Los antimaláricos actuales son producto de la investigación extranjera en países donde la malaria fue superada y sus líneas de investigación se dirigen a otros campos, por ello el Laboratorio de Investigación de Productos Naturales Antiparasitarios de la Amazonia (LIPNAA) de la Universidad Nacional de la Amazonia Peruana (UNAP) ubicado en una zona endémica de malaria, ha establecido una línea de investigación para la búsqueda, obtención y validación de nuevos agentes antimaláricos; en base a información etnobotánica de la selva amazónica que alberga las 2/3 partes de la biodiversidad del mundo. En el Perú existen 84 de las 117 zonas de vida del mundo y se tienen reportadas en el LIPNAA 170 especies de uso antimalarico por las etnias. En el presente trabajo de investigación se presentan los resultados correspondientes al estudio fitoquímico de la corteza de raíz de la especie amazónica Aspidosperma desmanthum Benth. ex Müll. Arg. de la familia Apocynaceae, de uso tradicional como antimalárico.

Aufbauend auf einen Datensatz von etwa 70 antimalaria-aktiven Verbindungen wurde mit Hilfe des CoMSIA-Verfahrens ein QSAR(Qantitative Structure Activity Relationship)-Modell erstellt, das in der Lage ist antiplasmodiale Aktivitäten von Verbindungen aus der Substanzklasse der Naphthylisochinolin-Alkaloide vorherzusagen. Da die behandelten Strukturen ein sehr kompliziertes konformatives Verhalten aufweisen, mussten für ein möglichst flexibles Alignment (unter Verwendung von FLEXS und GASP) eigene Abläufe entwickelt werden, die schließlich weitestgehend automatisiert werden konnten. Das erstellte Modell erlaubte es darüber hinaus, die für die Aktivität verantwortlichen strukturellen Merkmale zu identifizieren und so entscheidende Anregungen zur Vereinfachung des relativ komplizierten Grundgerüsts zu geben. Die Vorschläge wurden zu einem großen Teil bereits synthetisch verwirklicht, wobei die anschließend experimentell gefundenen Aktivitäten die vorher berechneten sehr gut bestätigten. Die neu entwickelten Substanzen befinden sich derzeit im Patentprüfungsverfahren. / Based on a dataset of about 70 antimalarially-active compounds a QSAR (Qantitative Structure Activity Relationship) model to predict antiplasmodial activities of aphthylisoquinoline alkaloids was developed, using the popular CoMSIA procedure. Since the structures under interest show a quite complicated conformational behaviour, the application of flexible alignment approaches (using FLEXS and GASP) hat to be developed and could be automated to the greatest possible extend. Additionally the QSAR made it possible to identify the structural features responsible for biological activity and decisive clues to simplify some of the quite complicated structural features of the substance class could be given. Some of the proposals made could already be synthetically realized and the experimental activities found confirmed the calculated ones quite satisfactorily.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Biology and Control of African Disease Vectors. Johannesburg, February 2012 / In Tanzania both Anopheles funestus and An. gambiae s.l. play a role as major vectors of malaria. Different species exist in the An. funestus group and the An. gambiae complex and play different roles in disease transmission. For malaria vector control programmes knowledge of vector species and their behaviour is key. A recent report on increased exophagy of An. funestus in southern rural Tanzania as a response to increased use of insecticide treated bed nets raised the question of whether there was misidentification of species and/or behavioural insecticide resistance playing a part. The present study used molecular tools to identify the species and determine human biting rates indoors and outdoors along with development and field evaluation of a new tool for sampling malaria vectors which is more effective than human landing catches. The results showed that the majority (96.2%) of the An. funestus group that were collected were An. funestus s.s. by PCR assay. Also, the exophagic proportion (45.9%) of An. funestus was lower than the endophagic proportion (54.1%), similar to other places in Africa, although in this study the difference was insignificant when untreated bed nets and treated bed nets were used. In addition, there was significant outdoor biting activity early in the evening that could lead to the malaria transmission cycle being unaffected by ITNs. The new trap, “Sticky Bucket Trap”, caught considerably fewer mosquitoes (14.2%) than that caught by human landing catches (85.8%), with statistical significance of P>0.05. These results imply that the sticky bucket trap is not a suitable substitute for human landing catches and some modifications are needed to make it more effective. Whereas indoor and outdoor proportions insignificant difference in feeding preference imply that both indoor and outdoor interventions should be used to control this vector.

ABSTRACT Introduction Under-five mortality is still a major cause of concern in Sub Saharan Africa and among the highest in the world. This is also exacerbated by the high prevalence and episodes of malaria in this age group, which accounts for 90% of all under-five deaths estimated in the region annually. The effect of detecting clustering of all cause and cause specific mortality and underlying factors is crucial for timely public health interventions. This is especially important for health authorities in Tanzania where under-five malaria attributable deaths accounts for 45% of the annual estimated mortality of 100, 000. Study objectives To estimate under-five mortality and analyze clustering of all cause and malaria specific mortality among under five children in Ifakara Demographic Surveillance System from 2002-2005. Methods Data from the Ifakara Health Research and Development Centre (IHRDC) were obtained for all under-five children who lived in 25 villages in the DSS from 2002 – 2005. Analyses for all cause and malaria cause specific under-five mortality were done using data collected from the DSS and verbal autopsy systems. Annual all cause and malaria specific mortality rates were calculated by dividing number of deaths and person years observed. Clustering of deaths for all cause and cause specific (malaria) in the 25 villages were analyzed using SaTScanTM version 7.0 software. A Poisson model was used to detect clusters with high rates in space and in space-time. Household assets and characteristics were used to construct a wealth index using Principal component analysis (PCA) in StataTM version9. The index was used to group households into five equal groups from poorest to least poor. Results Overall infants’ mortality was sixty-three times higher (326 per 1,000 person years) compared to children (5.1 per 1,000 person years) and with mortality rates between girls and boys were very similar, (15.8 and 14.8 per 1,000 person years). Year of death and place of death (village) were found to be significantly associated with malaria deaths. However, socio-economic status of parents in households where deaths occurred was not associated to malaria deaths in the DSS. A number of statistically significant clusters of all cause and cause specific malaria deaths were identified in several locations in the DSS. The located clusters imply that villages within the clusters have an elevated risk of under-five deaths. A space-time cluster of four villages with radius of 15.91 km was discovered with the highest risk (RR 2.71; P-value 0.020) of malaria deaths in 2004. Conclusion These findings demonstrate that there is non-random clustering of both all cause and malaria cause specific mortality in the study area. The high infant mortality results also suggest a careful examination of the data collection procedures in the DSS and require further studies to understand this pattern of mortality among the under-five population. Appropriate health interventions aimed at reducing burden of malaria should be strengthened in this part of rural Tanzania. There is need to replicate this study to other areas in the country.

Malaria is caused by Plasmodium parasites and is the world’s most devastating tropical infectious disease. The need for identifying novel drug targets is fuelled by an increased resistance of these parasites against available drugs. The human host red cell membrane plays an important role during invasion and subsequent development of the parasite within the red cell and undergoes several structural, functional and biochemical changes triggered by various protein-protein interactions between the parasite and the host cells. These interactions form a fundamental part of malaria research, since the parasite spends the pathogenic stage of its life cycle in the human erythrocyte. The Plasmodium kinome is complex and the exact role of protein phosphorylation in malaria parasites is not yet fully understood. This study aims to characterise the kinase domain of Plasmodium falciparum (3D7) Protein Kinase 8 (PfPK8), described as a putative protein on the Plasmodium falciparum database. PfPK8 is encoded by the PfB0150c gene (recently renamed as PF3D7_0203100) situated on chromosome 2 of the parasite genome. A 1 507bp section of the PfB0150c gene, containing a 822bp centrally located kinase domain was cloned into a pTriEx-3 expression vector. A soluble recombinant octa-histidine-tagged PfPK8 was expressed in Escherichia coli Rosetta 2 (DE3) cells, but with relatively low yield and purity.To improve the expression, a recombinant PfB0150c-baculovirus infected Spodoptera frugiperda (Sf9) insect cell system was attempted, but without success. A different tag was employed and glutathione-S-transferase-PfPK8 was successfully expressed in Escherichia coli Rosetta 2 (DE3) cells, with a higher yield and purity. Recombinant GST-PfPK8 was used in non-radioactive coupled spectrophotometric kinase assays in the presence of known kinase substrates casein, MBP and H1 to determine kinetic parameters of the enzyme. It phosphorylated all three substrates at a temperature of 37ºC and pH of 7.4. Recombinant GST-PfPK8 was inactive at a pH below 6 and most active at pH 7.4. The relative activity of the enzyme was highest at a temperature synonymous to a fever spike in a Plasmodium falciparum infected individual. Secondary structural analysis of PfPK8 revealed the position of a conserved substrate binding domain containing an ATP-binding site and binding loop within the kinase domain. The kinase domain of rPfPK8 was modelled using available crystal structures of its identified homologues. The gene is expressed throughout the intraerythrocytic stages of the parasite life cycle, as well as in gametocytes. Protein-protein binding studies revealed that host-parasite protein-protein interactions exist between rPfPK8 and erythrocyte membrane protein, band 3. Plasmodium falciparum PK8 could therefore play a role during invasion of host erythrocytes and during the intraerythrocytic development of the parasite, by phosphorylating red blood cell membrane proteins. This study provides the groundwork for future X-ray crystallographic studies to elucidate the structure of the enzyme, and for additional gene manipulation experiments to ascertain whether it is essential for parasite survival in all the intraerythrocytic stages and therefore a potential new drug target candidate.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg,in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, South Africa, 2015 / Plasmodium falciparum remains the most virulent cause of malaria. With increasing drug resistance to artemisinin and other antimalarial drugs, combined with an absence of an effective vaccine, there’s a critical need for new agents to complement existing treatment and prophylaxis. Therefore, the aim of the study was to evaluate the in vitro antimalarial activity and potential toxicity to mammalian cells of select synthetic and natural colourants, nucleoside and imidazo[1,2a]pyridine (IP) analogues on the erythrocytic stages of the 3D7 chloroquine-sensitive strain of P. falciparum. The P. falciparum 3D7 strain was maintained in vitro according to standard methods. Quinine and chloroquine were used as positive controls. The tritiated hypoxanthine incorporation assay was used for evaluating the ability of test compounds to inhibit the growth of P. falciparum. Active test compounds were tested in combination studies with quinine. Uninfected human red blood cell (RBC) toxicity was analysed spectrophotometrically. The ability of test compounds to inhibit -haematin formation, a metabolic pathway that sequesters toxic haem within the parasites, was determined. Cytotoxic activity of active compounds was evaluated on two human cell lines (HEK293 and K562) using the [3H]-thymidine incorporation assay. Data was analysed using the one-way ANOVA test and reported as the mean ± standard deviation of at least triplicate experiments and significant difference when p < 0.05. Of the 56 compounds tested, the synthetic colourants showed the most potent antimalarial activity. Methylene blue and safranin O were most potent with IC50 values of 4.19 ± 0.16 nM and 86.50 ± 2.61 nM, respectively, compared to quinine (IC50: 103.90 ± 8.30 nM), and displayed negligible toxicity to uninfected human RBCs. Combination studies with methylene blue and quinine demonstrated a synergistic interaction. Methylene blue also demonstrated the highest selectivity indices (480 and 968) compared to quine (180). Curcumin (diferuloylmethane), a natural extract was active (IC50: 2.29 ± 0.18 μg/ml) against P. falciparum, but significantly (p < 0.05) less potent than quinine. Curcumin was 78-fold more active in inhibiting -haematin formation than quinine, indicating of a possible mechanism of action. The most active nucleoside analogue, JLP118.1 (IC50: 1.79 ± 0.12 μM), demonstrated inhibitory activity against the trophozoite stage of P. falciparum. The imidazopyridine analogue, IP-4, displayed the least potent antimalarial activity (IC50: 15.3 ± 0.41 μM) of the synthetic compounds tested, with low selectivity indices < 1. The study has confirmed the potent antimalarial activity and relative safety of methylene blue as well as its potential as an antimalarial drug. The nucleoside and imidazopyridine analogues showed promising activity and with structural modification their potency and selectivity indices may be enhanced.

In order to employ the Sterile Insect Technique (SIT), biologically fit mosquitoes able to compete with their wild counterparts, suitable field sites for mass release of sterilized male mosquitoes, and appropriate methods of rearing genetic sex-separation (GSS) mosquito strains are required. The life history traits and biological fitness of four laboratory-reared, southern African Anopheles arabiensis strains were investigated. Despite being colonized at different times, the four strains demonstrated comparable levels of biological fitness. Three sites in the Kruger National Park were assessed as possible SIT field sites. Mosquito collections were conducted at each site during three summer months. Anopheles arabiensis was predominant at Malahlapanga during each collection period, establishing Malahlapanga as the most appropriate site for SIT field trials. A standard larval diet was shown to be appropriate for rearing An. arabiensis GSS. This work formed the laboratory basis for the evaluation of a SIT strategy for South Africa.

Faculty of Science School of Animal,Plant,Enviromental Sciences 0311595p NicrosM@social.mpu.gov.za / The antifolate combination of sulfadoxine and pyrimethamine (SP) is one of few remaining affordable drug combinations available for wide-scale treatment of uncomplicated Plasmodium falciparum malaria in Africa. In vivo studies of SP efficacy conducted during 1998, 2000 and 2002 at the Naas sentinel site in Mpumalanga province, South Africa, demonstrated a gradual non-significant increase in late treatment failure (LTF) and early treatment failure (ETF) resistance to SP, while gametocyte carriage increased significantly between 1998 and 2002 (p < 0.0001). This study aimed to determined and compare the frequency of dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) resistant haplotypes in P. falciparum parasites from patients treated with SP in three consecutive standardized in vivo therapeutic efficacy studies in Mpumalanga province, since implementation of SP as first line treatment in 1998, and to investigate associations between the presence of mutations and treatment outcomes after SP treatment. Four hundred-and-three samples were studied and 358 yielded polymerase chain reaction products. A novel high throughput sequence-specific oligonucleotide probe-based approach was used to examine the resistance status of the three in vivo P. falciparum populations. Screening for the presence of all known point mutations in dhfr and dhps genes revealed that only five dhfr and three dhps allelic haplotypes were present. In all the samples investigated, point mutations were identified only at codons 108, 51 and 59 of the dhfr gene and at codons 347 and 540 of the dhps gene. The prevalence of dhfr resistant haplotypes was 35.4% in 1998, 38.7% in 2000, and 41.0% in 2002, while the prevalence of dhps resistant haplotypes was 9.7% in 1998, 7.2% in 2000 and 41.6% in 2002, the latter representing a significant increase (p < 0.002). The prevalence in both dhfr and dhps gene resistant haplotypes were selected gradually during the three in vivo studies in Mpumalanga province. Infection with parasites having triple dhfr mutations and double dhps mutations, "the quintuple mutant", was associated with SP treatment failure (p < 0.001). Mutations at both dhfr and dhps loci may be important predictors of SP resistance in Mpumalanga province.

Faculty of Health Science School of Public Health 0418444d benido_impouma@yahoo.com / Study objectives The objective of this meta-analysis was to review the comparative efficacy and tolerance of sulfadoxine-pyrimethamine (SP) given alone or in combination with one (SPAS1) or three (SPAS3) doses of artesunate in children with uncomplicated P. falciparum malaria, aged 6 months to 10 years. Specifically, it assessed cure rate, fever and parasite clearance time, gametocyte carriage and tolerability. Methods The methodology used was a systematic review and a meta-analysis of four randomised controlled trials. The primary endpoint was the parasitological cure rate at day 28. Secondary endpoint included the parasitological cure rate at day 14, time to fever and parasite clearance, gametocyte carriage and occurrence of adverse events. Results Cure rate at day 28 corrected by PCR was 2.5 times higher in the combination of SPAS3 than in SP alone (pooled OR=2.55, 95% CI 1.93 to 3.37). There was no difference in cure rate at day 28 corrected by PCR between the combination of SPAS1 and SP alone (pooled OR=1.06 95% CI 0.98 to 1.15). Fever and parasite clearance times were significantly faster in both SPAS1 and SPAS3 compared to SP alone (p<0.001). By day 28 all children on the combination therapy were agametocytaemic as opposed to those on SP alone (p<0.001). All drug regimens were well tolerated and safe. Conclusion The combination of SPAS3 is more efficacious than SP alone in treatment of children with uncomplicated P. falciparum malaria. The combination is recommended for adoption as a replacement for SP alone in areas where malaria is endemic.

Thesis advisor: Sarah Babb / Since 2000, the global health management approach of health systems strengthening (HSS) has gained support from many local and international stakeholders. This thesis investigates the diffusion of the HSS norm in the global health community and within two disease programs of malaria and tuberculosis. I show how strong support for HSS by the global health community has been overwhelmed by coercive pressure from resourcewielding funding and governance structures. Drawing on organizational theory, I argue that global health organizations and experts have engaged in strategic social reconstruction and avoidance tactics to rationalize hypocrisy towards the HSS norm. / Thesis (BA) — Boston College, 2017. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: International Studies.