Tuberculosis (TB) is the second leading cause of death from an infectious disease worldwide and remains a major public health challenge, particularly in resource limited settings. Given the effectiveness of current treatment regimens, enhanced detection of disease in both low- and high-burden settings will be critical in making progress towards TB elimination.

As TB case rates have declined in high-income settings, TB control has centered on finding and treating individuals with non-infectious latent TB infection (LTBI) in order to prevent reactivation infectious TB disease. Since 2005, the Centers for Disease Control and Prevention has recommended use of interferon-gamma release assays (IGRAs), in vitro immuno-diagnostic tests that measure effector T-cell mediated interferon-gamma (IFN-gamma) response to M. tuberculosis specific antigens, could be used for targeted screening of LTBI in all circumstances in which the tuberculin skin test (TST) is used. We found that higher quantitative IFN-gamma results were associated with active tuberculosis and added clinical value to a prediction model incorporating conventional risk factors; however, in all settings and especially within low- and middle-income countries, IGRAs are inadequate rule-out or rule-in tests for active TB. Although IGRAs are widely used in high-income countries and numerous studies have evaluated their diagnostic performance for detection of LTBI, there is limited data on the precision of IGRA results. In the largest precision study of an IGRA to date, we found considerable variability in TB response measured by QuantiFERON-TB Gold In-Tube (QFT-GIT, Cellestis, Australia); test results should be interpreted cautiously among low-risk individuals with positive TB response less than 0.59 IU/ml.

In contrast to low TB burden, high income settings, TB control in low income settings focuses on early detection and treatment of individuals with active, infectious TB. In the WHO African Region, the incidence of multidrug resistant TB (MDR-TB) has tripled in the past 20 years and poses a major risk to regional TB control programs. Accurate, timely, and affordable drug susceptibility testing for patient management and in support of surveillance programs is urgently needed. In a politically unstable, high HIV prevalence region of southern Africa, we validated use of a low-cost, accelerated phenotypic method for MDR-TB detection, and provide the first report of the prevalence of MDR-TB from this country in 17 years.

Pertussis (whooping cough) is a respiratory infection caused by the bacterium Bordetella pertussis that results in prolonged cough illness often characterized by paroxysms, followed by an inspiratory “whoop”. Nearly all severe and fatal cases of pertussis occur in infants younger than three months of age, who are too young to be protected through active immunization. To help protect infants from pertussis during this vulnerable period in their first few weeks of life, women are recommended to receive a pertussis vaccine booster (Tdap) at the start of the third trimester of each pregnancy to optimize transplacental transfer of antibodies to the fetus. This recommendation was made by the Advisory Committee on Immunization Practices in 2013 based on immunogenicity data, and no studies in the United States have yet evaluated the effectiveness of this strategy in reducing pertussis incidence among infants.

The objectives of this research are to evaluate the effectiveness of prenatal Tdap vaccination in both preventing pertussis and reducing severity of illness in infants, characterize infants at greatest risk of pertussis in California, and identify risk factors for pertussis among Hispanic and non-Hispanic infants.

Chapter 1 summarizes the current literature and presents background information for pertussis and pertussis vaccines to provide the context for this research.

Chapter 2 evaluates the impact of prenatal Tdap vaccination on the risk of pertussis in infants within the first few weeks of life among the cohort of women who gave birth in California from January 1, 2013 through December 31, 2014. I demonstrate that Tdap vaccination received during pregnancy is 85% effective at preventing pertussis in infants younger than eight weeks of age compared to Tdap vaccination received postpartum. Additionally, I present data to support the current ACIP recommendation to administer Tdap between 27-36 weeks gestation.

Chapter 3 includes a retrospective evaluation of the cohort of infants in California who were reported with pertussis before three months of age from January 1, 2013 through December 31, 2014. I determine that infants whose mothers received Tdap vaccine during pregnancy had a lower risk of hospitalization or intensive care unit admission and had shorter hospital stays compared to infants born to unvaccinated mothers, indicating an impact of prenatal Tdap vaccination on pertussis severity. Tdap vaccination during pregnancy was determined to be 58% effective at preventing hospitalization among infants with pertussis.

Chapter 4 presents a case-control study in which I describe pertussis cases in infants younger than four months of age and evaluate maternal and infant characteristics associated with pertussis among Hispanic and non-Hispanic infants. Additionally, I evaluate the relationship between maternal parity, a proxy for the number of siblings, and risk of pertussis and determine that infants of all racial/ethnic groups whose mothers are of higher parity are more likely to be reported with pertussis in the first four months of life compared to first-born infants, with the odds of pertussis increasing with higher maternal parity. Case-infants of all racial/ethnic groups were also more likely to have younger mothers who were born in 1997 and later and who likely only ever received DTaP vaccines during childhood. Maternal age and parity largely explain the increased risk of pertussis observed among Hispanic infants.

Finally, chapter 5 summarizes the major findings of each chapter, provides a conclusion and suggests future directions.

The data used in this dissertation are both unique and robust as the dissertation includes matched surveillance, immunization and vital records data from the entire state of California for multiple years, capturing a peak in disease incidence. More pertussis hospitalizations and deaths have occurred in California in recent years than in any other state, so this is an ideal setting in which to evaluate severe cases of pertussis. Ultimately, this research will assist in targeting vaccination policies to prevent severe disease in the most vulnerable populations.

Dengue virus is a flavivirus of worldwide importance, with approximately 4 billion people across 128 countries at risk of dengue virus infection. Most cases present as classic dengue fever, a debilitating, but self-limited illness that manifests with high fever, retro-orbital pain, severe myalgia or arthralgia, and rash. However, in some cases, illness progresses to life-threatening dengue hemorrhagic fever or dengue shock syndrome. The identification of distinguishing clinical and laboratory features that occur during the early febrile phase of illness is important for developing a clinical prediction algorithm to differentiate dengue from other febrile illnesses, and severe dengue from mild dengue. In addition, the success of community-based programs for preventing dengue indicates that identifying environments that could benefit from intervention at a community level is critical in order to have the greatest impact and to target limited resources.

This dissertation focuses on data from two ongoing studies in order to investigate the epidemiologic, clinical and laboratory features of pediatric dengue in Nicaragua. Chapter 1 reports on the clinical and laboratory features of dengue virus infection in Nicaraguan children. The aims of the study were to examine the frequency of clinical signs and symptoms by day of dengue illness and to analyze the association of signs and symptoms with dengue virus infection during the early febrile phase of illness and over the course of illness.

Chapter 2 reports on the association of lower low-density lipoprotein cholesterol levels with severe dengue outcome. The aim of the study was to delineate the trajectories of cholesterol levels over time by dengue virus infection status in order to understand the effect of dengue virus infection on cholesterol metabolism. We also sought to delineate their trajectories by dengue severity and to assess the effect of cholesterol level at presentation on development of severe dengue.

Chapter 3 reports on individual-, household- and neighborhood-level determinants of dengue virus seropositivity in a community-based cohort. The aim of the study was to identify risk factors for DENV infection among children living in urban neighborhoods in Managua, Nicaragua. We also sought to determine the seroprevalence of dengue virus infection and identify individual- and household-level risk factors for dengue virus infection in neighborhood groups categorized by similar socioeconomic, infrastructural and ecological characteristics.

Together, these studies present important findings on the natural history of pediatric dengue in Nicaragua and provide the basis for future research to develop clinical prediction algorithms to discriminate dengue from other febrile illnesses, and severe dengue from mild dengue. They also reveal the importance of understanding neighborhood-level factors in targeting community-based programs to prevent dengue.

Cardiovascular Disease Outcomes from Use of Antiretroviral Agents Among HIV-infected Individuals

By

Kunchok Dorjee

Doctor of Philosophy in Epidemiology

University of California, Berkeley

Professor Arthur L. Reingold, Chair

Combination anti-retroviral therapy (ART) has improved the quality of life of Human Immunodeficiency Virus (HIV)-infected individuals, and has helped them to live longer. A better understanding of the adverse drug events associated with use of various antiretroviral (ARV) drugs can help in further improving the health outcomes of HIV-infected patients. In the past years, use of various individual and combinations of ARV agents have been shown to be associated with an increased risk of cardiovascular disease (CVD) among HIV-infected patients. Of the various ARV agents, the propensity of abacavir, a nucleoside reverse transcriptase inhibitor, to increase the risk of CVD has received particular attention because of its central role as an anchor drug in combination ART. This dissertation aims to investigate the risk of acute myocardial infarction (AMI) associated with use of various antiretroviral agents among HIV-infected individuals in the United States. It further presents the results of an in-depth analysis of the risk of CVD associated with use of abacavir among HIV-infected individuals. Additionally, the results of a systematic review and meta-analysis of epidemiologic studies that were carried out to assess the risk of CVD associated with abacavir use are also presented in this dissertation.

Because a number of covariates that could potentially confound the relationship between use of ARV agents and the risk of CVD may also lie on the causal pathway, and because these factors could potentially be influenced by receipt of the exposure in the past, marginal structural Cox proportional hazard models using stabilized inverse probability of treatment weights were used to investigate this relationship. Results from unweighted Cox regressions were additionally presented for comparison. For the systematic review and meta-analysis, random-effects models were used when there was heterogeneity of results across studies, and fixed-effects models were used when there was no heterogeneity. The fixed-effects and random-effects models were weighted by inverse of variance.

Chapter One of this dissertation describes the risk of AMI associated with use of several individual and combinations of ARV agents among HIV-infected patients receiving ART. The risk of AMI was elevated among HIV-infected individuals currently exposed to abacavir, lamivudine, didanosine, lopinavir, and darunavir. Of the ARV drug combinations, current exposure to combinations of abacavir+lamivudine+atazanavir, abacavir+lamivudine+darunavir, and tenofovir+emtricitabine+raltegravir was associated with an increased risk of developing AMI. There was a protective effect against AMI from exposures to tenofovir, emtricitabine, and efavirenz, as individual agents, and as a combination regimen. Chapter Two of this dissertation presents the results of a further investigation into the risk of ischemic CVD associated with use of abacavir among HIV-infected patients. The investigation showed an increased risk of CVD and AMI associated with current, recent, and cumulative exposure to abacavir. The results of a systematic review and a meta-analysis of the epidemiologic studies, presented in chapter three of this dissertation, confirmed that recent and cumulative exposure to abacavir were associated with an increased risk of CVD among HIV-infected individuals.

In sum, exposure to various individual and combinations of ARV agents was associated with an increased risk of CVD among HIV-infected individuals. Risk and benefits should be carefully weighed while formulating antiretroviral treatment regimens for HIV-infected patients. A trans-disciplinary approach in future studies with a translational focus to achieve a better understanding of the biological mechanisms underlying the observed risk of CVD from use of ARV agents shall help to inform future development of ARV drugs with better safety profile.

Pandemic influenza is currently one of the most visible public health threats of concern to the general public, and private businesses are an important part of pandemic preparedness. The health of communities is affected by the local economy, which is driven by the businesses in that economy. To date, public health authorities' efforts to engage businesses in pandemic influenza preparedness efforts have justified preparedness based on potential losses due to future, uncertain threats. However, this approach has not successfully engaged businesses on a broad scale. This dissertation proposes that a more effective way to engage the private sector may be to shift the conversation away from justifying preparedness only as a long-term insurance strategy and toward justifying it as an investment strategy with short-term benefits such as improved employee health during interpandemic cold and influenza seasons. The viability and acceptability of this new approach are explored here via three distinct but complementary studies using both quantitative and qualitative methods.

The first study, a prospective observational cohort study, examined the individual characteristics and situations that predicted changes in hand and respiratory hygiene and social distancing behaviors among university students during an interpandemic cold and influenza season. This analysis reveals that individuals have higher adherence to behaviors in situations such as when they are ill. Additionally, some individual characteristics predict higher behavior adherence. In particular, individuals who perceive peer expectations concerning adherence to hygiene behaviors tend to have better adherence to those behaviors over the course of a cold and influenza season.

The second study, a cost-effectiveness analysis of a hand and respiratory hygiene intervention among university students, assessed whether an intervention could be cost-effective in reducing influenza-like illness and associated time lost from productive activities during an interpandemic cold and influenza season. This analysis finds that hand and respiratory hygiene interventions can be cost-effective and may even become cost-saving during a severe cold and influenza season, especially using group-level interventions that may create peer expectations to influence behaviors.

Finally, the third study, an exploratory analysis based on key informant interviews with private sector business continuity managers, consultants, and public sector planners, examined private sector preparedness for pandemic influenza. This analysis assessed the key components of employer pandemic influenza preparedness plans, including whether short-term benefits are a consideration in business planning. The results indicate that the most important components of private sector pandemic influenza plans before and during the 2009 H1N1 influenza pandemic included communications and employee education around hygiene behaviors. Participants further identified that implementation of these initiatives during interpandemic cold and influenza seasons is of interest to organizations due to potential short-term and long-term benefits.

These results together provide evidence that education and provision of materials for hygiene behaviors at a group level can be cost-effective in reducing influenza-like illnesses during interpandemic cold and influenza seasons and are an acceptable strategy to the private sector. This provides a basis for the hypothesis that employer preparedness for public health events such as pandemic influenza can be justified as a short-term business investment strategy rather than only as a long-term insurance strategy.

Human immunodeficiency virus type 1 (HIV-1) exhibits extraordinary genetic diversity that is driven by high rates of mutation and recombination, coupled with elevated rates of viral turnover and the persistent nature of infection. Through these mechanisms, HIV-1 group M, the group largely responsible for the global pandemic, diversified into nine distinct subtypes and additional circulating recombinant forms. Subtype C HIV-1, which accounts for approximately 50% of the estimated 37 million individuals living with HIV/AIDS worldwide, predominates the epidemics in southern Africa and South / Southeast Asia. In contrast, subtype B HIV-1, predominant in the United States and Europe, comprises 12% of the global HIV-1 prevalence.

Current US and, increasingly, international guidelines recommend that HIV genotypic sequencing be performed for newly-identified HIV-positive patients, in order to identify genetic mutations that may confer drug resistance and pose a barrier to effective antiretroviral therapy (ART). This practice has resulted in the generation of enormous amounts of genotypic data often linked with clinical, demographic, and geospatial information. Analyses of these large datasets, utilizing sophisticated statistical and computational methods, has enabled identification of important and previously unrecognized trends in epidemiologic and vertical transmission, persistence of HIV within and among distinct risk groups, and the accumulation and propagation of ART resistance. Results of these analyses, in turn, advance treatment and care for persons living with HIV.

This thesis takes advantage of large, de-identified and anonymized datasets of HIV-1 genotypic and demographic information available through clinical testing and treatment programs in two distinct and epidemiologically important regions in the global HIV pandemic, northern California and southern Africa. Employing innovative and computationally intensive tools which combine experience from molecular biology, evolutionary biology and biostatistics, in-depth analyses were undertaken to explore and define risk factors, underlying epidemiologic features and clinical characteristics relevant to transmission and ART resistance. The final chapter provides a comprehensive literature review of the relevance of molecular epidemiologic principles and innovative biology to the complex and continued phenomenon of mother-to-child transmission of HIV, as a case study exemplifying the need for continued molecular studies grounded in solid epidemiologic principles. The findings presented here are ultimately intended to help guide domestic and global efforts to scale up ART treatment while considering well-known and lesser-known factors that may influence eventual clinical and epidemiologic outcomes.

Despite decades of prevention and treatment efforts, human immunodeficiency virus (HIV) infection is a global pandemic, with HIV/acquired immunodeficiency syndrome (AIDS) among the leading causes of death for children and adults in developing countries. HIV/AIDS disproportionately affects men who have sex with men (MSM), including in the United States, where MSM account for over half of all new HIV infections. Existing HIV prevention tools have not been sufficient to curb the pandemic, and biomedical prevention strategies, such as circumcision and microbicides, had not been shown to be effective in preventing HIV infection in MSM until 2010, when the multi-national iPrEx study found that daily oral preexposure prophylaxis (PrEP) using antiretroviral therapy (emtricitabine/tenofovir, or FTC/TDF) prevented HIV infection among MSM and transgender women.

Although the success of FTC/TDF PrEP in such trials has given new hope to the HIV prevention field, excitement has been tempered by concerns about low adherence to daily pill use among trial participants. Adherence, as measured by self-reported pill use, was high among participants in the iPrEx trial, but testing for evidence of the drug in blood specimens from a subset of participants indicated that actual pill use was much lower than reported pill use. Adherence is critical for PrEP effectiveness; iPrEx participants in whom antiretroviral drug was detected in blood experienced a substantially greater reduction in HIV risk than was seen overall. The iPrEx results suggest that low adherence may be a barrier to successful implementation of PrEP as an HIV prevention strategy among MSM. Identification of effective strategies for supporting the consistent use of prophylactic medications could provide much needed guidance in the development of interventions to support adherence to PrEP.

In addition to concerns about adherence, there is a widespread concern that PrEP could result in an increase in sexual risk behaviors by lowering users' perception of their risk of acquiring HIV infection; such risk compensation could reduce or even negate the benefits of PrEP. Foundational to most models of health behavior is the hypothesis that perceived risk is a primary motivation for self-protective behavior. Based on this theory, when a new prevention strategy, such as PrEP, is introduced, risk compensation could be prevented by sustaining individuals' perceptions of their own HIV risk. While the act of taking PrEP on a daily basis could result in risk compensation, it could also provide a daily opportunity to contemplate and manage HIV risk, thus motivating condom use and other self-protective behaviors. A better understanding of whether, and how, PrEP use affects sexual risk behavior could inform the development of risk-reduction interventions for individuals prescribed PrEP.

In addition to preventing HIV infection, there is evidence that FTC/TDF PrEP may also be protective against infection with Herpes simplex virus type 2 (HSV-2). HSV-2 is the primary cause of genital ulcer disease worldwide, and HSV-2 infection is known to increase the risk of sexual transmission and acquisition of HIV infection. In the CAPRISA 004 study, a topical 1% tenofovir gel used as PrEP against HIV infection also reduced the risk of HSV-2 acquisition by 51% in women. In the Partners PrEP study, oral FTC/TDF reduced HSV-2 acquisition by 35% among heterosexual couples. Protection against HSV-2 acquisition or expression could enhance the public health impact of PrEP, but it is unknown whether FTC/TDF has anti-herpetic properties in men who have sex with men.

The effectiveness of FTC/TDF PrEP in protecting against HIV infection depends on individual-level behaviors, including adherence and sustained risk reduction, as well as its effect on other sexually transmitted infections (STI) that increase the risk of HIV acquisition. Chapter 1 of this dissertation describes a systematic review that aimed to identify interventions that have been used to support adherence to daily oral medications prescribed to healthy or asymptomatic individuals. This review found evidence of interventions that demonstrated short-term improvements in medication adherence across a variety of prevention settings, thus identifying potential strategies for adherence support among PrEP users. Chapter 2 describes an analysis of data from the randomized phase of the iPrEx study to determine whether sexual risk compensation occurred among trial participants. Consistent with other PrEP studies, this analysis found no evidence of increased sexual risk behavior or STIs among participants who believed they had been assigned to the FTC/TDF arm and that the drug was highly effective. Finally, Chapter 3 describes an analysis that used data from the randomized phase of iPrEx to measure the effect of FTC/TDF on HSV-2 acquisition and expression among men who have sex with men. Although there was no effect of FTC/TDF on HSV-2 acquisition, participants receiving the active drug had a lower prevalence of herpetic ulcers than participants receiving the placebo. Taken together, these papers contribute to the HIV prevention field by providing guidance on behavioral support strategies for daily oral PrEP and identifying anti-herpetic properties of FTC/TDF that could increase its effectiveness as a novel prevention tool.

As of December 31, 2015, there were 15,995 diagnosed and reported persons living with human immunodeficiency virus (HIV) who were residents of San Francisco, California at time of diagnosis. Approximately one quarter of men who have sex with men (MSM) in San Francisco are HIV-positive. Despite a high HIV burden, a higher proportion of HIV-positive San Franciscans are virally suppressed (72% in 2014 in San Francisco) than nationally (55% nationally in 2013), and 81% of all cases in San Francisco had at least one HIV viral load or CD4 test in 2014 (versus 71% nationally in 2013). Recently, new strategies have emerged to prevent HIV transmission, including post-exposure prophylaxis, treatment as prevention and pre-exposure prophylaxis (PrEP). With these new tools, the end of the HIV epidemic is within reach. New HIV diagnoses in San Francisco are now half of what they were a decade ago, down from 528 new HIV diagnoses in 2006 to 255 in 2015. The Getting to Zero (GTZ) consortium, formed in San Francisco after World AIDS Day in 2013, which is comprised of representatives from the San Francisco Department of Public Health, the University of California San Francisco, public and private medical providers, community based organizations, other San Francisco government agencies and people living with HIV (PLWH), aims to get to zero new HIV transmissions, zero HIV deaths and zero HIV stigma in San Francisco. My dissertation is aligned with the mission of the GTZ consortium.

My dissertation seeks to address how the population dynamics of MSM (through migration and HIV serostatus) could affect the prevalence and incidence of HIV in San Francisco and may help explain why there is ongoing HIV transmission in the era of PrEP and treatment as prevention. The ability to migrate has increased for PLWH as survival markedly improved following introduction of combination antiretroviral therapy in 1996. Although there is a high frequency of migration among the general U.S. population and among PLWH in San Francisco, migration patterns of MSM in San Francisco have, to my knowledge, never been described. Output from the novel migration model outlined in Chapter 2 can be used to understand the dynamics of the MSM population in San Francisco and macro-level forces that could affect the prevalence and incidence of HIV in the population. Estimating the number of MSM by HIV status also allows researchers to have a denominator of this hidden population for use in estimating prevalence, incidence, service needs and funding allocations. Further, estimating the number of HIV-negative in-migrants relative to the overall MSM population is important because research has shown that recent MSM migrants to metropolitan areas are at increased risk of HIV acquisition because of higher risk behaviors. My results suggest that the overall MSM population and all the MSM subpopulations studied decreased in size from 2006 to 2014. Further, there were differences in migration patterns by race and by HIV serostatus.

Next, given the goal of eliminating all transmission of HIV, I assessed the association between knowledge of an HIV-negative partner’s PrEP use and reported condomless anal sex (CAS) among sexually active MSM in San Francisco. In 2010, the iPrEx trial showed that a daily dose of tenofovir disoproxil fumerate (TDF) and emtricitabine (FTC) for HIV-negative persons in HIV serodiscordant relationships could reduce HIV transmission by 42%. In 2012, the FDA approved daily FTC/TDF for HIV prophylaxis. Although no increase in CAS was observed in the iPrEx trial, there is concern that expansion of PrEP could result in reduced condom use. The study population for iPrEx, individuals who consented to participate in a randomized-controlled trial, likely did not represent the general population of MSM. Furthermore, participants knew that they could have been randomized to receive a placebo and, therefore, it is not surprising that in this setting, no increase in CAS was observed. Recent research has focused on PrEP use and CAS reported by HIV-negative individuals on PrEP; no research to my knowledge has focused on PLWH’s reported condom use during sex if they know that their HIV-negative partner is on PrEP. I found that there was a higher prevalence of reported CAS and insertive condomless anal sex (ICAS) in partnerships that were either seroconcordant or serodiscordant with PrEP, compared to partnerships that were serodiscordant without PrEP. There was evidence that men in this sample were adapting their condom use based on their sexual partner’s HIV status and PrEP use, and their own viral suppression status. Discordant partnerships with PrEP had an increased adjusted prevalence of reported CAS and ICAS.

I used causal inference methods to determine the effect of internalized HIV stigma (IHS) on viral suppression. Past research on IHS has focused on ART adherence as an outcome, and depression has been determined to be a mediator along this path. To my knowledge, no research has looked at the effect of IHS on viral suppression as an outcome. Research focusing on IHS in San Francisco has been limited to subgroups of HIV-positive individuals, such as homeless and marginally housed HIV-positive adults. Causal inference methods were used to estimate the counterfactual proportion of HIV-positive adults virally suppressed if all adults in HIV care in San Francisco did not experience IHS compared to the proportion of HIV-positive adults virally suppressed if all adults in HIV care in San Francisco did experience IHS, by following the causal roadmap. Three estimators were used to estimate the average treatment effect: simple substitution, inverse probability of treatment weighting, and targeted maximum likelihood estimation (TMLE). The results from each estimator were similar, and a statistically significant causal effect was observed for all. Using TMLE, the counterfactual proportion of adults virally suppressed would decrease by roughly 4.5% if all adults did not experience internalized HIV stigma as opposed to if all adults experience internalized HIV stigma.

Interactions between HIV infection and malaria in children living in sub-Saharan Africa in the era of widening access to improved interventions

by

Anne Frances Gasasira

Doctor of Philosophy in Epidemiology

University of California, Berkeley

Professor Arthur Reingold, Chair

Over 65% of the world's HIV-infected population lives in sub-Saharan Africa where 350 million people are exposed to infection with malaria. Thus any biological interactions between HIV and malaria would have major public health implications for this region. There is growing evidence that HIV infection leads to negative malaria outcomes and these effects may differ across different populations and epidemiologic settings. In non-pregnant adults and pregnant women, HIV infection increases the risk of asymptomatic parasitemia and clinical malaria. Additionally, in non-pregnant adults, the response to antimalarial therapy may be compromised by HIV infection. There are scanty data describing the effects of HIV infection on the risk of malaria and on malaria outcomes in children, the population at highest risk of clinical malaria and malaria mortality.

Over the last two decades, treatment and control of both HIV infection and malaria in sub-Saharan Africa have undergone major changes. There has been rapid scale-up of programs providing antiretroviral therapy (ART) and trimethoprim-sulfamethoxazole (TS) prophylaxis for HIV care and in parallel, wide scale implementation of malaria control interventions, such as artemisinin-combination therapies (ACTs) for treatment of uncomplicated malaria, indoor residual spraying, and widespread distribution of insecticide treated bednets. It is not clear, how these interventions singly or in combination might affect the interactions between HIV infection and malaria in different risk groups

The goal of this dissertation was to contribute to knowledge on the effects of HIV infection on malaria in children in the era of increasing access to improved HIV and malaria interventions. Data from HIV-infected and HIV-uninfected children enrolled in cohort studies in two settings of differing malaria transmission intensity in Uganda were utilized. The dissertation focused on the risk of malaria in HIV-infected children and antimalarial treatment outcomes in those with uncomplicated malaria. The objectives of studies that comprised this dissertation were 1) to assess the effect of daily TS prophylaxis on the risk of malaria in HIV-infected children, 2) to determine the association between daily TS prophylaxis and infection with antifolate-resistant malaria parasites, 3) to determine the risk factors for malaria in young HIV-infected children living in an area of high malaria transmission, and 4) to determine the efficacies and safety of ACTs for the treatment of uncomplicated malaria in HIV-infected children.

HIV-infected children receiving daily TS had an 80% reduced risk of clinical malaria compared to HIV-uninfected children not receiving TS. However, TS use was associated with infection with parasites with a rare mutation, dhfr 164L, known to mediate high-level antifolate resistance. Among HIV-infected children less than 38 months of age; low CD4 cell percentage, interruption of daily TS prophylaxis, and ART use were independent risk factors for malaria. We hypothesize that non-nucleoside reverse transcriptase inhibitors given as part of ART alter the metabolism of certain ACTs or TS leading to reduced malaria prophylaxis in those receiving these drugs concomitantly. Artemether-lumefantrine (AL), dihydroartemisinin-piperaquine (DP) and artesunate-amodaiquine (AS/AQ) were all 100% efficacious for malaria treatment, however compared to AL, AS/AQ was associated with a higher risk of neutropenia 45% vs. 17% (p=0.001), malaise 27% vs. 5% (p=0.010) and anorexia 24% vs. 5% (p=0.014). Adverse events associated with AS/AQ treatment were highest among those who were concomitantly receiving ART. In the setting of high malaria transmission intensity, DP was superior to AL in preventing recurrent parasitemia (7.1% vs. 34%, p <0.001).

Findings from these studies support the use of daily TS prophylaxis in HIV-infected children living in sub-Saharan Africa. However, there is need for continuous surveillance to monitor the emergence and spread of rare mutations that may be associated with widespread TS use. Artemether-lumefantrine and dihydroartemisinin-piperaquine appear to be safe and efficacious for the treatment of malaria in HIV-infected individuals, even among those concomitantly receiving ART. However the potential interactions between NNRTIs and other drugs given as part of HIV care and ACTs are an urgent concern. Studies in more diverse HIV-infected populations living in different malaria transmission settings are needed to confirm these findings.

Malaria and malnutrition are major causes of morbidity and mortality in children worldwide. Malnourished children may be at higher risk of malaria due to impaired immune response. Malnutrition and young age may alter the pharmacokinetics (PK) of antimalarial treatment thus potentially impacting treatment efficacy. Though malaria and malnutrition frequently coexist, results from previous studies that have investigated the association between these two co-morbidities are conflicting. No previous studies have evaluated the effect of malnutrition on response to treatment with artemisinin-based combination therapies (ACTs). Moreover, there are no other studies that have evaluated the effect of malnutrition on the PK of ACT regimens. This dissertation examines the following: 1) the magnitude of the difference between efficacy estimates derived from 3 analytical methods and discusses the optimal statistical approach for monitoring in vivo efficacy (chapter 2); 2) the effect of nutritional status on the response to treatment with artemisinin-combination therapy (ACT) in young Ugandan children with malaria (Chapter 3); and 3) the effect of nutritional status on the pharmacokinetics (PK) of two ACT treatment regimens (chapter 4).

Chapter 2 utilizes data from 29 clinical trials conducted in Africa and Thailand to compare the risk estimates of treatment failure, adjusted and unadjusted by genotyping, derived by 3 analytical methods; intention to treat (ITT), modified intention to treat (mITT) and per protocol (PP) analysis. Estimates of treatment failure were consistently higher when derived from the ITT or PP analyses compared to the mITT approach in both unadjusted and adjusted analyses. Poor patient adherence to follow-up, higher incidence of P. vivax relapse and high incidence of P. falciparum new infections were all factors contributing to differences in failure estimates. Because estimates of antimalarial clinical efficacy vary significantly depending on the analytical methodology from which they are derived, standardized analytical tools should be used to monitor temporal and spatial trends in antimalarial efficacy. Survival analysis is the preferred approach to monitor in vivo efficacy of malaria treatment.

Chapter 3 and 4 utilize data from the Tororo Child Cohort (TCC) Study conducted in Tororo, Uganda. In chapter 3, children aged 4 to 12 months diagnosed with uncomplicated malaria were randomized to either dihydroartemisinin-piperaquine (DP) or artemether-lumefantrine (AL) and followed for up to 2 years for repeated episodes of malaria. The primary exposure variables of interest were height-for-age (HAZ score) and weight-for-age (WAZ score) z-scores and outcomes included parasite clearance at day 2 and 3 and risk of recurrent parasitemia after 42 days of follow-up. HAZ and WAZ scores were not associated with a positive blood smear two days following treatment with DP or AL. In children treated with DP not on trimethoprim-sulfamethoxazole (TS) prophylaxis, a decreasing HAZ score was independently associated with a higher risk of recurrent parasitemia. However, statistical significance was reached only when comparing HAZ scores <-1 with those > 0 (HAZ ¡Ý -2 - <-1: HR=2.89, p=0.039; HAZ <-2: HR=3.18, p=0.022). Overall, DP and AL are effective antimalarial therapies in chronically malnourished children in a high transmission setting however, children taking DP with signs of mild to moderate chronic malnutrition not taking TS prophylaxis are at higher risk of recurrent parasitemia.

In chapter 4, PK samples were collected from a subset of patients ages 6 months to 2 years who were randomized to DP or AL and followed prospectively for multiple episodes of malaria providing a total of 214 treatments for DP and 243 treatments for AL for PK analysis. Primary exposure variables included stunting and underweight, (HAZ score of <-2 and WAZ score of <-2, respectively). Chronic malnutrition appeared to be associated with day 3 piperaquine concentrations in adjusted analyses with stunted children having lower concentrations than non-stunted children (OR=0.78, p=0.007). Stunting was associated with apparent clearance (CL/fpip) (OR=1.32, p=0.001) with stunted children having higher CL/fpip than non-stunted children which may be the consequence of a lower overall exposure to drug and is consistent with the lower piperaquine concentrations measured on day 3. Chronic malnutrition does not have an effect of piperaquine or lumefantrine concentrations at day 7 ¨C an important determinant for treatment response.

Overall, our results indicate that DP and AL are effective antimalarial treatments in very young chronically malnourished children. This is supported by the finding that chronic malnutrition does not have an effect on of piperaquine or lumefantrine day 7 concentrations, an indicator for treatment response. However, children taking DP not on TS prophylaxis may be at higher risk of recurrent parasitemia. Further studies should be conducted to justify these results and provide a definitive understanding of the causal relationship between malnutrition and malaria.