The World Health Organization (WHO) has outlined a TB-free World strategy that aims to reduce tuberculosis prevalence by 2015. That could be achieved through the development of a specific and rapid detection system. In countries with a high incidence of tuberculosis, detection is solely based on sputum smear microscopy using Ziehl-Neelsen stain and chest radiography. This is followed by cultivation, which requires up to 12 weeks for confirmation. The diagnostic test available is insensitive, laborious, lacks specificity and does not even give unequivocal proof of infection. Consequently, this study was conducted to obtain confirmatory diagnostic information from Ziehl-Neelsen stained smears on glass slides. The fast-grower, non-pathogenic Mycobacterium smegmatis was used as the model organism due to similarity to the pathogenic strain, Mycobacterium tuberculosis. Laser capture microscopy (LCM) is primarily used as a microdissection tool in studies involving tissues and membranes. This tec1mology was able to isolate intact individual cells fixed on archived glass slides regardless of how the smears and staining have been performed, confirmed by transmission electron microscopy (TEM) observation. Typically between 100 - 1,000 catapulted cells are enumerated using flow cytometry (FCM). A series of DNA extraction tec1miques which are heatshock, heat-shock followed by ethanol precipitation and QIAamp® DNA Micro kit were compared and optimized for the lowest number of post-catapult cells. To maintain the detection of lowest number of catapulted cells, the rpoB-gene specific primers were designed for amplification using nested real-time PCR. Routinely, this system was able to detect as few as 30 catapulted cells per assay. This result demonstrated that it is possible to isolate the bacteria from glass slides and subsequently perform downstream molecular applications regardless of any inhibitory factors. In conclusion, we strongly recommend that this system may offer improved specificity and speed of tuberculosis detection with lower risk of exposure to infection through the use of stained slides.

Climate change, globalisation and increased travel, increasing urban populations, overcrowding, continued poverty, and the breakdown of public health infrastructure are among the factors contributing to the 30-fold increase in total dengue incidence in the past 50 years. Consequently, with an estimated 40% of the world's population at risk of infection, dengue is now the world's most important mosquito-borne viral infection. However estimates of dengue transmissibility and burden remain ambiguous. Since the majority of infections are asymptomatic, surveillance systems substantially underestimate true rates of infection. With advances in the development of novel control measures and the recent licensing of the Sanofi Dengvaxia® dengue vaccine, obtaining robust estimates of average dengue transmission intensity is key for estimating both the burden of disease from dengue and the likely impact of interventions. Given the highly spatially heterogeneous nature of dengue transmission, future planning, implementation, and evaluation of control programs are likely to require a spatially targeted approach. Here we collate existing age-stratified seroprevalence and incidence data and develop catalytic models to estimate the burden of dengue as quantified by the force of infection and basic reproduction number. We identified a paucity of serotype-specific age stratified seroprevalence surveys in particular but showed that non-serotype specific data could give robust estimates of baseline transmission. Chapters explore whether estimates derived from different data types are comparable. Using these estimates we mapped the estimated number of dengue cases across the globe at a high spatial resolution allowing us to assess the likely impact of targeted control measures.

Sanitation programme monitoring is often limited to latrine access and coverage, with little emphasis on use of the facilities. This may be partially explained by the challenges associated with measuring individual and household latrine use. The conventional methods used each have their limitations. The overall goal of this research was to improve the methods for assessing latrine use in low-income countries and enhance our understanding of the patterns and determinants of latrine use in rural India. The evidence from a cross-sectional study to compare reported latrine use with a technology based measure, Passive Latrine Use Monitors, indicated that reported latrine use, though already suggesting low adoption, likely exaggerates the actual level of uptake of government constructed latrines in rural Odisha, India. Moderate agreement was obtained when comparing daily reported use during the previous 48 hours with the average daily PLUM count. Thus, if self-report measures are used, survey questions should focus on the 48 hours prior to the date of the survey rather than asking about “usual” latrine use behavior. The study also assessed patterns and determinants of individual latrine use over 12 months in the study population. Based on a prior 48 hour recall measure of reported use, we classified use into three categories—“never”, “sometimes” and “always/usually”. We also assessed consistency of latrine use across the dry cold, dry hot and rainy seasons. Overall, we found that latrine use was poor. There was significant seasonal variation in use. There was increased reported likelihood of consistently using the latrine among females and where latrines had a door and roof. Older age groups and an increase in household size were associated with a decreased reported likelihood of consistently always/usually using the latrine versus never using it. The leading reported reason for non-use of latrines was a preference for open defecation.

Background: Herpes zoster affects millions of people worldwide each year and many go on to suffer long-term pain, called postherpetic neuralgia (PHN). As zoster is common and PHN is difficult to treat, preventing zoster through vaccination is important. This thesis aims to better understand risk factors for zoster and PHN, in order to inform vaccination policy. Methods: Three large observational studies were carried out using primary care data from the UK Clinical Practice Research Datalink and linked secondary care data from the Hospital Episodes Statistics. First, a matched case-control study quantified the effects of possible risk factors for zoster and explored whether their effects differed by age group. Second, a descriptive study looked at antiviral prescription patterns and patient characteristics associated with antiviral receipt after zoster diagnosis. Third, a cohort study assessed risk factors for PHN and investigated whether their effects were modified by antiviral use. Results: The case-control study of zoster risk factors included 144,959 zoster patients and 549,336 controls and found an increased risk of zoster among patients with rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, chronic kidney disease, depression and type 1 diabetes; odds ratios ranged from 1.14 to 1.72. In general, the relative effects of these risk factors on zoster decreased with increasing age. In the descriptive study of antiviral use, of 142,216 zoster cases, only 58.1% received an antiviral prescription at zoster diagnosis. Antivirals were even under-prescribed among the immunosuppressed and older individuals, for whom guidelines recommend routine treatment. The cohort study of PHN risk factors identified 119,413 zoster patients, 5.8% of whom developed PHN. An increased risk of PHN was found among patients with rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, depression, type 2 diabetes, lower socioeconomic status, smoking and under- or overweight; odds ratios ranged from 1.13-1.82. Antiviral use was not associated with PHN risk overall. The zoster case-control and PHN cohort study showed that patients with severely immunosuppressive conditions were at greatest risk of both zoster and PHN. Conclusions: A number of patient characteristics and comorbidities were associated with increased zoster and PHN risks. Patients at highest risk of zoster and PHN are those of older age and those with immunosuppression; currently, patients with immunosuppression are not eligible for vaccination, highlighting a need for alternative risk reduction strategies in this group. Low antiviral use at zoster diagnosis suggests treatment guidelines be revised to encourage greater use, especially among the immunosuppressed and older individuals who are recommended, but not routinely given, antivirals. Research on the cost-effectiveness of vaccinating patients with specific risk factors is needed.

Children bear a considerable proportion of the impact of epidemics of influenza and measles and are a driving force in community-wide epidemics. This thesis considers this impact and the benefits of existing vaccination programmes. First, we estimated the impact of seasonal influenza outbreaks in primary schools. To estimate the health-related impact we started by conducting a review of the literature to determine the methods used to estimate health utilities in children and adolescents, establishing that a wide variety of systems have been used without clear guidance on an optimal method. Mean absence from school was 3.8 days (95% CI: 3.0-4.8) and 3.7 days (95% CI: 2.7-4.8) off work for caregivers. The mean loss in HRQoL was 2.1 QALDs (95% CI: 1.5-2.7). Next, we modelled the childhood influenza vaccination programme at a national- and communitylevel, exploring the impact of heterogeneous coverage. Nationally, a vaccination programme that focuses on primary school vaccination supplemented with fewer vaccinations in secondary schools is optimal from the perspective of the healthcare provider, but heterogeneous uptake within targeted populations consistently resulted in a larger total burden of disease at the community level, emphasising the importance of adherence to school-based vaccination policies. Two studies of the measles outbreaks in England between 2012 and 2013 were conducted to further the understanding of the impact of measles infection. Significant work/school absence was reported by individuals with infection and their caregivers. Infection was associated with a mean loss of 6.9 QALDs (95% CI: 5.8-8.0). The total economic cost of the outbreak was £4.4m in Merseyside alone, compared to a total cost of £182,909 over the previous five years to achieve herd immunity through the MMR vaccination programme. The findings demonstrate the importance of adherence to preventative vaccination programmes to reduce the potential for outbreaks of influenza and measles.

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