https://link.springer.com/content/pdf/10.1007%2Fs10156-009-0669-9.pdf

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.

Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax Gametocytes in Relation to Malaria Control and Elimination

The interruption of malaria transmission worldwide is one of the greatest challenges for international health and development communities. The current expert view suggests that, by aggressively scaling up control with currently available tools and strategies, much greater gains could be achieved against malaria, including elimination from a number of countries and regions; however, even with maximal effort we will fall short of global eradication. The Malaria Eradication Research Agenda (malERA) complements the current research agenda—primarily directed towards reducing morbidity and mortality—with one that aims to identify key knowledge gaps and define the strategies and tools that will result in reducing the basic reproduction rate to less than 1, with the ultimate aim of eradication of the parasite from the human population. Sustained commitment from local communities, civil society, policy leaders, and the scientific community, together with a massive effort to build a strong base of researchers from the endemic areas will be critical factors in the success of this new agenda.

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A research agenda to underpin malaria eradication.

Malaria is a major cause of death in tropical and sub-tropical countries, killing each year over 1 million people globally; 90% of fatalities occur in African children. Although effective ways to manage malaria now exist, the number of malaria cases is still increasing, due to several factors. In this emergency situation, prompt and effective diagnostic methods are essential for the management and control of malaria. Traditional methods for diagnosing malaria remain problematic; therefore, new technologies have been developed and introduced to overcome the limitations. This review details the currently available diagnostic methods for malaria.

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Malaria diagnosis: a brief review.

Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5′ and 3′ untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.

/pdf/new-insights-into-the-blood-stage-transcriptome-of-2erfyhspwu.pdf

New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

A small-scale systematic analysis of alternative splicing in Plasmodium falciparum.

Rhoptry-associated membrane antigen (RAMA) is an abundant glycophosphatidylinositol (GPI)-anchored protein that is embedded within the lipid bilayer and is implicated in parasite invasion. Antibody responses against rhoptry proteins are produced by individuals living in a malaria-endemic area, suggesting the immunogenicity of Plasmodium vivax RAMA (PvRAMA) for induction of immune responses during P. vivax infection. To determine whether PvRAMA contributes to the acquisition of immunity to malaria and could be a rational candidate for a vaccine, the presence of memory T cells and the stability of the antibody response against PvRAMA were evaluated in P. vivax-exposed individuals. The immunogenicity of PvRAMA for the induction of T cell responses was evaluated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs). High levels of interferon (IFN)-γ and interleukin (IL)-10 cytokines were detected in the culture supernatant of PBMCs, and the CD4+ T cells predominantly produced IL-10 cytokine. The levels of total anti-PvRAMA immunoglobulin G (IgG) antibody were significantly elevated, and these antibodies persisted over the 12 months of the study. Interestingly, IgG1, IgG2 and IgG3 were the major antibody subtypes in the response to PvRAMA. The frequency of IgG3 in specific to PvRAMA antigen maintained over 12 months. These data could explain the immunogenicity of PvRAMA antigen in induction of both cell-mediated and antibody-mediated immunity in natural P. vivax infection, in which IFN-γ helps antibody class switching toward the IgG1, IgG2 and IgG3 isotypes and IL-10 supports PvRAMA-specific antibody production.

/pdf/naturally-acquired-immune-response-against-plasmodium-vivax-seiy430qr1.pdf

Naturally-Acquired Immune Response against Plasmodium vivax Rhoptry-Associated Membrane Antigen.

The present invention provides an easy and rapid method for detecting/identifying the presence or absence of specific Plasmodium parasites and four species of malaria parasites in a human specimen, an anti-malaria measure support system, and a malaria infection-prevention/treatment system, which can contribute to practical diagnosis in a malaria endemic area. According to the present invention, using a genus-specific primer set that can detect four Plasmodium parasites that infect humans at a time, and the primer sets each specific to each of four species of Plasmodium parasites (P. falciparum, P. vivax, P. malariae, and P. ovale), the presence or absence of infection with these parasites can be detected/identified easily and rapidly.

Eun-Taek Han

Papers

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

A small-scale systematic analysis of alternative splicing in Plasmodium falciparum.

Naturally-Acquired Immune Response against Plasmodium vivax Rhoptry-Associated Membrane Antigen.

Primers for detecting plasmodium

Diagnosis of Malaria Infections in an Endemic Setting in Thailand