Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs

Background: The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag (R) system associated with the atmospheric generators for capnophilic bacteria Genbag CO2 (R) was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. Methods: The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour H-3-hypoxanthine uptake inhibition method using the Genbag CO2 (R) system and compared to controlled incubator conditions (5% CO2 and 10% O-2). Results: The counts per minute values in the control wells in incubator atmospheric conditions (5% CO2 and 10% O-2) were significantly higher than those of Genbag (R) conditions (2738 cpm vs 2282 cpm, p < 0.0001). The geometric mean IC50 estimated under the incubator atmospheric conditions was significantly lower for atovaquone (1.2 vs 2.1 nM, p = 0.0011) and higher for the quinolines: chloroquine (127 vs 94 nM, p < 0.0001), quinine (580 vs 439 nM, p < 0.0001), monodesethylamodiaquine (41.4 vs 31.8 nM, p < 0.0001), mefloquine (57.5 vs 49.7 nM, p = 0.0011) and lumefantrine (23.8 vs 21.2 nM, p = 0.0044). There was no significant difference of IC50 between the 2 conditions for dihydroartemisinin, doxycycline and pyrimethamine. To reduce this difference in term of anti-malarial susceptibility, a specific cut-off was estimated for each drug under Genbag (R) conditions by regression. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O-2), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2000 nM). Conclusions: The atmospheric generators for capnophilic bacteria Genbag CO2 (R) is an appropriate technology that can be transferred to the field for epidemiological surveys of drug-resistant malaria. The present data suggest the importance of the gas mixture on in vitro microtest results for anti-malarial drugs and the importance of determining the microtest conditions before comparing and analysing the data from different laboratories and concluding on malaria resistance

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

<p>Abstract</p> <p>Background</p> <p>Quinine is still recommended as an effective therapy for severe cases of <it>Plasmodium falciparum </it>malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na⁺/H⁺exchanger, <it>Pfnhe-1</it>, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of <it>Pfnhe </it>on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a <it>Pfnhe </it>polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between <it>Pfnhe </it>and QNR is investigated in a series of isolates from central Africa.</p> <p>Methods</p> <p>The sequence analysis of the polymorphisms at the <it>Pfnhe-1 </it>ms4760 microsatellite and the evaluation of <it>in vitro </it>quinine susceptibility (by isotopic assay) were conducted in 74 <it>P. falciparum </it>isolates from the Republic of Congo.</p> <p>Results</p> <p>Polymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the <it>Pfnhe-1 </it>ms4760 microsatellite were not associated with quinine susceptibility.</p> <p>Conclusions</p> <p>The polymorphism in the microsatellite ms4760 in <it>Pfnhe-1 </it>that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of <it>Pfnhe </it>as a molecular marker for QNR. The contribution of <it>Pfnhe </it>to the <it>in vitro </it>response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure.</p

Atorvastatin treatment is effective when used in combination with mefloquine in an experimental cerebral malaria murine model

<p>Abstract</p> <p>Background</p> <p>One of the major complications of <it>Plasmodium falciparum </it>infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine.</p> <p>Methods</p> <p>The efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental <it>Plasmodium berghei </it>ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining.</p> <p>Results</p> <p>AVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with MQ, rather than MQ alone, led to a significant delay in mouse death and had an effect on the onset of CM symptoms and on the level of parasitaemia. Histopathological findings show a correlation between brain lesions and CM onset. A neuronal anti-apoptotic effect of AVA in the AVA + MQ combination was not shown.</p> <p>Conclusions</p> <p>The combination of AVA and MQ therapy led to a significant delay in mouse mortality. There were differences in the incidence, time to cerebral malaria and the level of parasitaemia when the drug combination was administered to mice. When used in combination with MQ, AVA had a relevant effect on the in vivo growth inhibition and clinical outcome of <it>P. berghei </it>ANKA-infected mice.</p

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC(50)) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC(50 )for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O(2 )to 21% O(2 )conditions, no parasiticide effect of O(2 )concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O(2 )with an increase in the length of schizogony. The chloroquine IC(50 )at 10% O(2 )were significantly higher than those at 21% O(2), means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC(50 )> 100 nM) at 10% O(2), 17 were sensitive to chloroquine (IC(50 )< 100 nM) at 21% O(2). CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance

Bounding the Time Delay between High-energy Neutrinos and Gravitational-wave Transients from Gamma-ray Bursts

We derive a conservative coincidence time window for joint searches of gravita-tional-wave (GW) transients and high-energy neutrinos (HENs, with energies above 100GeV), emitted by gamma-ray bursts (GRBs). The last are among the most interesting astrophysical sources for coincident detections with current and near-future detectors. We take into account a broad range of emission mechanisms. We take the upper limit of GRB durations as the 95% quantile of the T90's of GRBs observed by BATSE, obtaining a GRB duration upper limit of ~150s. Using published results on high-energy (>100MeV) photon light curves for 8 GRBs detected by Fermi LAT, we verify that most high-energy photons are expected to be observed within the first ~150s of the GRB. Taking into account the breakout-time of the relativistic jet produced by the central engine, we allow GW and HEN emission to begin up to 100s before the onset of observable gamma photon production. Using published precursor time differences, we calculate a time upper bound for precursor activity, obtaining that 95% of precursors occur within ~250s prior to the onset of the GRB. Taking the above different processes into account, we arrive at a time window of tHEN - tGW ~ [-500s,+500s]. Considering the above processes, an upper bound can also be determined for the expected time window of GW and/or HEN signals coincident with a detected GRB, tGW - tGRB ~ tHEN - tGRB ~ [-350s,+150s]

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Recently, <it>Plasmodium falciparum </it>parasites bearing <it>Pfdhfr </it>I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves.</p> <p>Methods</p> <p>Thirty <it>Plasmodium falciparum </it>clinical isolates were collected in 2008 in the south-east of Madagascar. A part of <it>Pfdhfr </it>gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC_50sto pyrimethamine was performed on adapted isolates.</p> <p>Results</p> <p>Four different <it>Pfdhfr </it>alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of <it>P. falciparum </it>isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine <it>in vitro </it>susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC₅₀) < 10 nM. The geometric mean of IC₅₀of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC₅₀of the triple mutant parasite (13,804 nM).</p> <p>Conclusion</p> <p>The IC₅₀s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC₅₀s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the <it>Pfdhfr </it>I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.</p

Absence of association between pyronaridine in vitro responses and polymorphisms in genes involved in quinoline resistance in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The aim of the present work was to assess the <it>in vitro </it>cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>and <it>pfnhe</it>.</p> <p>Methods</p> <p>The <it>in vitro </it>chemosusceptibility profiles of 23 strains of <it>Plasmodium falciparum </it>were analysed by the standard 42-hour ³H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfnhe-1 </it>and <it>pfmrp </it>genes.</p> <p>Results</p> <p>The IC₅₀values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (<it>r²</it>= 0.20; <it>P </it>= 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC₅₀and responses to other anti-malarials. Significant associations were not found between pyronaridine IC₅₀and polymorphisms in <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>or <it>pfnhe-1</it>.</p> <p>Conclusion</p> <p>There was an absence of cross-resistance between pyronaridine and quinolines, and the IC₅₀values for pyronaridine were found to be unrelated to mutations in the transport protein genes <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfmrp </it>or <it>pfnhe-1</it>, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.</p

The effect of structure slope and packing arrangement on the hydraulic stability of geotextile sand container revetments

Thesis (MScEng)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Innovative and versatile coastal protection structures made of Geotextile Sand Containers (GSCs) are increasingly being incorporated into coastal management solutions because of their cost effective and environmentally friendly characteristics. This is as opposed to conventional ‘hard’ coastal protection solutions that utilise rocks and or concrete units to protect the coastline. With GSC structures being a relatively new coastal protection solution, few design and construction guidelines are available. Research into the behaviour of GSC structures under wave attack is on-going with particular emphasis on the hydraulic processes that affect GSC structures and cause them to fail. The use of GSC revetments as coastal protection solutions has become more popular in South Africa during recent times, particularly along the coastline of KwaZulu-Natal. However, the chosen design of these GSC revetments falls outside the range of applicability of the available design charts and stability equations. Therefore the hydraulic stability of these structures is largely unknown. The primary objective of this study is to investigate the effect of structure slopes and packing arrangements on the hydraulic stability of GSC revetments. The application of available design charts and stability equations was also evaluated. Two-dimensional physical modelling was undertaken and a total of 12 GSC revetment permutations were tested during the physical modelling test series. The results of the physical modelling showed that the structure slope had the most significant effect on the hydraulic stability. Steeper structure slopes were more hydraulically stable than gentler structure slopes. The packing arrangements of the GSCs had less of an effect on the hydraulic stability of the GSC revetments. Single layer GSC armour revetments matched or out-performed the equivalent double layer GSC revetments; while GSC revetments with GSCs orientated with the long axis perpendicular to the wave attack performed marginally better than the equivalent GSC revetments with GSCs orientated with the long axis parallel to the wave attack. The available design charts and stability equations were assessed against the results of the physical modelling and showed varying degrees of correlation. The stability equation proposed by Recio (2007) proved to be particularly accurate.AFRIKAANSE OPSOMMING: Innoverende en veelsydige kusbeskermingstrukture wat van geotekstielsandhouers (GSH’s) gemaak is, word al hoe meer by kusbestuursoplossings ingesluit weens die kostedoeltreffendheid en omgewingsvriendelike aard daarvan. Dít is in teenstelling met konvensionele ‘harde’ kusbeskermingsoplossings, wat van rotse en/of betoneenhede gebruik maak om die kuslyn te beskerm. Aangesien GSH-strukture ’n betreklik nuwe kusbeskermingsoplossing is, is weinig ontwerp- en konstruksieriglyne beskikbaar. Navorsing oor die werkverrigting van GSH-strukture onder golfaanslag duur voort, met bepaalde klem op die hidrouliese prosesse wat GSH-strukture beïnvloed en die werking daarvan benadeel. Die gebruik van GSH-bedekte hellings as kusbeskermingsoplossings het in die laaste tyd al hoe gewilder geword in Suid-Afrika, veral langs die kus van KwaZulu-Natal. Tog val die gekose ontwerp van hierdie GSH-bedekte hellings buite die toepaslikheidsbestek van die beskikbare ontwerpriglyne en stabiliteitsvergelykings. Die hidrouliese stabiliteit van hierdie strukture is dus grotendeels onbekend. Die hoofoogmerk van hierdie studie was om ondersoek in te stel na die effek van struktuurhellings en pakformasies op die hidrouliese stabiliteit van GSH-bedekte hellings. Die toepaslikheid van beskikbare ontwerpriglyne en stabiliteitsvergelykings is ook geëvalueer. Tweedimensionele fisiese modellering is onderneem en altesaam 12 GSH-bedekte hellings is gedurende die fisiese-modelleringstoetsreeks getoets. Die resultate van die fisiese modellering toon dat die struktuurhelling die beduidendste effek op hidrouliese stabiliteit het. Steiler struktuurhellings was hidroulies meer stabiel as platter hellings. Die pakformasies van die GSH’s blyk ’n kleiner effek op die hidrouliese stabiliteit van die GSH-bedekte hellings te hê. GSH-bedekte hellings wat met ’n enkele laag GSH’s versterk is, het ewe goed of beter presteer as die keermure met ’n dubbele laag GSH’s, terwyl GSH-bedekte hellings met die lang-as van die GSH’s loodreg op die rigting van die golfaanslag effens beter presteer het as dié met die lang-as parallel met die golfaanslag. Die beskikbare ontwerpriglyne en stabiliteitsvergelykings is geëvalueer aan die hand van die resultate van die fisiese modellering, en het ’n wisselende mate van korrelasie getoon. Veral die stabiliteitsvergelyking van Recio (2007) blyk besonder akkuraat te wees

Amorçage de fissures de corrosion sous contrainte du Zircaloy-4 recristallisé en milieu méthanol iodé

En situation d interaction pastille-gaine (IPG), les gaines de crayons combustible en alliage de zirconium sont susceptibles de rompre lors de transitoires de puissance incidentels dans les réacteurs à eau pressurisée, par un mécanisme de corrosion sous contrainte induite par l iode (CSC-I). Cette étude traite de l amorçage intergranulaire des fissures de CSC-I dans le Zircaloy-4 recristallisé, en milieu méthanol iodé à température ambiante, en s intéressant particulièrement aux paramètres mécaniques critiques et à la concentration en iode. Pour cela, une approche mêlant expériences et simulations numériques a été adoptée. Un modèle de comportement mécanique macroscopique de l alliage, viscoplastique et anisotrope, a été établi et validé sur une large gamme de sollicitations. Par la réalisation de nombreux essais de traction à vitesse de déformation imposée et de fluage en flexion quatre points, nous avons montré l existence d une concentration seuil I0 proche de 10-6 g.g-1 nécessaire à l apparition du dommage de CSC-I, mais également celle d une concentration de transition I1 proche de 2 10-4 g.g-1 au-delà de laquelle le mécanisme change, menant à un amorçage anticipé des fissures et une sensibilité réduite de l endommagement aux paramètres mécaniques. L importance de la concentration sur des paramètres tels que la densité des fissures, leur longueur moyenne et la vitesse de propagation intergranulaire et transgranulaire a été mise en évidence. Les résultats expérimentaux montrent que la déformation plastique macroscopique n est pas indispensable à l amorçage de fissures de CSC-I, pour un temps d essais suffisamment long en présence de contrainte. Son principal effet est de précipiter l apparition de fissures par la création de sites d amorçage, par rupture de la couche d oxyde et accumulation de contrainte intergranulaire. En dessous de I1 la détermination des déformations critiques à l amorçage montre un fort effet de vitesse. Dans ce domaine, une contrainte seuil de 100 MPa a été déterminée, bien inférieure à la limite élastique. L utilisation d éprouvettes entaillées et de la simulation numérique a permis de mettre en évidence un fort effet protecteur de l augmentation de la biaxialité des contraintes vis-à-vis de l amorçage, dans le domaine élastique comme dans le domaine plastique. Des éprouvettes préalablement irradiées aux protons à une dose de 2 dpa ont été testées dans les mêmes conditions que les éprouvettes non irradiées. La sensibilité accrue du matériau irradié à la CSC-I a pu être quantifiée et nous avons constaté qu un effet de concentration et un effet de vitesse de sollicitation subsistent après irradiation. L irradiation induit une localisation plus importante de la déformation menant à un amorçage prématuré des fissures, mais une sensibilité chimique plus importante, tendance à la piquration et décalage de I1, apparaît comme le principal responsable de la plus forte sensibilité de l irradié.During the pellet-cladding interaction, Zirconium-alloy fuel claddings might fail when subjected to incidental power transient in nuclear Pressurized Water Reactors, by Iodine-induced Stress Corrosion Cracking (I-SCC). This study deals with the intergranular initiation of I-SCC cracks in fully recrystallized Zircaloy-4, in methyl alcohol solution of iodine at room temperature, with the focus on critical mechanical parameters and iodine concentration. It was carried out with an approach mixing experiments and numerical simulations. An anisotropic and viscoplastic mechanical behavior model was established and validated over a wide range of loadings. With numerous constant elongation rate tensile tests and four points bending creep tests, the existence of a threshold iodine concentration I0 close to 10-6 g.g-1 was highlighted, necessary to the occurrence of I-SCC damage, along with a transition concentration I1 close to 2 10-4 g.g-1. Above I1 the mechanism changes, leading to a sped up crack initiation and a loss of sensitivity towards mechanical parameters. The importance of concentration on parameters such as crack density, crack average length and intergranular and transgranular crack velocities was evidenced. Experimental results show that plastic strain is not required for I-SCC crack initiation, if the test time is long enough in the presence of stress. Its main influence is to rush the occurrence of cracking by creating initiation sites, by way of breaking the oxide layer and building up intergranular stress. Below I1, the critical strains at initiation show a substantial strain rate sensitivity. In this domain, a threshold stress of 100 MPa was found, well below the yield stress. Thanks to the combined use of notched specimens and numerical simulations, a strong protective effect of an increasing stress biaxiality ratio was found, both in the elastic and plastic domains. Proton-irradiated samples, up to a dose of 2 dpa, were tested in the same conditions as fresh specimens. The higher I-SCC sensitivity of the irradiated material was measured and the effects of concentration and strain rate were found to remain. Irradiation leads to a higher strain localization causing early crack initiation, but the main reason for the higher sensitivity of the irradiated material seems to be a chemical one, with higher pitting occurrence and a shift of I1.TOULOUSE-INP (315552154) / SudocSudocFranceF

Developmental patterns of the invasive bramble (rubus alceifolius poiret, rosaceae) in Reunion Island: an architectural and morphometric analysis

International audienceThe aim of this study was to identify the developmental stages of Rubus alceifolius and to determine one or more characteristic morphological markers for each stage. The developmental reconstitution method used involved a detailed description of many individuals throughout the different stages of growth, from germination to the development of an adult shoot capable of fruiting. Results revealed that R. alceifolius passes through five developmental stages that can be distinguished by changes in several morphological markers such as internode length and diameter, pith diameter and plant shape. This analysis indicated that R. alceifolius has a heteroblastic developmental pattern, midway between that of a bush and a liana. Moreover, results showed that this species taps environmental resources early in its development, i.e. foliarization is high (the foliar component overrides the caulinary component) and an autotrophic stage is rapidly reached, whereas it ‘explores’ the environment during the adult stage, i.e. axialization is substantial (the caulinary component overrides the foliar component) and autotrophy occurs at a later stage. The morphological markers identified could benefit land‐use managers attempting to control this species before it reaches its optimum developmental stage