Molecular Determination of Point Mutation Haplotypes in the Dihydrofolate Reductase and Dihydropteroate Synthase of Plasmodium falciparum in Three Districts of Northern Tanzania

Abstract

The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2001 following 18 years of second-line use. The genetic determinants of in vitro resistance to the two drugs individually are shown to be point mutations at seven sites in the dihydrofolate reductase gene (dhfr) conferring resistance to pyrimethamine and five sites in the dihydropteroate synthase (dhps) gene conferring resistance to sulfadoxine. Different combinations of mutations within each gene confer differing degrees of insensitivity, but information about the frequency with which allelic haplotypes occur has been lacking because of the complicating effects of multiple infection. Here we used a novel highthroughput sequence-specific oligonucleotide probe-based approach to examine the present resistance status of three Plasmodium falciparum populations in northern Tanzania. By using surveys of asymptomatic infections and screening for the presence of all known point mutations in dhfr and dhps genes, we showed that just five dhfr and three dhps allelic haplotypes are present. High frequencies of both triple-mutant dhfr and doublemutant dhps mutant alleles were found in addition to significant interregional heterogeneity in allele frequency. In vivo studies have shown that the cooccurrence of three dhfr mutations and two dhps mutations in an infection prior to treatment is statistically predictive of treatment failure. We have combined data for both loci to determine the frequency of two-locus genotypes. The triple-dhfr/double-dhps genotype is present in all three regions with frequencies ranging between 30 and 63%, indicating that treatment failure rates are likely to be high.Sulfadoxine-pyrimethamine (SP) has now replaced chloroquine as the first-line curative antimalarial in much of East Africa. The earliest reports of emerging SP resistance in Africa were from Muheza district in Tanzania during 1994 and 1995 (32, 37). SP remains in use throughout Tanzania and was recently officially adopted as the national first-line treatment for nonsevere Plasmodium falciparum malaria. It is now a priority to learn how widespread genetic determinants of SP resistance currently are in the larger regions of northern Tanzania.To investigate this issue, we have carried out a populationbased genetic analysis of P. falciparum in the North and South Pare Mountains and Hai district, which are distinct geographical areas with their own microclimates and tribes. There had been no malaria research in these districts since 1965 until recent work showed relatively low levels of transmission, with an estimated entomological inoculation rate of 24 infective bites/person/year in Hai district (C. Drakeley and D. Chandramohan, unpublished data) compared with an entomological inoculation rate in the range of 34 to 405 infective bites/person/ year in Muheza district (14).It is widely understood that people self-treat with antimalarial drugs, which can be freely purchased (24); as a consequen...

Selection of antimalarial drug resistance after intermittent preventive treatment of infants and children (IPTi/c) in Senegal

“…DNA was extracted from positive P. falciparum blood spots by the Chelex-100 method described by Wooden and others 26 with some modifications described by Pearce and others 27 A nested polymerase chain reaction (PCR) (was used to amplify fragments of the Pfdhfr and Pfdhps genes as described by Alifrangis and others. 28 The 20-μL Pfdhfr/Pfdhps outer PCR mixture consisted of 0.3 mM of each dNTP, 0.25 μM of either primer set M1/M7 (Pfdhfr) or N1/N2 (Pfdhps), one unit …”

Prevalence of Single Nucleotide Polymorphisms in the Plasmodium falciparum Multidrug Resistance Gene (Pfmdr-1) in Korogwe District in Tanzania Before and After Introduction of Artemisinin-Based Combination Therapy

“…The extraction of DNA was performed by Chelex-100 method. 19,20 Laboratory isolate DNA was extracted using the NucleoSpin Blood QuickPure Kit (Macherey-Naegl GmbH & Co, Düren, Germany).…”

Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas

“…El programa de amplificación consistió en un ciclo inicial de desnaturalización a 94°C durante 3 minutos, 4 ciclos a 94°C durante un minuto, a 44°C durante 2 minutos y a 72°C durante un minuto, y posteriormente, 34 ciclos a 94°C durante un minuto, a 44°C durante un minuto y a 72°C durante un minuto, y una extensión final a 72°C durante 10 minutos (18). El producto obtenido en esta reacción (594 pb) fue sometido a digestión enzimática con la enzima Tsp509 I (20).…”

“…Para determinar los alelos de los codones 436, 437 y 540 del gen dhps se realizó una primera ronda de PCR (PCR universal) utilizando los iniciadores N1 y N2 (cuadro 1) con las mismas concentraciones de reactivos descritas para la PCR universal del gen dhfr y el mismo programa de amplificación, modificando únicamente la temperatura de alineamiento de los iniciadores que fue de 52°C (18).…”