Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis

Abstract

Speed is of the essence when evaluating an infant with symptoms consistent with sepsis. Because of the high morbidity and mortality associated with neonatal sepsis, infants receive multiple, broad-spectrum antibiotics before receiving finalized blood culture results. Incorporating an additional, reliable, yet rapid assay to detect bacteria directly from blood would facilitate timely diagnosis and appropriate care. To this end, we designed a real-time polymerase chain reaction (PCR) assay targeting the highly conserved 380 bases of 16S rDNA. DNA was extracted from whole-blood samples using a Qiagen column. The limit of detection for the TaqMan-based assay, using a Smartcycler instrument, was 40, 50, or 2000 colony-forming units per milliliter of blood (CFU/ml) of Escherichia coli, group B Streptococcus, and Listeria monocytogenes, respectively, when white blood cell counts were below 39,000/microl. Implementing this approach requires less than 4 hours for both sample preparation and real-time PCR compared with 1 to 2 days to detect growth in culture or 5 days to finalize no-growth culture results. There was an overall agreement between the results of culture and real-time PCR of 94.1% (80 of 85) in this study. These results suggest that molecular techniques can augment culture-based methods for diagnosing neonatal sepsis, especially in infants whose mothers have received intrapartum antibiotic prophylaxis.

Figure 1

Fluorescence measurements derived using the SmartCycler for 10-fold serial dilutions of GBS (5 to 50,000 CFU/ml) spiked into neonatal whole-blood samples and analyzed by 16S rDNA PCR. Site ID numbers represent samples tested in duplicate at the following concentrations: A1 to 2, 5 × 10⁴ CFU/ml; A3 to 4, 5 × 10³ CFU/ml; A5 to 6, 5 × 10² CFU/ml; A7 to 8, 50 CFU/ml; A9 to 10, 5 CFU/ml; and A11 to 12, negative controls lacking target DNA.

Figure 2

A plot comparing the ΔCT values of the Cx+/PCR+ specimens, in ascending order, with those of the Cx−/PCR− specimens, in descending order.