Molecular Identification of Infectious Etiologies in Necrotizing Granulomatous Lung Lesions Using FFPE Tissue: An Autopsy-Based Study

Abstract

Necrotizing granulomatous lung lesions have diverse infectious etiologies, and histology with Ziehl–Neelsen (ZN) staining is often insufficient for definitive diagnosis, particularly in formalin-fixed paraffin-embedded (FFPE) tissue. Among 2025 consecutive autopsy cases examined between 2011 and 2025, five cases showed necrotizing granulomatous lung lesions. DNA extracted from lesion-directed, manually trimmed FFPE tissue underwent PCR analysis targeting IS6110 and hsp65. Additional rpoB-based restriction fragment length polymorphism (RFLP) analysis was performed in suspected nontuberculous mycobacterial (NTM) infections, while one case with suspected parasitic infection underwent cox1 PCR and sequencing. Molecular analysis identified Mycobacterium tuberculosis complex (MTBC) in two cases, NTM in two cases, and Dirofilaria immitis in one case. rpoB-based RFLP demonstrated distinct patterns between the two NTM cases. ZN staining showed abundant bacilli in one MTBC case, rare bacilli in another, and negative results in the remaining cases. Lesion-directed manual trimming enabled successful amplification in all cases despite the use of archival FFPE tissue. Multi-target PCR analysis of lesion-enriched FFPE tissue provides a practical approach for etiological evaluation of necrotizing granulomatous lung lesions and may improve diagnostic characterization of paucibacillary or late-stage infectious lesions.