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.

Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species.

Kingella kingae is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of K. kingae infections. However, the present study shows that Kingella negevensis, a Kingella species newly identified in young children, harbors an identical Kingella RTX locus, raising the question of whether K. negevensis can be misidentified as K. kingae by clinical microbiology laboratories. In silico comparison of Kingella sp. RTX and groEL genes and in vitro studies provided evidence that targeting the rtxA and rtxB genes could not differentiate between strains of K. kingae and K. negevensis, whereas targeting the groEL gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting K. negevensis groEL (kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and Kingella-specific rtxB, K. kingae-specific groEL (kkgroEL), and kngroEL qPCR assays. Forty-two specimens were rtxB positive, including 41 that were also kkgroEL positive and 1 (the remaining one) that was kngroEL positive. Thus, this study discloses an invasive infection caused by K. negevensis in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of K. kingae infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by K. negevensis in humans.

A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation.

BACKGROUND: Outbreaks of Kingella kingae infection are an emerging public health concern among daycare attendees carrying epidemic clones in the oropharynx. However, genotyping of such epidemic clones from affected cases is limited by the low performance of current methods to detect K. kingae from blood samples and lack of specimens available from infected sites. We aimed at developing a modified multilocus sequence typing (MLST) method to genotype K. kingae strains from oropharyngeal samples without prior culture. We designed in silico MLST primers specific for K. kingae by aligning whole nucleotide sequences of abcZ, adk, aroE, cpn60, recA, and gdh/zwf genes from closely related species belonging to the Kingella and Neisseria genera. We tested our modified MLST protocol on all Kingella species and N. meningitidis, as well as 11 oropharyngeal samples from young children with sporadic (n = 10) or epidemic (n = 1) K. kingae infection. RESULTS: We detected K. kingae-specific amplicons in the 11 oropharyngeal samples, corresponding to sequence-type 6 (ST-6) in 6 children including the epidemic cases, ST-25 in 2 children, and 3 possible novel STs (ST-67, ST-68, and ST-69). No amplicon was obtained from other Kingella species and N. meningitidis. CONCLUSIONS: We herein developed a specific MLST protocol that enables genotyping of K. kingae by MLST directly from oropharyngeal samples. This discriminatory tool, with which we identified the first K. kingae outbreak caused by ST-6 in Europe, may be used in further epidemiological investigations.

Isolation and characterization of Kingella negevensis sp. nov., a novel Kingella species detected in a healthy paediatric population.

We herein report the isolation and characterization of 21 Gram-stain-negative strains cultivated from the oropharynx of healthy children in Israel and Switzerland. Initially described as small colony variants of Kingella kingae, phenotypic analysis, biochemical analysis, phylogenetic analysis based on sequencing of the partial 16S rRNA gene and five housekeeping genes (abcZ, adk, G6PD, groEL and recA), and whole genome sequencing and comparison between members of the genera Kingella and Neisseria provided evidence for assigning them to the genus Kingella. Cellular fatty acids included important amounts of C12 : 0, C14 : 0, C16 : 0 and C16 : 1n7. Digital DNA-DNA hybridization between the isolates Sch538T and K. kingae ATCC 23330T revealed relatedness of 19.9 %. Comparative analysis of 16S rRNA gene sequences available in GenBank allowed matches to strains isolated in the USA, suggesting a wider geographical distribution. A novel species named Kingella negevensis sp. nov. is proposed, as most strains have been isolated in the Negev, a desert region of southern Israel. The type strain is Sch538T (=CCUG 69806T=CSUR P957).

Paediatric Bone and Joint Infections in French Guiana: A 6 Year Retrospective Review.

The epidemiology of paediatric bone and joint infections from South America is poorly known. We herein report a retrospective study conducted in whole French Guiana from January 2010 to December 2015. Medical charts of 55 previously healthy children were analysed, identifying 27 with osteomyelitis, 22 with septic arthritis and 6 with multifocal infections and/or osteoarthritis. The male:female ratio was 2.2:1, and the mean age was 7.5 years. Eighty percent children were ≥36 months old who had predominantly osteomyelitis related to methicillin-susceptible Staphylococcus aureus (p < 0.05) in the course of neglected skin infections. Five children presented with multi-systemic infections resulting in one fatality, mainly caused by S. aureus producing Panton-Valentine leucocidin (p < 0.01). In contrast, children aged 6-36 months had more likely culture-negative infections (p < 0.05), septic arthritis and mild clinical and biological features. Further prospective studies are required to better guide rational diagnostic and therapeutic strategies.

Outbreaks of Kingella kingae Infections in Daycare Centers Suggest Tissue Tropism of the Causative Strains.

BACKGROUND: Although Kingella kingae is recognized as an important pediatric pathogen, our knowledge of the virulence factors involved in the invasion of specific host's tissues is limited. Outbreaks of K kingae infections in daycare centers represent natural experiments in which a single virulent strain, introduced into a cohort of susceptible young children, causes multiple infections. If K kingae strains exhibit tissue tropism, the syndromes observed in a given cluster of cases would be relatively homogeneous. METHODS: Clinical data of all the K kingae outbreaks known to date were gathered and analyzed. The clinical syndromes diagnosed in the affected attendees were classified as septic arthritis, osteomyelitis, tenosynovitis, soft tissue infection, bacteremia with no focal disease, endocarditis, and meningitis, and computed separately. To assess the similarity of the clinical syndromes detected within outbreaks, we used the Cramer V statistic, which is a measure of the association between 2 nominal variables and, for the purposes of the study, between the detected clinical syndromes and the outbreaks. RESULTS: A total of 23 outbreaks involving 61 attendees were identified. The mean±SD attack rate in the affected classrooms was 15.8% ± 4.8%, and the K kingae colonization rate among the attendees was 54.8% ± 25.3%. Seventy-two separate foci of infection were diagnosed. Osteomyelitis and septic arthritis were the most common clinical syndromes and were diagnosed in 26 children each, followed by tenosynovitis in 4 children. The clinical syndromes diagnosed among attendees to the same classroom showed a statistically significant tendency to be similar (P = .015). CONCLUSIONS: The distribution of clinical syndromes in clusters of K kingae infections differs from that of sporadic cases. The causative strains combine enhanced virulence and high transmissibility, and show tropism toward bones, joints, and tendon sheaths. This information can be used to identify virulence factors associated with invasion of these specific host tissues.

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Draft Genome Sequence of Kingella negevensis SW7208426, the First European Strain of K. negevensis Isolated from a Healthy Child in Switzerland.

We report here the draft genome of Kingella negevensis strain SW7208426, isolated from the oropharynx of a healthy 6-year-old boy in Geneva, Switzerland. To our knowledge, this is the first genome report of the newly described K. negevensis species from Europe.

An Outbreak of Kingella Kingae Infections Complicating a Severe Hand, Foot, And Mouth Disease Outbreak in Nice, France, 2016.

We report the investigation methods for the diagnosis of an epidemic and culture-negative Kingella kingae endocarditis complicating a severe outbreak of hand, foot and mouth disease in a childcare center. The diagnosis was confirmed by polymerase chain reaction testing performed from cardiac tissue. Our findings argue for the systematic investigation of K. kingae outbreaks by using molecular tools in such context.

Acute Septic Arthritis of the Knee Caused by Kingella kingae in a 5-Year-Old Cameroonian Boy.

Kingella kingae is an important cause of invasive infections in young children from Western countries. Although increasing reports indicate that this organism is the leading agent of bone and joint infections in early childhood, data on K. kingae infections from resource-limited settings are scarce, and none has yet been reported in Africa. We herein report the diagnostic and epidemiological investigations of the first case of K. kingae arthritis identified in a child from sub-Saharan Africa. A 5-year-old Cameroonian boy presented with a sudden painful limp which appeared in the course of a mild rhinopharyngitis. He lived in Cameroon where he had been vaccinated with BCG at birth and moved to France for holidays 4 days before consultation. There was no history of trauma and he did not have any underlying medical condition. Upon admission, he had a temperature of 36.7°C, and clinical examination revealed right-sided knee tenderness and effusion that was confirmed by ultrasound imaging. Laboratory results showed a white blood cell count of 5,700 cells/mm3, C-reactive protein level of 174 mg/L, and platelet count of 495,000 cells/mm3. He underwent an arthrocentesis and was immediately given intravenous amoxicillin-clavulanate. Conventional cultures from blood samples and synovial fluids were negative. Polymerase chain reaction (PCR) assay targeting the broad-range 16S rRNA gene and real-time quantitative PCR assays targeting Mycobacterium species were negative. Surprisingly, real-time PCR assays targeting the cpn60, rtxA, and rtxB genes of K. kingae were positive. Multicolor fluorescence in situ hybridization specific for K. kingae identified the presence of numerous coccobacilli located within the synovial fluid. Finally, multilocus sequence typing analysis performed on deoxyribonucleic acid directly extracted from joint fluid disclosed a novel K. kingae sequence-type complex. This case report demonstrates that K. kingae may be considered as a potential cause of septic arthritis in children living in sub-Saharan Africa, and hence the burden of K. kingae infection may be not limited to the Western countries. Further studies are required to determine the prevalence of K. kingae infection and carriage in Africa.

Patterns of Kingella kingae Disease Outbreaks.

BACKGROUND: Kingella kingae outbreaks occur sporadically in childcare centers but remain poorly understood and difficult to identify. METHODS: To provide the basis of a better knowledge of K. kingae outbreaks patterns that may help to guide identification and management strategies, we collected epidemiological, clinical and laboratory data from all reported K. kingae outbreaks, and those from 2 new Israel outbreaks in 2014. RESULTS: Nine outbreaks were identified in the USA, Israel and France from 2003 to 2014. Twenty-seven children with a median age of 14 ± 4.1 months were affected, male:female ratio of 1.4:1. Outbreaks demonstrated seasonal patterns from the 10th to the 45th weeks, a mean duration of 13.1 ± 8.4 days, a mean attack rate of 17.3 ± 5.1% and a case-fatality rate of 3.7% (1/27). Seventy-four percentage of children had fever (20/27), and the mean values of white blood cell count and C-reactive protein level were 14.6 ± 4.5 × 10/L and 23.8 ± 24.1 mg/L, respectively. Osteoarticular infections accounted for 88.9% of cases (24/27), bacteremia 7.4% (2/27), endocarditis 3.7% (1/27) and meningitis 3.7% (1/27). Specific real-time polymerase chain reaction demonstrated higher performance than culture methods in the diagnosis of case patients and investigations of oropharyngeal K. kingae carriage among close contacts, and multilocus sequence typing methods revealed that ST-6 and ST-25 invasive strains were responsible for multiple country-dependent outbreaks. Coviral infections were identified in the majority of K. kingae outbreaks, notably those causing oral ulcers. CONCLUSIONS: K. kingae outbreaks displayed severe K. kingae diseases that were poorly confirmed with culture methods. We argue for the use of genomic technologies to investigate further K. kingae outbreaks.

Kingella kingae DNA in langerhans cell histiocytosis of bone.

Langerhans cell histiocytosis of bone is a rare pediatric neoplastic disorder of unclear pathogenesis. We report the case of a 3-year-old girl who presented with Langerhans cell histiocytosis of the ilium in which Kingella kingae was detected. Our findings argue for the search for K. kingae by polymerase chain reaction in children with Langerhans cell histiocytosis of bone.

An outbreak of Kingella kingae infections associated with hand, foot and mouth disease/herpangina virus outbreak in Marseille, France, 2013.

BACKGROUND: Outbreaks of invasive Kingella kingae infections recently emerged as a new public health concern in daycare centers in Europe, USA and Israel. Despite this, no trigger factor has been yet identified, preventing the setting up of rational measures of control and prevention. We report an outbreak of K. kingae infections associated with hand, foot and mouth disease/herpangina outbreak, and we define the research and policy priorities. METHODS: From April 22 to May 07, 2013, 5 toddlers presented successive osteo-articular infections in a daycare center in Marseille, France. Real-time polymerase chain reaction targeting the cpn60 gene of K. kingae was used to investigate suspected cases and the prevalence of oropharyngeal K. kingae carriage of their close contacts. RESULTS: The attack rate of the K. kingae infections outbreak was 23.7% (5/21) with no fatality. Positive real-time polymerase chain reaction targeting the cpn60 gene of K. kingae confirmed the diagnosis in 3 cases and revealed a rate of K. kingae oropharynx carriage in the index classroom of 94.4% (17/18) among daycare attendees not given antibiotic during the previous month, and of 76.9% (10/13) among staff in close contact. The eradication rate of K. kingae was 21.4% (3/14) among classmates after oral administration of rifampicin, and eradication occurred spontaneously in 83.3% (5/6) of the staff. Clinical and epidemiological features of the herpangina outbreak were consistent with that of an emerging European Coxsackievirus-A6 outbreak. CONCLUSIONS: Hand, foot and mouth disease/herpangina virus outbreak enables triggering a K. kingae infections outbreak. Our findings offer support for new guidelines of K. kingae infections outbreaks management and emphasize the need for further research.

PTCH1 mutation and local aggressiveness of odontogenic keratocystic tumors in children: is there a relationship?

Keratocystic odontogenic tumors (KCOTs) are locally aggressive jaw lesions that may be related to PTCH1 mutations in isolation or in association with nevoid basal cell carcinoma syndrome. We sought to clarify the role of PTCH1 mutation in KCOT aggressiveness. We assessed cyst pathological characteristics, Ki-67 immunostaining, and somatic and germinal PTCH1 mutation in 16 KCOTs from 10 unrelated patients. Ten PTCH1 mutations were identified in 16 tumors. All tumors with PTCH1 mutations presented the criteria of pathological aggressiveness. We also noted the presence of a chorionic epithelial structure apparently acting as a secondary germinal center in these same tumors. Ki-67 immunostaining was not associated with PTCH1 mutation. KCOTs harboring the mutation display a chorionic epithelial structure that acts as a secondary germinal center. Genetic and microenvironmental factors might interact to propel tumor development.