Microbiological diagnosis of sepsis: comparison between real-time polymerase chain reaction and blood culture techniques

Background

A rapid microbiological diagnosis permits one to undertake an appropriate therapy against bacterial sepsis and contributes both to improve the healing possibilities and to lower the high costs of hospitalisation.

Materials and methods

Blood samples from hospitalised patients were inoculated into Bactec Plus Aerobic/F and Anaerobic bottles (standard inoculum; Becton Dickinson) and into an EDTA tube (3 ml). The Septifast Lys kit, Septifast Prep kit and LightCycler Septifast kit (Roche) were used in disrupting bacterial walls, and in extracting and amplifying bacterial DNA, respectively. Blood agar plates were from Becton Dickinson. Strains were identified by Vitek2 (Biomerieux) panels. Real-time PCR results were analysed by Septifast identification software. Blood cultures were incubated into a Bactec 9240 instrument and, if positive, aliquots were taken, plated onto blood agar plates and identified according to routine procedures. EDTA-blood samples were processed according to the LightCycler Septifast test.

Results

Among 147 samples from 51 patients with sepsis clinical signs, 30 were positive by Bactec and 27 by Septifast (which did not contain probes for three species isolated by cultural method). Concordance between the two methods was 76% for species identification. Different results between the two methods included 10 Bactec-positive and Septifast-negative samples (Acinetobacter baumannii was not amplified seven times, Gram-positive cocci were not determined by Septifast identification software three times) and 12 Bactec-negative and Septifast-positive samples (five Gram-positive cocci, three Pseudomonas aeruginosa, three Escherichia coli, one Klebsiella spp.), while four samples were positive by both methods but gave different species. The time response was evaluated: Septifast was faster than the traditional method in 97% cases.

Conclusion

Although the impossibility to determine antibiotic susceptibility represents a great limit of the Septifast technique, the short response time may contribute to undertake a rapidly tuned therapeutic regimen. Septifast Test appears to be a valid help in an early assessment of sepsis, along with cultural method.