**3.3 Microbiological diagnostic**

The microbiological diagnostic relies on specimens for culturing the involved pathogens. No expert recommends superficial wound swabs, because there is always an (inconstant) microbiome of multiple organisms in chronically open wounds. These superficial probes are frequently misleading, since they often represent colonizing species or contaminations [12] (unless the swab originates from mere pus). Clinicians should always aim for several deep samples of infected (intraoperative) tissues or bone. An optimal specimen would be deep, infected, and still vital tissue, with or without pus, to catch the anaerobic pathogens [12, 31]. The microbiological gold standard for DFO relies on a bone biopsy, which is also feasible outside of the operating theater; especially in patients with polyneuropathy, who feel almost no pain during the bed-side sampling [25]. The accuracy of the results is increased by taking at least two separate bone probes. If they show the same pathogen, we usually identify the pathogen of DFO [16]. Histology has no widely-accepted criteria for DFO. Characteristic findings are aggregates of inflammatory cells, bone lesions, fibrosis, and/or reactive bone formation. As with other orthopedic infections the results depend on the care with which intraoperative samples are obtained (to avoid contamination) and whether the patient was under active antibiotic therapy [32]. While newer molecular laboratory methods identify more pathogens from DFO, the IWGDF guidelines suggest sticking with conventional culture methods for the firstline identification [33]. This is because of their lower cost, the lack of evidence of any benefit to covering the additional isolates identified and the potential for incurring the adverse effects of unnecessarily broad-spectrum antibiotic therapy. Practically, the only serology with a theoretical use for diagnosing DFO are anti-streptolysin antibodies for beta-hemolytic streptococci. If they are positive [34], the clinicians can (retrospectively) diagnose a streptococcal infection, which might be more useful in acute and severe soft tissue diabetic foot infections than in chronic DFOs.
