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.

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.

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.

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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.

Sulphadoxine/pyrimethamine (SP) is often administered with quinine in the treatment of severe falciparum malaria to shorten the course of quinine. The efficacy of SP alone in the treatment of non-severe malaria has been declining rapidly in East Africa, raising concerns of the usefulness of a shortened course of quinine followed SP. We audited the efficacy of quinine/SP in the treatment of severe malaria in Kenyan children. Children with severe falciparum malaria were treated with parenteral quinine followed by a single oral dose of SP. A clinical evaluation was performed 3 weeks later in which a blood sample was obtained for full haemogram, blood slide and analysis of the parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) codons, mutations of which are associated with resistance to SP. A total of 452 children were enrolled, of whom 374 completed the study. Fifty-two (13.9%) children were parasitaemic by 3 weeks of whom 17 (4.5%) had fever as well. The treatment failure group had a significantly higher parasitaemia (129 61 vs. 43 339; P< . 1) and haemoglobin on admission, but only admission parasitaemia independently predicted treatment failure. Those with treatment failure had a significantly lower rise in haemoglobin at 3 weeks compared with treatment successes (9. vs. 1 . g/dl). Of the 76 parasite isolates collected before treatment, 4 (53%) were triple mutant DHFR-double DHPS (Tp-Db), the genotype most associated with SP resistance. Three weeks after SP treatment, the proportion of Tp-Db increased to 72% (31/43). The high treatment failure rate and proportion of parasites with Tp-Db negate the use of SP to shorten the course of quinine treatment in East Africa.

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

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).

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.

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 6 % of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S1 8N, N51I, C59R was observed. The quintuple mutant (dhfr-S1 8N, N51I, C59R and dhps-A437G or S436A, K54 E) considered as molecular marker for SP treatment failure was not found because dhps-K54 E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K54 E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

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

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

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.

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

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

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

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

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: 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. 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.

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.

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

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

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

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

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.

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

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

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

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

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

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.

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

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.

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.

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

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

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

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.

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

BACKGROUND: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution. METHODS AND FINDINGS: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 2 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations. CONCLUSIONS: Resistant dhps has emerged independently in multiple sites in Africa during the past 1 -2 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.

BACKGROUND: A study carried out in 2 3-2 5 in Southern Benin showed a day-28 sulphadoxine-pyrimethamine (SP) monotherapy failure rate greater than 4 %, while for SP combined with artesunate (SP-AS) the failure rate was 5.3%. Such a large difference could be explained by the relatively short 28-day follow-up period, with a substantial number of recurrent infections possibly occurring after day 28. This paper reports the treatment outcome observed in the same study cohort beyond the initial 28-day follow-up. METHODS: After the 28-day follow-up, children treated with either chloroquine alone (CQ), SP or SP-AS, were visited at home twice a week until day 9 after treatment. A blood sample was collected if the child had fever (axillary temperature > or =37.5 degrees C). Total clinical failure for each treatment group was estimated by combining all the early treatment failures and late clinical failures that occurred over the whole follow-up period, i.e. from day up to day 9 . Pre-treatment randomly selected blood samples were genotyped for the dhfr gene (59) and the dhps gene (437 and 54 ) point mutations related to SP resistance. RESULTS: The PCR-corrected clinical failure at day 9 was significantly lower in the SP-AS group (SP-AS: 2.7%, SP alone: 38.2%; CQ: 41.1%) (Log-Rank p < , 1). The most prevalent haplotype was dhfr Arg-59 with the dhps Gly-437 mutant and the dhps 54 wild type (85.5%). The dhps 54 mutation could be found in only three (8.3%) samples. CONCLUSION: Combining artesunate to SP dramatically increased the treatment efficacy, even when extending the follow-up to day 9 post-treatment, and despite the high percentage of failures following treatment with SP alone. Such a good performance may be explained by the low prevalence of the dhps 54 mutation, by the rapid parasite clearance with artesunate and by the level of acquired immunity.

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

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

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

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