A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

Using a multiple stage age of infection model, we derive an expression for the basic reproduction number, Ro. We apply this method to find Ro in analogous treatment models. We find, in the model without treatment, Ro depends only on the mean infective period, and not on the infective distribution. In treatment models, Ro depends on the mean infective and mean treatment period, as well as the distribution of the infective period, but not on the distribution of the treatment period. With an explicit formula for Ro and the final size relation, we provide a practical alternative to evaluating the effect of treatment and other control measures. We compare our models to previous models of SARS and TB. / Science, Faculty of / Mathematics, Department of / Graduate

Tuberculosis (TB) is the leading killer globally owing to an infectious disease. There is consequently an urgent need to develop novel TB drugs and shorter regimens to treat the causative agent, Mycobacterium tuberculosis, an imperative which demands the identification of new drug targets in essential mycobacterial pathways. To that end, the work presented in this dissertation aimed to functionally characterize ribF, an essential gene in the mycobacterial riboflavin (RF; vitamin B2) biosynthetic pathway. Given the role of RF as a core component of the essential flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it was hypothesized that silencing ribF would disrupt the biosynthesis of all flavoproteins, crippling numerous (essential) processes within the organism. Moreover, based on previous observations in Bacillus subtilis, it was predicted that the mycobacterial ribF homolog might play a role in regulating the rib operon (comprising a cluster of RF pathway genes) – either directly by binding to the FMN riboswitch, or indirectly through the production of FMN from RF, in turn enabling riboswitch-mediated repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro growth of M. smegmatis: CRISPRi-mediated depletion of ribF was bacteriostatic, resulting in a 10-fold growth inhibition in liquid media and corresponding to no reduction (0 log-fold change) in colony forming units (CFU). Moreover, targeted metabolomic analyses revealed that ribF depletion was associated with accumulation of 6,7-Dimethylribityllumazine (DMRL), suggesting that the disruption of RibF function blocked conversion of RF to the essential cofactors, FMN and FAD, in turn inhibiting cell growth. Notably, the lethality of ribF depletion could not be complemented chemically by exogenous supplementation of growth media with RF, FMN or FAD. Downregulation of ribF also caused enhanced susceptibility to the known cell wall-targeting agent, vancomycin, but not to the putative RibF domain inhibitor, thonzonium bromide, suggesting an alternative mechanism of action or impaired bacillary permeation. In summary, these data support the inferred essentiality of ribF in mycobacteria, in turn supporting future work which aims to target this enzyme for new TB drug discovery.

South Africa has one of the world’s highest burdens of TB, HIV/TB co-infection, and drug-resistant TB. Second-line TB treatment is less effective, more expensive, and more toxic than treatment for drug-sensitive TB. Nearly 1 in every 5 persons who starts treatment for drug-resistant TB in South Africa will die; 1 in every 3 persons who survives treatments experiences permanent, profound hearing loss. For decades there was little progress in TB research, however, and so treatment with old regimens continued despite safety concerns. In 2012 the US and European regulatory authorities approved a new drug, bedaquiline, but only for treatment in cases with no other options. In 2015, the South African Medicines Control Council approved bedaquiline for drug-resistant TB, but only a limited number of doses were approved in the 2016/2017 annual budget and the focus, again, was only for the patients who had no other options. In order to inform policy makers in planning and budgeting for drug-resistant TB treatment, the aim of this thesis was to determine whether the simple calculation that bedaquiline was too expensive relative to standard regimens using kanamycin was too simple. Particularly, given the high burden of adverse drug reactions (ADR) associated with kanamycin, would the inclusion of the cost and burden of ADR affect the incremental cost effectiveness ratio of a new treatment regimen where bedaquiline replaces kanamycin? Analysis of the national drug-resistant TB case register showed that mortality during second-line treatment was early, primarily in the first 6 months of treatment, even when patients do not have extensive drug resistance. HIV-positive patients not on anti-retroviral therapy (ART) at initiation of drug-resistant TB treatment have the highest risk of mortality. The high early mortality is a real risk that clinicians have to balance when deciding to initiate ART and effective second-line TB treatment both as quickly as possible. Daily injections coupled with taking more than 10 pills each day are a heavy burden for patient compliance, but also pose concerns in terms of overlapping and compounding toxicities; this burden was confirmed through a meta-analysis of the pooled frequency of adverse events among cohorts with at least 25% of the patients HIV-positive. A competing risk analysis of a cohort of drug-resistant TB patients from Johannesburg – addressing the reality that patients may not have experienced an ADR because they died rather than because they were at lower risk – indicated that HIV-infected patients who are not yet stable on ART and second-line TB treatment are at the highest risk of ADR. A Markov model built and parameterized using the data from the South African national TB programme indicates that bedaquiline for all drug-resistant TB led to a small gain in effectiveness at a cost that was under the costs of the drug itself, due to savings from daily injection visits. While cost-effective, it was not clear that South African policy makers needed to move beyond the offer of bedaquiline for patients with extensive drug resistance. However, the calculation, and the decision point, were different once the costs and disability associated with ADRs was included in the analysis. Bedaquiline-based regimens offer a cost-saving and more effective alternative to an injection-based regimen for drug-resistant TB patients treated in the public sector in South Africa.

Yes / SETTING: In 2005, in response to the increasing prevalence of rifampicin-resistant tuberculosis (RR-TB) and poor treatment outcomes, Rwanda initiated the programmatic management of RR-TB, including expanded access to systematic rifampicin drug susceptibility testing (DST) and standardised treatment.OBJECTIVE: To describe trends in diagnostic and treatment delays and estimate their effect on RR-TB mortality.DESIGN: Retrospective analysis of individual-level data including 748 (85.4%) of 876 patients diagnosed with RR-TB notified to the World Health Organization between 1 July 2005 and 31 December 2016 in Rwanda. Logistic regression was used to estimate the effect of diagnostic and therapeutic delays on RR-TB mortality.RESULTS: Between 2006 and 2016, the median diagnostic delay significantly decreased from 88 days to 1 day, and the therapeutic delay from 76 days to 3 days. Simultaneously, RR-TB mortality significantly decreased from 30.8% in 2006 to 6.9% in 2016. Total delay in starting multidrug-resistant TB (MDR-TB) treatment of more than 100 days was associated with more than two-fold higher odds for dying. When delays were long, empirical RR-TB treatment initiation was associated with a lower mortality.CONCLUSION: The reduction of diagnostic and treatment delays reduced RR-TB mortality. We anticipate that universal testing for RR-TB, short diagnostic and therapeutic delays and effective standardised MDR-TB treatment will further decrease RR-TB mortality in Rwanda.

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most prevalent infectious diseases inflicting humankind. The World Health Organization estimates that one third of the world's population, approximately 2.2 billion people, is infected with TB with a mortality of 1.7 million people annually. Currently, the WHO estimates that each year more than 9 million people develop TB. Bacille Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only licensed TB vaccine in the world. Clinical studies have shown childhood vaccination with BCG to be protective against disseminating and meningeal forms of TB. However, the efficacy of BCG against pulmonary TB in adults has been variable and inconsistent (0-80%). The objective of this study is to develop and test the efficacy of the B. abortus vaccine strain RB51 as a platform for expression of M. tuberculosis antigens (Ag85B, ESAT6 and Rv2660c) and induction of a protective immune response against M. tuberculosis and B. abortus challenge in mice. Here we report the construction of two recombinant strains of B. abortus vaccine strain RB51 capable of expressing mycobacterial antigens Ag85B, ESAT6 and Rv2660c. Our studies show that expression of mycobacterial antigens in strain RB51 lead to induction of antigen-specific immune responses characterized by secretion of IgG2a antibodies as well as of IFN- and TNF-α. Mice immunized with a combination of two strains of RB51 in equal numbers, one carrying Rv2660c-ESAT6 and another carrying Ag85B, led to a 0.90 log reduction in CFU burden with significance nearly reaching borderline (p = 0.052). However, when mice were primed with the same strains of RB51 and boosted with proteins Ag85B and ESAT6, a significant level of protection (1 log reduction) compared to the PBS vaccinated group was achieved. The protection levels conferred by this vaccination strategy was similar to that conferred by BCG vaccine. In conclusion, we have shown that recombinant RB51 strains expressing mycobacterial protective antigens result in stimulation of antigen specific immune response without altering the vaccine efficacy in protecting against the more virulent strain of B. abortus 2308. These recombinant vaccines could potentially be used to protect against M. tuberculosis infection. / Ph. D.

Introduction: Globally, one in four persons infected with the human immunodeficiency virus (HIV) who are living with the acquired immunodeficiency syndrome (AIDS) will die of tuberculosis (TB). The estimated number of HIV-infected children who die of tuberculosis, especially extrapulmonary TB (EPTB), in Haiti, is only loosely based on facts or investigation. This study proposes to describe demographics of children with EPTB in a pediatric TB hospital in Haiti. The objectives are two-fold. The first objective is to describe the population of children discharged from Grace Children's Hospital with a confirmed diagnosis of tuberculosis overall, and broken down by whether or not the child had an extrapulmonary manifestation of the TB disease. Specifically, we describe the demographic characteristics and the prevalence of HIV and other co-morbidities of the children, in-hospital mortality, and the diagnostic tools used to determine TB infection including the sputum test, and th e documentation of family members also infected. As part of the descriptive process, by examining those with only pulmonary TB (PTB) and EPTB separately, we investigate if they appear to be different sub-populations based on demographic characteristics and clinical measures. The second objective of this work is to determine if there is a positive association between HIV infection and the EPTB manifestation in children with a confirmed diagnosis of TB, both crudely and after adjusting for demographic variables and co-morbidities. Methods: A cross-sectional study design was used to review medical charts of clinically diagnosed pediatric TB cases for a five-year period (January 1, 1999 -- December 31, 2003). This included 492 pulmonary TB and 210 extrapulmonary TB cases. Variables measured included clinical measures and demographic characteristics. Results: Data for 615 hospitalized, clinically diagnosed pediatric TB cases were reviewed. There were 315 (51.4%) males and 298 (48.6%) females with a mean age of 5.40 years (range 0.17 - 14 years), with 214 (37.9%) of the patients aged 0-2 years. Percent males were 47.8% and 57.9% in PTB and EPTB groups respectively (p<0.05). One hundred and seven (17.4%) of patients were HIV positive. Three hundred eighty-eight (63%) of the patients had one or more additional co-morbidities: [anemia 299 (48.6%), intestinal parasites 93 (15.1%), malaria 58 (9.4%) and gastroenteritis 19 (3.1%)]. Nearly 85% of the children were undernourished. Eighty-three child patients (13.5%) died in the hospital. Children with EPTB were much more likely to be over the age of two (74% vs. 56% in PTB group), resulting in a highly significant Chi-square stati stic. The overall difference in mean age, however, was only borderline significant with children with EPTB being slightly older [p=0.059] and age was only weakly associated with TB group. They were much less likely to be HIV positive (8.6% vs. 22%, p<0.01). Children in the EPTB group were somewhat less likely to die in the hospital (10.0% vs. 15.4%, p=0.066). The OR was greater than 4 for HIV and was greater than 2 for poor nutrition status [p<0.01 for each]. Conclusion: There was no association in this model between EPTB and mortality. The apparent univariate association between EPTB and reduced mortality can be explained by lower prevalence of HIV and poor nutrition status in this sub-sample. This study has implications for hospital-based pediatric TB diagnosis and epidemiology in resource-poor countries.

Student number:0312412A Faculty of Health Sciences School of Public Health / Management of multidrug-resistant tuberculosis (MDR-TB) is more expensive, lengthy and is associated with less favourable outcomes and more adverse reactions than management of susceptible tuberculosis. The aim of this study was to review the management and treatment outcomes of registered MDR-TB patients hospitalized at Sizwe hospital during a five-year period. A cross-sectional study with both descriptive and analytic features was done on 237 MDR-TB patients hospitalized from the beginning of June 1998 to the end of May 2003. Data were analysed using SPSS version 12 Software. Main outcome measures were interim treatment outcomes at the end of hospitalization period. These outcomes comprised culture conversion rates, time to culture conversion, transfer out, interruption, and death rates. Multiple logistic regression analysis was performed to determine risk factors for poor treatment outcomes. These poor outcomes were defined as treatment interruption, failure and mortality rates. The burden of institutional care for MDR-TB patients in this setting was found to involve high numbers of MDR-TB patients for whom the allocated hospital beds were insufficient. Patients with primary MDR-TB, who had no history of nonadherence to treatment, were paradoxically more likely to be hospitalized shortly after diagnosis. Acquired MDR-TB patients were mostly managed as outpatients immediately after diagnosis only to be hospitalized later due to persistent nonadherence or disease severity. Overall, acquired MDR-TB patients were hospitalized in larger numbers than those with primary disease. This reflects the higher prevalence of acquired MDR-TB compared to primary MDR-TB. Page v Abstract Culture turnaround time was on average 19 days. The overall culture conversion rate of the hospitalized patients was low at 41.9 percent. This low culture conversion rate resulted in protracted hospitalization periods and high interim mortality rates. The mean duration of hospitalization, 3.52 months, correlated favourably with the time interval to the first culture conversion of 2.96 months. Hospitalization did not guarantee the expected adherence to treatment. Surgical interventions were done belatedly with resultant high mortality outcomes. The main reasons given by patients for refusing hospital treatment were visiting traditional healers, solving socioeconomic problems and attending to family matters. A large percentage of hospitalized patients were co-infected with HIV. HIV care and support was incomplete as antiretroviral drugs were not available at the hospital. Among the main findings of the study was the powerful influence HIV status had on poor hospitalization outcomes. Recommendations arising from the study include the need to provide ARVs at the Sizwe hospital. Admission and discharge guidelines aimed at ensuring adequate beds are reserved for deserving patients should be formulated. Continuing education for service providers must be encouraged and rewarded. Infection control procedures at both community and health institution level ought to be vigorously promoted. Patients known to be hopelessly non-adherent should at least be partially hospitalized in the interest of public health.

Tuberculosis (TB) is the leading cause of death due to a single infectious disease and remains a major threat to global public health. The increasing emergence of multi-drug resistance to current anti-TB drugs, exacerbated by the long treatment duration, highlights the need for new effective treatments or strategies to shorten the treatment duration, improve patient adherence and curb the alarming rates of resistance. A key challenge to current strategies employed in the development of anti-TB drugs is the complexity in TB disease pathology which presents as a wide spectrum of lesions in patients presenting with the disease. These lesions occur at different anatomical loci in the same individual and at different stages as the disease progresses. In addition, the interaction between the causative agent, Mycobacterium tuberculosis, and its obligate human host induces physiologic and metabolic changes in the infecting bacillus that are specific to each lesion compartment, and dynamic. This is likely to influence M. tuberculosis susceptibility to antibiotic treatment and, consequently, affect treatment duration and possibly the development of drug resistance. A major limitation in current strategies to address this problem is translation of in vitro compound potency to in vivo efficacy. To reach the target site, a drug must first distribute and accumulate in the lesion microenvironments where bacteria reside: the macrophage host cell and the caseum. This thesis focused on the development of an in vitro infection model that could be used to predict drug penetration into M. tuberculosis-infected macrophages. Of particular interest was the extent to which host intracellular drug concentrations translate into effective antimycobacterial activity. To this end, the thesis comprised three key aspects: (i) characterization of physicochemical properties, antimycobacterial activities and M. tuberculosis-mediated metabolism of selected antiTB compounds; (ii) determination of intracellular drug permeation in resting, activated and foamy macrophages; and (iii) determination of the correlation (or not) between intracellular drug concentration and effective M. tuberculosis growth inhibition. The highly lipophilic natural product, fusidic acid (FA), its known human metabolite, 3-ketofusidic (3-ketoFA or GKFA37), and two C-3 alkyl esters (GKFA16 and GKFA17) as FA prodrugs were utilized in the study. In addition, another chemical class, the less lipophilic benzoxazole-based oxime derivatives were also investigated. Moxifloxacin (MXF), levofloxacin (LVF), bedaquiline (BDQ), rifampicin (RIF) and clofazimine (CFZ) were included for reference as known anti-TB drugs with varying lipophilicities. In chapter 2, FA and derivatives showed potent antimycobacterial activity (~1 µM) with selectivity indices (SI) >20 against the THP-1 macrophage cell line. Predicted artificial membrane permeability assay (PAMPA) results suggested that FA and derivatives would readily permeate the cell membrane. M. tuberculosis metabolized the C-3 alkyl-ester prodrug GKFA17 to form both FA and 3-ketoFA, with complete hydrolysis of the prodrug. FA was metabolized to 3-ketoFA, but the low levels of the metabolite suggested that another unidentified metabolite, presumed to be 3-epifusidic acid (3-epiFA), was formed. In vitro assays revealed that the potent benzoxazole-based oxime carbamates (PMN1-201, PMN1-136 and PMN2-09) were rapidly hydrolyzed by M. tuberculosis and were also susceptible to spontaneous degradation in media, forming the poorly active corresponding free oximes (PMN1-199, PMN1-135 and PMN1-157). In chapter 3, the in vitro macrophage drug uptake assay showed that FA C-3 alkyl prodrugs, GKFA16 and GKFA17, accumulated in significantly higher amounts in resting macrophages in comparison to FA and GKFA37. Accumulation of MXF was comparable to the least accumulated FA derivative, GKFA37, and showed steady state intracellular concentrations over a 6-day period. While GKFA16 and GKFA17 showed continued increasing accumulation, intracellular concentrations of FA and GKFA37 decreased after 48 hours, suggesting a likely susceptibility to macrophage efflux. In infected macrophages, the presence of intracellular bacteria or increasing bacterial burden did not affect the host cell ability to accumulate the drugs. FA and derivatives exhibited bacteriostatic inhibition of intracellular mycobacterial growth. MXF showed a potent bactericidal effect, reducing intracellular bacterial counts significantly at 10x MIC, with complete sterilization at 50x MIC even though MXF accumulation was significantly less than that of FA alkyl esters. These results suggested that both the inherent activity of a compound and ability to accumulate within host cells drive cellular efficacy. Given that the C-3 alkyl ester prodrugs accumulated at significantly higher concentrations than FA and GKFA37, this demonstrates the limitations of this assay in ascertaining the impact of intracellular concentration on drug efficacy for bacteriostatic drugs while highlighting its ability to correlate drug penetration and intracellular activity for cidal drugs. The prodrug GKFA17 was shown to undergo metabolism in resting host cells and during infection to form FA and then 3-ketoFA. Therefore, the prodrug strategy could be used to increase intracellular exposure of FA as GKFA17 showed superior macrophage accumulation. Benzoxazole-based oxime carbamates and their corresponding free oximes failed to accumulate in host macrophages and this was corroborated by their failure to control host cell bacterial growth despite the potent in vitro activity against M. tuberculosis of the carbamates, suggesting that they are poorly permeable. Chapter 4 investigated drug permeation in different macrophage phenotypes known to exist in the granuloma during TB disease, including foamy and activated macrophages. The activation state of the host cell did not affect the ability to accumulate anti-TB drugs such as RIF and BDQ. However, FA and its prodrug GKFA17 were significantly reduced in M1 activated macrophages. Despite the significantly reduced intracellular concentration, activated macrophages treated with FA and derivatives showed superior intracellular M. tuberculosis growth inhibition, suggesting that macrophage activation potentiates the activity of these compounds. In order to assess the effect of foamy macrophage lipid bodies (LBs) on drug uptake and intracellular localization, oleic acid-induced foamy macrophages were treated with selected antiTB drugs and experimental compounds. FA and derivatives showed early increased accumulation in foamy cells compared to resting macrophages, while MXF, BDQ and RIF levels were not significantly changed. Intracellular:extracellular (I/E) ratios increased with increase in lipophilicity, with FA C-3 alkyl prodrugs exhibiting the highest I/E ratios of >100. Despite exhibiting increased foamy macrophage concentrations, FA and derivatives exhibited a similar reduction (bacteriostatic) in bacterial counts in both resting and foamy macrophages. The intracellular activity of RIF was also not affected by presence of LBsin foamy macrophages. BDQ, LVF and MXF, however, showed reduced intracellular efficacy against M. tuberculosis in foamy macrophages compared to resting macrophages, suggesting a role for LBs to impact intracellular drug distribution. In conclusion, this thesis demonstrates the potential utility in combining advanced analytical methods and an in vitro infection model to determine cellular drug permeation profiles that might be applied to prioritize compounds and combinations optimized for distribution to target bacterial populations. This will facilitate well-informed decision-making processes in progression of lead compounds in pre-clinical development and, therefore, may offer the potential to reduce high rates of attrition of compounds which enter clinical phase of development.

MSc (Med), Epidemiology and Biostatistics, Faculty of Health Sciences, University of the Witwatersrand, 2009 / South Africa is ranked fourth on the list of 22 high-burden TB countries in the world. Intensifying the prevalence of TB in South Africa is the high TB/HIV co-infection rate, with 44% of new TB patients testing positive for HIV. This burden is intense for rural communities due to poverty and return of people with TB/HIV co-infection who previously migrated for employment. In rural South Africa, TB is the leading cause of mortality in HIV-infected persons, but limited information is available about predictors of death. This study measures TB treatment outcomes in Rixile clinic and assesses predictors of TB mortality. Rixile HIV clinic is based in Tintswalo hospital, Acornhoek, Bushbuckridge, Mpumalanga province. This current study uses secondary data collected through a prospective cohort study conducted by PHRU and RADAR from March 2003 to March 2008 on 3 to 6 monthly intervals. Chi-square and logistic regression statistical tests were used to assess predictors of TB Mortality. TB mortality among study participants was 62.5% during the pre-ARV rollout period (March 2003- October 2005), and treatment completion was 31.7%. Some 5.8% participants interrupted treatment during the pre-ARV rollout period as compared to 4.5% during the ARV rollout period (November 2005- March 2008). TB mortality among study participants was 7.5% during ARV rollout and treatment completion increased to 84.4%. Factors associated with TB mortality were age (p=0.006), sex (p=0.017), BMI (p< 0.001), marital status (p=0.004), education (p=0.03), alcoholic beverages consumption (p=0.04), and ARV treatment (p<0.001). However, only age, sex, and ARV treatment were found to predict TB mortality. The proportion of TB treatment completion was higher and TB mortality was lower during ARV roll-out compared to pre-ARV roll-out. Being at the age of 40 to 75 years, not being on ARV treatment and male sex predicts TB mortality in this population. There is a need to expand ARV treatment and intensify TB care services for older people, particularly males living with HIV in this rural community.