Anderson, T.J., S. Nair, H. Qin, S. Singlam, A. Brockman, L. Paiphun, and F. Nosten, Are Transporter Genes Other than the Chloroquine Resistance Locus (pfcrt) and Multidrug Resistance Gene (pfmdr) Associated with Antimalarial Drug Resistance? Antimicrob Agents Chemother, 2005. 49(6): p. 2180-8.Pubmed
Mu et al. (Mu, J., M. T. Ferdig, X. Feng, D. A. Joy, J. Duan, T. Furuya, G. Subramanian, L. Aravind, R. A. Cooper, J. C. Wootton, M. Xiong, and X. Z. Su, Mol. Microbiol. 49:977-989, 2003) recently reported exciting associations between nine new candidate transporter genes and in vitro resistance to chloroquine (CQ) and quinine (QN), with six of these loci showing association with CQ or QN in a southeast Asian population sample. We replicated and extended this work by examining polymorphisms in these genes and in vitro resistance to eight drugs in parasites collected from the Thailand-Burma border. To minimize problems of multiple testing, we used a two-phase study design, while to minimize problems caused by population structure, we analyzed parasite isolates collected from a single clinic. We first examined associations between genotype and drug response in 108 unique single-clone parasite isolates. We found strong associations between single nucleotide polymorphisms in pfmdr and mefloquine (MFQ), artesunate (AS), and lumefantrine (LUM) response. We also observed associations between an ABC transporter (G7) and response to QN and AS and between another ABC transporter (G49) and response to dihydro-artemisinin (DHA). We reexamined significant associations in an independent sample of 199 unique single-clone infections from the same location. The significant associations with pfmdr-1042 detected in the first survey remained. However, with the exception of the G7-artesunate association, all other associations observed with the nine new candidate transporters disappeared. We also examined linkage disequilibrium (LD) between markers and phenotypic correlations between drug responses. We found minimal LD between genes. Furthermore, we found no correlation between chloroquine and quinine responses, although we did find expected strong correlations between MFQ, QN, AS, DHA, and LUM. To conclude, we found no evidence for an association between 8/9 candidate genes and response to eight different antimalarial drugs. However, the consistent association observed between a 3-bp indel in G7 and AS response merits further investigation.

Anderson, T.J.C., S. Nair, D. Sudimack, J.T. Williams, M. Mayxay, P.N. Newton, J.-P. Guthmann, F.M. Smithuis, T.T. Hien, I.V.F. van den Broek, N.J. White, and F. Nosten, Geographical Distribution of Selected and Putatively Neutral SNPs in Southeast Asian Malaria Parasites 10.1093/molbev/msi235. Mol Biol Evol, 2005. 22(12): p. 2362-2374.Pubmed
Loci targeted by directional selection are expected to show elevated geographical population structure relative to neutral loci, and a flurry of recent papers have used this rationale to search for genome regions involved in adaptation. Studies of functional mutations that are known to be under selection are particularly useful for assessing the utility of this approach. Antimalarial drug treatment regimes vary considerably between countries in Southeast Asia selecting for local adaptation at parasite loci underlying resistance. We compared the population structure revealed by 10 nonsynonymous mutations (nonsynonymous single-nucleotide polymorphisms [nsSNPs]) in four loci that are known to be involved in antimalarial drug resistance, with patterns revealed by 10 synonymous mutations (synonymous single-nucleotide polymorphisms [sSNPs]) in housekeeping genes or genes of unknown function in 755 Plasmodium falciparum infections collected from 13 populations in six Southeast Asian countries. Allele frequencies at known nsSNPs underlying resistance varied markedly between locations (FST = 0.18-0.66), with the highest frequencies on the Thailand-Burma border and the lowest frequencies in neighboring Lao PDR. In contrast, we found weak but significant geographic structure (FST = 0-0.14) for 8 of 10 sSNPs. Importantly, all 10 nsSNPs showed significantly higher FST (P < 8 x 10-5) than simulated neutral expectations based on observed FST values in the putatively neutral sSNPs. This result was unaffected by the methods used to estimate allele frequencies or the number of populations used in the simulations. Given that dense single-nucleotide polymorphism (SNP) maps and rapid SNP assay methods are now available for P. falciparum, comparing genetic differentiation across the genome may provide a valuable aid to identifying parasite loci underlying local adaptation to drug treatment regimes or other selective forces. However, the high proportion of polymorphic sites that appear to be under balancing selection (or linked to selected sites) in the P. falciparum genome violates the central assumption that selected sites are rare, which complicates identification of outlier loci, and suggests that caution is needed when using this approach.

Ashley, E.A., R. McGready, R. Hutagalung, L. Phaiphun, T. Slight, S. Proux, K.L. Thwai, M. Barends, S. Looareesuwan, N.J. White, and F. Nosten, A Randomized, Controlled Study of a Simple, Once-Daily Regimen of Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated, Multidrug-Resistant Falciparum Malaria. Clinical Infectious Diseases, 2005. 41(4): p. 425-432.Pubmed
Background. Dihydroartemisinin-piperaquine (DP) is a fixed-combination antimalarial drug increasingly deployed in Southeast Asia. The current regimen involves 4 doses given over 3 days. Simplification of the dose regimen should facilitate treatment adherence and thereby increase effectiveness. Methods. In a randomized, controlled, 3-arm trial conducted along the northwestern border of Thailand, the standard 4-dose course of DP (DP4) was compared to an equivalent dose given as a once-daily regimen (DP3) and to the standard treatment of mefloquine-artesunate (MAS3). Results. A total of 499 patients were included in the study. Times to fever and parasite clearance were similar in all groups. The PCR genotyping-adjusted cure rates at day 63 after treatment initiation were 95.7% (95% confidence interval [95% CI], 92.2%-98.9%) for MAS3, 100% for DP4, and 99.4% (95% CI, 98.1%-100%) for DP3. The DP4 and DP3 cure rates were significantly higher than that for MAS3 ( P=.008 and P=.03 , respectively). All regimens were well tolerated. There were 3 deaths (1 in the MAS3 group and 2 in the DP3 group), all of which were considered to be unrelated to treatment. Rates of other adverse events were comparable between the groups, except for diarrhea, which was more common in the DP4 group ( P=.05 vs. the MAS3 group). Conclusions. A once-daily, 3-dose regimen of DP is a highly efficacious treatment for multidrug-resistant falciparum malaria. This simple, safe, and relatively inexpensive fixed combination could become the treatment of choice for falciparum malaria.

Ashley, E., R. McGready, S. Proux, and F. Nosten, Malaria. Travel Medicine and Infectious Disease, 2005. In Press, Corrected Proof.Pubmed
Malaria is increasing worldwide due to the emergence and spread of drug resistant strains. This poses major health and economic problems for the population living in endemic areas and increases the risk of infections in travelers. The diagnosis of malaria relies on a biological proof of infection by microscopy or with a rapid test. The treatment must be initiated without delay preferably with an artemisinin containing regimen. Uncomplicated malaria can be treated with oral drugs while severe infections will be hospitalized and treated with injectables. Special attention will be given to the most susceptible groups: children and pregnant women.

Ashley, E.A. and N.J. White, Artemisinin-based combinations. Curr Opin Infect Dis, 2005. 18(6): p. 531-6.Pubmed
PURPOSE OF REVIEW: Artemisinin-based combination treatments have been the mainstay of treatment for falciparum malaria in Southeast Asia for more than 10 years and are now increasingly recommended as first-line treatment throughout the rest of the world. RECENT FINDINGS: A large multicentre radomised trial conducted in East Asia has shown a 35% reduction in mortality from severe malaria following treatment with parenteral artesunate compared with quinine. There is increasing evidence that artemisinin-based combination treatments are safe and rapidly effective. Artemether-lumefantrine (six doses) has been shown to be very effective in large trials reported from Uganda and Tanzania. A once daily three-dose treatment of dihydroartemisinin piperaquine, a newer fixed combination, was a highly efficacious and well tolerated treatment for multi-drug resistant falciparum malaria in Southeast Asia. SUMMARY: Early diagnosis and treatment of uncomplicated malaria with effective drugs remains a priority as part of a comprehensive malaria control strategy. Artemisinin-based combination treatments have consistently been shown to be highly effective and safe. The challenge is to make them accessible in tropical countries.

Carrara, V.I. and F. Nosten, Associations medicamenteuses dans le traitement du paludisme-le role des derives de l'artemisinine. La Lettre de l'Infectiologue, 2005. 20(5): p. 172-178.Pubmed

Dondorp, A., F. Nosten, K. Stepniewska, N. Day, and N. White, Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet, 2005. 366(9487): p. 717-25.Pubmed
BACKGROUND: In the treatment of severe malaria, intravenous artesunate is more rapidly acting than intravenous quinine in terms of parasite clearance, is safer, and is simpler to administer, but whether it can reduce mortality is uncertain. METHODS: We did an open-label randomised controlled trial in patients admitted to hospital with severe falciparum malaria in Bangladesh, India, Indonesia, and Myanmar. We assigned individuals intravenous artesunate 2.4 mg/kg bodyweight given as a bolus (n=730) at 0, 12, and 24 h, and then daily, or intravenous quinine (20 mg salt per kg loading dose infused over 4 h then 10 mg/kg infused over 2-8 h three times a day; n=731). Oral medication was substituted when possible to complete treatment. Our primary endpoint was death from severe malaria, and analysis was by intention to treat. FINDINGS: We assessed all patients randomised for the primary endpoint. Mortality in artesunate recipients was 15% (107 of 730) compared with 22% (164 of 731) in quinine recipients; an absolute reduction of 34.7% (95% CI 18.5-47.6%; p=0.0002). Treatment with artesunate was well tolerated, whereas quinine was associated with hypoglycaemia (relative risk 3.2, 1.3-7.8; p=0.009). INTERPRETATION: Artesunate should become the treatment of choice for severe falciparum malaria in adults.

Gupta, R.K., M. van Vugt, L. Paiphun, T. Slight, S. Looareesuwan, N.J. White, and F. Nosten, No Evidence of Cardiotoxicity of Atovaquone-Proguanil Alone or in Combination with Artesunate. Am J Trop Med Hyg, 2005. 73(2): p. 267-268.Pubmed
Combinations are set to become the mainstay in treatment and prophylaxis of malaria due to Plasmodium falciparum. Various antimalarials have been implicated in cardiotoxicity via prolongation of the QTc interval. Atovaquone-proguanil is an effective and increasingly popular antimalarial choice when used alone or with artesunate in areas of drug resistance. We report the results of an investigation carried out on the Thai-Burmese border in 42 patients randomized to receive either atovaquone-proguanil or atovaquone-proguanil-artesunate for three days. Electrocardiographic recordings were made at baseline and one hour after each dose. There was no statistically significant change in QTc interval between baseline and any subsequent readings in either treatment group or the cohort as a whole. We conclude that atovaquone-proguanil shows no evidence of cardiotoxicity either alone or when combined with artesunate.

Hutagalung, R., L. Paiphun, E.A. Ashley, R. McGready, A. Brockman, K.L. Thwai, P. Singhasivanon, T. Jelinek, N.J. White, and F.H. Nosten, A randomized trial of artemether-lumefantrine versus mefloquine-artesunate for the treatment of uncomplicated multi-drug resistant Plasmodium falciparum on the western border of Thailand. Malar J, 2005. 4(1): p. 46.Pubmed
BACKGROUND: The use of antimalarial drug combinations with artemisinin derivatives is recommended to overcome drug resistance in Plasmodium falciparum. The fixed combination of oral artemether-lumefantrine, an artemisinin combination therapy (ACT) is highly effective and well tolerated. It is the only registered fixed combination containing an artemisinin. The trial presented here was conducted to monitor the efficacy of the six-dose regimen of artemether-lumefantrine (ALN) in an area of multi-drug resistance, along the Thai-Myanmar border. METHODS: The trial was an open-label, two-arm, randomized study comparing artemether-lumefantrine and mefloquine-artesunate for the treatment of uncomplicated falciparum malaria with 42 days of follow up. Parasite genotyping by polymerase chain reaction (PCR) was used to distinguish recrudescent from newly acquired P. falciparum infections. The PCR adjusted cure rates were evaluated by survival analysis. RESULTS: In 2001-2002 a total of 490 patients with slide confirmed uncomplicated P. falciparum malaria were randomly assigned to receive artemether-lumefantrine (n=245) or artesunate and mefloquine (n=245) and were followed for 42 days. All patients had rapid initial clinical and parasitological responses. In both groups, the PCR adjusted cure rates by day 42 were high: 98.8% (95% CI 96.4, 99.6%) for artemether-lumefantrine and 96.3% (95% CI 93.1, 98.0%) for artesunate-mefloquine. Both regimens were very well tolerated with no serious adverse events observed attributable to either combination. CONCLUSION: Overall, this study confirms that these two artemisinin-based combinations remain highly effective and result in equivalent therapeutic responses in the treatment of highly drug-resistant falciparum malaria.

Kumkhaek, C., K. Phra-Ek, L. Renia, P. Singhasivanon, S. Looareesuwan, C. Hirunpetcharat, N.J. White, A. Brockman, A.C. Gruner, N. Lebrun, A. Alloueche, F. Nosten, S. Khusmith, and G. Snounou, Are Extensive T Cell Epitope Polymorphisms in the Plasmodium falciparum Circumsporozoite Antigen, a Leading Sporozoite Vaccine Candidate, Selected by Immune Pressure? J Immunol, 2005. 175(6): p. 3935-9.Pubmed
Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.

McGready, R., E.A. Ashley, E. Moo, T. Cho, M. Barends, R. Hutagalung, S. Looareesuwan, N.J. White, and F. Nosten, A Randomized Comparison of Artesunate-Atovaquone-Proguanil versus Quinine in Treatment for Uncomplicated Falciparum Malaria during Pregnancy. J Infect Dis, 2005. 192(5): p. 846-853.Pubmed
Background. There is no safe, practical, and effective treatment for pregnant women infected with multidrug-resistant Plasmodium falciparum. Methods. We recruited pregnant Karen women in the second or third trimesters of pregnancy who had uncomplicated falciparum malaria for a randomized, open-label trial with a restricted sequential trial design of 7 days of supervised quinine (SQ7) versus 3 days of artesunate-atovaquone-proguanil (AAP). Results. Eight-one pregnant women entered the study between December 2001 and July 2003; 42 were treated with SQ7 and 39 were treated with AAP. Fever, parasite clearance, and duration of anemia were significantly better with AAP; the treatment failure rate was 7 times lower (5% [2/39] vs. 37% [15/41]; relative risk, 7.1 [95% confidence interval, 1.7-29.2]; P=.001 ). There were no significant differences in birth weight, duration of gestation, or congenital abnormality rates in newborns or in growth and developmental parameters of infants monitored for 1 year. Conclusion. AAP is a well-tolerated, effective, practical, but expensive treatment for multidrug-resistant falciparum malaria during the second or third trimesters of pregnancy. Despite the small number of subjects, our results add to the growing body of evidence that AAP is safe for the mother and the fetus.

Nacher, M., R. McGready, C. Lermoo, J. Wichiponpiboon, and F. Nosten, Photoallergy to quinine. Trop Doct, 2005. 35(2): p. 117-8.Pubmed

Nash, D., S. Nair, M. Mayxay, P.N. Newton, J.P. Guthmann, F. Nosten, and T.J. Anderson, Selection strength and hitchhiking around two anti-malarial resistance genes. Proc Biol Sci, 2005. 272(1568): p. 1153-61.Pubmed
Neutral mutations may hitchhike to high frequency when they are situated close to sites under positive selection, generating local reductions in genetic diversity. This process is thought to be an important determinant of levels of genomic variation in natural populations. The size of genome regions affected by genetic hitchhiking is expected to be dependent on the strength of selection, but there is little empirical data supporting this prediction. Here, we compare microsatellite variation around two drug resistance genes (chloroquine resistance transporter (pfcrt), chromosome 7, and dihydrofolate reductase (dhfr), chromosome 4) in malaria parasite populations exposed to strong (Thailand) or weak selection (Laos) by anti-malarial drugs. In each population, we examined the point mutations underlying resistance and length variation at 22 (chromosome 4) or 25 (chromosome 7) microsatellite markers across these chromosomes. All parasites from Thailand carried the K76T mutation in pfcrt conferring resistance to chloroquine (CQ) and 2-4 mutations in dhfr conferring resistance to pyrimethamine. By contrast, we found both wild-type and resistant alleles at both genes in Laos. There were dramatic differences in the extent of hitchhiking in the two countries. The size of genome regions affected was smaller in Laos than in Thailand. We observed significant reduction in variation relative to sensitive parasites for 34-64 kb (2-4 cM) in Laos on chromosome 4, compared with 98-137 kb (6-8 cM) in Thailand. Similarly, on chromosome 7, we observed reduced variation for 34-69 kb (2-4 cM) around pfcrt in Laos, but for 195-268 kb (11-16 cM) in Thailand. Reduction in genetic variation was also less extreme in Laos than in Thailand. Most loci were monomorphic in a 12 kb region surrounding both genes on resistant chromosomes from Thailand, whereas in Laos, even loci immediately proximal to selective sites showed some variation on resistant chromosomes. Finally, linkage disequilibrium (LD) decayed more rapidly around resistant pfcrt and dhfr alleles from Laos than from Thailand. These results demonstrate that different realizations of the same selective sweeps may vary considerably in size and shape, in a manner broadly consistent with selection history. From a practical perspective, genomic regions containing resistance genes may be most effectively located by genome-wide association in populations exposed to strong drug selection. However, the lower levels of LD surrounding resistance alleles in populations under weak selection may simplify identification of functional mutations.

Nosten, F., [Malaria control in Plasmodium falciparum resistant to multi-therapy a field opinion]. Med Trop (Mars), 2005. 65(1): p. 91-2.Pubmed

Nosten, F., R. McGready, E. Ashley, and N.J. White, Malaria misconceptions. Lancet, 2005. 365(9460): p. 653.Pubmed

Stuetz, W., R. McGready, T. Cho, T. Prapamontol, H.K. Biesalski, K. Stepniewska, and F. Nosten, Relation of DDT residues to plasma retinol, a-tocopherol, and ß-carotene during pregnancy and malaria infection: A case–control study in Karen women in northern Thailand. Science of The Total Environment, 2005. In Press, Corrected Proof.Pubmed
Populations living in endemic malaria areas maybe exposed simultaneously to DDT and malaria infection. DDT may impair status of vitamins, which are implicated in the immunity and pathophysiology of malaria. To explore possible interactions, DDT residues, retinol, a-tocopherol, ß-carotene and cholesterol were measured in plasma samples of malaria-infected pregnant women (cases, n = 50) and age matched malaria-free controls (n = 58). DDT residues were found in all samples: mean (sd) total DDT levels of 29.7 and 32.7 ng/ml in cases and controls, respectively. Mean (sd) p,p'-DDT was higher in the controls than the cases (13.5 vs. 9.5 ng/ml, p = 0.006). Malaria infection was associated with lower mean (sd) plasma retinol (0.69 vs. 1.23 µmol/L) and cholesterol (2.62 vs. 3.48 mmol/L) compared to controls (p < 0.001). Mean (sd) plasma a-tocopherol (7.65 vs. 15.58 µmol/L) and a-tocopherol / cholesterol ratio (2.3 vs. 6.7 µmol/L/mmol/L) were significantly lower among the controls (p < 0.001). Mean (sd) plasma ß-carotene was low (< 0.3 µmol/L) in both groups, but higher among malaria cases (0.19 vs. 0.15 µmol/L). Plasma retinol among the controls showed highly significant positive correlations with individual DDT compounds, particularly with p,p'-DDT (r = 0.51, p < 0.001). Plasma a-tocopherol and ß-carotene seemed not to be affected by DDT residues.

van den Broek, I.V., U.A. Maung, A. Peters, L. Liem, M. Kamal, M. Rahman, M.R. Rahman, A.M. Bangali, S. Das, M. Barends, and A.M. Faiz, Efficacy of chloroquine + sulfadoxine-pyrimethamine, mefloquine + artesunate and artemether + lumefantrine combination therapies to treat Plasmodium falciparum malaria in the Chittagong Hill Tracts, Bangladesh. Trans R Soc Trop Med Hyg, 2005.Pubmed
Bangladesh faces growing levels of Plasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP). Alternative antimalarial therapies, particularly combination regimens, need to be considered. Therefore, the efficacy of three antimalarial combination therapies was assessed in Chittagong Hill Tracts. A total of 364 P. falciparum patients were recruited and randomly assigned to either CQ+SP, mefloquine+artesunate (MQ+AS) or lumefantrine+artemether (Coartem((c))). Results showed that CQ+SP therapy was less effective than the two artemisinin-based combination therapies. The day 42 PCR-corrected efficacy rate was 62.4% for CQ+SP, 100% for MQ+AS and 97.1% for Coartem. Failures occurred at a shorter interval after CQ+SP treatment than after Coartem. The artemisinin-based therapies effectively prevented development of gametocytes, whereas CQ+SP did not. All three therapies were well tolerated, although reports of mild complaints during treatment appeared higher with MQ+AS. We conclude that CQ+SP is not a viable option for replacing CQ monotherapy as first-line P. falciparum treatment in this area of Bangladesh. A change to artemisinin-based combination therapy is recommended. Both Coartem and MQ+AS appear to be good options, effective in curing P. falciparum malaria and in preventing recrudescences following treatment.