Malaria is a life threatening disease occurring mainly in the tropical regions of developing and under developed countries. Most of these affected countries have a low gross national income (GNI) per capita and cannot adequately afford widespread application of malaria surveillance and control programs. In such countries, due to limited access to diagnostic centers, patients often resort to self-treatment of suspected malaria cases.
Indiscriminate use of oral artemisinin based monotherapies has been identified as one of the factors that leads to drug resistance, which is a serious problem in malaria management. For example, a sulfadoxine-pyrimethamine combination, which is an effective treatment against multi drug resistant (MDR) malaria, has now been rendered useless in south-east Asia by its overuse and misuse (Choi et al., 2007). According to WHO, accurate and point of care (PoC) diagnosis of malaria are prerequisite in malaria management as these reduce evolution of multi drug resistant malaria caused by indiscriminate and overuse of drugs (Choi et al., 2007), lessen mismanagement of non-malaria fevers (Chanda et al., 2009), offer improved care of parasite positive patients and raise public trust in the efficacy of artemisinin-based combination therapy (ACT).
Most symptomatic malaria infections are preceded and followed by a PCR positive phase which is submicroscopic. The length of this submicroscopic period varies with the age of the patients. The sexual gametocytes responsible for disease transmission can also exist at submicroscopic levels. The group of people with these submicroscopic infections in
2 endemic areas are contributors to the human infectious reservoir. In order to make malaria control and elimination programs more effective further effort needs to be made towards diagnosing these submicroscopic infections. Sensitive malaria detection techniques can not only reduce the presumptive treatment associated with antimalarial drug resistance but also facilitate the treatment of submicroscopic infections that have so far been widely ignored.
In order to develop an efficient test for diagnosis of malaria, a profound understanding on various malaria related biomarkers is prerequisite. Among the biomarkers identified till date, Plasmodium species lactate dehydrogenase (LDH) has emerged as a highly promising target. It is the last enzyme in the glycolytic cycle of the parasite and is involved in the reduction of pyruvate to lactate thus regenerating the NAD+cofactor. During the intra-erythrocytic stages, the parasite principally relies on anaerobic respiration for ATP generation from glucose, leading to an over expression of enzymes involved in the glycolytic pathway (Roth, 1990). Parasite LDH differs structurally and kinetically from its human counterparts. Structurally it has been shown to exhibit unique epitopes present on the surface of the enzyme (Hurdayal et al., 2010). Kinetically it differs in the lack of substrate inhibition (Brown et al., 2004) and higher affinity towards a cofactor analogue APAD+ (3-Acetyl pyridine adenine dinucleotide) (Chaikuad et al., 2005). These differences make this parasite enzyme unique and easier to target using diagnostic methods.
At present, several analytical techniques exist for malaria diagnosis. However, these techniques are associated with certain limitations. Conventional malaria diagnosis techniques, like microscopy, ELISA and flow cytometry require skilled personnel and
expensive equipment facility, and thus these are difficult to use in PoC settings. The recent PoC malaria diagnostics largely belong to antibody based rapid diagnostic tests (RDTs).
While these portable RDTs have greatly benefitted malaria control and surveillance programs, they are still plagued with issues related to high cost, poor stability in tropical climates, being non-quantitative and incapable of differentiating different Plasmodium species. These RDTs are susceptible to malfunction at high temperature and humidity due to the use of protein based antibody, which is a labile bio recognition element (Chiodini et al., 2007). Hence, the current interest in malaria diagnosis focuses on the development of robust recognition materials that are stable in hot and humid climate due to obvious reason of the malaria prevalence in these climatic conditions.
DNA based probes have received wide interest for different diagnostics applications due to various positive traits such as, thermal stability, easy chemical synthesis and modification, and fairly low costs of production. DNA possesses its own set of intelligent material properties that could be selectively garnered to design viable bio recognition elements for specific sensor applications. The properties of DNA like, hybridization specificity, self-assembly, conductivity, and ease of functionalization add edge to its said bio recognition capabilities to develop tailored applications. This study aims to exploit the inherent property of DNA, and the intrinsic properties of advance nanomaterials for developing stable, sensitive, and specific sensing systems for Plasmodium species detection and differentiation. The following objectives are thus defined for this study:
4 Objectives of the study
1. Preparation of biomarker and control proteins: cloning, expression, purification, and characterization of Plasmodium falciparum, and human lactate dehydrogenases 2. Preparation of DNA based biorecognition elements: development of sensitive and
specific aptamer, and DNA based probes to target PfLDH
3. Application of developed biorecognition elements to detect the biomarker on optical and electrochemical platforms
This thesis has been subdivided into VII chapters as briefly described below. At the end of the chapters, a section describing the overall conclusion from the thesis and a critical evaluation on the work with the scope for future work has been included.