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Background: Plasmodium falciparum strains that are resistant to standard-dose chloroquine can be treated by higher chloroquine concentrations maintained for a longer time in vivo., Objectives: To determine the relative importance of chloroquine concentrations versus exposure time for elimination of chloroquine-susceptible and -resistant P. falciparum in vitro., Methods: Chloroquine-susceptible (3D7) and -resistant (FCR3) strains were exposed in vitro to 1, 2, 4, 8, 16 or 32 times their respective 90% inhibitory chloroquine concentrations for 3, 5, 7 or 14 days and then followed until recrudescence, or not, by 42 days after the end of exposure., Results: Exposure to chloroquine appeared to eliminate susceptible and resistant parasites, leaving small pyknotic apparently dead parasites. Chloroquine-susceptible and -resistant parasites recrudesced after 3 and 5 days of chloroquine exposure. Recrudescence occurred in one out of four 7 day exposure series but not after 14 days exposure. The median time to recrudescence was 13 to 28 days with a range of 8 to 41 days after the end of exposure. Time to recrudescence after the end of exposure increased with duration of exposure for susceptible and resistant strains (P < 0.001). Time to recrudescence did not correlate with concentrations greater than 1× IC90., Conclusions: Chloroquine-susceptible and -resistant P. falciparum probably become dormant. Elimination of dormant parasites is primarily dependent upon the duration of chloroquine exposure. Exposure to effective drug concentrations for 7 days eliminates most parasites in vitro. The results support in vivo data indicating that elimination of chloroquine-resistant P. falciparum correlates with Day 7 chloroquine concentrations., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Artesunate plus sulfadoxine-pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015-2016 after 3-4 years of ASP use, are reported., Methods: Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16-185, pfdhps 436-632 and K13 407-689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism., Results: Sulfadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively., Conclusion: The frequency of P. falciparum with reduced susceptibility to sulfadoxine-pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Higher chloroquine doses can effectively treat up to 93 to 96% of malaria infections caused by Plasmodium falciparum carrying the resistance-conferring chloroquine resistance transporter ( pfcrt ) 76T allele. The tolerability of 50 (double the standard dose) and 70 mg/kg total chloroquine doses were assessed in this study. Fifteen 4- to 8-year-old children with uncomplicated malaria were given 10 mg/kg of chloroquine twice daily for 2 days and 5 mg/kg twice daily on the third day. Fifteen additional children were given 5 mg/kg twice daily for 2 more days. Chloroquine concentrations, blood pressure, electrocardiograms (ECGs), parasite density, and adverse events were assessed until day 28. Both dosages were well tolerated, and symptoms resolved by day 3 in parallel with increasing chloroquine concentrations. The median corrected QT (QTc) interval was 12 to 26 ms higher at expected peak concentrations than at day 0 ( P  < 0.001). Pfcrt 76T was associated with delayed parasite clearance. Day 28 clinical and parasitological responses against P. falciparum with pfcrt 76T were 57% (4/7) and 67% (4/6) after treatment with 50 and 70 mg/kg, respectively. Dosages were well tolerated, and no severe cardiac adverse events occurred. The QTc interval increase was similar to that found in adults taking 25 mg/kg of chloroquine. (This study has been registered at ClinicalTrials.gov under identifier NCT01814423.)., (Copyright © 2020 American Society for Microbiology.)

Background: The fixed dose combination of artemether-lumefantrine (AL) is the most widely used treatment for uncomplicated Plasmodium falciparum malaria. Relatively lower cure rates and lumefantrine levels have been reported in young children and in pregnant women during their second and third trimester. The aim of this study was to investigate the pharmacokinetic and pharmacodynamic properties of lumefantrine and the pharmacokinetic properties of its metabolite, desbutyl-lumefantrine, in order to inform optimal dosing regimens in all patient populations., Methods and Findings: A search in PubMed, Embase, ClinicalTrials.gov, Google Scholar, conference proceedings, and the WorldWide Antimalarial Resistance Network (WWARN) pharmacology database identified 31 relevant clinical studies published between 1 January 1990 and 31 December 2012, with 4,546 patients in whom lumefantrine concentrations were measured. Under the auspices of WWARN, relevant individual concentration-time data, clinical covariates, and outcome data from 4,122 patients were made available and pooled for the meta-analysis. The developed lumefantrine population pharmacokinetic model was used for dose optimisation through in silico simulations. Venous plasma lumefantrine concentrations 7 days after starting standard AL treatment were 24.2% and 13.4% lower in children weighing <15 kg and 15-25 kg, respectively, and 20.2% lower in pregnant women compared with non-pregnant adults. Lumefantrine exposure decreased with increasing pre-treatment parasitaemia, and the dose limitation on absorption of lumefantrine was substantial. Simulations using the lumefantrine pharmacokinetic model suggest that, in young children and pregnant women beyond the first trimester, lengthening the dose regimen (twice daily for 5 days) and, to a lesser extent, intensifying the frequency of dosing (3 times daily for 3 days) would be more efficacious than using higher individual doses in the current standard treatment regimen (twice daily for 3 days). The model was developed using venous plasma data from patients receiving intact tablets with fat, and evaluations of alternative dosing regimens were consequently only representative for venous plasma after administration of intact tablets with fat. The absence of artemether-dihydroartemisinin data limited the prediction of parasite killing rates and recrudescent infections. Thus, the suggested optimised dosing schedule was based on the pharmacokinetic endpoint of lumefantrine plasma exposure at day 7., Conclusions: Our findings suggest that revised AL dosing regimens for young children and pregnant women would improve drug exposure but would require longer or more complex schedules. These dosing regimens should be evaluated in prospective clinical studies to determine whether they would improve cure rates, demonstrate adequate safety, and thereby prolong the useful therapeutic life of this valuable antimalarial treatment., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: KIB and NJW are members of the WHO Technical Expert Group (TEG) on Malaria Chemotherapy. KIB is also a member of the WHO TEG on Drug Resistance and Containment. KIB, NJW, JT and SP are members of the WHO Malaria Chemotherapy sub-group on dosage recommendations. GL, KH, FE and RB are employees of Novartis, the manufacturer of the drug that is the subject of this publication. EAA and NJW are members of the Editorial Board of PLOS Medicine. None of the authors declare any other conflict of interest.

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.

Background: To assess the effect on malaria prevalence, village specific monthly administrations of pyrimethamine, chlorproguanil, chloroquine or placebo were given to children in four previously treatment-naïve Liberian villages, 1976-78. Plasmodium falciparum in vivo resistance developed to pyrimethamine only. Selection of molecular markers of P. falciparum resistance after 2 years of treatment are reported., Methods: Blood samples were collected from 191 study children in a survey in 1978. Polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps, pfmrp1 and pfnhe1 genes were determined using PCR-based methods., Results: Pfcrt 72-76 CVIET was found in one chloroquine village sample, all remaining samples had pfcrt CVMNK. Pfmdr1 N86 prevalence was 100%. A pfmdr1 T1069 ACT→ACG synonymous polymorphism was found in 30% of chloroquine village samples and 3% of other samples (P = 0.008). Variations in pfnhe1 block I were found in all except the chloroquine treated village (P < 0.001). Resistance associated pfdhfr 108N prevalence was 2% in the pyrimethamine village compared to 45-65% elsewhere, including the placebo village (P = 0.001)., Conclusions: Chloroquine treatment possibly resulted in the development of pfcrt 72-76 CVIET. Selection of pfmdr1 T1069 ACG and a pfnhe1 block 1 genotypes indicates that chloroquine treatment exerted a selective pressure on P. falciparum. Pyrimethamine resistance associated pfdhfr 108N was present prior to the introduction of any drug. Decreased pfdhfr 108N frequency concurrent with development of pyrimethamine resistance suggests a non-pfdhfr polymorphisms mediated resistance mechanism.

Background: Artemether-lumefantrine (AL) was introduced for treatment of uncomplicated malaria in Guinea-Bissau in 2008. Malaria then resurged and recurrent malaria after treatment with AL and stock-outs of AL were common. This study therefore aimed to assess the efficacy of AL and identify an alternative second line antimalarial. Dihydroartemisinin-piperaquine (DP) was chosen as it has been shown to be safe and efficacious and to reduce the incidence of recurrent malaria., Methods and Findings: In a multicentre randomised open-label non-inferiority clinical trial, AL or DP were given over 3 days to children aged 6 months-15 years with uncomplicated P. falciparum mono-infection. Intake was observed and AL was given with milk. Children were seen on days 0, 1, 2 and 3 and then weekly days 7-42. Recurring P. falciparum were classified as recrudescence or new infections by genotyping. Between November 2012 and July 2015, 312 children were randomised to AL (n = 155) or DP (n = 157). The day 42 PCR adjusted per protocol adequate clinical and parasitological responses were 95% and 100% in the AL and DP groups respectively, Mantel-Haenszel weighted odds ratio (OR) 0.22 (95% CI 0-0.68), p = 0.022. In a modified intention to treat analysis in which treatment failures day 0 and reinfections were also considered as treatment failures adequate clinical and parasitological responses were 94% and 97% (OR 0.42 [95% CI, 0.13-1.38], p = 0.15). Parasite clearance and symptom resolution were similar with both treatments., Conclusions: Both treatments achieved the WHO recommended efficacy for antimalarials about to be adopted as policy. DP was not inferior to AL for treatment of uncomplicated P. falciparum malaria in Guinea-Bissau., Trial Registration: ClinicalTrials.gov NTC01704508., Competing Interests: The authors have declared that no competing interests exist.

Background: Due to development of multidrug-resistant Plasmodium falciparum new antimalarial therapies are needed. In Guinea-Bissau, routinely used triple standard-dose chloroquine remained effective for decades despite the existence of "chloroquine-resistant" P. falciparum. This study aimed to determine the in vivo efficacy of higher chloroquine concentrations against P. falciparum with resistance-conferring genotypes., Methods: Standard or double-dose chloroquine was given to 892 children aged <15 years with uncomplicated malaria during 3 clinical trials (2001-2008) with ≥ 35 days follow-up. The P. falciparum resistance-conferring genotype (pfcrt 76T) and day 7 chloroquine concentrations were determined. Data were divided into age groups (<5, 5-9, and 10-14 years) because concentrations increase with age when chloroquine is prescribed according to body weight., Results: Adequate clinical and parasitological responses were 14%, 38%, and 39% after standard-dose and 66%, 84%, and 91% after double-dose chloroquine in children aged <5, 5-9, and 10-14 years, respectively, and infected with P. falciparum genotypes conferring chloroquine resistance (n = 195, P < .001). In parallel, median chloroquine concentrations were 471, 688, and 809 nmol/L for standard-dose and 1040, 1494, and 1585 nmol/L for double-dose chloroquine., Conclusions: Chloroquine resistance is dose dependent and can be overcome by higher, still well-tolerated doses., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

In 2008, artemether-lumefantrine was introduced in Guinea-Bissau, West Africa, but quinine has also been commonly prescribed for the treatment of uncomplicated Plasmodium falciparum malaria. An efficacious high-dose chloroquine treatment regimen was used previously. Temporal and seasonal changes of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine, and quinine have been described. P. falciparum chloroquine resistance transporter (pfcrt) K76T, pfmdr1 gene copy numbers, pfmdr1 polymorphisms N86Y and Y184F, and pfmdr1 sequences 1034 to 1246 were determined using PCR-based methods. Blood samples came from virtually all (n=1,806) children<15 years of age who had uncomplicated P. falciparum monoinfection and presented at a health center in suburban Bissau (from 2003 to 2012). The pfcrt K76T and pfmdr1 N86Y frequencies were stable, and seasonal changes were not seen from 2003 to 2007. Since 2007, the mean annual frequencies increased (P<0.001) for pfcrt 76T (24% to 57%), pfmdr1 N86 (72% to 83%), and pfcrt 76+pfmdr1 86 TN (10% to 27%), and pfcrt 76T accumulated during the high transmission season (P=0.001). The pfmdr1 86+184 NF frequency increased from 39% to 66% (from 2003 to 2011; P=0.004). One sample had two pfmdr1 gene copies. pfcrt 76T was associated with a lower parasite density (P<0.001). Following the discontinuation of an effective chloroquine regimen, probably highly artemether-lumefantrine-susceptible P. falciparum (with pfcrt 76T) accumulated, possibly due to suboptimal use of quinine and despite a fitness cost linked to pfcrt 76T. (The studies reported here were registered at ClinicalTrials.gov under registration no. NCT00137514 [PSB-2001-chl-amo], NCT00137566 [PSB-2004-paracetamol], NCT00426439 [PSB-2006-coartem], NCT01157689 [AL-eff 2010], and NCT01704508 [Eurartesim 2012].)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine., (© The American Society of Tropical Medicine and Hygiene.)

Background: Since 2011, artesunate + sulphadoxine-pyrimethamine (ASP), instead of chloroquine, has been recommended for treatment of uncomplicated malaria in India. In Ujjain, central India, with an annual parasite index <0.1, the prevalence of drug-resistant Plasmodium falciparum is unknown. In other parts of India chloroquine and sulphadoxine-pyrimethamine-resistant P. falciparum is prevalent. The aim of this study was to determine the prevalence of anti-malarial drug resistance-associated genetic polymorphisms in P. falciparum collected in Ujjain in 2009 and 2010, prior to the introduction of ASP., Methods: Blood samples from 87 patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons Pfcrt 72-76, pfmdr1 1034-1246, pfdhfr 16-185, pfdhps 436-632 and pfnhe1 ms4760 haplotypes were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism, and pfmdr1 gene copy number by real-time PCR., Results: Sulphadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 75/78 (96%) and 70/78 (90%) samples, respectively, and pfdhps 437G was found in 7/77 (9%) samples. Double mutant pfdhfr 59R + 108 N were found in 62/76 (82%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/76 (8%) samples. Chloroquine-resistance-associated pfcrt 76 T was found in 82/87 (94%). The pfcrt 72-76 haplotypes found were: 80/84 (95%) SVMNT, 3/84 (4%) CVMNK and 1/84 (1%) CVMNT. Pfmdr1 N86 and 86Y were identified in 70/83 (84%) and 13/83 (16%) samples, respectively. Pfmdr1 S1034 + N1042 + D1246 were identified together in 70/72 (97%) of successfully sequenced samples. One pfmdr1 gene copy was found in 74/75 (99%) successfully amplified samples., Conclusion: This is the first characterization of key anti-malarial drug resistance-associated genetic markers among P. falciparum collected in Ujjain, Madhya Pradesh, India. The results indicate that the efficacy of standard dose chloroquine at the time of the study was likely to be poor, whereas ASP was likely to be efficacious, supporting the changed drug treatment policy. However, P. falciparum with reduced susceptibility to sulphadoxine-pyrimethamine is highly prevalent, highlighting the need for continuous surveillance of ASP efficacy in the study area.

Background: Plasmodium falciparum malaria is treated with 25 mg/kg of chloroquine (CQ) irrespective of age. Theoretically, CQ should be dosed according to body surface area (BSA). The effect of dosing CQ according to BSA has not been determined but doubling the dose per kg doubled the efficacy of CQ in children aged <15 years infected with P. falciparum carrying CQ resistance causing genes typical for Africa. The study aim was to determine the effect of age on CQ concentrations., Methods and Findings: Day 7 whole blood CQ concentrations were determined in 150 and 302 children treated with 25 and 50 mg/kg, respectively, in previously conducted clinical trials. CQ concentrations normalised for the dose taken in mg/kg of CQ decreased with decreasing age (p<0.001). CQ concentrations normalised for dose taken in mg/m(2) were unaffected by age. The median CQ concentration in children aged <2 years taking 50 mg/kg and in children aged 10-14 years taking 25 mg/kg were 825 (95% confidence interval [CI] 662-988) and 758 (95% CI 640-876) nmol/l, respectively (p = 0.67). The median CQ concentration in children aged 10-14 taking 50 mg/kg and children aged 0-2 taking 25 mg/kg were 1521 and 549 nmol/l. Adverse events were not age/concentration dependent., Conclusions: CQ is under-dosed in children and should ideally be dosed according to BSA. Children aged <2 years need approximately double the dose per kg to attain CQ concentrations found in children aged 10-14 years. Clinical trials assessing the efficacy of CQ in Africa are typically performed in children aged <5 years. Thus the efficacy of CQ is typically assessed in children in whom CQ is under dosed. Approximately 3 fold higher drug concentrations can probably be safely given to the youngest children. As CQ resistance is concentration dependent an alternative dosing of CQ may overcome resistance in Africa.

Background: Multidrug-resistant Plasmodium falciparum is a major threat to global malaria control. Parasites develop resistance by gradually acquiring genetic polymorphisms that decrease drug susceptibility. The aim of this study was to investigate the extent to which parasites with different genetic characteristics are able to withstand individual drug blood concentrations., Methods: We analyzed 2 clinical trials that assessed the efficacy and effectiveness of artemether-lumefantrine. As a proof of concept, we used measured day 7 lumefantrine concentrations to estimate the concentrations at which reinfections multiplied. P. falciparum multidrug resistance gene 1 (pfmdr1) genotypes of these parasites were then correlated to drug susceptibility., Results: Reinfecting parasites with the pfmdr1 N86/184F/D1246 haplotype were able to withstand lumefantrine blood concentrations 15-fold higher than those with the 86Y/Y184/1246Y haplotype., Conclusions: By estimating drug concentrations, we were able to quantify the contribution of pfmdr1 single-nucleotide polymorphisms to reduced lumefantrine susceptibility. The method can be applied to all long-half-life antimalarial drugs, enables early detection of P. falciparum with reduced drug susceptibility in vivo, and represents a novel way for unveiling molecular markers of antimalarial drug resistance.

Artemether-lumefantrine is currently first-line therapy of Plasmodium falciparum malaria in many countries. This report describes a treatment failure despite adequate drug concentrations in a traveller returning from sub-Saharan Africa. Genotyping confirmed recrudescence and suggested reduced sensitivity. Potential sub-optimal effect of artemether-lumefantrine highlights the need to follow non-immune individuals the weeks after treatment.

Background: In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras., Methods: Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods., Results: Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples., Conclusion: The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P. falciparum and P. vivax is therefore essential also in Honduras.

Background: Scarce data are available on Plasmodium falciparum anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of P. falciparum resistance associated polymorphisms in field isolates from southern Pakistan., Methods: Blood samples from 244 patients with blood-slide confirmed P. falciparum mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt K76T), multi drug resistance (pfmdr1 N86Y), dihydrofolate reductase (pfdhfr A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (pfdhps A436S, G437A and E540K) genes and pfmdr1 gene copy numbers were determined using PCR based methods., Results: The prevalence of pfcrt 76T and pfmdr1 86Y was 93% and 57%, respectively. The prevalence of pfdhfr double mutations 59R + 108N/51R + 108N was 92%. The pfdhfr triple mutation (51I, 59R, 108N) occurred in 3% of samples. The pfdhfr (51I, 59R, 108N) and pfdhps (437G, 540E) quintuple mutation was found in one isolate. Pfdhps 437G was observed in 51% and 540E in 1% of the isolates. One isolate had two pfmdr1 copies and carried the pfmdr1 86Y and pfcrt 76T alleles., Conclusions: The results indicate high prevalence of in vivo resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among P. falciparum in southern Pakistan.

Background: In 2008, Guinea-Bissau introduced artemether-lumefantrine for treatment of uncomplicated malaria. Previously, 3 times the standard dose of chloroquine, that was probably efficacious against Plasmodium falciparum with the resistance-associated chloroquine-resistance transporter (pfcrt) 76T allele, was routinely used. The present study compared the efficacy and tolerability of a double standard dose of chloroquine with the efficacy and tolerability of artemether-lumefantrine., Methods: In a randomized open-label clinical trial, artemether-lumefantrine or chloroquine (50 mg/kg) were given as 6 divided doses over 3 days to children aged 6 months--15 years who had uncomplicated P. falciparum monoinfection. Drug concentrations were measured on day 7. P. falciparum multidrug resistance gene N86Y and pfcrt K76T alleles were identified., Results: The polymerase chain reaction-adjusted day 28 and 42 treatment efficacies were 162 (97%) of 168 and 155 (97%) of 161, respectively, for artemether-lumefantrine and 150 (95%) of 158 and 138 (94%) of 148, respectively, for chloroquine. When parasites with resistance-associated pfcrt 76T were treated, the day 28 efficacy of chloroquine was 87%. No severe drug-related adverse events were detected. Symptom resolution was similar with both treatments., Conclusions: Both treatments achieved the World Health Organization-recommended efficacy for antimalarials that will be adopted as policy. High-dose chloroquine treatment regimes should be further evaluated with the aim of assessing chloroquine as a potential partner drug to artemisinin derivatives. Clinical trials registration. NCT00426439.

Background: Potentially 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/microl) 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)., Conclusions: In 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.

High chloroquine doses are commonly prescribed in Guinea-Bissau. Double-dose chloroquine has been shown to be more efficacious (92% efficacy) than the standard dose (80% efficacy). However, chloroquine is toxic when overdosed, and it was not known if the high doses prescribed in Guinea-Bissau were taken or whether they caused adverse effects. We aimed to determine the dosage of chloroquine commonly prescribed, the doses commonly taken, and whether concentration-dependent adverse events occurred in routine practice. Chloroquine prescriptions by eight physicians and chloroquine intake by 102 children were recorded. Chloroquine intake and adverse events were assessed by questioning. Chloroquine concentrations in whole blood were measured. The median total chloroquine dose prescribed and that reportedly taken were 81 and 77 mg kg(-1), respectively. The total dose was usually split into two to three daily doses of 6.6 mg kg(-1) each. These were taken unsupervised for a median of 5 days. Forty percent of the study children had chloroquine concentrations in the same range as those found in a previous study in which double the normal dose (50 mg kg(-1)) of chloroquine was taken. Only 3/102 children had Plasmodium falciparum in the blood at the time of diagnosis and treatment. No severe adverse events were reported. No adverse events were associated with higher chloroquine concentrations. High doses of chloroquine are commonly taken and well tolerated in Guinea-Bissau. Malaria diagnostics are poor, and chloroquine is commonly prescribed to children without parasitemia. Use of high-dose chloroquine is concurrent with an exceptionally low prevalence of chloroquine-resistant P. falciparum.

Recent studies suggest that chloroquine resistance is mediated by an energy-dependent saturable chloroquine efflux carrier. An alternative explanation is that resistance is mediated by a channel. In Guinea-Bissau high doses of chloroquine are effective, well-tolerated and commonly used. This suggests that chloroquine resistance can be overcome by higher doses. Research on the mechanism of chloroquine resistance is of utmost importance and should include the effect of higher doses.

The aim of the present study was to compare different doses of chloroquine (CQ) and amodiaquine (AQ) for the treatment of falciparum malaria in children. Children with Plasmodium falciparum monoinfection were allocated by block randomisation to treatment with CQ 50/kg mg or 25 mg/kg or AQ 15 mg/kg or 30 mg/kg. The main outcomes were the cumulative adequate clinical and parasitological response (ACPR) rates and the number of true recrudescences as determined by PCR. A total of 729 children were included. In an evaluability analysis, the PCR-uncorrected cumulative ACPR rates on Day 28 for the treatment groups CQ 50/kg mg or 25 mg/kg and AQ 15 mg/kg or 30 mg/kg were 90%, 76%, 92% and 94%, respectively; the PCR-adjusted ACPR rates on Day 28 were 92%, 80%, 94% and 94%, respectively. No differences in adverse effects were observed. AQ has a high cure rate given as 30 mg/kg and 15 mg/kg, although it is not superior to treatment with CQ 50 mg/kg. However, 25 mg/kg of CQ is less efficient. As an interim option, Guinea-Bissau could change the recommended first-line treatment of uncomplicated malaria to CQ 50 mg/kg, reserving AQ as a partner drug for a future combination therapy.