A comparison of the sensitivities of detection of Plasmodium falciparum gametocytes by magnetic fractionation, thick blood film microscopy, and RT-PCR

<p>Abstract</p> <p>Background</p> <p>The magnetic properties of <it>Plasmodium</it>-infected erythrocytes have been exploited for different clinical and research purposes. A recent study in a rural clinical setting in Papua New Guinea has demonstrated that <it>Plasmodium falciparum </it>gametocyte detection is facilitated by magnetic deposition microscopy but no study has yet determined the relative sensitivity and limit of detection of a magnetic fractionation technique. The present study compares the detection limit and sensitivity of a technique based on the use of commercially available magnetic fractionation columns with those for thick blood film microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) methods.</p> <p>Methods</p> <p>Gametocyte detection in six series of dilutions of cultured <it>P. falciparum </it>parasites with known gametocytaemia was conducted using magnetic fractionation, thick blood film, and RT-PCR techniques.</p> <p>Results</p> <p>The preparations obtained by the magnetic fractionation method were of thin film quality allowing easy gametocyte identification by light microscopy. Magnetic fractionation had a higher sensitivity and approximately two orders of magnitude better limit of detection than thick blood film microscopy. Gametocytes were also more readily detectable on the magnetically fractionated preparations. Magnetic fractionation had a similar limit of detection to that of RT-PCR.</p> <p>Conclusion</p> <p>Magnetic fractionation is a highly sensitive and convenient method for gametocyte detection in comparison with the standard thick blood film and RT-PCR methods, and could readily be adapted to field application.</p

Parameterization of high magnetic field gradient fractionation columns for applications with Plasmodium falciparum infected human erythrocytes

<p>Abstract</p> <p>Background</p> <p>Magnetic fractionation of erythrocytes infected with <it>Plasmodium falicparum </it>has several research uses including enrichment of infected cells from parasite cultures or enhanced detection of <it>P. falciparum </it>gametocytes. The aim of the present study was to quantitatively characterize the magnetic fractionation process and thus enable optimization of protocols developed for specific uses.</p> <p>Methods</p> <p>Synchronized cultures of <it>P. falciparum </it>parasites incubated with human erythrocytes were magnetically fractionated with commercially available columns. The timing of the fractionation experiments was such that the parasites were in second half of their erythrocytic life cycle with parasite densities ranging from 1 to 9%. Fractionations were carried out in a single pass through the columns. Cells were enumerated and differentiated in the initial samples as well as in the positive and negative fractions. The capture of cells by the fractionation column was described by a saturation binding model.</p> <p>Results</p> <p>The magnetic binding affinity to the column matrix was approximately 350 times greater for infected cells compared with uninfected cells. The purity of infected cells in the captured fraction was generally >80% but decreased rapidly (to less than 50%) when the number of infected cells that passed through the column was substantially decreased (to less than 9 ± 5 × 10⁵cells). The distribution of captured parasite developmental stages shifted to mature stages as the number of infected cells in the initial samples and flow rate increased. The relationship between the yield of infected cells in the captured fraction and flow rate of cells conformed to a complementary cumulative log-normal equation with flow rates >1.6 × 10⁵cells per second resulting in yields <50%.</p> <p>Conclusions</p> <p>A detailed quantitative analysis of a batchwise magnetic fractionation process for malaria infected erythrocytes using high gradient magnetic fractionation columns was performed. The models applied in this study allow the prediction of capture efficiency if the initial infected cell concentration and the flow rate are known.</p

A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity

<p>Abstract</p> <p>Background</p> <p>Recently developed Sybr Green-based <it>in vitro Plasmodium falciparum </it>drug sensitivity assays provide an attractive alternative to current manual and automated methods. The present study evaluated flow cytometry measurement of DNA staining with Sybr Green in comparison with the <it>P. falciparum </it>lactate dehydrogenase assay, the tritiated hypoxanthine incorporation assay, a previously described Sybr Green based plate reader assay and light microscopy.</p> <p>Methods</p> <p>All assays were set up in standardized format in 96-well plates. The 50% inhibitory concentrations (IC₅₀) of chloroquine, mefloquine and dihydroartemisinin against the laboratory adapted <it>P. falciparum </it>strains 3D7, E8B, W2mef and Dd2 were determined using each method.</p> <p>Results</p> <p>The resolution achieved by flow cytometry allowed quantification of the increase in individual cell DNA content after an incubation period of only 24 h. Regression, and Bland and Altman analyses showed that the IC₅₀values determined using the flow cytometry assay after 24 h agreed well with those obtained using the hypoxanthine incorporation assay, the <it>P. falciparum </it>lactate dehydrogenase assay, the Sybr Green plate reader assay and light microscopy. However the values obtained with the flow cytometry assay after 48 h of incubation differed significantly from those obtained with the hypoxanthine incorporation assay, and the <it>P. falciparum </it>lactate dehydrogenase assay at low IC₅₀values, but agreed well with the Sybr Green plate reader assay and light microscopy.</p> <p>Conclusions</p> <p>Although flow cytometric equipment is expensive, the necessary reagents are inexpensive, the procedure is simple and rapid, and the cell volume required is minimal. This should allow field studies using fingerprick sample volumes.</p

Tissue iron distribution assessed by MRI in patients with iron loading anemias

Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue's iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation

Encapsulation and Sustained Release of Curcumin using Superparamagnetic Silica Reservoirs

For controlled release and targeted delivery of curcumin in an aqueous medium a method of encapsulating curcumin and magnetic nanoparticles inside porous silica matrix has been developed. Curcumin and superparamagnetic nanoparticles are loaded inside porous silica in a single process. The graphic shows the TEM image of microtomed sample of Fe3O4 particles surrounded by a silica matrix

Stereological analysis of liver biopsy histology sections as a reference standard for validating non-invasive liver fat fraction measurements by MRI

© 2016 St. Pierre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background and Aims: Validation of non-invasive methods of liver fat quantification requires a reference standard. However, using standard histopathology assessment of liver biopsies is problematical because of poor repeatability. We aimed to assess a stereological method of measuring volumetric liver fat fraction (VLFF) in liver biopsies and to use the method to validate a magnetic resonance imaging method for measurement of VLFF. Methods: VLFFs were measured in 59 subjects (1) by three independent analysts using a stereological point counting technique combined with the Delesse principle on liver biopsy histological sections and (2) by three independent analysts using the HepaFat-Scan® technique on magnetic resonance images of the liver. Bland Altman statistics and intraclass correlation (IC) were used to assess the repeatability of each method and the bias between the methods of liver fat fraction measurement. Results: Inter-analyst repeatability coefficients for the stereology and HepaFat-Scan® methods were 8.2 (95% CI 7.7-8.8)% and 2.4 (95% CI 2.2-2.5)% VLFF respectively. IC coefficients were 0.86 (95% CI 0.69-0.93) and 0.990 (95% CI 0.985-0.994) respectively. Small biases (=3.4%) were observable between two pairs of analysts using stereology while no significant biases were observable between any of the three pairs of analysts using Hepa-Fat-Scan®. A bias of 1.4±0.5% VLFF was observed between the HepaFat-Scan® method and the stereological method. Conclusions: Repeatability of the stereological method is superior to the previously reported performance of assessment of hepatic steatosis by histopathologists and is a suitable reference standard for validating non-invasive methods of measurement of VLFF

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates. \ua9 2014 Macmillan Publishers Limited. All rights reserved

A Sub-Microscopic Gametocyte Reservoir Can Sustain Malaria Transmission

Novel diagnostic tools, including PCR and high field gradient magnetic fractionation (HFGMF), have improved detection of asexual Plasmodium falciparum parasites and especially infectious gametocytes in human blood. These techniques indicate a significant number of people carry gametocyte densities that fall below the conventional threshold of detection achieved by standard light microscopy (LM).To determine how low-level gametocytemia may affect transmission in present large-scale efforts for P. falciparum control in endemic areas, we developed a refinement of the classical Ross-Macdonald model of malaria transmission by introducing multiple infective compartments to model the potential impact of highly prevalent, low gametocytaemic reservoirs in the population. Models were calibrated using field-based data and several numerical experiments were conducted to assess the effect of high and low gametocytemia on P. falciparum transmission and control. Special consideration was given to the impact of long-lasting insecticide-treated bed nets (LLIN), presently considered the most efficient way to prevent transmission, and particularly LLIN coverage similar to goals targeted by the Roll Back Malaria and Global Fund malaria control campaigns. Our analyses indicate that models which include only moderate-to-high gametocytemia (detectable by LM) predict finite eradication times after LLIN introduction. Models that include a low gametocytemia reservoir (requiring PCR or HFGMF detection) predict much more stable, persistent transmission. Our modeled outcomes result in significantly different estimates for the level and duration of control needed to achieve malaria elimination if submicroscopic gametocytes are included.It will be very important to complement current methods of surveillance with enhanced diagnostic techniques to detect asexual parasites and gametocytes to more accurately plan, monitor and guide malaria control programs aimed at eliminating malaria

Sensitivity of Pine Island Glacier to observed ocean forcing

We present subannual observations (2009–2014) of a major West Antarctic glacier (Pine Island Glacier) and the neighboring ocean. Ongoing glacier retreat and accelerated ice flow were likely triggered a few decades ago by increased ocean-induced thinning, which may have initiated marine ice-sheet instability. Following a subsequent 60% drop in ocean heat content from early 2012 to late 2013, ice flow slowed, but by < 4%, with flow recovering as the ocean warmed to prior temperatures. During this cold-ocean period, the evolving glacier-bed/ice-shelf system was also in a geometry favorable to stabilization. However, despite a minor, temporary decrease in ice discharge, the basin-wide thinning signal did not change. Thus, as predicted by theory, once marine ice-sheet instability is underway, a single transient high-amplitude ocean cooling has only a relatively minor effect on ice flow. The long-term effects of ocean-temperature variability on ice flow, however, are not yet known