Validation of Pyrosequencing for accurate and high throughput estimation of allele frequencies in malaria parasites.

Institute for Immunology and Infection Research, School of Biological Sciences, The University of Edinburgh, Ashworth Laboratories, The Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK.
Molecular and Biochemical Parasitology (Impact Factor: 2.73). 05/2007; 152(2):213-9. DOI: 10.1016/j.molbiopara.2006.12.008
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    ABSTRACT: The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2-5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites' circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.
    PLoS ONE 01/2012; 7(9):e45861. · 3.53 Impact Factor
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    ABSTRACT: Background.Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug specific cost-benefit index, indicating the possible advantage for mutated parasites.Methods.We specifically studied pfmdr1 D1246Y in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine and chloroquine, as well as relative fitness were determined in two modified isogenic P. falciparum clones only differing in the pfmdr1 1246 position. Data were used to create a new comparative graph of the relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment.Results.Pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine, but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine exposed parasites. In vivo a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data.Conclusions.This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.
    Antimicrobial Agents and Chemotherapy 12/2012; · 4.57 Impact Factor
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    ABSTRACT: If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin Combination Treatment (ACT) version of artesunate + mefloquine (ATN + MF) to gain insights about their ability to compete with ATN + MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN + MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14 days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN + MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.
    Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 01/2013; · 3.22 Impact Factor