Malaria is an infectious disease caused by eukaryotic protists in the genus Plasmodium. Approximately half of the world's population is at risk of malaria. The burden of Plasmodium falciparum malaria has increased in recent years due to the emergence of resistant strains, which have even been documented in regions previously reported as malaria-free. Although malaria vaccine research has been conducted and has showed recent positive results, there still remains no effective vaccine to prevent malaria in clinical practice. According to the World Health Organization, prompt confirmation of malaria infection by microscopy and/or rapid diagnostic test (RDT) is critical to control the spreading of malaria and to prevent the evolution of drug resistant Plasmodia strains. However, malaria diagnosis remains a significant challenge as many malaria endemic regions have inadequate access to microscopy, and antibody-based RDTs are restricted by their stability under tropical temperatures and by their cost. The objective of this study was to develop a new approach to malaria diagnosis using DNA aptamers to recognise proteins encoded by Plasmodium. The research is divided into two parts. Firstly, DNA aptamers against the diagnostic markers, P. falciparum histidine-rich protein 2 (HRP2) and P. falciparum lactate dehydrogenase (PfLDH), were selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Secondly, a selected PfLDH aptamer was incorporated into a gold nanoparticle detection system to develop an aptamer-nanoparticle conjugate as a new approach towards malaria diagnosis. The identified HRP2 and PfLDH aptamers were characterised by isothermal titration calorimetry (ITC) for their affinity to targets and were observed to bind with nanomolar affinity. As PfLDH aptamers were observed to have a higher affinity to their target, PfLDH, their specificities were further characterised by ITC using human lactate dehydrogenases, hLDHA1 and hLDHB. The PfLDH aptamers were shown to be highly specific to PfLDH with no observed affinity to human LDHs. After further characterisation, PfLDH aptamer 2008s was chosen for the next stage of the research to be combined with a nanoparticle as a route towards diagnostic application. In the second part of this study, PfLDH aptamer 2008s was conjugated to gold nanoparticles (AuNPs) to create aptamer-AuNP conjugates (2008s-AuNP). The aptamer-AuNP conjugates were characterised by their tolerance in different pH and salt concentration and in their sensitivity to PfLDH. This new approach of malaria diagnosis was further validated by incubating the aptamer-AuNP conjugates with various proteins and colour changes were observed specifically upon incubation with PfLDH but not with other proteins. Hence, a Plasmodium specific aptamer-AuNP conjugate to the malaria diagnostic marker, pLDH, has been developed in this research. This work lays the foundation for further development of novel rapid diagnostic tests based on nucleic acid aptamers and nanotechnology for robust and cost-effective malaria diagnosis with potential benefit not only for malaria but in a plethora of diagnostic applications. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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.

No description available.

Data based abnormality detection is a growing research field focussed on extracting information from feature rich data. They are considered to be non-intrusive and non-destructive in nature which gives them a clear advantage over conventional methods. In this study, we explore different streams of data based anomalies detection. We propose extension and revisions to existing valve stiction detection algorithm supported with industrial case study. We also explored the area of image analysis and proposed a complete solution for Malaria diagnosis. The proposed method is tested over images provided by pathology laboratory at Alberta Health Service. We also address the robustness and practicality of the solution proposed. / Process Control

Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Malaria affects over half of the world's population and causes half a million deaths annually, especially in Sub-Saharan Africa. Four species of the apicomplexan Plasmodium parasite (P. falciparum, P. ovale, P. malariae and P. vivax) are responsible for malaria in Africa. Both parasite and host factors contribute to heterogeneity in the risk of developing malaria, clinical manifestation of the disease as well as the number of treatments required to clear parasites. The epidemiology of the different species, and the role of exposure to mixed-species Plasmodium co-infections in generating heterogeneity remains poorly studied. Being an obligate intracellular parasite the blood-stage life cycle of the Plasmodium parasite takes place in the erythrocytes of the human host. The surfaces of these erythrocytes are the medically important ABO blood group antigens that have been reported to influence the susceptibility or otherwise of an individual developing severe malaria. In this thesis I have considered the contributions of the species of Plasmodium parasites and the ABO blood group of the host in driving heterogeneity in human malaria. The aims of this thesis were to determine: (i) the seroepidemiology of the different Plasmodium species in two mesoendemic African populations (Zimbabwe and Sudan); (ii) to determine if heterogeneity in clinical presentations of malaria (history of fever, body temperature and parasitaemia) and response to drug treatment is related to exposure to single vs. mixed-Plasmodium species infection; (iii) the spatial and temporal dynamics of malaria prevalence and Plasmodium species distribution in a mesoendemic village in eastern Sudan; (iv) gene expression changes in 3D7 P. falciparum parasites as they infect erythrocytes of different ABO blood group donors. For aims (i to iii) I developed an enzyme-linked immunosorbent assay using antigens derived from Plasmodium merozoite surface protein 1, also known as MSP-119, to detect IgG antibodies to all four malaria parasite species in Zimbabwean and Sudanese populations. In the Zimbabwean study, plasma samples from 100 individuals each (aged 5-18 years) from three villages (Burma Valley, Mutoko and Chiredzi) were screened for exposure to Plasmodium parasites. In Daraweesh, Sudan, plasma samples from 333 individuals (aged 1-74 years) who had experienced a first malaria episode between 1990 and 2000 were recruited into the study. For study aim (iv) I cultured a single clone of 3D7 P. falciparum parasite using erythrocytes of individuals of different ABO blood group types, harvested parasite RNA and sequenced it to determine gene expression changes in the different hosts. I showed that human IgG antibodies to MSP-119 antigens of the four Plasmodium species are species-specific and do not cross-react. In both study populations almost all antibody responses involved P. falciparum, and single-species responses were almost exclusively directed against P. falciparum antigens. Mixed-species responses accounted for more than a third of responses, and were associated with chloroquine treatment failure, with significantly high proportion of individuals with mixed-species infections requiring repeated treatment with chloroquine/sulfadoxine-pyrimethamine for parasite clearance. This finding highlights the need for a sensitive method for detecting mixed-species malaria infections to enable the assessment of the true prevalence and magnitude of the disease burden caused by the non-falciparum species in endemic populations. Drug treatment failures associated with mixed species infections have significant impact on malaria morbidity and mortality. Treatment failure or partial parasite clearance has the potential to allow dormant liver stages of P. vivax and P. ovale to become a source of parasite reservoir for onward transmission. Furthermore, untreated low-grade chronic infections caused by P. malariae have been reported to cause systemic diseases many years after the primary infection. Spatial analysis of malaria epidemiology showed that malaria parasite transmission in Daraweesh was focal, and that infections are not randomly distributed in the village. Two space-time clusters of significantly increased malaria risk were identified (1993- 1999, and 1998-1999) with marked variations between households, but little or no variation in the species of Plasmodium over time. Similarly, multiple significant clusters were identified for the parasite species; three for P. falciparum, two for P. vivax and P. malariae, and one for P. ovale. These clusters had overlapping time frames, with some of the species significantly infecting the same households. This suggests that even in a small geographic area malaria transmission shows heterogeneity, and that such data can provide useful information to guide malaria control efforts. Finally, I demonstrated that 3D7 P. falciparum parasite growth was similar in the erythrocytes of different blood group donors, and provide preliminary data to show that the non-coding RNA gene, PF3D7_1370800, is differentially expressed in blood group A donors relative to blood groups B and O donors. Further research is needed to better understand the role of this gene in malaria pathology. All together, these findings will aid malaria researchers and other stakeholders in making informed choices about tools for diagnosing Plasmodium species, and control programmes targeting eradication of malaria caused by all Plasmodium species, as is the case of incorporating these findings into current malaria research in Sudan.

Optical microscopy is the simplest and the gold standard method adopted for the screening and subsequent diagnosis of various hematological and infectious diseases like malaria, sickle cell disease, tuberculosis etc. In addition to infectious disease diagnosis, its applications range from routine blood tests to the more sophisticated cancer biopsy sample analysis. Microscopy Tests (MTs) follow a common procedural workflow: (1) A technician prepares a smear of the given sample on a glass slide in a specific manner depending on the sample and the disease to be diagnosed; (2) The smeared slide is subsequently exposed to fixative agents and different histochemical stains specific to the diagnosis to be performed and (3) the prepared slide is then observed under a high quality bright- field bench-top microscope. An expert pathologist/cytologist is required to manually examine multiple fields-of-views of the prepared slide under appropriate magnification. Multiple re-adjustments in the focus and magnification makes the process of microscopic examination time consuming and tedious. Further, the manual intervention required in all the aforementioned steps involved in a typical MT, makes it inaccessible to rural/resource limited conditions and restricts the diagnostics to be performed by trained personnel in laboratory settings. To overcome these limitations, there has been considerable research interest in developing cost-effective systems that help in automating MTs. The work done in this thesis addresses these issues and proposes a two-step solution to the problem of affordable automation of MTs for cellular imaging and subsequent diagnostic assessment. The first step deals with the development of a low cost portable system that employs custom-built microscopy setup using o -the-shelf optical components, low cost motorized stage and camera modules to facilitate slide scanning and digital image acquisition. It incorporates a novel computational approach to generate good quality in-focus images, without the need for employing high-end precision translational stages, thereby reducing the overall system cost. The process of slide analysis for result generation is further automated by using image analysis and classification algorithms. The application of the developed platform in automating slide based quantitative detection of malaria is reported in this thesis. The second aspect of the thesis addresses the automation of slide preparation. A major factor that could influence the analysis results is the quality of the prepared smears. The feasibility of automating and standardizing the process of slide preparation using Microfluidics with appropriate surface fictionalization is explored and is demonstrated in the context of automated semen analysis. As an alternative to the mechanism of fixing the spermatozoa to the glass slide by smearing and chemical treatment with fixative, microfluidic chips pre-coated with adhesive protein are employed to capture and immobilize the cells. The subsequent histochemical staining is achieved by pumping the stains through the microfluidic device. The proof-of-principle experiments performed in this thesis demonstrate the feasibility of the developed system to provide an end-to-end cost-effective alternative solution to conventional MTs. This can further serve as an assistive tool for the pathologist or in some cases completely eliminate the manual intervention required in MTs enabling repeatability and reliability in diagnosis for clinical decision making

Made available in DSpace on 2015-04-11T13:38:41Z (GMT). No. of bitstreams: 1 Dissertacao-Janaina Roriz Hipolito.pdf: 4312682 bytes, checksum: ecb7ef268b16e006660e9770d5f663b4 (MD5) Previous issue date: 2006-07-28 / Fundação de Amparo à Pesquisa do Estado do Amazonas / A malária é um problema de saúde pública na região Amazônica, mais de 200 mil casos dessa doença ocorrem anualmente no Amazonas, sendo 80% deles causados pelo P. vivax, que vem apresentando índices crescentes de morbidade, principalmente associados à diminuição da sensibilidade aos antimaláricos. Dentre as estratégias para combate e controle da doença, a identificação rápida e precisa da espécie é ferramenta indispensável para um tratamento apropriado, diminuição do risco de transmissão e melhor entendimento da epidemiologia desses parasitas. A técnica microscópica da gota espessa é a principal para diagnóstico da malária, entretanto, outros métodos vêm sendo testados, principalmente os moleculares que tem se mostrado mais sensíveis e específicos para detectar e diferenciar as espécies em baixas parasitemias. Avanços desse método, como a PCR em tempo real, permitem que o resultado do teste seja detectado simultaneamente a amplificação, diminuindo o tempo gasto para a realização do diagnóstico. Com o intuito de detectar molecularmente a malária, verificando a presença de plasmódios, o diagnóstico molecular foi realizado através das técnicas de PCR em tempo real e nested-PCR, para se fazer uma comparação desses dois métodos com o diagnóstico microscópico da gota espessa em 300 amostras criopreservadas, 200 coletadas no dia inicial do tratamento (D0), das quais 88% (176/200) eram provenientes de pacientes de Manaus, as 24 amostras restantes (12%) eram provenientes de localidades do interior do Amazonas: São Gabriel da Cachoeira (09), Tefé (08), Humaitá (04) e Careiro (03). Apenas 9% dessas amostras tinham diagnóstico microscópico de monoinfecção por P. falciparum e 91% (182/200) por P. vivax, não havia nenhuma amostra mista pelo diagnóstico microscópico. O diagnóstico molecular por nested-PCR confirmou a presença de DNA de plasmódio em 100% das amostras monoinfectadas. Adicionalmente, foram observadas infecções mistas, co-infecção de P. falciparum e P. vivax, em 19% (38/200) destas amostras. O diagnóstico molecular por PCR em tempo real (Lightcycler, Roche®) foi realizado em apenas 17% (34/200) dessas amostras. A co-positividade (sensibilidade) dos testes para P. vivax foi em média 71% e a co-negatividade (especificidade) 92%, para P. falciparum a co-positividade foi 91% e a conegatividade 79%. A concordância entre os testes foi regular. As 100 amostras restantes haviam sido coletadas no sétimo dia (D7) de tratamento e eram negativas pela microscopia. O diagnóstico molecular demonstrou 21% de positividade. Este estudo mostrou que muitas infecções mistas vêm sendo subestimadas para fins de avaliação epidemiológica, demonstrando que a sensibilidade e especificidade do diagnóstico molecular são superiores a do teste microscópico. O diagnóstico molecular seria então mais indicado como teste complementar no diagnóstico de pacientes com baixas parasitemias, na análise da quantidade de portadores assintomáticos, em estudo de infecções criptônicas e na avaliação da negativação da parasitemia para monitoramento terapêutico, e em estudos que visem a diminuição da transmissão pela existência de prováveis gametócitos persistentes após o tratamento. Entretanto, esse método não é indicado para rotina de diagnóstico de malária, uma vez que os resultados positivos por essa técnica não significam necessariamente que o paciente desenvolva a doença.

No description available.

In Tanzania laboratory services were observed to be not providing the quality of services required. It is assumed that the perceived discrepancy between malaria diagnosis and confirming laboratory result might be attributed to incompetence of health personnel. Objective The objective of this research was to explore the competence and extend to which health personnel in Muhimbli hospital comply with procedural norms in malaria diagnosis. Methodology A quantitative approach of explorative descriptive design was used. A survey was done using observation guidelines based on existing policies and norms. Actual practice of malaria diagnosis compared with the policies and procedural norms. Result The data revealed that health personnel are not competence in malaria diagnosis. Conclusion Competence of health personnel is important in malaria diagnosis, a special guideline should be developed and in-service training be implemented to minimize errors in reporting for malaria investigation. / Health Studies / M. A. (Public Health)