Thierry Fandeur

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.

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.

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia

BackgroundThe 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.MethodsBlood 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.ResultsThe 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 pfmdr1 copy 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).ConclusionThis 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.

Efficacy and safety of dihydroartemisinin-piperaquine (artekin) in Cambodian children and adults with uncomplicated falciparum malaria

The safety and efficacy of a novel combination of dihydroartemisinin (DHA) and piperaquine, Artekin (Holleykin Pharmaceuticals), were assessed in 106 patients (76 children and 30 adults) with uncomplicated falciparum malaria from 2 remote areas in Cambodia. Age-based doses were given at 0, 8, 24, and 32 h. Mean total DHA and piperaquine doses were 9.1 and 73.9 mg/kg, respectively, for children and 6.6 and 52.9 mg/kg for adults. All patients became aparasitemic within 72 h. Excluding the results for 1 child who died on day 4, there was a 96.9% 28-day cure rate (98.6% in children and 92.3% in adults). Patients who had recrudescent infection received low doses of Artekin. Side effects were reported by 22 patients (21%) but did not necessitate premature cessation of therapy. Although Artekin is a promising and inexpensive option for antimalarial therapy, further efficacy and pharmacokinetic studies are needed, especially for its use in children.

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.).

Efficacy of artemether–lumefantrine for the treatment of uncomplicated falciparum malaria in northwest Cambodia

Objective  To determine the efficacy of artemether–lumefantrine malaria treatment, as an alternative to artesunate + mefloquine, which is becoming ineffective in some areas of the Thai–Cambodian border.

Large-scale malaria survey in Cambodia: Novel insights on species distribution and risk factors

BackgroundIn Cambodia, estimates of the malaria burden rely on a public health information system that does not record cases occurring among remote populations, neither malaria cases treated in the private sector nor asymptomatic carriers. A global estimate of the current malaria situation and associated risk factors is, therefore, still lacking.MethodsA large cross-sectional survey was carried out in three areas of multidrug resistant malaria in Cambodia, enrolling 11,652 individuals. Fever and splenomegaly were recorded. Malaria prevalence, parasite densities and spatial distribution of infection were determined to identify parasitological profiles and the associated risk factors useful for improving malaria control programmes in the country.ResultsMalaria prevalence was 3.0%, 7.0% and 12.3% in Sampovloun, Koh Kong and Preah Vihear areas. Prevalences and Plasmodium species were heterogeneously distributed, with higher Plasmodium vivax rates in areas of low transmission. Malaria-attributable fevers accounted only for 10–33% of malaria cases, and 23–33% of parasite carriers were febrile. Multivariate multilevel regression analysis identified adults and males, mostly involved in forest activities, as high risk groups in Sampovloun, with additional risks for children in forest-fringe villages in the other areas along with an increased risk with distance from health facilities.ConclusionThese observations point to a more complex malaria situation than suspected from official reports. A large asymptomatic reservoir was observed. The rates of P. vivax infections were higher than recorded in several areas. In remote areas, malaria prevalence was high. This indicates that additional health facilities should be implemented in areas at higher risk, such as remote rural and forested parts of the country, which are not adequately served by health services. Precise malaria risk mapping all over the country is needed to assess the extensive geographical heterogeneity of malaria endemicity and risk populations, so that current malaria control measures can be reinforced accordingly.

Origin and evolution of sulfadoxine resistant Plasmodium falciparum.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

pfcrt Polymorphism and Chloroquine Resistance in Plasmodium falciparum Strains Isolated in Cambodia

ABSTRACT Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.

Association of Severe Malaria with a Specific Plasmodium falciparum Genotype in French Guiana

Why severe Plasmodium falciparum malaria occurs in only a small percentage of patients is unclear. The possibility that specific parasite characteristics contribute to severity has been investigated in French Guiana, a hypoendemic area, where parasite diversity is low and all patients with severe cases are referred to a single intensive care unit. Parasite genotyping in geographically and temporally matched patients with mild and severe disease showed that the association of a specific msp-1 allele (B-K1) with a specific var gene (var-D) was overrepresented among patients with severe versus mild disease (47% vs. 3%, respectively; P<.001). Moreover, this genotype combination was consistently observed in the most severe clinical cases. Reverse-transcription polymerase chain reaction demonstrated programmed expression of var-D in vivo, which is consistent with its potential implication in severe disease. These results provide field evidence of an association of severe malaria with specific genetic characteristics of parasites and open the way for intervention strategies targeting key virulence factors of parasites.