Evaluation of a novel SYBR Green real-time PCR assay for the Klebsiella pneumoniae species complex: an accurate and fast molecular tool for public health

Klebsiella pneumoniae species complex (KpSC), importantly multidrug-resistant Klebsiella pneumoniae is a critical priority pathogen that causes serious infections in humans and animals. Laboratory-developed tests were used to detect specific phylogroups, particularly the KpSC, although the translational potential has yet to be assessed due to a lack of internal validation data. We investigated one novel SYBR Green real-time PCR method, the ZKIR (zur-khe intergenic region) assay, for identifying KpSC using human and animal KpSC (n = 53) and non-KpSC isolates (n = 39) at Vidyasagar University, West Bengal, India. Based on melt-curve analysis, we considered a cut-off Ct of <= 30 as positive results, showing a Tm of 79.3 +/- 2 degrees C for bacterial cultures. The assay demonstrated high sensitivity and specificity along with accuracy (> 90%) considering an automated culture identification system as standard. A very good concordance (Kappa = 0.89) was observed between the assay and culture results. Notably, four KpSC isolates tested negative in the assay, and one Pantoae agglomerans was misidentified as KpSC. Out of five discrepant results, the identification of three and two isolates were matched with automated culture and PCR assay by the 16S rRNA sequencing, respectively. The assay produced results by similar to 2 h at a cost of US$5 per sample. Accurate and rapid detection of KpSC at a low-cost encourages widespread use of the ZKIR real-time PCR assay, especially in resource-constrained public health laboratories, for early containment of such a potential biohazard.