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

Abstract

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.

Keywords: Kingella kingae; Kingella negevensis; RTX locus; groEL gene; malate dehydrogenase; mdh gene; pediatrics; real-time PCR.

FIG 1

Genomic architecture of the region carrying the mdh gene of Kingella kingae (Kkimdh). The genomic elements are not drawn to scale. The dashed lines indicate that the sensor histidine kinase may be encoded by either a single gene or two-component genes.

FIG 2

Neighbor-joining tree (40) based on the comparison of mdh nucleotide sequences from 18 genetic variants of Kingella kingae and their closest orthologs in Acinetobacter, Acidovorax, Polaromonas, and Neisseria species. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (41) and are in the units of the number of base substitutions per site. The scale bar indicates a 10% nucleotide sequence divergence. Bootstrap values (expressed as percentages of 1,000 replications) are showed next to the branches. Only bootstrap values greater than or equal to 70% were displayed. All positions containing gaps and missing data were eliminated. There was a total of 978 positions in the final data set. Numbers in parentheses next to the organism names are accession numbers.