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The usefulness of atovaquone-proguanil (AP) as an antimalarial treatment is compromised by the emergence of atovaquone resistance during therapy. However, the origin of the parasite mitochondrial DNA (mtDNA) mutation conferring atovaquone resistance remains elusive. Here, we report a patient-based stochastic model that tracks the intrahost emergence of mutations in the multicopy mtDNA during the first erythrocytic parasite cycles leading to the malaria febrile episode. The effect of mtDNA copy number, mutation rate, mutation cost, and total parasite load on the mutant parasite load per patient was evaluated. Computer simulations showed that almost any infected patient carried, after four to seven erythrocytic cycles, de novo mutant parasites at low frequency, with varied frequencies of parasites carrying varied numbers of mutant mtDNA copies. A large interpatient variability in the size of this mutant reservoir was found; this variability was due to the different parameters tested but also to the relaxed replication and partitioning of mtDNA copies during mitosis. We also report seven clinical cases in which AP-resistant infections were treated by AP. These provided evidence that parasiticidal drug concentrations against AP-resistant parasites were transiently obtained within days after treatment initiation. Altogether, these results suggest that each patient carries new mtDNA mutant parasites that emerge before treatment but are killed by high starting drug concentrations. However, because the size of this mutant reservoir is highly variable from patient to patient, we propose that some patients fail to eliminate all of the mutant parasites, repeatedly producing de novo AP treatment failures., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine (n = 96), fixed-dose artesunate-amodiaquine (n = 96), and sulfadoxine-pyrimethamine (n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC50) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC50s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC50s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex vivo, molecular, and pharmacological data is warranted.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

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 (GMIC(50)s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC(50)s 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 GMIC(50)s 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.

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-(3)H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC(50)) 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 (kappa = 0.88) between the two methods allowed comparison by determination of the IC(50)s. 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.

Many African countries currently use a sulfadoxine-pyrimethamine combination (SP) or amodiaquine (AQ) to treat uncomplicated Plasmodium falciparum malaria. Both drugs represent the last inexpensive alternatives to chloroquine. However, resistant P. falciparum populations are largely reported in Africa, and it is compulsory to know the present situation of resistance. The in vivo World Health Organization standard 28-day test was used to assess the efficacy of AQ and SP to treat uncomplicated falciparum malaria in Gabonese children under 10 years of age. To document treatment failures, molecular genotyping to distinguish therapeutic failures from reinfections and drug dosages were undertaken. A total of 118 and 114 children were given AQ or SP, respectively, and were monitored. SP was more effective than AQ, with 14.0 and 34.7% of therapeutic failures, respectively. Three days after initiation of treatment, the mean level of monodesethylamodiaquine (MdAQ) in plasma was 149 ng/ml in children treated with amodiaquine. In those treated with SP, mean levels of sulfadoxine and pyrimethamine in plasma were 100 microg/ml and 212 ng/ml, respectively. Levels of the three drugs were higher in patients successfully treated with AQ (MdAQ plasma levels) or SP (sulfadoxine and pyrimethamine plasma levels). Blood concentration higher than breakpoints of 135 ng/ml for MdAQ, 100 micro g/ml for sulfadoxine, and 175 ng/ml for pyrimethamine were associated with treatment success (odds ratio: 4.5, 9.8, and 11.8, respectively; all P values were <0.009). Genotyping of merozoite surface proteins 1 and 2 demonstrated a mean of 4.0 genotypes per person before treatment. At reappearance of parasitemia, both recrudescent parasites (represented by common bands in both samples) and newly inoculated parasites (represented by bands that were absent before treatment) were present in the blood of most (51.1%) children. Only 3 (6.4%) therapeutic failures were the result not of treatment inefficacy but of new infection. In areas where levels of drug resistance and complexity of infections are high, drug dosage and parasite genotyping may be of limited interest in improving the precision of drug efficacy measurement. Their use should be weighted according to logistical constraints.

A PCR-based technique using molecular beacons was developed to detect the chloroquine resistance-associated pfcrt K76T point mutation in Plasmodium falciparum. One hundred thirty African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This rapid and inexpensive genomic assay could expand the possibilities for monitoring chloroquine resistance.

FR160, a catechol iron chelator, and tetracyclines or norfloxacin exert in vitro additive or synergistic activity against a chloroquine-resistant Plasmodium falciparum clone. FR160 shows antagonistic effects in association with macrolides, ofloxacin, and rifampin.

A PCR-based technique using new fluorescent probes, called molecular beacons, was developed to detect the antifolate resistance-associated S108N point mutation in Plasmodium falciparum. One hundred African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This new molecular technique appears to be a promising tool for epidemiological studies.

The 50% inhibitory concentration (IC50s) of benflumetol (range, 12.5 to 240 nM; mean, 55.1 nM) for 158 Senegalese isolates were evaluated. Ten isolates (6%) showed decreased susceptibility to benflumetol. Benflumetol was slightly more potent against chloroquine-resistant isolates (P < 0.025). No correlation or weak correlations in the responses to benflumetol and pyrimethamine, chloroquine, amodiaquine, artemether, quinine, and pyronaridine were observed, and these correlations are insufficient to suggest cross-resistance. Benflumetol may be an important alternative drug for the treatment of chloroquine-resistant malaria.