Abstract:
This study reports a systematic follow up of genetic changes in the genes encoding
the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes
of Plasmodium falciparum, in isolates from three rural districts of South-eastern
Tanzania. The enzymes are the target of antimalarial drug, sulphadoxine-
pyrimethamine (SP). The population-wide analysis of resistance mutations in the dhfr
and dhps genes was applied to examine the influence of different drug use policies
and their potential for the selection for SP resistance. A total of 47 244 bloodspot
filter paper samples were collected from all individuals of all ages in randomly
selected households in a series of annual surveys conducted between 2000 - 2006.
Twenty percent (9 662) of all samples were found positive for P. falciparum infection
on microscopy and hence were used for the genetic studies. DNA was extracted from
P. falciparum-positxvz samples and dhfr and dhps genes were amplified by a nested
polymerase chain reaction (PCR) and resistance conferring point mutations
determined. Size polymorphisms at three sets of microsatellite loci linked to dhfr and
three other sets of unlinked microsatellite loci were analysed by PCR amplification
and electrophoresis on an automated sequencer. The influence of National treatment
policy on the parasite reservoir was profound. The change of first line therapy from
CQ to SP brought about highly significant increase of the frequencies of dhfr triple
and dhps double mutants. Artemisinin-based combination therapy (ACT-
SP+Artesunate) in Rufiji had a small and non-significant impact on the frequency of
dhps double and dhfr triple mutant alleles, but significantly disrupted their association. Z)A/r-l inked microsatellites revealed high diversity around the dhfr
sensitive alleles and significantly reduced diversity around mutant dhfr alleles. The
majority of triple mutant alleles had one flanking microsatellite haplotype which has
previously been shown to be derived from Southeast Asia, while the double mutant
alleles had multiple haplotypes which were independently derived. Distribution of
major lineages indicates that there is extensive genetic exchange among the
geographic regions. Unlinked microsatellites confirmed the extent of allele sharing among the regions and revealed a major trend for reduced transmission intensity, which was apparently independent of the ACT intervention.
