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

Abstract

Abstract 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.

Keywords

• Diabetic foot osteomyelitis
• Bone and ulcer bed biopsy
• Culture
• Molecular culture
• 16S rRNA gene amplicon sequencing