<i>Plasmodium falciparum</i>Clonal Population Dynamics during Malaria Treatment

Jafari, Sayeh; Bras, J. Le; Bouchaud, Olivier; Durand, Rémy

doi:10.1086/380910

Cited by 75 publications

(74 citation statements)

References 38 publications

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“…The presence of minority drug-resistant variants is consistent with results of other studies, which have shown patients with genotypicaly wild-type infections before therapy exhibiting genotypicaly mutant infections after unsuccessful chemotherapy (14,15). In Malawi, where chloroquine was replaced with sulfadoxine-pyrimethamine in 1993, the prevalence of the resistance marker pfcrt 76T, as determined by PCR, has been reported to have almost disappeared (6)(7)(8)(9).…”

Section: Discussionsupporting

confidence: 88%

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Juliano

Kwiek

Cappell

et al. 2007

Emerg. Infect. Dis.

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Genotyping of the chloroquine-resistance biomarker pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) suggests that, in the absence of chloroquine pressure, Plasmodium falciparum parasites in Malawi have reverted to chloroquine sensitivity. However, malaria infections in Africa are commonly polyclonal, and standard PCRs cannot detect minority genotypes if present in <20% of the parasites in an individual host. We have developed a multiple site-specifi c heteroduplex tracking assay (MSS-HTA) that can detect pfcrt 76T mutant parasites consisting of as little as 1% of the parasite population. In clinical samples, no pfcrt 76T was detected in 87 pregnant Malawian women by standard PCR. However, 22 (25%) contained minority-variant resistant genotypes detected by the MSS-HTA. These results were confi rmed by subcloning and sequencing. This fi nding suggests that the chloroquine-resistant genotype remains common in Malawians and that PCR-undetectable drug-resistant genotypes may be present in disease-endemic populations. Surveillance for minority-variant drugresistant mutations may be useful in making antimalarial drug policy. D rug-resistant Plasmodium falciparum malaria continues to be a growing health problem throughout most of the world (1). To combat this threat, governments and aid agencies need accurate drug resistance surveillance data. The World Health Organization has stressed the need for methods of detecting molecular markers of drug resistance that will be useful in predicting responses to both clinical and public health interventions (2). This has been diffi cult in highly malaria-endemic areas, where infections are almost always polyclonal (3,4). In patients with polyclonal infections, small drug-resistant parasite populations (minority variants) may be masked by larger drug-sensitive populations because standard PCRs are relatively insensitive to minority variants (2). Therefore, new methods capable of detecting these subpopulations may lead to better drug resistance surveillance and provide a better tool to predict outcome.We describe a new multiple site-specifi c heteroduplex tracking assay (MSS-HTA) for detecting the pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) K76T mutation. This mutation in a putative transporter gene is well-associated with chloroquine resistance in P. falciparum (5). The MSS-HTA was compared with a standard allele-restricted PCR (ARPCR) in clinical samples from Malawi, a country where standard PCR analyses and a recent clinical trial have suggested that chloroquine-resistant malaria has disappeared (6-9). Materials and Methods Study SamplesInformed consent, as approved by the ethics committees of the University of North Carolina and the Malawi College of Medicine/Ministry of Health, was obtained from all participants in this research study. The Malaria and HIV in Pregnancy Study (MHP) patient samples originated from a study of pregnant women attending Queen Elizabeth Central Hospital, an urban hospital in Blantyre. The complete characte...

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Section: Discussionsupporting

confidence: 88%

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Juliano

Kwiek

Cappell

et al. 2007

Emerg. Infect. Dis.

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“…A higher yield might have been obtained using an advanced method of extracting DNA. Even if DNA is successfully extracted, PCR may fail to detect all genotypes present in a mixed-clones infection both at baseline and at the time of parasite recurrence because some might be present below PCR detection level (Jafari et al 2004) or might be sequestered. However, some evidence indicates that symptomatic infections are less complex than asymptomatic ones.…”

Section: Discussionmentioning

confidence: 99%

Molecular genotyping to distinguish between recrudescents and new infections in treatment trials of Plasmodium falciparum malaria conducted in Sub‐Saharan Africa: adjustment of parasitological outcomes and assessment of genotyping effectiveness

Mugittu

Adjuik

Snounou

et al. 2006

Tropical Med Int Health

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SummaryMolecular genotyping of baseline and post-treatment recurrent Plasmodium falciparum is recommended to distinguish recrudescent from new infections. However, genotyping performance and adjustment of treatment outcomes have not been evaluated in large field trials. Parasitological outcomes were assessed in nine double-blinded trials of uncomplicated P. falciparum malaria in African children treated with artesunate/placebo plus standard monotherapies. Day 28 failure rates were adjusted by stepwise genotyping the P. falciparum glutamate rich protein (glurp), merozoite surface protein 1 (msp1) and 2 (msp2). We calculated overall and laboratory genotyping performance and compared unadjusted (crude) and PCR-adjusted outcomes. 3455 (93.6%) of 3691 enrolled patients were evaluable by Day 28. 767 (22%) had post-Day 14 recurrent parasitemias of which 686 could be genotyped: 246 were recrudescences, 286 new infections and 154 unresolved. The overall and laboratory genotyping performance were 69 (12-100)% and 78 (50-100)%, respectively. The mean Day 28 crude parasitological failure rate was 44 (range 3-87)%. PCR-adjusted rates were 36 (range 2-86)% if unresolved infections were counted as failures or 33 (range 2-86)% if excluded from analysis. The overall difference between crude Day 28 and Day 14 failure rates was 22% (95% CI 20.3, 24.6) but decreased to 14% (95% CI 12.1, 16.3) if unresolved infections are counted as failures, or to 11% (95% CI 9.8, 16.3) if unresolved infections are excluded from the analysis. Genotyping refined treatment outcomes but diligence is needed in sample collection and analysis to improve its performance. Our findings support the WHO recommendation of PCR genotyping in malaria clinical trials and suggest that stepwise genotyping of only two loci (msp2 and msp1 or glurp) can reliably discriminate recrudescences from new infections.

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“…DNA was prepared by Chelex extraction, as previously described (Plowe et al 1995). A fluorescent PCR analysed block 2 of the msp-1 and of msp-2 domains, as described (Jafari et al 2004). Primers were: msp-1 f-5¢-CACATGAAAGTTATCAAGAACTTGTC-3¢ 1993).…”

Section: Dna Preparation and Pcr Amplificationmentioning

confidence: 99%

“…Primers were: msp-1 f-5¢-CACATGAAAGTTATCAAGAACTTGTC-3¢ 1993). Amplification products were processed in an ABI Prism 310 Genetic analyser (Perkin Elmer Applied Biosystems) and analysed using Genescan software (Applied Biosystems) (Jafari et al 2004). Each genotype is characterized by its size and the area under the curve (AUC) of the peak corresponding to msp-1 or msp-2 PCR products.…”

Section: Dna Preparation and Pcr Amplificationmentioning

confidence: 99%

Dramatically decreased therapeutic efficacy of chloroquine and sulfadoxine‐pyrimethamine, but not mefloquine, in southern Benin

Aubouy¹,

Fiévet²,

Bertin³

et al. 2007

Tropical Med Int Health

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Summaryobjective To evaluate the in vivo therapeutic efficacy of chloroquine (CQ), sulfadoxinepyrimethamine (SP) and mefloquine (MQ) in children presenting with uncomplicated malaria in Benin.methods Drug efficacy was tested according to the WHO in vivo 28-day protocol. For failures that occurred after 7 days of follow-up, paired pre-and post-treatment blood samples were genotyped at msp1 and msp2 loci to distinguish new infections and recrudescent strains. Children enrolled were randomly assigned to a therapeutic group (CQ, n ¼ 14; SP, n ¼ 42; MQ, n ¼ 44). The number of CQ treatment was intentionally restricted after 1 month, as its use was considered to constitute a danger for children.results Chloroquine and SP showed very high failure rates (85.7% and 50%, respectively), whereas MQ treatment was successful in 97.5%. The molecular tool allowed to re-evaluate two new infections previously considered as failures.conclusions Chloroquine should no longer be used to treat children presenting with Plasmodium falciparum malaria in Benin.

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Plasmodium falciparumClonal Population Dynamics during Malaria Treatment

Cited by 75 publications

References 38 publications

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Molecular genotyping to distinguish between recrudescents and new infections in treatment trials of Plasmodium falciparum malaria conducted in Sub‐Saharan Africa: adjustment of parasitological outcomes and assessment of genotyping effectiveness

Dramatically decreased therapeutic efficacy of chloroquine and sulfadoxine‐pyrimethamine, but not mefloquine, in southern Benin

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