Rapid detection of a molecular marker for chloroquine-resistant falciparum malaria.
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ABSTRACT:
SUBMITTER: Durand R
PROVIDER: S-EPMC127343 | biostudies-literature | 2002 Aug
REPOSITORIES: biostudies-literature
Publications
Rapid detection of a molecular marker for chloroquine-resistant falciparum malaria.
Durand Rémy R Huart Virginie V Jafari Sayeh S Le Bras Jacques J
Antimicrobial agents and chemotherapy 20020801 8
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. ...[more]
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Project description: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.
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Project description:ObjectivesIn the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes.DesignWe conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment.SettingThe study took place in the town of Farafenni and surrounding villages in the Gambia.ParticipantsParticipants were 500 children aged 6 mo to 10 y with uncomplicated P. falciparum malaria.InterventionsChildren were randomised to receive CQ, SP, or CQ/SP.Outcome measuresOutcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes.ResultsAfter SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP.ConclusionsParasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo.
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Project description:ObjectivesIn the Gambia, the combination of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) has replaced CQ monotherapy for treatment of malaria caused by Plasmodium falciparum. We measured the efficacy of the combination CQ/SP, and the prevalence of parasites carrying alleles associated with resistance to CQ or SP.DesignWe conducted a single-blind, randomised, controlled trial to compare the efficacy of CQ/SP to that of SP or CQ alone.SettingThe study took place in the town of Farafenni and surrounding villages in the Gambia.ParticipantsParticipants were children aged 12 mo to 10 y presenting as outpatients with uncomplicated P. falciparum malaria.Interventions500 children were randomised to receive CQ, SP, or CQ/SP as supervised treatment and actively followed over 28 d.Outcome measuresPrimary outcome was parasitaemia at any time during follow-up. Secondary outcomes were PCR-confirmed recrudescent infections among treatment failures, and clinical failure requiring rescue medication by day 28. Pretreatment parasite isolates from 161 patients were tested for the presence of resistance-associated genetic markers.ResultsThe prevalence of parasitological failure by day 28 for the CQ group was 60.3%, compared to 17.6% for SP (odds ratio [OR], 0.106; 95% confidence interval [CI], 0.057-0.194; p < 0.001) and 13.9% for CQ/SP (OR versus CQ, 0.140; 95% CI, 0.078-0.250; p < 0.001). There was no difference between the SP and CQ/SP groups (OR, 1.324; 95% CI, 0.705-2.50). The projected prevalence of PCR-corrected treatment failure was 30.2, 6.06, and 3.94% in the CQ, SP, and CQ/SP groups, respectively. The pfdhfr-triple mutant and pfdhps-437G mutation were common, with prevalences of 67.4 and 51.2%, respectively. Pretreatment carriage of pfdhps-437G and of multidrug-resistant parasite genotypes was associated with treatment failure in the SP group, but not in the CQ or CQ/SP groups.ConclusionsThe combination of CQ/SP was an efficacious treatment for uncomplicated malaria in Gambian children in this study, but the frequent occurrence of multidrug-resistant parasites suggests that this observed efficacy is not sustainable.
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Project description:Falciparum malaria is a significant problem for Afghan refugees in Pakistan. Refugee treatment guidelines recommended standard three-day chloroquine treatment (25 mg/kg) for first episodes and extended five-day treatment (40 mg/kg) for recrudescent infections, based on the assumption that a five-day course would more likely achieve a cure. An in-vivo randomized controlled trial was conducted among refugees with uncomplicated falciparum malaria to determine whether five-day treatment (CQ40) was more effective than standard treatment (CQ25).142 falciparum patients were recruited into CQ25 or CQ40 treatment arms and followed up to 60 days with regular blood smears. The primary outcome was parasitological cure without recrudescence. Treatment failures were retreated with CQ40. PCR genotyping of 270 samples, from the same and nearby sites, was used to support interpretation of outcomes.84% of CQ25 versus 51% of CQ40 patients experienced parasite recrudescence during follow-up (adjusted odds ratio 0.17, 95%CI 0.08-0.38). Cure rates were significantly improved with CQ40, particularly among adults. Fever clearance time, parasite clearance time, and proportions gametocytaemic post-treatment were similar between treatment groups. Second-line CQ40 treatment resulted in higher failure rates than first-line CQ40 treatment. CQ-resistance marker pfcrt 76T was found in all isolates analysed, while pfmdr1 86Y and 184Y were found in 18% and 37% of isolates respectively.CQ is not suitable for first-line falciparum treatment in Afghan refugee communities. The extended-dose CQ regimen can overcome 39% of resistant infections that would recrudesce under the standard regimen, but the high failure rate after directly observed treatment demonstrates its use is inappropriate.
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Project description:BackgroundWestern Cambodia is the epicentre of Plasmodium falciparum multidrug resistance and is facing high rates of dihydroartemisinin-piperaquine treatment failures. Genetic tools to detect the multidrug-resistant parasites are needed. Artemisinin resistance can be tracked using the K13 molecular marker, but no marker exists for piperaquine resistance. We aimed to identify genetic markers of piperaquine resistance and study their association with dihydroartemisinin-piperaquine treatment failures.MethodsWe obtained blood samples from Cambodian patients infected with P falciparum and treated with dihydroartemisinin-piperaquine. Patients were followed up for 42 days during the years 2009-15. We established in-vitro and ex-vivo susceptibility profiles for a subset using piperaquine survival assays. We determined whole-genome sequences by Illumina paired-reads sequencing, copy number variations by qPCR, RNA concentrations by qRT-PCR, and protein concentrations by immunoblotting. Fisher's exact and non-parametric Wilcoxon rank-sum tests were used to identify significant differences in single-nucleotide polymorphisms or copy number variants, respectively, for differential distribution between piperaquine-resistant and piperaquine-sensitive parasite lines.FindingsWhole-genome exon sequence analysis of 31 culture-adapted parasite lines associated amplification of the plasmepsin 2-plasmepsin 3 gene cluster with in-vitro piperaquine resistance. Ex-vivo piperaquine survival assay profiles of 134 isolates correlated with plasmepsin 2 gene copy number. In 725 patients treated with dihydroartemisinin-piperaquine, multicopy plasmepsin 2 in the sample collected before treatment was associated with an adjusted hazard ratio (aHR) for treatment failure of 20·4 (95% CI 9·1-45·5, p<0·0001). Multicopy plasmepsin 2 predicted dihydroartemisinin-piperaquine failures with 0·94 (95% CI 0·88-0·98) sensitivity and 0·77 (0·74-0·81) specificity. Analysis of samples collected across the country from 2002 to 2015 showed that the geographical and temporal increase of the proportion of multicopy plasmepsin 2 parasites was highly correlated with increasing dihydroartemisinin-piperaquine treatment failure rates (r=0·89 [95% CI 0·77-0·95], p<0·0001, Spearman's coefficient of rank correlation). Dihydroartemisinin-piperaquine efficacy at day 42 fell below 90% when the proportion of multicopy plasmepsin 2 parasites exceeded 22%.InterpretationPiperaquine resistance in Cambodia is strongly associated with amplification of plasmepsin 2-3, encoding haemoglobin-digesting proteases, regardless of the location. Multicopy plasmepsin 2 constitutes a surrogate molecular marker to track piperaquine resistance. A molecular toolkit combining plasmepsin 2 with K13 and mdr1 monitoring should provide timely information for antimalarial treatment and containment policies.FundingInstitut Pasteur in Cambodia, Institut Pasteur Paris, National Institutes of Health, WHO, Agence Nationale de la Recherche, Investissement d'Avenir programme, Laboratoire d'Excellence Integrative "Biology of Emerging Infectious Diseases".
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Project description:Malaria is a major public health problem in tropical and subtropical countries, including India. This study elucidates the cause of chloroquine treatment failure (for Plasmodium falciparum infection) before the introduction of artemisinin combination therapy. One hundred twenty-six patients were randomized to chloroquine treatment, and the therapeutic efficacy was monitored from days 1 to 28. An in vitro susceptibility test was performed with all isolates. Parasitic DNA was isolated, followed by PCR and restriction digestion of different codons of the pfcrt gene (codons 72 to 76) and the pfmdr1 gene (N86Y, Y184F, S1034C, N1042D, and D1246Y). Finally, sequencing was done to confirm the mutations. Forty-three (34.13%) early treatment failure cases and 16 (12.69%) late treatment failure cases were observed after chloroquine treatment. In vitro chloroquine resistance was found in 103 isolates (81.75%). Twenty-six (60.47%) early treatment failure cases and 6 (37.5%) late treatment failure cases were associated with the CVMNK-YYSNY allele (the underlined amino acids are those that were mutated). Moreover, the CVIEK-YYSNY allele was found in 8 early treatment failure (18.60%) and 2 late treatment failure (12.5%) cases. The presence of the wild-type pfcrt (CVMNK) and pfmdr1 (YYSNY) double mutant allele in chloroquine-nonresponsive cases was quite uncommon. In vivo chloroquine treatment failure and in vitro chloroquine resistance were strongly correlated with the CVMNK-YYSNY and CVIEK-YYSNY haplotypes (P < 0.01).
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