Plasmodium falciparum present in blood samples collected before and 3 weeks after treatment with either pyrimethamine-sulfadoxine or chlorproguanil-dapsone was analyzed for variants of the genes coding for the target enzymes of antifolate drugs, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Fragments of the genes were amplified by polymerase chain reactions, and variants were identified by specific restriction endonuclease digestion. Treatment with either drug combination selected for the variants Ile51, Arg59, and Asn1 8 of DHFR, which have been associated with in vitro resistance to pyrimethamine and cycloguanil. The genotype Ser436, Gly437, and Glu54 of DHPS was selected by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment, showing that a combination of these three variants is important for in vivo resistance to sulfadoxine in the area studied.

Plasmodium falciparum present in blood samples collected before and 3 weeks after treatment with either pyrimethamine-sulfadoxine or chlorproguanil-dapsone was analyzed for variants of the genes coding for the target enzymes of antifolate drugs, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Fragments of the genes were amplified by polymerase chain reactions, and variants were identified by specific restriction endonuclease digestion. Treatment with either drug combination selected for the variants Ile51, Arg59, and Asn1 8 of DHFR, which have been associated with in vitro resistance to pyrimethamine and cycloguanil. The genotype Ser436, Gly437, and Glu54 of DHPS was selected by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment, showing that a combination of these three variants is important for in vivo resistance to sulfadoxine in the area studied.

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.

In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 1 8-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 1 8.

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.

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.

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.

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.

The main objective of this work was to determine the prevalence of mutations in genes coding for the dihydropteroate synthase (DHPS) and the dihydrofolate reductase (DHFR) enzymes which are implicated in resistance of P. falciparum to antifolate (pyrimethamine-sulfadoxine (P/S)). In this study, 117 human blood samples were collected at Franceville located in the region of Haut-Ogooue (South-eastern Gabon). In this area, a relatively low level of sensitivity of Plasmodium falciparum to P/S has been reported with 18.2% of RII and 12.1% of RI resistance. A nested polymerase chain reaction was used to amplify a fragment of the DHFR gene containing codon 1 8, where a point mutation causing a Serine (wild type) to Asparagine or to a Threonine (resistant types) change occurs in pyrimethamine resistant parasites. Eleven DHFR fragments were sequenced and mutations occurring at codons 51, 59 and 1 8 were analysed. The DHPS gene was amplified by polymerase chain reaction (PCR) and sequenced directly or after cloning. Variant amino acid residues 436, 437, 54 , 581, 613 associated with sulfadoxine resistance were analysed. The analysis of codon 1 8 of the DHFR gene was undertaken for 81 isolates. More than one DHFR P. falciparum genotype was present in 64% of the samples. We showed that 47% of 141 DHFR gene PCR products had Serine (wild phenotype), and 52% had Asparagine. We found one isolate with the Thr-1 8 confirmed by sequencing of the PCR product. Triple, double and single DHFR mutant at positions 51, 59 and 1 8 were found. Only codons 436 and 437 of the 38 analysed sequences of the DHPS gene revealed point mutations. These results have been compared with those reported from different sites in Africa, Asia or South-America.

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.

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.

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

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.

We have determined the relationship between point mutations in the gene that encodes the sulfa target, dihydropteroate synthase (DHPS) and the chemosensitivity profile to sulfadoxine and dapsone in 67 isolates from Kilifi, Kenya. We assessed the presence of mutations at codons 436, 437, 54 , 581, and 613 of dhps. The results showed that the dhps genotype had a strong influence on the sensitivity to sulfadoxine and dapsone, but that the correlation was far from perfect. Eleven isolates carried a wild-type dhps allele, but were resistant to sulfadoxine (IC(5 ) values >1 microg/ml), and 4/28 isolates were classed as sensitive to sulfadoxine (IC(5 ) values <1 microg/ml), but carried a triple mutant (436/437/613) allele of dhps. These data show that in low folate medium in vitro, the dhps genotype alone did not account completely for sulfadoxine or dapsone resistance; other factors such as the utilisation of exogenous folate must also be considered

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.

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.

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.

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.

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.

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.

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

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

The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2 1 following 18 years of second-line use. The genetic determinants of in vitro resistance to the two drugs individually are shown to be point mutations at seven sites in the dihydrofolate reductase gene (dhfr) conferring resistance to pyrimethamine and five sites in the dihydropteroate synthase (dhps) gene conferring resistance to sulfadoxine. Different combinations of mutations within each gene confer differing degrees of insensitivity, but information about the frequency with which allelic haplotypes occur has been lacking because of the complicating effects of multiple infection. Here we used a novel high-throughput sequence-specific oligonucleotide probe-based approach to examine the present resistance status of three Plasmodium falciparum populations in northern Tanzania. By using surveys of asymptomatic infections and screening for the presence of all known point mutations in dhfr and dhps genes, we showed that just five dhfr and three dhps allelic haplotypes are present. High frequencies of both triple-mutant dhfr and double-mutant dhps mutant alleles were found in addition to significant interregional heterogeneity in allele frequency. In vivo studies have shown that the cooccurrence of three dhfr mutations and two dhps mutations in an infection prior to treatment is statistically predictive of treatment failure. We have combined data for both loci to determine the frequency of two-locus genotypes. The triple-dhfr/double-dhps genotype is present in all three regions with frequencies ranging between 3 and 63%, indicating that treatment failure rates are likely to be high.

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.

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.

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.

Short Communication: no abstract

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.

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.

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.

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.

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

Both 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.

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.

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

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).

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.

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.

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.

Background: 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.

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.

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.

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

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

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 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.

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

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

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

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

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

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

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.

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.

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.

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

BACKGROUND: In 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 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.

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

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

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

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.

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

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

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

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

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

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

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.

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.

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

Background: 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.

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

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

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

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

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

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