Deep 16S rDNA Sequencing of Chronic Subdural Hematomas Suggests Involvement of Bacterial Infection in Recurrences

BACKGROUND AND OBJECTIVES: Chronic subdural hematoma (CSDH) is an encapsulated blood collection between the dura mater and arachnoid that often follows mild head trauma. It involves pronounced inflammation and angiogenesis, and recurrence remains common despite advances in surgical management. Subclinical bacterial involvement has been hypothesized as a potential factor in these recurrent cases. We aimed to determine whether bacteria are present in CSDH outer membranes and to define accompanying histological and transcriptomic changes. METHODS: A total of 39 Formalin-fixed paraffin-embedded outer-capsule specimens from 19 patients (17 primary, 22 recurrent) underwent: (1) ultra-deep Nanopore 16S rDNA profiling; (2) histomorphology with Gram staining, lipopolysaccharide immunohistochemistry and quantitative immune-cell counts; and (3) RNA-seq of 3 matched primary-recurrent pairs, followed by differential-expression and pathway analysis. RESULTS: Gram staining revealed bacteria in 2 recurrent samples (5%) from a single patient, whereas lipopolysaccharide immunohistochemistry was negative in all cases. Notably, 16S rDNA sequencing detected bacterial DNA exclusively in recurrent lesions (6 samples from 4 patients), with none in primary cases ( P = .02, χ 2 test). The identified bacterial genera included Staphylococcus , Neisseria, Prevotellamassilia , and Paracoccus . Histopathological evaluation showed no significant differences in eosinophils, myeloperoxidase-positive cells, CD3-positive T-cells, or CD20- positive B-cells when comparing primary with recurrent lesions, or infected to uninfected membranes. Unsupervised clustering of matched primary and recurrent CSDH samples revealed distinct transcriptomic profiles, identifying 184 differentially expressed genes (including consistent Toll-like receptor 4 upregulation) and highlighting pathways related to development, protein biosynthesis, and wound healing. CONCLUSION: These findings suggest that bacterial DNA is present in a subset of recurrent CSDH, suggesting that bacteria could be involved in recurrences. Further research with larger cohorts is needed to determine whether antimicrobial or anti-inflammatory strategies might help reduce recurrence.