Malaria is still a major concern in Swaziland despite recent reports of significant reduction of disease incidence by more than 95% . Selection of effective and affordable anti-malarials for treatment and control remain extremely important and complicated in this era of emergence of P. falciparum parasites resistant to multiple anti-malarials. CQ failure in Swaziland was first reported in 1987 . More than twenty years later, Swaziland retained CQ as a first-line anti-malarial for treating uncomplicated malaria. Although SP has not been used as a first-line anti-malarial in Swaziland, it is still of interest to determine whether parasites remain susceptible to the drug especially following reports of resistance from neighbouring countries. SP is used as a second-line anti-malarial for CQ resistant (CQR) parasites.
The high prevalence of the pfcrt CVIET haplotype, which originated in CQR parasites from Southeast Asia [30, 31], and the absence of CQR haplotype SVMNT (South American) among isolates examined in this study is consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. Reports of SVMNT occurrence described in isolated areas of East Africa are probably a result of resistance to AQ or its metabolite desethyl-amodiaquine (DEAQ) following use of AQ as monotherapy . High prevalence of 76T mutations both in 1999 and 2007 suggests that CQ resistance is widespread in Swaziland. Studies have also shown that acquisition of 76T mutations favours severity and multiplicity of malaria infection  and, together with pfmdr1 mutations, is associated with life-threatening complications such as severe anaemia (Hb<5 g/dl) in young children .
No published data are available on the in vivo efficacy of CQ in Swaziland. Evidence of heavy over-prescription is described here, in which only six of 252 patients treated for malaria in 2007 proved to be parasite positive by both retrospective slide-reading and PCR. Previous studies elsewhere have shown that the 76T and 86Y mutations occur among parasites surviving CQ treatment, including those responsible for production of transmissible gametocytes [35, 36]. In CQ-treated children, parasites with pfmdr1 86Y and pfcrt 76T are transmitted more frequently than other genotypes and this mechanism probably supports continued circulation of these genotypes . Further studies among malaria patients in Swaziland are required to estimate any impact of these mutations on treatment outcome. The pfmdr1-1246Y allele, found at 49% prevalence among 1999 isolates, was absent among 2007 isolates, suggesting withdrawal of CQ use may lead to further beneficial changes in the parasite population in Swaziland.
The novel 86F mutation in pfmdr1 identified in two samples from this study has not previously been described in field-collected parasites. In the isolates described here, the phenylalanine at position 86 is encoded by the codon UUU, which has two base changes from the wild-type asparagine codon AAU, but only a single base-change from the common CQ-resistance-associated tyrosine codon, UAU. The 86F allele is therefore likely to have arisen from the 86Y form of pfmdr1 by a single point mutation, rather than directly from the wild-type 86N form, which would require a minimum of two changes. This is consistent with in vitro observations .
The absence of the wild-type pfmdr1-NYD haplotype in 2007 and its low prevalence among 1999 isolates suggest that CQ resistance had developed over a long time. The high prevalence of the pfmdr1-NFD haplotype in 2007 and absence of the YYY haplotype is of interest, as studies elsewhere in Africa have reported selection for the NFD allele of pfmdr1 and loss of the YYY allele following treatment with artemether-lumefantrine (AL) [12, 13]. Since there is no history of AL use in Swaziland, this pattern may be a result of the impact of drug policy changes in neighbouring South Africa, KwaZulu-Natal (KZN) and Mozambique. Swaziland is a very small country and the influence of drug policies in Mozambique and South Africa (KZN) cannot be ruled out. KZN introduced AL in 2001 while Mozambique introduced artesunate-sulphadoxine/pyrimethamine (AS-SP) combination therapy in 2002 and artesunate/AQ in 2004. Thus, deployment of AL in KZN and the introduction of AS-SP treatment policy in Mozambique could have influenced selection of 86N parasites through successful transmission compared to their mutant counter-parts. However, this phenomenon can only be confirmed through analysis of a larger number of samples from Swaziland. Transmission studies reported a four-fold reduction in transmissibility to mosquitoes of the 86Y allele compared to the 86N allele following addition of artesunate to CQ . The same study reported an association between CQ monotherapy and increased 86Y transmissibility. Other studies have shown increased tolerance of laboratory parasite clones to mefloquine and halofantrine, (which are related to lumefantrine) if they carried the 86N allele  or the 1034S/1042N/1246D haplotype . Therefore, the prevalence of NFD haplotypes in Swaziland has to be closely monitored as it could compromise the effectiveness of combinations involving lumefantrine in the future. Unfortunately, data on the prevalence of NFD and YYY haplotypes in KZN following AL implementation in 2001 are not available.
Regional drug pressure, as well as prescription of SP to CQ-resistant cases probably partly explains the high prevalence of dhfr triple-mutant haplotypes (82%) observed in 1999 in Swaziland. The prevalence of the dhfr-CIRNI triple-mutant haplotype in Zone 1 in Southern Mozambique, the zone closest to Swaziland, was 47.5% in 1999  which is lower than that observed in Swaziland, suggesting only partial influence from Mozambique, possibly because SP was in use in Swaziland as a second-line drug. In contrast, there was a high prevalence of the triple-mutant haplotype at Masvold Hospital in northern KZN  in 1999. The results given here suggest that the prevalence of dhfr mutations in Swaziland was high compared to that in neighbouring countries. Thus, regional drug pressure alone may not explain allele prevalence in Swaziland. SP drug pressure in Swaziland may also have been further enhanced by the use of other anti-folate compounds, such as the combination antibiotic cotrimoxazole, which have cross-resistance with SP. As HIV infection rates are high in Swaziland, cotrimoxazole is commonly prescribed as prophylaxis against opportunistic infections to HIV patients , and may have contributed to persistence of CIRNI triple-mutant malaria parasites in Swaziland.
The results presented here strongly support the recent move to replace CQ with the highly efficacious artemisinin-combination therapies (ACTs) in Swaziland. The replacement of presumptive treatment with a quick, simple and cheaper diagnostic method such as rapid diagnostic tests (RDTs) to reduce inappropriate prescription of drugs should reduce the cost of ACT drug and further delay development of resistance. Deployment of AL in Swaziland is likely to further reduce prevalence of YYY haplotype but could increase the NFD haplotype, and this may help preserve good AQ efficacy [12, 13, 41], as will the recent withdrawal of AQ use in neighbouring Mozambique. In the absence of the pfmdr1 YYY haplotype, AQ shows in vivo and vitro efficacy against parasite clones with the CVIET pfcrt haplotype [13, 42]; parasites of this genotype are shown here to be abundant in Swaziland.