Dhfr Mutations Research Articles

Carriage of Mutant Dihydrofolate Reductase and Dihydropteroate Synthase Genes among Plasmodium falciparum Isolates Recovered from Pregnant Women with Asymptomatic Infection in Lagos, Nigeria

Objective: To assess N51I, C59R and S108N polymorphisms of dihydrofolate reductase (dhfr) and A437G and K540E of dihydropteroate synthase (dhps) genes of P. falciparum isolates recovered from pregnant women with asymptomatic malaria in a coastal setting in Nigeria. Subjects and Methods: A total of 107 consenting and consecutively enrolled pregnant women (mean age ± standard deviation, 26.6 ± 4.5 years) attending antenatal care at the Iru/Victoria Island Primary Health Centre, Lagos, were screened for peripheral malaria by microscopy, by a histidine-rich protein-2-based rapid diagnostic test (RDT) and by polymerase chain reaction (PCR) using finger-pricked and dot blood samples. DNA was extracted from the blood and used for dhfr and dhps gene polymorphism analyses by PCR and restriction fragment length polymorphism. The sociodemographic and parasite data obtained were analysed. Results: Of the 107 patients, 34 (31.8%), 46 (43%) and 40 (37.4%) were found to be P. falciparum infected using microscopy, RDT and corrected RDT-PCR, respectively (p < 0.05). The prevalence of P. falciparum isolates with mutant and mixed genotypes of dhfr at codons 51, 59 and 108 was 70, 75 and 80%, respectively, and the triple mutation in the homozygous form was 35%. The prevalence of the homozygous quintuple dhfr plus dhps mutant was 5%, while that of the P. falciparum isolates with mutant or mixed genotypes of dhps at codons 437 and 540 was 37.5 and 22.5%, respectively. Conclusion: This study revealed the emergence of the K540E mutation among the parasite population in Lagos. However, it supports the implementation of the intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine with continuous effectiveness monitoring in the study area.

Declining efficacy of artesunate plus sulphadoxine-pyrimethamine in northeastern India.

BackgroundAnti-malarial drug resistance in Plasmodium falciparum in India has historically travelled from northeast India along the Myanmar border. The treatment policy for P. falciparum in the region was, therefore, changed from chloroquine to artesunate (AS) plus sulphadoxine-pyrimethamine (SP) in selected areas in 2005 and in 2008 it became the first-line treatment. Recognizing that resistance to the partner drug can limit the useful life of this combination therapy, routine in vivo and molecular monitoring of anti-malarial drug efficacy through sentinel sites was initiated in 2009.MethodsBetween May and October 2012, 190 subjects with acute uncomplicated falciparum malaria were enrolled in therapeutic efficacy studies in the states of Arunachal Pradesh, Tripura, and Mizoram. Clinical and parasitological assessments were conducted over 42 days of follow-up. Multivariate analysis was used to determine risk factors associated with treatment failure. Genotyping was done to distinguish re-infection from recrudescence as well as to determine the prevalence of molecular markers of antifolate resistance among isolates.ResultsA total of 169 patients completed 42 days of follow-up at three sites. The crude and PCR-corrected Kaplan-Meier survival estimates of AS + SP were 60.8% (95% CI: 48.0-71.4) and 76.6% (95% CI: 64.1-85.2) in Gomati, Tripura; 74.6% (95% CI: 62.0-83.6) and 81.7% (95% CI: 69.4-89.5) in Lunglei, Mizoram; and, 59.5% (95% CI: 42.0-73.2) and 82.3% (95% CI: 64.6-91.6) in Changlang, Arunachal Pradesh. Most patients with P. falciparum cleared parasitaemia within 24 hours of treatment, but eight, including three patients who failed treatment, remained parasitaemic on day 3. Risk factors associated with treatment failure included age < five years, fever at the time of enrolment and AS under dosing. No adverse events were reported. Presence of dhfr plus dhps quintuple mutation was observed predominantly in treatment failure samples.ConclusionAS + SP treatment failure was widespread in northeast India and exceeded the threshold for changing drug policy. Based on these results, in January 2013 the expert committee of the National Vector Borne Disease Control Programme formulated the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast. Continued monitoring of anti-malarial drug efficacy is essential for effective malaria control.

Origin of robustness in generating drug-resistant malaria parasites.

Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.

Substantial increase in mutations in the genes pfdhfr and pfdhps puts sulphadoxine–pyrimethamine‐based intermittent preventive treatment for malaria at risk in Burkina Faso

Sulphadoxine-pyrimethamine (SP) is widely used as intermittent preventive treatment (IPT) for malaria in pregnant women in Sub-Saharan Africa. There are reports of wide-spread SP resistance in countries where SP had once been used as a first-line treatment. It is unclear whether the development of SP resistance also affects countries where SP is mainly used in the context of IPT, as is the case in Burkina Faso. To assess the efficacy of SP-based IPT, we monitored the prevalence of SP conferring genetic mutations in the genes dhfr and dhps in Plasmodium falciparum populations in a rural area of Burkina Faso over a period of 13years. Molecular epidemiological study consisted of six consecutive cross-sectional surveys of rainy and dry seasons (2009-2012). Data from the rainy season in 2000 served as a baseline. Mutations in dhfr and dhps associated with SP resistance were analysed by pyrosequencing in 861 parasite-positive samples. The prevalence of the SP resistance conferring triple dhfr mutation 51I, 59R, 108N increased from 1.3% in the rainy season of 2000 to 35.3% in 2009, and 54.3% in 2011 (P≤0.001). Comparing rainy and dry seasons, we observed an increasing step-like pattern with higher prevalence of the dhfr triple mutant in the respective dry season compared with the preceding rainy season. The proportion of the dhps 437Gly mutation in the rainy season of 2000 was 53.2% and subsequently increased to 77.6% in 2009 (P≤0.001). The increase in molecular markers linked with SP resistance jeopardises the efficacy of IPTp and the planned IPTi interventions in Burkina Faso, calling for careful monitoring of genotypic resistance markers and in vivo validation of IPT efficacy.

CoMFA and docking study of 2,N6-disubstituted 1,2-dihydro-1,3,5-triazine-4,6-diamines as novel PfDHFR enzyme inhibitors for antimalarial activity

A three-dimensional quantitative structure–activity relationship (3D-QSAR) study was performed on 1,3,5-triazine derivatives which were based on Ala16Val+Ser108Thr mutant DHFR inhibitors of Plasmodium falciparum clone (FCR-3). Comparative Molecular Field Analysis (CoMFA) was carried out for designing novel PfDHFR enzyme inhibitors. It is shown that the steric and electrostatic properties by CoMFA contours can be related to the PfDHFR inhibitory activity. Glide-XP of Schrödinger was used for docking of PfDHFR inhibitors into the putative binding sites of the PfDHFR.

Prevalence of sulfadoxine–pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia

Ethiopia changed the first-line anti-malarial drug for uncomplicated Plasmodium falciparum malaria from sulfadoxine–pyrimethamine (SP) to Coartem® in 2004 following nation-wide assessment of the efficacy of both drugs in 2003. This study was conducted to assess the prevalence of sulfadoxine–pyrimethamine resistance-associated mutations in dhfr and dhps genes of P. falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia. A total of 165 blood spot samples were collected from patients infected with P. falciparum in Bahir Dar Health Center in 2005 (n=78) and 2008 (n=87) using Whatman (3M) filter papers. The three dhfr codons (dhfr108, dhfr 51 and dhfr 59) and the two dhps codons (dhfr 437 and 540) which are believed to determine SP resistance were detected by using nested PCR-based dot blot-hybridization technique. In dhfr, only the dhfr59Arg mutant-type showed statistically significant reduction from 80.3% in 2005 to 56.4% in 2008 (p<0.01) with a significant increase of the wild type dhfr59Cys haplotypes from 4.9% in 2005 to 29.5% in 2008 (p<0.01). The double mutants dhfr108Asn/51Ile were detected at rate of 98.4% in 2005 and 98.7% in 2008. A significant decrease in the triple dhfr (108Asn/51Ile/59Arg) mutation was observed from 2005 (78.6%) to 2008(56.4%) (p<0.01). The quadruple mutations of dhfr (108Asn/51Ile/59Arg)/dhps437Gly were significantly declined from 78.6% in 2005 to 53.8% in 2008 (p<0.01) while quintuple mutations (dhfr (108Asn/51Ile/59Arg)/dhps437Gly/dhps540Glu) showed a reduction from 60.6% to 37.2% after three years (p<0.01). In conclusion, the decline in the prevalence of dhfr/dhps combination mutations might indicate the re-emergence of sensitive parasites in the population following SP withdrawal. Therefore, further monitoring and assessment is important to determine the feasibility of re-introduction of SP alone or in combination as a more affordable and safer drug in the future in Ethiopia.

Ordered Accumulation of Mutations Conferring Resistance to Sulfadoxine-Pyrimethamine in the Plasmodium falciparum Parasite

Monitoring the prevalence of drug resistant Plasmodium falciparum is essential for effective malaria control. Resistance to pyrimethamine and sulfadoxine increases as mutations accumulate in the parasite genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), respectively. Although parasites are exposed to these antifolate drugs simultaneously, it remains virtually unknown whether dhfr and dhps mutations accumulate along interrelated paths. We investigated the order of step-wise accumulation in dhfr and dhps by cumulative analyses using binomial tests in 575 P. falciparum isolates obtained from 7 countries in Asia and Melanesia. An initial step in the accumulation of mutations preferentially occurred in dhfr (2 mutations), followed by 1 mutation in dhps. In a subsequent step, mutations were estimated separately for 5 dhfr/dhps-resistant lineages identified using 12 microsatellites flanking dhfr and dhps. Among these lineages, we found 3 major mutational paths, each of which follows a unique stepwise trajectory to produce the most highly resistant form with 4 mutations in dhfr and 3 in dhps. The ordered accumulation of mutations in dhfr and dhps elucidated here will assist in predicting the status and progression of antifolate resistance in malaria-endemic regions where antifolate drugs are used for intermittent preventive treatment.

A clinical and molecular study of artesunate + sulphadoxine-pyrimethamine in three districts of central and eastern India

BackgroundArtesunate + sulphadoxine-pyrimethamine (AS + SP) is recommended throughout India as the first-line treatment for uncomplicated falciparum malaria. Due to the presence of several eco-epidemiological zones of malaria and variable drug pressure, it is necessary to evaluate the efficacy of this combination in different regions of India. The objective of this study was to use clinical and molecular methods to monitor the efficacy of AS + SP in three diverse sites.MethodsThe study was undertaken in three high endemic sites of central and eastern India. Patients with uncomplicated falciparum malaria were enrolled and followed for 28 days. Molecular genotyping was conducted for merozoite surface protein (msp1 and msp2) to differentiate between re-infection and recrudescence and for the dhfr and dhps genes to monitor antifolate drug resistance.ResultsIn all, 149 patients were enrolled at the three sites. The crude cure rates were 95.9%, 100%, and 100% in Ranchi, Keonjhar, and West Garo Hills respectively. PCR-corrected cure rates were 100% at all sites. In dhfr, 27% of isolates had triple mutations, while 46% isolates were double-mutants. The most prevalent mutation was S108N followed by C59R. 164 L mutation was observed in 43/126 (34%) isolates. In dhps, most (76%) of the isolates were wild-type. Only 2.5% (2/80) isolates showed double mutation. dhfr-dhps two locus mutation were observed in 16% (13/80) isolates. Parasite clearance time was not related with antifolate mutations.ConclusionsAS + SP combination therapy remained effective against falciparum malaria despite common mutations promoting resistance to antifolate drugs. Although the prevalence of double and triple mutations in dhfr was high, the prevalence of dhfr-dhps two locus mutations were low. Even isolates with dhfr triple and dhfr-dhps two locus mutations achieved adequate clinical and parasitological response.

Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen

BackgroundDespite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region.MethodsFive polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz.ResultsHigh diversity was seen in non-drug genes, pfg377 (He = 0.66), msp-2 (He = 0.80) and three microsatellites on chr 8, 7.7 kb (He = 0.88), 4.3 kb (He = 0.77) and 0.8 kb (He = 0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p < 0.05). Genetic differentiation between populations was low (most pair-wise FST values <0.03), indicating extensive gene flow between the parasites in the three sites.There was a high prevalence of mutations in pfmdr1, pfcrt and dhfr; with four mutant pfmdr1 genotypes (NFCDD[57%], NFSND[21%], YFCDD[13%] and YFSND[8% ]), two mutant pfcrt genotypes (CVIET[89%] and SVMNT[4%]) and one mutant dhfr genotype (ICNI[53.7%]). However, no dhps mutations were detected.ConclusionThe high diversity of P. falciparum in Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate + sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT.

Prevalence of antifolate resistance mutations in Plasmodium falciparum isolates in Afghanistan

BackgroundArtesunate plus sulphadoxine-pyrimethamine (AS+SP) is now first-line treatment for Plasmodium falciparum infection in several south Asian countries, including Afghanistan. Molecular studies provide a sensitive means to investigate the current state of drug susceptibility to the SP component, and can also provide information on the likely efficacy of other potential forms of artemisinin-combination therapy.MethodsDuring the years 2007 to 2010, 120 blood spots from patients with P. falciparum malaria were obtained in four provinces of Afghanistan. PCR-based methods were used to detect drug-resistance mutations in dhfr, dhps, pfcrt and pfmdr1, as well as to determine copy number of pfmdr1.ResultsThe majority (95.5%) of infections had a double mutation in the dhfr gene (C59R, S108N); no mutations at dhfr positions 16, 51 or 164 were seen. Most isolates were wild type across the dhps gene, but five isolates from the provinces of Kunar and Nangarhar in eastern Afghanistan had the triple mutation A437G / K540E / A581G; all five cases were successfully treated with three receiving AS+SP and two receiving dihydroartemisinin-piperaquine. All isolates showed the pfcrt SVNMT chloroquine resistance haplotype. Five of 79 isolates had the pfmdr1 N86Y mutation, while 52 had pfmdr1 Y184F; positions 1034, 1042 and 1246 were wild type in all isolates. The pfmdr1 gene was not amplified in any sample.ConclusionsThis study indicates that shortly after the adoption of AS+SP as first-line treatment in Afghanistan, most parasites had a double mutation haplotype in dhfr, and a small number of isolates from eastern Afghanistan harboured a triple mutation haplotype in dhps. The impact of these mutations on the efficacy of AS+SP remains to be assessed in significant numbers of patients, but these results are clearly concerning since they suggest a higher degree of SP resistance than previously detected. Further focused molecular and clinical studies in this region are urgently required.

Historical shifts in Brazilian P. falciparum population structure and drug resistance alleles.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

Malaria treatment failure with novel mutation in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene in Kolkata, West Bengal, India

The aim of this work was to define the cause of sulfadoxine/pyrimethamine (SP) treatment failure in Plasmodium falciparum infections in a malaria-endemic zone of India. Samples were collected from 176 patients in Kolkata from November 2008 to July 2009. In vitro susceptibility testing was performed on all isolates. Parasite DNA was extracted, and PCR and restriction fragment length polymorphism (RFLP) analysis of different codons of the dhfr gene (16, 51, 59, 108 and 164) and dhps gene (436, 437, 540, 581 and 613) were performed. Finally, sequencing of the products was performed to confirm the mutations. The in vivo treatment response to SP among the 176 patients was determined. A novel mutation of isoleucine was observed at codon 108 of the dhfr gene, which was highly correlated with in vitro SP resistance as well as early treatment failure. A double dhfr mutation (108I+51I) was observed in 77.3% of isolates, and triple mutation of the dhps gene was observed in 18.2% of isolates. In this endemic zone, SP treatment failure is due to a novel dhfr mutation (108I+51I) and any one of the dhps mutations (S436A, A437G, A581G or A613T/S). An increase in these mutations was highly correlated with SP resistance (P<0.0001).

Increased prevalence of dhfr and dhps mutants at delivery in Malawian pregnant women receiving intermittent preventive treatment for malaria

Increased prevalence of dhfr and dhps mutants at delivery in Malawian pregnant women receiving intermittent preventive treatment for malaria

A Community-Randomized Evaluation of the Effect of Intermittent Preventive Treatment in Infants on Antimalarial Drug Resistance in Southern Tanzania

Intermittent preventive treatment in infants (IPTi) is the administration of sulfadoxine-pyrimethamine (SP) at 2, 3, and 9 months of age to prevent malaria. We investigated the influence of IPTi on drug resistance. Twenty-four areas were randomly assigned to receive or not receive IPTi. Blood collected during representative household surveys at baseline and 15 and 27 months after implementation was tested for SP and resistance markers. The frequency of SP in blood was similar in the IPTi and comparison areas at baseline and at 15 months. dhfr and dhps mutations were also similar at baseline and then increased similarly in both arms after 15 months of SP-IPTi. First-line treatment was switched from SP to artemether-lumefantrine before the final survey, when SP positivity fell among infants in comparison areas but increased in IPTi areas. This was accompanied by an increase in dhfr but not dhps mutations in IPTi areas (P = .004 and P = .18, respectively). IPTi did not increase drug pressure or the selection on dhfr and dhps mutants, when SP was the first-line malaria treatment. Introduction of artemether-lumefantrine was followed by an increase in dhfr mutations, consistent with weak selection attributable to SP-IPTi, but not by an increase in dhps mutations, suggesting a fitness cost of this mutation.

Low Prevalence of Pneumocystis pneumonia (PCP) but High Prevalence of Pneumocystis dihydropteroate synthase (dhps) Gene Mutations in HIV-Infected Persons in Uganda

Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

BackgroundMalaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti.MethodsDNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum.ResultsThirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59).ConclusionThis study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.

Distribution of Drug Resistance Genotypes in Plasmodium falciparum in an Area of Limited Parasite Diversity in Saudi Arabia

Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent.

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia

A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (He) of microsatellites around mutant dhfr alleles (He=0.312; n=60) was lower (P⩽0.05) than that around the wild-type allele (He=0.834; n=116). Also, the mutant dhfr isolates showed high He for dhps (He=0.80) and the non-drug resistance locus Pfg377 (He=0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.

Differences in selective pressure on dhps and dhfr drug resistant mutations in western Kenya

BackgroundUnderstanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions.MethodsThis study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized.ResultsBy 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate.ConclusionsThe complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations.

Association between prevalence of pyrimethamine resistance and double mutation in pfdhfr gene in West Bengal, India

Objective To find whether antifolate drug (pyrimethamine) resistance has occurred in the two malaria endemic zones (Kolkata and Purulia) of West Bengal, India. Methods Parasitic bloods were collected from patients of Kolkata and Purulia, in vitro susceptibility test were performed in those 90 isolates. Now parasitic DNA was isolated by phenol chloroform extraction method and then polymerase chain reaction and restriction fragment length polymorphism analysis of different codons of pfdhfr gene (51, 59, and 108) were assessed in Plasmodium falciparum isolates from patients in India. Results Among 45 isolates from Kolkata dhfr mutant isolates at codons 108, 51 and 59 were found in 71.11%, 100% and 15.55% isolates respectively while in Purulia mutation found in those codons were 42.22%, 57.77% and 0%, respectively. In Kolkata, the isolate having double mutation (N108 + 51I) were resistant to pyrimethamine (P < 0.01) whereas in Purulia N108 mutation is occurred with both mixed and mutant 51I mutation. Conclusions Our present findings implicate that due to enormous drug (pyrimethamine) pressure double mutation with dhfr S108N/T and N51I was highly correlated (P < 0.01) with in vitro pyrimethamine resistance in two malaria endemic region of West Bengal. The increase in the number of dhfr mutations was strongly correlated to resistance to pyrimethamine.