Comparative Analysis of Several Detection Methods for Mycobacterium Tuberculosis in Paraffin-Embedded Tissues

Abstract Objective: To evaluate the diagnostic value of real-time quantitative PCR (RT-qPCR) and metagenomics next-generation sequencing (mNGS) in detecting Mycobacterium tuberculosis (MTB) in paraffin-embedded tissue samples. Methods: Twenty paraffin-embedded tissue samples diagnosed with chronic granulomatous inflammation were selected. RT-qPCR and mNGS were performed on these samples, and Ziehl-Neelsen acid-fast staining was conducted for comparison. Results: Among the 20 specimens, 10 cases were identified as Mycobacterium tuberculosis complex (MTBC) by both RT-qPCR and mNGS, with a concordance rate of 100%. Acid-fast staining results differed from both molecular methods in four samples. Among the 10 RT-qPCR-negative samples, mNGS detected no mycobacteria in 6 cases, while non-tuberculous mycobacteria (NTM) were identified in 4 cases (including Mycobacterium wolinskyi, Mycobacterium gallinarum, and Mycobacterium kansasii). Acid-fast staining results differed from mNGS in one sample. Conclusion: Compared to acid-fast staining, RT-qPCR demonstrated higher sensitivity in detecting MTBC and can be used as a routine tool for rapid detection of MTB DNA in paraffin-embedded tissues. mNGS, when economically feasible, can serve as an important method for detecting non-tuberculous mycobacteria or other pathogens.