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A prospective study was undertaken to assess the diagnostic value of various rapid diagnostic tests for tuberculosis in pleural fluid, and to assess the sensitivity and speed of a radiometric mycobacterial culture system (BACTEC, Johnson Laboratories). Patients presenting to the Department of Medicine at Groote Schuur Hospital with pleural effusions for diagnostic pleural aspiration and biopsy over a 6 month period were entered into the study. Because the incidence of tuberculous effusions was observed to be high in this population (65% of 94 patients), patients from the Department of Radiotherapy with proven malignant disease and the development of new pleural effusions requiring diagnostic or therapeutic aspiration were included in the study in order to increase the number of control patients without tuberculosis. The 111 patients (17 of whom were recruited from the Department of Radiotherapy) were divided into 4 diagnostic categories: tuberculosis - 62 patients, malignant - 28 patients, miscellaneous conditions - 10 patients, and undiagnosed - 11 patients (3 of whom probably had tuberculosis). There were 59 male patients. The racial distribution was 11 whites, 51 of mixed race, and 49 blacks. Exudative pleural effusions were present in 109 patients. Closed pleural biopsies with the Abrams needle were performed on 100 patients using a modified version of the standard technique whereby larger specimens were obtained by stripping pleura off the chest wall. Seven pleural biopsies were reported as inadequate by the pathologist and the diagnostic yield of the procedure was 63%. Tuberculosis was confirmed histologically or by culture in 62 patients. The age distribution of these patients was bimodal, with most cases occuring in the third decade. The presentation was usually acute, with 60% of patients being symptomatic for less than 4 weeks. Granulomata were found on initial pleural biopsy in 52 cases (84%). Pleural biopsy culture was positive in 44 cases (71%). The radiometric culture system tested (12B BACTEC) yielded the same number (14) of positive cultures as conventional mycobacterial culture media in pleural fluid, but was almost twice as fast. Bedside inoculation of pleural fluid into 13A BACTEC bottles more than doubled the yield in the 24 patients tested (11 positive cultures compared with 4 each for conventional and 12B BACTEC media, p=0.046). The rapid diagnostic tests assessed on pleural fluid were adenosine deaminase (ADA), an antigen (BCG) capture enzymelinked immunosorbent assay (ELISA), and a specific DNA probe after amplification with the polymerase chain reaction. ADA was found to have a sensitivity of 0.77 and a specificity of 0.83 in the 109 patients tested, and values were significantly higher in tuberculosis patients compared with the other three diagnostic categories (p< 0.001 ). The ELISA test was performed on 103 patients and showed a sensitivity of only 0.26 and a specificity of 0.72. The DNA probe was performed on 43 patients, and had a sensitivity of 0.93 with a specificity of 0.43. Contamination of samples or latent tuberculous infection may have been responsible for the poor specificity of the DNA probe.

The relevance of the use of molecular tools in the global epidemiology of Mycobacterium tuberculosis complex (MTBC) cannot be undermined. Molecular epidemiological studies of the MTBC in Nigeria are not extensive, and to date, there has only been one detailed report. More strains are therefore needed to be genotyped in order to give a clear indication of disease transmission chains and to highlight routes of infection particularly with respect to zoonotic tuberculosis. This study therefore focuses on the identification and genotyping of MTBC isolates in south western Nigeria, with emphasis on interactions occurring at the human/livestock interface. The molecular epidemiology of M. bovis strains in Hluhluwe-iMfolozi Park in South Africa was also undertaken. Prior to this study, a pilot study was initially done to establish techniques, using samples from Belgium. Mycobacterium bovis strains were first identified in Belgium using the Multiple locus [variable number of tandem repeats] (MLVA) technique and analysis was done using capillary electrophoresis. In this study, the Belgium isolates were repeated using MLVA and analysis by agaorse gel electrophoresis and the two analysis techniques compared. Human isolates (136) and livestock isolates from cattle (50), pigs (12) and goats (5) isolated in Nigeria were also used and species identification of the members of the MTBC were done using the deletion analysis PCR technique amplifying RD1mic, RD2seal, RD4 andRD9 regions as well as spoligotyping. Seventy four positive MTBC strains (humans and livestock) were genotyped using 16 VNTR loci. The discriminatory ability of the 16 loci MLVA was compared with spoligotype data on 33 MTBC strains. Mycobacterium bovis isolates from buffalo in HluhluweiMfolozi Park (HiP) South Africa, were also genotyped using the 16 loci MLVA and spoligotyping. Results indicated that agarose based MLVA is as discriminatory as the capillary based MLVA. Furthermore, the relevance of molecular techniques in the rapid identification and genotyping of members of the MTBC, especially in a tuberculosis endemic setting like Nigeria, is also highlighted. This was clearly seen in the identification of undescribed spoligopatterns of the LAM 10-CAM M. tuberculosis strains in humans as well as the identification of undescribed M. bovis spoligopatterns in livestock isolates. The prevalent M. bovis strain (SB0944) in Nigeria was also identified in a human isolate. Also, two classical M. bovis strains were identified in two human isolates obtained from cattle traders, thus suggesting the influence of close interaction between infected animals and man as a means of zoonotic tuberculosis transmission. Mycobacterium tuberculosis was also identified in three isolates, from cattle, pig and goat; with the goat isolate having a spoligopattern (EAI5) typical of strains indigenous to East Africa and India. This study demonstrated the prevalent strains of M. bovis and M. tuberculosis circulating in Nigeria with SB0944 the predominant M. bovis spoligotype and LAM10-CAM the predominant M. tuberculosis spoligotype. The MLVA results revealed the occurrence of interspecies transmission of mycobacterial species, which was seen as isolates from different animal species having identical VNTR profiles and thus belonging to the same genotype. In the HiP, two strains of M. bovis were identified, a strain previously described in cattle and buffalo in other regions of South Africa and a new undescribed strain, thus giving an indication of the circulating strains in HiP and also suggesting possible sources of introduction of novel species in HiP. The relevance of a detailed molecular epidemiological study was clearly demonstrated in both Nigeria and HiP. Strain relatedness and interactions occurring at human/livestock interface and domestic/wild life interface could also clearly be demonstrated. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Mycobacterium avium, an opportunistic human pathogen, infects between 25 and 50% of advanced-stage acquired immuno-deficiency syndrome (AIDS) patients in the United States. M. avium has been isolated from many environmental sources including: natural waters, soils, and aerosols. M. avium has also been recovered from within municipal and hospital drinking water systems. Rhesus macaques (Macaca mulatta) infected with the simian HIV analog, SIV, have been shown to acquire M. avium infections from potable water. Reduced-aggregate fractions (cell suspensions free of large aggregates) of Mycobacterium avium were exposed to chlorine, monochloramine, chlorine dioxide, and ozone and kinetics of disinfection measured. Chlorine disinfection kinetics was also measured in M. avium cultures grown in biofilms. M. avium exhibited a high resistance to chlorine compared to E. coli. M. avium CT99.9% (disinfectant concentration x time to 3 logs cell inactivation) values were between 571- and 2318 -times those of E. coli. Clinical isolates of M. avium showed 0.24 and 2.5-fold increase in resistance to chlorine compared to their pulsed-field-gel-electrophoresis- (PFGE) matched environmental isolates. M. avium strains exhibited a mixed response to exposure to monochloramine. The CT99.9% values of three strains (2 clinical, 1 environmental) were between 6.3- and 23.5- times that of E. coli. Two strains (1 clinical, 1 environmental) exhibited CT99.9% values approximately the same as E. coli, a difference from all the other disinfectants which were much less effective on M. avium than on E. coli. M. avium strains exhibited a high resistance to chlorine dioxide when compared to E.coli. M. avium CT99.9% values of between 133- and 706- times higher that that of E. coli. In the paired isolates tested, the clinical isolate was 5.3 times more resistant than the matched environmental isolate. M. avium exhibited a high resistance to ozone when compared to E. coli. M. avium strains exhibited a CT99.9% value of between 52 and 90 times higher that that of E. coli. In the paired isolates tested, the clinical isolate was nearly identical as judged by CT99.9% values. M. avium strain 5002 exhibited an unusual disinfection kinetics curve. Disinfection rate increased by a non-logarithmic factor, indicating that inactivation efficiency was increasing over time. M. avium strain 1060 showed between a 17% decrease to a 265% increase in CT99.9% value when grown as biofilms as opposed to suspension. Due to the large variance in biofilm density and and CT99.9% values, any conclusions based on these experiments should be considered tentative at best. M. avium's resistance to chlorine and chlorine dioxide approaches that of the protozoan cysts of Giardia muris and Entamoeba hystolytica. M. avium is much less resistant, relatively, to monochloramine possessing values similar to E. coli. Ozone resistance of M. avium is two orders of magnitude greater than E. coli and one order of magnitude of less than G. muris cysts. A critical concentration threshold level for chlorine dioxide was found. That is, there was no linear relationship between concentration of chlorine dioxide and cell inactivation. Initial experiments using a range of concentrations from 0.1 ppm to 0.5 ppm chlorine dioxide showed a biphasic curve with the inflection point (indicating the critical concentration) between 0.3 and 0.4 ppm chlorine dioxide. / Master of Science

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mycobacterium ulcerans is an acid-fast bacillus that is the causative agent of Buruli ulcer, a necrotizing skin disease. The transmission route for M. ulcerans is unknown, but many insects have been posited as part of the web, including Belostomatids, Naucorids, and Culicids. Aedes albopictus was selected for use in a set of experiments where the first-generation larvae were inoculated with M. ulcerans, and mosquitoes were reared throughout the third generation to interrogate presence and quantity of the bacteria. Using qPCR, second and third generations displayed positivity (22% and 5.6% respectively). 16S V4 sequencing was used to obtain microbiota for all life stages as well as environmental samples, and many relationships between generations, life stages, and treatments displayed statistical significance in alpha diversity, beta diversity, and relative abundance of microbiomes. This study opens multiple avenues of further investigation into the transmission web of Buruli ulcer.

Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.

Buruli ulcer disease (BUD) remains a ‘mysterious disease’ due to the unknown mode of M. ulcerans transmission and pathogenesis. To understand these, it is important to determine the reservoir of the organism in its natural environments, and stress response and interactions of M. ulcerans in its natural niche and during infection of a host. The major virulence factor of M. ulcerans is mycolactone, a lipid cytotoxin that is encoded on a giant plasmid pMUM001. Genetic analysis suggests that plasmid pMUM001 was acquired by M. ulcerans during evolution from its progenitor, M. marinum. Coincidental evolution of virulence hypothesis suggests that many microbes evolve to acquire traits to outcompete or overcome biotic and abiotic forces during their normal life cycle in the outside-host environment, which can confer virulence during infection of a human host. Hence in this study, we exposed M. ulcerans to selective abiotic forces such as UV, and dynamic oxygen and temperature conditions to determine their effect on M. ulcerans growth, and mycolactone and global gene expression. We also studied the role of mycolactone in determining polymicrobial interaction of M. ulcerans in its natural aquatic habitat by exposing mycolactone coated and uncoated slides in M. ulcerans endemic and non-endemic aquatic locations and determining differences in microbial community composition between them. Further, we studied quorum quenching ability of mycolactone against an opportunistic pathogen, S. aureus. The results obtained showed that exposure of M. ulcerans to abiotic stresses such as higher temperature and lower than optimal oxygen conditions modulate its global and mycolactone gene expression. Further, we also showed that mycolactone can impact overall microbial community structure in a polymicrobial environment in its natural, aquatic habitat. Mycolactone also effected virulence and quorum sensing in an opportunistic pathogen, S. aureus, without inhibiting its growth. These findings are important as they provide insight toward potential reservoirs or environmental niches which may harbor M. ulcerans and inform new potential mechanisms of pathogenesis. Further, our novel research of synergistic or antagonistic interactions within the complex polymicrobial communities colonizing skin and aquatic habitats is a powerful approach in determining M. ulcerans colonization efficiency, resiliency, and transmission mechanisms.

No description available.

No description available.