Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission

<p>Abstract</p> <p>Background</p> <p>Aggregated haemozoin crystals within malaria-infected erythrocytes confer susceptibility of parasitized cells to a magnetic field. Here the utility of this method for diagnosis of human malaria is evaluated in a malaria-endemic region of Papua New Guinea (PNG).</p> <p>Methods and findings</p> <p>Individuals with <it>Plasmodium falciparum </it>malaria symptoms (n = 55) provided samples for conventional blood smear (CBS) and magnetic deposition microscopy (MDM) diagnosis. Standard Giemsa staining and light microscopy was performed to evaluate all preparations. <it>Plasmodium falciparum </it>parasitaemia observed on MDM slides was consistently higher than parasitaemia observed by (CBS) for ring (CBS = 2.6 vs. MDM = 3.4%; t-test P-value = 0.13), trophozoite (CBS = 0.5 vs. MDM = 1.6%; t-test P-value = 0.01), schizont (CBS = 0.003 vs. MDM = 0.1%; t-test P-value = 0.08) and gametocyte (CBS = 0.001 vs. MDM = 0.4%; t-test P-value = 0.0002) parasitaemias. Gametocyte prevalence determined by CBS compared to MDM increased from 7.3% to 45%, respectively.</p> <p>Conclusion</p> <p>MDM increased detection sensitivity of <it>P. falciparum</it>-infected, haemozoin-containing erythrocytes from infected humans while maintaining detection of ring-stage parasites. Gametocyte prevalence five-fold higher than observed by CBS suggests higher malaria transmission potential in PNG endemic sites compared to previous estimates.</p

Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

Christopher King and colleagues found that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes, suggesting that a PvDBP-based vaccine may reduce blood stage Plasmodium vivax infection

Expanding the Antimalarial Drug Arsenal—Now, But How?

The number of available and effective antimalarial drugs is quickly dwindling. This is mainly because a number of drug resistance-associated mutations in malaria parasite genes, such as crt, mdr1, dhfr/dhps, and others, have led to widespread resistance to all known classes of antimalarial compounds. Unfortunately, malaria parasites have started to exhibit some level of resistance in Southeast Asia even to the most recently introduced class of drugs, artemisinins. While there is much need, the antimalarial drug development pipeline remains woefully thin, with little chemical diversity, and there is currently no alternative to the precious artemisinins. It is difficult to predict where the next generation of antimalarial drugs will come from; however, there are six major approaches: (i) re-optimizing the use of existing antimalarials by either replacement/rotation or combination approach; (ii) repurposing drugs that are currently used to treat other infections or diseases; (iii) chemically modifying existing antimalarial compounds; (iv) exploring natural sources; (v) large-scale screening of diverse chemical libraries; and (vi) through parasite genome-based (“targeted”) discoveries. When any newly discovered effective antimalarial treatment is used by the populus, we must maintain constant vigilance for both parasite-specific and human-related factors that are likely to hamper its success. This article is neither comprehensive nor conclusive. Our purpose is to provide an overview of antimalarial drug resistance, associated parasite genetic factors (1. Introduction; 2. Emergence of artemisinin resistance in P. falciparum), and the antimalarial drug development pipeline (3. Overview of the global pipeline of antimalarial drugs), and highlight some examples of the aforementioned approaches to future antimalarial treatment. These approaches can be categorized into “short term” (4. Feasible options for now) and “long term” (5. Next generation of antimalarial treatment—Approaches and candidates). However, these two categories are interrelated, and the approaches in both should be implemented in parallel with focus on developing a successful, long-lasting antimalarial chemotherapy

Correction: PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0139253.]

Data from: PCR-Free enrichment of mitochondrial DNA from human blood and cell lines for high quality next-generation DNA sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases

An ON-OFF Magneto-Optical Probe of Anisotropic Biofluid Crystals: A β-Hematin Case Study

We have designed, developed, and evaluated an innovative portable magneto-optical detector (MOD) in which a light beam with variable polarization passes through a fluid sample immersed in a variable magnetic field. The light intensity is measured downstream along the forward scattering direction. The field is turned on and off through the in-and-out motion of nearby permanent magnets. As a result, for sufficiently, magnetically, and optically anisotropic samples, the optical absorption is sensitive to changes in the light polarization. Both detection and characterization applications are, therefore, available. For instance, both the degree of malaria infection and hemozoin crystalline properties can be measured and studied, respectively. We present experimental results for synthetic hemozoin and describe them in terms of the basic physics and chemistry underlying the correlations of the directions of the external magnetic field and the light beam polarization. We connect this work to a commercialized product for malaria detection and compare it with other magneto-optical instruments and methods. We conduct tests of absorption parameters and the electric polarizability tensor, and we discuss the connection to magnetic and electric dipole moments

Growth of Plasmodium falciparum in response to a rotating magnetic field

Abstract Background Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite’s haemozoin to inhibit parasite viability. Results Paramagnetic haemozoin crystals, a byproduct of the parasite’s haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most affected by the rotating magnetic fields (p = 0.0075) as compared to a static magnetic field of the same strength. Parasite growth in the presence of a static magnetic field appears to aid parasite growth. Conclusions Sequestration of the toxic haem resulting from haemoglobin digestion is key for the parasites’ survival and the focus of almost all existing anti-malarial drugs. Understanding how the parasites create the haemozoin molecule and the disruption of its creation aids in the development of drugs to combat this disease

Application of magnetic cytosmear for the estimation of Plasmodium falciparum gametocyte density and detection of asexual stages in asymptomatic children

Research Article published by Malaria JournalBackground: Conventional malaria parasite detection methods, such as rapid diagnostic tests (RDT) and light microscopy (LM), are not sensitive enough to detect low level parasites and identification of gametocytes in the peripheral blood. A modified and sensitive laboratory prototype, Magnetic Deposition Microscopy (MDM) was developed to increase the detection of sub-microscopic parasitaemia and estimation of gametocytes density in asymptomatic school children. Methods: Blood samples were collected from 303 asymptomatic school children from seven villages in Bagamoyo district in Tanzania. Participants were screened for presence of malaria parasites in the field using RDT and MDM whereas further examination of malaria parasites was done in the laboratory by LM. LM and MDM readings were used to calculate densities and estimate prevalence of asexual and sexual stages of the parasite. Results: Plasmodium falciparum parasites (asexual and sexual stages) were detected in 23 (7.6 %), 52 (17.2 %), and 59 (19.5 %) out of 303 samples by LM, RDT and MDM respectively. Gametocytes were detected in 4 (1.3 %) and 12 (4.0 %) out of the same numbers of samples by LM, and MDM, respectively. Likewise, in vitro results conducted on two laboratory strains of P. falciparum, 3D7 and NF54 to assess MDM sensitivity on gametocytes detection and its application on concentrating gametocytes indicated that gametocytes were enriched by MDM by 10-fold higher than LM. Late stages of the parasite strains, 3D7 and NF54 were enriched by MDM by a factor of 20.5 and 35.6, respectively. MDM was more specific than LM and RDT by 87.5 % (95 %, CI 71.2–89.6 %) and 89.0 % (95 % CI 82.9–91.4) respectively. It was also found that MDM sensitivity was 62.5 % (95 % CI 49.5–71.8) when compared with RDT while with LM was 36.5 % (95 % CI 32.2–60.5). Conclusions: These findings provide strong evidence that MDM enhanced detection of sub-microscopic P. falciparum infections and estimation of gametocyte density compared to current malaria diagnostic tools. In addition, MDM is superior to LM in detecting sub-microscopic gametocytaemia. Therefore, MDM is a potential tool for low-level parasitaemia identification and quantification with possible application in malaria transmission research