New Real-Time PCR-Based Method for Kingella kingae DNA Detection: Application to Samples Collected from 89 Children with Acute Arthritis

Abstract

Inoculation of blood culture vials with joint fluid samples has revealed the important pathogenic role of Kingella kingae in pediatric arthritis. However, recent studies based on broad-range 16S ribosomal DNA PCR and real-time PCR without a probe suggest that conventional methods remain suboptimal. We developed a new real-time PCR method with a probe that is highly specific for K. kingae and applied it to joint fluid samples collected from 89 children with suspected arthritis admitted to our institution during a 2-year period. Real-time PCR was also applied to blood samples obtained before surgery and to joint drainage fluid samples obtained during several days after surgery. Thirty-six (40%) of the 89 cases of suspected septic arthritis had positive culture. Staphylococcus aureus was the main isolate (n ‫؍‬ 19/36, 53%), followed by K. kingae (n ‫؍‬ 7/36, 19%). Specific real-time PCR identified K. kingae in 24 of the 53 culture-negative cases. Thus, K. kingae was present in 31 (52%) of the 60 documented cases, making it the leading pathogen. Real-time PCR on all 15 blood DNA extracts from patients with K. kingae infection was negative, demonstrating that joint fluid positivity did not result from DNA circulating in blood. Real-time PCR amplification of drainage fluid samples showed that the pathogen could be detected for up to 6 days after antibiotic initiation. K. kingae real-time PCR applied to DNA extracted from joint fluid samples, but not from blood samples, markedly improved the etiological diagnosis of septic arthritis in children. Retrospective diagnosis is feasible for up to 6 days after treatment initiation.

Kingella kingae: Carriage, Transmission, and Disease

“…Of note, the PCR assay detected K. kingae DNA sequences in all 7 culture-positive samples and was negative in all those in which other microorganisms were recovered by culture, confirming the specificity of the molecular assay. Altogether, K. kingae was detected in 31 (52%) of 60 bacteriologically proven cases (15).…”

“…The disease generally involves the large weight-bearing knee, ankle, or hip joints in over 80% of cases (15,102,(157)(158)(159)(160), followed by the wrist, shoulder, and elbow (102,(160)(161)(162)(163)(164)(165)(166)(167)(168)(169). However, the metacarpophalangeal, sternoclavicular, tarsal, and sacro-iliac joints (148,(170)(171)(172)(173), which are rarely affected by other bacterial species, are involved with unusual frequency in K. kingae arthritis (18).…”

“…Ilharreborde et al enrolled 89 children with presumptive joint infections, inoculated synovial fluid aspirates into BCVs, and analyzed the specimens by real-time PCR employing primers that amplify K. kingae's cpn60 gene (15). The diagnosis of suppurative arthritis was established by culture in 36 samples (40%), of which 7 grew K. kingae, whereas the NAA test disclosed 24 additional cases of K. kingae infection among the 53 children in whom the exudate cultures were negative.…”

“…The number, however, has sharply increased in recent years, jumping to 105 in the following decade and 83 in the short period from January 2010 through August 2014 (determined by a PubMed search with "Kingella kingae" and "Moraxella kingii"), and has firmly established the status of K. kingae as a common agent of bacteremia with no focus (also called occult bacteremia) (11,12) and the predominant etiology of joint and bone infections in 6-to 36-month-old children (13)(14)(15)(16). This increasing detection of K. kingae does not indicate that the bacterium is really a novel human pathogen.…”

Major Intercontinentally Distributed Sequence Types of Kingella kingae and Development of a Rapid Molecular Typing Tool

“…The optimization of conventional culture techniques and the development of current molecular techniques, such as real-time PCR, led to the recognition of K. kingae as the major pathogen causing osteoarticular infections (OAIs) in children younger than 4 years in many countries (3)(4)(5)(6) and as a common etiology of occult bacteremia (7) and, more rarely, of endocarditis in children and adults (3,7,8). Moreover, six outbreaks of invasive K. kingae infections in daycare centers in the United States, Israel, and France were recently described (8)(9)(10)(11)(12), with an average infection rate of around 20% (9)(10)(11) and up to an 85% carriage rate among healthy attendees (11), suggesting the increased colonization fitness, transmissibility, and invasiveness of some K. kingae clones.…”