Ronan Jambou

Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6

Artemisinin derivatives are an essential component of treatment against multidrug-resistant Plasmodium falciparum malaria. We aimed to investigate in-vitro resistance to artemisinin derivatives in field isolates. In-vitro susceptibility of 530 P falciparum isolates from three countries (Cambodia, French Guiana, and Senegal) with different artemisinin use was assessed with an isotopic microtest. Artemether IC50 up to 117 and 45 nmol/L was seen in French Guiana and Senegal, respectively. DNA sequencing in a subsample of 60 isolates lends support to SERCA-PfATPase6 as the target for artemisinins. The S769N PfATPase6 mutation, noted exclusively in French Guiana, was associated with raised (>30 nmol/L) artemether IC50s (p<0.0001, Mann-Whitney). All resistant isolates came from areas with uncontrolled use of artemisinin derivatives. This rise in resistance indicates the need for increased vigilance and a coordinated and rapid deployment of drug combinations.

Identification of a family of Rab G-proteins in Plasmodium falciparum and a detailed characterisation of pfrab6

As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.

Invasion of Africa by a single pfcrt allele of South East Asian type

BackgroundBecause of its dramatic public health impact, Plasmodium falciparum resistance to chloroquine (CQ) has been documented early on. Chloroquine-resistance (CQR) emerged in the late 1950s independently in South East Asia and South America and progressively spread over all malaria areas. CQR was reported in East Africa in the 1970s, and has since invaded the African continent. Many questions remain about the actual selection and spreading process of CQR parasites, and about the evolution of the ancestral mutant gene(s) during spreading.MethodsEleven clinical isolates of P. falciparum from Cambodia and 238 from Africa (Senegal, Ivory Coast, Bukina Faso, Mali, Guinea, Togo, Benin, Niger, Congo, Madagascar, Comoros Islands, Tanzania, Kenya, Mozambique, Cameroun, Gabon) were collected during active case detection surveys carried out between 1996 and 2001. Parasite DNA was extracted from frozen blood aliquots and amplification of the gene pfcrt exon 2 (codon 72–76), exon 4 and intron 4 (codon 220 and microsatellite marker) were performed. All fragments were sequenced.Results124 isolates with a sensitive (c76/c220:CVMNK/A) haplotype and 125 isolates with a resistant c76/c220:CVIET/S haplotype were found. The microsatellite showed 17 different types in the isolates carrying the c76/c220:CVMNK/A haplotype while all 125 isolates with a CVIET/S haplotype but two had a single microsatellite type, namely (TAAA)3(TA)15, whatever the location or time of collection.ConclusionThose results are consistent with the migration of a single ancestral pfcrt CQR allele from Asia to Africa. This is related to the importance of PFCRT in the fitness of P. falciparum point out this protein as a potential target for developments of new antimalarial drugs.

World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility.

Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy.

Understanding the persistence of plague foci in Madagascar.

Plague, a zoonosis caused by Yersinia pestis, is still found in Africa, Asia, and the Americas. Madagascar reports almost one third of the cases worldwide. Y. pestis can be encountered in three very different types of foci: urban, rural, and sylvatic. Flea vector and wild rodent host population dynamics are tightly correlated with modulation of climatic conditions, an association that could be crucial for both the maintenance of foci and human plague epidemics. The black rat Rattus rattus, the main host of Y. pestis in Madagascar, is found to exhibit high resistance to plague in endemic areas, opposing the concept of high mortality rates among rats exposed to the infection. Also, endemic fleas could play an essential role in maintenance of the foci. This review discusses recent advances in the understanding of the role of these factors as well as human behavior in the persistence of plague in Madagascar.

Countrywide Survey Shows Very High Prevalence of Plasmodium falciparum Multilocus Resistance Genotypes in Cambodia

ABSTRACT 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.

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

Anti‐malaria antibody‐producing B cell frequencies in adults after a Plasmodium falciparum outbreak in Madagascar

The central highlands of Madagascar offer a unique opportunity to explore the malaria immune memory, as the last murderous epidemic in the study area occurred 8 years ago. Quantification of the circulating memory B lymphocytes reacting to Plasmodium falciparum was assessed among 14 Madagascans by using a limiting dilution assay, applied to the EL4 culture system, which leads to activation, proliferation and differentia lion into antibody‐secreting cells (ASC) of most peripheral B cells. This system allowed us to observe, without any malaria‐specific restimulalion, a geometric mean frequency of one anti‐P. falciparum ASC among 2992 circulating B cells, except for one Madagascan who did not have any detectable ASC. A geometric mean frequency of one anti‐P. falciparum ASC among 1403 was obtained for six malaria hyperimmune Cameroonians, but conversely, no anti‐malaria ASC was detected in the blood of six malaria non‐immune French control subjects. Anti‐P. falciparum ASC frequencies and serum specific antibodies were strongly related. Our results indicate that anti‐malaria ASC are still present in peripheral blood of Madagascan subjects, who have not been exposed to P. falciparum for several years. These responder B cells reflect the malaria B cell memory acquired during the last epidemic.

Identifying Relapsing Fever Borrelia, Senegal

We describe a nested polymerase chain reaction for the identification of Borrelia species from serum of patients with unidentified fevers. This technique, based on single nucleotide polymorphisms of the 16S ribosomal RNA gene, was used to test blood samples from 7,750 patients, 33 of whom were diagnosed with spirochete infections. Borrelia crocidurae was the only species identified.

Validity of Lot Quality Assurance Sampling to optimize falciparum malaria surveys in low-transmission areas

To control the reappearance of malaria in the Madagascan highlands, indoor house-spraying of DDT was conducted from 1993 until 1998. Before the end of the insecticide-spraying programme, a surveillance system was set up to allow rapid identification of new malaria epidemics. When the number of suspected clinical malaria cases notified to the surveillance system exceeds a predetermined threshold, a parasitological survey is carried out in the community to confirm whether or not transmission of falciparum malaria is increasing. Owing to the low specificity of the surveillance system, this confirmation stage is essential to guide the activities of the control programme. For this purpose, Lot Quality Assurance Sampling (LQAS), which usually requires smaller sample sizes, seemed to be a valuable alternative to conventional survey methods. In parallel to a conventional study of Plasmodium falciparum prevalence carried out in 1998, we investigated the ability of LQAS to rapidly classify zones according to a predetermined prevalence level. Two prevalence thresholds (5% and 15%) were tested using various sampling plans. A plan (36, 2), meaning that at least 2 individuals found to be positive among a random sample of 36, enabled us to classify a community correctly with a sensitivity of 100% and a specificity of 94%. LQAS is an effective tool for rapid assessment of falciparum malaria prevalence when monitoring malaria transmission.