Drug resistance in Plasmodium falciparum affects prevention of malaria in pregnancy. In a cross-sectional study of 53 pregnant Ghanaian women, P. falciparum dihydrofolate reductase (DHFR) gene mutations urled with pyrimethamine resistance were assessed and associations with pyrimethamine intake were analyzed. P. falciparum infected 69% of women without pyrimethamine use, 59% of those who had a history of pyrimethamine consumption but a negative urine test, and 53% of individuals with a positive urine test. Eighty-one percent, 43%, and 74% of the isolates contained the mutations Asn-1 8, Ile-51, and Arg-59, respectively. Thr-1 8 occurred in 8%. Pyrimethamine use was associated with increased frequencies of Asn-1 8 and Arg-59 but not of Ile-51 or Thr-1 8. In women with prophylaxis, wild-type parasites were absent and anemia tended to be more common with an increasing number of DHFR gene mutations. Pyrimethamine appears to be not adequately effective in this part of Ghana, most likely due to the predominance of resistant parasites. Selection for resistance following insufficient prophylaxis could possibly affect the efficacy of future intermittent sulfadoxine-pyrimethamine treatment.

During an epidemic of Plasmodium falciparum malaria in Chogoria, Kenya, P. falciparum DNA was collected from 24 cases of severe malaria admitted to hospital for parenteral quinine treatment. These patients had all failed first- (chloroquine) and second-line (sulfadoxine-pyrimethamine or amodiaquine) drug treatments. Twenty-two (92%) of the 24 patients sampled carried parasites with the (Asn)86(Tyr) point mutation in the pfmdr1 gene (chromosome 5), 2 (83%) had an (Asp)1246(Tyr) mutation and 18 (82%) had both of these mutations. These alleles are both reported to be associated with chloroquine-resistance. Polymorphisms in the cg2 gene (chromosome 7) are also associated with chloroquine resistance, and 18 (75%) of the 24 parasite samples each had the cg2 and pfmdr1 polymorphisms. These 18 samples also had the mutations associated with resistance to pyrimethamine and sulfadoxine: (Asn)51(Ile), (Cys)59(Arg) and (Ser)1 8(Asn) of gene dhfr (chromosome 4) and (Ala)437(Gly) and (Lys)54 (Glu) of dhps (chromosome 8), respectively. Genotyping of the parasites from all 24 patients revealed extensive diversity in the sequences for the merozoite surface antigens (MSA-1 and MSA-2) and the glutamate-rich protein (GLURP) and indicated that each sample contained more than one parasite clone. Although samples from non-admitted malaria cases were not available, it appears that drug resistance may have played an important role in the development of severe malaria in this epidemic.

The increasing resistance of Plasmodium falciparum in the treatment of uncomplicated malaria with pyrimethamine/sulphadoxine has been associated in several studies with the occurrence of point mutations in the genes of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). In this study, the prevalence of these mutations was examined in samples from south-east Mauritania, where atypically strong rainfalls in 1998 and 1999 led to a severe outbreak of falciparum malaria. We analysed 386 samples by polymerase chain reaction (PCR) for infection with P. falciparum, of which 162 (41.97%) were positive. These isolates were examined for point mutations in the genes of DHFR (codons 16, 51, 59, 1 8 and 164) and DHPS (codons 436, 437, 54 , 581 and 613) by nested PCR and subsequent mutation-specific restriction enzyme digest. We found a low overall prevalence of DHFR gene mutations (up to 18.6% of isolates), but a high overall prevalence of DHPS gene mutations (up to 49.1% of isolates). Thus, emerging resistance to antifolate drugs may be expected to develop soon in the investigated area. This study demonstrates the utility of simple, relatively rapid and inexpensive molecular methods and their application in surveillance programmes. Testing for prevalence of point mutations conferring antifolate resistance might help to identify the developing of drug resistance at a very early stage.

BACKGROUND: Resistance to the affordable malaria treatments chloroquine and pyrimethamine-sulfadoxine is seriously impeding malaria control through treatment in east Africa. We did an open, alternate drug allocation study to assess the efficacy of chlorproguanil-dapsone in the treatment of falciparum malaria clinically resistant to pyrimethamine-sulfadoxine. METHODS: Children younger than 5 years with non-severe falciparum malaria, attending Muheza district hospital in Tanzania, were treated with the standard regimen of pyrimethamine-sulfadoxine. Patients whose clinical symptoms resolved but who remained parasitaemic 7 days after pyrimethamine-sulfadoxine were followed up for 1 month. Clinical malaria episodes were retreated with either single dose pyrimethamine-sulfadoxine or a 3-day regimen of chlorproguanil-dapsone. Those with parasitaemia after 7 days were treated with chlorproguanil-dapsone. Parasite DNA was collected on day 7 after first treatment with pyrimethamine-sulfadoxine and we looked for point mutations in the genes encoding dihydrofolate reductase (dhfr) and dyhydropteroate synthetase (dhps). FINDINGS: 36 children were enrolled and treated with pyrimethamine-sulfadoxine. On day 7, 192 (55%) of 348 had cleared parasitaemia. Of the remaining 156 parasitaemic children, 14 (9 %) were followed up to day 28, and 92 (66%) of 14 developed clinical malaria. These 92 patients were alternately retreated with either pyrimethamine-sulfadoxine (46) or chlorproguanil-dapsone (46). 28 (61%) of 46 children retreated with pyrimethamine-sulfadoxine were still parasitaemic at day 7, compared with three (7%) of 44 [corrected] children retreated with chlorproguanil-dapsone. Resistance to pyrimethamine-sulfadoxine increased from 45% (156/348) at the first treatment to 61% (28/46) after retreatment. 83 of 85 parasite isolates collected after the first pyrimethamine-sulfadoxine treatment, and before and after the second treatments with pyrimethamine-sulfadoxine and chlorproguanil-dapsone showed triple-mutant dhfr alleles, associated with a variety of dhps mutations. INTERPRETATION: Most patients treated with pyrimethamine-sulfadoxine, who remain parasitaemic at day 7, develop new malaria symptoms within 1 month. Chlorproguanil-dapsone was a practicable therapy under these circumstances. Analysis of parasite dhfr and dhps before and after treatment supports the view that pyrimethamine-sulfadoxine resistance in this part of Africa is primarily due to parasites with three mutations in the dhfr domain.

The DNA sequence of the dihydrofolate reductase (dhfr) gene, a molecular marker for pyrimethamine resistance, was determined for 178 field isolates of Plasmodium falciparum collected along the east-west axis in southern Cameroon. The proportion of isolates having the wild-type dhfr allele varied from 48.1% in the east (city of Bertoua) to 11.3-15.7% in central provinces (Yaounde and Eseka) and % in the littoral region (port city of Douala). Isolates with a single Asn-1 8 mutation or double mutations (Ile-51 or Arg-59 and Asn-1 8) constituted approximately 1 % of the samples. Isolates with triple mutations (Ile-51, Arg-59, and Asn-1 8) were present in an equal proportion (48.1%) as the wild-type isolates in the east (Bertoua), while triple mutations predominated in Yaounde (62.3%), Eseka (62.7%), and Douala (78.9%). The distribution of triple dhfr mutations along the east-west axis in southern Cameroon suggests the presence of a decreasing gradient from the west coastal region to the central region and then to the east towards the interior of the country.

In Liberia, little information is available on the efficacy of antimalarials against Plasmodium falciparum malaria. We measured parasitological resistance to chloroquine and sulfadoxine-pyrimethamine (SP) in Harper, south-west Liberia in a 28-d study in vivo. A total of 5 patients completed follow-up in the chloroquine group, and 66 in the SP group. The chloroquine failure rate was 74. % (95% confidence interval [95% CI] 59.7-85.4%) after 14 d of follow-up and 84. % (95% CI 7 .9-92.8%) after 28 d (no polymerase chain reaction [PCR] analysis was performed to detect reinfections in this group). In the SP group, the failure rate was 48.5% (95% CI 36.2-61. %) after 14 d and 69.7% (95% CI 57.1-8 .4%) after 28 d, readjusted to 51.5% (95% CI 38.9-64. %) after taking into account reinfections detected by PCR. Genomic analysis of parasite isolates was also performed to look for point mutations associated with resistance. Genotyping of parasite isolates revealed that all carried chloroquine-resistant K-76T mutations at gene pfcrt, whereas the triple mutation (S1 8N, N511, C59R) at dhfr and the A437G mutation at dhps, both associated with resistance to SP, were present in 84% and 79% of pretreatment isolates respectively. These results seriously question the continued use of chloroquine and SP in Harper and highlight the urgency of making alternative antimalarial therapies available. Our study confirms that resistance to chloroquine may be high in Liberia and yields hitherto missing information on SP.

Treatment with the novel antifolate drug combination chlorproguanil-dapsone effectively cleared asymptomatic Plasmodium falciparum infections in 246 (93.5%) of 263 children in the Usambara Mountains of Tanzania during the course of a 2-week follow-up. Samples from 71 recurrent infections, collected over a 9-week follow-up, showed selection for parasites with the triple mutant Ile(51)-Arg(59)-Asn(1 8) in dihydrofolate reductase. There was no selection for mutations in dihydropteroate synthetase, the target enzyme of dapsone. Search for complete identity in the highly polymorphic genes coding for merozoite surface proteins 1 and 2 in parasite samples collected before and after treatment indicated that the majority of recurrent parasitemias were new infections. These observations on selection in Tanzania and the lack of selection reported from a less endemic area suggest that the active metabolite of chlorproguanil, which has a short half-life in the blood, may persist in the liver, where it exerts selective pressure on growing preerythrocytic stages.

OBJECTIVES: To assess the relationship between the presence of DHFR and DHPS mutations in Plasmodium falciparum, parasite in vitro resistance, and in vivo efficacy of sulfadoxine-pyrimethamine (SP) treatment. PATIENTS AND METHODS: Measurement of SP treatment efficacy in malaria-infected children in Gabon was combined with in vitro tests of susceptibility to pyrimethamine and cycloguanil, and molecular genotyping at several DHFR and DHPS loci of parasites isolated before treatment. DHFR was studied at codons 1 8, 51, and 59, whereas DHPS gene was typed at positions 436, 437, 54 and 581. RESULTS: SP treatment was effective in 86% of children by day 28. Seventy-five percent of isolates were in vitro resistant to pyrimethamine and 65.5% to cycloguanil. No mutation was detected at codons 54 and 581 of the DHPS gene. Most isolates (71.8%) presented with the triple mutant DHFR genotype, whereas 64.3% combined at least three DHFR and one DHPS mutations. The increase in the number of DHFR mutations was associated with an increase in in vitro resistance to pyrimethamine and cycloguanil; three DHFR mutations conferred pyrimethamine and to a lesser extent cycloguanil resistance. Treatment failures only occurred with isolates presenting at least two DHFR mutations (S1 8N and C59R) and one DHPS mutation (S436A or A437G), but SP treatment of infections with such parasites gave treatment success in 82. % of children. CONCLUSIONS: DHFR mutations that lead to high-level in vitro resistance to pyrimethamine plus 1-2 DHPS mutations are not sufficient to induce in vivo failure of SP treatment in young children from Gabon

Malawi changed its national policy for malaria treatment in 1993, becoming the first country in Africa to replace chloroquine by sulfadoxine and pyrimethamine combination (SP) as the first-line drug for uncomplicated malaria. Seven years after this change, we investigated the prevalence of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) mutations, known to be associated with decreased sensitivity to SP, in 173 asymptomatic Plasmodium falciparum infections from Salima, Malawi. A high prevalence rate (78%) of parasites with triple Asn-1 8/Ile-51/Arg-59 dhfr and double Gly-437/Glu-54 dhps mutations was found. This 'quintuple mutant' is considered as a molecular marker for clinical failure of SP treatment of P. falciparum malaria. A total of 11 different dhfr and dhps combinations were detected, 3 of which were not previously reported. Nineteen isolates contained the single Glu-54 mutant dhps, while no isolate contained the single Gly-437 mutant dhps, an unexpected finding since Gly-437 are mostly assumed to be one of the first mutations commonly selected under sulfadoxine pressure. Two isolates contained the dhps single or double mutant coupled with dhfr wild-type. The high prevalence rates of the three dhfr mutations in our study were consistent with a previous survey in 1995 in Karonga, Malawi, whereas the prevalences of dhps mutations had increased, most probably as a result of the wide use of SP. A total of 52 P. falciparum isolates were also investigated for pyrimethamine and sulfadoxine/pyrimethamine activity against parasite growth according to WHO in vitro standard protocol. A pyrimethamine resistant profile was found. When pyrimethamine was combined with sulfadoxine, the mean EC(5 ) value decreased to less than one tenth of the pyrimethamine alone level. This synergistic activity may be explained by sulfadoxine inhibition of dhps despite the double mutations in the dhps genes, which would interact with pyrimethamine acting to block the remaining folate despite dhfr mutations in the low p-aminobenzoic acid and low folic acid medium mixed with blood.

Plasmodium falciparum resistance to sulfadoxine/pyrimethamine (S/P) is due to mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhfr) genes. Large-scale screening of the prevalence of these mutations could facilitate the surveillance of the level of S/P resistance in vivo. The prevalence of mutations in dhfr and dhps in relation to S/P efficacy was studied in four sites of differing endemicity in Sudan, Mozambique, and Tanzania. The sites were organized in order of increasing resistance and a significant increase in the prevalence of triple mutations in codons c51, c59, and c1 8 of dhfr was observed. A similar trend was observed when dhfr genotypes were combined with c437 of dhps. Since the differences in S/P resistance between the sites were minor, but nevertheless revealed major differences in dhfr genotype prevalence, the role of dhfr as a general molecular marker seems debatable. The differences may reflect variation in the duration and magnitude of S/P usage (or other antifolate drugs) between the sites. Thus, triple dhfr mutations may prove suitable only as a general guideline for detecting emerging S/P resistance in areas where S/P has been introduced recently. However, changes in susceptibility within the same area with moderate levels of resistance may be possible by longitudinal surveillance of a subset of dhfr/dhps mutations that has been associated with S/P resistance in vivo in a defined location.

BACKGROUND: Sulfadoxine-pyrimethamine was first introduced for treatment of malaria in Africa during the early 198 s for cases when chloroquine treatment failed, and has since become the first-line treatment in many countries. Resistance to sulfadoxine-pyrimethamine is now increasing, especially in southeast Africa. METHODS:We characterised genetic change in dhfr and dhps genes in the Plasmodium falciparum population of KwaZulu-Natal, South Africa, during 1995-99, a period of rapid deterioration of the effectiveness of sulfadoxine-pyrimethamine. We assessed the evolutionary origin of the resistance by analysing polymorphic microsatellite repeats in the flanking region of the dhfr and dhps genes, which show whether resistance alleles originated through shared or independent ancestral mutation events. We then assessed the current extent of dispersal of dhfr and dhps resistance alleles by doing the same analysis in P falciparum sampled from communities in the Kilimanjaro region of northern Tanzania in 2 1. FINDINGS: The large genetic change during 1995-99 in KwaZulu-Natal, South Africa, in both the health facility and the wider community surveys, was at the dhps locus, apparently because resistance at dhfr was established before 1995. The allelic determinants of resistance in this province share a common evolutionary origin with those found in Kilimanjaro, Tanzania, even though the two sites are 4 km apart. INTERPRETATION: Three resistant dhfr alleles, and one resistant dhps allele, each derived from independent ancestral lineages, have been driven through through southeast Africa. The movement by the dhfr alleles (pyrimethamine resistance) preceded that of the dhps allele (sulfadoxine resistance). Our findings emphasise that gene flow rather than new mutations has been the most common originator of resistance in African countries.

AIMS To assess resistance to chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) in a Sudanese parasite population. METHODS Recurrent security problems in Akuem, Sudan, prevented us from conducting a classical in vivo treatment efficacy study. Instead we genotyped key mutations in the chloroquine resistance transporter (pfcrt), the multidrug resistance gene (pfmdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps). We genotyped the K76T mutation in pfcrt and the N86Y mutation in (pfmdr) by restriction digestion of fluorescent end-labelled polymerase chain reaction (PCR) products, while we genotyped codons 16, 51, 59, 1 8 and 164 in dhfr and codons 436, 437, 54 , 581 and 613 in dhps by primer extension in 1 blood samples. RESULTS Sixty-three percent of parasites carried the 76T mutation at pfcrt critical for CQ resistance, while 31% carried the 86Y mutation at pfmdr that is associated with, although not essential, for CQ resistance. We found five dhfr alleles: 6 % of infections contained wild-type dhfr alleles, 3% had one mutation, 34% had two mutations, while 3% had three mutations. We found three dhps alleles: 47% were wild type, 44% had one mutation, while 9% had two mutations. CONCLUSIONS We expect high levels of treatment failure (RI-RIII) with CQ (2 -4 %) and predict efficient treatment with SP. However, dhfr alleles with three mutations (51I, 59R, 1 8N) are present as are dhps alleles with two mutations (437G, 54 E). Successful treatment with SP is therefore likely to be short-lived

A total of 7 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), trimethoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated with PYR and TRM drug responses (r = .93 and .85). Isolates with wild-type alleles showed mean half inhibitory concentrations (IC5 +/- SD) of .1 +/- .1 and .15 +/- . 6 microg/1 microl for PYR and TRM. Parasites with mutations at codons 1 8 and 51 alone or combined with codon 59 have IC5 of 11.46 +/- .86 (PYR) and 2.9 +/- .59 microg/1 microl (TRM). For both drugs, the differences in the mean IC5 between wild and mutant parasites were statistically significant (P < . 1). Isolates with mixed wild and mutant alleles showed an intermediate level of susceptibility. Our data show partial cross-resistance between PYR/TRM and SDX/SMX (r = .85 and .65). Correlation was not observed between different dhps genotypes and the in vitro outcome to SDX and SMX (r = .3 and .34). The lack of correlation could be due to folates and para-aminobenzoic acid in the RPMI medium and the serum used to supplement the cultures.

The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2 1 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 high-throughput 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 double-mutant 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 3 and 63%, indicating that treatment failure rates are likely to be high.

Surveillance of molecular markers for key mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) has been proposed as a means of predicting sulfadoxine/ pyrimethamine (SP) treatment outcomes in Africa. This study assessed the association between DHFR and DHPS mutations and standardized clinical outcomes in children treated with SP for uncomplicated malaria in Kampala, Uganda. Two mutations (DHFR Asn-1 8 and Ile-51) were too common to be useful predictors. Three other mutations (DHFR Arg-59, DHPS Gly-437, and DHPS Glu-54 ) were associated with clinical treatment failure after 14 days, although associations were not significant. When follow-up was extended to 28 days and genotyping was used to distinguish recrudescence from new infections, associations were significantly strengthened. The presence of both the DHFR Arg-59 and DHPS Glu-54 mutations had the strongest association with clinical treatment failure (odds ratio = 1 .7, P = . 9). These results support a previously proposed method of predicting clinical outcomes based on the prevalence of these two mutations.

Atovaquone-proguanil has recently been introduced for the treatment and prophylaxis of malaria. However, resistance of Plasmodium falciparum is increasingly reported. We assessed P. falciparum polymorphisms associated with resistance to atovaquone (cytochrome b, cytb) and to cycloguanil, the active compound of proguanil (dihydrofolate reductase, dhfr) in 1 isolates from northern Ghana. None of these exhibited cytb codon 268 mutations. Moreover, no dhfr V16A, S1 8T or I164L mutations urled with cycloguanil resistance were detected. However, dhfr triple mutants (S1 8N-I51L-C59R) conferring resistance to proguanil and sulphadoxine-pyrimethamine were seen in 51% of the isolates. In northern Ghana, P. falciparum cytb codon 268 mutations associated with atovaquone resistance are absent. Although proguanil appears to act synergistically with atovaquone in a way different from its antifolate property, the abundance of dhfr polymorphisms will likely compromise the prevention of dissemination of atovaquone-resistant parasites once emerged.

The antifolate drugs sulphadoxine and pyrimethamine are used for treatment of chloroquine-resistant Plasmodium falciparum in Africa. Resistance to pyrimethamine has been associated with point mutations in the dhfr-gene and resistance to sulphadoxine with mutations in the dhps-gene. There is concern that the use of the antifolates trimethoprim and sulphamethoxazole for treatment of other infectious diseases will result in the selection of malaria parasites with mutations in these genes. In Guinea-Bissau, where sulfonamide and trimethoprim-containing drugs have been used extensively, we decided to assess the prevalence of mutations in the dhfr-and dhps-gene in P. falciparum isolated from children suffering from acute malaria and to assess the resistance patterns to trimethoprim/sulphamethoxazole in Escherichia coli isolated from the same patients. A thick film and a blood sample for polymerase chain reaction (PCR) were obtained from 1 children attending the Bandim Health Centre in Bissau with symptoms compatible with malaria. Furthermore, a stool sample was collected from the same children and cultured for E. coli. Of the cultured E. coli, 67% were resistant both to sulfonamides and trimethoprim, 4% to sulfonamides alone, 3% to trimethoprim alone while 26% were fully sensitive to both drugs. PCR was successfully performed in 97 blood samples. Of these, 41% had triple mutations at the dhfr-gene (at codons 51, 59 and 1 8), and 15% had triple mutations plus mutation at codon 437 in the dhps-gene. Only 45% harboured the wild-type dhfr-gene. Thus both bacterial resistance and mutations in the parasitic genes were common, but not urled in the individual child. As sulphadoxine-pyrimethamine has only been used as a second line treatment for chloroquine resistant malaria in Guinea-Bissau for a few years, it is worrying to find a high prevalence of mutations in the parasitic genes coding for resistance to these drugs. Therefore, restricting the use of sulphadoxine-pyrimethamine for the treatment of chloroquine resistant malaria might not be sufficient to prevent the development of resistance in the parasites as long as antifolate drugs are used extensively.

Prior to the 2 1 malarial treatment policy change in Tanzania, we conducted trials to assess the efficacy of sulfadoxine-pyrimethamine (SP) and the usefulness of molecular markers in monitoring resistance. A total of 383 uncomplicated Plasmodium falciparum malaria patients (between 6 and 59 months old) were treated with SP and their responses were assessed. Mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes in admission day blood samples were analyzed. Results indicated that 85.6% of the patients showed an adequate clinical response, 9.7% an early treatment failure, and 4.7% a late treatment failure. The quintuple mutant genotype (pfdhfr 51 Ile, 59 Arg, and 1 8 Asn and pfdhps 437 Gly and 54 Glu) showed an association with treatment outcome (odds ratio = 2.1; 95% confidence interval = .94-4.48, P = . 45). The prevalence of the triple pfdhfr mutant genotype (51 Ile, 59 Arg, and 1 8 Asn) at a site of high SP resistance (23.6%) was four times higher compared with that observed at sites of moderate SP resistance (6.8-14.4%) (P = . 1). The genotype failure index calculated by using this marker was invariable (1.96-2.1) at sites with moderate SP resistance, but varied (3.4) at a site of high SP resistance. In conclusion, our clinical and molecular findings suggest that SP may have a short useful therapeutic life in Tanzania; thus, its adoption as an interim first-line antimalarial drug. The findings also point to the potential of the triple pfdhfr mutant genotype as an early warning tool for increasing SP resistance. These data form the baseline SP efficacy and molecular markers profile in Tanzania prior to the policy change.

None

Point mutations in the genes for dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) of Plasmodium falciparum isolates are associated with sulfadoxine/pyrimethamine (SP) treatment failure, respectively. This study was conducted to assess the prevalence of SP resistance in P. falciparum isolates collected at the Jimma Health Center in southwestern Ethiopia. In this study, the genetic profile of P. falciparum isolates with respect to DHFR and DHPS genes was assessed in 124 individuals. The prevalence of single, double, and multiple mutations in these genes was calculated. The sequence profile showed that all samples carried a double mutation at the positions 51 and 1 8 (I51N1 8) in the DHFR gene. Sixty-seven (54. 3%) of the isolates had an additional third mutation at position 59, resulting in the triple mutant I51R59N1 8. All isolates carried mutations G437 and E54 in the DHPS gene. Two isolates (1.61%) had additional mutations at codon 581 (A581).

Markers of Plasmodium falciparum resistance to chloroquine (CQ) and pyrimethamine-sulfadoxine (PYR-SDX) are widespread in areas where malaria is endemic. In an area where the use PYR-SDX is negligible, the Ashanti Region of Ghana, West Africa, adult individuals were enrolled in an analysis of CQ- and PYR-SDX-associated molecular resistance markers in 2 1 (n = 177) and 2 3 (n = 18 ). Parasite prevalence, as assessed by PCR assays, were 56.5 and 48.8% in 2 1 and 2 3, respectively. A high frequency of CQ, PYR, and SDX resistance markers was observed, whereby, as a weak trend, the frequency was higher in 2 3. The quintuple combination of three pfdhfr mutations and two pfdhps mutations has previously been recognized to be the most important determinant of PYR-SDX resistance. Approximately 6 % of parasite carriers harbored fourfold mutated parasites, indicative of a considerable risk for a switch to high-level PYR-SDX resistance in an area where the rate of PYR-SDX use is low. Among the factors contributing to the high frequency of PYR-SDX resistance-associated mutations are background use of PYR-SDX, past use of PYR for malaria prophylaxis, cross-resistance of trimethoprim with PYR, and the sufficient biological fitness of resistant parasites in the absence of drug pressure.

Both use of sulphadoxine-pyrimethamine (SP) and SP-resistance of Plasmodium falciparum are increasing in sub-Saharan Africa. Mutations in the P. falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes can predict treatment failure of SP, however, the degree of this relationship varies regionally. In northern Ghana, pre-treatment dhfr/dhps genotypes were examined in 126 children and associations with PCR-corrected SP treatment outcome and gametocyte carriage were analysed. SP treatment failure within 4 weeks of follow-up occurred in 28%. Among all pre-treatment isolates, the dhfr triple mutation (Ile-51 + Arg-59 + Asn-1 8) was detected in 47%. Compared with dhfr wildtype parasites, the presence of the dhfr triple mutation increased the risk of treatment failure tenfold. Likewise, parasite clearance was delayed in the presence of dhfr variants. Dhfr mutants and dhps Gly-437 were selected in treatment failure isolates. Gametocytaemia 1 week following treatment was strongly associated with dhfr mutations. Remarkably, this was also true for the prevalence of gametocytes at recruitment. Dhps alleles did neither influence treatment outcome nor gametocyte carriage. In northern Ghana, the prevalence of the dhfr triple mutation can be used as a tool to screen for and to monitor SP resistance. The lack of association between dhps alleles and SP treatment outcome suggests a minor role of these molecular markers in this region at present.

Sulfadoxine-pyrimethamine (SP) has been widely used in recent years to treat acute uncomplicated Plasmodium falciparum malaria. Risk factors for SP therapeutic failure include young age, subtherapeutic SP concentrations, and resistance-conferring genetic mutations in parasite target enzymes. A substantial proportion of patients are able to clear genetically highly resistant P. falciparum genotypes. To determine whether blood SP concentrations independently affect the patient's ability to clear resistant genotypes, we compared SP pharmacokinetics of cases of adequate clinical and parasitological response (ACPR) with cases of treatment failure (TF). When patients with ACPR and TF were compared, mean values were similar for the day 3 blood pyrimethamine (2 5 ng/ml versus 172 ng/ml; P = .25) and estimated maximum sulfadoxine (79 +/- 6.52 versus 69 +/- 6.27 mug/ml; P = .6 ) concentrations, for sulfadoxine terminal-phase elimination half-lives (7.15 versus 6.41 days; P = .42), and for the extents of sulfadoxine absorption (areas under the concentration-time curve of 932 +/- 1 versus 888 +/- 78.9 mug day ml(-1); P = .72). Among patients infected with the quintuple resistant parasites, day 3 blood pyrimethamine concentrations were higher in those who cleared the infection than in those who did not (3 5 +/- 35.4 versus 228 +/- 21.7 ng/ml; P = . 37). Within this subgroup, this finding remained significant after adjusting for endogenous folate levels, age, site, and resistance-conferring mutations (odds ratio: 1. 11 [1. 3 to 1. 24]; P = . 18). However, as a subgroup analysis, our biologically plausible observation that higher blood pyrimethamine concentrations enhance the ability of patients to clear resistant P. falciparum should be interpreted with caution and needs further validation.

BACKGROUND: Trimethoprim-sulfamethoxazole (TS) prophylaxis is recommended for persons living with human immunodeficiency virus infection and acquired immunodeficiency syndrome in Africa. TS and the antimalarial combination sulfadoxine-pyrimethamine (SP) share mechanisms of action and resistance patterns, and concerns about the impact of TS resistance on SP efficacy have contributed to reluctance to implement TS prophylaxis in Africa. METHODS: To determine whether TS prophylaxis impairs SP efficacy for treatment of uncomplicated falciparum malaria, we conducted a randomized, controlled, open-label study of TS prophylaxis. Two hundred and forty children 5-15 years old were randomized in a 2 : 1 fashion to receive either thrice-weekly TS for 12 weeks or no prophylaxis and were treated with SP for subsequent episodes of malaria. The incidence of malaria, SP efficacy, and the prevalence of parasite mutations that confer antifolate drug resistance were measured. RESULTS. TS prophylaxis had a 99.5% protective efficacy against episodes of clinical malaria, with 97% efficacy against infection. Four SP treatment failures occurred in the control group, and none occurred in the TS group. No evidence was seen for selection by TS of antifolate resistance-conferring mutations in parasite dihydrofolate reductase or dihydropteroate synthase during subclinical infections. CONCLUSIONS. In this setting of low antifolate resistance, TS was highly effective in preventing falciparum malaria infection and disease and did not appear to select for SP-resistant parasites.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 1 9 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-1 8/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-54 ) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Senegal recently (2 4) switched to sulfadoxine-pyrimethamine (SP) with amodiaquine as first line therapy for malaria in response to increasing chloroquine resistance. In anticipation of emerging resistance to SP as a result of this change in drug pressure, we set out to define the baseline prevalence of SP-associated mutations in the dhfr and dhps genes in Plasmodium falciparum using geographically diverse and longitudinally collected samples. A total of 153 blood samples were analysed from patients (5 years or older) with mild malaria after informed consent was obtained. Longitudinal samples were collected between 2 and 2 3 in Pikine, a suburb of Dakar. Geographically diverse site sampling was carried out in 2 3. The mutation prevalence in DHFR codons 51, 59 and 1 8 is 65%, 61% and 78% in Pikine, 2 3. The overall prevalence of the triple mutation that is associated with high-level pyrimethamine resistance is 61%. The mutation prevalence rate in DHPS codons 436 and 437 is 21% and 4 %, respectively. There is significant geographic variation in genotypic resistance, as samples from Pikine in 2 3 had higher mutation prevalence in the pfdhfr and pfdhps genes compared to samples from Tambacounda (P < . 15). In summary, this study demonstrates a high background prevalence of SP resistance mutations already present in P. falciparum in Senegal.

Both northern and southern Sudan are deploying artemisinin-based combinations against uncomplicated Plasmodium falciparum malaria (artesunate+sulfadoxine-pyrimethamine [AS+SP] in the north, artesunate+amodiaquine [AS+AQ] in the south). In 2 3, we tested the efficacy of 3 day AS+SP and AS+AQ regimens in vivo in the isolated, seasonally endemic Nuba Mountains region (the first study of AS combinations in southern Sudan). We also analysed pre-treatment blood samples for mutations at the P. falciparum chloroquine transporter (Pfcrt) gene (associated with CQ resistance), and at the dihydrofolate reductase (Dhfr) gene (associated with pyrimethamine resistance). Among 161 randomized children under 5 years, PCR-corrected cure rates after 28 days were 91.2% (52/57, 95% CI 8 .7-97.1) for AS+SP and 92.7% (51/55, 95% CI 82.4-98. ) for AS+AQ, with equally rapid parasite and fever clearance. The Pfcrt K76T mutation occurred in 9 . % (144/16 ) of infections, suggesting CQ would work poorly in this region. Overall, 82.5% (132/16 ) carried mutations at Dhfr (N51I, C59R or S1 8N, but not I164L), but triple mutants (more predictive of in vivo SP failure) were rare (3.1%). CQ use should be rapidly discontinued in this region. SP resistance may propagate rapidly, and AS+AQ is likely to be a better long-term option, provided AQ use is limited to the combination.

We assessed the efficacy of trimethoprim/sulfamethoxazole (TRM/SMX) in vivo in relation to the frequency of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) alleles in 45 Sudanese malaria patients. Plasma levels of TRM, SMX, and acetylsulfamethoxazole (AcSMX) were measured before treatment and at days 3, 7, and 14 or upon recrudescence to ascertain drug absorption. Forty patients (89%) had an adequate clinical response, one patient (2%) had an early treatment failure response, while four patients (8%) showed late treatment failure responses. Genotyping of merozoite surface protein 1, MSP-1, MSP-2, and glutamate-rich protein before treatment and upon recrudescence showed that all recurring parasites were recrudescences. The plasma levels of TRM, AcSMX, and SMX indicated adequate drug absorption in all patients. This suggests parasite resistance as a cause of treatment failure. The presence of dhfr Ile 51 and Asn 1 8 alone or coupled with dhps Ala-436 among parasites that were cleared after treatment indicates that these alleles alone are insufficient to cause in vivo resistance. However, the presence of the triple mutant dhfr (Ile-51/Arg-59/Asn-1 8) with the dhps Gly-437 genotype in all recurring infections, suggests the importance of codon 59 and 437 alleles in susceptibility to TRM/SMX. However, the number is too little to make firm conclusions

In November of 2 , Uganda changed its anti-malarial policy to replace chloroquine (CQ) with a combination of CQ and sulphadoxine-pyrimethamine (SP) as the first line agents. Information was limited on the efficacy of either drug. The present study was designed to provide baseline information on the efficacy of SP and the prevalence of molecular markers that are associated with SP resistance. Blood samples were collected on filter paper from 169 consenting patients who were diagnosed with malaria. Patients were treated with SP and followed for 14 days using the WHO clinical guidelines. The samples were analysed for molecular resistance markers and correlation of the molecular markers with clinical findings was assessed. SP monotherapy was efficacious for 14 of 163 (85.9%) treated patients. We found a high level of mutations in alleles which have previously been reported to be associated with SP resistance, but there was no correlation between clinical outcomes and molecular markers. With the exception of codon S1 8 in dhfr (dhfr S1 8N was at 94.9%), frequencies of dihydropteroate synthase (dhps) mutant and mixed alleles combined (A437G 89% and K54 E 83.9%) were higher than those of dihydrofolate reductase (dhfr) (N51I 58.4%, C59R 31.3%).

Sulfadoxine-pyrimethamine (SP) is still a useful drug to combat chloroquine-resistant Plasmodium falciparum malaria in Cameroon. Because of several disadvantages of the in vivo test and in vitro drug sensitivity assays, molecular assays are an alternative laboratory tool to monitor the evolution of antifolate resistance, especially over the entire country that is characterized by several epidemiologic strata and malaria transmission patterns. In this study, 1,43 blood samples from either symptomatic children or asymptomatic carriers were collected from 14 sites throughout the country between 1999 and 2 3 for the analysis of dihydrofolate reductase (dhfr) sequence. Of 1,368 samples (95.7%) that were successfully amplified, 1,18 were analyzed by direct sequencing of the polymerase chain reaction product, and 188 were analyzed by restriction enzymes. The prevalences of the wild-type, single Asn-1 8 mutation, double Arg-59/Asn-1 8 mutations, double Ile-51/Asn-1 8 mutations, triple Ile-51/Arg-59/Asn-1 8 mutations, and mixed alleles were 2 .8%, 2.8%, 5.7%, .8%, 62.2%, and 7.6%, respectively. The proportions of triple dhfr mutations were > 6 % at all study sites, with the exception of the eastern province (42% triple mutants in Bertoua in 1999) and the northern provinces (11-35% triple mutants in Ngaoundere, Garoua, and Maroua). In these two provinces, the proportion of mutant parasites increased significantly (P < . 5) over the period of 2-4 years. Furthermore, there was a higher proportion (P < . 5) of wild-type parasites in the northern provinces, compared with the rest of the country. The geographic mapping of molecular markers offers a novel tool for monitoring the epidemiology of drug-resistant malaria.

We determined the baseline frequency distribution of mutant alleles of genes associated with resistance to chloroquine and sulfadoxine-pyrimethamine in Plasmodium falciparum isolates in Bangui, Central African Republic. Mutant alleles of the P. falciparum chloroquine resistance transporter (pfcrt) gene were found in all samples and the frequency of the deduced CIET pfcrt haplotype was high (45%). The most common allele of the P. falciparum multidrug resistance 1 (pfmdr1) gene among the field isolates of P. falciparum was 86Y (21.9%). The 1246Y allele was also common (18. %). Of the 167 P. falciparum isolates in which the dihydrofolate reductase gene was studied, only 11 carried the wild-type allele (6.6%) whereas many (5 .3%) were quadruple mutants (5 R, 51I, 59R, 1 8N). The frequency of the 436A mutant allele of the dihydropteroate synthase gene was high (74.3%), but the frequencies of the 437G (18.6%) and 54 E (5.2%) mutant alleles were low. Molecular analyses of antimalarial drug-resistant alleles of P. falciparum isolates in Bangui strongly suggest the widespread distribution of chloroquine and pyrimethamine resistance and to a lesser extent sulfadoxine resistance.

Sulfadoxine-pyrimethamine (SP) is the first line antimalarial treatment in the Democratic Republic of Congo. Using polymerase chain reaction, we assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 1 8, 51, 59) and dihydropteroate synthase (dhps) (codons 437, 54 ) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulfadoxine, respectively. Four hundred seventy-four patients were sampled in Kilwa (N = 138), Kisangani (N = 112), Boende (N = 1 6), and Basankusu (N = 118). The proportion of triple mutations dhfr varied between sites but was always > 5 %. The proportion of dhps double mutations was < 2 %, with some sites as low as .9%. A quintuple mutation was present in 12.8% (16/125) samples in Kilwa; 11.9% (13/1 9) in Kisangani, 2.9% (3/1 2) in Boende, and .9% (1/112) in Basankusu. These results suggest high resistance to pyrimethamine alone or combined with sulfadoxine. Adding artesunate to SP does not seem a valid alternative to the current monotherapy.

Sulfadoxine-pyrimethamine (SP) is the first line antimalarial treatment in the Democratic Republic of Congo. Using polymerase chain reaction, we assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 1 8, 51, 59) and dihydropteroate synthase (dhps) (codons 437, 54 ) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulfadoxine, respectively. Four hundred seventy-four patients were sampled in Kilwa (N = 138), Kisangani (N = 112), Boende (N = 1 6), and Basankusu (N = 118). The proportion of triple mutations dhfr varied between sites but was always > 5 %. The proportion of dhps double mutations was < 2 %, with some sites as low as .9%. A quintuple mutation was present in 12.8% (16/125) samples in Kilwa; 11.9% (13/1 9) in Kisangani, 2.9% (3/1 2) in Boende, and .9% (1/112) in Basankusu. These results suggest high resistance to pyrimethamine alone or combined with sulfadoxine. Adding artesunate to SP does not seem a valid alternative to the current monotherapy.

BACKGROUND: In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) is frequent and intense in some areas. METHODS: In 1 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. RESULTS: P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. CONCLUSION: These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region.

OBJECTIVES: In the Gambia, the combination of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) has replaced CQ monotherapy for treatment of malaria caused by Plasmodium falciparum. We measured the efficacy of the combination CQ/SP, and the prevalence of parasites carrying alleles associated with resistance to CQ or SP. DESIGN: We conducted a single-blind, randomised, controlled trial to compare the efficacy of CQ/SP to that of SP or CQ alone. SETTING: The study took place in the town of Farafenni and surrounding villages in the Gambia. PARTICIPANTS: Participants were children aged 12 mo to 1 y presenting as outpatients with uncomplicated P. falciparum malaria. INTERVENTIONS: 5 children were randomised to receive CQ, SP, or CQ/SP as supervised treatment and actively followed over 28 d. OUTCOME MEASURES: Primary outcome was parasitaemia at any time during follow-up. Secondary outcomes were PCR-confirmed recrudescent infections among treatment failures, and clinical failure requiring rescue medication by day 28. Pretreatment parasite isolates from 161 patients were tested for the presence of resistance-associated genetic markers. RESULTS: The prevalence of parasitological failure by day 28 for the CQ group was 6 .3%, compared to 17.6% for SP (odds ratio [OR], .1 6; 95% confidence interval [CI], . 57- .194; p < . 1) and 13.9% for CQ/SP (OR versus CQ, .14 ; 95% CI, . 78- .25 ; p < . 1). There was no difference between the SP and CQ/SP groups (OR, 1.324; 95% CI, .7 5-2.5 ). The projected prevalence of PCR-corrected treatment failure was 3 .2, 6. 6, and 3.94% in the CQ, SP, and CQ/SP groups, respectively. The pfdhfr-triple mutant and pfdhps-437G mutation were common, with prevalences of 67.4 and 51.2%, respectively. Pretreatment carriage of pfdhps-437G and of multidrug-resistant parasite genotypes was associated with treatment failure in the SP group, but not in the CQ or CQ/SP groups. CONCLUSIONS: The combination of CQ/SP was an efficacious treatment for uncomplicated malaria in Gambian children in this study, but the frequent occurrence of multidrug-resistant parasites suggests that this observed efficacy is not sustainable.

The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2 2 malaria transmission season. Sixty-two percent of the isolates collected were from patients with severe malaria and 38% were from patients with mild malaria. In vitro activities of chloroquine, quinine, cycloguanil, atovaquone, mefloquine, halofantrine, and artesunate were evaluated. The prevalence of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and dihyropteroate synthetase (dhps) genes and the P. falciparum chloroquine resistance transporter (Pfcrt) gene associated with cycloguanil, pyrimethamine, sulfadoxine, and chloroquine resistance were estimated. The genetic polymorphism of the parasite populations was evaluated by analysis of the highly polymorphic regions of merozoite surface protein 1 (msp1) block 2 and msp2. Seventy percent of the isolates were assessed by an in vitro assay. Fifty-two percent of the isolates were chloroquine resistant, 45% were cycloguanil resistant, and 24% were atovaquone resistant. Four percent had low susceptibility to quinine. The Pfcrt and dhfr mutations were associated with in vitro chloroquine- and antimetabolic drug-resistant isolates, respectively. Approximately 7 % of the isolates contained two or more clones. Genetic diversity of P. falciparum was high. The prevalence of allelic family K1 of msp1 was 68%. Isolates of P. falciparum were highly resistant to chloroquine, cycloguanil and atovaquone. The transmission rate of malaria in Dakar is low but a high degree of genetic polymorphism can increase severe malaria, as shown by persons coming to Dakar from areas highly endemic for malaria. Areas with urban malaria should use vector control measures and efficient chemoprophylaxis for non-immune populations.

Sulfadoxine-pyrimethamine (SP), the current first line antimalarial drug in Tanzania, is compromised by evolution and spread of mutations in the parasite's dhfr and dhps genes. In the present study we established the baseline frequencies of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) mutant genotypes and their potential for predicting the in vivo efficacy of SP in Mlandizi, Tanzania. The efficacy of SP treatment was by following 116 children with uncomplicated falciparum malaria for 14 days after treatment. Infected blood samples were collected on filter paper at days , 3, 7 and 14. Parasite genomic DNAwas extracted and point mutations at positions 51, 59, 1 8 and 164 of the dhfr gene and at 581, 54 and 437 of the dhps gene were analysed by nested Polymerase Chain Reaction/ Restriction Fragment Length Polymorphism. Out of 116 children enrolled, 98 (86%) of eligible children demonstrated an adequate clinical response by day 14. There were 7.3 % early and 6.7% late therapeutic failures. At day , only 8. % (4/5 ) the parasites showed no mutation at the dhfr locus; for dhps this was 73%. Triple mutant dhfr alleles (Ile 51, Arg 59, Asn 1 8) occurred in 47%, double mutant dhps (Gly 437, Glu 54 ) alleles in 7.9%. No mutation was detected at codon 164 of the dhfrgene. The presence of triple dhfrmutant alleles was related to clinical failure, but did not show significant association (Fisher exact test, P= .166, OR 2.15 .776.2 ). The higher rates of mutation on the dhfr do not spell a bright future for SP treatment in Tanzania. It is rational to think of an alternative first line antimalarial drug, while retaining SP for malaria intermittent treatment in pregnancy.

OBJECTIVE: To compare the efficacy of atovaquone-proguanil (AP) and sulphadoxine-pyrimethamine (SP) in the treatment of malarial anaemia in Zambian children. METHODS: An individually randomised, double-blind, controlled trial was undertaken in Zambian children with moderately severe anaemia and Plasmodium falciparum parasitaemia. The main trial endpoint was treatment failure defined as a need for blood transfusion or treatment with quinine, persistent anaemia or death within 14 days from the start of treatment. Secondary endpoints were parasitological and haematological findings 14 or 28 days after the start of treatment. RESULTS: A total of 128 children with a packed cell volume of <21% and >9% and P. falciparum parasitaemia received treatment with AP and 127 treatment with SP. Treatment failure occurred in 28 children (22%) who received SP and in 1 (8%) who received AP (OR: 3.34, 95% CI: 1.54, 7.21). Ten children required blood transfusion, all of whom were in the SP treatment group. Six children died, five of whom were in the AP group; none of the deaths were considered to be related directly to treatment. CONCLUSIONS: Atovaquone-proguanil proved more effective than SP in the treatment of malarial anaemia in an area with a modest level of SP resistance. AP is no longer available through the Malarone Donation Programme and is too expensive for routine use in Africa. However, this study has shown that in an area with a modest level of resistance to SP, use of a more effective antimalaria reduces the need for blood transfusion in children with malarial anaemia.

The impact of vector control measures on the evolution of antimalarial drug resistance is an important issue for malaria control programs. We investigated whether the in vivo efficacy of chloroquine (CQ) in children aged 6-59 months with uncomplicated malaria differed in 9 villages that had benefited from long-term use of insecticide-treated curtains (ITCs) and in 9 nearby non-ITC villages. We also compared the prevalence of genetic markers of resistance to CQ and sulfadoxine-pyrimethamine (SP) between the two groups of villages. The study enrolled 1, 35 children with uncomplicated malaria and 231 infected but asymptomatic children. After taking account of re-infections, the proportions of children who experienced clinical failure after treatment with CQ were 14% and 19% in ITC and non-ITC villages, respectively (OR = .68; 95% CI: .39, 1.18). Parasitologic failure was observed in 49% of children in ITC villages and 58% of children in non-ITC villages (OR = .71 95%CI: .44, 1.13). The proportion of symptomatic children who harbored parasites carrying the pfcrt-76T allele was 43% in ITC villages and 4 % in non-ITC villages (OR = 1. 9; 95%CI: .8 , 1.5 ). The pfmdr1-86Y allele was detected in 31% and 29% of children in the two groups of villages (OR = 1.14; 95%CI: .75, 1.72). Triple mutations in the dhfr gene were observed in 12% of children in both groups. No double mutations in the dhps gene were observed. Similar results were observed in asymptomatic children. In this setting, ITC use was not associated with increased circulation of parasites resistant to standard antimalarial drugs, or with a greater risk of treatment failure among children less than 5 years of age.

Sulfadoxine-pyrimethamine efficacy was determined with a 28-day follow-up in 97 children between 6 months and 15 years of age. The polymerase chain reaction (PCR)-corrected treatment failure was 8.2% and the uncorrected was 21.6%. The presence of the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) mutations urled to sulfadoxine-pyrimethamine resistance before and after treatment was determined by PCR-restriction fragment length polymorphism (RFLP) and by a fluorogenic PCR assay. Before treatment, the prevalence of the triple DHFR mutations was higher among the patients having had a recurrent parasitemia (either recrudescence or new infection; 28.6% versus 9.3%), although the difference was not significant (P = .1). The double mutation Ala-436/Gly-437 was observed in 67% of samples, whereas no Glu-54 mutation was found. After treatment, the triple DHFR mutation was found in 76.2% of patients with recurrent parasitemia, recrudescence, and new infection alike. Such high prevalence of mutant parasites indicates that sulfadoxine-pyrimethamine should not be used as monotherapy.

Over a 2-year study period, three methods [a test of therapeutic efficacy, an in-vitro assay, and sequencing of the parasites' dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes] were used to monitor sulfadoxine-pyrimethamine (SP) resistance in the Plasmodium falciparum strains infecting young children near Abidjan, the largest city in Cote d'Ivoire. Overall, 118 children aged<5 years and infected with P. falciparum were treated with SP. Twenty-one (23.5%) of the 89 children who completed the 14 days of follow-up were categorized as therapeutic failures. When P. falciparum isolates from the 61 children with adequate parasitaemias were investigated in the in-vitro assay, 24 (39.5%) were found to be highly resistant to pyrimethamine, each having a median inhibitory concentration (IC5 ) of at least 2 nM. Polymorphism analysis of gene fragments of 118 P. falciparum isolates (one from each child enrolled in the study) revealed that 46 (39%) of the isolates had mutant dhfr and 111 (94%) had mutant dhps. The mutations mainly affected DHFR codon 1 8 (39% of the isolates) and DHPS codons 436 (65%), 437 (52%) and 613 (27%). Of the 37 DHFR mutant isolates from children who completed follow-up, 21 were from children with therapeutic failure, indicating that mutant DHFR was associated with resistance to pyrimethamine in vivo (kappa= .61). A mutant dhfr genotype was also found to be strongly associated with resistance to pyrimethamine in vitro (kappa= .79). There was, however, little evidence of an association between SP efficacy and dhps genotype (kappa= . 4). Resistance to SP appears to be an increasing problem in southern Cote d'Ivoire and one which may now justify a change away from this drug combination as the first- or second-line treatment for P. falciparum malaria in this area.

Placental sequestration of Plasmodium falciparum in pregnancy may impair the usefulness of molecular markers of sulfadoxine-pyrimethamine resistance. In 3 infected, delivering women, the concordance of PCR-restriction fragment length polymorphism-derived parasite resistance alleles in matched samples from placenta and circulation was 83 to 98%. Sulfadoxine-pyrimethamine resistance typing in peripheral blood is reasonably representative of P. falciparum infecting pregnant women.

BACKGROUND: In the last five years, countries have been faced with changing their malaria treatment policy to an artemisinin-based combination therapy (ACT), many with no national data on which to base their decision. This is particularly true for a number of West African countries, including Guinea, where these studies were performed. Two studies were conducted in 2 4/2 5 in programmes supported by Medecins Sans Frontieres, when chloroquine was still national policy, but artesunate (AS)/sulphadoxine-pyrimethamine (SP) had been used in refugee camps for two years. METHODS: In Dabola (central Guinea), 22 children aged 6-59 months with falciparum malaria were randomized to receive either AS/amodiaquine (AQ) or AS/SP. In vivo efficacy was assessed following the 2 3 World Health Organization guidelines. In a refugee camp in Laine (south of Guinea), where an in vivo study was not feasible due to the unstable context, a molecular genotyping study in 16 patients assessed the prevalence of mutations in the dihydrofolate reductase (dhfr) (codons 1 8, 51, 59) and dihydropteroate synthase (dhps) (codons 436, 437, 54 ) genes of Plasmodium falciparum, which have been associated with resistance to pyrimethamine and sulphadoxine, respectively. RESULTS: In Dabola, after 28 days of follow-up, Polymerase Chain Reaction (PCR)-adjusted failure rates were 1. % (95%CI -5.3) for AS/AQ and 1. % (95%CI -5.5) for AS/SP. In the refugee camp in Laine, the molecular genotyping study found three dhfr mutations in 85.6% (95%CI 79.2-9 .7) patients and quintuple dhfr/dhps mutations in 9.6% (95%CI 5.2-15.9). CONCLUSION: Both AS/AQ and AS/SP are highly efficacious in Dabola, whereas there is molecular evidence of established SP resistance in Laine. This supports the choice of the national programme of Guinea to adopt AS/AQ as first line antimalarial treatment. The results highlight the difficulties faced by control programmes, which have gone through the upheaval of implementing ACTs, but cannot predict how long their therapeutic life will be, especially in countries which have chosen drugs also available as monotherapies.

The prevalence of point mutations associated with resistance to sulfadoxine and pyrimethamine was determined by sequencing the fragments of genes encoding dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), respectively, in 59 isolates collected during 2 2-2 3 in São Tomé and Príncipe to develop an early warning system of drug-resistant P. falciparum. Almost all isolates (57 of 59, 97%) were dhfr mutant. The majority of the isolates (43 of 59, 73%; 16 with mixed alleles) carried the triple dhfr mutations (Ile-51/Arg-59/Asn-1 8). The presence of dhps mutations were only limited to positions 436 and 437. The pure mutant codon Gly-437 was present in 39 out of 59 isolates (66%), in association with Ser-436 (n=34) or Ala-436 (n=5). Mixed isolates displayed different combinations of 436 (Ser, Ala ou Phe) and 437 (Gly, with or without Ala) alleles. None of the isolates carried mutant 54 , 581 or 613 codons. A total of 39 isolates (66%) were characterized as quadruple mutants (i.e. triple dhfr mutations + Gly-437 dhps mutant allele). Monitoring the possible emergence of isolates carrying multiple dhfr and dhps mutations, in particular codon Glu-54 , may be used as an early warning system which indicates a decrease in sulfadoxine-pyrimethamine efficacy.

We assessed the presence of point mutations associated with resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in 178 Plasmodiumfalciparum infections from three geographically distinct sites in Malawi. We confirm that CQ-resistance mutations are now rare in Malawi, being detectable at very low frequencies (2-4%) in infections from two of the three study sites. We also show that over 9 % of infections from each of the three study sites carry a set of three dihydrofolate reductase (dhfr) and two dihydropteroate synthase (dhps) mutations strongly associated with SP treatment failure. In this short communication, we present these molecular data and discuss their implications for Malawi's first-line antimalarial treatment policy.

BACKGROUND: Plasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate. METHODS: A total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa. RESULTS: The frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/1 8N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/1 8N + 437G/54 E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected. CONCLUSION: The observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

The frequency and distribution of mutations in Plasmodium falciparum, dihydrofolate reductase and dihydropteroate synthase genes were analyzed, using the polymerase chain reaction and restriction fragment length polymorphism methodology, in infected blood samples from Mozambican children living in Maputo, before and seven days after treatment with sulfadoxine/pyrimethamine (S/P). The results showed the occurrence of point mutations in the genes studied and the presence of combinations of three alleles in dhfr (51Ile, 59Arg and 1 8Asn) and "quintuple" mutant (dhfr 51Ile, 59Arg, 1 8Asn and dhps 437Gly, 54 Glu). Both of these situations were associated with seven-day therapeutic failure, following treatment with S/P. These findings show the importance of studying S/P resistance in Mozambique, and how molecular markers for antimalarial resistance can provide important data for national malaria control policy.

OBJECTIVE: To test the efficacy of sulfadoxine-pyremethamine (SP) monotherapy and establish the prevalence of mutations in dhfr and dhps in Brazzaville, Congo. METHOD: We recruited 97 patients aged 6-59 months with uncomplicated malaria who attended Tenrikyo public health centre. Eighty-three were followed until day 28. SP efficacy was determined by the WHO 28-day test and analysis of mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes. RESULTS: There were seven (8.4%) early treatment failures, 23 late treatment failures (27.7%), nine (1 .8%) late parasitological failures and 44 (53%) adequate clinical and parasitological responses (ACPR). After polymerase chain reaction (PCR) analysis of 64 available samples, the corrected results there were 44 (68.8%) ACPR and 19 recrudescent cases (31.2%). Approximately, 97.5% of samples bore the Asn51Ile mutation, 66.2% the Cys59Arg mutation and 98.8% the Ser1 8Asn mutation. Mutations of dhps at positions 437 (Ala-Gly) and 436 (Ser-Ala) were found in 85% and 12.5% of samples. Quadruple mutations (pfdhfr triple mutations in codons 51, 59 and 1 8+ pfdhps mutation in 437) were found in 42 samples (52.5%) and associated with treatment failures. CONCLUSION: This high level of treatment failures and mutations in both genes calls for the urgent application of the new policy for malaria treatment to delay the spread of SP resistance.

Background. Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance. Methodology/Principal Findings. We conducted on a yearly basis a retrospective survey over a ten-year period that included two successive treatment policies, namely quinine during 199 ?1994, and chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) as first and second line treatments, respectively, during 1995?1999. Molecular beacon based genotyping, gene sequencing and microsatellite analysis showed a low prevalence of Pfcrt and Pfdhfr-ts resistance alleles of Southeast Asian origin by the end of 1994 and their effective dissemination within one year of CQ and SP implementation. The Pfcrt resistant allele rose from 9% to 46% prevalence during the first year of CQ reintroduction, i.e., after a mean of 1.66 CQ treatment courses/person/year. The Pfdhfr-ts triple mutant rose from % to 2 % by end 1996, after a mean of .35 SP treatment courses/person in a 16-month period. Both resistance alleles were observed at a younger age than all other alleles. Their spreading was associated with enhanced in vitro resistance and rapidly translated in an increased incidence of clinical malaria episodes during the early post-treatment period. Conclusion/Significance. In such a highly endemic setting, selection of drug-resistant parasites took a single year after drug implementation, resulting in a rapid progression of the incidence of clinical malaria during the early post-treatment period. Controlled antimalarial use at the community level did not prevent dissemination of resistance haplotypes. This data pleads against reintroduction of CQ in places where resistant allele frequency has dropped to a very low level after CQ use has been discontinued, unless drastic measures are put in place to prevent selection and spreading of mutants during the post-treatment period.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are enzymes of central importance in parasite metabolism. The dhfr and dhps gene mutations are known to be associated with sulphadoxine/pyrimethamine (SP) resistance. OBJECTIVE: To investigate the effects of dhfr/dhps mutations on parasite characteristics other than SP resistance. METHOD: Parasite infections obtained from 153 Sudanese patients with uncomplicated falciparum malaria treated with SP or SP + chloroquine, were successfully genotyped at nine codons in the dhfr/dhps genes by PCR-ELISA. RESULTS & CONCLUSION: Mutations were detected in dhfr at N51I, S1 8N and C59R, and in at dhps at A/S436F, A437G, K54 E and A581G, the maximum number of mutations per infection were five. Based on number of mutant codons per infection (multiplicity of mutation, MOM), the infections were organized into six grades: wild-types (grade ; frequency, . 3) and infections with MOM grades of 1 to 5, with the following cumulative frequency; .97, .931, .866, .719, .121, respectively. There was no significant association between the MOM and SP response. Importantly, immunity, using age as a surrogate marker, contributed significantly to the clearance of parasites with multiple dhfr/dhps mutations. However, these mutations have a survival advantage as they were associated with increased gametocytogenesis. The above implications of dhfr/dhps mutations were associated with MOM 2 to 5, regardless of the gene/codon locus.

BACKGROUND: In malaria endemic areas children may recover from malaria after chemotherapy in spite of harbouring genotypically drug-resistant Plasmodium falciparum. This phenomenon suggests that there is a synergy between drug treatment and acquired immunity. This hypothesis was examined in an area of moderately intense transmission of P. falciparum in Tanzania during a drug trail with sulphadoxine-pyrimethamine (SP) or amodiaquine (AQ). METHODS: One hundred children with uncomplicated malaria were treated with either SP or AQ and followed for 28 days. Mutations in parasite genes related to SP and AQ-resistance as well as human sickle cell trait and alpha-thalassaemia were determined using PCR and sequence-specific oligonucleotide probes and enzyme-urled immunosorbent assay (SSOP-ELISA), and IgG antibody responses to a panel of P. falciparum antigens were assessed and related to treatment outcome. RESULTS: Parasitological or clinical treatment failure (TF) was observed in 68% and 38% of children receiving SP or AQ, respectively. In those with adequate clinical and parasitological response (ACPR) compared to children with TF, and for both treatment regimens, prevalence and levels of anti-Glutamate-rich Protein (GLURP)-specific IgG antibodies were significantly higher (P < . 1), while prevalence of parasite haplotypes associated with SP and AQ resistance was lower (P = . 2 and P = . 7, respectively). Interestingly, anti-GLURP-IgG antibodies were more strongly associated with treatment outcome than parasite resistant haplotypes, while the IgG responses to none of the other 11 malaria antigens were not significantly associated with ACPR. CONCLUSION: These findings suggest that GLURP-specific IgG antibodies in this setting contribute to clearance of drug-resistant infections and support the hypothesis that acquired immunity enhances the clinical efficacy of drug therapy. The results should be confirmed in larger scale with greater sample size and with variation in transmission intensity.

BACKGROUND: In Tanzania, drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2 1. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes. METHODS: In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps, conferring SP resistance, as well in cytb which is urled to resistance to atovaquone. RESULTS: Pfcrt T76 occurs in 5 % and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected. CONCLUSION: These findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy.

Chloroquine has been the first line drug of treatment for malaria in Zimbabwe until a recent adoption of an interim policy to treat using a combination of chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). We examined the prevalence of parasites with mutations associated with resistance to the drug combination in three areas that have been previously described to differ in malaria endemicity. Our results show that the parasite population from the three areas had a high prevalence of molecular markers of resistance to chloroquine and pyrimethamine. The prevalence of crt (K76T) was 64, 82, and 92% for Chiredzi, Kariba, and Bindura, respectively. On the dhfr locus, the presence of triple mutations (codons 51, 59, and 1 8) was approximately 5 % for all the three locations. On the other hand, the prevalence of dhps mutations (codons 436, 437, and 54 ) was low accounting for less than 2 % in all the areas. Studies reported here demonstrate widespread prevalence of molecular markers associated with chloroquine and pyrimethamine resistance and should be taken into consideration for further refinement of malaria control strategies in Zimbabwe. The design and implementation of successful control strategies would benefit from understanding the prevalence of mutations associated with drug resistance in parasite populations.

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum is encoded by a number of mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes. Here, we have characterized point mutations in dhfr and dhps and microsatellite loci around dhfr on chromosome 4 and dhps on chromosome 8 as well as neutral markers on chromosomes 2 and 3 in 332 samples from Yaoundé, Cameroon. The triple mutant dhfr haplotype that originated in Southeast Asia is the most predominant in this sample set, but we also find additional independent haplotypes at low frequency and an incipient process of genetic differentiation among alleles of Southeast Asian origin. As reported for other African populations, we find evidence of a selective sweep for resistant dhfr mutants in this Cameroonian population due to drug selection. Although we find evidence for a selective sweep in dhps mutants associated with SP resistance, the dynamics of dhps mutants appear different than those observed for dhfr mutants. Overall, our results yield support for the use of microsatellite markers to track resistant parasites; however, the detection of resistant dhfr alleles in low frequency, the evidence of divergence among dhfr alleles that share a common evolutionary origin, and the distinct dynamics of resistant dhps alleles emphasize the importance of comprehensive, population-based investigations to evaluate the effects of drug selection on parasite populations

None

We assessed the effect of daily cotrimoxazole, essential for HIV care, on development of antifolate-resistant Plasmodium falciparum, naso-pharyngeal Streptococcus pneumoniae (pneumococcus), and commensal Escherichia coli. HIV-positive subjects with CD4 cell count < 35 cells/muL (lower-CD4; N = 692) received cotrimoxazole; HIV-positive with CD4 cell count > or = 35 cells/muL (higher-CD4; N = 336) and HIV-negative subjects (N = 132) received multivitamins. Specimens were collected at baseline, 2 weeks, monthly, and at sick visits during 6 months of follow-up to compare changes in resistance, with higher-CD4 as referent. P. falciparum parasitemia incidence density was 16 and 156/1 person-years in lower-CD4 and higher-CD4, respectively (adjusted rate ratio [ARR] = .11; 95% confidence interval [CI] = . 6- .15; P < . 1) and 97/1 person-years in HIV-negative subjects (ARR = .62; 95% CI = .44- .86; P = 5). Incidence density of triple and quintuple dihydrofolate-reductase/dihydropteroate-synthetase mutations was 9 % reduced in lower-CD4 compared with referent. Overall, cotrimoxazole non-susceptibility was high among isolated pneumococcus (92%) and E. coli (76%) and increased significantly in lower-CD4 subjects by Week 2 (P < . 5). Daily cotrimoxazole prevented malaria and reduced incidence of antifolate-resistant P. falciparum but contributed to increased pneumococcus and commensal Escherichia coli resistance.

Resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP) emerged in Plasmodium falciparum from Asia in the 196 s and subsequently spread to Africa. It is not known whether alleles that confer SP resistance also arose independently in Africa. We defined the coding region and microsatellite haplotypes of dhfr alleles in P. falciparum collected in Kilifi, Kenya, during 1987--2 6, which spans the period when SP was first introduced. Isolates that carried a double-mutant or triple-mutant dhfr allele were detected at a low frequency, even during 1987--1988. Each of 2 double mutants carried a unique haplotype, and both were related to wild-type haplotypes from the same population. The number of isolates that carried a triple-mutant dhfr allele increased rapidly after introduction of SP and shared the haplotype of the triple mutant derived form Asia. We observed no triple-mutant alleles with haplotypes related to those of the Africa-derived wild-type and double-mutant alleles.

ABSTRACT: BACKGROUND: Since the late 198 s a series of malaria epidemics has occurred in western Kenya highlands. Among the possible factors that may contribute to the highland malaria epidemics, parasite resistance to antimalarials has not been well investigated. METHODS: Using parasites from highland and lowland areas of western Kenya, we examined key mutations associated with Plasmodium falciparum resistance to sulfadoxine - pyrimethamine and chloroquine, including dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps), chloroquine resistance transporter gene (pfcrt), and multi-drug resistance gene 1 (pfmdr1). RESULTS: We found that >7 % of samples harbored 76T pfcrt mutations and over 8 % of samples harbored quintuple mutations (51I/59R/1 8N pfdhfr and 437G/54 E pfdhps ) in both highland and lowland samples. Further, we did not detect significant difference in the frequencies of these mutations between symptomatic and asymptomatic malaria volunteers, and between highland and lowland samples. CONCLUSIONS: These findings suggest that drug resistance of malaria parasites in the highlands could be contributed by the mutations and their high frequencies as found in the lowland. The results are discussed in terms of the role of drug resistance as a driving force for malaria outbreaks in the highlands.

None

We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine-pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 1 % efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > . 5). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < . 1), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < . 1). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.

The prevalence and frequency of the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) mutations associated with sulfadoxine-pyrimethamine (SP) resistance at 13 sentinel surveillance sites in southern Mozambique were examined regularly between 1999 and 2 4. Frequency of the dhfr triple mutation increased from .26 in 1999 to .96 in 2 3, remaining high in 2 4. The dhps double mutation frequency peaked in 2 1 ( .22) but declined to baseline levels ( . 7) by 2 4. Similarly, parasites with both dhfr triple and dhps double mutations had increased in 2 1 ( .18) but decreased by 2 4 ( . 5). The peaking of SP resistance markers in 2 1 coincided with a SP-resistant malaria epidemic in neighboring KwaZulu-Natal, South Africa. The decline in dhps (but not dhfr) mutations corresponded with replacement of SP with artemether-lumefantrine as malaria treatment policy in KwaZulu-Natal. Our results show that drug pressure can exert its influence at a regional level rather than merely at a national level.

BACKGROUND: Intermittent preventive treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a potential malaria control strategy. There is concern about the impact that increasing in vivo resistance to SP has on the efficacy of IPTi, as well as about the potential contribution of IPTi to increases in resistance. METHODS: We compared the frequency of clinical episodes of malaria caused by P. falciparum parasites with mutations in dhfr and dhps among sick children who received SP or placebo in the context of a randomized, double-blind, placebo-controlled IPTi trial in Mozambique. RESULTS: Half of the children who received placebo harbored quintuple-pure mutant parasites. Nevertheless, the protective efficacy of IPTi within the 35 days after the third dose was 7 .8% (95% confidence interval [CI], 4 .7%-85.6%). Between month 2 after the third IPTi dose and the end of the follow-up period, children receiving SP harbored more dhps codon 437 mixed infections (odds ratio [OR], 1 .56 [95% CI, 1.3 -86.14]) and fewer dhps double-pure mutant parasites (OR, .43 [95% CI, .22- .84]) than did placebo recipients. CONCLUSIONS: IPTi appears to be associated with some changes in the prevalence of genotypes involved in SP resistance. In the face of a high prevalence of quintuple-mutant parasites, SP exhibited a high level of efficacy in the prevention of new episodes of malaria in infants.

BACKGROUND: In late 2 2, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2 4, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the Plasmodium falciparum genes Pfdhfr, Pfdhps and Pfcrt before and a year after the introduction of SP. METHODS: Samples were collected during two cross sectional surveys in early 2 2 and 2 3 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhica district, Southern Mozambique. Out of these, 171 and 173 P. falciparum positive samples were randomly selected to measure the frequency of resistance- related haplotypes in Pfdhfr, Pfdhps and Pfcrt based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA. RESULTS: The frequency of the SP-resistance associated Pfdhps double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < . 1), while the triple mutant Pfdhfr haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = .4 5) after one year with SP as first-line treatment in the study area. Conversely, the combined Pfdhfr/Pfdhps quintuple mutant haplotype increased from 8% to 26% (P = . 5). The frequency of the chloroquine resistance associated Pfcrt-CVIET haplotype was above 9 % in both years. CONCLUSION: These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple Pfdhfr/Pfdhps haplotypes found here as early as 2 2 most likely cause ideal conditions for the development of artesunate tolerance in the P. falciparum populations and may even endanger the sensitivity to the second-line drug, Coartem.

SUMMARY: In the Sahel, most malaria deaths occur among children 1-4 years old during a short transmission season. A trial of seasonal intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine (SP) and a single dose of artesunate (AS) showed an 86% reduction in the incidence of malaria in Senegal but this may not be the optimum regimen. We compared this regimen with three alternatives. METHODS: 21 2 children aged 6-59 months received either one dose of SP plus one dose of AS (SP+1AS) (the previous regimen), one dose of SP plus 3 daily doses of AS (SP+3AS), one dose of SP plus three daily doses of amodiaquine (AQ) (SP+3AQ) or 3 daily doses of AQ and AS (3AQ+3AS). Treatments were given once a month on three occasions during the malaria transmission season. The primary end point was incidence of clinical malaria. Secondary end-points were incidence of adverse events, mean haemoglobin concentration and prevalence of parasites carrying markers of resistance to SP. FINDINGS: The incidence of malaria, and the prevalence of parasitaemia at the end of the transmission season, were lowest in the group that received SP+3AQ: 1 % of children in the group that received SP+1AS had malaria, compared to 9% in the SP+3AS group (hazard ratio HR .9 , 95%CI .6 , 1.36); 11% in the 3AQ+3AS group, HR 1.1 ( .76-1.7); and 5% in the SP+3AQ group, HR .5 ( .3 - .81). Mutations associated with resistance to SP were present in almost all parasites detected at the end of the transmission season, but the prevalence of Plasmodium falciparum was very low in the SP+3AQ group. CONCLUSIONS: Monthly treatment with SP+3AQ is a highly effective regimen for seasonal IPT. Choice of this regimen would minimise the spread of drug resistance and allow artemisinins to be reserved for the treatment of acute clinical malaria.

BACKGROUND: Although malaria treatment aims primarily to eliminate the asexual blood stages that cause illness, reducing the carriage of gametocytes is critical for limiting malaria transmission and the spread of resistance. METHODS: Clinical and parasitological responses to the fixed-dose combination of sulfadoxine and pyrimethamine in patients with uncomplicated falciparum malaria were assessed biannually since implementation of this treatment policy in 1998 in Mpumalanga Province, South Africa. RESULTS: Despite sustained cure rates of > 9 % (P = .14), the duration of gametocyte carriage increased from 3 to 22 weeks (per 1 person-weeks) between 1998 and 2 2 (P < . 1). The dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance were the most important drivers of the increased gametocytemia, although these mutations were not associated with increased pretreatment asexual parasite density or slower asexual parasite clearance times. The geometric mean gametocyte duration and area under the gametocyte density time curve (per 1 person-weeks) were 7. weeks and 6 .8 gametocytes/microL per week, respectively, among patients with wild-type parasites, compared with 45.4 weeks (P = . 16) and 1212 gametocytes/microL per week (P = . 14), respectively, among those with parasites containing 1-5 dhfr/dhps mutations. CONCLUSIONS: An increased duration and density of gametocyte carriage after sulfadoxine-pyrimethamine treatment was an early indicator of drug resistance. This increased gametocytemia among patients who have primary infections with drug-resistant Plasmodium falciparum fuels the spread of resistance even before treatment failure rates increase significantly.

The S1 8N, C59R, and N51I mutations in the Plasmodium falciparum gene that encodes dihydrofolate reductase, dhfr, confer resistance to pyrimethamine and are common in Africa. However, the I164L mutation, which confers high-level resistance, is rarely seen. We found a 14% prevalence of the I164L mutation among a sample of 51 patients with malaria in Kabale District in southwest Uganda in 2 5 and a 4% prevalence among 72 patients with malaria in the neighboring district of Rukungiri during the same year. Surveillance at 6 sites across Uganda during 2 2-2 4 reported a single case of infection involving an I164L mutant, also in the southwest, suggesting that this is a regional hot spot. The spatial clustering and increasing prevalence of the I164L mutation is indicative of local transmission of the mutant. Targeted surveillance is needed to confirm the extent of the spread of the I164L mutation and to monitor the impact of I164L on the efficacy of antifolates for intermittent preventive treatment of pregnant women and/or infants with falciparum malaria.

BACKGROUND: In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha. METHODS: A representative sample of 2,78 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion. RESULTS: Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-1 8 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-54 ) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-1 8 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-1 8, Arg-59 and (or) Ile-51, was also absent. CONCLUSION: This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll.

OBJECTIVES: To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin. METHODS: Seventy-one samples of blood from children admitted to the Pediatric Emergency Unit of Uige Provincial Hospital in 2 4 were screened for resistance mutations at pfcrt, pfmdr1, pfdhfr, pfdhps and pfATPase6. RESULTS: Mutations in pfcrt (codon76), pfmdr1 (codon86), pfdhfr (codons 51, 59, 1 8) and pfdhps (codons 436, 437) were common. Among the 66 isolates for which we were able to determine complete genetic information 13.7% carried all seven of these mutations. Flanking microsatellite analysis revealed the triple mutant pfdhfr was derived from the southeast Asian lineage, while the N51I+S1 8N double mutant pfdhfr alleles are a local origin. pfATPase6 mutations were rare and S769N was not found. CONCLUSION: The parasite population of Uige Angola has high frequency mutations in pfcrt, dhfr and dhps associated with resistance to chloroquine and sulphadoxine pyrimethamine, reflecting past reliance on these two drugs which were the mainstay of treatment until recently. Our findings show that drug resistance in Uige has occurred through a combination of local drug pressure and the regional and international dispersal of resistance mutant alleles.

Sulfadoxine-Pyrimethamine (S-P) still used in some parts of the country was suggested as the second line drug to amodiaquine following widespread failure of chloroquine in Cameroon in 2 2. We investigated the efficacy of S-P and determined the baseline mutations on marker genes for folate metabolism (dhfr and dhps) in the forest and Guinea-Savanna ecozones of Cameroon, as a way of tracking resistance in patients aged between .5 and 1 years in Limbe (n=138), Nkambe (n=1 3), Fontem (n=1 ) and Dschang (n=93 ). Filter paper blood sample were collected prior to treatment and on clinical failure days to determine the prevalence of molecular markers of resistance and to assess the mutation rates on the folate metabolising genes by restriction fragment length polymorphism assays or dot-blot assays with 32-P labeled mutation-specific probes. Sequencing using the dideoxy-chain termination method by PCR was conducted to confirm doubtful cases. Late parasitological failure (LPF) was higher in Limbe (3 .6%) compared to Nkambe 1 .3% (p= . 1). The prevalence of the 437-Gly mutation though lower in Nkambe, 57.6%, than in Limbe, 6 % were statistically not different (p= .2). All genotypes with the 1 8N mutation also carried the 51-Ile and 59-Arg mutations. All sensitive alleles (S1 8) also carried the amino acids, 51-Asn and 59-Cys. S-P is no longer efficacious in Limbe and Nkambe, Cameroon for treating uncomplicated malaria in children below 1 years. Instead 437G rather than the 1 8 N of Plasmodium falciparum may be determinant as the marker for tracking the spread of SP resistance in Cameroon.

he aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (2 in Madagascar and 6 in the Comoros Islands) from 2 6 to 2 8. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.

Background Information on the epidemiology of malaria is essential for designing and interpreting results of clinical trials of drugs, vaccines and other interventions. As a background to the establishment of a site for anti-malarial drugs and vaccine trials, the epidemiology of malaria in a rural site in central Ghana was investigated. Methods Active surveillance of clinical malaria was carried out in a cohort of children below five years of age (n = 335) and the prevalence of malaria was estimated in a cohort of subjects of all ages (n = 1484) over a 12-month period. Participants were sampled from clusters drawn around sixteen index houses randomly selected from a total of about 22, houses within the study area. The child cohort was visited thrice weekly to screen for any illness and a blood slide was taken if a child had a history of fever or a temperature greater than or equal to 37.5 degree Celsius. The all-age cohort was screened for malaria once every eight weeks over a 12-month period. Estimation of Entomological Inoculation Rate (EIR) and characterization of Anopheline malaria vectors in the study area were also carried out. Results The average parasite prevalence in the all age cohort was 58% (95% CI: 56.9, 59.4). In children below five years of age, the average prevalence was 64% (95% CI: 61.9, 66. ). Geometric mean parasite densities decreased significantly with increasing age. More than 5 % of all children less than 1 years of age were anaemic. Children less than 5 years of age had as many as seven malaria attacks per child per year. The attack rates decreased significantly with increasing cut-offs of parasite density. The average Multiplicity of Infection (MOI) was of 6.1. All three pyrimethamine resistance mutant alleles of the Plasmodium falciparum dhfr gene were prevalent in this population and 25% of infections had a fourth mutant of pfdhps-A437G. The main vectors were Anopheles funestus and Anopheles gambiae and the EIR was 269 infective bites per person per year. Conclusion The transmission of malaria in the forest-savanna region of central Ghana is high and perennial and this is an appropriate site for conducting clinical trials of anti-malarial drugs and vaccines.

OBJECTIVES: Resistance to pyrimethamine in Plasmodium falciparum is conferred by mutations in the gene encoding dihydrofolate reductase (DHFR). It is known that DHFR double mutants have evolved independently in multiple geographic areas, whereas the triple mutant prevalent in Africa appears to have originated in south-east Asia. In this study, we investigated whether other triple mutants may have evolved independently in Africa. METHODS: We determined the DHFR genotypes and haplotypes of five microsatellite loci flanking the DHFR locus between 4.49 kb upstream and 1.48 kb downstream of 159 isolates collected from three African countries (Republic of Congo, Ghana and Kenya). RESULTS: The CIRNI type of DHFR triple mutant (with mutations underlined at amino acid positions 51, 59 and 1 8) was predominant in the Republic of Congo (82%) and Ghana (81%) and was the second most prevalent in Kenya (27%), where the CICNI type of DHFR double mutant was dominant. Three distinct microsatellite haplotypes were identified in the DHFR triple mutant. One haplotype was identical to that originating in south-east Asia. The other two haplotypes occurred in Ghana and Kenya, which were unique, previously undescribed and identical to those of the two DHFR double mutants found in the same locations. CONCLUSIONS: This study presents strong evidence for the unique, multiple independent evolution of pyrimethamine resistance in Africa. Indigenous evolution of the triple mutant from the double mutant appears to have occurred in a step-wise manner in Kenya and Ghana or in nearby countries in east and west Africa

BACKGROUND: The spread of resistance to chloroquine (CQ) led to its withdrawal from use in most countries in sub-Saharan Africa in the 199 s. In Malawi, this withdrawal was followed by a rapid reduction in the frequency of resistance to the point where the drug is now considered to be effective once again, just nine years after its withdrawal. In this report, the polymorphisms of markers associated with CQ-resistance against Plasmodium falciparum isolates from coastal Kenya (Kilifi) were investigated, from 1993, prior to the withdrawal of CQ, to 2 6, seven years after its withdrawal. Changes to those that occurred in the dihydrofolate reductase gene (dhfr) that confers resistance to the replacement drug, pyrimethamine/sulphadoxine were also compared. METHODS: Mutations associated with CQ resistance, at codons 76 of pfcrt, at 86 of pfmdr1, and at codons 51, 59 and 164 of dhfr were analysed using PCR-restriction enzyme methods. In total, 4 6, 24 and 323 isolates were genotyped for pfcrt-76, pfmdr1-86 and dhfr, respectively. RESULTS: From 1993 to 2 6, the frequency of the pfcrt-76 mutant significantly decreased from around 95% to 6 %, while the frequency of pfmdr1-86 did not decline, remaining around 75%. Though the frequency of dhfr mutants was already high (around 8 %) at the start of the study, this frequency increased to above 95% during the study period. Mutation at codon 164 of dhfr was analysed in 2 6 samples, and none of them had this mutation. CONCLUSION: In accord with the study in Malawi, a reduction in resistance to CQ following official withdrawal in 1999 was found, but unlike Malawi, the decline of resistance to CQ in Kilifi was much slower. It is estimated that, at current rates of decline, it will take 13 more years for the clinical efficacy of CQ to be restored in Kilifi. In addition, CQ resistance was declining before the drug's official withdrawal, suggesting that, prior to the official ban, the use of CQ had decreased, probably due to its poor clinical effectiveness.

Background: Antifolate drugs have an important role in the treatment of malaria. Polymorphisms in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes cause resistance to the antifol and sulpha drugs, respectively. Rwanda has the highest levels of antimalarial drug resistance in Africa. Methods: We correlated the efficacy of chlorproguanil-dapsone+artesunate (CPG-DDS+A) and amodiaquine+sulphadoxine-pyrimethamine (AQ+SP) in children with uncomplicated P. falciparum malaria with Pfdhfr and Pfdhps mutations, known to confer reduced drug susceptibility, in two areas of Rwanda. Results: In the Eastern Province, where cure rates were low, over 75% of isolates had >/=3 Pfdhfr mutations and 2 or 3 Pfdhps mutations and 11% had the Pfdhfr 164-Leu polymorphism. In the Western province, where cure rates were significantly higher (p< . 1), the prevalence of multiple resistance mutations was lower and the Pfdhfr I164L polymorphism was not found. The risk of treatment failure following AQ+SP more than doubled for each additional Pfdhfr resistance mutation (OR=2.4; 95% CI 1. 1 to 5.55; p= . 48) and each Pfdhps mutation (OR=2.1; 95% CI 1.21 to 3.54; p= . 8). The risk of failure following CPG-DDS+A treatment was 2.2 times higher (95% CI 1.34 to 3.7) for each additional Pfdhfr mutation, whereas there was no association with mutations in the Pfdhps gene (p= .13). Conclusion: The Pfdhfr 164-Leu polymorphism is prevalent in Eastern Rwanda. Antimalarial treatments with currently available antifol-sulpha combinations are no longer effective in Rwanda because of high level resistance.

We conducted a prevalence study of mutations in Plasmodium falciparum that are associated with antimalarial drug resistance at a rural site in Karonga near Malawi's northern border with Tanzania. We found a higher prevalence of the key chloroquine resistance-conferring mutation in the pfcrt gene (K76T) at this site in comparison with the prevalence in Blantyre, a city in the south of Malawi, far from an international border (9%vs. %; P < . 5). In contrast we found a lower prevalence of the quintuple dhfr/dhps mutation, which is highly predictive of SP treatment failure, at the Karonga site compared to Blantyre (76%vs. 88%; P < . 5). The prevalence of the K76T pfcrt mutation at two Tanzanian sites close to the border with Malawi was recently reported to be over 5 %. Our findings suggest a considerable 'leakage' of parasite antimalarial drug resistance across the border between two countries with different national malaria control policies and with different levels of resistance. Neighbouring countries should consider implementing common regional rather than national malaria treatment policies to prevent the spread of antimalarial drug resistance alleles across their borders.

BACKGROUND: To update the National Malaria Control Programme of Mali on the efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria. METHODS: During the malaria transmission seasons of 2 2 and 2 3, 455 children--between six and 59 months of age, with uncomplicated malaria in Kolle, Mali, were randomly assigned to one of three treatment arms. In vivo outcomes were assessed using WHO standard protocols. Genotyping of msp1, msp2 and CA1 polymorphisms were used to distinguish reinfection from recrudescent parasites (molecular correction). RESULTS: Day 28 adequate clinical and parasitological responses (ACPR) were 14.1%, 62.3% and 88.9% in 2 2 and 18.2%, 6 % and 85.2% in 2 3 for chloroquine, amodiaquine and sulphadoxine-pyrimethamine, respectively. After molecular correction, ACPRs (cACPR) were 63.2%, 88.5% and 98. % in 2 2 and 75.5%, 85.2% and 96.6% in 2 3 for CQ, AQ and SP, respectively. Amodiaquine was the most effective on fever. Amodiaquine therapy selected molecular markers for chloroquine resistance, while in the sulphadoxine-pyrimethamine arm the level of dhfr triple mutant and dhfr/dhps quadruple mutant increased from 31.5% and 3.8% in 2 2 to 42.9% and 8.9% in 2 3, respectively. No infection with dhps 54 E was found. CONCLUSION: In this study, treatment with sulphadoxine-pyrimethamine emerged as the most efficacious on uncomplicated falciparum malaria followed by amodiaquine. The study demonstrated that sulphadoxine-pyrimethamine and amodiaquine were appropriate partner drugs that could be associated with artemisinin derivatives in an artemisinin-based combination therapy.

BACKGROUND: Over the last years, significant progress has been made in the comprehension of the molecular mechanism of malaria resistance to drugs. Together with in vivo tests, the molecular monitoring is now part of the survey strategy of the Plasmodium sensitivity. Currently, DNA-microarray analysis allows the simultaneous study of many single nucleotide polymorphisms (SNP) of Plasmodium isolates. In December 2 5, the International Federation of the Red Cross distributed two million three hundred thousand long-lasting insecticide nets to pregnant women and mothers of under five years children in the whole Niger. Then, Niger adopted artemisinin-based combination therapy as first-line treatment. METHODS: Thirty four SNPs of pfcrt, pfdhfr, pfdhps, pfmdr and pfATPase were analysed by DNA-microarray and PCR/RFLP in two villages - Zindarou and Banizoumbou - with different durations of malaria transmission. The main objective of the study was to measure the dynamics of Plasmodium falciparum resistant strains and associated factors. RESULTS: This study shows a global and clear increase of the drug-resistance associated molecular markers frequencies during a relatively short-time period of four years. Markers associated with resistance to chloroquine and sulphonamids were more frequently found in the short transmission zone than in the long transmission one. The pfcrt76T mutation is significantly more present at Banizoumbou than Zindarou (38.3% vs 25.2%, p = . 13). This work allowed the screening of several field strains for five SNPs of PfATPase6 gene. The pfATPase6S769N, candidate mutation of resistance to artemisinin was not found. However the pfATPsaeA623E mutation was found in 4.7% of samples. CONCLUSION: A significant increase of several SNPs frequencies was highlighted over a four-year period. The polymorphism of five PfATPase6 gene SNPs was described. The global, large and fast increase of the molecular resistance is discussed in the context of current changes of health policy and malaria control in Niger.

BACKGROUND: The long terminal half life of piperaquine makes it suitable for intermittent preventive treatment for malaria but no studies of its use for prevention have been done in Africa. We did a cluster randomized trial to determine whether piperaquine in combination with either dihydroartemisin (DHA) or sulfadoxine-pyrimethamine (SP) is as effective, and better tolerated, than SP plus amodiaquine (AQ), when used for intermittent preventive treatment in children delivered by community health workers in a rural area of Senegal. METHODS: Treatments were delivered to children 3-59 months of age in their homes once per month during the transmission season by community health workers. 33 health workers, each covering about 6 children, were randomized to deliver either SP+AQ, DHA+PQ or SP+PQ. Primary endpoints were the incidence of attacks of clinical malaria, and the incidence of adverse events. RESULTS: 1893 children were enrolled. Coverage of monthly rounds and compliance with daily doses was similar in all groups; 9 % of children received at least 2 monthly doses. Piperaquine combinations were better tolerated than SP+AQ with a significantly lower risk of common, mild adverse events. 1 3 episodes of clinical malaria were recorded during the course of the trial. 68 children had malaria with parasitaemia >3 /microL, 29/671 (4.3%) in the SP+AQ group, compared with 22/6 4 (3.6%) in the DHA+PQ group (risk difference .47%, 95%CI -2.3%,+3.3%), and 17/618 (2.8%) in the SP+PQ group (risk difference 1.2%, 95%CI -1.3%,+3.6%). Prevalences of parasitaemia and the proportion of children carrying Pfdhfr and Pfdhps mutations associated with resistance to SP were very low in all groups at the end of the transmission season. CONCLUSIONS: Seasonal IPT with SP+PQ in children is highly effective and well tolerated; the combination of two long-acting drugs is likely to impede the emergence of resistant parasites. TRIAL REGISTRATION: ClinicalTrials.gov NCT 52962 .

ABSTRACT: BACKGROUND: The development of Plasmodium falciparum 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 2 7 Swaziland still relied on CQ as first-line anti-malarial drug. METHODS: Parasite DNA was amplified from P. falciparum isolates from Swaziland collected in 1999 (thick smear blood slides) and 2 7 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing. RESULTS: Retrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2 7 carried detectable P. falciparum. The pfcrt haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 7 % (n=64) in 1999 and 83% (n=6) in 2 7. Prevalence of the pfmdr1-86N allele was 24% in 1999 and 67% in 2 7. A novel substitution of phenylalanine for asparagine at codon 86 of pfmdr1 (N86F) occurred in two of 51 isolates successfully amplified from 1999. The pfmdr1-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2 7. The 86N/184F/1246D pfmdr1 haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2 7 isolates. The pfdhfr triple-mutant 16C/51I/59R/1 8N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n=34) and 2 7 (5 %, n=6), as was the wild-type 431I/436S/437A/54 K/581A/613A haplotype of pfdhps (1 % and 93% respectively in 1999 and 2 7). The quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2 7 isolates. CONCLUSIONS: The prevalence of pfcrt and pfmdr1 alleles reported in this study is consistent with a parasite population under sustained CQ drug pressure. The low prevalence of dhps-437G and dhps-54 E 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.

BACKGROUND: Sulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended for intermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment of malaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated with mutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6-59 month children with uncomplicated malaria and in asymptomatic 2-1 month old infants. METHODOLOGY AND PRINCIPAL FINDINGS: An open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacy protocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 1 months old asymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to the high failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and a review of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-corrected early treatment failure was 38.8% (95% CI 26.8-5 .8) and total failures by day 28 were 82.2% (95% CI 72.5-92. ). There was no significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carried parasites with mutations at codons 51, 59 and 1 8 in the dhfr gene and 63% carried a double mutation at codons 437 and 54 . 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotype maybe associated with earlier treatment failure. CONCLUSION: In northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study found a new combination of parasite mutations that maybe associated with increased and earlier failure. TRIAL REGISTRATION: ClinicalTrials.gov NCT 361114.

In January 2 7, Tanzania replaced sulfadoxine-pyrimethamine (SP) with artemether-lumefantrine for treatment of uncomplicated malaria. This study examined the impact of widespread SP use on molecular markers of Plasmodium falciparum drug resistance in blood samples from persons living in two villages in Korogwe District, Tanzania, from 2 3 through 2 7. The prevalence of the P. falciparum dihydropteroate synthase (Pfdhps) gene 581G mutation increased from 12% in 2 3 to 56% in 2 7 (P < . 1), resulting in an increase in the triple mutant Pfdhps haplotype SGEGA from 8% to 32% (P < . 1). In contrast, the chloroquine-sensitive P. falciparum chloroquine resistance transporter (Pfcrt) CVMNK haplotype increased from 6% to 3 % (P < . 1). The dramatic increase of the triple Pfdhps mutant SGEGA haplotype may endanger the continued use of SP for intermittent presumptive treatment of pregnant women (IPTp). Further studies are needed to determine the importance of Pfdhps SGEGA haplotype parasites on the efficacy of SP for IPTp.

BACKGROUND: The efficacy of amodiaquine (AQ), sulphadoxine-pyrimethamine (SP) and the combination of SP+AQ in the treatment of Cameroonian children with clinical malaria was investigated. The prevalence of molecular markers for resistance to these drugs was studied to set the baseline for surveillance of their evolution with time. METHODS: Seven hundred and sixty children aged 6-59 months with uncomplicated falciparum malaria were studied in three ecologically different regions of Cameroon - Mutengene (littoral equatorial forest), Yaoundé (forest-savannah mosaic) and Garoua (guinea-savannah). Study children were randomized to receive either AQ, SP or the combination AQ+SP. Clinical outcome was classified according to WHO criteria, as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The occurrence of mutations in pfcrt, pfmdr1, dhfr and dhps genes was studied by either RFLP or dot blot techniques and the prevalence of these mutations related to parasitological and therapeutic failures. RESULTS: After correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 7 .1% and 8 .9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 8 .3%, 67.5% and 76.2% in Yaoundé respectively. High levels of Pfcrt 76T (87.11%) and Pfmdr1 86Y mutations (73.83%) were associated with quinoline resistance in the south compared to the north, 31.67% (76T) and 22. 8% (86Y). There was a significant variation (p < . 1) of the prevalence of the SGK haplotype between Garoua in the north (8.33%), Yaoundé (36.29%) in the savannah-forest mosaic and Mutengene (66.41%) in the South of Cameroon and a weak relation between SGK haplotype and SP failure. The 54 E mutation on the dhps gene was extremely rare ( .3%) and occurred only in Mutengene while the pfmdr1 1 34K and 1 4 D mutations were not detected in any of the three sites. CONCLUSION: In this study the prevalence of molecular markers for quinoline and anti-folate resistances showed high levels and differed between the south and north of Cameroon. AQ, SP and AQ+SP treatments were well tolerated but with low levels of efficacy that suggested alternative treatments were needed in Cameroon since 2 5.

Being given that point mutations affecting Plasmodium genes are correlated to phenotype resistance to antimalarials in Plasmodium falciparum isolates, molecular markers can be used for monitoring drug resistance in a country. From February, to December 2 6, blood samples were collected in Côte d?Ivoire in order to evaluate the polymorphism of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps), Plasmodium falciparum chloroquine resistance transporter (pfcrt) and Plasmodium falciparum multidrug resistance-1 (pfmdr-1) genes. The analysis of 144 DNA fragments of P. falciparum isolates revealed the presence of the mutant pfcrt (T76), dhfr-ts and pfmdr-1 in 94 (65.3%), 39 (27.1%) and 112 (77.8%) samples respectively. The frequency distribution of mutations in dhfr was 46.4% for dhfr-N1 8 allele, 22.6% for dhfr-I51 and 31% for dhfr-R59. As for pfmdr-1, the mutant-type samples showed the following haplotypes: 65 single mutations, Y86/Y184 (n=14), N86/F184 (n = 51), and 47 double mutations Y86/F184. For dhps gene, only 7 (6.6%) of 1 6 DNA fragments were wild-type, while 99 (93.4%) samples were mutant dhps. Mutations occurred only in positions 436 (42.9%), 437 (4 .3%), and 613 (16.8%). Finally, in vivo and in vitro chloroquine and antifolate resistance found in previous studies in Côte d?Ivoire which led to a change in favour of the use of the ACTs as recommended by WHO, is confirmed by a high level of mutations affecting pfcrt, dhfr-ts and dhps genes.

Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes. Methods Blood samples were collected in July and December 2 4 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions. Results An increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci. Conclusions This study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal.

ABSTRACT: BACKGROUND: Intermittent preventive treatment in infants (IPTi) with sulphadoxine-pyrimethamine (SP) given during routine vaccinations is efficacious in preventing malaria disease and shows no interaction with the vaccines. However, there is a fear that IPTi may result in a rapid increase of parasite resistance to SP. METHODS: To evaluate the impact of IPTi on SP-resistance point mutations, the 22 health sub-districts in the district of Kolokani, Mali, were randomized in a 1:1 ratio and starting in December 2 6, IPTi with SP was implemented in 11 health sub-districts (intervention zone), while the other 11 health sub-districts served as the control (non-intervention zone). Blood smears and blood dots on filter paper were obtained from children aged -5 years, randomly selected in each of heath sub-districts during two cross-sectional surveys. The first survey was conducted in May 2 7 before the start of the transmission season to collect baseline prevalence of the molecular markers of resistance to SP and the second in December 2 7 after the end of the transmission season and one year after implementation of IPTi. A total of 427 and 923 randomly selected blood samples from the first and second surveys respectively were analysed by PCR for dhfr and dhps mutations. RESULTS: Each of the three dhfr mutations at codons 51, 59 and 1 8 was present in 35% and 57% of the samples during the two surveys with no significant differences between the two zones. Dhps mutations at codons 437 and 54 were present respectively in about 2 % and 1% of the children during the two surveys in both zones at similar proportion. The prevalence of quadruple mutants (triple dhfr-mutants + dhps-437G) associated with in-vivo resistance to SP in Mali after one year implementation of IPTi was also similar between the two zones (11.6% versus 11.2%, p = .9 ) and to those obtained at baseline survey (1 .3% versus 8.1%). CONCLUSION: This study shows no increase in the frequency of molecular markers of SP resistance in areas where IPTi with SP was implemented for one year.

Accumulation of mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) is strongly associated with sulfadoxine-pyrimethamine (SP) treatment failure. Routine surveillance for these resistance markers was conducted annually at 26 sentinel sites in Maputo Province, Mozambique, before and after the phased deployment of artesunate plus SP (AS-SP), with 15,758 children sampled between 2 4 and 2 8. Mean asexual parasite prevalence, polymerase chain reaction (PCR) corrected, decreased from 44.2% in 2 4 to 3.8% in 2 8 (P < . 1). Among the 2, 12 PCR-confirmed falciparum samples, the dhfr triple mutation remained close to fixation, whereas both dhps double and dhfr/dhps "quintuple" mutations increased from 11. % in 2 4, to 75. % by 2 8 (P < . 1). Adding artesunate to SP did not retard the spread of SP-resistant parasites. The high "quintuple" mutation prevalence suggests a limited useful therapeutic lifespan of AS-SP for treating uncomplicated malaria, and may curb efficacy of SP-monotherapy for intermittent preventive treatment in Mozambique.

BACKGROUND: One of the major problems to the treatment of malaria is the emergence and spread of parasite resistant to antimalarial drugs. Due to increased chloroquine (CQ) resistance, the antifolate combinations are becoming important in the chemotherapy of falciparum malaria. However, resistance to antifolate exists and they are still effective in the above combinations. This study aimed at determining the prevalence of antimalarial drug resistance markers in P. falciparum isolates, involving the detection of mutations at the mdr 1- 86 which associates with amodiaquine resistance, and dhfr mutations associated with SP resistances. METHODS: The dot-blot/ probe hybridization, which is more sensitive and specific; it detects parasitaemia of less than 1 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture, was adopted to determine multi-drug resistance (mdr1-86) to show the correlation of Amodiaquine (AQ) resistance and PCR/ RFLP adopted to determine dihydrofolate reductase (dhfr) baseline resistance to Sulphadoxine- Pyrimethamine (SP) resistance in Nubian region of southern Sudan. A randomized open label trial of Artesunate (AS) + SP and AS+ SP was carried out in children less than 5 years. Molecular analysis of filter paper preserved blood samples collected was carried out to provide a baseline estimate of allele prevalences. RESULTS: Baseline of the allele prevalence of the mdr1 86 locus in the AS+ AQ was successful for 8 isolates: 71(8.11%) carried parasites harbouring the mdr1-86 Tyr resistance allele, while 7 (89.19%) carried mdr1-86 Asn sensitivity allele and 2 (2.7%) were of mixed infection, having both resistance and wild type allele. Overall, the prevalence of the dhfr point mutation, codon 51, 59 and 1 8: 82.5% (132/16 ) carried mutations at dhfr (N51I, C59R or S1 8N), but triple mutants were rare (3.1%) in the AS + SP arm. CONCLUSION: The research provides the evidence that mutations present in dhfr and mdr1 86 has a significant effect on the type of treatment following SP and AQ chemotherapy. SP resistance may spread rapidly, and AS + AQ is likely to be a better option, provided AQ use is restricted to the combination. The significance of the study shows that definitely combination of drugs improves SP therapy at the study site.

Background: It is argued that, the efficacy of anti-malarials could be prolonged through policy-mediated reductions in drug pressure, but gathering evidence of the relationship between policy, treatment practice, drug pressure and the evolution of resistance in the field is challenging. Mathematical models indicate that drug coverage is the primary determinant of drug pressure and the driving force behind the evolution of drug resistance. These models show that where the basis of resistance is multigenic, the effects of selection can be moderated by high recombination rates, which disrupt the associations between co-selected resistance genes. Methods: To test these predictions, dhfr and dhps frequency changes were measured during 2 -2 1 while SP was the second-line treatment and contrasted these with changes during 2 1-2 2 when SP was used for first-line therapy. Annual cross sectional community surveys carried out before, during and after the policy switch in 2 1 were used to collect samples. Genetic analysis of SP resistance genes was carried out on 4,95 Plasmodium falciparum infections and the selection pressure under the two policies compared. Results: The influence of policy on the parasite reservoir was profound. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr (N51I,C59R,S1 8N) allele (conferring pyrimethamine resistance) increased by 37% - 63% and the frequency of the double A437G, K54 E mutant dhps allele (conferring sulphadoxine resistance) increased 2 %-3 %. A strong association between these unurled alleles also emerged, confirming that they are co-selected by SP. Conclusion: The national policy change brought about a shift in treatment practice and the resulting increase in coverage had a substantial impact on drug pressure. The selection applied by first-line use is strong enough to overcome recombination pressure and create significant urlage disequilibrium between the unurled genetic determinants of pyrimethamine and sulphadoxine resistance, showing that recombination is no barrier to the emergence of resistance to combination treatments when they are used as the first-line malaria therapy.

A prospective cohort design was used to measure the association between daily cotrimoxazole-prophylaxis and infection with Plasmodium falciparum containing mutations associated with antifolate resistance among persons infected with human immunodeficiency virus (HIV) in Tororo and Busia District, in eastern Uganda. Of 149 cases of P. falciparum parasitemia diagnosed, 147 (99%) (smears from participants taking prophylaxis = 91 and smears from those not taking cotrimoxazole prophylaxis = 56) were successfully assessed for mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations associated with antifolate resistance. Prevalences of the dhfr pure triple mutant (74% and 7 %; P = .71), the dhps pure double mutant (95% and 88%; P = .21), and the dhfr/dhps pure quintuple mutant (73% and 64%; P = .36), were not significantly different between those taking and those not taking cotrimoxazole-prophylaxis, respectively. The overall prevalence of the pure quintuple mutant in this study was 69%, which is among the highest in Africa. Although resistance rates of P. falciparum to antifolate drugs are high, cotrimoxazole-prophylaxis in HIV-infected persons was not associated with a higher prevalence of mutations associated with antifolate resistance.

Background Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2 6. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. Methods Plasmodium falciparum samples collected in Luanda, in 2 7, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. Results The most prevalent polymorphisms identified were pfdhfr 1 8N (1 %), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (54 E in 1 %, 581G in 7% of samples). Conclusion This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

BACKGROUND: Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to Plasmodium falciparum infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2 6 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in pfdhfr and pfdhps genes associated to P. falciparum resistance to SP before the introduction of S/P IPT in children. METHODS: The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in pfdhfr and pfdhps genes was carried out in 452 P. falciparum blood samples by PCR RFLP. RESULTS: For pfdhfr gene, 9 ,3% of the samples carried the mutation 51I, with 7.5% of mixed infections; 51% carried wild type allele 59C, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 1 8N. Concerning, pfdhps gene, 83,1% were mutant type 437G with 11% mixed infections , while 87% of the studied isolates were wild type for codon 54 . DISCUSSION: This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51I and 1 8N in pfdhfr, and 437G in pfdhps) was found, besides a low prevalence of the quintuple mutation. CONCLUSION: The data showed that the implementation IPT using SP in children needs to be reviewed.