Prevalence and Genotypes of Mycobacteria Species in Formalin-Fixed Paraffin-Embedded Tissue Samples in a Tertiary Laboratory, Northern Pretoria, South Africa

Background/Objectives: Granulomatous inflammation in tissue biopsies presents a diagnostic challenge, particularly in differentiating between Mycobacterium tuberculosis complex (MTB) and non-tuberculous mycobacteria (NTM) in high-HIV-prevalence areas. This study evaluates the prevalence and species distribution of mycobacteria in formalin-fixed, paraffin-embedded (FFPE) tissues from patients with granulomatous inflammation and correlates these findings with demographic and clinical data. Methods: Conducted at the NHLS-Dr George Mukhari Tertiary Laboratory, the retrospective descriptive study analysed 37 FFPE samples (January 2019–December 2020) after obtaining ethical approval and collecting clinicopathological data. Histopathological assessments using H&E and Ziehl–Neelsen (ZN) stains were carried out, and mycobacterial species were identified utilising multiplex PCR with reverse hybridisation (GenoType MTBC/CM/AS assays). Results: The cohort had an average age of 38.59 years, with a predominance of females (59.5%) and a high HIV positivity rate (62.2%). The most common biopsy site was lymph nodes (32.4%). All samples exhibited necrotising granulomas, but only 8.1% displayed acid-fast bacilli (AFB) on ZN staining. Molecular testing revealed mycobacterial DNA in 75.7% of samples, identifying NTM alone in 50.0%, MTB alone in 10.7%, and both in 28.6%. M. fortuitum was the most frequently recognised species. Importantly, there was no significant association between mycobacterial detection and HIV status (p > 0.05). Conclusions: The findings demonstrate that molecular methods substantially improve mycobacterial detection rates and speciation. This underscores the diagnostic limitations of ZN staining and highlights the importance of molecular diagnostics for accurately identifying bacterial aetiology, crucial for appropriate antimicrobial therapy in immunocompromised patients.