Eric Legrand

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.

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.).

Identification of a Mutant PfCRT-Mediated Chloroquine Tolerance Phenotype in Plasmodium falciparum

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

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.

Tuberculosis in the Caribbean: Using Spacer Oligonucleotide Typing to Understand Strain Origin and Transmission

We used direct repeat (DR)-based spacer oligonucleotide typing (spoligotyping) (in association with double-repetitive element–polymerase chain reaction, IS6110-restriction fragment length polymorphism [RFLP], and sometimes DR-RFLP and polymorphic GC-rich sequence-RFLP) to detect epidemiologic links and transmission patterns of Mycobacterium tuberculosis on Martinique, Guadeloupe, and French Guiana. In more than a third of the 218 strains we typed from this region, clusters and isolates shared genetic identity, which suggests epidemiologic links. However, because of limited epidemiologic information, only 14.2% of the strains could be directly linked. When spoligotyping patterns shared by two or more isolates were pooled with 392 spoligotypes from other parts of the world, new matches were detected, which suggests imported transmission. Persisting foci of endemic disease and increased active transmission due to high population flux and HIV-coinfection may be linked to the recent reemergence of tuberculosis in the Caribbean. We also found that several distinct families of spoligotypes are overrepresented in this region.

In Vitro Monitoring of Plasmodium falciparum Drug Resistance in French Guiana: a Synopsis of Continuous Assessment from 1994 to 2005

ABSTRACT Implemented as one arm of the malaria control program in French Guiana in the early 1990s, our laboratory has since established in vitro profiles for parasite drug susceptibility to a panel of eight antimalarials for more than 1,000 Plasmodium falciparum isolates from infected patients. The quinine-doxycycline combination was introduced in 1995 as the first-line drug treatment against uncomplicated P. falciparum malaria, replacing chloroquine, and the first-line drug combination was changed to the artemether-lumefantrine combination in 2002. Resistance to chloroquine declined 5 years after it was dropped in 1995 as the first-line drug, but unlike similar situations in Africa, there was a rapid halt to this decline. Doxycycline susceptibility substantially decreased from 2002 to 2005, suggesting parasite selection under quinine-doxycycline drug pressure. Susceptibility to mefloquine decreased from 1997 onward. Throughout the period from 1994 to 2005, most isolates were sensitive in vitro to quinine, amodiaquine, and atovaquone. Susceptibility to amodiaquine was strongly correlated with that to chloroquine and to a lesser extent with that to mefloquine and halofantrine. Susceptibilities to mefloquine and to halofantrine were also strongly correlated. There were two alerts issued for in vitro artemether resistance in the period from 2002 to 2003 and again in 2005, both of which could be associated with the presence of an S769N polymorphism in the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-type P. falciparum ATPase6 (PfATPase6) gene. Analysis of susceptibility to lumefantrine, conducted for the first time in 2005, indicates an alarming rate of elevated 50% inhibitory concentrations. In vitro monitoring of parasite drug susceptibility should be pursued to further document the consequences of specific drug policies on the local parasite population and, in particular, to establish profiles of susceptibility to individual components of drug combinations to provide early warning signs of emerging parasite resistance.

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes. Methods/Principal Findings Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported. Conclusions/Significance The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.

Multiple independent introductions of Plasmodium falciparum in South America

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase.

BackgroundMalaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity.MethodsPlasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed.ResultsNo variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium. vivax; and three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species.ConclusionThe results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale.

Resistance to Dihydroartemisinin

The original title of our article was “Lack of Plasmodium falciparum in Vitro and Genomic Resistance to Dihydroartemisinin in Travelers Returning to France from Africa.” EID’s shortening of the title (1) led to the perception that the letter title was misleading, but it was not on purpose. We have recently tested the 50% inhibitory concentration for artemether of the S769N PfATPase6 isolate that we had kept in liquid nitrogen, and it showed susceptibility. We underline that the previously reported clinical or parasitologic failures to some artemisinin-based combination therapies (2,3) were not synonymous with the emergence of resistance to artemisinin compounds. In the study by Grandesso et al., a combination of artesunate plus amodiaquine was given to children <5 years of age who lived in an area in which amodiaquine alone was ineffective to adequately treat uncomplicated falciparum malaria in 1 of 3 cases at day 28 (2). Such a combination (artesunate plus amodiaquine) was nearly equivalent in 1 of 3 cases to a 3-day artesunate monotherapy, which may fail to completely cure children because of the short half-life of artesunate. In the study by Bukirwa et al., no recrudescence occurred in patients treated with artesunate plus amodiaquine and only 2 of 199 patients treated with artemether plus lumefantrine experienced recrudescence at day 28 (3). As Birkiwa et al. themselves acknowledged, artemether plus lumefantrine was not administered with food, and it is known that lumefantrine is absorbed better when it is taken with a small amount of fat. Thus, the clinical failures observed did not necessarily reflect P. falciparum resistance to artemisinin compounds.