Use of next generation sequencing to detect biofilm bacteria in a patient with pedicle screw loosening after spine surgery: a case report

Title: Use of next generation sequencing to detect biofilm bacteria in a patient with pedicle screw loosening after spine surgery: a case report
Yijuan Xu1, Trine Rolighed Thomsen1,2, Jan Lorenzen1, Kathrin Chamaon3, Per Trobisch4, Steffen Drange3
1. Danish Technological Institute, Aarhus, Denmark
2. Center for Microbial Communities, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark
3. Otto-von-Guericke University Magdeburg, Department of Orthopedic Surgery, Magdeburg, Germany
4. Eifelklinik St. Brigida, Simmerath, Germany

Aim: ”Hidden deep implant-related infection is believed to be linked to pedicle screw loosening after spine surgery. Low-grade bacterial infection can be hard to diagnose and may be undetected by conventional culture based methods. Next generation sequencing (NGS) could help to uncover hidden bacterial infections as a possible cause for implant loosening. This case report describes the use of NGS in the diagnostic work-up of a patient with pedicle screw loosening after spine surgery.”

Method: ”A 60 y/o male had to undergo revision spine surgery for pedicle screw loosening and adjacent segment disease 3 years after primary surgery performed because of osteochondrosis L3-S1 and spinal canal stenosis. In addition, the patient had left and right knee prostheses, and suffered from arterial hypertension and hyperuricemia. At revision surgery, samples from pedicle screw canals were collected for microbiological diagnosis. The removed screws were sonicated, and sonication fluids were cultured and sent for molecular analysis. For culturing Schaedler agar, thioglycollate medium and bouillon bottles under aerobic and anaerob conditions were used. Upon growth in bouillon differentiation on Schaedler agar followed. Isolates were identified by MALDI-TOF .
For the molecular detection, DNA was extracted using MolYsis complete5 (Molzym, Germany). The V1-3 region of 16S rRNA gene was PCR-amplified with bacterial primers 27F and 534R (30 cycles) and paired-end sequenced on Miseq DNA sequencer (v3 chemistry, 2×300 bp). Paired-end reads were trimmed (trimmomatic v.0.32) and merged (FLASH v.1.2.11). The reads were screened for potential PhiX contamination (USEARCH v.7.0.1090), clustered into operational taxonomic units (sequence identity ≥ 97%) using USEARCH, and subsequently classified using the RDP classifier with the MiDAS database (v.1.20).”


Results: “Clinically there were no signs of local or general infection. Serum parameters were normal (C-reactive protein 0.7 mg/L, WBC 6.2 Gpt/L) at revision surgery. No other infectious foci were noticed. Histology showed no signs of infection. Routine microbial culturing was negative. However, long-term cultures detected Propionibacterium acnes. Molecular analysis revealed presence of Corynebacterium species. The patient received oral clindamycine 600 mg 3 times/day for 6 weeks; 10 months later he had no problems from the lumbar spine. Follow-up CT scan showed no recurrence of pedicle screw loosening.”

Conclusions: ”Molecular analysis using NGS may be useful to detect hidden low grade infection especially in the setting of pedicle screw loosening after spine surgery. It could be a crucial additional method in combination with routine clinical microbiology to improve our understanding of implant loosening.”