This is a 1 year follow-up cohort study. Stump wound healing, postoperative antibiotic therapy will be assessed at the secondary end point of the study.
In this preliminary study we report the incidence of positive cultures in section’s osseous slice biopsy (SOB) taken at the level of major limb amputation and compare the microbiology of positive cultures with percutaneous bone biopsy cultures performed at distal infected site. We also analyze the relationship between diabetes and microbiological culture results.
Population
In this multicenter prospective cohort study we included adult patients who underwent a major amputation of lower limb in the Hospital of Lithuanian University of Health Sciences Kauno klinikos, Kaunas Clinical Hospital and Republican Hospital of Kaunas between 2012 and 2013. The only exclusion criterion was the refusal to participate in the study. Patients had a complete medical history taken, full examination and tests performed. The following data were collected: age, sex, diabetes status and glycemic control, presence of peripheral vascular disease, osteoarthritis, level of infection, context of trauma, level of amputation, immunosuppression, bedridden disability, comorbidity, duration and history of the pathology, reason of the amputation, time from the decision to amputate to surgical procedure, history of previous amputations, total duration of antibiotic therapy before the amputation, antibiotic-free interval before amputation, inflammatory markers (C-reactive protein), renal function (creatinine, estimated glomerular filtration rate), and antibiotic use before and after hospitalization.
Peripheral vascular disease was assumed to be present if 1) dorsal-pedal and posterior-tibial pulses were absent and/or 2) pathological results were evident on Doppler UltraSound, peripheral arteriography and/or 3) there was a previously made diagnosis of peripheral vascular disease by vascular surgeon by using the methods mentioned above. Comorbidity status was evaluated on the presence of cardiac, renal or hepatic insufficiency. Patients were considered to have had an antibiotic-free interval, if they had not received any systemic antibiotic for at least 2 weeks before the amputation. An informed consent form had to be completed by every patient who met the inclusion criteria. The study protocol was approved by Lithuanian Ethics Committee (Number BE-2-23, 2012 May 14).
Specimen collection
Concomitant SOB and percutaneous bone biopsy of the distal site were performed during limb amputation. A new surgical setup and new instruments were used, to try and decrease the likelihood of cross-contamination during surgery. Percutaneous bone biopsy was performed in the surgical room using an 11-gauge biopsy needle inserted through a 5–10-mm skin incision at least 20 mm from the ulcer periphery, to avoid contamination by the colonizing flora, following the methodology described in the literature [2]. During the amputation, in the surgical room we performed SOB at the level of major limb amputation from the amputated limb part using Liston bone cutting forceps and removing the bone fragments (cortical bone and bone marrow for each biopsy). Three bone fragments were obtained for both SOBs and percutaneous distal biopsies, two of which underwent the microbiological assessment, the rest bone fragment - histopathological assessment.
Microbiological assessment
Specimens were immediately placed into Amies transport medium (Brescia, Italy), brought to the microbiology laboratory within 1 h after sampling. The samples were inoculated directly into 5 % sheep blood agar (BBL, USA), chocolate agar (BBL, USA), Mac Conkey agar plates (Oxoid, UK), Schaedler agar (BBL, USA) and thioglycollate broth. Sheep blood and chocolate agar plates were incubated at 35 °C in an atmosphere containing 5 % CO2 and Mac Conkey agar plates – at 35 °C for 18–24 h. If culture was negative at the first observation, sheep blood and chocolate agar plates were re-examined after a second 24 h incubation. Thioglycollate broth was incubated at 35 °C temperature for 5 days. Schaedler agar was incubated at 35 °C in an anaerobic workstation Bug Box (UK) for 2 weeks. Maldi-TOF-MS (BRUKER) mass spectrometry was used for microorganism identification. Analysis of the number of colony-forming units per culture was performed by means of a counting frame for bone biopsy cultures. A positive culture was defined as the identification of at least 1 bacteria not belonging to the skin flora, at least 2 bacteria belonging to the skin flora (CoNS (coagulase negative staphylococci), Corynebacterium spp) with the same antibiotic susceptibility profiles or the same bacteria belonging to the skin flora in two different sites. A doubtful culture was defined as the identification of one bacteria belonging to the skin flora in one site. The dominant pathogen required at least a three times higher growth in bone cultures than other microorganisms. All positive cultures were frozen at −60 °C for further research. The isolated pathogens from SOB and distal site biopsy, which happened to be the same species were compared using genotyping (pulsed field gel electrophoresis) to confirm the same strain.
Histopathological assessment
Bone fragment was fixed in 4 % paraformaldehyde, decalcified in 10 % HCl and embedded in paraffin for histological analysis. 3-μm thick sections were made, stained with hematoxylin-eosin.
Histopathological findings for the signs of infection were evaluated. Histopathological findings in bone specimens were defined as acute osteomyelitis when necrosis, destroyed bone and infiltrations of polymorphonuclear granulocytes at cortical sites and inside the bone marrow were present. Congestion or thrombosis of medullary or periosteal small vessels was also a frequent finding. It was defined as chronic osteomyelitis when there was destroyed bone and infiltrations of lymphocytes, histiocytes and/or plasmatic cells at cortical sites and inside the bone marrow. All cases of osteomyelitis exhibited areas of fibrosis in variable forms and medullar oedema.
Statistical analyses
Fisher’s exact test and Student’s test were used to compare the populations and microbiological results, all the variables had normal distribution. The statistical significance level was set at P < 0.05.