Rapid identification of sepsis-causing pathogens with polymerase chain reaction and microarray-based assay

Background

During the course of a bacterial infection, a rapid identification of causative agents is necessary for the determination of effective treatment options. We developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 19 sepsis-causing pathogens at species level, as well as coagulase-negative staphylococci and Enterobacteriaceae at taxon level.

Materials

One hundred and fifty-four positive and 15 negative blood culture samples were collected to evaluate the assay performance. For the analysis, DNA was automatically extracted from the samples.

Methods

The broad-range PCR primer mixture was designed using conservative regions of topoisomerase gene subunits gyrB and ign from various bacteria. The primer design allowed the use of a novel DNA amplification method producing labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed against species-specific or taxon-specific variable regions of the gyrB and ign genes flanked by the primers. As a microarray platform for the probes, we applied TubeArray that is a microreaction vial containing a microarray at the bottom. To indicate the detection of antimicrobial resistance, we included mecA-specific primers and probes in the same assay. Furthermore, the software for automated data analysis was provided.

Results

Comparison of the assay results with the gold standard culturing method revealed sensitivity of 98% and specificity of 93%. When the mecA identification was correlated with Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus genus detection by the specific probes, accurate information about the association of the mecA gene with staphylococci was provided. The results from these 15 samples were in line with the antimicrobial susceptibility testing (oxacillin susceptibility testing).

Conclusion

The results from the method were available 3 hours after the positive blood culture result. Up to 24 samples could be processed simultaneously. The assay therefore provides rapid and reliable data, which can guide antimicrobial treatment decisions in a timely manner. We have further broadened the pathogen panel for the detection of 50 bacterial species; 24 at the species level and at least 26 species at the taxon level. The described panel is now commercially available from Mobidiag Ltd (Helsinki, Finland) under the product name Prove-it™ Bacteria.