Polymorphism in drug resistance genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India

Sharma, Divya; Lather, Manila; Mallick, Prashant K.; Adak, T.; Dang, Amita Suneja; Valecha, Neena; Singh, O. P.

doi:10.1186/s13071-015-1080-2

Cited by 13 publications

(13 citation statements)

References 32 publications

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“…Malaria is still a major public health burden in many countries, and the most lethal malaria parasite is Plasmodium falciparum ( Pf ) [ 1 , 2 ]. An understanding of the gene function of Pf and the performance of pathogen genetic manipulations is essential for anti-malaria drug and malaria vaccine development.…”

Section: Resultsmentioning

confidence: 99%

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

Tong

Pan

et al. 2016

Parasites Vectors

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BackgroundA highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits the size of knock-in DNA fragments. Because of the inefficient end joining (EJ) DNA repair mechanism of Pf, a suicide-rescue approach could be used to address the challenges. Cas9 nuclease and sgRNA were co-expressed from a single plasmid (suicide vector) with one selectable marker, and the donor DNA was ligated into the other plasmid (rescue vector) containing only the ampicillin-resistance gene (AmpR) and a ColEl replication origin (ori). Nonetheless, whether this approach can mediate even the regular gene editing in Pf remains unknown. This study aimed to demonstrate the basic gene editing function of this Cas9-mediated suicide-rescue system.FindingsThe suicide and rescue vectors were constructed and co-transfected into Pf3D7. This system worked as expected when used to disrupt the Pfset2 gene and to insert a green fluorescent protein-renilla luciferase (gfp-ruc) fusion gene cassette of 3334 base pairs (bp) into the Pf47 locus, demonstrating that the suicide vector actually induced double-strand breaks (DSBs) and that the rescue vector functioned without maintenance via drug selection.ConclusionsThe adapted marker-free CRISPR/Cas9 system with only a single episome-selectable marker performs well as the current systems for general gene editing which lays a solid foundation for further studies including consecutive gene manipulations and large gene knock-ins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1487-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

Tong

Pan

et al. 2016

Parasites Vectors

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“…2001 who also reported that the N86Y mutation modulates a higher level of CQ resistance when present with the mutant K76T pfcrt genotype 34 . Mutations in the pfdhfr and pfdhps genes are associated with SP resistance and have been reported in most parts of the country 25 , 26 , 35 – 38 . In this study, a high level (78%) of the pfdhfr mutation (A 16 N 51 R 59 S 108 I 164, A 16 N 51 C 59 N 108 I 164, A 16 N 51 R 59 N 108 I 164 ) and a low level (6%) of pfdhps mutations (S 436 G 437 K 540 A 581 A 613, A 436 G 437 E 540 A 581 A 613 ) was recorded.…”

Section: Discussionmentioning

confidence: 99%

Prevalence of mutations linked to antimalarial resistance in Plasmodium falciparum from Chhattisgarh, Central India: A malaria elimination point of view

Patel

Bharti

Bansal

et al. 2017

Sci Rep

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Antimalarial drug resistance is a major global challenge in malaria control and elimination. Mutations in six different genes of Plasmodium falciparum (crt, mdr1, dhfr, dhps, ATPase6 and K-13 propeller) that confer resistance to chloroquine, sulphadoxine-pyrimethamine and artemisinin-based combination therapy were analyzed in samples from Chhattisgarh. Seventy-eight percent of the samples were found to have a pfcrt mutation (53% double, 24% triple and 1% single mutant), and 59% of pfmdr1 genes were found to have an N86Y mutation. Double mutations were recorded in pfdhfr gene among 76% of the samples while only 6% of the samples harbored mutant genotypes in pfdhps. No mutation was found in the K-13 propeller gene, while only one sample showed a mutant genotype for the PfATPase6 gene. The Tajima test confirmed that there is no role of evolutionary natural selection in drug resistance, and gene pairwise linkage of disequilibrium showed significant intragenic association. The high level of pfcrt mutations suggests that parasite resistance to chloroquine is almost at a fixed level, whereas resistance to SP is evolving in the population and parasites remain sensitive to artemisinin derivatives. These findings provide potential information and understanding of the evolution and spread of different drug resistance alleles in Chhattisgarh.

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“…DNA isolation from the dried blood spots was conducted according to the manufacturer’s protocol (EURx GeneMATRIX Bio-Trace DNA Purification Kit). For amplification of Pfdhfr and Pfdhps genes, nested PCR assay was performed as described elsewhere [ 7 ]. Briefly, Pfdhfr gene was amplified by using the primers AMP-1 F and AMP-2R, which was followed by the nested reaction with primers M1 and M5.…”

Section: Methodsmentioning

confidence: 99%

“…The 728-bp fragment of the second amplicon covered the codons, which are related with resistance to sulfadoxine. Finally, the purified PCR products were sequenced in both strands and the single nucleotide polymorphisms (SNPs) were analysed [ 7 ]. The sequences of Pfdhfr and Pfdhps genes were submitted to GenBank under the accession numbers MG254624–MG254671 and MG254672–MG254719, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, encoding the related DHFR and DHPS enzymes in the folate-pathway, cause resistance against pyrimethamine and sulfadoxine, respectively [ 6 ]. Notably, SP resistance is initiated by single polymorphisms, and augmented by accumulation of mutations in Pfdhfr and Pfdhps genes [ 7 ]. Five mutations have been widely reported in Africa, which are N51I, C59R, S108N in Pfdhr ; with A437G and K540E in Pfdhps gene [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

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Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo

Ruh

Bateko

İmir

et al. 2018

Malar J

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BackgroundPoint mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to sulfadoxine-pyrimethamine (SP) occur at increasing rates. The present study aimed to identify Pfdhfr and Pfdhps mutations in P. falciparum isolates recovered from women who received two doses of SP during pregnancy in Bandundu, the Democratic Republic of Congo (DRC).MethodsA total of 48 women with confirmed P. falciparum infection were enrolled in the study. Finger-prick blood samples that were collected on filter paper at the time of delivery were used for DNA isolation. Pfdhfr and Pfdhps genes were amplified by a nested PCR protocol. DNA sequencing was performed on both strands, and the point mutations were analysed.ResultsAll of the 48 (100.0%) P. falciparum isolates carried at least one polymorphism in both genes. The wild-type haplotypes of Pfdhfr (CNCSI [C50, N51, C59, S108, I164]) and Pfdhps (SAKAA [S436, A437, K540, A581, A613]) were not observed in the study. In Pfdhfr, N51I (85.4%), C59R (60.4%), and S108N (100.0%) polymorphisms were detected. Triple mutation (CIRNI) (mutant amino acids are underlined) was the most prevalent (47.9%) Pfdhfr haplotype. In the study, all P. falciparum isolates (100.0%) harboured the A437G allele in Pfdhps gene. Also, K540E and A581G polymorphisms were observed in one (2.1%) isolate. Single mutant haplotype (SGKAA) was detected in 97.9% of the isolates. Mutant Pfdhfr and Pfdhps allele combinations revealed quintuple (CICNI-SGEGA; 2.1%), quadruple (CIRNI-SGKAA; 47.9%), triple (CICNI-SGKAA; 35.4%, CNRNI-SGKAA; 12.5%), and double (CNCNI-SGKAA; 2.1%) haplotypes.ConclusionsIn the study, the rate of SGEGA haplotype was low (2.1%). Although K540E and A581G alleles are more common in Eastern Africa, a distinct lineage of SGEGA is also present in the DRC, which is located in Central Africa. This haplotype is associated with decreased efficacy of SP in pregnant women and infants, therefore, it should be carefully considered in the DRC and SP resistance should be routinely monitored.

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Polymorphism in drug resistance genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India

Cited by 13 publications

References 32 publications

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

Prevalence of mutations linked to antimalarial resistance in Plasmodium falciparum from Chhattisgarh, Central India: A malaria elimination point of view

Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo

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