Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells

<p>Abstract</p> <p>Background</p> <p>Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary.</p> <p>Methods</p> <p>A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from <it>Plasmodium falciparum </it>cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates.</p> <p>Results</p> <p>In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures.</p> <p>Conclusion</p> <p>The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.</p

A distinct peripheral blood monocyte phenotype is associated with parasite inhibitory activity in acute uncomplicated Plasmodium falciparum malaria.

Monocyte (MO) subpopulations display distinct phenotypes and functions which can drastically change during inflammatory states. We hypothesized that discrete MO subpopulations are induced during malaria infection and associated with anti-parasitic activity. We characterized the phenotype of blood MO from healthy malaria-exposed individuals and that of patients with acute uncomplicated malaria by flow cytometry. In addition, MO defense function was evaluated by an in vitro antibody dependent cellular inhibition (ADCI) assay. At the time of admission, the percentages and absolute numbers of CD16+ MO, and CCR2+CX3CR1+ MO, were high in a majority of patients. Remarkably, expression of CCR2 and CX3CR1 on the CD14(high (hi)) MO subset defined two subgroups of patients that also differed significantly in their functional ability to limit the parasite growth, through the ADCI mechanism. In the group of patients with the highest percentages and absolute numbers of CD14(hi)CCR2+CX3CR1+ MO and the highest mean levels of ADCI activity, blood parasitemias were lower (0.14+/-0.34%) than in the second group (1.30+/-3.34%; p = 0.0053). Data showed that, during a malaria attack, some patients' MO can exert a strong ADCI activity. These results bring new insight into the complex relationships between the phenotype and the functional activity of blood MO from patients and healthy malaria-exposed individuals and suggest discrete MO subpopulations are induced during malaria infection and are associated with anti-parasitic activity

Molecular and immunological analyses of confirmed Plasmodium vivax relapse episodes

International audienceBackground: Relapse infections resulting from the activation hypnozoites produced by Plasmodium vivax and Plasmodium ovale represent an important obstacle to the successful control of these species. A single licensed drug, primaquine is available to eliminate these liver dormant forms. To date, investigations of vivax relapse infections have been few in number.Results: Genotyping, based on polymorphic regions of two genes (Pvmsp1F3 and Pvcsp) and four microsatellite markers (MS3.27, MS3.502, MS6 and MS8), of 12 paired admission and relapse samples from P. vivax-infected patients were treated with primaquine, revealed that in eight of the parasite populations in the admission and relapse samples were homologous, and heterologous in the remaining four patients. The patients’ CYP2D6 genotypes did not suggest that any were poor metabolisers of primaquine. Parasitaemia tended to be higher in the heterologous as compared to the homologous relapse episodes as was the IgG3 response. For the twelve pro- and anti-inflammatory cytokine levels measured for all samples, only those of IL-6 and IL-10 tended to be higher in patients with heterologous as compared to homologous relapses in both admission and relapse episodes.Conclusions: The data from this limited number of patients with confirmed relapse episodes mirror previous observations of a significant proportion of heterologous parasites in relapses of P. vivax infections in Thailand. Failure of the primaquine treatment that the patients received is unlikely to be due to poor drug metabolism, and could indicate the presence of P. vivax populations in Thailand with poor susceptibility to 8-aminoquinolines