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      Identification of pyrimethamine- and chloroquine-resistant Plasmodium falciparum in Africa between 1984 and 1998: genotyping of archive blood samples

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          Abstract

          Background

          Understanding the geographical distribution of drug resistance of Plasmodium falciparum is important for the effective treatment of malaria. Drug resistance has previously been inferred mainly from records of clinical resistance. However, clinical resistance is not always consistent with the parasite's genetic resistance. Thus, molecular identification of the parasite's drug resistance is required. In Africa, clinical resistance to pyrimethamine (Pyr) and chloroquine (CQ) was evident before 1980 but few studies investigating the genetic resistance to these drugs were conducted before the late 1990s. In this study, genotyping of genes involved in resistance to Pyr and CQ was performed using archive blood samples from Africa between 1984 and 1998.

          Methods

          Parasite DNA was extracted from P. falciparum-infected blood smears collected from travellers returning to Japan from Africa between 1984 and 1998. Genotypes of the dihydrofolate reductase gene ( dhfr) and CQ-resistance transporter gene ( pfcrt) were determined by polymerase chain reaction amplification and sequencing.

          Results

          Genotyping of dhfr and pfcrt was successful in 59 and 80 samples, respectively. One wild-type and seven mutant dhfr genotypes were identified. Three dhfr genotypes lacking the S108N mutation (N RSI, ICSI, IRSI; amino acids at positions 51, 59, 108, and 164 with mutations underlined) were highly prevalent before 1994 but reduced after 1995, accompanied by an increase in genotypes with the S108N mutation. The dhfr IRNI genotype was first identified in Nigeria in 1991 in the present samples, and its frequency gradually increased. However, two double mutants ( IC NI and N RNI), the latter of which was exclusively found in West Africa, were more frequent than the IRNI genotype. Only two pfcrt genotypes were found, the wild-type and a Southeast Asian type (CV IET; amino acids at positions 72-76 with mutations underlined). The CV IET genotype was already present as early as 1984 in Tanzania and Nigeria, and appeared throughout Africa between 1984 and 1998.

          Conclusions

          This study is the first to report the molecular identification of Pyr- and CQ-resistant genotypes of P. falciparum in Africa before 1990. Genotyping of dhfr and pfcrt using archive samples has revealed new aspects of the evolutionary history of Pyr- and CQ-resistant parasites in Africa.

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          Most cited references38

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          Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum.

          Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.
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            Intercontinental spread of pyrimethamine-resistant malaria.

            Here we present molecular evidence demonstrating that malaria parasites bearing high-level pyrimethamine resistance originally arrived in Africa from southeast Asia. The resistance alleles carried by these migrants are now spreading across Africa at an alarming rate, signaling the end of affordable malaria treatment and presenting sub-Saharan Africa with a public health crisis.
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              Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum.

              We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS; EC 1.5.1.3 and EC 2.1.1.45, respectively) from two pyrimethamine-resistant clones of Plasmodium falciparum, HB3 and 7G8. We have also derived the sequence of the DHFR portion of the gene, by amplification using polymerase chain reaction, for the pyrimethamine-sensitive clone 3D7 and the pyrimethamine-resistant strains V-1, K-1, Csl-2, and Palo-alto. The deduced protein sequence of the resistant DHFR portion of the enzyme from HB3 contained a single amino acid difference from the pyrimethamine-sensitive clone 3D7. It is highly likely that this difference is involved in the mechanism of drug resistance in HB3. The sequence of the DHFR gene from other pyrimethamine-resistant strains contains the same amino acid difference from the sensitive clone 3D7. However, they all differ at one other site that may influence pyrimethamine resistance. The DHFR-TS gene is present as a single copy on chromosome 4 in all pyrimethamine-sensitive and pyrimethamine-resistant isolates tested. Therefore, the molecular basis of pyrimethamine resistance in the parasites tested is not amplification of the DHFR-TS gene.
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                Author and article information

                Journal
                Malar J
                Malar. J
                Malaria Journal
                BioMed Central
                1475-2875
                2011
                31 December 2011
                : 10
                : 388
                Affiliations
                [1 ]Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
                [2 ]Laboratory of Malariology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
                [3 ]Department of International Affairs and Tropical Medicine, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan
                Article
                1475-2875-10-388
                10.1186/1475-2875-10-388
                3309963
                22208458
                4735e917-666e-40d2-a572-2888b751cc1b
                Copyright ©2011 Saito-Nakano et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 September 2011
                : 31 December 2011
                Categories
                Research

                Infectious disease & Microbiology
                pyrimethamine,pfcrt,plasmodium falciparum,archive sample,chloroquine,dhfr,africa,drug resistance

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