Concordance between culture, Molecular Culture and Illumina 16S rRNA gene amplicon sequencing of bone and ulcer bed biopsies in people with diabetic foot osteomyelitis.

BACKGROUND: In clinical practice the diagnosis of diabetic foot osteomyelitis (DFO) relies on cultures of bone or ulcer bed (UB) biopsies, of which bone biopsy is reference standard. The slow growth or fastidious nature of some bacteria, hamper expeditious detection and identification. Rapid molecular techniques may solve both issues, but their additional value for everyday practice is unknown. We investigated the concordance between conventional culture, the molecular techniques Molecular Culture (MC), and illumina 16S rRNA gene amplicon (16S) sequencing in people with DFO. METHODS: In the BeBoP trial, bone and UB biopsies were obtained from people with DFO who visited Amsterdam UMC. These biopsies were analysed using 1) conventional culture, 2)MC, a rapid broad range PCR analysing the 16S-23S ribosomal-interspace-region, and 3) 16S sequencing, and evaluated concordance among these techniques. RESULTS: We analysed 20 samples (11 bone and 9 UB) of 18 people. A total of 84 infectious agents were identified, 45 (54%) by all techniques, an additional 22 (26.5%, overall 80.5%) by both MC and 16S, and the remaining 16 species by culture and MC or 16S, or by a single method only. MC and 16S identified anaerobes not detected by culturing in 5 samples, and the presence of bacteria in 7 of 8 culture-negative (6 bone, 2 UB) samples. CONCLUSION: The high level of concordance between MC and 16S and the additional ability of molecular techniques to detect various bacteria not detected by culturing opens up prospects for routine use of fast molecular techniques, in clinical settings including DFO. TRIAL REGISTRATION: The BeBoP trial is retrospectively registered on 05-03-2019 in Netherlands Trial Register: NL 7582.

Cold plasma treatment is safe for diabetic foot ulcers and decreases Staphylococcus aureus bacterial load.

AIM: Cold atmospheric plasma (CAP) has antimicrobial properties. We studied the safety of a novel CAP device (PLASOMA prototype; Plasmacure, The Netherlands) that is simple to use and could be applied at a patient's home for the treatment of diabetic foot ulcers (DFUs). Secondary objectives were to investigate the effect of CAP on bacterial load and on ulcer size. METHOD: We included subjects with non-infected, superficial DFUs and treated them with CAP on a daily basis for 10 days. The primary endpoint was the occurrence of serious adverse device effects (SADE). We defined safety as: ≤10% of patients experiencing a SADE other than infection (non-infectious SADE), and ≤60% of patients developing infection of the foot (infectious serious adverse event (SAE)). RESULTS: We enrolled 20 patients. No SADE occurred, but three infectious SAEs occurred at the site of application within one month of treatment; three SAEs unrelated to treatment occurred, and 55% of subjects reported transient mild adverse device effects. Staphylococcus aureus bacterial load decreased directly after CAP application (p=0.01). The mean decrease of ulcer surface area was 43% (95% confidence interval: 20.2%-65.9%). CONCLUSION: CAP treatment in DFUs was safe and well tolerated. Ulcer size and Staphylococcus aureus colonisation decreased during treatment.

The association between bacteria and outcome and the influence of sampling method, in people with a diabetic foot infection.

PURPOSE: Different bacteria lead to divers diabetic foot infections (DFIs), and some bacteria probably lead to higher amputation and mortality risks. We assessed mortality and amputation risk in relation to bacterial profiles in people DFI and investigated the role of sampling method. METHODS: We included people (> 18 years) with DFI in this retrospective study (2011-2020) at a Dutch tertiary care hospital. We retrieved cultures according to best sampling method: (1) bone biopsy; (2) ulcer bed biopsy; and (3) swab. We aggregated data into a composite determinant, consisting of unrepeated bacteria of one episode of infection, clustered into 5 profiles: (1) Streptococcus and Staphylococcus aureus; (2) coagulase-negative Staphylococcus, Cutibacterium, Corynebacterium and Enterococcus; (3) gram-negative; (4) Anaerobic; and (5) less common gram-positive bacteria. We calculated Hazard Ratio's (HR's) using time-dependent-Cox regression for the analyses and investigated effect modification by sampling method. RESULTS: We included 139 people, with 447 person-years follow-up and 459 episodes of infection. Sampling method modified the association between bacterial profiles and amputation for profile 2. HR's (95% CI's) for amputation for bacterial profiles 1-5: 0.7 (0.39-1.1); stratified analysis for profile 2: bone biopsy 0.84 (0.26-2.7), ulcer bed biopsy 0.89 (0.34-2.3), swab 5.9*(2.9-11.8); 1.3 (0.78-2.1); 1.6 (0.91-2.6); 1.6 (0.58-4.5). HR's (95% CI's) for mortality for bacterial profiles 1-5: 0.89 (0.49-1.6); 0.73 (0.38-1.4); 2.6*(1.4-4.8); 1.1(0.58-2.2); 0.80(0.19-3.3). CONCLUSIONS: In people with DFI, there was no association between bacterial profiles in ulcer bed and bone biopsies and amputation. Only in swab cultures, low-pathogenic bacteria (profile 2), were associated with a higher amputation risk. Infection with gram-negative bacteria was associated with a higher mortality risk. This study underlined the possible negative outcome of DFI treatment based on swabs cultures.

Using a BonE BiOPsy (BeBoP) to determine the causative agent in persons with diabetes and foot osteomyelitis: study protocol for a multicentre, randomised controlled trial.

BACKGROUND: Diabetic foot osteomyelitis (DFO) poses a major disease burden. It can generally be treated with long-term antibacterial therapy. International guidelines recommend to base antibacterial therapy choices on percutaneous bone biopsy culture, while in practice, therapy is frequently based on (less invasive) ulcer bed cultures. It is currently unknown if treatment outcomes of DFO differ depending on the chosen diagnostic strategy. METHODS: The BeBoP trial is a multicentre; randomised controlled; physician-, researcher- and subject-blinded; clinical trial comparing two diagnostic strategies in persons with DFO. Culture-directed antibacterial therapy will be based on either percutaneous bone biopsy culture results (intervention group) or ulcer bed biopsy culture results (comparison group). We will enrol 80 subjects with diabetes mellitus (≥ 18 years) and DFO, and we will use block randomisation stratified per centre to randomise them in a 1:1 allocation. The primary outcome is remission of DFO 12 months after enrolment. The secondary outcomes are the time to remission, signs of inflammation or ulceration at the primary location of infection at 6 and 12 months, microbiological and molecular profiles of culture outcomes, surgical interventions including amputation, total antibacterial therapy duration, infection-free survival days, adverse events, quality of life and survival. We will compare the outcomes by intention-to-treat and per-protocol analysis. DISCUSSION: We aim to compare clinical remission in persons with DFO treated with antibacterial therapy based on either percutaneous bone biopsy culture results or ulcer bed biopsy culture results. TRIAL REGISTRATION: Netherlands Trial Register NL 7582 . Registered on 05 March 2019.

Specialised orthotic care to improve functioning in adults with neuromuscular disorders: protocol of a prospective randomised open-label blinded end-point study.

INTRODUCTION: People suffering from leg muscle weakness caused by neuromuscular disorders (NMDs) are often provided with leg orthoses to reduce walking problems such as increased walking effort, diminished walking speed, reduced balance and falls. However, evidence for the effectiveness of leg orthoses to improve walking in this patient group is limited and there is an absence of standardised practice in orthotic prescription. In 2012 a Dutch multidisciplinary guideline was developed aimed to standardise the orthotic treatment process in NMD. Although application of the guideline in expert centres (specialised orthotic care) seems beneficial regarding clinical effectiveness, larger studies are necessary to confirm results and investigate cost-effectiveness. Therefore, this study aims to examine the effectiveness and cost-effectiveness of specialised orthotic care compared with usual orthotic care in adults with slowly progressive NMD. METHODS AND ANALYSIS: A prospective randomised open-label blinded end-point study will be performed, in which 70 adults with slowly progressive NMD are randomly assigned to specialised orthotic care (intervention) or usual orthotic care (control). Outcome measures are assessed at baseline and at 3 and 6 months follow-up. The primary endpoints are gross walking energy cost (J/kg/m) assessed during a 6 min walk test and achievement of personal goals, measured with the Goal Attainment Scale. Secondary endpoints include walking speed, gait biomechanics, stability, physical functioning, falls and fear of falling, perceived fatigue and satisfaction. For the economic evaluation, societal costs and health-related quality of life will be assessed using cost questionnaires and the 5-Level version of EuroQol 5 Dimension, retrospectively. ETHICS AND DISSEMINATION: The study is registered in the Dutch trial register (NL 7511) and the protocol has been approved by the Medical Ethics Committee of the Academic Medical Center in Amsterdam. Results will be presented at national and international scientific conferences and disseminated through peer-reviewed journals and media aimed at a broad audience including patients.