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Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross.

ABSTRACT: Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) are major determinants of verapamil (VP)-reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 × Dd2 cross, we show that the P. falciparum multidrug resistance gene 1 (pfmdr1) interacts with the South-East Asia-derived mutant pfcrt haplotype to modulate CQR levels. A novel chromosome 7 locus is predicted to contribute with the pfcrt and pfmdr1 loci to influence CQR levels. Chemoreversal via a wide range of chemical structures operates through a direct pfcrt-based mechanism. Direct inhibition of parasite growth by these reversal agents is influenced by pfcrt mutations and additional loci. Direct labelling of purified recombinant PfMDR1 protein with a highly specific photoaffinity CQ analogue, and lack of competition for photolabelling by VP, supports our QTL predictions. We find no evidence that pfmdr1 copy number affects CQ response in the progeny; however, inheritance patterns indicate that an allele-specific interaction between pfmdr1 and pfcrt is part of the complex genetic background of CQR.

SUBMITTER: Patel JJ

PROVIDER: S-EPMC3091165 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross.

Patel Jigar J JJ Thacker Drew D Tan John C JC Pleeter Perri P Checkley Lisa L Gonzales Joseph M JM Deng Bingbing B Roepe Paul D PD Cooper Roland A RA Ferdig Michael T MT

Molecular microbiology 20100929 3

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) are major determinants of verapamil (VP)-reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 × Dd2 cross, we show that the P. falciparum multidrug ...[more]

PMID: 20807203

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Project description:BACKGROUND:Reduced artemisinin susceptibility and artemisinin-based combination therapy (ACT)-resistance against Plasmodium falciparum and chloroquine (CQ)-resistant P. vivax malaria has been reported in Vietnam. Two therapeutic efficacy studies were conducted in Thuan Bac District (Ninh Thuan Province, Vietnam) in 2015 and 2016 to determine the extent of reduced artemisinin susceptibility and ACT resistant falciparum malaria, and CQ-resistant vivax malaria were present. METHODS:Twenty-seven patients with falciparum malaria were randomized to receive artesunate alone (AS?~?4 mg/kg/day) for 4 days followed by dihydroartemisinin (DHA) (2.2 mg/kg)-piperaquine (PPQ) (18 mg/kg) daily for 3 days or artemether (AM) (1.7 mg/kg)-lumefantrine (LUM) (12 mg/kg) twice daily for 3 days. Sixteen subjects with vivax malaria received CQ (total 25 mg/kg over 3 days). The therapeutic efficacy study for treating falciparum malaria was complemented with molecular analysis for artemisinin and piperaquine resistance, and in vitro drug susceptibility testing. Patient's drug exposure following both falciparum and vivax treatment studies was determined. RESULTS:Twenty-five of 27 patients treated with the artemisinin regimens completed the 42-day follow-up period. None had parasites present on day 3 after commencing treatment with no incidence of recrudescence (100% curative rate). One patient on AS?+?DHA-PPQ was lost to follow-up and one patient had Plasmodium falciparum and Plasmodium vivax infection on day 0 by PCR. Of the vivax patients, 15 of 16 completed CQ treatment and two had a recurrence of vivax malaria on day 28, a failure rate of 13.3% (2/15). No mutations in the Pfkelch-13 gene for artemisinin resistance or exo-E415G gene polymorphism and amplification in plasmepsins 2 and 3 for piperaquine resistance were observed. In vitro testing of patient's falciparum parasites indicated susceptibility (low IC50 nM values) to dihydroartemisinin, lumefantrine, piperaquine and pyronaridine. Patient's drug exposure to artesunate and lumefantrine was comparable to published data, however, blood CQ concentrations were lower. CONCLUSIONS:Clinical findings, molecular analysis and in vitro testing revealed that the falciparum parasites at Phuoc Chien Commune were artemisinin susceptible. The clinical failure rate of the 15 vivax patients who completed CQ treatment was 13%. Further studies are required to determine whether CQ-resistant vivax malaria is present at the commune.

Project description:We used a high-density tiling array to estimate genetic recombination rate among 32 independent recombinant progeny of a P. falciparum genetic cross (7G8 M-CM-^W GB4). We detected 3184 segregating multi-probe single-feature polymorphisms (mSFPs) and 638 recombination events (496 excluding those from subtelomeric regions). These data, in combination with results from 254 previously reported microsatellites, enabled us to construct a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb/cM (12.8 kb/cM excluding subtelomeric regions) and identified 54 hotspots, some of which occurred in genes encoding surface antigens or proteins with repetitive motifs that might play a role in genetic recombination in the parasite. Motifs enriched in hotspots were also identified. In agreement with results from a previous cross (HB3 M-BM-4 Dd2), there was positive correlation between sizes of individual chromosomes and their recombination events. These results show that the P. falciparum genome is highly recombinogenic, providing an important genetic basis for parasite survival under various selection pressures. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination frequency observed. Ten microgram of genomic DNA, extracted and purified from 3D7 (reference), thirty-two P. falciparum independent recombinant progeny of the 7G8 x GB4 cross, and the two parental lines (Hayton, 2008), were hybridized to the PFSANGER GenechipM-BM-. (Affymetrix, Inc., Santa Clara, CA, USA). The scanned image CEL files were first processed using the RMA method, then averaged and compared with reference genome 3D7, and lastly assigned either 7G8 or GB4 alleles based on similarities to the two parental lines. Total of 35 genomic DNA samples (biological replicates: 6 for 3D7, 4 for 7G8, 4 for GB4, and 2 for Pf_WE2). The supplementary file 'GSE25656_QuantNormData_Log2_AllSamples.txt' contains the RMA-normalized data for all of the samples. The supplementary files 'GSE25656_chr*' contain the parental allele assignment of each chromosome and include probe-level annotation.

Project description:Bhutan has made substantial progress in reducing malaria incidence. The national guidelines recommend chloroquine (CQ) and primaquine (PQ) for radical cure of uncomplicated Plasmodium vivax, but the local efficacy has not been assessed. The impact of cases imported from India on the genetic make-up of the local vivax populations is currently unknown.Patients over 4 years of age with uncomplicated P. vivax mono-infection were enrolled into a clinical efficacy study and molecular survey. Study participants received a standard dose of CQ (25 mg/kg over 3 days) followed by weekly review until day 28. On day 28 a 14-day regimen of PQ (0.25 mg/kg/day) was commenced under direct observation. After day 42, patients were followed up monthly for a year. The primary and secondary endpoints were risk of treatment failure at day 28 and at 1 year. Parasite genotyping was undertaken at nine tandem repeat markers, and standard population genetic metrics were applied to examine population diversity and structure in infections thought to be acquired inside or outside of Bhutan.A total of 24 patients were enrolled in the clinical study between April 2013 and October 2015. Eight patients (33.3 %) were lost to follow-up in the first 6 months and another eight patients lost between 6 and 12 months. No (0/24) treatment failures occurred by day 28 and no (0/8) parasitaemia was detected following PQ treatment. Some 95.8 % (23/24) of patients were aparasitaemic by day 2. There were no haemolytic or serious events. Genotyping was undertaken on parasites from 12 autochthonous cases and 16 suspected imported cases. Diversity was high (H E 0.87 and 0.90) in both populations. There was no notable differentiation between the autochthonous and imported populations.CQ and PQ remains effective for radical cure of P. vivax in Bhutan. The genetic analyses indicate that imported infections are sustaining the local vivax population, with concomitant risk of introducing drug-resistant strains.

Dataset Information

Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross.

Publications

Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross.

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