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<p>Abstract</p> <p>Background</p> <p>The development of <it>Plasmodium falciparum </it>resistance to chloroquine (CQ) has limited its use in many malaria endemic areas of the world. However, despite recent drug policy changes to adopt the more effective artemisinin-based combination (ACT) in Africa and in the Southern African region, in 2007 Swaziland still relied on CQ as first-line anti-malarial drug.</p> <p>Methods</p> <p>Parasite DNA was amplified from <it>P. falciparum </it>isolates from Swaziland collected in 1999 (thick smear blood slides) and 2007 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing.</p> <p>Results</p> <p>Retrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2007 carried detectable <it>P. falciparum</it>. The <it>pfcrt </it>haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 70% (n = 64) in 1999 and 83% (n = 6) in 2007. Prevalence of the <it>pfmdr1</it>-86N allele was 24% in 1999 and 67% in 2007. A novel substitution of phenylalanine for asparagine at codon 86 of <it>pfmdr1 </it>(N86F) occurred in two of 51 isolates successfully amplified from 1999. The <it>pfmdr1</it>-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2007. The 86N/184F/1246D <it>pfmdr1 </it>haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2007 isolates. The <it>pfdhfr </it>triple-mutant 16C/51I/59R/108N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n = 34) and 2007 (50%, n = 6), as was the wild-type 431I/436S/437A/540K/581A/613A haplotype of <it>pfdhps </it>(100% and 93% respectively in 1999 and 2007). The quintuple-mutant haplotype <it>pfdhfr/pfdhps</it>-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2007 isolates.</p> <p>Conclusions</p> <p>The prevalence of <it>pfcrt </it>and <it>pfmdr1 </it>alleles reported in this study is consistent with a parasite population under sustained CQ drug pressure. The low prevalence of dhps-437G and dhps-540E mutations (ISGEAA) and the rarity of quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA suggest that SP retains some efficacy in Swaziland. Anti-malarial policy changes in neighbouring countries may have had an impact on the prevalence of molecular markers of anti-malarial resistance in Swaziland, and it is hoped that this new information will add to understanding of the regional anti-malarial resistance map.</p>