Amoxicillin-Clavulanic Acid Empirical Oral Therapy for the Management of Children with Acute Haematogenous Osteomyelitis

According to the Guidelines of the European Society of Pediatric Infectious Diseases (ESPID), in low methicillin-resistant Staphylococcus aureus (MRSA) prevalence settings, short intravenous therapy is recommended in uncomplicated cases of acute haematogenous osteomyelitis (AHOM), followed by empirical oral therapy, preferentially with first/second-generation cephalosporin or dicloxacillin or flucloxacillin. However, several practical issues may arise using some of the first-line antibiotics such as poor palatability or adherence problems. Clinical, laboratory and therapeutic data from children with AHOM hospitalized in one Italian Paediatric Hospital between 2010 and 2019 were retrospectively collected and analyzed. The aim of the study was to highlight the extent of the use and the possible role of amoxicillin-clavulanic acid in the oral treatment of children with AHOM. Two hundred and ten children were included. S.aureus was identified in 42/58 children (72.4% of identified bacteria); 2/42 S.aureus isolates were MRSA (4.8%). No Kingella kingae was identified. Amoxicillin-clavulanic acid was the most commonly used oral drug (60.1%; n = 107/178) and it was associated with clinical cure in all treated children. Overall, four children developed sequelae. One (0.9%) sequela occurred among the 107 children treated with amoxicillin-clavulanic acid. Our results suggest that amoxicillin-clavulanic acid might be an option for oral antibiotic therapy in children with AHOM.


Introduction
Acute haematogenous osteomyelitis (AHOM) is the most common musculoskeletal infection in children [1]. In most cases, AHOM is the consequence of hematogenous diffusion of a bacterial pathogen, commonly occurring in children under five years of age and in males [2]. Other risk factors for AHOM include history of recent trauma, recent febrile episodes or upper respiratory tract infections, prematurity, congenital or acquired immunodeficiency, or sickle cell disease [3]. Staphylococcus aureus is the most commonly isolated pathogen, accounting for 70-90% of AHOM culture-positive cases [4]. Other microorganisms include Group-A Streptococcus pyogenes, Streptococcus pneumoniae, and Gram-negative rods [5]. Kingella kingae is an emergent pathogen, particularly in children aged <4 years [6]. Community-acquired methicillin-resistant S. aureus (MRSA) infections are increasingly reported in the United States, while low rates are observed in most European countries [7,8]. Moreover, an increasing frequency of Panton-Valentine leukocidin (PVL)-producing S. aureus isolates is reported [9,10]. Since microbiological diagnosis is obtained in about one-third of children, most cases undergo an empirical antibiotic therapy, based on individual clinical features, age, and local epidemiological data. In low/intermediate MRSA prevalence settings, antistaphylococcal penicillin (i.e., oxacillin or flucloxacillin), a cephalosporin, or clindamycin are the recommended first-line treatment. Historically, AHOM was treated with intravenous (IV) antibiotics for several weeks [1]. However, accumulating data support the use of short IV therapy followed by oral antibiotic therapy in uncomplicated cases [11][12][13][14][15] since this regimen appears to be as effective as longer antibiotic IV courses with no increased risk of complications or sequelae. Short IV therapy (about seven days) is now adopted by several centers in Europe [14] and the US [15]. The European Society of Paediatric Infectious Diseases (ESPID) guidelines for Bone and Joint infections [1] recommend switching from cefazolin to cephalexin or cefuroxime, IV clindamycin to oral clindamycin, and IV ceftriaxone + oxacillin to oral equivalents, such as dicloxacillin or flucloxacillin. At the same time, amoxicillin-clavulanic acid is considered an alternative option. However, "thorough evidence is lacking, and the tolerance is worse" [1,[13][14][15]. However, several practical issues may arise. Some antibiotics considered as the first-choice drug by the ESPID guidelines may have low bone penetration (i.e., cephalexin, cefadroxil), poor palatability, adherence issues with four daily administrations (i.e., dicloxacillin, flucloxacillin), or poor tolerability (i.e., risk of a severe rash or Clostridium difficile diarrhea using clindamycin) [16][17][18]. Amoxicillin-clavulanic acid therapy would be more feasible in children. However, only a few studies are available in children with AHOM [4,5,19], and bone penetration varies from 10 to 20% [20][21][22]. The aim of our study was to evaluate the management and outcome of AHOM in a third-level Italian university hospital over ten years, with particular consideration to the possible role of amoxicillin-clavulanic acid in the empirical oral therapy in children with AHOM.

Results
Overall, 210 children were included in the study (Table 1).
Blood culture was performed in 85/210 (40.5%) children and yielded positive results in 36/85 (42.4%) cases. Culture on pus/biopsy was performed in 45/210 (21.4%) cases; one pathogen was isolated in 18/45 (40.0%) cases. PCR assay on a blood sample was performed in 119/210 (56.7%) cases and was positive in 8/119 (6.7%) children. PCR assay on pus/biopsy was performed in 53/210 (25.2%) children and was positive in 30/53 (56.6%) children. A positive result both in PCR and culture assays were obtained very few patients. In particular, in 28 children, both culture and PCR were positive and consistent (24 S. aureus positive), in 25 children, culture was positive but PCR was negative (21 S aureus positive), and, in five, culture was negative and PCR was positive (2 S aureus positive results)".

Imaging Studies
Conventional radiography was performed in 185/210 children (88.1%). A radiographic image compatible with AHOM was obtained in 76/185 (41.1%) children. Magnetic resonance imaging was performed in 197/210 cases (93.8%), and images compatible with a diagnosis of AHOM were obtained in 196/197 cases (99.5%). Computed tomography scan was performed in 16/210 children (7.6%), and images compatible with AHOM diagnosis were observed in 14/16 cases (87.5%). Bone scintigraphy was performed in 8/210 children (3.8%), and results compatible with the diagnosis of osteomyelitis were detected in all cases.

Follow-Up Results
Seventeen out of 210 children (8.1%) were switched to second-line IV therapy for clinical failure, and all of them resolved after the second-line treatment. Treatment failure with the need of rehospitalization was observed in five children (2.4%), and three of these children were treated with amoxicillin-clavulanic acid (3/107; 2.8%). Four children (1.9%) developed sequela including two children with angular deformity and two with limited articular movement. One (0.9%) sequela occurred among the 107 children treated with amoxicillin-clavulanic acid.

Discussion
The present study is a continuation of a previous one published in 2017 [23] and confirm other epidemiological data present in literature [24]. As expected, the microbiological diagnosis was reached in about one third of children, and S.aureus was the most commonly identified pathogen, being about 70% of the total. Moreover, the prevalence of MRSA infection is confirmed to be low in our setting (only 2/42 S. aureus isolates). Prevalence of MRSA strains was in line with those previously reported in Europe. In the United States the incidence of community-acquired MRSA (CA-MRSA) is increased [25], but in Europe CA-MRSA is still uncommon and below 2% [26]. The reason for the low number of positive blood PCR in comparison to the positive blood culture in our study is unclear and more studies are needed at this regard.
The preferred IV antibiotic regimen was oxacillin plus third-generation cephalosporin, adopted in more than 60% of children, while the most commonly prescribed oral antibiotic was amoxicillin-clavulanic acid, which was prescribed in a similar rate (60%). Our data demonstrate the extensive use of amoxicillin-clavulanic acid (>60%). Probably physicians considered its good activity against methicillin-sensitive S. aureus (MSSA), pharmacokinetics/pharmacodynamics profile and the low severe event event rate [16,27]. Interestingly, among children treated with amoxicillin-clavulanic acid, only three out of 107 needed rehospitalization, and only one (0.9%) sequela occurred. Similarly, in a recent Danish study, amoxicillin-clavulanic acid was administered to 82 children (42 children with osteomyelitis [OM] 40 children with SA) for three weeks after IV cefuroxime for at least three days [13]. Relapse was reported in two children (2%) with AHOM, and permanent sequelae were observed in two children (2%) [13] (Table 3). In Filleron et al. study, 176 children were included in the retrospective study: 56 with OM, 95 with SA and 25 with both OM and SA. Oral therapy with amoxicillin-clavulanic acid was administered in 82/176 children (46.6%) [14]. Sequelae were observed in two children (1%) [14] (Table 3). Roul-Levy et al. retrospectively compared two groups of children treated for AHOM (n = 45) [19]. One group was treated with oral amoxicillin-clavulanic acid (without IV therapy), and the other group had received two to four days of IV therapy (Cefamandolo) followed by oral amoxicillin-clavulanic acid for four weeks. Treatment failure was observed in four children (8.9%) (one in the oral group and three in the IV group) [19] (Table 3). Complexly, these results evidence a large use of amoxicillin-clavulanic acid in children with AHOM, although this regimen is not the first-choice therapeutic option for oral therapy, according to the international guideline recommendations. Apparently, this drug seems to be administered to young children more easily than other molecules recommended as first line therapy. Available data suggest good efficacy.
Our study has several limitations due to its retrospective nature. Moreover, 31% of children did not underwent any microbiological tests. Another limitation is that the prevalence of MRSA varies from region to region, and therefore the observed low prevalence may not be the same throughout the nation. Another limitation is that a PCR using specific primers for K. kingae with a higher sensitivity than the broad-range 16S rDNA real-time PCR used in this study was available only starting from 2019 (performed only in 10 children). In addition, K. kingae is a fastidious to grow bacterium. This may explain the fact that no K. kingae was identified in our dataset.
In conclusion, in our dataset amoxicillin-clavulanic acid was used in two-thirds of children and associated with clinical cure and no failure in all of them. This finding is confirmed in larger studies [13,14,19], and suggests that amoxicillin-clavulanic acid might by recommended for oral antibiotic therapy in children with AHOM.

Definitions
AHOM was defined as any bone infection with a period between symptoms onset and diagnosis < two weeks [28,29]. Osteomyelitis was diagnosed in the presence of clinical (fever, swelling, warmth, pain, movements limitations) and radiological signs compatible with AHOM with or without bacterial isolation [26].
AHOM was defined complicated in the presence of sepsis, septic shock, arthritis, cellulitis, sub-periosteal or muscle abscess, deep vein thrombosis [26] pathological fracture, septic emboli or hospitalization in the intensive care unit [30].

Study Design and Population
A retrospective study was conducted in a single tertiary center, evaluating data of all children aged between one month and 18 years referred to the Meyer Paediatric University Hospital, Florence, Italy, from 1 January 2010, to 31 December 2019, with discharge code with the diagnosis of osteomyelitis (ICD code 10 M86.00-86.99), following The World Health Organization International Classification of Disease (WHO ICD-10). Inclusion criteria were: diagnosis of AHOM, as defined above; age between one month and 18 years. Exclusion criteria were: age ≤ 30 days, congenital or acquired immunodeficiency, underlying bone disease, carrier of prosthetic material, open fracture or surgery at the infection's site or in an adjacent area, isolated septic arthritis (SA) (with no evidence of osteomyelitis in the adjacent area), hospital-acquired infections. The study is a continuation of a previous study published in 2017 [23]. Clinical, laboratory and therapeutic data of children with AHOM were collected and analyzed, as previously described [23]. In particular, clinical specimens for cultural analysis were collected into transport vials and inoculated onto an array of culture media suitable for detection of bacteria, and fungi (Columbia blood agar, chocolate agar, Schaedler CNA agar, Shaedler KKV agar, and sabouraud dextrose agar) after an enrichment step at 35 • C in thioglycollate broth and nutrient broth. Cultures were carried out at 35 • C for 48 h under aerobic (Columbia blood agar and sabouraud dextrose agar), 5% CO 2 enriched (chocolate agar) or anaerobic (Schaedler CNA agar and Shaedler KKV agar) conditions. Identification of microbial isolates and antimicrobial susceptibility testing were carried out by the Vitek2 automated system (bioMérieux, Craponne, France).
Broad-range 16S rDNA real-time PCR followed by PCR product sequencing was performed as previously prescribed [23]. Bacterial genomic DNA was extracted from 200 µL of biological samples using the QIAmp DNA Easy Blood & Tissue kit (Qiagen, Venlo, The Netherlands), according to the manufacturer's instructions. RT-PCR for several bacteria (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Kingella kingae) were performed using specific primers and probes, as previously described [23]. All reactions were performed in triplicates. A negative control (no template) and a positive control for each pathogen were included in every run. DNA was amplified in an ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA, brand of ThermoFisher, Waltham, MA, USA) using the following cycling parameters: 95 • C for 10 min followed by 45 cycles of a two-stage temperature profile of 95 • C for 15 s and 60 • C for 1 min.

Conflicts of Interest:
The authors have no conflict of interest to disclose.