS on D3, data from D3 were used.
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 Sequential Organ Failure Assessment 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 points 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 were classified at the time of VAP diagnosis into those with sepsis, those with severe sepsis and those with septic shock, which were defined according to international criteria [14, 15].
Patients' progress was followed until the 28th day (D28) after the diagnosis of VAP. Patients who survived until follow up were counted as survivors. Assuming crude mortality, patients who died before D28 were nonsurvivors. 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-one patients enrolled from October 2003 to August 2005 constituted the study population. The research protocol was reviewed and approved by the Human Research Committee from the Hospital de Clínicas de Porto Alegre, 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, on D3, on D4, and weekly until D28. The recorded data included age, sex, cause of ICU admission, arterial partial pressure of oxygen/fraction of inspired oxygen, APACHE II score, Sequential Organ Failure Assessment score, CPIS, comorbidities including chronic obstructive pulmonary disease, whether an active smoker, history of congestive heart failure, history of malignancy, immunosuppression, albumin, use of histamine type-2 receptor 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 the QEA culture or from blood), by at least one antimicrobial administered by the onset of VAP, determined by the sensitivity pattern in the antibiogram [16]. Treatment was considered adequate when cultures were negative.
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, and then stored at -80°C in the Hospital de Clínicas de Porto Alegre research laboratory. Assays were performed in batches at the end of the study period.
Copeptin measurements were performed in D0 and D4 samples using a new sandwich immunoluminometric assay, as described recently [17]. Briefly, two polyclonal antibodies to the C-terminal region (covering amino acids 132 to 164 of pre-proAVP) were used. One antibody is bound to polystyrene tubes, and the other is labeled with acridinium ester for chemiluminescence detection. The assay requires 50 μl serum or plasma and yields results within 3 hours. In contrast to measurements of mature AVP, no extraction step prior to measurement is needed and the analyte shows ex vivo stability for at least 7 days at room temperature and for 14 days at 4°C. The assay has a functional assay sensitivity (defined as the lowest value with an interassay coefficient of variation <20%) of 2.25 pmol/l. The median copeptin level in 359 healthy individuals in previous investigations was 4.2 pmol/l [17].
Copeptin measurements were performed in the Research Department of BRAHMS AG (Biotechnology Centre, Hennigsdorf/Berlin, Germany). Laboratory measurements were performed in a blinded fashion without knowledge of the clinical status of the patient.
Statistical analysis
Continuous baseline data are expressed as the means ± standard deviation. Categorical variables were compared with the chi-squared test. Comparison of the copeptin levels between survivors and nonsurvivors was analyzed by the Mann–Whitney test. Comparison of the copeptin levels in different septic status patients was analyzed by the Kruskal–Wallis test. 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. We performed logarithmic transformation of copeptin values in the regression models, since they have a nonparametric distribution. In a 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.
