Early Diagnosis of Ventilator-Associated Pneumonia (Materials and Methods)

20 Dec
2013

Early Diagnosis of Ventilator-Associated Pneumonia (Materials and Methods)Patients

One hundred thirty-two mechanically ventilated patients were studied from February 1992 to August 1994 at the ICU, Edouard Herriot Hospital-Lyon (France). All these patients were suspected of having nosocomial pneumonia: they all had fever (>=38.5°C), purulent tracheal aspirates, leukocytosis (10,000 cells/per cubic millimeter), and new or persistent radiographic lung infiltrates. Antibiotic therapy, when instituted, was administered for a minimum duration of 72 h. Ninety-eight patients had not received any antimicrobial during the 3 days or more preceding fibroscopy. Sixty-five patients were receiving antibiotics (for >72 h) when the procedure was performed. No patient received topical prophylactic antibiotic.

Sampling Technique

One hundred sixty-three BALs and PSBs were performed by fiberoptic bronchoscopy (Olympus BF P 20D; Olympus Optical Corp of America; New Hyde Park, NY). During the procedures, the patients were placed on a fraction of inspired oxygen (FI02) of 1.0, sedated with midazolam, and paralyzed with pancuronium bromide. Topical anesthesia was not used. Heart rate, arterial pressure, and arterial oxygen saturation were monitored during the investigation.

The trachea was aspirated before introducing the bronchoscope. The PSB was inserted in the bronchoscope channel. The bronchoscope was advanced through an adapter (Bodai Suction Safe Y; Sontek Medical; Lexington, Mass) into the desired bronchial segment as suspected on the chest radiograph. Then, the PSB catheter was placed out of the bronchoscope, the polyethylene glycol plug on the tip was ejected by protruding the brush (Ventimed; Marsannay-la-Cote, France) which was manipulated and rotated to be placed in contact with the secretions. The brush was retracted and the catheter was removed. The extremity of the brush was placed into a sterile tube that contained 1 mL of saline solution. The sample was transported to the laboratory within 20 min of collection.

The BAL was performed by injecting 2 or 3 aliquots of 50 mL sterile saline solution through the bronchoscope. The fluid was then withdrawn by hand suction into syringes for infusion. Approximatively 30% of instilled fluid was retrieved. The BAL fluid was filtered and pooled. Gram’s and Giemsa stains, count of polymorphonuclear neutrophils (PMN), and IC were performed using standard methods.’

Bacteriologic Analysis

The tube that contained the brush was vortexed, then the fluid was diluted to obtain concentrations of 10″\ 10 , and 10′. The sample was inoculated on Columbia agar, chocolate agar, trypticase soy, McConkey, and Sabouraud agar. The bacterial colonies were counted and identified by conventional techniques.

Cytologic Analysis

The cytologic study of BAL was performed on a homogeneous sample. This sample was centrifuged for 10 min at 8,000 g, and the BAL fluid was stained using Gram’s and Wright stains. For BAL specimens, a total cell count was performed on aliquots of resuspended BAL using a hemocytometer counting chamber. Slides were stained with Wright and Gram’s stains. A differential cell count (squamous epithelial cells, alveolar macrophages, and neutrophils) was obtained by counting 100 cells twice on the Wright stain slide at low magnification (x25). Screening for microorganisms was performed at high magnification (1,000-fold) on the Wright stain slide. If this was positive, the Gram’s stain was then examined at high magnification (xlOO) to determine the morphologic features and the percentage of ICs (intracytoplasmic microorganisms within macrophages and neutrophils) by counting 100 cells. To minimize the variability dependent on the operator’s skill, all of the cytologic procedures were performed by the same microbiologist.

The percentage of ciliated cells was noted in BAL samples. Samples containing more than 1% ciliated cells were considered as contaminated and were excluded.

Diagnostic Categories

The diagnosis of pneumonia was based on positive results of PSB culture (cutoff >103 cfu/mL) and clinical outcome consistent with bacterial pneumonia while receiving appropriate antibiotic therapy for organisms cultured at a significant growth in PSB. Pneumonia was excluded if there were these criteria: negative or nonsignificant growth in culture of PSB and full recovery without antibiotic therapy or without changes in the antimicrobial therapy initiated at least 72 h before the appearance of radiologic infiltrates or diagnosis of another disease of the chest accounting for the chest radiograph abnormality.

The therapeutic strategy based on the results of the determination of ICs in BAL fluid was to administer immediate empiric antimicrobial therapy if the percentage of infected cells was 5% or greater (based on previous studies). Patients were also treated if PSB was 103 cfu/mL or greater at day 1 or day 2. When the percentage of ICs was less than 5%, no immediate empirical antimicrobial therapy was instituted unless there was evidence of septic shock or severe hypoxemia. In most of the cases, patients were initially treated with ceftazidime and vancomycin according to the bacterial ecology of our ICU. Antimicrobial chemotherapy was secondarily adapted to the results of PSB cultures.

Statistical Analysis

Results were expressed as mean±SD. An estimation of sensitivity, specificity, and positive and negative predictive values was established using standard formulas. Comparison between the groups was made using the Mann-Whitney U test. A p value of less than 0.05 was considered significant. An ROC curve expresses the relationship between sensitivity and specificity: it is a graphic with 1-specificity and sensitivity as coordinates. In this study, this curve was used to discriminate between bacterial pneumonia and absence of bacterial pneumonia. Each point of the ROC curve corresponds to a cutoff value. The best cutoff value is a compromise between sensitivity and specificity. A perfect test has an AUC of 1.0, and the top of the curve grazes the upper left comer of the graphic.

The degree of concordance between cytologic examination of the BAL sample and definite diagnosis was established using the Co-hen-kappa coefficient. If the value of this coefficient is 1.0, there is a high degree of concordance.

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