Dhps Genes Research Articles

Absence of dihydropteroate synthase gene mutations inPneumocystis jiroveciiisolated from Swedish patients

Pneumocystis jirovecii remains an important cause of pneumonia in the immunocompromised host, with the largest group of patients at risk for P. jirovecii pneumonia (PCP) in Sweden being those with haematological diseases. Widespread prophylaxis and treatment for P. jirovecii with sulfa-containing drugs have effectively decreased the incidence of PCP, but concerns have been raised about the possible emergence of P. jirovecii isolates that are resistant to these drugs. Two point mutations in the gene coding for the dihydropteroate synthase enzyme (DHPS) in P. jirovecii have been shown to be associated with prior exposure to sulfa drugs. We retrospectively studied the occurrence of P. jirovecii DHPS mutations in isolates recovered from 103 Swedish patients. The DHPS gene, including the polymorphic positions 165 and 171, were amplified and sequenced by pyrosequencing technology. All the clinical specimens showed a wild-type pattern indicating that the occurrence of P. jirovecii DHPS mutations in Sweden is very low or absent.

Molecular epidemiology of Plasmodium vivax anti-folate resistance in India

BackgroundSulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent.MethodsMicroscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes.ResultsSequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p < 0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent.ConclusionStudy suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.

Plasmodium vivax: Prevalence of mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium vivax clinical isolates from Pakistan

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine–pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR–RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I 13P 33F 57S 58T 61S 117I 173/A 383A 553 (43.9%), I 13P 33F 57S 58T 61 N 117I 173/A 383A 553 (33.6%) and I 13P 33F 57 R 58T 61 N 117I 173/A 383A 553 (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.

Effect of trimethoprim-sulphamethoxazole on the risk of malaria in HIV-infected Ugandan children living in an area of widespread antifolate resistance

BackgroundDaily trimethoprim-sulfamethoxazole (TS) protects against malaria, but efficacy may be diminished as anti-folate resistance increases. This study assessed the incidence of falciparum malaria and the prevalence of resistance-conferring Plasmodium falciparum mutations in HIV-infected children receiving daily TS and HIV-uninfected children not taking TS.Materials and methodsSubjects were 292 HIV-infected and 517 uninfected children from two cohort studies in Kampala, Uganda observed from August 2006 to December 2008. Daily TS was given to HIV-infected, but not HIV-uninfected children and all participants were provided an insecticide-treated bed net. Standardized protocols were used to measure the incidence of malaria and identify markers of antifolate resistance.ResultsSixty-five episodes of falciparum malaria occurred in HIV-infected and 491 episodes in uninfected children during the observation period. TS was associated with a protective efficacy of 80% (0.10 vs. 0.45 episodes per person year, p < 0.001), and efficacy did not vary over three consecutive 9.5 month periods (81%, 74%, 80% respectively, p = 0.506). The prevalences of dhfr 51I, 108N, and 59R and dhps 437G and 540E mutations were each over 90% among parasites infecting both HIV-infected and uninfected children. Prevalence of the dhfr 164L mutation, which is associated with high-level resistance, was significantly higher in parasites from HIV-infected compared to uninfected children (8% vs. 1%, p = 0.001). Sequencing of the dhfr and dhps genes identified only one additional polymorphism, dhps 581G, in 2 of 30 samples from HIV-infected and 0 of 54 samples from uninfected children.ConclusionDespite high prevalence of known anti-folate resistance-mediating mutations, TS prophylaxis was highly effective against malaria, but was associated with presence of dhfr 164L mutation.

Tracing the origins and signatures of selection of antifolate resistance in island populations of Plasmodium falciparum

BackgroundResistance 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.MethodsBlood samples were collected in July and December 2004 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.ResultsAn 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.ConclusionsThis 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.

Co-existence of Dihydrofolate Reductase (dhfr108) Gene with Plasmodium falciparum Chloroquine Resistance Transporter Gene (Pfcrt T76) in P. falciparum Isolates from Gezira State, Central Sudan~!2009-07-01~!2009-09-06~!2010-05-11~!

Malaria parasite multi-drug resistance poses serious health problems in tropical countries. The aim of this study was to assess the Sulfadoxine-pyrimethamine (S/P) resistance of Plasmodium falciparum parasite in central Sudan, using the molecular markers. The genotying of P. falciparum parasite from forty-four patients using RFLP and PCR showed that the polymorphism of dhfr gene was in codons 51, 59 and 108. In codon 51; two strains (4.5%) were mutant type; 3 (6.8%) were found as mixed infection (both mutant and wild types) and 28 (63.6%) were found as wild type. One sample (2.2 %) was dhfr 59 mutant and 31 (70.4. %) were wild type, while 14 (31.8%) were dhfr 108 mutant; three (6.8%) were found as mixed infection and 24 (54.5%) were wild types. The Screening of dhps 540 polymorphisms of the gene revealed that 2 (4.5%) were found as mixed infection, and 42 (95.5%) as wild type. Fifteen samples were analyzed for Pfcrt T76, and Pfmdr-1 Y 86 for CQ resistant polymorphisms from the current study, the result showed that 33.3% were found to be mutant at dhfr 108 and PfcrtT76 genes reflecting the link between dhfr108 and Pfcrt76 genes. In conclusion, the polymorphism in the dhfr and dhps genes in central Sudan are increasing, but less abundant compared to the neighboring countries. However, the current studies indicate the link between dhfr108 and Pfcrt76 genes. Therefore, further study is need for using the S/P in areas that confirmed with chloroquine resistant strains.

Active case detection, treatment of falciparum malaria with combined chloroquine and sulphadoxine/pyrimethamine and vivax malaria with chloroquine and molecular markers of anti-malarial resistance in the Republic of Vanuatu

BackgroundChloroquine-resistant Plasmodium falciparum was first described in the Republic of Vanuatu in the early 1980s. In 1991, the Vanuatu Ministry of Health instituted new treatment guidelines for uncomplicated P. falciparum infection consisting of chloroquine/sulphadoxine-pyrimethamine combination therapy. Chloroquine remains the recommended treatment for Plasmodium vivax.MethodsIn 2005, cross-sectional blood surveys at 45 sites on Malo Island were conducted and 4,060 adults and children screened for malaria. Of those screened, 203 volunteer study subjects without malaria at the time of screening were followed for 13 weeks to observe peak seasonal incidence of infection. Another 54 subjects with malaria were followed over a 28-day period to determine efficacy of anti-malarial therapy; chloroquine alone for P. vivax and chloroquine/sulphadoxine-pyrimethamine for P. falciparum infections.ResultsThe overall prevalence of parasitaemia by mass blood screening was 6%, equally divided between P. falciparum and P. vivax. Twenty percent and 23% of participants with patent P. vivax and P. falciparum parasitaemia, respectively, were febrile at the time of screening. In the incidence study cohort, after 2,303 person-weeks of follow-up, the incidence density of malaria was 1.3 cases per person-year with P. vivax predominating. Among individuals participating in the clinical trial, the 28-day chloroquine P. vivax cure rate was 100%. The 28-day chloroquine/sulphadoxine-pyrimethamine P. falciparum cure rate was 97%. The single treatment failure, confirmed by merozoite surface protein-2 genotyping, was classified as a day 28 late parasitological treatment failure. All P. falciparum isolates carried the Thr-76 pfcrt mutant allele and the double Asn-108 + Arg-59 dhfr mutant alleles. Dhps mutant alleles were not detected in the study sample.ConclusionPeak seasonal malaria prevalence on Malo Island reached hypoendemic levels during the study observation period. The only in vivo malaria drug efficacy trial thus far published from the Republic of Vanuatu showed chloroquine/sulphadoxine-pyrimethamine combination therapy for P. falciparum and chloroquine alone for P. vivax to be highly efficacious. Although the chloroquine-resistant pfcrt allele was present in all P. falciparum isolates, mutant alleles in the dhfr and dhps genes do not yet occur to the extent required to confer sulphadoxine-pyrimethamine resistance in this population.

Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan

BackgroundAnalysis of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations in Plasmodium vivax wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the dhfr and dhps genes in P. vivax clinical isolates circulating in two malaria endemic areas in Afghanistan.MethodsP. vivax clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of the pvdhps genes using PCR-RFLP methods.ResultsAll 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of pvdhfr and pvdhps haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I13P33F57S58T61N 117I173/A383A553 (6.4%).Double (I13P33S57R58T61N117I173/A383A553) and triple mutant haplotypes (I13P33S57R 58T61N117I173/G383A553) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates.ConclusionThe present study shows a limited polymorphism in pvdhfr from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the dhfr and dhps genes in both P. vivax and Plasmodium falciparum isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of P. vivax and P. falciparum molecular markers are needed to monitor the development of resistance to SP in the region.

Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas

Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

Efficacy of amodiaquine, sulphadoxine-pyrimethamine and their combination for the treatment of uncomplicated Plasmodium falciparum malaria in children in Cameroon at the time of policy change to artemisinin-based combination therapy.

BackgroundThe 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.MethodsSeven 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.ResultsAfter correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 70.1% and 80.9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 80.3%, 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 22.08% (86Y). There was a significant variation (p < 0.001) 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 540E mutation on the dhps gene was extremely rare (0.3%) and occurred only in Mutengene while the pfmdr1 1034K and 1040D mutations were not detected in any of the three sites.ConclusionIn 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 2005.

High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh

In Bangladesh, despite the official introduction of artemisinin combination therapy in 2004, chloroquine+sulfadoxine/pyrimethamine has been used for the treatment of uncomplicated malaria. To assess the distribution of pfcrt, pfmdr1, dhfr, and dhps genotypes in Plasmodium falciparum, we conducted hospital- and community-based surveys in Bandarban, Bangladesh (near the border with Myanmar) in 2007 and 2008. Using nested PCR followed by digestion, 139 P. falciparum isolates were genotyped. We found fixation of a mutation at position 76 in pfcrt and low prevalence of a mutation at position 86 in pfmdr1. In dhfr, the highest pyrimethamine resistant genotype quadruple mutant was found in 19% of isolates, which is significantly higher prevalence than reported in a previous study in Khagrachari (1%) in 2002. Microsatellite haplotypes flanking dhfr of the quadruple mutants in Bangladesh were identical or very similar to those found in Thailand and Cambodia, indicating a common origin for the mutant in these countries. These observations suggest that the higher prevalence of the dhfr quadruple mutant in Bandarban is because of parasite migration from Myanmar. However, continuous use of sulfadoxine/pyrimethamine would have also played a role through selection for the dhfr quadruple mutant. These results indicate an urgent need to collect molecular epidemiological information regarding dhfr and dhps genes, and a review of current sulfadoxine/pyrimethamine usage with the aim of avoiding the widespread distribution of high levels of resistant parasites in Bangladesh.

High prevalence of sulfadoxine/pyrimethamine-resistant alleles of Plasmodium falciparum isolates in pregnant women at the time of introduction of intermittent preventive treatment with sulfadoxine/pyrimethamine in Gabon

The frequency of dhfr and dhps point mutations was assessed in Plasmodium falciparum isolates from pregnant women in Libreville. PCR-restriction fragment length polymorphism of polymorphic codons of the dhfr gene (51, 59 and 108) and the dhps gene (436, 437 and 540) was performed in matched peripheral and placental blood samples. The proportion of multiple mutations was high (98%), and was not different between women with and without a history of intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp/SP). The prevalence of triple dhfr mutation was 80%, and that of quadruple and quintuple mutations was 53% and 22%, respectively. The Glu540 mutation was present in two isolates. The concordance of resistant alleles in matched peripheral and placental isolates was >90% for both genes. These findings underline the need for a regular assessment of the relationship between the presence of resistant isolates and in vitro/in vivo IPTp/SP efficacy, and evaluation of an alternative drug.

Seasonal distribution of anti-malarial drug resistance alleles on the island of Sumba, Indonesia.

BackgroundDrug resistant malaria poses an increasing public health problem in Indonesia, especially eastern Indonesia, where malaria is highly endemic. Widespread chloroquine (CQ) resistance and increasing sulphadoxine-pyrimethamine (SP) resistance prompted Indonesia to adopt artemisinin-based combination therapy (ACT) as first-line therapy in 2004. To help develop a suitable malaria control programme in the district of West Sumba, the seasonal distribution of alleles known to be associated with resistance to CQ and SP among Plasmodium falciparum isolates from the region was investigated.MethodsPlasmodium falciparum isolates were collected during malariometric surveys in the wet and dry seasons in 2007 using two-stage cluster sampling. Analysis of pfcrt, pfmdr1, pfmdr1 gene copy number, dhfr, and dhps genes were done using protocols described previously.Results and DiscussionThe 76T allele of the pfcrt gene is nearing fixation in this population. Pfmdr1 mutant alleles occurred in 72.8% and 53.3%, predominantly as 1042D and 86Y alleles that are mutually exclusive. The prevalence of amplified pfmdr1 was found 41.9% and 42.8% of isolates in the wet and dry seasons, respectively. The frequency of dhfr mutant alleles was much lower, either as a single 108N mutation or paired with 59R. The 437G allele was the only mutant dhps allele detected and it was only found during dry season.ConclusionThe findings demonstrate a slighly higher distribution of drug-resistant alleles during the wet season and support the policy of replacing CQ with ACT in this area, but suggest that SP might still be effective either alone or in combination with other anti-malarials.

Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in China

In China, Chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) were abandoned for the treatment of falciparum malaria 20 years ago due to resistance. Subsequent field studies showed a trend of declining CQ and SP resistance in the country. The main purpose of this study was to analyse the molecular markers of antimalarial resistance and thereby to assess the possibility of reintroduction of CQ or SP for falciparum malaria treatment. Plasmodium falciparum field isolates were collected in 2006-2007 from Hainan and Yunnan provinces, China. Nested PCR-sequencing assays were applied to analyse the SNPs in four genes: P. falciparum chloroquine resistance transporter (pfcrt) gene, multi-drug resistance 1 (pfmdr1) gene, dihydrofolate reductase (dhfr) gene and dihydropteroate synthetase (dhps) gene. We found the widespread presence of point mutations in the dhfr and dhps genes which are associated with SP treatment failure. The molecular analyses also showed the fairly high prevalence of point mutation in the pfcrt gene which is linked to CQ resistance. The results of the present study indicate that CQ and SP should not be reintroduced for falciparum malaria treatment in the near future in China.

Multiple Origins and Regional Dispersal of Resistant dhps in African Plasmodium falciparum Malaria

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. 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 20 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. Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 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.

Extended high efficacy of the combination sulphadoxine-pyrimethamine with artesunate in children with uncomplicated falciparum malaria on the Benin coast, West Africa

BackgroundA study carried out in 2003–2005 in Southern Benin showed a day-28 sulphadoxine-pyrimethamine (SP) monotherapy failure rate greater than 40%, 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.MethodsAfter 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 90 after treatment. A blood sample was collected if the child had fever (axillary temperature ≥37.5°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 0 up to day 90. Pre-treatment randomly selected blood samples were genotyped for the dhfr gene (59) and the dhps gene (437 and 540) point mutations related to SP resistance.ResultsThe PCR-corrected clinical failure at day 90 was significantly lower in the SP-AS group (SP-AS: 2.7%, SP alone: 38.2%; CQ: 41.1%) (Log-Rank p < 0,001). The most prevalent haplotype was dhfr Arg-59 with the dhps Gly-437 mutant and the dhps 540 wild type (85.5%). The dhps 540 mutation could be found in only three (8.3%) samples.ConclusionCombining artesunate to SP dramatically increased the treatment efficacy, even when extending the follow-up to day 90 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 540 mutation, by the rapid parasite clearance with artesunate and by the level of acquired immunity.

High Resistance of Plasmodium falciparum to Sulphadoxine/Pyrimethamine in Northern Tanzania and the Emergence of dhps Resistance Mutation at Codon 581

BackgroundSulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended for intermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment of malaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated with mutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6–59 month children with uncomplicated malaria and in asymptomatic 2–10 month old infants.Methodology and Principal FindingsAn open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacy protocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 10 months old asymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to the high failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and a review of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-corrected early treatment failure was 38.8% (95% CI 26.8–50.8) and total failures by day 28 were 82.2% (95% CI 72.5–92.0). There was no significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carried parasites with mutations at codons 51, 59 and 108 in the dhfr gene and 63% carried a double mutation at codons 437 and 540. 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotype maybe associated with earlier treatment failure.ConclusionIn northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study found a new combination of parasite mutations that maybe associated with increased and earlier failure.Trial RegistrationClinicalTrials.gov NCT00361114

Submicroscopic Gametocytes and the Transmission of Antifolate-Resistant Plasmodium falciparum in Western Kenya

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

Novel point mutations in sulfadoxine resistance genes of Plasmodium falciparum from India

Point mutations in the dhfr and dhps genes of Plasmodium falciparum are associated with pyrimethamine and sulfadoxine resistance respectively. In this study we have analyzed these genes from Bikaner (situated in North-West region of India), where both uncomplicated and severe manifestations of P. falciparum malaria are seen. A majority of isolates showed double mutant allele for DHFR. In contrast, the only reported mutation present in DHPS was A437G in few isolates. In addition, three novel non-synonymous mutations were observed in the PfDHPS from this region viz., S587F, N666K and C668W. The mutations at the 666 and 668 codon seem to form a bend in the big loop region of the DHPS enzyme and may affect the binding of the drug to the enzyme. Molecular docking of sulfadoxine to this mutated structure indicates reduction in its binding affinity to this enzyme.

Molecular characterization of antifolates resistance-associated genes, (dhfr and dhps) in Plasmodium vivax isolates from the Middle East

BackgroundIn Iran, co-infections of Plasmodium vivax and Plasmodium falciparum are common and P. vivax infections are often exposed to sulphadoxine-pyrimethamine (SP). In the present study, the frequency distribution of mutations associated to SP resistance was investigated in pvdhfr and pvdhps genes from field isolates.MethodsClinical isolates of P. vivax were collected in two different malaria endemic regions in northern and south-eastern Iran, between 2001 and 2006. All 189 collected isolates were analysed for SNP/haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of pvdhps genes using nested PCR-RFLP methodsResultsAll 189 examined isolates were found to carry wild-type amino acids at positions 13, 33, 61 and 173, while 57L and 58R and 117N mutations in pure form was detected among 1.1%, 17.5% and 26% examined samples, respectively, with no polymorphisms in different loci of dhps genes. Based on size polymorphism of pvdhfr genes at repeat region, among northern isolates, the frequency distribution for type A and B were 2.2% and 97.8% respectively. However, in southern samples the prevalence of type A, B and C were 7%, 89.5% and 7.7%, respectively. Mixed genotype infections (type B and C) were detected in only 4.2% (6/143) of southern, but in none of the northern isolates. The combination of pvdhfr and pvdhps haplotypes among all 189 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were I13P33F57S58T61S117I173/A383A553 (65.6%) and I13P33F57S58T61N117I173 (16.4%). Two other alleles with one point mutation I13P33F57R58T61S117I173/A383A553 and two mutations I13P33F57R58T61N117I173/A383A553 accounted for 7.4% and 9.5% of the total isolates.ConclusionThe present molecular data provide important information for making decisions on population based drug use in Iran. In addition, since October 2005, with more availability of SP as first-line treatment, P. vivax isolates are more exposed to SP and the selection or spread of resistant pvdhfr and pvdhps alleles might increase in the near future in this region.