On 11 July 2016, a 5-year-old Cameroonian boy was admitted to the emergency department at the Dracenie Hospital in the region Provences-Alpes-Cote d'Azur, France, due to a painful limp that appeared in the morning. He lived in Cameroon where he had been vaccinated with BCG at birth, and moved to Southeastern France for holidays 4 days before consultation. A mild rhinopharyngitis had occurred the previous week, but as the symptoms were mild, no treatment had been undertaken. There was no history of trauma, and he did not have any underlying medical condition. Upon admission to hospital, the child had a temperature of 36.7 C and refused to walk. Clinical examination revealed right-sided knee tenderness and effusion. Neither skin rash nor oral ulcerations were noted. Laboratory results showed an elevated C-reactive protein (CRP) level at 174 mg/L, with normal white blood cell count of 5,700 cells/mm3 and platelet count of 495,000 cells/mm3. Ultrasound imaging confirmed effusion of the right knee, whereas conventional radiograph showed no significant abnormality. The child underwent an arthrocentesis, and mildly opaque and yellowish liquid was extracted, suggesting a septic arthritis of the right knee. Consequently, the child was immediately given intravenous amoxicillin-clavulanate 100 mg/kg three doses daily during 3 days.
Because conventional cultures applied for Gram-positive, Gram-negative, mycobacterial species, and fungi from the joint fluid and blood samples were negative, joint specimens were sent in dry ice to the molecular diagnosis laboratory of the URMITE unit in Marseille, where bacterial deoxyribonucleic acid (DNA) was extracted directly from the joint fluid. Polymerase chain reaction (PCR) assay targeting the broad-range bacterial 16S rRNA gene and qPCR assays targeting both Mycobacterium species and Mycobacterium tuberculosis complex were negative. Given the age of the patient, K. kingae was also sought by using specific qPCR assay targeting the K. kingae cpn60 (groEL) gene. Surprisingly, this specific K. kingae assay was positive, as well as qPCR assays targeting the Kingella-specific rtxA and rtxB genes, thus confirming the diagnosis of septic arthritis caused by K. kingae. The organism was also identified by multicolor fluorescence in situ hybridization specific for K. kingae (Figures S1 and S2 in Supplementary Material), which revealed the presence of large numbers of viable coccobacilli located within the synovial fluid (Figure 1). Cardiac investigations ruled out endocarditis. A switch to oral amoxicillin-clavulanate 100 mg/kg three doses daily was then undertaken on 15 July 2016 and was planned for a total duration of 2 weeks. Despite these recommendations, the treatment was continued for another 2 months in Cameroon. During the final follow-up 3 months postoperatively, clinical examination revealed a normal knee status with a normal range of motion.
Thereafter, MLST studies using a modified protocol specific for K. kingae was performed on bacterial DNA extracted directly from the joint fluid as previously described. Five alleles were unambiguously identified, namely, adk-2, aroE-2, cpn60-2, zwf-13, and recA-2. Unexpectedly, 14 single nucleotide variants of the abcZ allele were identified from nucleotides 6-447 (Figure S3 in Supplementary Material; Table 1). To estimate the between-strain relatedness and define an MLST scheme for K. kingae, a different allele number was given to each distinct sequence within a locus, and a distinct sequence-type (ST) number was attributed to each distinct allele combination. K. kingae isolates were then grouped into ST-complexes (STcs) if they differed at no more than one locus from at least one other member of the group. Among the 70 STs of K. kingae that are documented in the multilocus sequence database (MLST) of the Institut Pasteur database (), ST-26, which belongs to the highly invasive STc-25, was the closest ST by sharing four alleles, namely, adk-2, cpn60-2, gdh/zwf-13, and recA-2 with the causative strains that were herein identified (Table 2). Although analysis of the combination produced by the five unambiguous alleles indicated that the causative K. kingae strains belongs to a novel ST, the presence of multiple abcZ alleles does not allow to precisely define it. Moreover, in the MLST scheme of K. kingae, founder genotypes of STcs were defined as the ST of the STc with the highest number of neighboring STs [, Table 3]. Consequently, although analysis of the combination produced by the five unambiguous alleles indicated that the causative K. kingae strains belong also to a novel STc, no specific denomination is yet possible. Moreover, since each of these housekeeping genes is present in one copy in the whole genome of K. kingae, these findings suggested co-infection by strains belonging to distinct STs.