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      Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

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          Abstract

          Background

          With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene ( pfmdr1) copy number, mutations and the chloroquine resistance transporter gene ( pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations.

          Methods

          Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 ( msp2) genotyping was done to select monoclonal infections for copy number analysis.

          Results

          The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (× 2 = 96.31, p <0.001) and pfcrt K76 (× 2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (× 2 = 38.52, p <0.001) and pfcrt T76 (× 2 = 43.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (× 2 = 7.39,p=0.060; × 2 = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (× 2 = 20.75, p < 0.001).

          Conclusion

          Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

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          Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

          The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.
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            A molecular marker for chloroquine-resistant falciparum malaria.

            Chloroquine-resistant Plasmodium falciparum malaria is a major health problem, particularly in sub-Saharan Africa. Chloroquine resistance has been associated in vitro with point mutations in two genes, pfcrt and pfmdr 1, which encode the P. falciparum digestive-vacuole transmembrane proteins PfCRT and Pgh1, respectively. To assess the value of these mutations as markers for clinical chloroquine resistance, we measured the association between the mutations and the response to chloroquine treatment in patients with uncomplicated falciparum malaria in Mali. The frequencies of the mutations in patients before and after treatment were compared for evidence of selection of resistance factors as a result of exposure to chloroquine. The pfcrt mutation resulting in the substitution of threonine (T76) for lysine at position 76 was present in all 60 samples from patients with chloroquine-resistant infections (those that persisted or recurred after treatment), as compared with a base-line prevalence of 41 percent in samples obtained before treatment from 116 randomly selected patients (P<0.001), indicating absolute selection for this mutation. The pfmdr 1 mutation resulting in the substitution of tyrosine for asparagine at position 86 was also selected for, since it was present in 48 of 56 post-treatment samples from patients with chloroquine-resistant infections (86 percent), as compared with a base-line prevalence of 50 percent in 115 samples obtained before treatment (P<0.001). The presence of pfcrt T76 was more strongly associated with the development of chloroquine resistance (odds ratio, 18.8; 95 percent confidence interval, 6.5 to 58.3) than was the presence of pfmdr 1 Y86 (odds ratio, 3.2; 95 percent confidence interval, 1.5 to 6.8) or the presence of both mutations (odds ratio, 9.8; 95 percent confidence interval, 4.4 to 22.1). This study shows an association between the pfcrt T76 mutation in P. falciparum and the development of chloroquine resistance during the treatment of malaria. This mutation can be used as a marker in surveillance for chloroquine-resistant falciparum malaria.
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              Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi.

              In 1993, Malawi became the first African country to replace chloroquine with sulfadoxine-pyrimethamine nationwide in response to high rates of chloroquine-resistant falciparum malaria. To determine whether withdrawal of chloroquine can lead to the reemergence of chloroquine sensitivity, the prevalence of the pfcrt 76T molecular marker for chloroquine-resistant Plasmodium falciparum malaria was retrospectively measured in Blantyre, Malawi. The prevalence of the chloroquine-resistant pfcrt genotype decreased from 85% in 1992 to 13% in 2000. In 2001, chloroquine cleared 100% of 63 asymptomatic P. falciparum infections, no isolates were resistant to chloroquine in vitro, and no infections with the chloroquine-resistant pfcrt genotype were detected. A concerted national effort to withdraw chloroquine from use has been followed by a return of chloroquine-sensitive falciparum malaria in Malawi. The reintroduction of chloroquine, ideally in combination with another antimalarial drug, should be considered in areas where chloroquine resistance has declined and safe and affordable alternatives remain unavailable.
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                Author and article information

                Journal
                Malar J
                Malar. J
                Malaria Journal
                BioMed Central
                1475-2875
                2013
                30 October 2013
                : 12
                : 377
                Affiliations
                [1 ]Epidemiology Department, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, PO Box LG581, Legon, Ghana
                [2 ]Centre for Tropical Clinical Pharmacology and Therapeutics, University of Ghana Medical School, PO Box GP4236, Accra, Ghana
                [3 ]United States Naval Medical Research Unit No.3, Cairo, Egypt
                Article
                1475-2875-12-377
                10.1186/1475-2875-12-377
                3819684
                24172030
                d091a752-9fc8-485e-9968-7091c816467a
                Copyright © 2013 Duah 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
                : 20 August 2013
                : 28 October 2013
                Categories
                Research

                Infectious disease & Microbiology
                anti-malarial drug resistance,plasmodium falciparum chloroquine resistance transporter gene (pfcrt),plasmodium falciparum multidrug resistance gene (pfmdr1),molecular markers,ghana

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