{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T04:05:32Z","timestamp":1784261132632,"version":"3.55.0"},"reference-count":21,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T00:00:00Z","timestamp":1784246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>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\u2013December 2020) after obtaining ethical approval and collecting clinicopathological data. Histopathological assessments using H&amp;E and Ziehl\u2013Neelsen (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 &gt; 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.<\/jats:p>","DOI":"10.3390\/diagnostics16142234","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T03:12:38Z","timestamp":1784257958000},"page":"2234","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Prevalence and Genotypes of Mycobacteria Species in Formalin-Fixed Paraffin-Embedded Tissue Samples in a Tertiary Laboratory, Northern Pretoria, South Africa"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6999-6836","authenticated-orcid":false,"given":"Moshawa C.","family":"Khaba","sequence":"first","affiliation":[{"name":"Department of Anatomical Pathology, Dr George Mukhari Academic Laboratory, National Health Laboratory Service, Ga-Rankuwa 0208, South Africa"},{"name":"Department of Anatomical Pathology, Sefako Makgatho Health Sciences University, Pretoria 0204, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3820-1882","authenticated-orcid":false,"given":"Lubabalo","family":"Ndam","sequence":"additional","affiliation":[{"name":"Department of Anatomical Pathology, Sefako Makgatho Health Sciences University, Pretoria 0204, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7666-8416","authenticated-orcid":false,"given":"Ezintle","family":"Mhlaba","sequence":"additional","affiliation":[{"name":"Department of Anatomical Pathology, Sefako Makgatho Health Sciences University, Pretoria 0204, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6653-1469","authenticated-orcid":false,"given":"Lucia N.","family":"Mhlongo","sequence":"additional","affiliation":[{"name":"Department of Anatomical Pathology, Dr George Mukhari Academic Laboratory, National Health Laboratory Service, Ga-Rankuwa 0208, South Africa"},{"name":"Department of Anatomical Pathology, Sefako Makgatho Health Sciences University, Pretoria 0204, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9500-2815","authenticated-orcid":false,"given":"Ndivhuho A.","family":"Makhado","sequence":"additional","affiliation":[{"name":"Department of Microbiological Pathology, Sefako Makgatho Health Sciences University, Pretoria 0204, South Africa"},{"name":"Department of Medical Microbiology, Dr George Mukhari Academic Laboratory, National Health Laboratory Services, Ga-Rankuwa 0208, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1099\/mic.0.000601","article-title":"Microbe profile: Mycobacterium tuberculosis: Humanity\u2019s deadly microbial foe","volume":"164","author":"Gordon","year":"2018","journal-title":"Microbiology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1080\/00365540500444652","article-title":"Non-tuberculous mycobacterial infections","volume":"38","author":"Petrini","year":"2006","journal-title":"Scand. J. Infect. Dis."},{"key":"ref_3","unstructured":"World Health Organization (2023). Global Tuberculosis Report 2023, World Health Organization. Report 20-23."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"649","DOI":"10.3346\/jkms.2016.31.5.649","article-title":"Diagnosis and treatment of nontuberculous mycobacterial lung disease","volume":"31","author":"Kwon","year":"2016","journal-title":"J. Korean Med. Sci."},{"key":"ref_5","first-page":"379","article-title":"Comparison of Ziehl-Neelsen stain and TB-PCR on detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues of chronic granulomatous inflammation","volume":"37","author":"Cho","year":"2003","journal-title":"Korean J. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1016\/j.jmoldx.2011.02.004","article-title":"Rapid, sensitive, and specific detection of Mycobacterium tuberculosis complex by real-time PCR on paraffin-embedded human tissues","volume":"13","author":"Lee","year":"2011","journal-title":"J. Mol. Diagn."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"a1657","DOI":"10.4102\/SAJHIVMED.v25i1.1657","article-title":"Southern African HIV Clinicians Society guideline on the management of non-tuberculous mycobacteria in people with HIV","volume":"25","author":"Dawood","year":"2024","journal-title":"S. Afr. J. HIV Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1128\/CMR.00042-10","article-title":"HIV and tuberculosis: A deadly human syndemic","volume":"24","author":"Kwan","year":"2011","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"628","DOI":"10.4269\/ajtmh.20-0973","article-title":"Prevalence of nontuberculous mycobacterium infections versus tuberculosis among autopsied HIV patients in Sub-Saharan Africa: A systematic review and meta-analysis","volume":"104","author":"Chiang","year":"2021","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Meghdadi, H., Khosravi, A.D., Ghadiri, A.A., Sina, A.H., and Alami, A. (2015). Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning. Front. Microbiol., 6.","DOI":"10.3389\/fmicb.2015.00675"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1111\/j.1469-0691.2009.03014.x","article-title":"Clinical manifestations of nontuberculous mycobacteria infections","volume":"15","author":"Tortoli","year":"2009","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Negi, S.S., Singh, P., and Sharma, K. (2023). Molecular diagnosis of tuberculosis. Diagnosis of Mycobacterium, Springer Nature.","DOI":"10.1007\/978-981-99-5624-1_6"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1002\/path.1728","article-title":"Modified immunohistological staining allows detection of Ziehl-Neelsen-negative Mycobacterium tuberculosis organisms and their precise localization in human tissue","volume":"205","author":"Ulrichs","year":"2005","journal-title":"J. Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1111\/j.1574-695X.2012.00987.x","article-title":"Diagnosis of extrapulmonary tuberculosis by PCR","volume":"66","author":"Mehta","year":"2012","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1093\/cid\/ciaa1125","article-title":"Treatment of nontuberculous mycobacterial pulmonary disease: An official ATS\/ERS\/ESCMID\/IDSA clinical practice guideline","volume":"71","author":"Daley","year":"2020","journal-title":"Clin. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"913","DOI":"10.3346\/jkms.2005.20.6.913","article-title":"Diagnosis and treatment of nontuberculous mycobacterial pulmonary diseases: A Korean perspective","volume":"20","author":"Koh","year":"2005","journal-title":"J. Korean Med. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ratnatunga, C.N., Lutzky, V.P., Kupz, A., Doolan, D.L., Reid, D.W., Field, M., Bell, S.C., Thomson, R.M., and Miles, J.J. (2020). The Rise of Non-Tuberculosis Mycobacterial Lung Disease. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00303"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e5479","DOI":"10.7717\/peerj.5479","article-title":"Epidemiology of and risk factors for extrapulmonary nontuberculous mycobacterial infections in Northeast Thailand","volume":"6","author":"Chetchotisakd","year":"2018","journal-title":"PeerJ"},{"key":"ref_19","first-page":"e91532","article-title":"Disseminated Extrapulmonary Tuberculosis and Pulmonary Mycobacterium avium Complex Co-infection in a Newly Diagnosed HIV Patient: A Case Report","volume":"17","author":"Lopez","year":"2025","journal-title":"Cureus"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"100464","DOI":"10.1016\/j.ijregi.2024.100464","article-title":"Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures","volume":"13","author":"Opperman","year":"2024","journal-title":"IJID Reg."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"12002","DOI":"10.1038\/s41598-017-12175-z","article-title":"Non-tuberculous Mycobacteria isolated from Pulmonary samples in sub-Saharan Africa\u2014A Systematic Review and Meta Analyses","volume":"7","author":"Okoi","year":"2017","journal-title":"Sci. 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