Volume of blood required to detect common neonatal pathogens

Objective: To determine the minimum volume of blood and the absolute number of organisms required for detection of bacteremia and fungemia by an automated calorimetric blood culture system (BacT/Alert, Organon Teknika). Design: Common neonatal pathogens, Escherichia coli, Streptococcus agalactiae (group B streptococcus (GBS): one American Type Culture Collection (ATCC) strain and one clinical isolate), Staphylococcus epidermidis, and Candida albicans, were seeded into blood to produce bacteremia or fungemia with low colony counts (1 to 3 colony-forming units (CFU) per milliliter) and ultra- low colony counts (<1 CFU/ml). For each organism, 96 culture bottles were inoculated with either 0.25, 0.5, 1.0, or 4.0 ml of the two seeded blood concentrations. Blood culture bottles were incubated in the BacT/Alert device for 5 days, and time to positivity was noted when applicable. All bottles were subcultured on plated media. Data analysis: The Poisson statistic was used to calculate the probability of finding at least one viable CFU per inoculated culture bottle. The fraction of culture bottles with positive findings per group was divided by the probability of one or more organisms present to give the positivity index. Results: Plated subculture identified no growth of organisms not detected by the calorimetric detection system. The false-positive rate for the automated device was less than 1%. The positivity index for the GBS clinical isolate was 1.13, for the GBS ATCC isolate 0.96, for S. epidermidis 0.94, for C. albicans 0.97, and for E. coli 0.95. There was a statistically significant difference with time to positivity and inocula volume (p <0.01), but the difference was not clinically important. Conclusions: If one or two viable colony-forming units are in the blood inoculated into culture media, the BacT/Alert system will detect growth rapidly. Because there appears to be a sizable subset of neonates who are at risk of sepsis with a colony count less than 4 CFU/ml then a 0.5 ml inoculum of blood into the culture media is inadequate for sensitive and timely detection of bacteremia. One to two milliliters of blood should increase microorganism recovery in the face of low-colony-count sepsis.