Validation of polymerase chain reaction in experimental sepsis diagnosis beyond blood culture

Background

A positive blood culture (BC) is considered the gold-standard method for sepsis diagnosis, although its sensibility is low (10% to 30%) – which demands a better diagnostic tool to limit broad-spectrum antibiotic use in the majority of patients without culture-based sepsis diagnosis. Besides, after microbial invasion, bacteria can remain dead or fragmented in the circulation, thus limiting BC efficiency. Herein we evaluated the PCR diagnostic efficacy under live, dead and fragmented bacteria contents in the bloodstream.

Methods

Wistar rats were distributed into three groups (n = 20/group) based on live, dead and DNA inoculations. Another lipopolysaccharide (LPS) + DNA group (1 mg/kg LPS injection plus 4 hours later DNA injection, n = 10) was designed for DNA detection under an induced inflammatory state. Live, dead and extracted DNA forms of Pseudomonas aeruginosa (ATCC 27853), 10⁷ colony-forming units/ml/100 g body weight, were injected into the tail vein of respective groups. Blood samples were collected after 20 minutes (n = 10) and 6 hours (n = 10) from all groups except for the LPS + DNA group (6 hours), and were submitted to a nested PCR assay using general and specific primers. BC was performed only in the live group.

Results

In the live group at 20 minutes the sensibility was 100% by both BC and PCR, and at 6 hours the sensitivity was 60% to BC and 80% to PCR. In the dead group, the PCR sensitivity was 90% at 20 minutes and 50% at 6 hours. In the DNA group, the sensitivity was 90% at 20 minutes and 40% at 6 hours, and in the LPS + DNA group at 6 hours the sensitivity was 40%.

Conclusion

The sensitivity of the PCR was as effective as BC in 20 minutes and superior in 6 hours. Besides, the PCR assay was able to detect circulating dead bacteria and bacterial DNA in the blood, which is not possible by the BC method. These findings suggest that the live bacteria remains for a short period of time in the bloodstream as compared with dead and DNA bacteria, and a systemic inflammation state seems to not interfere with the PCR assay. Besides, the PCR tool with specific primers can be a useful method for sepsis diagnosis in the negative blood culture conditions as well as in specific bacterial surge events in the ICU, thus improving the antibiotic usage potentials.