A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment for the Degree of Master of Medicine In the branch of Anatomical Pathology Johannesburg 2015 / Background: Mycobacterium tuberculosis (Mtb) poses a major global health problem. According to the World Health Organization, South Africa is a country with one of the highest reported incidence rates of this disease. Key to overcoming this preventable and treatable disease lies in establishing a reliable and rapid diagnostic approach. Aims and Objectives: This study aims to investigate the validity of the mycobacterium polymerase chain reaction (PCR) assay applied to formalin-fixed, paraffin-embedded tissue in which the histology showed granulomatous inflammation with no demonstrable acid-fast bacilli. Methods: A retrospective, cross sectional and non-interventional study was conducted on 121 histopathology cases showing granulomatous inflammation with a negative Ziehl-Neelsen (ZN) stain. The mycobacterium PCR results obtained in these cases were compared against the results of mycobacterium culture obtained from a specimen derived from the same or related site as the biopsy. Results: The mean age of the study population was 35.3 years and the study cohort included 63 males and 58 females. The sensitivity of nested mycobacterium PCR (detecting the 133 base pair product of the heat shock protein 65 kilo Dalton gene), was 64.1% and the specificity was 68.2%. The positive and negative predictive values were 49% and 80% respectively. Twenty six of the 121 cases studied had a false positive result (21.5%). CONCLUSION: There are many factors that may influence the result of a PCR assay and the interpretation thereof. Some of these factors include the inability of the test to distinguish between live and dead bacilli, the high risk of carry over contamination, and the paucibacillary nature of certain samples with an unequal distribution of the few bacilli that may be present. Although the sensitivity and specificity of mycobacterium PCR on paucibacillary, formalin-fixed, paraffin embedded tissue is suboptimal, the interpretation of these results must be performed in conjunction with the overall clinical presentation of the patient. / MT2016

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L'analyse de la structure cristallographique de l'hémoglobine tronquée N de Mycobacterium tuberculosis (trHbN) a mis en évidence deux tunnels hydrophobes reliant le site actif à l'extérieur de la protéine. TrHbN a la capacité de protéger la respiration aérobique en phase stationnaire de Mycobacterium bovis de l'inhibition par le NO, possiblement grâce à son activité NO-dioxygénase. De plus, trHbN présente une des affinités pour l'oxygène les plus élevées observées pour une globine (K<i = 9 nM). Ces caractéristiques pourraient dépendre des tunnels de trHbN qui, par leur topologie, leur taille et leur caractère hydrophobe, pourraient servir à la migration sélective de ligands apolaires. Pour étudier cette hypothèse, des mutants aux entrées des tunnels obstruées ont été conçus in silico dans un premier temps, par une approche rationnelle. L'effet de ces mutations a été étudié par dynamique moléculaire d'une part et par spectroscopie et enzymologie d'autre part. Cette approche a montré que ces mutations n'ont pas perturbé le repliement de la protéine, la dynamique des résidus impliqués dans la stabilisation de ligands exogènes ou les caractéristiques spectrales de trHbN. De plus, la stabilisation d'une molécule d'eau au site distal est demeurée l'étape limitante de l'association de ligands à Thème. L'obstruction partielle à l'addition du NO à trHbNFen02 pour les mutants du tunnel court indique que celui-ci, ainsi que des voies de migration autres que les tunnels cristallographiques sont impliqués dans la migration de substrats apolaires au site distal.

No / Background: Ethiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages re-sponsible for TBLN in Southwest Ethiopia.Methods:A total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) (n = 167) and qPCR-based single nucleotide polymorphism (n = 38). Isolates were classified into main phylogenetic lineages and families by using the re-ference strain collections and identification tools available at MIRU-VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF. Results: The majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2(n = 44), Ethiopia_3 (n = 34) and Ethiopia_H37Rv-like (n = 30). Other sub-lineages included URAL (n = 18), S(n = 17), Uganda I (n = 16), LAM (n = 13), X (n = 5), TUR (n = 5), Uganda II (n = 4) and unknown (n = 19).Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2(Beijing) were represented by 3 and 1 isolates respectively.M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance. Conclusions: In TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The contribution of M. bovis in TBLN infection is minimal. / This work was supported by the Mycobacteriology Unit of Instituteof Tropical Medicine, Antwerp, Belgium and interuniversity coopera-tion between Jimma University and Flemish Universities (VLIR-OUSproject).

The primary purpose of this investigation is not only to improve the present technique of culture of Mycobacterium tuberculosis, but also to make a comparative study of the media use, namely, Veterans Administration modification of Trudeau's medium, Lowenstein's egg medium, and that developed by the author, in order to find which, if any, of these will most easily and effectively meet the needs of the hospital laboratory.