Rapid Detection of Hypervirulent Klebsiella pneumoniae by Multiplex PCR and Nucleic Acid Chromatography: Evaluation of Diagnostic Performance and Clinical Utility.

BACKGROUND AND AIM: Hypervirulent Klebsiella pneumoniae (hvKp) causes severe community-acquired infections with metastatic complications. The string test is widely used as a phenotypic marker; however, its diagnostic accuracy is limited. This study aimed to improve hypervirulent phenotype identification by combining the string test with rapid multiplex polymerase chain reaction (PCR) targeting hvKp-associated virulence genes and detection by nucleic acid chromatography.

METHODS

We established a multiplex PCR and nucleic acid chromatography system capable of detecting three virulence genes (prmpA, iucA-PP2, and peg-344-PP2). We included 119 cases of bacteremia caused by isolates identified as K. pneumoniae by a parCgene sequence analysis. The presence of the three virulence genes was assessed using both singleplex and multiplex PCR. Multiplex PCR products were detected using agarose gel electrophoresis and nucleic acid chromatography. Clinical characteristics were compared between the hypervirulent and non-hypervirulent phenotype groups. Strains with the disseminated lesion, abscess formation, or death during the first 30 days were classified as hypervirulent phenotype.

RESULTS

Isolates that were string test-negative but carried hvKp-associated virulence genes were identified, suggesting that reliance on phenotypic methods alone may lead to underdiagnosis of hypervirulent strains. In a receiver operating characteristic curve analysis, positivity for three or more markers among the four variables (string test and the three virulence-associated genes) yielded a sensitivity of 0.84 and specificity of 0.90. High concordance rates were also observed between singleplex and multiplex PCR using either gel electrophoresis or DNA chromatography.

CONCLUSIONS

HvKp-associated genes can be detected more rapidly by combining multiplex PCR with nucleic acid chromatography than by conventional gel electrophoresis-based methods. This approach also demonstrated high sensitivity and may be helpful for potential clinical risk assessment of hvKp infections.