Malaria is a deadly disease that causes nearly one million deaths each year. Understanding the demographic history of the malaria parasite Plasmodium falciparum and the genetic basis of its adaptations to antimalarial treatments and the human immune system is important for developing methods to control and eradicate malaria. To study the long-term demographic history and recent effective size of the population in order to identify genes under selection more efficiently and predict the effectiveness of selection, in Chapter 2 we sequenced the complete genomes of 25 cultured P. falciparum isolates from Senegal. In addition, in Chapter 3 we estimated temporal allele frequencies in 24 loci among 528 strains from the same population across six years. Based on genetic diversity of the genome sequences, we estimate the long-term effective population size to be approximately 100,000, and a major population expansion of the parasite population approximately 20,000-40,000 years ago. Based on temporal changes in allele frequencies, however, the recent effective size is estimated to be less than 100 from 2007-2011. The discrepancy may reflect recent aggressive efforts to control malaria in Senegal or migration between populations.

Malaria is a preventable and treatable disease caused by infection with Plasmodium parasites. Complex socioeconomic and political factors limit access to vector control and antimalarial drugs, and an estimated 600,000 people die from malaria every year. Rising drug resistance threatens to make malaria untreatable. As for all new traits, resistance is limited by fitness, and a small number of pathways are heavily favored by evolution. These pathways are targets for drug discovery. Pairing compounds active against the wild-type and the small emerging resistant population, a strategy we termed "targeting resistance," could block the rise of competitively viable resistance.

Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

No description available.

Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World Health Organization (WHO) has endorsed artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Currently recommended ACTs are artemether-lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine. This study sought to review evidence of the efficacy and safety of different non-artemisinin antimalarials in combination with artesunate, artemether or dihydroartemisinin for the treatment of uncomplicated P. falciparum malaria in non-pregnant adults and children. The search for randomized controlled trials (RCTs) was conducted in the Cochrane Central Register for Controlled Trials (CENTRAL), MEDLINE, EMBASE and in ClinicalTrials.gov in January 2009. The eligibility and the methodological quality of trials were assessed and data were extracted, using standard forms. Data were captured and analyzed in Review Manager Software, versions 4.2 and 5.0. The outcomes assessed were: treatment failure, fever and parasite clearance time, calculating the relative risk (RR) and a weighted mean difference (WMD) with a 95% confidence interval and p-values, indicating statistical significance at 0.05. Thirty-seven trials with 6862 participants were included. Artesunate combined with amodiaquine had a statistically significant lower risk of treatment failure compared to the combination of artesunate with sulfadoxine-pyrimethamine (RR=0.57, 95% CI [0.33, 0.97], p=0.04, seven trials, N=1341). In addition, treatment with artesunate plus mefloquine was significantly associated with a lower risk of treatment failure compared to artesunate plus azithromycin (RR=0.04, 95% CI [0.00, 0.64], p=0.02, one trial, N=54). There was no significant difference when either mefloquine or atovaquone-proguanil were combination partners with artesunate (RR=2.6, 95% CI [0.93; 7.24], p=0.07, one trial, N=1066). When artesunate was combined with chloroquine, primaquine or azithromycin and compared with artesunate monotherapy, there was no statistically significant difference in the risk of unadjusted treatment failure. Each of these comparisons had one trial each. Artesunate plus chloroquine was quicker at clearing fever compared to artesunate plus sulfadoxinepyrimethamine (WMD= -7.20, 95% CI [-12.53, -1.87], one trial, N=132). Few trials adequately reported adverse events. There was no significant difference observed in the risk of adverse events between artesunate plus amodiaquine compared with artesunate monotherapy, however, adverse events were significantly less in artesunate plus amodiaquine compared to artesunate plus methylene-blue. Artesunate plus amodiaquine on the other hand had significantly more adverse events reported compared to artesunate plus sulfadoxine-pyrimethamine. The findings of this study support the implementation of artemisinin-based combination therapy for the treatment of uncomplicated malaria. Most crucially, this review found a greater advantage of combining amodiaquine with artesunate compared to sulfadoxine-pyrimethamine. The efficacy of artesunate plus mefloquine was superior to that of artesunate plus azithromycin. Furthermore, the combination of artemisinins with chloroquine, primaquine and azithromycin has shown very low efficacy and these combination therapies should not be recommended. The reporting of efficacy was not standardized as many trials did not differentiate between re-infections and recrudescences. Adverse events were also not adequately reported. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Pyrimethamine efficacy was investigated in vitro on the blood asexual stages, the sexual stages and exflagellation in Plasmodium falciparum. Gametocytogenesis was stimulated following the standard methods on five isolates of Plasmodium falciparum. From these five isolates, RSA 2, 3 and 5 produced gametocytes which reached maturity within seven days and the gametocytes were able to exflagellate. Isolate MW2 produced young gametocytes which disappeared within ten days. NF54 produced mature gametocytes which lasted for 24 hours only. There were no statistically significant differences between the static and the synchronization methods of gametocyte stimulation for any of the isolates. The effect of pyrimethamine was investigated by adding a known concentration of the drug (For RSA 2, MW2 and NF54, l00nmol/ℓ; RSA 3 and 5, 3000nmol/ℓ pyrimethamine) to the culture medium for seven days during gametocyte stimulation. The results of this investigation show that there was gametocytocidal activity on the isolates that were used and pyrimethamine also had a schizontocidal action on NF54 and the young gametocytes of this isolate were destroyed by the drug. At concentrations which were inhibitory to asexual parasites, the drug had a sporontocidal effect on isolate RSA 2 but not on isolate RSA 5. The pyrimethamine MIC values for asexual parasites ranged from 300nmol/ℓ to > 3000nmol/ℓ (RSA 2 and 5 were not inhibited at 3000nmol/ℓ ). These results are consistent with those found in previous studies when pyrimethamine resistance was first detected in South Africa. The chloroquine MICs indicate a good correlation with the results obtained from previous drug sensitivity tests for all the isolates examined using both the 48-hour in vitro test and isotope incorporation for growth assessment. The isobolograms constructed to determine relationship between chloroquine and pyrimethamine indicated no synergism for isolates RSA 2 and 5, but the Σ relative IC[50]s indicated a weak synergism. Both the isobolograms and the Σ relative IC[50]s for the isolates RSA 6, 9 and 14 indicated an antagonistic action between chloroquine and pyrimethamine. The results obtained from this study have important implications for malaria control in South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Background Recent malaria epidemics in KwaZulu-Natal indicate that effective anti-malarial therapy is essential for malaria control. Although artemether-lumefantrine has been used as firstline treatment for uncomplicated Plasmodium falciparum malaria in northern KwaZulu- Natal since 2001, its efficacy has not been assessed since 2002. The objectives of this study were to quantify the proportion of patients treated for uncomplicated P. falciparum malaria with artemether-lumefantrine who failed treatment after 28 days, and to determine the prevalence of molecular markers associated with artemether-lumefantrine and chloroquine resistance. Methods An observational cohort of 49 symptomatic patients, diagnosed with uncomplicated P. falciparum malaria by rapid diagnostic test, had blood taken for malaria blood films and P. falciparum DNA polymerase chain reaction (PCR). Following diagnosis, patients were treated with artemether-lumefantrine (Coartem®) and invited to return to the health facility after 28 days for repeat blood film and PCR. All PCR P. falciparum positive samples were analysed for molecular markers of lumefantrine and chloroquine resistance. Results Of 49 patients recruited on the basis of a positive rapid diagnostic test, only 16 were confirmed to have P. falciparum by PCR. At follow-up, 14 were PCR-negative for malaria, one was lost to follow-up and one blood specimen had insufficient blood for a PCR analysis. All 16 with PCR-confirmed malaria carried a single copy of the multi-drug resistant (mdr1) gene, and the wild type asparagine allele mdr1 codon 86 (mdr1 86N). Ten of the 16 samples carried the wild type haplotype (CVMNK) at codons 72-76 of the chloroquine resistance transporter gene (pfcrt); three samples carried the resistant CVIET allele; one carried both the resistant and wild type, and in two samples the allele could not be analysed. ii Conclusions The absence of mdr1 gene copy number variation detected in this study suggests lumefantrine resistance has yet to emerge in KwaZulu-Natal. In addition, data from this investigation implies the possible re-emergence of chloroquine-sensitive parasites. Results from this study must be viewed with caution, given the extremely small sample size. Recommendations A larger study is needed to accurately determine therapeutic efficacy of artemetherlumefantrine and resistance marker prevalence. The high proportion of rapid diagnostic test false-positive results requires further investigation. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2013.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

Effective malaria vaccine might help improve control strategies against malaria, but the complexity of interactions between the parasite and its hosts poses challenges. The asexual blood stage P. falciparum antigen Pf332 has potentials as one of the proteins in understanding the complex host-parasite interactions. The interest in Pf332 as a target for parasite neutralizing antibodies, evolved from previous studies demonstrating that Pf332-reactive antibodies inhibits parasite growth in vitro. The presence of natural P. falciparum infection also indicated that Pf332 has the ability to induce protective antibodies. In paper I, we identified and characterized the immunogenicity of a C-terminal region of Pf332. Immunological analyses carried out with this fragment revealed that rabbit anti-C231 antibodies possess parasite in vitro inhibitory capabilities. In paper II, the functional activity of C231 specific antibodies was confirmed with human-affinity purified antibodies, where the antibodies inhibited late stage parasite development, by the presence of abnormal parasites and disintegrated red cell membranes. Epidemiological data from malaria endemic area of Senegal (Paper III & IV), showed that antibodies were reactive with two different fragments of Pf332 (C231 and DBL). Distribution of anti-C231 antibodies in the IgG subclasses, gave similar levels of IgG2 and IgG3. The levels of anti-C231 antibodies were associated with protection from clinical malaria, but with DBL reactive antibodies IgG3 was associated with protection from clinical malaria. We hereby conclude that antigen Pf332 contains immunogenic epitopes, and is a potential target for parasite neutralizing antibodies. The Pf332 protein should thus be considered as a candidate antigen for inclusion in a subunit P. falciparum malaria vaccine. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.