Detection of Microbial Presence in Post-Mortem Formalin-Fixed Paraffin

Date of Award

6-2026

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Brian Balin, PhD

Second Advisor

Minal Mulye, PhD

Third Advisor

Jocelyn Lippman-Bell, PhD

Abstract

Introduction: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β plaques and neurofibrillary tangles. Increasing evidence suggests that microbial pathogens may contribute to AD pathogenesis through persistent immune activation and inflammatory signaling following entry into the brain. However, the presence and spatial distribution of microbial organisms within vulnerable brain regions remain unclear. This study investigated the detection and regional localization of Chlamydia pneumoniae (Cpn), Borrelia burgdorferi (Bbg), and Mycobacterium avium subspecies paratuberculosis (MAP) in post-mortem AD/dementia and non-AD brain tissue using RNAscope in situ hybridization.

Materials & Methods: Formalin-fixed paraffin-embedded (FFPE) human brain tissue sections from 20 total cases (10 AD/dementia and 10 non-AD controls) were analyzed. Three anatomically vulnerable brain regions associated with memory and higher-level cognitive function were examined: middle hippocampus, frontal cortex, and amygdala. RNAscope 2.5 HD Red assays with organism-specific probes targeting microbial RNA were performed alongside positive, negative, and no-probe controls to confirm assay specificity and RNA integrity. Twenty randomized fields per tissue section were evaluated using semi-quantitative scoring analysis. Initial image analysis and scoring procedures were performed under blinded conditions prior to unblinding of tissue groups.

Results: Positive RNAscope labeling for MAP, Cpn, and Bbg was identified within the middle hippocampus, frontal cortex, and amygdala. Signal intensity and distribution varied between tissue cases, organisms, and brain regions. Frontal cortex tissue sections generally demonstrated the strongest and most consistent labeling patterns across iv analyzed organisms, whereas Cpn-associated labeling appeared comparatively sparse relative to MAP and BBg labeling. Labeling was observed in both AD/dementia and non-AD control tissues, with heterogeneous microbial-associated labeling patterns identified across all analyzed tissue groups and anatomical regions.

Discussion: This study demonstrates the successful application of RNAscope in archival FFPE human brain tissue for the detection and semi-quantitative assessment of microbial RNA. The detection and localization of MAP, Cpn, and Bbg within anatomically vulnerable brain regions supports continued investigation into microbial-associated neuroinflammation and the potential role of infection in Alzheimer’s disease progression.

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