It has been proposed that polymorphisms of the Merozoite Surface Protein 1 and 2 (MSP1 and MSP2) and the Glutamate Rich Protein (GLURP) genes can be considered as genetic markers for the genotyping of field populations of Plasmodium falciparum. During a field study on in vivo drug resistance against chloroquine, sulphadoxine/pyrimethamine (S/P) and cotrimoxazole in West Uganda, sensitive and resistant isolates were collected from patients by fingerprick for genotyping. 59 (72.8%) of the 81 P. falciparum samples isolated at day showed multiclonal infection with 2-7 clones. Among the isolates we investigated, presence of the allelic family MAD2 of MSP1 at day was significantly (P = . 41) associated with decreased resistance to antimalarials. Use of this method in a field study on in vivo drug resistance demonstrates another potential application of genotyping as a tool for epidemiological investigations.

To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 19 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days. Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect DHFR and DHPS mutations on filter paper blood samples from pretreatment and post-treatment infections. Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed. Post-PS treatment infections had significantly higher rates of DHFR mutations at codons 1 8 and 59. No significant selection for DHPS mutations was seen. Pyrimethamine-sulfadoxine is highly efficacious in Mali, and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance, rapid selection of DHFR mutations supports their role in PS failure.

A total of 252 children were enrolled in a drug trial to assess the effect of minimal doses of sulfadoxine (Sdx) and pyrimethamine (Pyr). Parasite samples isolated from these patients were analyzed before and after treatment to investigate the level of drug-resistant strains. The parasite genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were assayed for point mutations that are associated with resistance against drugs. Before treatment, Pyr(r) genotypes of the DHFR gene were found in 42% of all samples, 8% of the patients harbored a mixed parasite population and 5 % had a sensitive DHFR genotype. In terms of the DHPS gene, we found mutations in 45% of the parasites. Twenty-four percent had a Ser(436) mutation, and 26% had a Gly(437) mutation. Recrudescent parasites were highly enriched for both Pyr(r) and Sdx(r) strains after treatment (P < . 1 and P = . 29, respectively).

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 24 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 2 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (3 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were "pure" triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (2 .7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L54 E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.

Mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) are associated with in vitro resistance to sulfadoxine and pyrimethamine, respectively. The response of 75 patients to sulfadoxine-pyrimethamine was determined, and the genes of the corresponding Plasmodium falciparum isolates were sequenced. Of 12 different unmixed allelic combinations, the triple dhfr mutation Asn-1 8/Arg-59/Ile-51 was observed in all patients responding with early treatment failure. Some, but not all, patients with an adequate clinical response also harbored isolates with the triple dhfr mutation. Higher initial parasitemia and fever distinguished these 2 patient groups. The dhps genotype apparently had no influence on the clinical outcome. The other dhfr alleles with 1 or 2 mutations and the wild-type allele were found in patients with an adequate clinical response. The triple dhfr mutation is one of the genetic determinants associated with in vivo resistance to sulfadoxine-pyrimethamine.

A prospective study was conducted to measure the selective effect of pyrimethamine prophylaxis on point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR). A total of 1 9 Malian children were given pyrimethamine weekly for 5 weeks. P. falciparum infections were analyzed by polymerase chain reaction for DHFR mutations, which were dramatically more frequent among prophylaxis-breakthrough infections than at baseline: the prevalence of Asn-1 8 rose from 13% to 1 %, Ile-51 from 4% to 5 %, and Arg-59 from 11% to 9 %. Eight persistent infections lacking detectable DHFR mutations at baseline developed multiple mutations within 1 week of the patients' starting pyrimethamine prophylaxis. Microsatellite analysis found no evidence of clonal identity among baseline and breakthrough infections. Analysis of these data demonstrates that under prophylaxis conditions, pyrimethamine is strongly selective for DHFR mutations, which arise extremely rapidly under drug pressure, even when undetectable in the initial infection. These findings have implications for prophylaxis regimens with other antifolate drugs.

ACKGROUND: 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.

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.

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.

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.

Considerable levels of resistance to sulfadoxine-pyrimethamine (SP) have been reported in Plasmodium falciparum in north-eastern Tanzania, and the identification of a suitable antimalarial to replace SP is now a high priority. We conducted a trial in July 2 to determine the efficacy of proguanil (PG) plus dapsone (DS), compared with that of SP, for the treatment of asymptomatic falciparum infection. A total of 22 children with parasitaemia > or = 2 per microL completed the study; 112 had received a single dose of SP (dosage calculated for pyrimethamine 1.25 mg/kg and sulfadoxine 25 mg/kg) and 1 8 had taken PG 1 mg/kg with DS 2.5 mg/kg each day for 3 days. Clearance of asexual parasites at day 7 was 14.3% with SP, but 93.5% with PG-DS. The remarkably high failure rate with SP was not associated with occurrence of leucine substitution at position 164 of the dhfr gene. Both treatment regimens were well tolerated. Compared with available data on another antifolate combination, chlorproguanil-dapsone ('Lapdap'), PG-DS was slightly but significantly inferior in achieving parasite clearance (99.5% versus 93.5%). The estimated cost of a 3-day course of PG-DS treatment for a child weighing 18 kg is US $ .15. With the rising incidence of SP-resistant P. falciparum infection, PG-DS could provide an effective, affordable and already available therapeutic alternative for malaria in East Africa at least until chlorproguanil-dapsone is registered.

Several in vitro studies have shown the correlation between mutations in dhfr and dhps genes and resistance to pyrimethamine/sulfadoxine (PYR/SDX) combination, but the in vivo correlates of these mutations with PYR/ SDX efficacy have not been investigated fully. We assessed PYR/SDX efficacy in relation to the frequency of dhfr and dhps mutations in 37 Plasmodium falciparum isolates sampled before treatment. Plasma levels of SDX measured at days , 3, 7, and 14 ascertained drug absorption. Point mutations were detected only at codons 51 and 1 8 of dhfr and codon 436 of dhps. The frequency of dhfr 51/1 8 and dhps 436 mutations was 79% and 8%. The plasma levels of SDX indicated adequate drug absorption by all patients. The presence of Ile 51 and Asn 1 8 mutations among parasites that cleared after treatment indicates that these mutations alone are insufficient to cause in vivo resistance. In all recrudescent parasites, however, the presence of Ile 51/Asn 1 8 dhfr mutations was coupled with the dhps Ala 436. The findings suggest that the presence of Ile 51/Asn 1 8 dhfr mutations and Ala 436 dhps confers decreased susceptibility of P. falciparum to PYR/SDX in areas of low endemicity.

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.

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.

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.

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.

The selection of point mutation at codon 164 (from isoleucine to leucine) of the dihydrofolate reductase (DHFR) enzyme in Plasmodium falciparum is associated with high sulfadoxine /pyrimethamine (SP) resistance. Using the yeast expression system that allows the detection of dhfr allele present at low level, the presence of this mutation had previously been reported between 1998-1999 in Muheza, Tanzania, an area of high SP resistance. Eighty five P. falciparum isolates, obtained from the same area between 2 2 and 2 3, were analysed for the presence of Leu-164 mutation, using standard protocol based on PCR-RFLP. None of the isolates had the Leu-164 mutation.

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.

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.

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

The current first-line and second-line drugs for Plasmodium falciparum malaria in South Sudan, chloroquine and sulfadoxine-pyrimethamine (SP), were evaluated and compared with amodiaquine, in an MSF-Holland-run clinic in eastern Upper Nile, South Sudan from June to December 2 1. Patients with uncomplicated malaria and fever were stratified by age group and randomly allocated to one of 3 treatment regimes. A total of 342 patients was admitted and followed for 14 d after treatment. The dropout rate was 1 .2%. Of those who completed the study, 1 4 were treated with chloroquine (25 mg/kg, 3 d), 1 2 with SP (25 mg/kg sulfadoxine and 1.25 mg/kg pyrimethamine, single dose) and 1 1 with amodiaquine (25 mg/kg, 3 d). Adequate clinical response was observed in 88.5% of patients treated with chloroquine, 1 % of patients treated with SP and 94.1% of patients treated with amodiaquine. In children aged < 5 years, the success rate was lower: 83.3% for chloroquine and 93. % for amodiaquine. In adults no treatment failures were found, but children aged 5-15 years showed intermediate levels. In addition, we determined the initial genotypes of dhfr and dhps of 44 isolates from the SP-treated group and > 8 % were found to be wild type for dhfr and 1 % for dhps. Two percent of isolates had a single mutation and 16% had double mutations of dhfr. These data are in full agreement with the clinical effectiveness of SP. A change in malaria treatment protocols for South Sudan is recommended.

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.

Sulphadoxine/pyrimethamine (SP) has become the first-line treatment of uncomplicated malaria in a number of African countries. Molecular surveillance of resistance-mediating mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) has been proposed as a means of predicting SP treatment outcomes, but optimal methods of surveillance in different populations have not been well established. To investigate the relationship between molecular markers of SP resistance, host immunity, and response to therapy, we evaluated the association between the presence of five key dhfr and dhps mutations at enrollment and clinical outcome in children and adults treated with SP for uncomplicated malaria in Kampala, Uganda. Clinical treatment failure was 11% at 14 days, increasing to 3 % at 28 days, after excluding new infections. Outcomes varied markedly based on the number of dhfr and dhps mutations and on the age of treated subjects. All infections with less than two dhfr/dhps mutations were successfully treated. Treatment failure associated with any two, three, or four dhfr/dhps mutations occurred in nine of 24 (38%) children up to 5 years, but not in older patients ( /2 ). In the presence of all five mutations, treatment failure occurred equally in children aged 5 years or younger [7/16 (44%)] and in older patients [8/16 (5 %)]. Our results showed that age, a surrogate marker of antimalarial immunity, had a major impact on the relationship between polymorphisms in SP target enzymes and treatment outcomes. The use of molecular markers of SP resistance to predict treatment failure rates should take age into account.

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.

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.

We conducted a prevalence study of mutations in Plasmodium falciparum that are associated with antifolate resistance in Blantyre, Malawi. The dihydrofolate reductase 164-Leu mutation, which confers resistance to both pyrimethamine and chlorproguanil, was found in 4.7% of the samples. Previously unreported mutations in dihydropteroate synthase were also found.

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).

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 report 12 uncomplicated falciparum-malaria cases from semi-immune people from Central Africa treated with sulfadoxine/pyrimethamine (Fansidar) in a Spanish hospital. We resolved by PCR-RFLP the mutations in dhfr and dhps genes related to resistance to antifolate drugs. The 12 patients presented high frequencies of combined mutations in both genes but they were completely cured after treatment.

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.

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.

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.

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

Drugs with long elimination half-lives such as Sulfadoxine -Pyrimethamine (SP) maintain sub-curative levels in blood for a long time such that in high malaria transmission areas, re-infecting parasites are continuously under selection pressure for resistant genotypes.Objective: To assess SP efficacy and post therapeutic in-vivo selection for Plasmodium falciparum check for this species in other resources dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) mutations associated with SP resistance.Design: SP efficacy trial with prospective follow up for selection of parasites with DHFR and DHPS mutations at re-infection in the resistance selection period (RSP).Subjects: Children aged < 5 years attending the outpatient reproductive and child health clinic of Kibaha district hospital with uncomplicated malaria fulfilling the inclusion criteria for efficacy trials in holoendemic settings.Main outcome measures: Clinical & parasitological efficacy, pre-treatment and post-treatment prevalence of P. falciparum DHFR & DHPS mutations.Results: Very high (98.2%) clinical & parasitological cure rates. DHFR single, double or triple mutations occurred in 46.7% of pre-treatment infections; triple c1 8/51/59 & double c1 8/51 mutations being commonest. Few (15.9%) DHPS mutations occurred in pre-treatment infections at c436 and c437. DHFR & DHPS mutations were significantly higher in post- than pre- SP treatment parasites. In a Poisson regression analysis, DHFR mutations at c1 8, c51 & c59 and the exclusive c1 8/51/59 triple mutations were strongly associated with exposure to SP at re-infection. Conclusion: DHFR & DHPS mutations associated with SP resistance exist in P. falciparum infections in a background of high SP efficacy. Despite optimal dosage, in holoendemic areas, these mutations will be selected by SP at re-infection; cumulatively shortening the useful therapeutic life of SP due to resistance.

Analysis of Plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in Djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic cannot be attributed to a sudden increase in drug resistance of local parasite populations.

The purpose of this prospective cohort study was to assess the effect of cotrimoxazole prophylaxis taken by human immunodeficiency virus (HIV)-infected persons on the selection of sulfadoxine-pyrimethamine (SP)-resistant malaria parasites among HIV-uninfected household members. A total of 2,567 HIV-uninfected persons from 6 5 households were followed and blood specimens were collected each time an episode of Plasmodium falciparum malaria was diagnosed. Study participants were living in households where HIV-infected persons were either taking (exposed) or not taking (unexposed) cotrimoxazole prophylaxis. From all malaria episodes diagnosed, 5 % of the specimens were randomly selected and tested for the presence of five key mutations known to mediate resistance to SP (dihydrofolate reductase [dhfr] Asn-1 8, Ile-51, and Arg-59, and dihydropteroate synthase [dhps] Gly-437 and Glu-54 ). Plasmodium falciparum isolates were recovered from 163 specimens in the exposed households and 113 specimens in the unexposed households, with similar proportions containing the dhfr triple mutant (37% versus 45%; P = .18), the dhps double mutant (64% versus 62%; P = .81), and the dhfr/dhps quintuple mutant (3 % versus 32%; P = .74). The HIV-uninfected persons living with HIV-infected household members taking cotrimoxazole prophylaxis had a lower incidence of malaria (incidence rate ratio [IRR] = .64, 95% confidence interval [CI] = .5 - .83, P = . 1) and fewer malaria episodes due to parasites containing the dhfr/dhps quintuple mutant (IRR = .61, 95% CI = .41- .91, P = . 14). Cotrimoxazole prophylaxis taken by HIV-infected persons did not select for SP-resistant malaria parasites among HIV-uninfected household members, and was associated with a lower overall incidence of SP-resistant malaria among household members.

BACKGROUND: Recent clinical trials from Uganda have shown that the risk of failure following antimalarial therapy varies geographically. We tested the hypothesis that geographic differences in the response to therapy could be explained by differences in the prevalence of known molecular markers of drug resistance. METHODS: Samples from 2 84 patients treated with chloroquine (CQ) plus sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) plus SP were tested for the presence of known molecular markers of resistance. Differences in the risk of treatment failure across 6 sites were compared, and age and complexity of infection were controlled for. RESULTS: The prevalence of molecular markers of drug resistance was high at all of the sites: 61%-91% of patients were infected with parasites containing the pfcrt Thr-76 mutation and dhfr/dhps quintuple mutation. The risk of treatment failure decreased with increasing transmission intensity for both CQ plus SP (73% to 19%) and AQ plus SP (38% to 2%). Restricting the analyses to patients infected with parasites containing all 6 mutations of interest did not affect these trends. CONCLUSIONS: The risk of treatment failure was inversely proportional to transmission intensity and was not explained by differences in molecular markers of antimalarial drug resistance. Our findings strongly suggest that geographic differences in response to antimalarial therapy in Uganda are primarily mediated by acquired immunity associated with malaria transmission intensity, rather than by parasite factors.

BACKGROUND: Sulphadoxine-pyrimethamine (SP) has been and is currently used for treatment of uncomplicated Plasmodium falciparum malaria in many African countries. Nevertheless, the response of parasites to SP treatment has shown significant variation between individuals. METHODS: The genes for dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) were used as markers, to investigate parasite resistance to SP in 141 children aged less than 5 years. Parasite DNA was extracted by Chelex method from blood samples collected and preserved on filter papers. Subsequently, polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) were applied to detect the SP resistance-associated point mutations on dhfr and dhps. Commonly reported point mutations at codons 51, 59, 1 8 and 164 in the dhfr and codons 437, 54 and 581 in the dhps domains were examined. RESULTS: Children infected with parasites harbouring a range of single to quintuple dhfr/dhps mutations were erratically cured with SP. However, the quintuple dhfr/dhps mutant genotypes were mostly associated with treatment failures. High proportion of SP resistance-associated point mutations was detected in this study but the adequate clinical response (89.4%) observed clinically at day 14 of follow up reflects the role of semi-immunity protection and parasite clearance in the population. CONCLUSION: In monitoring drug resistance to SP, concurrent studies on possible confounding factors pertaining to development of resistance in falciparum malaria should be considered. The SP resistance potential detected in this study, cautions on its useful therapeutic life as an interim first-line drug against malaria in Tanzania and other malaria-endemic countries.

BACKGROUND: Sulfadoxine-pyrimethamine has been widely used as first-line therapy for uncomplicated malaria throughout sub-Saharan Africa. Recent studies conducted in Asia and Africa suggest the triple-mutant dhfr genotype (51I/59R/1 8N) may have been generated as a single event in Southeast Asia, with subsequent spread of the single lineage to the African continent, but this hypothesis needs further validation. METHODS: Direct sequencing of polymerase chain reaction (PCR) products, pyrosequencing, and cloning of PCR products were utilized to identify mutations in dhfr. To investigate the evolutionary history of dhfr alleles, we assayed microsatellite loci flanking dhfr along chromosome 4. RESULTS: A total of 15 of 479 samples from western Kenya showed the presence of I164L, in 5 different genotypes. We document C5 R in 2 of our samples. Using microsatellite markers, we show 2 haplotypes for both the 51I/1 8N/164L and 51I/59R/1 8N/164L genotypes. Our results also show multiple lineages for the triple-mutant dhfr genotype in Africa. CONCLUSIONS: These findings highlight the importance of local characterization of alleles before molecular surveillance of drug-resistant alleles is considered in different endemic settings and populations.

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.

We evaluated associations between key polymorphisms in target genes and responses to treatment with sulfadoxine-pyrimethamine (SP) or amodiaquine (AQ) for uncomplicated Plasmodium falciparum malaria in Bobo-Dioulasso, Burkina Faso. Presence of the dihydrofolate reductase (dhfr) 1 8N or 59R mutations (but not dhfr 51I or dihydropteroate synthetase [dhps] 437G) and P. falciparum chloroquine resistance transporter (pfcrt) 76T or P. falciparum multidrug resistance 1 (pfmdr1) 86Y or 1246Y mutations (but not pfmdr1 184F) predicted recrudescence after treatment with SP and AQ, respectively. Treatment led to significant increases in the prevalence of the same mutations (except 1246Y) in new infections that presented after therapy. The dhfr 164L and dhps 54 E mutations were not seen in any isolates. These results clarify the key roles of a small number of mutations in P. falciparum resistance to SP and AQ in west Africa.

ulfadoxine-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 .77<OR>6.2 ). 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.

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.

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.

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: Usefulness of sulfadoxine-pyrimethamine as first-line therapy for uncomplicated Plasmodium falciparum malaria and intermittent preventive treatment in pregnancy throughout sub-Saharan Africa is compromised by the spread of dhfr alleles associated with pyrimethamine resistance. A predominant haplotype associated with the N51I+C59R+S1 8N triple-mutant dhfr allele has been reported recently in 4 African countries. A more comprehensive picture of the evolution of this mutant allele in Africa is lacking. METHODS: Seventy-five P. falciparum isolates carrying the wild-type dhfr allele and 2 4 carrying the triple-mutant dhfr allele from 11 African countries were selected. The genetic diversity of the chromosomes bearing these alleles was analyzed with 4 microsatellite markers closely urled to the dhfr gene. RESULTS: Seventy-three different 4-locus haplotypes carrying the wild-type dhfr allele were found. By contrast, 175 (85%) of 2 4 isolates carrying the triple-mutant dhfr allele shared a unique haplotype, identical to the one identified in Thailand. For the remaining triple-mutant isolates and one isolate with the quadruple-mutant dhfr allele (N51I+C59R+S1 8N+I164L), haplotypes were closely related to the predominant haplotype by mutation or recombination. CONCLUSIONS: Migration of parasites carrying an ancestral triple-mutant dhfr allele drives the spread of dhfr alleles associated with pyrimethamine resistance throughout West and Central Africa.

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.

The rapidly changing epidemiology of antifolate-resistant Plasmodium falciparum in Africa requires monitoring. The present study was designed to assess the degree of association between the clinical and parasitological response to sulfadoxine-pyrimethamine and allelic combinations of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. Of 357 children who completed the 14-day follow-up, an adequate clinical and parasitological response was observed in 316 patients (88.5%) and early and late failures occurred in 18 (5%) and 23 (6.4%, mostly due to recrudescence) patients, respectively. The majority of clinical isolates were characterized as "quadruple" (n=196, 55.2%; N51I-C59R-S1 8N in DHFR and A437G in DHPS) or "triple" mutants (n=97, 27.3%; N51I-C59R-S1 8N in DHFR and wild-type DHPS; S1 8N+N51I or C59R in DHFR and A437G in DHPS). Wild-type, single mutation, and double mutation were observed in 29, 2 , and 13 parasites, respectively. The comparison of different sets of mutations and early or late failures did not reveal any molecular marker associated with treatment outcome when the follow-up period was limited to 14 days (P> . 5). In this study, the determination of dhfr-dhps genotypes was of limited value to predict the treatment outcome in individual patients, mostly due to few treatment failures and few wild-type haplotypes. Further monitoring will be required to define the relationship between clinical response to SP therapy and parasite genotypes in our epidemiological setting.

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.

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 2002-2003 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.

. 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 beaconbased 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.

Recently, Plasmodium falciparum bearing dihydrofolate reductase (DHFR) I164L was isolated from Africa. Quadruple mutations containing I164L confer high-level resistance to antifolate antimalarials. We prospectively measured the effect of co-trimoxazole (CTX) prophylaxis on P. falciparum antifolate resistance development among HIV-infected persons. HIV-positive patients with CD4 cell count < 35 cells/microl (n=692) received CTX; HIV-positive patients with CD4 cell count > or = 35 cells/microl (n=336) and HIV-negative patients (n=132) received multivitamins. Malaria microscopy-positive samples (n=413) and selected microscopy-negative/PCR-positive samples (n=76) were analysed for DHFR mutations at baseline and during six months follow up. We identified I164L in 14 patients. Seven were malaria microscopy-positive: two failed sulfadoxine-pyrimethamine (SP). Among seven microscopy-negative/PCR-positive patients, none developed patent infections with I164L. I164L was not associated with high-level SP resistance or poor outcome among adults living where malaria is highly endemic. Surveillance to monitor spread of I164L is critical, especially among children and pregnant women, who are potentially a source for I164L amplification.

Objective To determine the relationship between mutations in dhfr and dhps and SP treatment failure in Plasmodium falciparum malaria in the Democratic Republic of the Congo (DRC). Methods Therapeutic efficacy trial was conducted in Rutshuru, Eastern DRC, between June and September 2 2, comparing sulfadoxine-pyrimethamine (SP), SP plus amodiaquine (AQSP) and artesunate plus SP (ASSP) regimens for treating malaria in children under 5 years old. We genotyped 212 samples for mutations associated with SP resistance and investigated their association with treatment failure. Results In the SP arm, 61% of the subjects experienced treatment failure after 14 days. The failure rate was lower in the combination arms (AQSP: 32%, ASSP: 21%). The dhfr-1 8 and dhfr-51 mutations were nearly universal while 89% of the samples had at least one additional mutation at dhfr-59, dhps-437 or dhps-54 . Dhps mutations had a bigger impact on treatment failure in children with high parasite density: for children with a parasite density <45 parasites/mul, the risk of treatment failure was 37% for mutations at dhps-437 and dhps-54 mutation and 21% for neither mutation [risk difference (RD) = 17%, 95% CI: -3%, 36%]. In children with a parasite density >45 parasites/mul, the treatment failure risk was 58% and 8% for children with both mutations or neither mutation, respectively (RD = 51%, 95% CI: 34%, 67%). Conclusions Dhps-437 and dhps-54 are strongly associated with SP treatment failure and should be evaluated further as a method for surveillance of SP-based therapy in DRC.

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.

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.

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.

BACKGROUND: In Malawi, there has been a return of Plasmodium falciparum sensitivity to chloroquine (CQ) since sulfadoxine-pyrimethamine (SP) replaced CQ as first line treatment for uncomplicated malaria. When used for prophylaxis, Amodiaquine (AQ) was associated with agranulocytosis but is considered safe for treatment and is increasingly being used in Africa. Here we compare the efficacy, safety and selection of resistance using SP or CQ+SP or artesunate (ART)+SP or AQ+SP for the treatment of uncomplicated falciparum malaria. METHODOLOGY AND FINDINGS: 455 children aged 1-5 years were recruited into a double-blinded randomised trial comparing SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p< . 1). AQ+SP had an ACPR rate of 97%, higher than CQ+SP (81%) and ART+SP (7 %), p< . 1. Nineteen children developed a neutropenia of </= .5x1 (3) cells/microl by day 14, more commonly after AQ+SP (p = . 3). The mutation pfcrt 76T, associated with CQ resistance, was detected in none of the pre-treatment or post-treatment parasites. The prevalence of the pfmdr1 86Y mutation was higher after treatment with AQ+SP than after SP, p = . 2. CONCLUSIONS: The combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found evidence that AQ may exert selective pressure for resistance associated mutations many weeks after treatment. This study confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of pfcrt 76T after treatment with CQ or AQ. Given the safety record of AQ when used as a prophylaxis, our observations of marked falls in neutrophil counts in the AQ+SP group requires further scrutiny. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN22 75368.

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.

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pfdhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanil-dapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolate-artemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2 3 and 2 5.

Letter

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.

Genetic polymorphisms in Plasmodium falciparum are associated with resistance to a number of drugs, but data on their prevalence are limited from many areas. We explored the prevalence of key polymorphisms in patients presenting with malaria in Kinshasa. Prevalences of pfcrt K76T; pfmdr1 N86Y; pfdhfr N51I, C59R, and S1 8N; and pfdhps A437G were well above 5 % and of pfmdr1 Y184F, N1 42D, and D1246Y; pfdhfr I164L; and pfdhps K54 E were low. These results suggest an intermediate level of resistance to aminoquinoline and antifolate antimalarials in Kinshasa compared with other areas of Africa

Typing of polymorphic microsatellites that are urled to drug resistance genes has shed light on the origin and pattern of spread of some anti-malarial drugs. Recent surveys revealed spread of a high-level pyrimethemine resistant lineage of Plasmodium falciparum, of Asian origin, across Africa. Here, we examined mutations in dihydrofolate reductase, dhfr [chromsosome 4], the dihydropteroate synthase, dhps [chromosome 8] associated with resistance to sulfadoxine-pyrimethamine (SP), and neighboring microsatellites among P. falciparum isolates in Asar village, eastern Sudan. This area lies at the fringes of malaria endemicity, where the remote P. falciparum parasites have some distinct genetic characteristics. Overall, 89% (84/94) of the examined isolates carried double mutations at dhfr (N51I and S1 8N), but the 59R and I164L mutations were not seen. Similarly, the majority, 43% (35/81) of the isolates carried double mutations at dhps (437G, 54 E). Analysis of neighboring microsatellites revealed one major dhfr haplotype with mutations (51I, 1 8N) and one dhps haplotype with mutations (436S, 437G, 54 E). These haplotypes differ from the major ones thought to drive resistance to SP across Africa. The resistant haplotypes of dhfr and dhps, in Asar, share some microsatellites with the wild genotypes suggesting that they were generated locally. Among isolates successfully examined, 4 % shared identical haplotypes of the 2 loci, comprising a dominant resistant lineage. Undoubtedly, this lineage plays an important role in clinical failure to SP in this area.

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: Single nucleotide polymorphisms (SNPs) in the dhfr and dhps genes are associated with sulphadoxine-pyrimethamine (SP) treatment failure and gametocyte carriage. This may result in enhanced transmission of mutant malaria parasites, as previously shown for chloroquine resistant parasites. In the present study, we determine the association between parasite mutations, submicroscopic P. falciparum gametocytemia and malaria transmission to mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: Samples from children treated with SP alone or in combination with artesunate (AS) or amodiaquine were genotyped for SNPs in the dhfr and dhps genes. Gametocytemia was determined by microscopy and Pfs25 RNA-based quantitative nucleic acid sequence-based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays. We observed no wild type infections, 66.5% (127/191) of the infections expressed mutations at all three dhfr codons prior to treatment. The presence of all three mutations was not related to higher Pfs25 QT-NASBA gametocyte prevalence or density during follow-up, compared to double mutant infections. The proportion of infected mosquitoes or oocyst burden was also not related to the number of mutations. Addition of AS to SP reduced gametocytemia and malaria transmission during follow-up. CONCLUSIONS/SIGNIFICANCE: In our study population where all infections had at least a double mutation in the dhfr gene, additional mutations were not related to increased submicroscopic gametocytemia or enhanced malaria transmission. The absence of wild-type infections is likely to have reduced our power to detect differences. Our data further support the use of ACT to reduce the transmission of drug-resistant malaria parasites.

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.

n 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.

The 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.

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: 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.

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.

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 Cote 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 Cote 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.

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.

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.

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.

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.

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.