Molecular diagnosis of polymicrobial brain abscesses with 16S-rDNA-based next-generation sequencing.

OBJECTIVES: Brain abscesses lead to high mortality despite antibiotic and surgical treatment. Identification of causative bacteria is important to guide antibiotic therapy, but culture-based methods and molecular diagnostics by Sanger sequencing of 16S PCR products are hampered by antibiotic treatment and the often polymicrobial nature of brain abscesses. We have applied 16S-rRNA-based next-generation sequencing (NGS) for metagenomic analysis of intracranial abscess (brain and epidural) and meningitis samples. METHODS: Seventy-nine samples from 54 patients with intracranial abscesses or meningitis were included. DNA was subjected to 16S PCR. Amplicons were analysed with the Illumina MiSeq system, sequence reads were blasted versus the NCBI 16S bacterial database and analysed using MEGAN software. Results were compared to those of gram-staining, culture and Sanger sequencing. RESULTS: The NGS workflow was successful for 51 intracranial abscesses (46 brain and five epidural) and nine meningitis samples. Inclusion of (mono)bacterial meningitis samples allowed us to establish a cut-off criterion for the exclusion of contaminating sequences. In total 86 bacterial taxa were identified in brain abscesses by NGS, with Streptococcus intermedius and Fusobacterium nucleatum as most prevalent species; Propionibacterium and Staphylococcus spp. were associated with epidural abscesses. NGS identified two or more bacterial taxa in 31/51 intracranial abscesses, revealing the polymicrobial nature of these infections and allowing the discrimination of up to 16 bacterial taxa per sample. CONCLUSION: These results extend earlier studies showing that NGS methods expand the spectrum of bacteria detected in brain abscesses and demonstrate that the MiSeq platform is suitable for metagenomic diagnostics of this severe infection.

An in vitro analysis of the size and shape of cryolesions for facet joint denervation.

OBJECTIVES: Lumbar facet joint syndrome (LFJS) is the cause of lower back pain in 15-54% of the patients. Clinical studies of cryotherapy for LFJS have reported promising outcomes. However, few studies have focused on the technical aspects of cryoneurolysis for LFJS. The aim of the study was to determine the size and shape of cryolesions in vitro and to determine how they are affected by the duration of freezing, size of the cryoprobe and distance and angulation to an osseous boundary layer. MATERIALS AND METHODS: Two different cryolesion generators were used. Cryolesions were generated in tempered physiologic NaCl solution in the vicinity of an osseous surface. The size of the cryoprobes, duration of freezing, distance to the bone surface and angulation of the probe were studied. Cryolesions were recorded with a video camera during their emergence. Images at distinct time points were analysed using digital image processing software. RESULTS: The probe size, the system in use and the duration of the freezing cycle were the main determinants for the size of the cryolesion. The vicinity of the osseous boundary resulted in a modest increase in the size of the cryolesion. Angulation of the cryoprobe towards the osseous boundary is of minor importance for the size of the contact area to the nerve. CONCLUSION: For cryoneurolysis of LFJS, duration of freezing, temperature and probe size are the main determinants of lesion size and thus the probability of success of the procedure. A tangential approach of the probe is not essential.