Derecho a la salud

The research undertaken in this dissertation was designed to expand upon epidemiological findings of reduced malaria infection in people with iron deficiency and potentially increased malaria susceptibility following iron supplementation, attempting to understand this relationship on a cellular level.

This was initiated with a study enrolling people from UNC hospitals who were iron deficient or iron replete and using their RBCs to conduct in vitro assays comparing various aspects of parasite growth (Chapter 2). We determined that parasite growth was significantly reduced in RBCs from iron deficient individuals, and we went on to characterize this reduced growth as involving both reduced invasion into as well as replication within iron deficient RBCs, but not altered parasite maturation. In RBCs from iron deficient and iron replete donors who received iron supplementation, there were no differences. We further characterized increased parasite growth and invasion in young RBCs and put forth a model hypothesizing that RBC population dynamics influenced malaria susceptibility, not just simple iron withholding as is the case with many other pathogens such as bacteria. In our model, presence of iron deficient RBCs limits infection potential due to their reduced susceptibility to parasite infection, and when someone receives iron they begin to erythropoiese at elevated rates, such that they have a much higher level of young RBCs in circulation which are highly susceptible to malaria infection. Above-average numbers of young RBCs and replacement of protective iron deficient RBCs would thus constitute a period of increased malaria risk before finally, RBC populations would normalize as would infection

susceptibility.

In anticipation of testing this model using RBCs from relevant populations in malaria endemic areas, we next sought to develop a method for preserving valuable RBCs to allow for prolonged usage in in vitro malaria experiments (Chapter 3), as established malaria culture protocols require freshly drawn RBCs (utilized within 2-4 weeks from blood draw). We first characterized growth, invasion, and replication

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within RBCs stored over time, confirming significant reductions in parasite growth and invasion in RBCs stored longer than 2 weeks. We tested several different RBC storage medias, but did not find any improved RBC viability. However, we did find that biopreservation of RBCs in liquid nitrogen provided a means for preserving RBCs for prolonged periods without any detriment to in vitro parasite growth indices.

We next conducted a field study partnering with clinical trial research of iron supplementation in anemic Gambian children aged 6-24 months (Chapter 4). We attempted to better characterize the degree of protection from iron deficiency as well as changes in RBC population dynamics following iron

supplementation that influence parasite growth. This work was all conducted using in vitro malaria culture as a proxy measure of malaria susceptibility, both because clinical malaria incidence is very low in The Gambia currently, and because this method provided a better means of systematically measuring malaria infection potential without biases from anti-malarial preventative measures (provided to trial participants in accordance with ethical safety precautions). We determined that RBCs from anemic children significantly reduced parasite growth and invasion, and that parasite growth rates specifically correlated with

hemoglobin concentration within our study population. In fact, within our population of anemic children, the parasite growth reduction was on par with or greater than growth reduction observed in RBCs from those children with sickle-trait. Following iron supplementation, we observed significant increases in parasite growth and invasion in subject RBCs, over and above growth in non-anemic controls. These growth changes paralleled increases in circulating reticulocytes and other measures of young RBC populations following iron supplementation, fitting with our proposed model of how RBC population structures might influence overall infection susceptibility.

Briefly, regarding the data from this study, it should be mentioned why we have ultimately focused on anemia (as defined by hemoglobin values <11g/dl) and thus hemoglobin as the primary variable influencing parasite growth, as opposed to the degree of iron deficiency – especially when we have spent so much time throughout this dissertation discussing the importance of iron deficiency specifically. It is true anemia can have multiple causes beyond iron deficiency and the term anemia is less specific than iron deficiency anemia. There are a number of reasons for our shift towards using the term anemia. First, in our linear regression modeling examining which variables correlated with parasite growth rates, we did

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not find any measurements relating to iron status were significant (i.e. ferritin, serum iron, soluble transferrin receptor (sTfR), hepcidin, transferrin saturation). We believe this is due to the second point – that it is notoriously difficult to measure iron deficiency in populations with high levels of inflammation, such as seen with children exposed to many infectious organisms and less sterile environments (as is common in developing countries). This influences accurate measurement of iron status because several markers of iron status are also acute phase proteins that rise with inflammation, thus falsely portraying the true iron status of an iron deficient individual simultaneously experiencing inflammation.

Understanding this point, it is not surprising that we found no significant correlation of parasite growth rates with iron markers, because so many of the children in our study do have high levels of inflammation (specifically, 24.9% had CRP >5mg/dl at baseline and 61.5% had AGP levels >1 at baseline). Multiple definitions of iron deficiency anemia have been proposed to more accurately measure iron status in the presence of inflammation. We thoroughly examined the data our study population to enumerate the number of children fitting into several well-known or practical definitions of iron deficiency anemia (based on ferritin, CRP, hepcidin, and Hgb response to iron supplementation) and consistently found that approximately 60% of our population had iron deficiency based on these measurements (discussed in Chapter 4). However, taking into account other very relevant variables, nearly every child could be categorized as having iron deficiency anemia specifically. In particular, we found MCV to be below normal levels (<70fL) in 83.8% of the population at baseline and sTfR:log ferritin ratios to be >2 in every single participant (ratios above 2 being highly indicative of iron deficiency anemia as soluble transferrin receptor levels rise in the presence of iron deficiency and ferritin levels drop). Thus, we remain confident in saying that our study population of anemic children was in fact suffering from iron deficiency anemia. This discussion of measurements of iron deficiency anemia closely relates to the final point of why we focused on using the terms hemoglobin and anemia as impacting parasite growth rates in presentation of our results – because hemoglobin is a simple measurement which allows for very practical and easy translation to the field setting in malaria endemic areas where it would be desirable to predict malaria susceptibility and the impact of iron supplementation. Hemoglobin measurements can be made on-the- spot with relatively simple and inexpensive diagnostic tools, rather than the complex and expensive diagnostic machines required for measuring complete iron panels which are nearly entirely absent from

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the developing world with the exception of research laboratories. Our results show that hemoglobin measurement alone is sufficient to predict malaria susceptibility, as measured by our in vitro growth data, thus providing a more practical option for researchers and clinicians who have continuously struggled to measure iron deficiency and understand the relationship between iron status markers and malaria susceptibility.

Finally, we also partnered with a clinical trial of iron supplementation in pregnant women, in order to investigate the same malaria-related safety questions in this population (Chapter 5). Not all participants were anemic, but we still found significantly reduced parasite growth in RBCs from study subjects at baseline, compared to growth in RBCs from non-anemic, non-pregnant donors. Again, parasite growth rates correlated significantly with hemoglobin concentration, and again, the growth reductions seen with anemia status were on par with growth reductions observed in RBCs from donors harboring the sickle- trait genotype. We attempted to further characterize mechanisms of reduced invasion into RBCs from anemic donors, but did not find the invasion deficit was due to either specific PfRh proteins being able to “identify” differences in iron replete versus iron deficient RBCs, nor to merozoite attachment differences. Yet again, we observed significantly elevated parasite growth rates in subject RBCs following iron supplementation with growth rate changes paralleling increases in young RBC populations in circulation. Analyses for the pregnant women iron supplementation study are ongoing, but preliminarily they clearly match our findings from the study involving anemic children undergoing iron supplementation and also fit our larger model describing the relationship between iron deficiency, iron supplementation, and malaria susceptibility.