Nutritional zinc deficiency, immune capacity and malaria : a study on mediators of immunity to malaria caused by Plasmodium falciparum in African children

This thesis aimed at investigating the role of genetic and nutritional factors that affect the immune response to malaria in Tanzanian children. The introductory chapter (Chapter 1) reviews the importance of nutritional deficiencies, particularly of zinc, and presents the hypothesis that such deficiencies lead to impaired immunity and contribute to the burden of malaria. The chapter also describes current efforts to prevent malaria through intermittent preventive treatment, both in infants (IPTi) and pregnant women (IPTp). Sulfadoxinepyrimethamine is still used for first-line treatment of uncomplicated malaria, or, in many countries, to prevent malaria and anaemia in pregnancy. In malaria endemic areas, development of resistance to previously valuable antimalarial drugs has been continuously reported for decades. Thus our initial longitudinal study aimed at measuring the prevalence of resistance-associated mutations on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes (dhfr and dhps) that confer parasite resistance to sulphadoxinepyrimethamine (SP) that was used as an interim antimalarial drug after chloroquine resistance. Although SP resistance-associated point mutations were highly prevalent, we observed an adequate parasite response to SP (Chapter 2). We speculated that the impact of the dhfr and dhps mutations on SP resistance may be dependent at least in part on the protective immunity that has developed in response to frequent exposure to infection and may be weighed down by host immunity in endemic areas and thus impacts in the continued use of the drug for treatment of malaria. The impact of other drugs with similar mechanisms of action used as antibiotics in selecting mutations responsible for SP resistance needs therefore to be studied for their modulating activity of the immune response. These findings underscore the relevance to further study the crucial involvement of the immune system in the development of protection against malaria but also affecting the efficacy of treatment modalities of malaria in various African conditions. In the subsequent cross-sectional studies, we assessed the effect of deficiencies of zinc and magnesium as well as iron deficiency anaemia on malaria-specific cytokine responses indicative of innate immunity to Plasmodium falciparum (Chapter 3). In this study, we used Plasmodium falciparum-parasitised red blood cells (pRBCs) as antigens for in vitro stimulation of peripheral blood mononuclear cells (PBMCs). Cytokines were measured in the supernatant of cultured PBMCs after 24 hours of stimulation. Zinc deficiency was associated with a marked increase in monocyte-derived TNF-α concentration in children with malarial infection but not in their uninfected peers. In children with malarial infection, iron deficiency anaemia was associated with elevated concentrations of TNF-α, whereas magnesium deficiency in children without malaria seemed to be associated with increased concentrations of IL-10. Our findings reflected plasticity in cytokine profiles of monocytes reacting to malaria infection under conditions of different nutrient deficiencies. Following the observation of the variable impact of micronutrients on innate cytokines, we evaluated the profile of both type I and type II cytokines and whether they were influenced by nutritional and malaria status (Chapter 4). The cytokine measurements were performed at day 7 of stimulation anticipating that this timing was optimal for measuring effects on these cytokines mainly derived from activated T-cells. The results indicated a variable influence of nutrient deficiencies on increased cytokine response with zinc deficiency and iron deficiency anemia having greater impact on type I and magnesium deficiency on type II cytokines. The subsequent study evaluated the plasma levels of naturally acquired antimalarial antibodies of variousIgG subclasses plus the total IgG and IgM levels and whether they were associated with zinc deficiency based on preceding chapters (Chapter 5). The results indicated a high variability in antibody levels with zinc deficiency, varying with age of the affected child. IgG3 appeared to be predominant across all age subgroups within These studies demonstrate that despite antimalarial drug resistance, there is a potential for optimizing the immunological protective capacity in the population to confer parasite clearance that can be variably influenced by micronutrient status. Improving nutritional status in this population could be rewarding not only to increase protection to malaria but possibly also to other infections. <br/

Optimization of a protocol for extraction of Plasmodium falciparum RNA from infected whole blood samples for use in DNA microarrays

This study was carried out to determine the efficiency of two reagents, RNAlater and RNAwiz, for their ability to stabilize Plasmodium falciparum RNA in infected whole blood and saponin lysed parasitepellets for use in DNA microarrays. Eight infected blood samples were stored in each of the reagents, and RNA extracted at days 0 and 56 post collection. RNA yields and quality were compared at thedifferent time points between the two test reagents. We show that for both reagents, higher RNA yields and quality is obtained when RNA is isolated immediately after sample collection (day 0), however,results show that RNAwiz storage provides a marginally higher RNA yield compared to RNAlater storage. Our results indicate that whole blood gave slightly higher RNA yields with superior quality ascompared to saponin lysed samples when such whole blood samples are stored in RNA wiz, but not in RNAlater. From our results, we recommend RNAwiz as a better reagent for use in storage of wholeinfected blood intended for extraction of P. falciparum RNA for DNA microarrays and other sensitive techniques

THE CONTRIBUTION OF PARENTAL SOCIO-ECONOMIC STATUS ON PUPILS' KCPE PERFORMANCE IN MWEA-EAST DISTRICT, KIRINYAGA COUNTY, KENYA

The purpose of this study was to investigate parental socio-economic contribution and its implications on pupils’ Kenya Certificate of Primary Education (KCPE) performance in primary schools in Murinduko Zone, Mwea-East District. The objectives of the study included: to find out the extent to which the parents' level of income affects pupils performance, to analyze the effects of parents' level of education on pupils' KCPE performance in Murinduko zone, to investigate the manner in which the parents' political involvement affects pupils' KCPE performance in Murinduko Zone. The study employed descriptive research design while stratified random sampling was used to collect data. The data collection instruments were questionnaires, interview schedules and observations. The data was analyzed qualitatively and quantitatively using SPSS statistical package. The study was modeled by Abraham Maslow's hierarchy of need theory. The study established that most of the learners hailed from poor backgrounds and hence affected the KCPE performance. Most of the parents had attained only the primary level of education and were primary school drop-outs. Political wrangles between parents and the administration also affected the performance of pupils. Child labour is very prevalent and had a very great influence in the KCPE performance where children are exposed to it in order to subsidize family income. The main recommendations include; the government should continue offering Free Primary Education (FPE) and increase funding for building facilities like computer laboratories and libraries, public private partnership should be encouraged to increase resources in the primary education sector, the government should also consider giving stipends to pupils in hard to reach areas such as those in slums and marginalized zones this area so as to reduce drop-out rates and boost participation rates, need to enact and enforce legislation so as to curb child labour.  Article visualizations

Multiplicity of infections and level of recrudescence in Plasmodium falciparum malaria in Mlimba, Tanzania

Polymorphism and antigenic variation are important biological survival strategies of malaria parasites determining the episode, outcome and implications of treatment interventions. In P. falciparum, polymorphic antigens are associated with the asexual blood-stage; merozoite surface protein 2 (MSP2). The MSP2 genes have been invaluable in post-treatment discrimination of parasite resurgence fromnew infection, especially in high transmission areas. We performed polymerase chain reaction (PCR) on DNA extracted from blood samples of 141 malaria-infected infants, followed by restriction fragmentlength polymorphism (RFLP) of PCR products. The findings showed multiplicity of infections of single to six infections with an average of 2.58 infections per patient. Single infections of either 3D7 or FC27allelic families of the MSP2 gene occurred in 51 patients (50.5%) out of all PCR-RFLP successful samples (n = 101). Out of 15 (10.6%) follow up samples with resurgent parasitaemia, 3 (20%) sampleshad recrudescent infections while 12 (80%) had variable results. Our findings provide an insight on the prevalence of the genetic determinants of suphadoxine-pyrimethamine (SP) resistance in Mlimba during the study period, and in the face of rapidly spreading resistance, calls for the periodic surveillance in order to timely detect early warning signal of the deteriorating SP cure rate

Intronic variants in the long non-coding RNA CDKN2B-AS1 are strongly associated with the risk of coronary artery disease in the Northern Tribes of Tanzania

Introduction: Sub-Saharan Africa (SSA) is facing a rising epidemic of non-communicable diseases including the coronary artery disease (CAD) ranking at the top of the list. Chromosome locus 9p21.3 containing CDKN2B antisense RNA 1 (CDKN2B-AS1), identified in many genome-wide association studies for coronary artery disease (CAD), encompasses multiple single nucleotide polymorphisms (SNPs). This study aimed to conduct the first genetic study evaluating the common polymorphisms in 9p21.3 locus in Tanzanian CAD patients from different regions of Tanzania and their associations with CAD risk factors. Material and Methods: A total of 90 patients from Northern region (N-CAD) of Tanzania and 65 patients from other regions (South, East, West and Central) (R-CAD) were included in the study. Further the biochemical analysis the genotyping of common variants was performed with the LightSNiP typing assay using qRT-PCR method.&nbsp; Results: Our analyses revealed that both genotype and allele frequencies of rs10757274, rs10757278 and rs10811656 were significantly different between the groups (p&lt;0.05, respectively). We identified that one previously undescribed three-marker haplotype (rs1333049, rs10757274 and rs10757278) encompassing CDKN2B-AS1 was overrepresented (G-G-G, the risk haplotype, p&lt;0.05) in N-CAD group compared to R-CAD group. The AUC of a risk model based on non-genetic factors was 0.730 (0.654-0.797) and the combination with genetic risk factors improved the AUC to 0.784 (95%CI=0.713-0.844, p&lt;0.0001). Conclusion: Our results identified the presence of a novel three-marker haplotype having a significant association with CAD in Northern Tanzania. Moreover, combination of the nongenetic and genetic risk models were demonstrated to indicate good diagnostic accuracy for CAD in Northern Tanzania

Effect of Preventive Supplementation with Zinc and other Micronutrients on Non-Malarial Morbidity in Tanzanian Pre-School Children: A Randomized Trial.

The efficacy of preventive zinc supplementation against diarrhea and respiratory illness may depend on simultaneous supplementation with other micronutrients. We aimed to assess the effect of supplementation with zinc and multiple micronutrients on diarrhea and other causes of non-malarial morbidity. Rural Tanzanian children (n = 612) aged 6-60 months and with height-for-age z-score < -1.5 SD were randomized to daily supplementation with zinc (10 mg) alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Children were followed for an average of 45 weeks. During follow-up, we recorded morbidity episodes. We found no evidence that concurrent supplementation with multi-nutrients influenced the magnitude of the effect of zinc on rates of diarrhea, respiratory illness, fever without localizing signs, or other illness (guardian-reported illness with symptoms involving skin, ears, eyes and abscesses, but excluding trauma or burns). Zinc supplementation reduced the hazard rate of diarrhea by 24% (4%-40%). By contrast, multi-nutrients seemed to increase this rate (HR; 95% CI: 1.19; 0.94-1.50), particularly in children with asymptomatic Giardia infection at baseline (2.03; 1.24-3.32). Zinc also protected against episodes of fever without localizing signs (0.75; 0.57-0.96), but we found no evidence that it reduced the overall number of clinic visits. We found no evidence that the efficacy of zinc supplements in reducing diarrhea rates is enhanced by concurrent supplementation with other micronutrients. By reducing rates of fever without localizing signs, supplementation with zinc may reduce inappropriate drug use with anti-malarial medications and antibiotics. ClinicalTrials.gov NCT00623857

Potential of genomic approaches in conservation of plant and animal biodiversity in Africa: A review

In Africa, status of biodiversity conservation of many plants and animals is questionable as this is considered to be caused by limited and lack of authentic information concerning genetic diversity. This has led to a considerable compromise of conservation decisions in Africa. As a result, lack of reliable information continues to cause a great effect on the long-term security of species of plants and animals. Current advancement in genomics has proved to play a vital role in conservation of plant and animal biodiversity. It produces genetic data that helps researchers to understand the interaction between ecosystem and organisms, also among organisms themselves. The information extracted from plants and animals via genomics techniques can be used to develop good approaches for biodiversity conservation. Despite its usefulness, there is a limited awareness on the application of potential genomics in plants and animals conservation in many developing countries, especially in Africa. The aim of this review is to raise awareness and catalyse the application of genomics techniques in rejuvenation and conservation of plants and animals in Africa. Precisely, the paper addresses the efficacy of potential genomics in plants and animals conservation; and seeks to show how Africa can benefit from genomics technology. About 62 peer-reviewed articles were reviewed. This current review has shown that genomics helps to identify good genes for fitness, and develops tools to monitor and conserve plants and animals biodiversity. The review recommends that regardless of the limitation of genomics application in biodiversity conservation in Africa, African researchers must consider using this technology for better conservation of plants and animals biodiversity

Are we prepared for emerging and re-emerging diseases? Experience and lessons from epidemics occurred in Tanzania during the last five decades

This paper reviews preparedness for containing and controlling emerging and re-emerging diseases drawing lessons from disease events that occurred in animal and human populations in the last five decades (1961-2011). A comprehensive analysis based on retrieval and analysis of grey and published literature as well as reported cases was carried out to document type and trend of occurrence of emerging and re-emerging infectious diseases in different parts of Tanzania. Overall, the majority of diseases reported in the country were viral in nature followed by bacterial diseases. The trend for the occurrence shows a number of new emerging diseases as well as re-occurrence of old diseases in both animal (domestic and wild) and human populations. In humans, the major disease epidemics reported in the last five decades include cholera, influenza A H1N1, plague and rubella. In animals, the major epidemic diseases reported were Contagious Bovine Pleuropneumonia, Contagious Caprine Pleuropneumonia, Peste des petits ruminants and Giraffe Ear and Skin Diseases. Some epidemics have been reported in both human and animal populations including Rift Valley fever and anthrax.&nbsp; The emergence of the &lsquo;fit-for purpose&rsquo; approaches and technologies such as the discipline of One Health, use of participatory epidemiology and disease surveillance and mobile technologies offers opportunity for optimal use of limited resources to improve early detection, diagnosis and response to disease events and consequently reduced impact of such diseases in animal and human populations

Insecticidal decay effects of long-lasting insecticide nets and indoor residual spraying on Anopheles gambiae and Anopheles arabiensis in Western Kenya

BackgroundIndoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are the first-line tools for malaria prevention and control in Africa. Vector resistance to insecticides has been extensively studied, however the insecticidal effects of the nets and sprayed walls on pyrethroid resistant mosquitoes has not been studied thoroughly. We evaluated the bioefficacy of LLINs of different ages and lambda-cyhalothrin (ICON 10cs) on the sprayed mud walls for a period of time on malaria vector survivorship.MethodsWHO tube bioassay was performed using diagnostic doses of lambda-cyhalothrin (0.05%), permethrin (0.75%) and deltamethrin (0.05%). Cone bioassays were conducted on netting materials from 0 to 3 years old long-lasting insecticide-impregnated nets. Wall bioassays were performed monthly on mud slabs sprayed with lambdacyhalothrin over a period of seven months. All bioassays used An. gambiae mosquitoes collected from the field and the laboratory susceptible reference Kisumu strain. Concentration of the insecticides on the netting materials was examined using the gas chromatography method. Mosquitoes were identified to species level using PCR and genotyped for the kdr gene mutation frequencies.ResultsWHO bioassays results showed that populations from five sites were highly resistant to the pyrethroids (mortalities ranged from 52.5 to 75.3%), and two sites were moderately resistant to these insecticides (80.4 - 87.2%). Homozygote kdr mutations of L1014S ranged from 73 to 88% in An. gambiae s.s. dominant populations whereas L1014S mutation frequencies were relatively low (7-31%) in An. arabiensis dominant populations. There was a significant decrease (P &lt; 0.05) in mosquito mortality with time after the spray with both lambda-cyhalothrin (75% mortality after six months) and with the age of LLINs (60% mortality after 24 month). Field collected mosquitoes were able to survive exposure to both IRS and LLINs even with newly sprayed walls (86.6-93.5% mortality) and new LLINs (77.5-85.0% mortality), Wild mosquitoes collected from the field had significantly lower mortality rates to LLINs (59.6-85.0%) than laboratory reared susceptible strain (100%). Insecticide concentration decreased significantly from 0.14 μg/ml in the new nets to 0.077 μg/ml in nets older than 18 months (P &lt; 0.05).ConclusionThis study confirms that insecticide decay and developing levels of resistance have a negative contribution to reduced efficacy of ITN and IRS in western Kenya. These factors contribute to decreased efficacy of pyrethroid insectides in ongoing malaria control programs. In order to mitigate against the impact of insecticide resistance and decay it is important to follow the WHO policy to provide the residents with new LLINs every three years of use while maintaining a high level of LLINs coverage and usage. There is also need for urgent development and deployment of non-pyrethroid based vector control tools