A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene of Kingella kingae and Application to 201 Pediatric Clinical Specimens

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting the groEL gene and the RTX locus of K. kingae However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack specificity because they could not distinguish between K. kingae and the recently described Kingella negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingaegroEL-based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of the K. kingae mdh gene from 20 distinct sequence types of K. kingae This novel K. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children between 7 months and 7 years old.