Pfmdr1 86Y Research Articles

Selection of Parasites with Diminished Drug Susceptibility by Amodiaquine‐Containing Antimalarial Regimens in Uganda

Amodiaquine (AQ) is paired with artesunate (AS) or sulfadoxine-pyrimethamine (SP) in recommended antimalarial regimens. It is unclear how readily AQ resistance will be selected with combination chemotherapy. We collected 61 Plasmodium falciparum samples from a cohort of Ugandan children randomized for treatment with AQ-SP, AS-AQ, or artemether-lumefantrine (AL) for uncomplicated malaria. In vitro susceptibility to monodesethylamodiaquine (MDAQ) was measured with a histidine-rich protein 2-based enzyme-linked immunosorbent assay, and potential resistance-mediating polymorphisms in pfmdr1 were evaluated. Parasites collected from patients treated with AQ-SP or AS-AQ within the prior 12 weeks were less susceptible to MDAQ (n = 18; mean of the median inhibitory concentration [IC(50)], 62.9 nmol/L; range, 12.7-158.3 nmol/L) than were parasites from those not treated within 12 weeks (n = 43; mean IC(50), 37.5 nmol/L; range, 6.3-184.7 nmol/L; P=.009) or only from those patients in the treatment arm that did not receive AQ (n = 12; mean IC(50), 28.8 nmol/L; range, 6.3-121.8 nmol/L; P = .004). The proportion of strains with polymorphisms expected to mediate diminished response to AQ (pfmdr1 86Y and 1246Y) increased after AQ therapy, although differences were not statistically significant. Prior therapy selected for diminished response to MDAQ, which suggests that AQ-containing regimens may rapidly lose efficacy in Africa. The mechanism of diminished MDAQ response is not fully explained by known mutations in pfmdr1.

Prevalence of Antimalarial Drug Resistance Mutations in Plasmodium vivax and P. falciparum from a Malaria-Endemic Area of Pakistan

To study drug resistance in Bannu district, a malaria-endemic area in Pakistan, molecular-based analyses were undertaken. In Plasmodium vivax, antifolate resistance mutations were detected in pvdhfr gene codons 57, 58, and 117, with a 117N mutation frequency of 93.5%. All P. falciparum isolates exhibited double 59R + 108N mutations in pfdhfr, whereas the triple mutant 59R + 108N + 437G haplotype was found in 31.8% isolates. Furthermore, all (100%) P. falciparum isolates exhibited the key chloroquine resistance mutation, pfcrt 76T, which is also associated with resistance to amodiaquine. Additionally, pfmdr1 86Y and D1042Y mutations were, respectively, detected in 32% and 9% isolates. These results indicate an emerging multi-drug resistance problem in P. vivax and P. falciparum malaria parasites in Pakistan.

No Seasonal Accumulation of Resistant P. falciparum when High-Dose Chloroquine Is Used

BackgroundPotentially chloroquine resistant P. falciparum, identified by the 76T haplotype in the chloroquine resistance transporter (pfcrt 76T), are highly prevalent throughout Africa. In Guinea-Bissau, normal and double dose chloroquine have respective efficacies of 34% and 78% against P.falciparum with pfcrt 76T and approximately three times the normal dose of chloroquine is routinely taken. Proportions of pfcrt 76T generally increase during high transmission seasons, as P.falciparum with pfcrt 76T commonly survive treatment with normal dose chloroquine. In Guinea-Bissau, there should be no seasonal increase of pfcrt 76T if the high doses of CQ commonly used are effective.Methods and Findings P. falciparum parasite density, age, sex, the proportion of chloroquine resistance associated haplotypes pfcrt 76T and P. falciparum multidrug resistance gene 1 86Y were assessed in 988 samples collected from children between 2002 and 2007. There was no seasonal accumulation of any allele. During the high and low transmission periods the pfcrt 76T proportions were 24% (95% CI, 21–27%) and 26% (95% CI, 20–33%). There was no significant change of pfcrt 76T (OR 1.05, 95% CI; 0.94–1.16 p = 0.39) or pfmdr1 86Y (OR 0.92, 95%CI; 0.83–1.01 p = 0.08) proportions between 2003 and 2007. Lower median parasite density (P.falciparum/µl) was associated with pfcrt 76T (15254 [95% CI, 12737–17772]; n = 164) compared to pfcrt 76K (18664 [95% CI, 16676–20653]; p = 0.003; n = 591). Similarly, pfmdr1 86Y was associated with a lower median parasite density (16320 [95% CI, 13696–18944]; n = 224) compared to pfmdr1 86N, (18880 [95% CI, 16701–21059]; P = 0.018; n = 445).ConclusionsIn contrast to the rest of Africa, P. falciparum parasites resistant to normal dose chloroquine do not have a selective advantage great enough to become the dominant P.falciparum type in Guinea-Bissau. This is most likely due to the efficacy of high-dose chloroquine as used in Guinea-Bissau, combined with a loss of fitness associated with pfcrt 76T.

In‐vivo efficacy of amodiaquine‐artesunate in children with uncomplicated Plasmodium falciparum malaria in western Kenya

To assess the efficacy of amodiaquine-artesunate in an area with high chloroquine resistance in western Kenya. Twenty-eight day in-vivo efficacy trial of amodiaquine-artesunate in 103 children aged 6-59 months in western Kenya with smear-confirmed uncomplicated Plasmodium falciparum malaria. The 28-day uncorrected adequate clinical and parasitological response (ACPR) was 69.0%, with 15.5% Late Clinical Failure and 15.5% Late Parasitologic Failure rates. The PCR-corrected 28-day ACPR was 90.2%. Clinical risk factors for recurrent infection (recrudescences and reinfections) were lower axillary temperature at enrollment and low weight-for-age Z-score. The presence of single nucleotide polymorphisms pfcrt 76T and pfmdr1 86Y at baseline was associated with increased risk of recurrent infections, both reinfections and recrudescences. Although artemether-lumefantrine (Coartem) is the first line ACT in Kenya, amodiaquine-artesunate is registered as an option for treatment of uncomplicated P. falciparum and remains an effective alternative to Coartem in western Kenya. Continued amodiaquine monotherapy in the private sector may jeopardize the future use of amodiaquine-artesunate as an alternative artemisinin-based combination therapy.

Complexity ofPlasmodium falciparumClinical Samples from Uganda during Short‐Term Culture

We cultured Plasmodium falciparum parasites from 98 Ugandan children with malaria and determined the complexity of infection (COI) on the basis of msp-2 polymorphisms daily for 9 days. The mean COI decreased during culture from 1.73 to 1.56. New strains appeared after day 0 in 20 cultures. Strains disappeared after day 0 in 56% of 45 cultures that were initially mixed; persisting strains more commonly had wild-type dhfr (C59) and dhps (K540) sequences and mutant pfmdr1 (86Y) sequences. Thus, initial genotypes offer an imperfect representation of clinical COI. Loss of strains in culture may be due to diminished fitness of some drug-resistant strains.

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002–2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt– pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai–Laos border and no cases of QN resistance were found in this study.

Association of pfcrt But Not pfmdr1 Alleles with Chloroquine Resistance in Iranian Isolates of Plasmodium falciparum

This study was designed to analyze the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) mutations as markers of chloroquine (CQ) resistance in 200 blood samples collected from malaria patients in south-eastern Iran during 2002-2005. Among these, 25 (post-treatment) fulfilled the 28-day follow-up study. A high number of Iranian P. falciparum (97%) strains harbored quadruple mutations at codons 76T, 220S, 326D, and 356L. All post-treatment isolates harbored the mutant allele 76T, but low rates of the mutant allele 86Y (44%) of the pfmdr1 gene were detected. No wild haplotype of pfcrt (72-CVMNKAQNIR-371) was found in post-treatment samples; however, 56% of clinical "failure" samples carried the wild type of pfmdr1 (NYSND). The present results suggest a strong association between pfcrt 76T, but not pfmdr1 86Y mutation and in vivo CQ resistance. Furthermore, we found the CQ resistance-associated SVMNT haplotype, which previously had been seen in South American isolates. Although Iran is located more proximally to Southeast Asia than to South America, no CQ resistance-associated CVIET haplotye has been observed in this region. Therefore, these results were not consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. In conclusion, P. falciparum mutations associated with resistance to CQ are abundant in south-eastern Iran and this finding strongly supports that CQ as the first line drug is inadequate for treatment of uncomplicated falciparum malaria in Iran.

Sulfadoxine-Pyrimethamine–Based Combinations for Malaria: A Randomised Blinded Trial to Compare Efficacy, Safety and Selection of Resistance in Malawi

BackgroundIn Malawi, there has been a return of Plasmodium falciparum sensitivity to chloroquine (CQ) since sulfadoxine-pyrimethamine (SP) replaced CQ as first line treatment for uncomplicated malaria. When used for prophylaxis, Amodiaquine (AQ) was associated with agranulocytosis but is considered safe for treatment and is increasingly being used in Africa. Here we compare the efficacy, safety and selection of resistance using SP or CQ+SP or artesunate (ART)+SP or AQ+SP for the treatment of uncomplicated falciparum malaria.Methodology and Findings455 children aged 1–5 years were recruited into a double-blinded randomised trial comparing SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p<0.001). AQ+SP had an ACPR rate of 97%, higher than CQ+SP (81%) and ART+SP (70%), p<0.001. Nineteen children developed a neutropenia of ≤0.5×103 cells/µl by day 14, more commonly after AQ+SP (p = 0.03). The mutation pfcrt 76T, associated with CQ resistance, was detected in none of the pre-treatment or post-treatment parasites. The prevalence of the pfmdr1 86Y mutation was higher after treatment with AQ+SP than after SP, p = 0.002.ConclusionsThe combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found evidence that AQ may exert selective pressure for resistance associated mutations many weeks after treatment. This study confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of pfcrt 76T after treatment with CQ or AQ. Given the safety record of AQ when used as a prophylaxis, our observations of marked falls in neutrophil counts in the AQ+SP group requires further scrutiny.Trial RegistrationControlled-Trials.com ISRCTN22075368

Chloroquine‐resistance molecular markers (Pfcrt T76 and Pfmdr‐1 Y86) and amodiaquine resistance in Burkina Faso

We investigated the relationship between the two main molecular markers for chloroquine resistance (Pfcrt T76 and Pfmdr-1 Y86) and the clinical efficacy of amodiaquine in Burkina Faso. Before treatment, the prevalence of Pfcrt T76, Pfmdr-1 Y86 or both mutations in the same infection was significantly higher in patients who experienced a recrudescence than in those who successfully responded to the treatment. Therefore, these two molecular markers could be useful in monitoring amodiaquine resistance, particularly in countries where this drug is used in combination with artesunate as first- or second-line treatment.

Prevalence of mutations associated with antimalarial drugs in Plasmodium falciparum isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran

BackgroundThis work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran.MethodsThe prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005.ResultsAll P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas.ConclusionFinding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria.

PREVALENT PFMDR1 N86Y MUTANT PLASMODIUM FALCIPARUM IN MADAGASCAR DESPITE ABSENCE OF PFCRT MUTANT STRAINS

We assessed the status of point mutations associated with chloroquine resistance in pfcrt codon 76 and in pfmdr1 codon 86 among Plasmodium falciparum isolates from symptomatic patients in 3 sites in Madagascar. The in vitro susceptibility of P. falciparum isolates to quinoline-containing drugs was also determined. All isolates (N = 117) successfully typed were pfcrt wild-type, except one from Tsiroanomandidy (1 of 27). However, 67.5% (95% CI: 58.2-75.9%) of these isolates contained mutant pfmdr1 86Y. The pfmdr1 N86Y mutation is associated with higher mefloquine susceptibility, but it did not affect the sensitivity of parasites to chloroquine or quinine. Our findings demonstrate that pfmdr1 mutant P. falciparum are prevalent in Madagascar and confirm the low prevalence of pfcrt mutant P. falciparum after 60 years of chloroquine use. They provide additional field-based evidence for increased mefloquine susceptibility in pfmdr1 mutant P. falciparum and are suggestive of the intrahost selection of pfmdr1 mutant parasites.

Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria

BackgroundChloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of parasite populations is, therefore, important to decide whether chloroquine could be re-introduced.MethodsTo estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1 N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET) method on a real-time PCR instrument.Results116 children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at enrollment and during follow-up for identification of parasite carrying the chloroquine resistant transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in both pre- and post-treatment isolates. No association was observed between age or initial level of parasitaemia and chloroquine treatment outcome.ConclusionThe result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1 mutation detection and also give further evidence to the reliability of the pfcrt T76 point mutation as a molecular marker for CQ resistance.

Selection of pfmdr1 mutations after amodiaquine monotherapy and amodiaquine plus artemisinin combination therapy in East Africa

Despite the pharmacodynamic advantages with artemisinin-based combination therapy (ACT) and some potentially opposite molecular mechanisms of tolerance to amodiaquine (AQ)/desethylamodiaquine (DEAQ) and artesunate (ART), there is a risk for rapid decay in efficacy if the two drugs are unable to ensure mutual prevention against a selection and spread of drug-resistant parasites. We have studied if mutations in the pfcrt and pfmdr1 genes selected in recurrent infections after AQ monotherapy are also selected after AQ plus ART combination therapy. Samples for molecular analysis were derived from three clinical trials on children <5 years old with uncomplicated Plasmodium falciparum malaria; one AQ monotherapy study conducted in Kenya 2003 and two AQ plus ART combination therapy studies conducted in Zanzibar 2002–2003 and 2005, respectively. The PCR-adjusted treatment failure rates in the three studies were 19%, 8% and 9%, respectively. After monotherapy there was a significant selection of pfcrt 76T in re-infections (OR not calculable; p = 0.048) and of pfmdr1 86Y in recrudescent infections (OR 8.0; p = 0.048). No such selection was found after combination therapy. A selection of pfmdr1 1246Y and the pfmdr1 haplotype (a.a 86, 184, 1246) YYY was found in recrudescent infections both after monotherapy (OR 7.6; p = 0.009 and OR 3.1; p = 0.029) and combination therapy in 2005 (OR 3.6; p = 0.017 and OR 5.4; p < 0.001). Hence, pfmdr1 1246Y with synergistic or compensatory addition of pfmdr1 86Y and 184Y appears to be involved in AQ/DEAQ resistance and treatment failure. Our results suggest that ART may protect against a selection of these SNPs initially, but maybe not after continuous drug pressure in a population. However, treatment failure rate and spread of pfmdr1 SNPs may remain at a low level because of the suggested opposite selection by ART and the pharmacodynamic advantages with ACT.

Chloroquine resistant P. falciparum prevalence is low and unchanged between 1990 and 2005 in Guinea-Bissau: An effect of high chloroquine dosage?

Chloroquine resistant malaria was first reported in Guinea-Bissau in 1990 but chloroquine remains the most commonly used antimalarial in the country. Since 1990, we have conducted nearly annual standardized WHO in vitro micro-tests to assess chloroquine resistance. We have identified pfcrt 76T and other genetic polymorphisms in samples from 1992, 1993, 1995, 2004 and 2005. We have also monitored drug prescriptions for febrile illnesses. The mean proportion of in vitro tests indicating chloroquine resistance was 33% (range 14–54%) with the exception of an outlying value year 2000. The proportion of chloroquine resistant P. falciparum detected by in vitro testing did not increase over time. Pfcrt 76T was associated with chloroquine resistance but pfmdr1 86Y was not. The mean pfcrt 76T prevalence varied between 13% and 38%. The prevalence of SNPs at Pfcrt positions 76, 271, 326 and pfmdr1 position 86 did not change significantly between 1992 and 2005. In a health centre the median chloroquine dose prescribed for febrile illnesses between 1994 and 2003 was 63 mg/kg. The genetic basis of chloroquine resistance appears to be the same in Guinea-Bissau as in other countries. Despite that, the prevalence of chloroquine resistant P. falciparum has not gradually increased between 1990 and 2005 in Guinea-Bissau. Chloroquine is commonly prescribed at more than double the normal dose in Guinea Bissau. It has previously been hypothesized that treatment with high doses of chloroquine may be effective. We discuss the possibility that the delayed spread of chloroquine resistant P. falciparum in Guinea-Bissau is the result of treatment with high and effective doses of chloroquine.

Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparum in south-west Tanzania

BackgroundIn Tanzania, drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes.MethodsIn samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps, conferring SP resistance, as well in cytb which is linked to resistance to atovaquone.ResultsPfcrt T76 occurs in 50% and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected.ConclusionThese findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy.

High prevalence of drug-resistance mutations in Plasmodium falciparum and Plasmodium vivax in southern Ethiopia

BackgroundIn 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.MethodsIn 100 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.ResultsP. 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.ConclusionThese 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.

ASSOCIATION BETWEEN MUTATIONS IN PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER AND P. FALCIPARUM MULTIDRUG RESISTANCE 1 GENES AND IN VIVO AMODIAQUINE RESISTANCE IN P. FALCIPARUM MALARIA–INFECTED CHILDREN IN NIGERIA

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

ROLES OF SPECIFIC PLASMODIUM FALCIPARUM MUTATIONS IN RESISTANCE TO AMODIAQUINE AND SULFADOXINE-PYRIMETHAMINE IN BURKINA FASO

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) 108N 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 540E 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.

Field-based evidence for linkage of mutations associated with chloroquine ( pfcrt/ pfmdr1) and sulfadoxine–pyrimethamine ( pfdhfr/ pfdhps) resistance and for the fitness cost of multiple mutations in P. falciparum

Mutations in the Plasmodium falciparum pfcrt gene on chromosome 7 and possibly mutations in pfmdr1 on chromosome 5 have a role in conferring resistance against chloroquine (CQ), as do mutations of pfdhfr on chromosome 4 and pfdhps on chromosome 8 in terms of resistance against sulfadoxine/pyrimethamine (SP). The additive role of multiple mutations in the development of resistance to each drug suggests a non-random occurrence. In this study, parasite isolates were obtained from 50 patients with uncomplicated P. falciparum malaria from rural Eastern Sudan, an endemic setting with minimal overlap of infection. The parasite isolates were genotyped for detection of 12 alleles in CQ and SP resistance genes. Our main findings were: (1) the frequency of mutant alleles, pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, pfdhfr S108N, pfdhps K540E and pfdhps A581G were; 0.90, 0.86, 0.84, 0.84, 0.80 and 0.20, respectively. (2) No mutations were detected for the pfdhfr loci A16V, C59R and I164L, and for pfdhps loci S436A, A437G and A613S. (3) There was a statistically significant association between the mutations in: (i) the CQ resistance (CQR) genes, pfcrt T76 and pfmdr1 Y86 ( P ≤ 0.001), (ii) the SP resistance (SPR) genes, pfdhfr I51, pfdhfr N108 and pfdhps E540 ( P ≤ 0.001–0.04) and (iii) the CQ “i” and SP “ii” resistance genes ( P = 0.001) 4. The fitness cost of multiple mutations was revealed by a significantly reduced parasite density of isolates bearing the mutant alleles ( P = 0.048). However, the significantly higher gametocyte carriage rate among isolates with resistance mutations ( P = 0.001) is possibly an evolutionary mechanism for survival of mutant parasites.

Quinoline resistance associated polymorphisms in the pfcrt, pfmdr1 and pfmrp genes of Plasmodium falciparum in Iran

Plasmodium falciparum resistance to chloroquine (CQ) has been documented in Iran since the early 1980s and has since gradually increased. Iran is therefore reviewing its national drug policy for malaria control. We describe the prevalence of single nucleotide polymorphisms (SNP) associated with quinoline drug resistance in south eastern Iran. Pre-treatment blood from patients with uncomplicated but symptomatic P. falciparum infection was analysed. Polymorphisms at codons 76, 152, 163 and 220 of the pfcrt gene (chloroquine resistance transporter) and at codons 86, 184, 1034, 1042 and 1246 of the pfmdr1 gene (multidrug resistance) were determined by PCR–RFLP and sequencing. In addition, SNPs on a recently described multidrug resistance protein ( pfmrp) and a microsatellite (MS-4760) in the pfnhe-1 (sodium hydrogen exchanger) gene associated with quinoline and quinine resistance, respectively, were investigated for the first time in field samples not from Thailand. pfcrt 76T was found in 99% and pfmdr1 86Y in 72% of the samples. pfmrp 191H and 437S associated with decreased quinoline response were found to be absolutely linked at a frequency of 13.6%. The pfnhe-1 MS-4760 one repeat allele associated to quinine response in vitro was also detected. Sequencing of the pfcrt 72–76 haplotype revealed that SVMNT was the most common allele as previously observed in India. This suggests that pfcrt found in the Iranian P. falciparum population may have the same origin as in the P. falciparum populations in India but different from that normally found in south east Asia. In conclusion, the frequencies of quinoline resistance associated gene polymorphisms in this region suggest a population that has been significantly selected for by the long use of CQ.