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Standard diagnostics used in longitudinal antimalarial studies are unable to characterize the complexity of submicroscopic parasite dynamics, particularly in high transmission settings. We use molecular markers and amplicon sequencing to characterize post-treatment stage-specific malaria parasite dynamics during a 42 day randomized trial of 3- versus 5 day artemether-lumefantrine in 303 children with and without HIV (ClinicalTrials.gov number NCT03453840). The prevalence of parasite-derived 18S rRNA is >70% in children throughout follow-up, and the ring-stage marker SBP1 is detectable in over 15% of children on day 14 despite effective treatment. We find that the extended regimen significantly lowers the risk of recurrent ring-stage parasitemia compared to the standard 3 day regimen, and that higher day 7 lumefantrine concentrations decrease the probability of ring-stage parasites in the early post-treatment period. Longitudinal amplicon sequencing reveals remarkably dynamic patterns of multiclonal infections that include new and persistent clones in both the early post-treatment and later time periods. Our data indicate that post-treatment parasite dynamics are highly complex despite efficacious therapy, findings that will inform strategies to optimize regimens in the face of emerging partial artemisinin resistance in Africa.

Artemisinin-based combination therapies (ACTs) are the primary treatment for malaria. It is essential to characterize the pharmacokinetics (PKs) and pharmacodynamics (PDs) of ACTs in vulnerable populations at risk of suboptimal dosing. We developed a population PK/PD model using data from our previous study of artemether-lumefantrine in HIV-uninfected and HIV-infected children living in a high-transmission region of Uganda. HIV-infected children were on efavirenz-, nevirapine-, or lopinavir-ritonavir-based antiretroviral regimens, with daily trimethoprim-sulfamethoxazole prophylaxis. We assessed selection for resistance in two key parasite transporters, pfcrt and pfmdr1, over 42-day follow-up and incorporated genotyping into a time-to-event model to ascertain how resistance genotype in relation to drug exposure impacts recurrence risk. Two hundred seventy-seven children contributed 364 episodes to the model (186 HIV-uninfected and 178 HIV-infected), with recurrent microscopy-detectable parasitemia detected in 176 episodes by day 42. The final model was a two-compartment model with first-order absorption and an estimated age effect on bioavailability. Systemic lumefantrine exposure was highest with lopinavir-ritonavir, lowest with efavirenz, and equivalent with nevirapine and HIV-uninfected children. HIV status and lumefantrine concentration were significant factors associated with recurrence risk. Significant selection was demonstrated for pfmdr1 N86 and pfcrt K76 in recurrent infections, with no evidence of selection for pfmdr1 Y184F. Less sensitive parasites were able to tolerate lumefantrine concentrations ~ 3.5-fold higher than more sensitive parasites. This is the first population PK model of lumefantrine in HIV-infected children and demonstrates selection for reduced lumefantrine susceptibility, a concern as we confront the threat to ACTs posed by emerging artemisinin resistance in Africa.

BackgroundArtemether-lumefantrine (AL) is the most widely used artemisinin-based combination therapy in Sub-Saharan Africa and is threatened by the emergence of artemisinin resistance. Dosing is suboptimal in young children. We hypothesized that extending AL duration will improve exposure and reduce reinfection risks.MethodsWe conducted a prospective, randomized, open-label pharmacokinetic/pharmacodynamic study of extended duration AL in children with malaria in high-transmission rural Uganda. Children received 3-day (standard 6-dose) or 5-day (10-dose) AL with sampling for artemether, dihydroartemisinin, and lumefantrine over 42-day clinical follow-up. Primary outcomes were (1) comparative pharmacokinetic parameters between regimens and (2) recurrent parasitemia analyzed as intention-to-treat.ResultsA total of 177 children aged 16 months to 16 years were randomized, contributing 227 total episodes. Terminal median lumefantrine concentrations were significantly increased in the 5-day versus 3-day regimen on days 7, 14, and 21 (P < .001). A predefined day 7 lumefantrine threshold of 280 ng/mL was strongly predictive of recurrence risk at 28 and 42 days (P < .001). Kaplan-Meier estimated 28-day (51% vs 40%) and 42-day risk (75% vs 68%) did not significantly differ between 3- and 5-day regimens. No significant toxicity was seen with the extended regimen.ConclusionsExtending the duration of AL was safe and significantly enhanced overall drug exposure in young children but did not lead to significant reductions in recurrent parasitemia risk in our high-transmission setting. However, day 7 levels were strongly predictive of recurrent parasitemia risk, and those in the lowest weight-band were at higher risk of underdosing with the standard 3-day regimen.Clinical trial registrationClinicalTrials.gov number NCT03453840.

BackgroundThe choice of malaria treatment for HIV-infected pregnant women receiving efavirenz-based antiretroviral therapy must consider the potential impact of drug interactions on antimalarial exposure and clinical response. The aim of this study was to investigate the effects of efavirenz on artemether-lumefantrine (AL) because no studies have isolated the impact of efavirenz for HIV-infected pregnant women.MethodsA prospective clinical pharmacokinetic (PK) study compared HIV-infected, efavirenz-treated pregnant women with HIV-uninfected pregnant women in Tororo, Uganda. All women received the standard 6-dose AL treatment regimen for Plasmodium falciparum malaria with intensive PK samples collected over 21 days and 42-days of clinical follow-up. PK exposure parameters were calculated for artemether, its active metabolite dihydroartemisinin (DHA), and lumefantrine to determine the impact of efavirenz.ResultsNine HIV-infected and 30 HIV-uninfected pregnant women completed intensive PK evaluations. Relative to controls, concomitant efavirenz therapy lowered the 8-hour artemether concentration by 76% (P = 0.013), DHA peak concentration by 46% (P = 0.033), and day 7 and 14 lumefantrine concentration by 61% and 81% (P = 0.046 and 0.023), respectively. In addition, there were nonsignificant reductions in DHA area under the concentration-time curve0-8hr (35%, P = 0.057) and lumefantrine area under the concentration-time curve0-∞ (34%, P = 0.063) with efavirenz therapy.ConclusionsPregnant HIV-infected women receiving efavirenz-based antiretroviral therapy during malaria treatment with AL showed reduced exposure to both the artemisinin and lumefantrine. These data suggest that malaria and HIV coinfected pregnant women may require adjustments in AL dosage or treatment duration to achieve exposure comparable with HIV-uninfected pregnant women.

BackgroundLumefantrine is a long-acting antimalarial drug with an elimination half-life of over 3 days and protein binding of 99 percent. Correlation of lumefantrine concentrations from capillary plasma via fingerprick (Cc) versus venous plasma (Cv) remains to be defined.MethodsVenous and capillary plasma samples were collected simultaneously from children, pregnant women, and non-pregnant adults at 2, 24, 120hr post last dose of a standard 3-day artemether-lumefantrine regimen they received for uncomplicated malaria. Some of the enrolled children and pregnant women were also HIV-infected. Samples were analyzed via liquid chromatography tandem mass spectrometry. Linear regression analysis was performed using the program Stata® SE12.1.ResultsIn children, the linear regression equations for Cc vs Cv at 2, 24, and 120hr (day 7) post dose are [Cc] = 1.05*[Cv]+95.0 (n = 142, R2 = 0.977), [Cc] = 0.995*[Cv]+56.7 (n = 147, R2 = 0.990) and [Cc] = 0.958*[Cv]+18.6 (n = 139, R2 = 0.994), respectively. For pregnant women, the equations are [Cc] = 1.04*[Cv]+68.1 (n = 43, R2 = 0.990), [Cc] = 0.997*[Cv]+37.3 (n = 43, R2 = 0.993) and [Cc] = 0.941*[Cv]+11.1 (n = 41, R2 = 0.941), respectively. For non-pregnant adults, the equations are [Cc] = 1.05*[Cv]-117 (n = 32, R2 = 0.958), [Cc] = 0.962*[Cv]+9.21 (n = 32, R2 = 0.964) and [Cc] = 1.04*[Cv]-40.1 (n = 32, R2 = 0.988), respectively. In summary, a linear relationship with a slope of ~1 was found for capillary and venous lumefantrine levels in children, pregnant women and non-pregnant adults at 2hr, 24hr and 120hr post last dose, representing absorption, distribution, and elimination phases.ConclusionsCapillary and venous plasma concentration of lumefantrine can be used interchangeably at 1:1 ratio. Capillary sampling method via finger prick is a suitable alternative for sample collection in clinical studies.

BackgroundThe antiretroviral drug nevirapine and the antimalarial artemisinin-based combination therapy artemether-lumefantrine are commonly co-administered to treat malaria in the context of HIV. Nevirapine is a known inhibitor of cytochrome P450 3A4, which metabolizes artemether and lumefantrine. To address the concern that the antiretroviral nevirapine impacts the antimalarial artemether-lumefantrine pharmacokinetics, a prospective non-randomized controlled study in children presenting with uncomplicated malaria and HIV in sub-Saharan Africa was carried out.MethodsParticipants received artemether-lumefantrine (20/120 mg weight-based BID) for 3 days during nevirapine-based antiretroviral therapy (ART) co-administration (158-266 mg/m2 QD). HIV positive participants who were not yet on ART drugs were also enrolled as the control group. The target enrollment was children aged 3-12 years (n = 24 in each group). Intensive pharmacokinetics after the last artemether-lumefantrine dose was assessed for artemether, its active metabolite dihydroartemisinin, and lumefantrine. Pharmacokinetic parameters (area under the plasma concentration vs. time curve (AUC), maximum concentration and day 7 lumefantrine concentrations) were estimated using non-compartmental methods and compared to controls.ResultsNineteen children (16 on nevirapine and three not on ART) enrolled. Fifteen of the 16 (aged 4 to 11 years) on nevirapine-based ART were included in the pharmacokinetic analysis. Due to evolving WHO HIV treatment guidelines, insufficient children were enrolled in the control group (n = 3), so the pharmacokinetic data were compared to a historical control group of 20 HIV-uninfected children 5-12 years of age who also presented with malaria and underwent identical study procedures. Decreases of pharmacokinetic exposure [as estimated by AUC (AUC0-8hr)] were marginally significant for artemether (by -46%, p = 0.08) and dihydroartemisinin (-22%, p = 0.06) in the children on nevirapine-based ART, compared to when artemether-lumefantrine was administered alone. Similarly, peak concentration was decreased by 50% (p = 0.07) for artemether and 36% (p = 0.01) for dihydroartemisinin. In contrast, exposure to lumefantrine increased significantly in the context of nevirapine [AUC0-120hr:123% (p

BackgroundMalaria remains endemic in Afghanistan. National control and prevention strategies would be greatly enhanced through a better ability to forecast future trends in disease incidence. It is, therefore, of interest to develop a predictive tool for malaria patterns based on the current passive and affordable surveillance system in this resource-limited region.MethodsThis study employs data from Ministry of Public Health monthly reports from January 2005 to September 2015. Malaria incidence in Afghanistan was forecasted using autoregressive integrated moving average (ARIMA) models in order to build a predictive tool for malaria surveillance. Environmental and climate data were incorporated to assess whether they improve predictive power of models.ResultsTwo models were identified, each appropriate for different time horizons. For near-term forecasts, malaria incidence can be predicted based on the number of cases in the four previous months and 12 months prior (Model 1); for longer-term prediction, malaria incidence can be predicted using the rates 1 and 12 months prior (Model 2). Next, climate and environmental variables were incorporated to assess whether the predictive power of proposed models could be improved. Enhanced vegetation index was found to have increased the predictive accuracy of longer-term forecasts.ConclusionResults indicate ARIMA models can be applied to forecast malaria patterns in Afghanistan, complementing current surveillance systems. The models provide a means to better understand malaria dynamics in a resource-limited context with minimal data input, yielding forecasts that can be used for public health planning at the national level.

BackgroundMalaria remains endemic in Afghanistan. National control and prevention strategies would be greatly enhanced through a better ability to forecast future trends in disease incidence. It is, therefore, of interest to develop a predictive tool for malaria patterns based on the current passive and affordable surveillance system in this resource-limited region.MethodsThis study employs data from Ministry of Public Health monthly reports from January 2005 to September 2015. Malaria incidence in Afghanistan was forecasted using autoregressive integrated moving average (ARIMA) models in order to build a predictive tool for malaria surveillance. Environmental and climate data were incorporated to assess whether they improve predictive power of models.ResultsTwo models were identified, each appropriate for different time horizons. For near-term forecasts, malaria incidence can be predicted based on the number of cases in the four previous months and 12 months prior (Model 1); for longer-term prediction, malaria incidence can be predicted using the rates 1 and 12 months prior (Model 2). Next, climate and environmental variables were incorporated to assess whether the predictive power of proposed models could be improved. Enhanced vegetation index was found to have increased the predictive accuracy of longer-term forecasts.ConclusionResults indicate ARIMA models can be applied to forecast malaria patterns in Afghanistan, complementing current surveillance systems. The models provide a means to better understand malaria dynamics in a resource-limited context with minimal data input, yielding forecasts that can be used for public health planning at the national level.

BackgroundArtemisinins are primarily responsible for initial parasite clearance. Antimalarial pharmacokinetics (PK), human immunodeficiency virus (HIV) infection, and antiretroviral therapy have been shown to impact treatment outcomes, although their impact on early parasite clearance in children has not been well characterized.MethodsParasite clearance parameters were generated from twice-daily blood smears in HIV-infected and HIV-uninfected Ugandan children treated with artemether-lumefantrine (AL). Artemether and dihydroartemisinin (DHA) area-under-the-curve from 0-8 hours (AUC0-8hr) after the 1st AL dose was compared with AUC0-8hr after the last (6th) dose in a concurrently enrolled cohort. The association between post-1st dose artemisinin AUC0-8hr and parasite clearance was assessed.ResultsParasite clearance was longer in HIV-infected versus HIV-uninfected children (median, 3.5 vs 2.8 hours; P = .003). Artemether AUC0-8hr was 3- to 4-fold lower after the 6th dose versus the 1st dose of AL in HIV-infected children on nevirapine- or lopinavir/ritionavir-based regimens and in HIV-uninfected children (P ≤ .002, 1st vs 6th-dose comparisons). Children on efavirenz exhibited combined post-1st dose artemether/DHA exposure that was significantly lower than those on lopinavir/ritonavir and HIV-uninfected children. Multiple regression analysis supported that the effect of artemether/DHA exposure on parasite clearance was significantly moderated by HIV status.ConclusionsParasite clearance rates remain rapid in Uganda and were not found to associate with PK exposure. However, significant decreases in artemisinin PK with repeated dosing in nearly all children, coupled with small, but significant increase in parasite clearance half-life in those with HIV, may have important implications for AL efficacy, particularly because reports of artemisinin resistance are increasing.

BackgroundThe optimal treatment of malaria in human immunodeficiency virus (HIV)-infected children requires consideration of critical drug-drug interactions in coinfected children, as these may significantly impact drug exposure and clinical outcomes.MethodsWe conducted an intensive and sparse pharmacokinetic/pharmacodynamic study in Uganda of the most widely adopted artemisinin-based combination therapy, artemether-lumefantrine. HIV-infected children on 3 different first-line antiretroviral therapy (ART) regimens were compared to HIV-uninfected children not on ART, all of whom required treatment for Plasmodium falciparum malaria. Pharmacokinetic sampling for artemether, dihydroartemisinin, and lumefantrine exposure was conducted through day 21, and associations between drug exposure and outcomes through day 42 were investigated.ResultsOne hundred forty-five and 225 children were included in the intensive and sparse pharmacokinetic analyses, respectively. Compared with no ART, efavirenz (EFV) reduced exposure to all antimalarial components by 2.1- to 3.4-fold; lopinavir/ritonavir (LPV/r) increased lumefantrine exposure by 2.1-fold; and nevirapine reduced artemether exposure only. Day 7 concentrations of lumefantrine were 10-fold lower in children on EFV vs LPV/r-based ART, changes that were associated with an approximate 4-fold higher odds of recurrent malaria by day 28 in those on EFV vs LPV/r-based ART.ConclusionsThe choice of ART in children living in a malaria-endemic region has highly significant impacts on the pharmacokinetics and pharmacodynamics of artemether-lumefantrine treatment. EFV-based ART reduces all antimalarial components and is associated with the highest risk of recurrent malaria following treatment. For those on EFV, close clinical follow-up for recurrent malaria following artemether-lumefantrine treatment, along with the study of modified dosing regimens that provide higher exposure, is warranted.

Artemether-lumefantrine is a first-line regimen for the treatment of uncomplicated malaria during the second and third trimesters of pregnancy. Previous studies have reported changes in the pharmacokinetics and clinical outcomes following treatment with artemether-lumefantrine in pregnant women compared to nonpregnant adults; however, the results are inconclusive. We conducted a study in rural Uganda to compare the pharmacokinetics of artemether-lumefantrine and the treatment responses between 30 pregnant women and 30 nonpregnant adults with uncomplicated Plasmodium falciparum malaria. All participants were uninfected with HIV, treated with a six-dose regimen of artemether-lumefantrine, and monitored clinically for 42 days. The pharmacokinetics of artemether, its metabolite dihydroartemisinin, and lumefantrine were evaluated for 21 days following treatment. We found no significant differences in the overall pharmacokinetics of artemether, dihydroartemisinin, or lumefantrine in a direct comparison of pregnant women to nonpregnant adults, except for a statistically significant but small difference in the terminal elimination half-lives of both dihydroartemisinin and lumefantrine. There were seven PCR-confirmed reinfections (5 pregnant and 2 nonpregnant participants). The observation of a shorter terminal half-life for lumefantrine may have contributed to a higher frequency of reinfection or a shorter posttreatment prophylactic period in pregnant women than in nonpregnant adults. While the comparable overall pharmacokinetic exposure is reassuring, studies are needed to further optimize antimalarial efficacy in pregnant women, particularly in high-transmission settings and because of emerging drug resistance. (This study is registered at ClinicalTrials.gov under registration no. NCT01717885.).

ObjectivesArtesunate plus amodiaquine is used for malaria treatment in regions with overlapping HIV endemicity. Co-administration of artesunate/amodiaquine with antiretroviral therapy (ART) may result in drug-drug interactions, but minimal data exist. This study evaluated the impact of nevirapine-based ART, containing a backbone of zidovudine and lamivudine, on the disposition of amodiaquine and its active metabolite, desethylamodiaquine (DEAQ).MethodsThis was an open-label, parallel-group pharmacokinetic comparison between HIV-infected, adult subjects receiving steady-state nevirapine-based ART (n = 10) and ART-naive subjects (control group, n = 11). All subjects received a loose formulation of artesunate/amodiaquine (200/600 mg) daily for 3 days, with serial pharmacokinetic sampling over 96 h following the final dose of artesunate/amodiaquine. Amodiaquine and DEAQ were quantified using a validated HPLC method with UV detection. Pharmacokinetic parameters were determined using standard non-compartmental methods.ResultsExposures to both amodiaquine and DEAQ were significantly lower in the nevirapine-based ART group compared with the control group (amodiaquine AUC₀₋₂₄ 145 versus 204 ng·h/mL, P = 0.02; DEAQ AUC₀₋₉₆ 14,571 versus 21,648 ng·h/mL, P < 0.01). The AUCDEAQ/AUC(amodiaquine) ratio was not different between groups (ART group 116 versus control group 102, P = 0.67).ConclusionsSubjects on nevirapine-based ART had lower exposure to both amodiaquine and DEAQ (28.9% and 32.7%, respectively). Consequently, this may negatively impact the effectiveness of artesunate/amodiaquine in HIV-infected individuals on this ART combination.

Sickle cell trait (HbAS) is the best-characterized genetic polymorphism known to protect against falciparum malaria. Although the protective effect of HbAS against malaria is well known, the mechanism(s) of protection remain unclear. A number of biochemical and immune-mediated mechanisms have been proposed, and it is likely that multiple complex mechanisms are responsible for the observed protection. Increased evidence for an immune component of protection as well as novel mechanisms, such as enhanced tolerance to disease mediated by HO-1 and reduced parasitic growth due to translocation of host micro-RNA into the parasite, have recently been described. A better understanding of relevant mechanisms will provide valuable insight into the host-parasite relationship, including the role of the host immune system in protection against malaria.

Abstract Sickle cell trait (HbAS) is the best-characterized genetic polymorphism known to protect against falciparum malaria. Although the protective effect of HbAS against malaria is well known, the mechanism(s) of protection remain unclear. A number of biochemical and immune-mediated mechanisms have been proposed, and it is likely that multiple complex mechanisms are responsible for the observed protection. Increased evidence for an immune component of protection as well as novel mechanisms, such as enhanced tolerance to disease mediated by HO-1 and reduced parasitic growth due to translocation of host micro-RNA into the parasite, have recently been described. A better understanding of relevant mechanisms will provide valuable insight into the host-parasite relationship, including the role of the host immune system in protection against malaria.

BackgroundAlthough dihydroartemisinin-piperaquine (DP) is used primarily in children, pharmacokinetic/pharmacodynamic (PK/PD) data on DP use in young children are lacking.MethodsWe conducted a prospective PK/PD study of piperaquine in 107 young children in Uganda. Samples were collected up to 28 days after 218 episodes of malaria treatment, which occurred during follow-up periods of up to 5 months. Malaria follow-up was conducted actively to day 28 and passively to day 63.ResultsThe median capillary piperaquine concentration on day 7 after treatment was 41.9 ng/mL. Low piperaquine concentrations were associated with an increased risk of recurrent malaria for up to 42 days, primarily in those receiving trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. In children not receiving TMP-SMX, low piperaquine concentrations were only modestly associated with an increased risk of recurrent malaria. However, for children receiving TMP-SMX, associations were strong and evident for all sampling days, with PQ concentrations of ≤ 27.3 ng/mL on day 7 associated with a greatly increased risk of recurrent malaria. Notably, of 132 cases of recurrent malaria, 119 had detectable piperaquine concentrations at the time of presentation with recurrent malaria.ConclusionsThese piperaquine PK/PD data represent the first in children

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

BackgroundThe antiretroviral drug efavirenz (EFV) and the antimalarial artemisinin-based combination therapy artemether-lumefantrine (AL) are commonly co-administered to treat HIV and malaria. EFV is a known inducer of cytochrome P450 3A4, which converts artemether to dihydroartemisinin (DHA) that is also active and metabolizes longer acting lumefantrine (LR). A study in healthy volunteers was completed to address the concern that EFV impacts AL pharmacokinetics (PKs).MethodsAdults received AL (80/480 mg twice daily) for 3-days before and during EFV co-administration (600 mg daily for 26 days) with intensive PK for artemether, DHA, and LR conducted after the last AL dose for each period. EFV PK was evaluated with and without AL. PK parameters were estimated using noncompartmental methods.ResultsTwelve subjects completed the 2-period study. PK exposure for artemether, DHA, and LR [as estimated by the area under the concentration time curve (AUClast)] decreased or trended toward decrease with EFV, compared with when administered alone [-51% (P = 0.084), -46% (P = 0.005), and -21% (P = 0.102), respectively]. Day-7 LR levels, previously deemed predictive of treatment success, were 46% lower (P = 0.002) with EFV, but the LR half-life was unchanged. EFV PK exposure was minimally altered after AL co-administration [AUC0-24 hrs decreased by 17% (P = 0.034)].ConclusionsExposure to DHA, but not LR, was significantly lower during EFV-AL co-administration compared with that during administration of AL alone. These findings may have implications for the treatment efficacy of AL, particularly in children. However, the observed modest changes probably do not warrant dosage adjustment during co-administration of AL with EFV.

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

BackgroundClinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.Methodology/principal findingsWe compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31-0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70-1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37-0.88 for females).Conclusions/significanceThis study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria.

BackgroundClinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.Methodology/principal findingsWe compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31-0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70-1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37-0.88 for females).Conclusions/significanceThis study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria.