Recent advances in sequencing technology and computing power mean that we are in an unprecedented position to analyse large viral sequence datasets using state-of-the-art methods, with the aim of better understanding pathogen evolution and epidemiology. This thesis concerns the evolutionary analysis of rapidly evolving RNA viruses, with a focus on avian influenza and the use of Bayesian methodologies which account for uncertainty in the evolutionary process. As avian influenza viruses present an epidemiological and economic threat on a global scale, knowledge of how they are circulating and evolving is of substantial public health importance. In the first part of this thesis I consider avian influenza viruses of haemagglutinin (HA) subtype H7 which, along with H5, is the only subtype for which highly pathogenic influenza has been found. I conduct a comprehensive phylogenetic analysis of available H7 HA sequences to reveal global evolutionary relationships, which can help to target influenza surveillance in birds and facilitate the early detection of potential pandemic strains. I provide evidence for the continued distinction between American and Eurasian sequences, and suggest that the most likely route for the introduction of highly pathogenic H5N1 avian influenza to North America would be through the smuggling of caged birds. I proceed to apply novel methods to better understand the evolution of avian influenza. Firstly, I use an extension of stochastic mutational mapping methods to estimate the dN/dS ratio of H7 HA on different neuraminidase (NA) subtype backgrounds. I find dN/dS to be higher on the N2 NA background than on N1, N3 or N7 NA backgrounds, due to differences in selective pressure. Secondly, I investigate reassortment, which generates novel influenza strains and precedes human influenza pandemics. The rate at which reassortment occurs has been difficult to assess, and I take a novel approach to quantifying reassortment across phylogenies using discrete trait mapping methods. I also use discrete trait mapping to investigate inter-subtype recombination in early HIV-1 in Kinshasa, the epicentre of the HIV-1 group M epidemic. In the final section of the thesis, I describe a method whereby epidemiological parameters may be inferred from viral sequence data isolated from different infected individuals in a population. To conclude, I discuss the findings of this thesis in the context of other evolutionary and epidemiological studies, suggest future directions for avian influenza research and highlight scenarios in which the methods described in this thesis might find further application.

Microscopical examination of <I>Candida albicans</I> grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of <I>C. albicans</I> followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in <I>C. albicans</I> was attenuated by gadolinium ions and by verapamil suggesting that calcium uptake may be involved in thigmotropic regulation. Thigmotropism was also observed for the first time in three genera of dermatophytic fungi <I>(Epidermophyton, Trichophyton </I>and <I>Microsporum)</I> and two saprophytic fungi <I>(Mucor mucedo </I>and <I>Neurospora crassa</I>). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca²⁺ appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca²⁺sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to <I>C. albicans</I> and <I>S. cerevisiae</I> using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in <I>C. albicans</I> as compared with <I>S. cerevisiae. </I>Aequorin was reconstituted in protein extracts of <I>C. albicans</I> and <I>S. cerevisiae</I> by supplementing them with coelenterazine. The level for <I>C. albicans </I>was ten times higher than for <I>Neurospora crassa</I>, the only filamentous fungus to be transformed with this gene so far. Aequorin was successfully reconstituted in transformed living cells, and the luminescence levels were sufficiently high to be detected when external Ca²⁺ was added to the growth medium. Transformed <I>C. albicans</I> cells undergoing the dimorphic transition from yeast-to-hyphal form exhibited higher resting levels of luminescence indicating that cells induced to form hyphae have higher [Ca²⁺] than yeast cells. The work presented in this thesis presents first evidence of construction of strains expressing the luminescence photoprotein aequorin in a pathogenic fungus. This method provides a non-toxic, non-invasive method for monitoring [Ca²⁺] in <I>C. albicans.</I>

The dependency of maize (Zea mays L.) and bambara groundnut (Vigna subterranea) grown in acid tropical soils, on the mycorrhizal condition for improved growth and nutrient uptake, and the effects of various management practices on root infection by vesicular-arbuscular mycorrhizal fungi in maize plants were investigated. The effect of soil pH on mycorrhiza formation in perennial ryegrass and maize was also investigated using a temperate soil in which pH levels had been maintained in the field over a long period. Infection of roots of maize and bambara groundnut plants by vesicular-arbuscular mycorrhizal (VAM) fungi indigenous to the tropical soils, resulted in increased plant dry weights as well as increased uptake of nutrients. The population of VAM fungi in the soils consisted of several species in the genera Glomus and Gigaspora. The foliar application of two systemic fungicides (Triforine and calixin) to maize plants resulted in reduced infection with the application of calixin compared to control plants. Root infection by VAM fungi was not different from that of control plants when triforine was applied. Two phosphorus sources (triple superphosphate and Ghafsa phosphate rock) applied to soils at rates equivalent to 22kg ha-1 and 44kg ha-1 had varied effects on VAM fungal infection in maize roots. Phosphate rock applied at both rates and triple superphosphate applied at the lower rate (22kg ha -1) increased root infection compared to that for triple superphosphate at the higher rate. It was suggested that the increased availability of the relatively soluble triple superphosphate was responsible for the reduced infection. The effect of lime application on VAM fungal infection was dependent on the type of phosphorus fertilizer applied to the soils. Increased root infection occurred when lime was applied in addition to triple superphosphate in comparison to phosphate rock. Without lime application, increased infection occurred when phosphate rock was applied compared to triple superphosphate. In the temperate soil with pH maintained over a long period, no VAM fungal infection was found at pH values below 5.0 when ryegrass was the host, although infection occurred when maize was the host plant. There was an effect of host plant on the infectivity of the fungi present in the soils with low pH but infection was low in comparison with that in soil at pH values above 5.0 indicating that management practices which result in soil pH changes may influence mycorrhizal associations in different plant species to different extents. The application of phosphate rock was beneficial to mycorrhiza formation and it was suggested that fertilizer practices which involve the use of phosphate rock could confer additional benefits that can be derived from increased mycorrhiza formation.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfillment of the requirements for the degree of Master of Science in Medicine in Pharmaceutical Affairs Johannesburg, May 2012 / ABSTRACT Introduction Nosocomial infections which occur after 72 hours in hospitalised neonates cause morbidity and mortality particularly in very low birth weight neonates admitted to a neonatal intensive care unit (NICU). Prolonged hospitalisation and use of sophisticated, expensive antibiotics lead to spiraling costs. Prevention of nosocomial infections are of the essence to contain expenditure and prevent life-threatening complications in vulnerable neonates. A prospective, descriptive study was undertaken to determine the cost of antibiotics used in the neonatal unit at Kalafong Hospital for nosocomial infections. Patients and Methods Neonates with nosocomial infections admitted consecutively to the neonatal unit were studied prospectively by documenting the birth weight, site of infection, pathogen, outcome, admission to the NICU and antibiotics administered. The cost related to antibiotic use was determined for each antibiotic, for individual neonates (expressed as the mean and standard deviation) and for the group as a whole. Results Over a period of seven months (1/1/2011 - 31/7/2011) 682 neonates with a mean birth weight of 2375g, ±868g were admitted to the neonatal unit for ~72 hours of whom 53/682 (7.8%) developed a nosocomial infection and of the 53 who developed a nosocomial infection, eight demised (15.1 %). Of the remaining 629 neonates who did not develop a nosocomial infection, 15/629 (2.4%) demised (p=0.7). Nosocomial infection occurred in 21/36 (58%) neonates <1 OOOg vs 22/646 (3.4%) ~1 OOOg (p<0.01 ).Of 199/682 neonates admitted to the NICU, 42/199 (21.1 %) developed a nosocomial infection vs 11/483 (2 .3%) not admitted to the NICU (p=<0.01 ). Of 22 pathogens cultured from blood, coagulase negative Staphylococcus aureus was the most common (7/22). The total cost of second line antimicrobials (meropenem, vancomycin and fluconazole) for the study period of seven months was R27 032.00 of which an amount of R1 0 321.00 was spent on neonates weighing <1000g. The mean cost per neonate was R563.77±283 for meropenem (n=51), R70.23±32 for vancomycin (n=5) and R78.66±53 for fluconazole (n=6) of which drug wastage comprised at least 50% in each instance. Conclusions Extremely low birth weight ( <1 OOOg) and admission to the NICU place neonates at risk of nosocomial infection at Kalafong Hospital. Meropenem was the most commonly used second line) antibiotic followed by vancomycin and fluconazole. Pharmaceutical curtailment of expenditure generated by nosocomial infections should be addressed by the manufacture of vials with a lower concentration of drug for neonates to minimise wastage.

Thesis (M.Sc.(Med.)(Epidemiology and Biostatistics)), Faculty of Health Sciences, University of the Witwatersrand,2009 / BACKGROUND Studies suggest an association between Bacterial Vaginosis (BV) and HIV infection; however, its temporal effect has not been greatly investigated. METHODS This is a secondary data analysis of a cohort study: set out to describe the association between BV infection and HIV acquisition. There were 750 participants enrolled in the primary cohort study. The main exposure, BV, was measured from a gram stain slide prepared from a vaginal swab. The slide was read in a laboratory qualitatively and scored by Nugents scoring. A score of 7 or above was considered positive for BV. The outcome variable (HIV) was determined by dual rapid tests and confirmed in the laboratory by a third generation ELISA. Descriptive statistics was done to describe demographic characteristics and the prevalence of BV and STIs. HIV incidence rate was calculated. Kaplan Meier survival time analysis and log rank test for significance were performed. Cox regression (univariate and multivariate) was done to determine association of BV with HIV infection. RESULTS The baseline prevalence of BV was 52 %, 95 % CI; 45 – 59. There were 21 HIV seroconversions experienced of which 7 had BV results missing and were excluded in the analysis. The remaining 14 seroconversions were followed for a mean time of 0.40 of a year and accumulated follow up time at risk of 286 person years, this represented an HIV incidence rate of 4.9 per 100 person years of follow up, 95 % CI: 2.9 – 8.27. Kaplan Meier curves revealed a higher risk of HIV-1 acquisition among women who were BV positive than the women who were BV negative. A log rank test showed that the v probability of seroconversion was different among the women depending on BV status, chi-square value 3.8, p 0.05. Controlling for confounding variables, seroconversion was high, but not significant, among BV positive women, adjusted hazard ratio 3.21; 95 % CI; 0.85-12.12, p value 0.08. CONCLUSION This study suggests that BV increases HIV seroconversion risk though statistical significance was not achieved. Vaginal cleansing education, screening and treating women with BV could maintain normal vaginal flora and reduce their susceptibility to HIV.

Bacterial resistance to antibiotics has presented increasing challenges in healthcare and the management of infection. This has resulted in alternative and traditional products that are used in other cultures being considered as an alternative to topical antibiotics. Honey, particularly Manuka honey is a product which has gained credibility as an antibacterial agent in a healthcare environment. The aim of this study was to investigate the antimicrobial capacity of syrups and honeys from different floral sources on antibiotic sensitive and resistant bacteria isolated from a clinical environment. The antimicrobial activity of seven Manuka honeys, seven honeys from other floral sources and two syrups were assayed against antibiotic sensitive and resistant isolates of 'Staphylococcus aureus', 'Enterococcus species', 'Escherichia coli' and 'Pseudomonas aeruginosa' using agar diffusion and microbroth methods to determine minimum inhibitory concentrations. These assays demonstrated both the superior antimicrobial activity of the Manuka products and highlighted differences in susceptibility between sensitive and resistant strains within organism groups. Clinical grade Manuka honeys were used to study the effect of bioload on antimicrobial efficacy on isolates from clinical polymicrobial wound populations. This demonstrated that it was the direct physical contact with the organism and not the microbial bioload which influences antimicrobial efficacy. Bacteria may form biofilms when they come into contact with an adherent surface. Organisms in biofilms have greater resistance to antimicrobials and are recognised clinically as a feature of chronically infected wounds. The ability of medical grade Manuka honey to remove established biofilms from a variety of surfaces was investigated. The results indicated potential activity but were inconsistent due to the fragility of biofilm adherence to artificial surfaces. To better emulate a clinical environment a wound model was designed using cooked meat and the polymicrobial bacterial populations from clinical wounds. The results of these experiments showed the Manuka honeys to have a bacteriostatic effect on the biofilms with no contamination of the surrounding honey medium. Chemical analysis of the honey products was performed using thin layer chromatography (TLC) and diffusion ordered spectroscopy nuclear magnetic resonance (DOSY NMR). TLC demonstrated the presence of antimicrobial fractions but insufficient material was yielded for further analysis and identification using NMR. Using DOSY NMR directly on the untreated honey products enabled characterisation of the products, identifying aromatic compounds in the Manuka products which are reputed to have antimicrobial activity. There did not appear to be any single constituent proportional to the antimicrobial UMF rating (Unique Manuka Factor) of the Manuka products where a high rating indicates a high level of antibacterial activity. The results suggest that it is a combination of compounds which confer the antimicrobial properties of the Manuka products. In conclusion this study demonstrated the superior antimicrobial activity of Manuka honey compared to syrups and honey from other floral sources and that this activity is likely due to a number of aromatic compounds present only in the Manuka products. Clinical grade Manuka honey appears to have bactericidal activity upon planktonic organisms with mainly bacteriostatic activity on biofilms grown on a wound model.

Mycobacterium abscessus is an emerging pathogen with infections increasing worldwide, especially among Cystic Fibrosis (CF) patients. During my PhD, I studied key aspects of the biology of M. abscessus spp.; particularly, I studied host-pathogen interactions, antimicrobial resistance mechanisms, and genetic determinants of virulence. First, I performed phenotypic characterization of M. abscessus spp. clinical isolates obtained from CF patients classified according to subspecies and clustering. I found clustered isolates, representing probable transmission events, were phenotypically distinct from sporadic isolates and showed adaptation phenotypes associated with chronic lung infection, such as enhanced intracellular survival, increased antibiotic resistance, and metabolic adaptations to the host environment. Second, I assessed the role of an inserted element containing an active methyltransferase in M. a. massiliense. Infection experiments with an isolate containing the inserted element (BIR1049wt) and a knockout strain (BIR1049Δ1809078_1815649) showed decreased survival of BIR1049Δ1809078_1815649 within macrophages. RNAseq analysis showed a distinct gene expression pattern between both isolates, with a number of mycobacterial virulence factors upregulated in BIR1049wt. Third, I studied heritable non-mutational antibiotic resistance mechanisms in M. abscessus to linezolid and clofazimine. For both antibiotics, I found clonal isolates of M. abscessus spp. with varying susceptibilities and different gene expression patterns, suggesting transcriptional regulation of antibiotic resistance. Mutation- mediated resistance to clofazimine was also found due to mutations in two transcriptional regulators predicted to regulate efflux pumps. Last, I evaluated the potential of repurposing a kinase inhibitor (compound H) in clinical trials for the treatment of cancer and CF, to treat M. abscessus infection. I found compound H enhanced killing of intracellular M. abscessus in macrophages through stimulation of autophagy and lysosomal function. I further studied over 60 chemical analogues of compound H in order to find a more active and specific compound for M. abscessus infection.

Monitoring the epidemic of Human Immunodeficiency Virus (HIV) infection plays a vital role in tracking the leading edge of HIV transmission and designing intervention programs both at individual and population level. At individual level, it is imperative to identify newly infected individuals to reduce onwards transmission. At population level, knowledge on the HIV incidence is essential to monitor the spread of the epidemic and plan/evaluate HIV prevention programs. This dissertation will examine the way in which biomarker data can be used to monitor the HIV epidemic. There are two primary aims of this thesis: a) to investigate the use of biomarkers in quantifying the recency of HIV infection at individual level and b) to estimate quantities such as mean window period and testing rate that are the building blocks for estimating HIV incidence at population level. We apply and further develop existing statistical methods to answer the research questions of interest. At individual level, we investigate the use of one or more biomarkers to quantify the recency of HIV infection. We propose a novel approach to make probabilistic statements on the recency of HIV infection by combining the knowledge on the growth of such biomarkers with observations from a newly diagnosed individual. Univariate and bivariate non-linear mixed-effects models are implemented in a fully Bayesian framework. A simulation study is conducted to investigate the biomarkers’ features that affect the accuracy of the estimation of recency. The research findings suggest that rapidly evolving biomarkers of antibody response, such as LAg Avidity, provide reliable estimates of the probability of recency. The proposed methods are applied to a panel of individuals for whom information on various biomarkers is given along with an estimated date of detectable infection. At population level, we focus on estimating two fundamental ingredients, the mean window period and the HIV testing rate, required for estimating HIV incidence using biomarker data. We compare commonly used statistical methods and explore the use of multi-state models in estimating the mean window period of the fourth generation Architect Avidity. We further investigate the factors that are associated with the probability of having an HIV test and the HIV testing rate using surveillance data. Logistic and count regression models using the Generalized Estimating Equations (GEE) approach are employed to make inference at population level about the probability of testing and HIV testing rate respectively.

Introduction: An amebic liver abscess is the most common presentation of extraintestinal amebiasis. This condition is the result of a parasite infection caused by Entamoeba histolytica. Materials and Methods: We report a case of a 53-year-old male who presented with abdominal pain in the right upper quadrant, jaundice, and a 10-kg weight loss within a 1-month span. Results and Conclusion: A wide range of symptoms and findings in the imaging tests suggestive of neoplasia, elevated levels of CA 19-9 and CA 125, and the presentation of biliary peritonitis as a complication makes this case a challenge for its approach and management. / Revisión por pares

Hemodialysis patients are at high risk of acquiring a blood stream infection (BSI), the second leading cause of death in this population. The purpose of this project was to create a clinical practice guideline (CPG) based on current evidence-based practice (EBP) that would bring a cohesiveness to the policies and provide an auditing tool to monitor infection control practices. Current literature supports the bundle approach, a small set of EBPs combined as a group of recommended interventions that apply to a specific patient population with the goal of improved delivery of care. The hemodialysis bundle project incorporated the theory of planned behavior to create a set of evidence-based interventions developed from an in-depth review of current, peer-reviewed studies. Three experts reviewed the CPG using the Appraisal of Guidelines for Research and Evaluation Instrument II; the scores from the 6 domains showed approval of the guideline as it was created with a score of greater than 90%. The three experts were chosen because they are responsible for updating and writing policies for the hemodialysis units. The creation of a CPG to improve infection control practices might benefit hemodialysis staff by providing an organized and cohesive method of following current policies. The new CPG might impact social change by applying current EBP to a clinical practice with end results of improving hemodialysis care and patient outcomes.