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Ventilator-associated pneumonia is the first or second most commonly diagnosed nosocomial infection in the PICU. Centers for Disease Control diagnostic criteria include clinical signs or symptoms in conjunction with a “positive” tracheal aspirate, defined as more than 104 colony-forming units/mL of bacteria on quantitative culture and/or more than 25 polymorphonuclear neutrophils per low-power field on Gram stain. We hypothesized that tracheal aspirate cultures and Gram stains would not correlate with clinical signs and symptoms and would therefore not distinguish between colonization and infection.Prospective observational study.PICU in an academic tertiary care center.Children intubated more than 48 hours.Sequential tracheal aspirate quantitative cultures and Gram stains in conjunction with daily collection of concordant clinical signs and symptoms.Time since intubation correlated strongly (p < 0.001) with the proportion of positive (> 104 colony-forming units/mL) tracheal aspirate quantitative cultures, but Centers for Disease Control–defined clinical signs or symptoms of ventilator-associated pneumonia, either singly or in combination, did not. Use of in-line suction catheters versus new, sterile catheters to obtain tracheal aspirates was associated with significantly greater proportion of positive tracheal aspirate bacterial cultures (p < 0.001). Most subjects had more than 25 polymorphonuclear neutrophils per low-power field on Gram stain; polymorphonuclear neutrophils on Gram stain correlated with positive bacterial culture (p = 0.04). Seventy-seven percent of the bacterial isolates detected in positive quantitative cultures were “pathogens.” Antibiotic use at the time tracheal aspirates were obtained was associated with a lower frequency of positive quantitative cultures only with antibiotic regimens that included cefepime.Positive bacterial cultures of tracheal aspirates increase rapidly after intubation and usually include bacteria considered to be pathogens. Tracheal aspirate cultures and Gram stains do not appear to distinguish between infection and colonization. Antibiotic regimens that include cefepime decrease the frequency of positive cultures, but the significance of this is unclear.