Bacterial Genotyping of Central Nervous System Tuberculosis in South Africa: Heterogenic Mycobacterium tuberculosis Infection and Predominance of Lineage 4

Abstract

Tuberculous meningitis (TBM), the most severe extrapulmonary manifestation of tuberculosis, is caused by the pathogen Mycobacterium tuberculosis The M. tuberculosis complex includes seven lineages, all described to harbor a unique geographical dissemination pattern and clinical presentation. In this study, we set out to determine whether a certain M. tuberculosis lineage demonstrated tropism to cause TBM in patients from Cape Town, South Africa. DNA was extracted from formalin-fixed paraffin-embedded central nervous system (CNS) tissue from a unique neuropathological cohort of 83 TBM patients, collected between 1975 and 2012. M. tuberculosis lineages 1, 2, 3, and 4 were determined using an allele-specific PCR and Sanger sequencing. Of the 83 patient specimens tested, bacterial characterization could be performed on 46 specimens (55%). M. tuberculosis lineage 4 was present in 26 patient specimens (56%), and non-lineage 4 was identified in 10 cases (22%). Moreover, genomic heterogeneity was detected in the CNS specimens of 7 adults and 3 children. We could show that infection of the CNS is not restricted to a single M. tuberculosis lineage and that even young children with rapid progression of disease can harbor more than one M. tuberculosis lineage in the CNS.

Keywords: CNS tuberculosis; Mycobacterium tuberculosis lineage; childhood tuberculosis; genomic heterogeneity; genotyping; mixed infection; tuberculous meningitis.

FIG 1

Distribution of lineages between 1975 and 2012. Identification of lineage 4, non-lineage 4, or heterogenic infection in patients <5 years of age (small blue circles), ages 5 to 14 years (medium green circles), and >14 years of age (large red circles) per year between 1975 and 2012. Number indicates multiple patients at a specific time point for the youngest group. No association was found between presence of a specific lineage and year of admission.

FIG 2

Neuropathological findings in three children with tuberculous meningitis and heterogenic infection. Characteristics of the three cases presented are specified in Table 2. (A) Case 1, medulla oblongata; multiple intraparenchymal granulomas. H&E, ×40 magnification. (B) Case 1, medulla oblongata; granulomas with lymphocytes (chronic inflammation). H&E, ×100 magnification. (C) Case 1, leptomeninges; lymphocytes (chronic inflammation), no granulomas present. H&E, ×100 magnification. (D) Case 2, leptomeninges; poorly formed granulomatous inflammation. H&E, ×100 magnification. (E) Case 2, leptomeninges; lymphocytes (open arrow, chronic inflammation) and macrophages (closed arrow). H&E, ×400 magnification. (F) Case 2, leptomeninges; fibrosis and vascular proliferation (arrow). Reticulin silver stain, ×100 magnification. (G) Case 3, brain parenchyma and leptomeninges; poorly formed granuloma (closed arrow) and foreign body giant cell (open arrow). H&E, ×100 magnification. (H) Case 3, brain parenchyma and leptomeninges; Langhans giant cell (arrow). H&E, ×400 magnification. (I) Case 3, brain parenchyma and leptomeninges; fibrosis (arrow) and vascular proliferation. Reticulin silver stain, ×100 magnification. (J) Case 3, brain parenchyma; infarct. H&E, ×40 magnification. (K) Case 3, brain parenchyma; infarct with vascular proliferation. Reticulin silver stain, ×40 magnification. (L) Case 3, brain parenchyma; infarct with vascular proliferation (arrows). Reticulin silver stain, ×100 magnification.