Loop mediated isothermal amplification: a novel diagnostic tool for malaria elimination

Abstract

Malaria elimination, as outlined by the World Health Organization, is an enticing and challenging goal for many malaria endemic regions worldwide. By definition, malaria elimination is fundamentally different from malaria control, as the focus shifts from detection of parasites in symptomatic patients to detection and clearance of all infections. This includes active case detection of asymptomatic persons with sub-microscopic parasitemias who have been shown to be able to maintain the cycle of malaria transmission. However, the diagnostic tools available for detection of sub-patent, low level parasitemia are limited. While expert microscopy has been shown to have a sensitivity of 100% at 50 parasites/ µL, the sensitivity decreases significantly under field laboratory conditions limiting the ability to detect low level parasitemia of all Plasmodium species. Rapid diagnostic tests are simple to perform and inform the management of patent clinical cases of malaria. However, they are insufficiently sensitive at parasitemias less than 200 parasites/ µL and particularly so for non-falciparum Plasmodium species, limiting their role in detecting low level parasitemia. PCR is the gold standard, and most sensitive, diagnostic modality, with the ability to detect less than 1 parasite/ µL and is readily available in reference laboratories. However, the technical requirements and expertise required for performing PCR limits is ability to be performed in resource limited settings. Therefore there is a critical need to improve the diagnostic repertoire available for detection of low level parasitemia in resource limited, malaria elimination settings. Loop mediated isothermal amplification (LAMP) is a molecular diagnostic modality that has the potential to meet this need. Firstly, although inherently non-quantitative, LAMP is an isothermal process relying on the Bacillus stearothermophilus (Bst) polymerase enzyme and does not require cyclical temperature changes inherent to PCR. This reduces the logistic impediments to field adaptation of a LAMP assay. Secondly, a positive LAMP reaction results in the formation of a magnesium pyrophosphate precipitate which is detectable visually, by turbidimetry or using metal ion detectors such as calcein, hydroxynaphthol blue and pico-green. LAMP end products have also been visualised using melting curve analysis, a bioluminescent output in real time (BART), a lateral flow dipstick or a portable fluorescence detection unit (realAmp) thereby simplifying the interpretation of assay results without the need for expensive probes and gel electrophoresis. Thirdly, LAMP has been shown to have the ability to detect low level parasitemia, to detect all the Plasmodium species and to be able to be performed in resource limited settings. However, among the limitations to currently available formats of LAMP are that it has limited capacity for high throughput processing of samples, for example from cross sectional surveys or surveillance studies, as might be required in malaria elimination settings. In addition, although there are LAMP primers available for detecting all the Plasmodium species causing human infection, their sensitivity for detecting low level parasitemia remains limited, particularly for non-falciparum species. The hypothesis tested in this thesis is that LAMP can be applied as a high-throughput molecular diagnostic tool capable of detecting species-specific low level parasitemia and has the capacity to be performed in resource limited settings, towards the goal of malaria elimination. The specific aims were 1) to optimise LAMP as a high-throughput assay (HtLAMP), 2) to compare HtLAMP to microscopy, rapid diagnostic tests (RDTs) and PCR for diagnosing mixed malaria species infections, 3) to develop sensitive LAMP primers for the specific detection of non-falciparum Plasmodium species and 4) to validate the HtLAMP assay for its ability to be performed in resource limited settings This thesis describes the successful optimisation of LAMP into a high-throughput, simple, platform for the molecular diagnosis of human Plasmodium parasites and its validation in a resource-limited laboratory. It also outlines the process by which novel, highly sensitive LAMP primers were developed for the detection of Plasmodium vivax and Plasmodium knowlesi species. To my knowledge, these are the most sensitive LAMP primers described in the literature to date. Taken together, this high-throughput LAMP platform for the identification of non-falciparum Plasmodium species has the potential to make a significant contribution to the molecular diagnostic tools available for detecting Plasmodium parasites for the purpose of malaria elimination.