Jacques Le Bras

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.

Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa.

Resistance of Plasmodium falciparum to many therapeutic agents is an increasing problem in most endemic areas. The role of the mdr-like gene products of P. falciparum in resistance to quinoline-containing compounds is not clear. The purpose of this study was to further examine the role of pfmdr1 in drug resistance in fresh clinical isolates originating from Africa. Drug susceptibility testing (chloroquine, mefloquine, halofantrine and quinine) and a molecular analysis of pfmdr1 was completed for 51 fresh clinical isolates. A statistical association between the chloroquine sensitivity phenotype and an intragenic allele of pfmdr1 was noted at a position, amino acid 86, which was previously associated with chloroquine resistance. There was little variation in the other intragenic alleles previously associated with chloroquine resistance. No correlation between pfmdr1 intragenic allelic variation and susceptibility to mefloquine, halofantrine or quinine was found. There was no association between gene copy number of pfmdr1 and any drug resistant phenotype in an analysis of selected isolates. This, along with other data, suggests that mefloquine resistance may have arisen by two different mechanisms in African and Southeast Asian isolates. Much more variability in the polyasparaginated region of the pfmdr1 gene was noted in this study than previously reported. In addition, fingerprint analysis using multiplex PCR revealed considerable genetic variability among these isolates.

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.

The mechanisms of resistance to antimalarial drugs in Plasmodium falciparum

Drug‐resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South‐east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple‐drug resistance develops against well‐tolerated drugs. Plasmodium falciparum drug‐resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i) impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii) one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii) the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv) treatment with sulphadoxine–pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino‐alcohols (quinine, mefloquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug‐resistance encountered in the forests of South‐east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine‐resistant prevalence seems to stabilize at the same level as chloroquine‐sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.

Severe Imported Falciparum Malaria: A Cohort Study in 400 Critically Ill Adults

Background Large studies on severe imported malaria in non-endemic industrialized countries are lacking. We sought to describe the clinical spectrum of severe imported malaria in French adults and to identify risk factors for mortality at admission to the intensive care unit. Methodology and Principal Findings Retrospective review of severe Plasmodium falciparum malaria episodes according to the 2000 World Health Organization definition and requiring admission to the intensive care unit. Data were collected from medical charts using standardised case-report forms, in 45 French intensive care units in 2000–2006. Risk factors for in-hospital mortality were identified by univariate and multivariate analyses. Data from 400 adults admitted to the intensive care unit were analysed, representing the largest series of severe imported malaria to date. Median age was 45 years; 60% of patients were white, 96% acquired the disease in sub-Saharan Africa, and 65% had not taken antimalarial chemoprophylaxis. Curative quinine treatment was used in 97% of patients. Intensive care unit mortality was 10.5% (42 deaths). By multivariate analysis, three variables at intensive care unit admission were independently associated with hospital death: older age (per 10-year increment, odds ratio [OR], 1.72; 95% confidence interval [95%CI], 1.28–2.32; P = 0.0004), Glasgow Coma Scale score (per 1-point decrease, OR, 1.32; 95%CI, 1.20–1.45; P<0.0001), and higher parasitemia (per 5% increment, OR, 1.41; 95%CI, 1.22–1.62; P<0.0001). Conclusions and Significance In a large population of adults treated in a non-endemic industrialized country, severe malaria still carried a high mortality rate. Our data, including predictors of death, can probably be generalized to other non-endemic countries where high-quality healthcare is available.

ANALYSIS OF PFCRT POINT MUTATIONS AND CHLOROQUINE SUSCEPTIBILITY IN ISOLATES OF PLASMODIUM FALCIPARUM

Recent transfection based studies demonstrated that cg2, a candidate gene for chloroquine resistance in Plasmodium falciparum, was not the resistance determinant. A further analysis of the initial 36 kb locus comprising the cg2 gene led to the discovery of another gene, pfcrt, which was absolutely associated with chloroquine resistance in forty parasite lines [Fidock DA, Nomura T, Talley AT, Su XZ, Cooper R, Dzekunov SM, Ferdig MT, Ursos LMB, Sidhu ABS, Naudé B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000;6:861-71]. The aim of this study was to evaluate, in 146 unselected clinical isolates obtained mostly from non-immune travellers returning from various endemic countries to France in years 1995-1999, the association between in vitro chloroquine resistance and the sequence of a part of the pfcrt gene. For comparison, the determination of the cg2 kappa and the pfmdr1 codon 86 genotypes were also performed on the same isolates. As determined by an isotopic semi-microtest, 70 isolates were susceptible to chloroquine (50% inhibitory concentration<80 nM) and 76 were resistant. The amplification of a portion of the pfcrt gene spanning codons 72-76, followed by sequencing showed three distinct genotypes: one type associated with susceptible isolates, one type associated mostly with resistant isolates and one type found in a resistant isolate originating from South America. Three different zones could be defined according to the status of codon 76. For 50% inhibitory concentration values< or =40 nM (n=47), all isolates but one had K76 (wild type). For 50% inhibitory concentration values located between 40 and 60 nM, isolates had either K76 (n=5) or K76T (mutant type) (n=6). For 50% inhibitory concentration values>60 nM (n=88), all isolates had K76T. A lack of a strong association between the pfmdr1 N86Y mutation and in vitro chloroquine resistance was observed. Cg2 genotypes were less strongly linked than pfcrt genotypes with in vitro chloroquine susceptibility in isolates located below 40 and above 60 nM. Further studies are needed to determine the reliability of the pfcrt gene as a genetic marker for chloroquine resistance.

Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

ABSTRACT The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC50s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

Plasmodium falciparum Clonal Population Dynamics during Malaria Treatment

We have developed a new fragment-analysis method to enumerate the clones and to quantify their proportions within Plasmodium falciparum isolates. We prospectively enrolled 20 adult patients with uncomplicated malaria who were returning to France from various sub-Saharan countries, from January 2000 through July 2001. The analysis of clonal populations was performed on blood samples obtained at 10 times: 1 before treatment with oral quinine and 9 during the first 96 h of the treatment. The resistance genotypes pfcrt and dhfr were determined for chloroquine and antifolinics. Multiple P. falciparum genotypes were detected in 19 (95%) of 20 patients: 2, 3, 4, and 5 genotypes were found in 4, 9, 4 and 2 patients, respectively. Disappearance and reappearance of some clones within a few hours was observed. Individual clones represented 0.4%-99.4% of total parasitemia. Surprisingly, in 10 of 15 subjects tested, resistance genotypes varied according to the time of blood collection. These findings may have important implications with regard to the interpretations of resistance studies.

Decreased In Vitro Susceptibility of Plasmodium falciparum Isolates to Artesunate, Mefloquine, Chloroquine, and Quinine in Cambodia from 2001 to 2007

ABSTRACT This study describes the results of in vitro antimalarial susceptibility assays and molecular polymorphisms of Plasmodium falciparum isolates from Cambodia. The samples were collected from patients enrolled in therapeutic efficacy studies (TES) conducted by the Cambodian National Malaria Control Program for the routine efficacy monitoring of artemisinin-based combination therapy (ACT) (artesunate-mefloquine and artemether-lumefantrine combinations). The isolates (n = 2,041) were obtained from nine sentinel sites during the years 2001 to 2007. Among these, 1,588 were examined for their in vitro susceptibilities to four antimalarials (artesunate, mefloquine, chloroquine, and quinine), and 851 isolates were genotyped for single nucleotide polymorphisms (SNPs). The geometric means of the 50% inhibitory concentrations (GMIC50s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC50s for isolates from participants who failed artesunate-mefloquine therapy were significantly higher than those for patients who were cured (P, <0.001). In vitro correlation of artesunate with the other drugs was observed. The distributions of the SNPs differed between eastern and western Cambodia, suggesting different genetic backgrounds of the parasite populations in these two parts of the country. The GMIC50s of the four drugs tested increased significantly in eastern Cambodia during 2006 to 2007. These results are worrisome, because they may signal deterioration of the efficacy of artesunate-mefloquine beyond the Cambodian-Thai border.

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.