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Phenotypic and genotypic analysis of in vitro-selected artemisinin-resistant progeny of Plasmodium falciparum.

ABSTRACT: Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC(50)s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

SUBMITTER: Tucker MS

PROVIDER: S-EPMC3256069 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Phenotypic and genotypic analysis of in vitro-selected artemisinin-resistant progeny of Plasmodium falciparum.

Tucker Matthew S MS Mutka Tina T Sparks Kansas K Patel Janus J Kyle Dennis E DE

Antimicrobial agents and chemotherapy 20111114 1

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resi ...[more]

PMID: 22083467

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Project description:BackgroundHigh rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.MethodsTo examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.ResultsThe characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.ConclusionsSurveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Project description:The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites.

Project description:BACKGROUND:Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections. METHODS:We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test. FINDINGS:In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0-3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50-0·87; p<0·0001). INTERPRETATION:The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed. FUNDING:Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.

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Phenotypic and genotypic analysis of in vitro-selected artemisinin-resistant progeny of Plasmodium falciparum.

Publications

Phenotypic and genotypic analysis of in vitro-selected artemisinin-resistant progeny of Plasmodium falciparum.

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