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Malaria represents one of the oldest documented diseases among humans and even today organisms in the genus Plasmodium kill more people than any other infectious disease, especially in tropical and subtropical areas. The four most common species which infect humans are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malaria. Of these four species, Plasmodium falciparum and Plasmodium vivax account for 95 percent of infections globally. Microscopy has been used since early days for the diagnosis of malaria because this method is simple, does not require highly equipped facilities, and in most cases enables differentiation among the species causing malaria in humans when performed by skilled microscopy readers. However, this method has been misleading in identifying parasite species, especially in the case of low level parasitemia, a mixed parasite infection, or modification by drug treatment as well as in placental malaria. Malaria rapid diagnostic tests (RDT) have played a major role in malaria management; particularly in providing blood based diagnosis in remote locations where microscopy based diagnosis is unavailable. These diagnostic tests are fast and easy to perform and do not require electricity or specific equipment. As part of strengthening malaria diagnostics in Malawi, the Ministry of Health and Population strongly recommends the use of malaria RDT’s at all levels of the health care delivery system. However, malaria microscopy remains a gold standard test for malaria. All patients (regardless of age) with suspected uncomplicated malaria should have a confirmed diagnosis with malaria RDT before anti-malaria treatment is administered. Based on field performance evaluations that assessed performance, quality control and production capacities of the manufacturing companies of malaria RDT’s, the Ministry of Health and Population recommended two brands of Histidine Rich Protein 2 (HRP-2), RDT’s for use in Malawi. These are SD Bioline malaria Ag Pf and the New Paracheck malaria Ag Pf. All these RDT’s are able to detect only P. falciparum. However, other species have been reported to exist in the country and there is a need to find proper RDT’s which will be able to detect all other species including P. falciparum. The main aim of this study was to evaluate Paramax-3 Pf/Pv/Pan RDT (Zephyr Biomedicals, India), if used in Malawi, could be able to detect and identify the different species of Plasmodium causing malaria in Malawi. The study recruited a total of 250 adult and infants at Bwaila Hospital in Lilongwe, Malawi. Study results showed that the overall sensitivity and specificity of the Paramax-3 RDT used in the study were 100 percent and 83 percent respectively. However, it was observed that the RDT test was not able to identify the P. ovale, and in some cases, the RDT test was positive for P. falciparum when the PCR identified the species as P. ovale. No P. vivax was detected both by RDT and PCR. This study was able to detect and identify the presence of P. malaria and P. ovale in Malawi apart from the P. falciparum. There were no significant differences between microscopy results compared to both the RDT and the PCR, with 94 percent and 98 percent sensitivities of R1 and R2 compared to RDT, as well as 94 percent and 96 percent sensitivities for R1 and R2 compared to PCR respectively. Both R1 and R2 had low specificities for example, R1 had 72 percent and R2 had 80 percent compared to RDT. Comparing R1 and R2 to PCR, the sensitivities were 64.9 percent and 67.2 percent respectively. However, the readers had difficulties differentiating the different species microscopically. The history of anti-malaria treatment had no significant effect on the outcome of the results in both the RDT and PCR.

Introduction: Plasmodium falciparum is the dominant cause of severe malaria in humans, with the highest number of global deaths occurring in Africa and Southeast Asia. Even though artemisinin-derivative combination therapies are readily available in Africa there are numerous reports of poor quality products which may lead to the development of drug resistance in Plasmodium parasites. Other aspects such as counterfeit medicines with sub-therapeutic levels of active ingredients may also be a contributing factor. The exact mechanism of P. falciparum drug resistance is still poorly understood, but numerous proteins linked to multi-drug resistance have been identified. The aim of this study was to ascertain whether clofazimine can act as a chemotherapeutic sensitiser as reported, and to determine its ability to alter the expression of chloroquine resistance gene products in vitro. Materials and Methods: In this study the 50% inhibitory concentrations of (IC50) humic and fulvic acid, the 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor mevinolin (lovastatin) and a riminophenazine antibiotic clofazimine were established using SYBR® green dye and fluorometric assays using chloroquine sensitive (3D7), chloroquine sensitive- pyrimethamine resistant (HB3) and chloroquine resistant (W2) parasites which demonstrate mefloquine, antifolate and chloroquine resistance respectively. Eflornithine (DFMO) is a polyamine synthesis inhibitor that was used in sensitivity assays for comparison. The selective toxicity of each of these agents was determined by Sulforhodamine B (SRB) colorimetric assays in hepatocarcinoma cells (HepG2). Flow cytometry techniques were employed to establish any alterations in life-cycle progression. Comparative proteomics of possible resistance proteins expressed on the membrane of the parasite food vacuole was conducted on W2, 3D7 and clofazimine-treated W2 strains of P. falciparum. Food vacuoles isolated by MidiMACS magnetic purification were separated using one dimensional gel electrophoresis, followed by in-gel trypsinisation, sample clean-up, fractionation by nano-liquid chromatography and mass spectrometric analysis by Matrix-Assisted Laser Desorption/Ionisation (MALDI) and Time of Flight (TOF) assays. Proteins were further identified in silico through the use of proteomic databases and homology comparison software. Results: Mevinolin showed poor antiplasmodial efficacy (IC50 of 1.19 x105 ± 1.02 nM), in comparison to artemisinin and chloroquine (32.61 ± 1.03 nM and 8.36 ± 1.03 nM, respectively) in 3D7 strains. Clofazimine showed greater antiplasmodial efficacy than DFMO in W2 strains (IC50 of 272.00 ± 1.04 and 3.39 x105 ± 1.06 nM, respectively). W2 strains were confirmed to be less susceptible to chloroquine (p<0.001). All test compounds showed decreased toxicity for model mammalian cells (p<0.001), except for mevinolin. Clofazimine (constant dose 375 nM) sensitised W2 strains to the actions of chloroquine (p<0.05), decreasing the IC50 of chloroquine by 40 nM. Food vacuoles were successfully harvested (confirmed by light microscopy), but quantities were below optimum value for reliable proteomic analysis and samples appeared to be contaminated with other cellular debris. Consequently proteins previously reported to be linked to drug resistance were not positively identified, while proteins involved in trafficking, motility and invasion of parasites were abundant and identified. Conclusion: The identification of novel antimalarials and the establishment of compounds that can be used in combination therapies to reverse resistance in P. falciparum parasites could greatly benefit communities suffering from the deadly malaria pandemic. Further research is required to establish the feasibility of clofazimine, or its derivatives, as adjunct therapy for the potential to decrease parasites’ active efflux of antimalarials. / Dissertation (MSc)--University of Pretoria, 2016. / Pharmacology / Unrestricted

A hallmark trait of P. falciparum malaria is sequestration, in which parasite infected erythrocytes (IEs) adhere to the vasculature, causing organ failure and death. Current antimalarials only kill the parasites, necessitating development of anti-adhesion drugs. Using our two-step approach, we can efficiently screen for anti-adhesion small molecules. Screenings of 75libraries using Bio-Plex 200 identified the most active TPI libraries, which were deconvoluted to single compounds. Screenings library TPI 1319 yielded 3 inhibiting non-optimized compounds, each of which inhibits binding between two receptors, CSA and ICAM1, and their binding PfEMP1 domains. Two compounds deconvoluted from TPI 2103 prevent binding between PfEMP1 and ICAM1. Cytoadhesion assays with live IEs support the results seen with Bio-Plex, with best hits showing inhibition below 200 nM. Cytotoxicity testing of active compounds showed minimaltoxicity. Identified hits appear to be amenable to Structure Activity Relationship studies to develop powerful anti-adhesion drugs to treat severe malaria. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Parasites causing avian malaria belong to the group Haemosporida, which represents a monophyletic group of dixenic protists within Apicomplexa. Their asexual reproduction takes place in a vertebrate intermediate host, and the formation of gametes and sporogony occur in blood-sucking dipteran insects, which are the definitive hosts of these parasites. Three main genera (​Plasmodium​, Haemoproteus ​and ​Leucocytozoon​) are found mostly in their avian hosts. We focused on the Haemosporida of wild birds and their transmission by insect vectors in natural populations, which had previously been a neglected area. Our results were obtained both by traditional methods (investigation of infections by microscopy of blood smears) and mainly by molecular methods (e.g. nested PCR) centered around work with unique haplotypes of the haemosporid lineages. The aim of our work was to determine the range of possible insect vectors of avian haemosporidians in the territory of the Czech Republic, taking into account the specificity of the parasites within these vectors, and to describe the diversity of haemosporidians in the populations of their bird intermediate hosts. We chose four different species of birds from four orders (Passeriformes, Strigiformes, Accipitriformes, and Galliformes). As potential vectors of avian...

Includes bibliographical references / [No subject] Malaria infection caused by the apicomplexa pathogen Plasmodium falciparum has a high rate of resistance to existing anti-malarial drugs. The World Health Organisation recommended interventions are unlikely to eliminate the growth of resistance and it would therefore be prudent to continue the search for new drug targets for the continued combatting of malaria. Plasmodium falciparum is parasitic on the host for its metabolites and therefore inhibiting the transportation of glutamine from the host, has long been considered a potential strategy for combating the spread of infection. The recently sequenced Plasmodium falciparum genome has however shown that pathways for independent survival are also conserved. Therefore, combating the spread of Plasmodium falciparum in the human host, in addition to inhibiting the transportation of glutamine, will also require the inhibition of the de novo expression of essential amino acids within the Plasmodium falciparum cell. This could be achieved by inhibiting the glutamine synthetase gene, which is an essential step in the tri-carboxylic acid cycle.

Includes bibliographical references / In addition to treating symptomatic patients, malaria prevention and elimination requires giving antimalarial drugs to asymptomatic or uninfected individuals. This shifts the harm-benefit balance and heightens the importance of accurately defining drug safety. Large data sets, including those pooled from multiple sources, are needed to understand uncommon adverse drug reactions. Interpreting individual studies , comparisons between studies and pooled datasets can be compromised, however, by inadequate or varied methods of safety data collection. Specifically, questioning methods may influence participants' reports of medical history, adverse events (AEs) and non-study medications. A Cochrane systematic review synthesised literature on research comparing methods for eliciting AEs from trial participants . A global online survey investigated how antimalarial researchers collect these data, and mixed-methods were used to identify barriers to accurate and complete reporting in South African and Tanzanian antimalarial-antiretroviral drug interaction trials. Focus group discussions were conducted in Ghana, Kenya and Uganda with women in a drugs exposure pregnancy registry to examine barriers to reporting at antenatal clinics, and how they might be overcome. The review included thirty-three studies in various therapeutic areas showing that more specific questioning increases the number of AEs reported by trial participants. Survey responses of 52 antimalarial researchers in 25 countries evidenced a range of methods to obtain AEs, medical histories and non-study drug reports. Qualitative data revealed that the trial context is influential and that detailed questioning facilitated participants' recognition and consideration of what to report. Non-reporting is due to forgetting, not knowing drug names, considering which information is relevant or significant to themselves or trial/healthcare workers, the potential consequences of reporting, and perceiving verbal responses inferior to what blood test results can show. Pregnant women's improved relationship with antenatal staff facilitated information-sharing and registry tools helped overcome problems with recall and naming of medicines. This project provides evidence of the substantial impact of different questioning methods on safety assessments . The results should contribute to developing a framework for researchers when planning globally-relevant, yet context-specific, antimalarial drug safety data collection strategies, and enhance efforts to pool data from multiple sources.

Introducción: Malaria representa una emergencia médica debido a la posible complicación y muerte del paciente cuando este no fue tratado apropiadamente. Malaria severa y/o complicada (MSC) es causada casi exclusivamente por Plasmodium falciparum. Uno de los factores de riesgo asociado con MSC es el tratamiento inadecuado de los casos de malaria no complicada (MNC). Objetivos: Se genotipificó a los genes dihidrofolato reductasa (Pfdhfr) y dihidropteroato sintasa (Pfdhps) en muestras de 60 pacientes con MSC. La resistencia al tratamiento combinado sulfadoxina-pirimetamina (SP) es causado principalmente por mutaciones puntuales en esos genes Diseño de estudio: Los pacientes con MSC de este estudio fueron enrolados durante el brote de malaria de 1998, cuando SP era la primera línea de tratamiento. Materiales y métodos: Se usó el método de secuenciamiento de Sanger para la identificación de los polimorfismos en el gen Pfdhfr y los métodos PCR-RFLP y PCR alelo-específico para el gen Pfdhps. Resultados: Se encontró que el 84% de las muestras tenían el genotipo del parásito cuádruple mutante N51I/S108N/I164L/inserción repetición Bolivia, y el 16% restante el genotipo mutante simple S108N. Con respecto al gen Pfdhps, encontramos cuatro genotipos, siendo el triple mutante A437G/K540E/A581G el más frecuente (78%). Conclusiones: Observamos que las mutaciones I164L de Pfdhfr y K540E de Pfdhps en los casos de MSC fueron más del doble de frecuente comparado con los reportes publicados en casos de MNC en la misma área y periodo de estudio. / --- Introduction: Malaria represents a medical emergency because it may rapidly progress to complication and death without prompt and appropriate treatment. Severe and/or complicated malaria (SCM) is almost exclusively caused by Plasmodium falciparum. One of the risk factors associated with SCM is an inappropriate treatment of the noncomplicated malaria (NCM). Objectives: We genotyped the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes from 60 SCM patients. Resistance to SP in P. falciparum is caused mainly by specific mutations at those genes. Study design: SCM patients of this study were enrolled during the malaria outbreak in 1998, when sulfadoxine/pyrimethamine (SP) was the first line of treatment. Material and methods: We used a Sanger sequencing approach for the identification of polymorphisms at Pfdhfr gene codons, and in the case of Pfdhps gene we used a PCRRFLP and PCR allele-specific methodology. Results: We found that 84% of samples harbored a quadruple mutant genotype N51I/S108N/I164L/insertion Bolivia repeat, and the left 16% of the sample contained an infection with a simple mutant genotype (S108N). Regarding the Pfdhps gene, we found four genotypes, the triple mutant genotype A437G/K540E/A581G was the more frequent (78%). Conclusions: We observed that the mutations I164L and K540E, known as highly predictor to SP resistance, in this group of patients with SCM were twice of frequency of the mutations from patients with NCM from published reports, also in the same area and period of study. / Tesis

A research report submitted to Faculty of Health Sciences in partial fulfilment of the requirements for the degree of Master of Science in Epidemiology in the field of Implementation Science, School of Public Health University of The Witwatersrand. Johannesburg June 2018. / Background Intermittent Preventive Therapy using sulfadoxine pyrimethamine (IPTp-SP) is a malaria control strategy to reduce cases of malaria in malaria endemic countries. However, the administration of the recommended three doses of IPTp throughout the stages of pregnancy still remains low in Nigeria even though, Nigeria is a malaria endemic country. Quality improvement approach has been demonstrated to improve practice among healthcare providers. This study therefore used a quality improvement approach to train and coach healthcare providers in order to improve the administration of the recommended doses of IPTp to pregnant women receiving antenatal care (ANC) services. Methods A quasi-experimental study design was carried out to evaluate the effect of quality improvement approach consisting of training and coaching of healthcare providers to improve the administration of IPTp during ANC service. Primary Healthcare Centre (PHC) Samaru was purposively selected and twelve healthcare providers participated in the study. ANC daily register was reviewed pre-intervention, intervention and post-intervention period of the study. Data were analysed using line graphs and run charts. Results A total of 36 ANC visit weeks were observed between 21st November 2016 and 27th July 2017. The mean level for IPTp1 administration pre-intervention was 105.85% (SD: 29.28) and 75.20% (SD: 16.89) for IPTp2+. The levels of IPTp1 administration were relatively stable from Week 1 to Week 10 although, there was overestimation of IPTp1 as 8 of the 16 Weeks in the pre-intervention period i.e. Weeks 3, 5, 7, 8, 11, 13, 15 and 16 all had more than 100% of eligible women administered IPTp1. The patterns of IPTp2+ administrations shows the levels of IPTp2+ administration were erratic. There was evidence indicating the process of IPTp1 was relatively stable post-intervention as the data crosses the median line only six times i.e. 7 runs. This indicates that the process of IPTp1 was within normal variation over the post-intervention period. There was an upward shift showing immediate improvement of the administration of IPTp2+ post-intervention although, there was a non-random variation in the administration of IPTp2+. The iv improvements of IPTp were not sustainable due to stock-outs. The quality of the ANC daily register was poor. Conclusion The integrated training and coaching intervention approach improved the administration of the recommended three doses of IPTp within the context of a PHC. These findings should be interpreted with caution as the impact of the intervention may not have reached its full impact due to the short post-intervention assessment. Stock-outs remains a huge barrier to the administration of IPTp under DOT during ANC services. The data quality of the ANC daily register improved post-intervention however, there were still slight errors thus, indicating that healthcare providers need constant coaching. It is important to integrate training and coaching of healthcare providers in order to have desired and sustained outcomes. Keywords: Malaria in pregnancy, IPTp, IPTp-SP, SP, ANC, Pregnant women, Healthcare providers, administration, QI, integrated training and coaching. / LG2018

Malaria is arguably one of the most overwhelming infectious diseases throughout the world's existence. The most virulent parasite, Plasmodium falciparum, has a redundancy of invasion proteins, allowing it to switch between different receptors on the host red blood cell. These invasion proteins are stored in the apical organelles, the rhoptries and micronemes, but very little is known about how newly synthesized proteins are transported to these organelles. The hypothesis in this study was that a common protein is involved in trafficking invasion proteins from the trans-Golgi network and PfSORTILIN was investigated as a potential escorter protein. The CCys domain of PfMAEBL, a rhoptry protein, and the prodomain of PfAMA-l, a microneme protein, have been implicated in trafficking to the apical organelles. These domains and the VPS 10 domain of PfSORTILIN were cloned into expression vectors encoding a GST- or Histag. Recombinant proteins were expressed in E. coli and purified by affinity chromatography on glutathione- or Ni-particles. In vitro binding assays were performed, which showed that PfSORTILIN VPS 10 bound to PfMAEBL ccys but not to the PfAMA-1 prodomain, suggesting that PfSORTILIN is a rhoptry protein escorter and is not involved in microneme trafficking. To identify novel binding partners of PfSORTILIN VPS 10, the protein was biopanned against a P. falciparum phage display library. No binding partners were identified, most likely because the library is not schizont-stage specific, which is when PfSORTILIN and invasion proteins are predominantly expressed. The results from this study were integrated with other studies and a trafficking model for PfMAEBL was proposed. This study enhances our knowledge of trafficking pathways and suggests that PfSORTILIN may serve as a common rhoptry protein escorter. / MT 2019