Malarial Diagnosis with Deep Learning and Image Processing Approaches

Malaria is a mosquito-borne disease that has killed an estimated a half-a-million people worldwide since 2000. It may be time consuming and costly to conduct thorough laboratory testing for malaria, and it also requires the skills of trained laboratory personnel. Additionally, human analysis might make mistakes. Integrating denoising and image segmentation techniques with Generative Adversarial Network (GAN) as a data augmentation technique can enhance the performance of diagnosis. Various deep learning models, such as CNN, ResNet50, and VGG19, for recognising the Plasmodium parasite in thick blood smear images have been used. The experimental results indicate that the VGG19 model performed best by achieving 98.46% compared to other approaches. This study demonstrates the potential of artificial intelligence to improve the speed and precision of pathogen detection which is more effective than manual analysis

Dissecting the CD8+ T cell responses to pre-erythrocytic malaria antigens

The pre-erythrocytic stages of malaria are the preliminary steps to a disease of massive global health importance. Following transmission of sporozoites by mosquito bite to a human host, a clinically silent period of parasite development in the liver preludes the harmful blood stages that characterise malarial infection. Creating a vaccine that targets these stages is a very attractive notion as it would prevent this burden of disease. Protection against the pre-erythrocytic stages has been shown to be dependent on CD8+ T cells. However, very few antigens that induce anti-Plasmodium CD8+ T cell responses have been identified, especially those expressed by the parasite when developing inside hepatocytes. Using mouse models, experimental genetics and bioinformatics tools, I present work that has progressed our understanding of CD8+ T cells induced in the preerythrocytic stages of malaria and assessed the ability of antigen-specific CD8+ T cells to protect against subsequent challenge following vaccination. I have investigated and compared the differences in CD8+ T cell responses to a sporozoite (Circumsporozoite Protein) and a vacuolar membrane liver stage protein (Upregulated in Infectious Sporozoites gene 4) and showed that despite a divergence in immunogenicity when immunising with radiation attenuated sporozoites, both types of antigen are equally protective when mice are vaccinated with viral vectors to induce large antigen-specific CD8+ T cell populations. The natural immunogenicity of the liver stage antigen does not improve when liver stage development is extended by using drug prophylaxis. Additionally, I have compared the protection induced by liver stage antigens expressed constitutively after hepatocyte invasion and those expressed only after at least 12 hours post invasion. I have shown that some protection can be induced by the mid-late expressed Liver Specific Proteins 1 and 2, suggesting that these antigens are effectively presented and recognised by CD8+ T cells. This highlights the potential for the incorporation of liver stage antigens into next-generation malaria vaccines. 4 Additionally, I have investigated the role of the immunodominant CD8+ T cell epitope of Circumsporozoite Protein and showed that a significant level of protection is mediated by CD8+ T cells specific for this epitope. Nonetheless, following multiple immunisations with a parasite lacking this epitope, sterile protection can still be achieved, suggesting other antigens are important for parasite-induced protection. Thus, finally I go on to identify a number of novel CD8+ T cell epitopes from antigens expressed in the sporozoite and liver stage parasite, to further broaden our view of the CD8+ T cell responses induced during the pre-erythrocytic stages of malaria

Exploring the Plasmodium falciparum Transcriptome Using Hypergeometric Analysis of Time Series (HATS)

Malaria poses a significant public health and economic threat in many regions of the world, disproportionately affecting children in sub-Saharan Africa under the age of five. Though success has been celebrated in lowering infection rates, it remains a serious challenge, causing at least 200 million infections and 655,000 deaths per year, with deleterious effects on economic growth and development. Investigation of the malaria parasite Plasmodium falciparum has entered the post-genomics age, with several strains sequenced and many microarray gene expression studies performed. Gene expression studies allow a full sampling of the genomic repertoire of a parasite, and their detailed analysis will prove invaluable in deciphering novel parasite biology as well as the modes of action of antimalarial drug resistance. We have developed a computational pipeline that converts a series of fluorescence readings from a DNA microarray into a meaningful set of biological hypotheses based on the comparison of two lines, generally one that is drug sensitive and one that is drug resistant. Each step of the computational pipeline is described in detail in this thesis, beginning with data normalization and alignment, followed by visualization through dimensionality reduction, and finally a direct analysis of the differences and similarities between the two lines. Comparisons and analyses were performed at both the individual gene and gene set level. An important component of the analytical methods we have developed is a suite of visualization tools that help to easily identify outliers and experimental flaws, measure the significance of predictions, show how lines relate and how well they can be aligned, and demonstrate the results of an analysis. These visualization tools should be used as a starting point for further biological study to test the resulting hypotheses. We also developed a software tool, Gene Attribute and Set Enrichment Ranking (GASER), which combines a wealth of genomic data from the TDR Targets web site along with expression data from a variety of sources, and allows researchers to create sophisticated weighted queries to undercover potential drug targets. Queries in our system can be updated in real time, along with their accompanying gene and gene set lists. We analyzed all possible pair-wise combinations of 11 parasite lines to create baseline distributions for gene and gene set enrichment. Using the baseline as a comparison, we identified and discarded spurious results and recognized stochastic genes and gene sets. We analyzed three major sets of parasite lines: those involving manipulation of the multidrug resistance-1 (PfMDR1) transporter, a key resistance determinant; those involving manipulation of the P. falciparum chloroquine resistance transporter (PfCRT), another important resistance determinant; and finally a set of parasites that had varying sensitivity to artemisinins. This analysis resulted in a rich library of high scoring genes that may merit further exploration as potential modes of action of resistance. More specifically, we found that manipulation of pfcrt expression resulted in an up-regulation of tRNA synthetases, which might serve to increase protein production in response to reduced amino acid availability from degraded hemoglobin. We observed that a copy number increase in pfmdr1 resulted in increases in glycerophospholipid metabolism and up-regulation of a number of ABC transporters. Finally, when comparing artemisinin sensitive to artemisinin tolerant lines, we found an increased abundance of redox metabolites and the transcripts involved in redox regulation, and significant reduction in transcription and altered expression of transcripts encoding for core histone proteins. These alterations could help confer an increased tolerance to drug induced redox perturbation by lowering endogenous redox stress. We also offer a robust computational tool, Hypergeometric Analysis of Time Series (HATS), to handle challenging biological questions related to comparison of time series experiments. Our pipeline provides a rigorous method for aligning expression experiments and then determining which genes and gene sets differ most between them. The changes in gene expression level between drug-sensitive and drug-resistant lines offer important clues in our quest for understanding mechanisms of resistance and identifying new drug targets. Our pipeline allows for comparison of future lines with our base set and holds potential for other organisms, especially those similar to Plasmodium with a strong time-dependent component. The full excel files of all the analyses performed in this thesis can be found at: (http://www.fidock.org/dan)

Bioinformatics Applications Based On Machine Learning

The great advances in information technology (IT) have implications for many sectors, such as bioinformatics, and has considerably increased their possibilities. This book presents a collection of 11 original research papers, all of them related to the application of IT-related techniques within the bioinformatics sector: from new applications created from the adaptation and application of existing techniques to the creation of new methodologies to solve existing problems

A Comparative Study of the Pathology and Pathophysiology of Severe Malaria in a Non-Human Primate Model

With the rise in plasmodial drug resistance throughout the world, a dwindling arsenal of anti-malarials and a predilection for use of less than ideal murine models, the need for effective, applicable and established animal models through which to understand severe malarial disease and test potential treatments is becoming increasingly crucial. Plasmodium coatneyi was discovered in the Philippines in the early 1960s in Rhesus macaque monkeys and has been evaluated sporadically as a potential platform for comparative studies of Plasmodium falciparum induced severe and cerebral malaria in humans. Using 30 years worth of archival samples at the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, compounded with data and material from a series of prospective experimental studies, the work in this thesis sought to fully characterize and describe the pathology and pathobiology of the P. coatneyi/Rhesus macaque model. This included a thorough examination of the parasite host interaction, clinical symptomology, hematology and clinical pathology, gross pathology, histopathology, immunohistochemistry, cerebrospinal fluid cytokine profiles, transmission and scanning electron microscopy and atomic force microscopy of the disease in 45 adult rhesus macaques. The study further compared P. coatneyi with existing P. falciparum infected human material maintained at the National University of Singapore’s Vivax laboratory and Oxford University’s Ultrastructural Morphology Group, Nuffield Division of Clinical Laboratory Sciences. The study culminated in a successful clinical drug trial which evaluated the efficacy of varying doses of methylene blue as a radical cure for malaria in this non-human primate model. In many ways, the model mirrors P. falciparum, however the benefit of this evaluation is that it highlights not only the similarities but also the divergences. The successful completion of this description of the pathology and pathobiology of the P. coatneyi/Rhesus macaque model of severe and cerebral malaria adds a complex but effective animal model to the armamentarium in the ongoing war against malaria

Drug action and resistance in malaria parasites: experimental genetics models and biochemical features of fast acting novel antimalarials

Resistance to antimalarial drugs inevitably follows their deployment in malaria endemic parts of the world. For instance, current malaria control efforts which significantly rely on artemisinin combination therapies (ACTs) are being threatened by the emergence of resistance to artemisinins and ACTs. Understanding the role of genetic determinants of artemisinin resistance is therefore important for implementation of mitigation strategies. Moreover, elucidating the mode of action for drugs that are in advanced stages of development is specifically critical as drug resistance mechanisms can be prospectively predicted and possible means of surveillance put in place. In the present work, CRISPR-Cas9 genome editing has been used to engineer candidate artemisinin resistance mutations (Kelch13 and UBP-1) in the rodent malaria parasite Plasmodium berghei. The role of these mutations in mediating artemisinin (and chloroquine) resistance under both in vitro and in vivo conditions has been assessed which up until now, has either remained un-validated (UBP-1) or debated (Kelch13, under in vivo conditions) in human infecting Plasmodium falciparum. The results have provided an in vivo model for understanding and validating artemisinin resistance phenotypes which just like their Plasmodium falciparum equivalents do not just mediate resistance phenotypes, but also carry accompanying fitness costs. In addition to the above findings, biochemical and drug inhibition studies have been carried out to demonstrate that small molecule inhibitors targeting ubiquitin hydrolases (to which UBP-1 is a class member) display activity in human and rodent infecting malaria parasites in vitro and in vivo. These inhibitors also show evidence of ability to potentiate artemisinin action which can be exploited to overcome the emerging resistance as combination partner drugs. Untargeted metabolomic screens have also been used to characterize the mode of action of lead antimalarial drug candidates that are emerging from the Novartis Institute of Tropical Diseases drug discovery pipeline. A common biochemical and metabolic profile of these compounds which display a very fast parasite killing rate is presented and can hopefully be used to identify compounds that can achieve a similar feat. Moreover, these profiles have pointed to possible mode of action for novel drugs whose mechanistic mode of parasite killing is still unknown or disputed

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Autophagy - A Double-Edged Sword

The chapters in this book review the latest advances in the molecular mechanisms of autophagy, highlighting some of the most challenging research topics. The focus is mainly on how this basic cell defense mechanism comes into play in various pathologies, including liver diseases, myopathies, infectious diseases, cancers and neurodegenerative diseases. In these diseases, the contradictory autophagy roles of cell survival versus cell death emphasize the necessity of taking into account this double-edged nature in future development of already promising, autophagy- modulating, therapies

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe