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Parasite drug resistance is partly conferred by single-nucleotide polymorphisms (SNPs), and monitoring them has been proposed as an alternative to monitoring drug resistance. Therefore, techniques are required to facilitate analyses of multiple SNPs on an epidemiological scale. We report a rapid and affordable microarray technique for application in epidemiological studies of malaria drug resistance. We have designed a multiwell microarray that is used in conjunction with PCR-amplified target genes implicated in the drug resistance of malaria with subsequent one-tube minisequencing using two fluorochromes. The drug-resistance-associated genes pfdhfr, pfdhps, pfcrt, pfmdr1 , and pfATPase were amplified and analyzed for cultured Plasmodium falciparum strains and from field samples. We obtained a specificity of 94%, and comparison of field sample results to those of restriction fragment length polymorphism (RFLP) typing resulted in an overall consistency of >90%, except for pfdhfr51 , for which most discrepancies were due to false determinations by RFLP of mixed infections. The system is sufficiently sensitive to assay parasites in clinical malaria cases and in most asymptomatic cases, and it allows high throughput with minimal hands-on time. The cost for the assay has been calculated as 0.27 euros/SNP (US$0.33), which is below the cost incurred with other systems. Due to the simplicity of the approach, newly identified SNPs can be incorporated rapidly. Such a monitoring system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.

The genetic variability and population structure of Plasmodium falciparum are key factors in malaria control strategies. Studies have suggested no P. falciparum population structure although linkage disequilibrium was observed in some African areas. We have assessed length polymorphism at 6-22 microsatellites in four urban and rural sites (Djibouti, Dakar, Niamey, and Zouan-Hounien, n = 240 blood samples). Results have shown a P. falciparum population structure in Africa (Fst = 0.17-0.24), lower genetic diversity in Djibouti (He = 0.53) than in the other sites (He = 0.73-0.76), and 3) significant linkage disequilibrium in Djibouti. These results could be related to geographic isolation and low flow of parasites between sites. They also suggest a potential effect of rural suburbs to generate genetic diversity in towns. This could affect the dispersal of selected drug resistance and should be considered when adapting urban malaria control strategies.

Experimental tools to identify human red blood cells (RBC) prone to mechanical retention upstream from the spleen venous sinus inter-endothelial slits are currently suboptimal. We designed a micro-bead device mimicking the geometry of the human narrow and short inter-endothelial slits. Upon filtration through a mixture of 5-25 μm diameter micro-beads, Plasmodium falciparum-hosting RBC (Pf-RBC) were retained in a parasite developmental stage-dependent way, the retention rates of a subset of ring-RBC being similar in micro-beads and in isolated-perfused human spleens. We found that this retention might be linked principally to the reduced surface-area-to-volume ratio of Pf-RBC. Interestingly, other rigid RBC, such as heat-treated RBC, and RBC from hereditary spherocytosis patients were also retained in micro-beads without any hemolysis. Micro-beads allow (i) depletion of heterogeneous RBC population from its rigid-RBC subpopulation ii) characteriziation of distinct molecular signatures of rigid versus deformable RBC subpopulations. This simple method portends wide medical applications, such as improving the quality of stored RBC concentrates prior to transfusion.

The atovaquone-proguanil combination (Malarone) has been introduced in French Guiana for prophylaxis and second-line treatment for Plasmodium falciparum malaria in 2002. We report here a treatment failure in a patient who was given a second-line atovaquone-proguanil treatment. A nonimmune P.

Cambodia is located in an area of resistance to multiple antimalarials and has been the first country to implement the systematic use of an artesunate-mefloquine combination as first-line treatment for Plasmodium falciparum malaria. Little is known, however, about the prevalence of resistance mutations within the natural parasite populations, impeding rational drug policy in this context. Using direct sequencing of PCR products, we have analyzed sequence polymorphism of the dihydrofolate reductase-thymidylate synthase, dihydropteroate synthetase, and multidrug resistance 1 genes in a large number of clinical P. falciparum isolates collected in various areas of Cambodia. This highlighted a 100% prevalence of haplotypes with multiple mutations in the target genes of antifolates after more than a decade without use of antifolates for malaria therapy. A high prevalence of mutations in Pfmdr1 , including mutations associated with decreased in vitro susceptibility to mefloquine and quinine, was also observed. In addition, novel, low-frequency mutations were detected in Pfmdr1 . Our findings show an alarming rate of multilocus resistance genotypes in Cambodia, requiring diligent surveillance and imposing limitations on possible future drug combinations.

International audience; A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

International audience; A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed

International audience; A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra-erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity-purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf155-RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross-reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood-stage antigens is discussed.

Plasmodium falciparum causes malaria infections in its human host. Its wide distribution in tropical countries is a major world health problem. Before a vaccine can be produced, the identification and characterization of parasite antigens is necessary. This can be achieved by the cloning and subsequent analysis of genes coding for parasite antigens1–4. Recently established cDNA banks allow the expression of cDNA derived from the simian parasite Plasmodium knowlesi5 and P. falciparum6,7 in Escherichia coli. Recombinants encoding parasite antigens have been identified by immunodetection in both banks. Two of them contain repetitive units of 11 (ref. 7) or 12 (ref. 5) amino acids. We describe here the construction of an expression bank made directly from randomly generated fragments of P. falciparum genomic DNA. We detect several clones which react strongly with human African immune sera. One clone expresses an antigenic determinant composed of occasionally degenerated repeats of a peptide nonamer.

Background The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy. Methods Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR. Results The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09–29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24–4.44], N = 109, p = 0.969). Conclusion This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.