Epidemiology and Management of Acute Haematogenous Osteomyelitis in a Tertiary Paediatric Center

Background: Paediatric acute hematogenous osteomyelitis (AHOM) is a serious disease requiring early diagnosis and treatment. To review the clinical presentation, management and organisms responsible for AHOM, and to explore risk factors for complicated AHOM, a large cohort referring to a single center over a 6-year period was evaluated. Methods: Data from children with AHOM, hospitalized between 2010 and 2015, and aged > 1 month, were retrospectively collected and analyzed. Results: 121 children (median age 4.8 years; 55.4% males) were included. Fever at onset was present in 55/121 children (45.5%); the lower limb was most frequently affected (n = 68/121; 56.2%). Microbiological diagnosis (by culture and/or polymerase chain reaction (PCR)) was reached in 33.3% cases. Blood and pus/biopsy culture sensitivities were 32.4% and 46.4%, respectively. PCR sensitivity was 3.6% (2/55) on blood, and 66.6% (16/24) on pus/biopsy sample. Staphylococcus aureus was the most commonly identified pathogen (n = 20); no methicillin-resistant Staphylococcus aureus (MRSA) was isolated, 10.0% (n = 2) strains were Panton-Valentine-Leukocidin (PVL) producer; 48.8% (59/121) cases were complicated. At univariate analysis, factors associated with complicated AHOM were: recent fever episode, fever at onset, upper limb involvement, white blood count (WBC) ≥ 12,000/µL, C reactive protein (CRP) ≥ 10 mg/L, S. aureus infection. At multivariate analyses S. aureus infection remained the only risk factor for complicated AHOM (aOR = 3.388 (95%CI: 1.061–10.824); p-value = 0.039). Conclusions: In this study microbiological diagnosis was obtained in over one third of cases. Empiric treatment targeting methicillin-sensitive Staphylococcus aureus seems to be justified by available microbiological data.


Introduction
Most acute pediatric osteomyelitis are haematogenous infections with an estimated incidence of 8 cases per 100,000 children/year [1,2]. Children below 5 years of age and males are more frequently affected [3]. Early detection of acute hematogenous osteomyelitis (AHOM) is crucial, given that a delay in the diagnosis of only 5 days is a major risk factor for complications [4]. Even if mortality is rare, permanent disabilities can occur, such as growth arrest with limb length discrepancy or deformity [5]. AHOM etiology is changing according to bacterial susceptibility pattern modifications over time, vaccination programs, and implementation of cultural and polymerase chain reaction (PCR) techniques. Staphylococcus aureus (S. aureus) is the most commonly isolated pathogen [6], accounting for 25-60% of cases of AHOM. Other microorganisms include Group-A Streptococcus pyogenes, Streptococcus pneumoniae, and Gram-negative rods [5]. Kingella kingae is an emergent pathogen [4,7]. Infections sustained by community acquired methicillin resistant S. aureus (MRSA) are increasing, particularly in the United States [8]. On the other hand, in Europe community acquired-MRSA is still identified at low rates, while Panton-Valentine-Leukocidin (PVL) producing S. aureus isolates are increasingly reported [9]. The empirical therapeutic choice is based on local epidemiological data [4]. An anti-staphylococcal penicillin, i.e., oxacillin or flucloxacillin, or a cephalosporin are generally recommended as first line treatment [4]. Some expert suggest the use of other drugs, including antibiotics effective towards MRSA, depending on local MRSA prevalence [10]. The duration and routes of administration of antibiotics is currently under debate [11]. Historically, AHOM was treated with intravenous (IV) antibiotics for several weeks [12]. More recently, a randomized trial showed that children who displayed a good clinical response after 2-4 days of IV treatment and shifted to oral treatment for further 20 days had not different outcomes when compared to children treated with continued IV therapy for 30 days [13]. A similar approach, using a short IV therapy (about 7 days) has been adopted by several centers in Europe [14] and USA [15], suggesting that short IV therapy may be as effective as longer courses with no increased risk of sequelae. Given the changes in immunization programs, the emergence of MRSA, availability of molecular diagnostics and changing trends in management, a review of the epidemiology of this important disease is needed. Therefore, we evaluated data from a large cohort of otherwise healthy children referring to a single center over a 6-year period.

Methods
Aim of the present study was to evaluate retrospectively the management and outcome of AHOM in a large single tertiary center over a 6-year period, with particular consideration to possible risk factors for complicated AHOM.

Setting
Meyer Children's Hospital is a tertiary pediatric university hospital with 200 beds and serves as the main referral center for Tuscany and surrounding regions [16]. The hospital comprises several paediatric departments, including general orthopedic, surgical, intensive care, infectious diseases and other specialist units, working in a multidisciplinary team. Tuscany has a population of three million and six hundred thousand people, 6.2% of the Italian population, with 565,886 people under the age of 18 years [16].

Definitions
AHOM was defined as any bone infection presenting with a time period between diagnosis and symptom onset <2 weeks [10,17]. AHOM was diagnosed in the presence of clinical features (fever, swelling, warmth, pain, restriction of movement) and compatible radiologic imaging with or without bacteriological isolation from blood or bone sample [14].

Complicated Osteomyelitis
AHOM was defined as complicated when the child developed sepsis, septic shock, or arthritis, cellulitis, sub-periosteal or muscle abscess, deep venous thrombosis (DVP) [14], pathologic fracture, septic emboli or when the child necessitated admission to the intensive care unit (ICU) [4].

Study Design and Population
A retrospective single center study was conducted, evaluating data from all children aged between 1 month and 18 years, consecutively admitted to Meyer Children's Hospital with a discharge code consistent with the diagnosis of osteomyelitis (ICD codes M86.00-86.99) , according to the World Health Organization International Classification of Diseases (WHO ICD-10), between 1 January 2010 and 31 December 2015. Data from these children were independently reviewed by two authors. Inclusion criteria was AHOM, as defined above, and exclusion criteria were: age ≤ 30 days; congenital or acquired immunodeficiency, underlying bone disease; carrier of prosthetic materials; hospital acquired infection, previous history of skin wound, open fracture or surgery at the site of bone infection or in contiguous areas, septic arthritis (i.e., no adjacent osteomyelitis on imaging). Only cases fulfilling the above criteria were included in the study. Data were collected from medical records, electronic records for laboratory, and radiology results. Demographic and clinical details, microbiological and radiologic results, and clinical management including type, route and duration of antibiotic treatment, need for surgery, and readmission to hospital within 6 months of initial diagnosis were entered into an electronic database. Reasons for changes in the antibiotic regimen were also recorded (i.e., clinical and/or radiological failure; switch from IV to oral therapy; drug toxicity; switch from empirical therapy to a targeted antimicrobial therapy after bacterial isolation).
Surgery was performed in children not responding to medical treatment, or needing draining an abscess or in children with adjacent arthritis needing draining and joint lavage.

Laboratory and Microbiological Investigations
All the laboratory tests were performed in the same laboratory at the Author's Institution using standardized techniques and according to manufacturer's instructions. Hematological parameters (WBC, CRP, ESR) were collected at admission before administration of any antimicrobial therapy. In particular CRP in serum or heparinized plasma was detected by means of particle enhanced immunonephelometry on the Dimension Vista™ System equipped with the Dimension Vista™ System Flex ® reagent cartridge (Siemens Healthcare Diagnostics, Marburg, Germany). The Analytical Measurement Range (AMR) is 0.29-19.0 mg/dL. ESR was measured using a capillary micro-photometer method, and expressed in mm/hour. The AMR is 2-120 mm/h. Blood cultures and cultures from bone or joint fluid samples were processed using standard methods. All samples were processed for detection of common human bacteria (or cultured for detection of MRSA isolates) using rich and selective culture media. After 48 hours of incubation in aerobic atmosphere at 37 • C, plates were read in order to detect the presence of pathogens. Identification of bacteria isolates was based on typical colony morphology on selective culture media (Mannitol Salt 2 Agar, Mac Conkey Agar, bioMérieux, (Geneva, Switzerland)) or on chromogenic culture media (chromID ® CARBA SMART, bioMérieux and BBL™ CHROMagar™ MRSA II, Becton Dickinson, (Becton Dickinson, Lincoln Park, NJ, USA). In order to confirm species-level identification mass spectrometry analysis was performed using MALDI-TOF (VITEK ® MS, bioMérieux). Organisms were identified phenotypically and confirmed using traditional methods or the Vitek2 gram positive card (bioMérieux). Antibiotic susceptibility testing was performed using an automated system (Vitek2 AST-P612 card, bioMérieux) and the Kirby-Bauer disk diffusion method in accordance with the guidelines of the Clinical and Laboratory Standards Institute. Antibiotic susceptibility was evaluated using the automated system VITEK ® 2 (bioMérieux) with the card AST-P632 for the several antibiotics. The European committee on antimicrobial susceptibility testing (EUCAST) clinical breakpoints were used as interpretation criteria. The pathogenicity of S. aureus infections is related to various bacterial surface components and to extracellular proteins such as PVL. In order to determine the potential virulence of MRSA strains, a specific PCR assay for the presence of the gene (lukS-lukF) encoding for the PVL was set up following a previously published protocol [18].
Universal real-time PCR assay targeting the gene coding for 16S ribosome RNA coupled with sequencing of amplified products was performed on blood and/or tissue samples, as previously described [19,20].

Statistical Methods
Data were reported as median and interquartile range (IQR) or absolute numbers and percentages. Non parametric Mann-Whitney test, Fisher's exact test or Chi Square test were used to compare continuous or categorical variables, as appropriate. All significant tests were two-sided. Uni-and multi-variate logistic regression analyses were performed to investigate the association between several parameters and risk of complicated AHOM, calculating odds ratios (ORs) and 95% confidence intervals (CIs). Variables included in univariate analyses were gender, age, site of infection (upper limb, lower limb, other site), previous trauma, previous fever episode, clinical characteristics at the time of admission, WBC (≥ or <12,000 cell/µL) CRP (≥ or <10 mg/L), ESR (≥ or <20 mm/h); initial X-ray (positive or negative); initial ultrasound (US) (positive or negative); initial MRI (positive or negative); positive microbiological culture or PCR on blood or pus; infection sustained by S. aureus vs. other/unknown; first antibiotic treatment duration (≥ or <10 days) and type of first antibiotic regimen. Factors significantly associated to complicated AHOM at univariate analysis were included in the multivariate analyses. All statistical analyses were carried out using the SPSS (Statistical Package for the Social Sciences, SSPS Inc., Chicago, IL, USA) for Windows software program version 19.0. A p-value < 0.05 was considered significant. The study was approved by the Ethics Committee at the authors' institution (121/2016).
Fifty With respect to uncomplicated cases, complicated cases more frequently involved the upper limb (27.1% vs. 8.1%; p-value = 0.005) and more frequently reported a recent episode of fever (69.5% vs. 40.3%; p-value = 0.001). We observed a higher proportion of complications when long bone were involved vs. non long bone (p-value = 0.021). Children with complicated AHOM presented more frequently fever at onset (59.3% vs. 32.3%; p-value = 0.002) ( Table 1). Other factors associated with complications were, WBC ≥ 12,000/µL, CRP ≥ 10 mg/L, a positive microbiological test, infection sustained by S. aureus and a positive MRI. In the multivariate analyses only infection sustained by S. aureus was associated with risk of complicated AHOM (OR = 3.388 (1.061-10.824); p-value = 0.039) ( Table 2).

Follow-Up Results
9/121 (7.4%) children were switched to a second IV therapy for clinical failure with subsequent complete resolution; 4 children out of 121 (3.3%) were hospitalized twice for clinical relapse. Median follow-up of the study children was 6.7 months (IQR 3.3-8.9 months). Two children (1.6%) developed mild sequelae (angular deformity).

Discussion
Availability of local data is important in order to optimize local therapeutic protocols [10]. To our knowledge this is one of the largest studies on paediatric AHOM performed in Europe. Similar cohort studies have been previously conducted in Spain [14], and France [21], as well as in other extra-European countries including Israel [22], Thailand [23], Australia [3], and the United States [24].
Results from these studies highlight differences in local microbiological data which may be, at least partly, influenced not only by geographical variations, but also by the availability of sophisticated microbiological/PCR assays. As an example, the reported prevalence of Kingella kingae infection ranges from 0% up to 82% children with osteo-articular infection [25,26], probably because its identification is challenging and requires targeted aerobic blood culture vials or real-time PCR technique.
Our study confirms epidemiological data previously well described in literature [5]: AHOM is more commonly reported in males and in young children, it more frequently involves the lower limb and long bones, and the patient's history is commonly positive for a recent trauma or a febrile episode. In our dataset fever at admission was present only in 45% of children. This finding is in accordance with that one reported by Dartnell et al. [17] who observed fever as the presenting symptom only in 61.7% of children, while pain and swelling and erythema where present in 81.1% and 70% of the cases, respectively. We also comfirmed that WBC has a low sensitivity compared to ESR and CRP. Similarly, in other studies, leukocytosis has been reported only in 36% of children, but increased ESR was observed in 91% of children and CRP in 81% of them [27]. As expected, we observed a low sensitivity of X-ray imaging [28] and US [4].
Microbiological identification of the causative pathogen in our dataset was consistent with previous results [14]: a microorganism was identified bin more than one third of cases. The sensitivity of blood and pus culture was 32.4% and 46.4%, respectively. The sensitivity of the PCR assay was only 3.6% on blood sample and reached 64.0% on pus/biopsy sample, underlying the importance of performing PCR assay on pus/biopsy specimens.
S. aureus was the most commonly identified etiological pathogen, but no MRSA strain was isolated. This result is in contrast with data reported from the United States [8,29] but is consistent with other European findings [14,16,17,30]. It should be noticed that, in our dataset 10.0% S. aureus strains were PVL producers. Both cases were complicated. In Europe PVL producing S. aureus, even if most commonly methicillin sensitive, has been associated with more severe infections, therefore treatment should be aggressive and should include an antibiotic with antitoxin effect (i.e., clindamycin, linezolid, or rifampicin) [9,31]. We observed that commonly prescribed antibiotics in our dataset included glycopeptides, carbapenems, or quinolones or a double therapy with cephalosporin plus one penicillin. Since these regimens are not recommended as first line treatment for AHOM in children, interventions aimed to promote a more appropriate use of antibiotics in our setting are needed.
Our study has several limitations, due to its retrospective nature. Moreover, only 59.5% of the children underwent at least one microbiological investigation before commencing an antibiotic therapy and follow-up was limited to 6 months. Another limitation is that although PCR16S is commonly used in our Center to identify bacterial infection, a specific real time PCR for K. kingae is not available. This may have led to an underestimation of K. kingae infections.

Conclusions
Complications occur frequently in children with AHOM and are strongly related to S. aureus infection. In this study microbiological diagnosis was obtained in over one third of cases. Empiric treatment targeting methicillin-sensitive Staphylococcus aureus seems to be justified by available microbiological data.
Author Contributions: Elena Chiappini, Caterina Camposampiero, and Simone Lazzeri contributed in the acquisition of data, analysis and interpretation of data. Elena Chiappini, Luisa Galli, and Caterina Camposampiero participated in the design of the study and performed the statistical analysis. Elena Chiappini, Maurizio De Martino and Luisa Galli conceived the study and participated in its coordination. All authors read and approved the final version of the present manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.