The results of this study demonstrate clearly that use of SP for the treatment of P falciparum
malaria is dangerous in the area where our study was done. SP had no effect on the course of illness in four cases, three in the symptomatic group and one in the asymptomatic group, who developed severe disease within 3 days of post treatment. The efficacy of SP resulted in similarly poor parasitologic clearance in 2–10 month old asymptomatic infants when compared with 6–59 month old symptomatic children. The WHO recommends changing first line treatment when day 28 failure rates exceed 10%
. In this study, early treatment failures of symptomatic children were 38.8% and by day 28 a staggering 82% required rescue treatment. Shortly after the study was stopped artemether-lumefantrine (ALU) became the first-line treatment for uncomplicated malaria in Tanzania, although SP remains widely available through private drug stores and is frequently used.
The primary purpose of this study was to determine if SP was more efficacious in asymptomatic parasitaemic infants than in symptomatic children even though the drug was failing as a treatment for the latter group. This has been shown to be the case for pregnant women in areas of moderate SP resistance where SP may still be effective for treatment or prevention even though it fails when used for the treatment of symptomatic children
. Because the study had to be terminated prematurely on the grounds of the unexpectedly high failure rates in symptomatic children and asymptomatic infants, it was underpowered to show a difference between the two study groups. Estimates of efficacy had confidence intervals of 10% and 20% in the symptomatic and asymptomatic study groups respectively and the study was powered to detect only a 30% or more difference between groups. Nevertheless, the findings suggest that there was no major difference in response to SP between symptomatic older children and asymptomatic infants contrasting with the major differences observed in comparisons between asymptomatic pregnant women and symptomatic children. This difference may be due to the fact that infants in the second half of the first year of life, in contrast to pregnant women, have less naturally acquired immunity to malaria. In pregnant women, but not infants, naturally acquired immunity may be sufficient to clear low density infections when parasites are exposed to an only partially effective drug. Evidence from high transmission sites suggests that maternal protective factors do protect infants against recrudescence
, however, how these protective factors compare to maternal immunity itself is not known. Although the study split children into symptomatic and asymptomatic groups the difference between these groups may only be that the asymptomatic children were at an earlier stage of the disease. In children in a moderate transmission setting in Uganda approximately 50% of asymptomatic parasitaemic children progressed to clinical disease within 30 days of detection
. This group of children, however, is relevant for this study as they represent the population who would be treated with IPTi. This study examined the treatment effect of SP and did not address the ability for SP to prevent blood stage infection, i.e. the prophylactic effect
. Whether SP has any efficacy in the study area when used for IPTi (both treatment and prophylactic effects
) will be determined in an ongoing IPTi study that will be reported in early 2009 (clintrial.gov identifier: NCT00158574).
Within 8 years the level of SP resistance in the study area has increased at an alarming rate. The day 28 ACPR has decreased from 55% in 1999 
to the current situation of 18%. This change is likely to have been driven, at least in part, by the use of SP as first line treatment for malaria from 2001 to 2007. Now that first line treatment for malaria has changed to combination therapy across most of sub- Saharan Africa, the selection pressure for resistance to SP will have been reduced. Nevertheless the rapid increase in SP resistance is a concern and an alternative drug needs to be developed for all SP-based prevention programs.
The molecular work was limited by the number of clones in the samples and the sensitivity of the methods to detect masked clones. In the symptomatic group 4 new infections were detected in the first 3 days post-treatment. This is a highly unlikely result and is probably due to masked clones resistant to SP becoming dominant post treatment, a common criticism of PCR-correction. This problem was also likely to interfere with the interpretation of new infections when comparing the symptomatic and asymptomatic groups as the symptomatic group was more likely to have heavier parasite densities and more clones. Thus, in the symptomatic group a higher proportion of recrudescences would be wrongly called new infections. The number of clones detected in samples was high and in many cases these represented multiple clones recrudescing reflecting the high transmission and high prevalence of resistance genotypes circulating in the study area.
Further in vivo
efficacy studies of SP are unjustifiable in areas where the level of SP resistance is known to be high. In such situations, studies of molecular markers of resistance play a key role in measuring patterns of resistance as there is strong evidence that mutations in dhfr
genes are associated with P. falciparum
resistance to SP. Previous reports from Africa have demonstrated an association between moderate levels of resistance to SP and triple mutation in the dhfr 
and double mutations in the dhps
and this association was found in our study. In South East Asia the presence of high level resistance to antifolates has been associated with the presence on an additional mutation at position 164 on the dhfr
. In the face of the very high level of SP resistance found in Hale we considered it possible that parasites carrying this mutation had emerged in north-eastern Tanzania. However, this did not turn out to be the case and no parasites carrying the 164 mutation were found. Instead 55% of haplotypes, when they could be ascertained, in addition to the 437 and 540 mutations had a mutation at codon 581 in the parasite dhps
gene and there was some evidence that the presence of this mutation in parasites at the start of treatment was associated earlier treatment failure. Biological plausibility of this is backed by the findings that the 581G single mutant offers resistance in vitro
and that similar mutations in bacteria produce resistance to sulfa drugs
. The dhps
triple is recognized in South America, where it is associated with SP treatment failure
. Parasites carrying the 581G mutation are widely distributed in Tanzania () and elsewhere in Africa (). The evidence of prevalence of this mutation is patchy with few studies looking at this locus and those that have show considerable differences in prevalence between countries. It is likely that the large drug pressure caused when SP was the first line drug for uncomplicated malaria that has driven the rise in frequency of first the dhps
double and now the more resistant dhps
triple mutation. This early failure may prove to be pertinent in the case of IPTp. ter Kuile and colleagues
found that SP IPTp had reasonable protective efficacy against low birth-weight even in areas where the day 14 treatment failure rate of SP in symptomatic children was as high as 35%. However, they observed that the duration of prophylaxis of SP had reduced from 4 to 2 weeks. In areas where the 581 mutation is present in conjunction with the triple dhfr
and double dhps
mutants, it is possible that the duration of prophylaxis given by SP will be further reduced and this may affect the protective efficacy of SP preventative strategies. Further work is needed to determine the geographical spread of this mutant and if the presence of this mutation in combination with the dhfr
triple and dhps
double confers increased and earlier failure of cases treated with SP.