Antoine Berry

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

Increased Tolerance to Artemisinin in Plasmodium falciparum Is Mediated by a Quiescence Mechanism

ABSTRACT Artemisinin (ART)-based combination therapies (ACTs) are the first-line drugs—and often the last treatments—that can effectively cure Plasmodium falciparum infections. Unfortunately, the decreased clinical efficacy of artesunate, one of the major ART derivatives, was recently reported along the Thailand-Cambodia border. Through long-term artemisinin pressure in vitro, we have obtained an ART-tolerant strain that can survive extremely high doses of ART. We showed that drug pressure could induce a subpopulation of ring stages into developmental arrest, which can explain the ART tolerance in P. falciparum. We also observed interesting transcriptomic modifications possibly associated with the acquisition of ART tolerance. These modifications include the overexpression of heat shock and erythrocyte surface proteins and the downexpression of a cell cycle regulator and a DNA biosynthesis protein. This study highlights a new phenomenon in the Plasmodium response to ART that may explain the delayed clearance of parasites after artesunate treatment observed on the Thailand-Cambodia border and that provides important information for achieving a better understanding of the mechanisms of antimalarial resistance.

Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia

ABSTRACT The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.

A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms.

BACKGROUND Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species

BackgroundParasite concentration methods facilitate molecular, biochemical and immunological research on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS® columns for the purification of human Plasmodium species is presented. This method was useful for the concentration/purification of either schizonts or gametocytes.Results and conclusionsThe magnetic removal of non-parasitized red blood cells (in vivo and in vitro) using magnetic columns (MACS) was evaluated. This easy-to-use technique enriched schizonts and gametocytes from Plasmodium falciparum in vitro cultures with a very high degree of purity. In addition, all haemozoin-containing stages (schizonts and/or gametocytes) from the peripheral blood of infected patients could be concentrated using this method. This method is particularly useful for the concentration of non-falciparum species, which do not grow in culture and are otherwise difficult to obtain in large amounts.

Trioxaquines Are New Antimalarial Agents Active on All Erythrocytic Forms, Including Gametocytes

ABSTRACT Malaria is the third most significant cause of infectious disease in the world. The search for new antimalarial chemotherapy has become increasingly urgent due to parasite resistance to classical drugs. Trioxaquines are synthetic hybrid molecules containing a trioxane motif (which is responsible for the antimalarial activity of artemisinin) linked to an aminoquinoline entity (which is responsible for the antiplasmodial properties of chloroquine). These trioxaquines are highly potent against young erythrocytic stages of Plasmodium falciparum and exhibit efficient activity in vitro against chloroquine-sensitive and -resistant strains of P. falciparum (50% inhibitory concentration, 4 to 32 nM) and are also active in vivo against P. vinckei petteri and P. yoelii nigeriensis in suppressive and curative murine tests. The trioxaquine DU1302 is one of these promising antimalarial agents. The present study confirms the absence of toxicity of this drug on cell lines and in a mice model. Moreover, DU1302 exhibits potent activity against gametocytes, the form transmitted by mosquitoes, as killing of the gametocytes is essential to limit the spread of malaria. The ease of chemical synthesis of this trioxaquine prototype should be considered an additional advantage and would make these drugs affordable without perturbations of the drug supply.

Comparison between Two Amplification Sets for Molecular Diagnosis of Toxoplasmosis by Real-Time PCR

ABSTRACT PCR is now commonly applied to the diagnosis of toxoplasmosis. Although several methods are available, comparative studies are few, making it difficult to compare the performance of each technique. We compared the sensitivities of two real-time PCR assays through a prospective study on fetuses, neonates, and immunocompromised patients and on the ocular diagnosis of toxoplasmosis. The first system targeted the widely used B1 gene (GenBank accession number AF179871) while the second (RE) targeted a more recently described sequence repeated roughly 200 to 300 times (GenBank accession number AF146527). We demonstrated that molecular diagnosis requires the duplication of PCR assays, especially with the B1 system, as only one PCR was positive in 33.3% of cases. Our study showed that the RE target was more sensitive for all biological samples (amniotic fluid, placenta, aqueous humor, whole blood, and cerebrospinal and bronchoalveolar fluids) and significantly improved the performance of the diagnosis of toxoplasmosis. Taking into consideration all clinical samples, the mean gain in the crossing point value was 4.2 ± 1.7 cycles and was even more significant for amniotic fluid (5.8 ± 1.7 cycles).

Routine Identification of Medical Fungi by the New Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight System with a New Time-Effective Strategy

ABSTRACT We report here a clinical evaluation of the Vitek MS system for rapid fungal identification. A strategy that uses a single deposit without prior protein extraction was utilized to save time and money. Clinical isolates from the Toulouse University hospital were used to evaluate the performance of the Vitek MS compared to that of both routine laboratory techniques and Vitek2. The Vitek MS performed well in the identification of yeasts and Aspergillus fungi (93.2% of correct identifications).

IL-13 induces expression of CD36 in human monocytes through PPARgamma activation.

The class B scavenger receptor CD36 is a component of the pattern recognition receptors on monocytes that recognizes a variety of molecules. CD36 expression in monocytes depends on exposure to soluble mediators. We demonstrate here that CD36 expression is induced in human monocytes following exposure to IL‐13, a Th2 cytokine, via the peroxisome proliferator‐activated receptor (PPAR)γ pathway. Induction of CD36 protein was paralleled by an increase in CD36 mRNA. The PPARγ pathway was demonstrated using transfection of a PPARγ expression plasmid into the murine macrophage cell line RAW264.7, expressing very low levels of PPARγ, and in peritoneal macrophages from PPARγ‐conditional null mice. We also show that CD36 induction by IL‐13 via PPARγ is dependent on phospholipase A2 activation and that IL‐13 induces the production of endogenous 15‐deoxy‐Δ12,14‐prostaglandin J2, an endogenous PPARγ ligand, and its nuclear localization in human monocytes. Finally, we demonstrate that CD36 and PPARγ are involved in IL‐13‐mediated phagocytosis of Plasmodium falciparum‐parasitized erythrocytes. These results reveal a novel role for PPARγ in the alternative activation of monocytes by IL‐13, suggesting that endogenous PPARγ ligands, produced by phospholipase A2 activation, could contribute to the biochemical and cellular functions of CD36.

Simultaneous detection of gastrointestinal pathogens with a multiplex Luminex-based molecular assay in stool samples from diarrhoeic patients

We have evaluated the multiplex molecular method xTAG(®) Gastrointestinal Panel (GPP) for detecting pathogens in stool samples of diarrhoeic patients. We collected 440 samples from 329 patients (male:female ratio of 1.2:1), including 102 immunosuppressed adults, 50 immunosuppressed children, 56 children attending the neonatal unit and 121 children attending the emergency unit. Of these, 176 samples from 162 patients were xTAG(®) GPP positive (102 viruses, 61 bacteria and 13 parasites) and the assay was more sensitive than the conventional test for detecting rotavirus (p <0.01), noroviruses (p <0.0001), Salmonella spp. (p <0.001), Campylobacter spp. (p <0.001) and toxigenic Clostridium difficile (p 0.005). The predominant pathogens were viruses (23.2%), with rotavirus (15.9%) being the most common. Bacterial agents were detected in 13.9%; the most common was Salmonella spp. (4.8%). Parasites were detected in 2.9%; Cryptosporidium spp. (2%) was the most common. There were 31 co-infections (7% of samples), involving two pathogens in 23 (5.2%) and three pathogens in eight (1.8%) samples. There were 113 (92.6%) positive samples from the children attending the emergency unit, 25 (17%) positive samples from immunosuppressed adults, 22 (25.3%) positive samples from immunosuppressed children and 16 (19%) positive samples from children attending the neonatal unit. The low turnaround time and technical hands-on time make this multiplex technique convenient for routine use. Nevertheless, conventional bacterial culture and parasitological stool examination are still required to detect other pathogens in specific cases and to determine susceptibility to antibiotics.