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OBJECTIVE:Adequate resources are required to rapidly diagnose and treat pediatric musculoskeletal infection (MSKI). The workload MSKI consults contribute to pediatric orthopaedic services is unknown as prior epidemiologic studies are variable and negative work-ups are not included in national discharge databases. The hypothesis was tested that MSKI consults constitute a substantial volume of total consultations for pediatric orthopaedic services across the United States. STUDY DESIGN:Eighteen institutions from the Children's ORthopaedic Trauma and Infection Consortium for Evidence-based Study (CORTICES) group retrospectively reviewed a minimum of 1 year of hospital data, reporting the total number of surgeons, total consultations, and MSKI-related consultations. Consultations were classified by the location of consultation (emergency department or inpatient). Culture positivity rate and pathogens were also reported. RESULTS:87,449 total orthopaedic consultations and 7,814 MSKI-related consultations performed by 229 pediatric orthopaedic surgeons were reviewed. There was an average of 13 orthopaedic surgeons per site each performing an average of 154 consultations per year. On average, 9% of consultations were MSKI related and 37% of these consults yielded positive cultures. Finally, a weak inverse monotonic relationship was noted between percent culture positivity and percent of total orthopedic consults for MSKI. CONCLUSION:At large, academic pediatric tertiary care centers, pediatric orthopaedic services consult on an average of ~3,000 'rule-out' MSKI cases annually. These patients account for nearly 1 in 10 orthopaedic consultations, of which 1 in 3 are culture positive. Considering that 2 in 3 consultations were culture negative, estimating resources required for pediatric orthopaedic consult services to work up and treat children based on culture positive administrative discharge data underestimates clinical need. Finally, ascertainment bias must be considered when comparing differences in culture rates from different institution's pediatric orthopaedics services, given the variability in when orthopaedic physicians become involved in a MSKI workup.

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.