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Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including β-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in β-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to β-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in β-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a β-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of β-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level β-lactam resistance. Loss of GdpP function thus increases tolerance to β-lactams that can lead to its therapy failure and can permit β-lactam resistance to occur more readily.

Intermittent preventive treatment (IPT) with dihydroartemisinin-piperaquine (DP) is highly protective against malaria in children, but is not standard in malaria-endemic countries. Optimal DP dosing regimens will maximize efficacy and reduce toxicity and resistance selection. We analyze piperaquine (PPQ) concentrations (n = 4573), malaria incidence data (n = 326), and P. falciparum drug resistance markers from a trial of children randomized to IPT with DP every 12 weeks (n = 184) or every 4 weeks (n = 96) from 2 to 24 months of age (NCT02163447). We use nonlinear mixed effects modeling to establish malaria protective PPQ levels and risk factors for suboptimal protection. Compared to DP every 12 weeks, DP every 4 weeks is associated with 95% protective efficacy (95% CI: 84-99%). A PPQ level of 15.4 ng/mL reduces the malaria hazard by 95%. Malnutrition reduces PPQ exposure. In simulations, we show that DP every 4 weeks is optimal across a range of transmission intensities, and age-based dosing improves malaria protection in young or malnourished children.

Artemether-lumefantrine (AL) is a first-line agent for uncomplicated malaria caused by Plasmodium falciparum. The WHO recommends periodic therapeutic efficacy studies of antimalarial drugs for the detection of malaria parasite drug resistance and to inform national malaria treatment policies. We conducted a therapeutic efficacy study of AL in a high malaria transmission region of northern Zambia from December 2014 to July 2015. One hundred children of ages 6 to 59 months presenting to a rural health clinic with uncomplicated falciparum malaria were admitted for treatment with AL (standard 6-dose regimen) and followed weekly for 5 weeks. Parasite counts were taken every 6 hours during treatment to assess parasite clearance. Recurrent episodes during follow-up (n = 14) were genotyped to distinguish recrudescence from reinfection and to identify drug resistance single nucleotide polymorphisms (SNPs) and multidrug resistance protein 1 (mdr1) copy number variation. Day 7 lumefantrine concentrations were measured for correspondence with posttreatment reinfection. All children who completed the parasite clearance portion of the study (n = 94) were microscopy-negative by 72 hours. The median parasite elimination half-life was 2.7 hours (interquartile range: 2.1-3.3). Genotype-corrected therapeutic efficacy was 98.8% (95% CI: 97.6-100). Purported artemisinin and lumefantrine drug resistance SNPs in atp6, 3D7_1451200, and mdr1 were detected but did not correlate with parasite recurrence, nor did day 7 lumefantrine concentrations. In summary, AL was highly effective for the treatment of uncomplicated falciparum malaria in northern Zambia during the study period. The high incidence of recurrent parasitemia was consistent with reinfection due to high, perennial malaria transmission.

Dihydroartemisinin-piperaquine (DHA-PQ) provides highly effective therapy and chemoprevention for malaria in pregnant African women. PQ concentrations of >10.3 ng/ml have been associated with reduced maternal parasitemia, placental malaria, and improved birth outcomes. We characterized the population pharmacokinetics (PK) of PQ in a post hoc analysis of human immunodeficiency virus (HIV)-infected and -uninfected pregnant women receiving DHA-PQ as chemoprevention every 4 or 8 weeks. The effects of covariates such as pregnancy, nutritional status (body mass index [BMI]), and efavirenz (EFV)-based antiretroviral therapy were investigated. PQ concentrations from two chemoprevention trials were pooled to create a population PK database from 274 women and 2,218 PK observations. A three-compartment model with an absorption lag best fit the data. Consistent with our prior intensive PK evaluation, pregnancy and EFV use resulted in a 72% and 61% increased PQ clearance, compared to postpartum and HIV-uninfected pregnant women, respectively. Low BMI at 28 weeks of gestation was associated with increased clearance (2% increase per unit decrease in BMI). Low-BMI women given DHA-PQ every 8 weeks had a higher prevalence of parasitemia, malaria infection, and placental malaria compared to women with higher BMIs. The reduced piperaquine exposure in women with low BMI as well as during EFV coadministration, compared to pregnant women with higher BMIs and not taking EFV, suggests that these populations could benefit from weekly instead of monthly dosing for prevention of malaria parasitemia. Simulations indicated that because of the BMI-clearance relationship, weight-based regimens would not improve protection compared to a 2,880 mg fixed-dose regimen when provided monthly. (The clinical trials described in this paper have been registered at ClinicalTrials.gov under identifiers NCT02163447 and NCT02282293.).

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.

BackgroundDihydroartemisinin-piperaquine (DHA-PQ) is highly efficacious as intermittent preventive therapy for malaria during pregnancy (IPTp). Determining associations between piperaquine (PQ) exposure, malaria risk, and adverse birth outcomes informs optimal dosing strategies.MethodsHuman immunodeficiency virus-uninfected pregnant women (n = 300) were enrolled in a placebo-controlled trial of IPTp at 12-20 weeks' gestation and randomized to sulfadoxine-pyrimethamine every 8 weeks, DHA-PQ every 8 weeks, or DHA-PQ every 4 weeks during pregnancy. Pharmacokinetic sampling for PQ was performed every 4 weeks, and an intensive pharmacokinetic substudy was performed in 30 women at 28 weeks' gestation. Concentration-effect relationships were assessed between exposure to PQ; the prevalence of Plasmodium falciparum infection during pregnancy; outcomes at delivery including placental malaria, low birth weight, and preterm birth; and risks for toxicity. Simulations of new dosing scenarios were performed.ResultsModel-defined PQ target venous plasma concentrations of 13.9 ng/mL provided 99% protection from P. falciparum infection during pregnancy. Each 10-day increase in time above target PQ concentrations was associated with reduced odds of placental parasitemia, preterm birth, and low birth weight, though increases in PQ concentrations were associated with QT interval prolongation. Modeling suggests that daily or weekly administration of lower dosages of PQ, compared to standard dosing, will maintain PQ trough levels above target concentrations with reduced PQ peak levels, potentially limiting toxicity.ConclusionsThe protective efficacy of IPTp with DHA-PQ was strongly associated with higher drug exposure. Studies of the efficacy and safety of alternative DHA-PQ IPTp dosing strategies are warranted.Clinical trials registrationNCT02163447.

Study of the clinical effects of combination therapy for malaria is aided by the ability to measure concentrations of individual partner drugs. Existing methods for measurement of the antimalarial drug lumefantrine (LF) in dried blood spots (DBS) on filter paper rely on chemical pretreatment of the paper to facilitate drug elution. However, in the absence of pretreatment, DBS may still offer some utility for semi-quantitative measurements and pharmacokinetic-pharmacodynamic (PK-PD) analyses. We present a method for semi-quantitation of LF in DBS on untreated filter paper using liquid chromatography tandem mass spectrometry. Optimal recovery was achieved by extraction with acetone-water-formic acid (90:5:5). The range of quantitation was 100-20,000ng/ml. Mean intra- and inter-day accuracy values were 86.6% (coefficient of variation [CV]: 10.1%) and 91.8% (CV: 16.1%), therefore we propose the assay as semi-quantitative. Clinical application was demonstrated in exploratory PK-PD analyses of a drug efficacy trial of artemether-lumefantrine in children with uncomplicated falciparum malaria using post-treatment day 7 samples, parasite clearance times estimated from serial blood smears, and recurrence of malaria out to 35days. The median day 7 concentration among children (n=71) was 111ng/ml (interquartile range: 100-194ng/ml). We used a truncated calibration curve of 100-5000ng/ml for calculations due to low observed concentrations. Calculations using the full calibration curve yielded similar values (+1% avg. deviation). Controlling for participant age, sex, and parasite burden, each log increase in LF day 7 concentration corresponded to a decrease of 7.1h in mean parasite clearance time (95% confidence interval: 0.1-14.3h, P=0.05). A nested case-control study of participants (n=18) with and without recurrent malaria showed mean post-treatment day 7 concentrations of 181ng/ml and 235ng/ml, respectively, but the difference was not significant (P=0.64). A method for semi-quantitation of LF from post-treatment day 7 collections of DBS on untreated filter paper demonstrated clinical application in exploratory PK-PD analyses of parasite clearance and reinfection. Use of DBS will endure in certain study settings by virtue of their ease of collection and resilience. Their utility should continue to be explored as our instruments gain in sensitivity and as clinical pharmacology inquiries are pursued to the field.

BackgroundA monthly treatment course of dihydroartemisinin-piperaquine (DHA-PQ) effectively prevents malaria during pregnancy. However, a drug-drug interaction pharmacokinetic (PK) study found that pregnant human immunodeficiency virus (HIV)-infected women receiving efavirenz-based antiretroviral therapy (ART) had markedly reduced piperaquine (PQ) exposure. This suggests the need for alternative DHA-PQ chemoprevention regimens in this population.MethodsEighty-three HIV-infected pregnant women who received monthly DHA-PQ and efavirenz contributed longitudinal PK and corrected QT interval (QTc) (n = 25) data. Population PK and PK-QTc models for PQ were developed to consider the benefits (protective PQ coverage) and risks (QTc prolongation) of alternative DHA-PQ chemoprevention regimens. Protective PQ coverage was defined as maintaining a concentration >10 ng/mL for >95% of the chemoprevention period.ResultsPQ clearance was 4540 L/day. With monthly DHA-PQ (2880 mg PQ), 96% of women, respectively. All regimens were safe, with ≤2% of women predicted to have ≥30 msec QTc increase.ConclusionsFor HIV-infected pregnant women receiving efavirenz, low daily DHA-PQ dosing was predicted to improve protection against parasitemia and reduce risk of toxicity compared to monthly dosing.Clinical trials registrationNCT02282293.

Abscisic acid (ABA) is an ancient stress hormone and is detectable in a wide variety of organisms where it regulates innate immunity and inflammation. Previously, we showed that oral supplementation with ABA decreased parasitemia in a mouse model of malaria, decreased liver and spleen pathology and reduced parasite transmission to mosquitoes. Here, we report that higher circulating ABA levels were associated with a reduced risk of symptomatic malaria in a cohort of Plasmodium falciparum-infected Ugandan children. To understand possible mechanisms of ABA protection in malaria, we returned to our mouse model to show that ABA effects on Plasmodium yoelii 17XNL infection were accompanied by minimal effects on complete blood count and blood chemistry analytes, suggesting a benefit to host health. In addition, orally delivered ABA induced patterns of gene expression in mouse liver and spleen that suggested enhancement of host anti-parasite defenses. To test these inferences, we utilized passive immunization and knockout mice to demonstrate that ABA supplementation increases circulating levels of protective, parasite-specific IgG and requires caspase-1 to reduce parasitemia. Collectively, ABA induces host responses that ameliorate infection and disease in an animal model and suggest that further studies of ABA in the context of human malaria are warranted.

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.

ObjectivesThe selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model.MethodsPharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7.ResultsMeropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone.ConclusionsSimulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

BackgroundIn a recent trial of intermittent preventive treatment in pregnancy (IPTp) in Uganda, dihydroartemisinin-piperaquine (DP) was superior to sulfadoxine-pyrimethamine (SP) in preventing maternal and placental malaria.MethodsWe compared genotypes using sequencing, fluorescent microsphere, and quantitative polymerase chain reaction assays at loci associated with drug resistance in Plasmodium falciparum isolated from subjects receiving DP or SP.ResultsConsidering aminoquinoline resistance, DP was associated with increased prevalences of mutations at pfmdr1 N86Y, pfmdr1 Y184F, and pfcrt K76T compared to SP (64.6% vs 27.4%, P < .001; 93.9% vs 59.2%, P < .001; and 87.7% vs 75.4%, P = .03, respectively). Increasing plasma piperaquine concentration at the time of parasitemia was associated with increasing pfmdr1 86Y prevalence; no infections with the N86 genotype occurred with piperaquine >2.75 ng/mL. pfkelch13 propeller domain polymorphisms previously associated with artemisinin resistance were not identified. Recently identified markers of piperaquine resistance were uncommon and not associated with DP. Considering antifolate resistance, SP was associated with increased prevalence of a 5-mutation haplotype (pfdhfr 51I, 59R, and 108N; pfdhps 437G and 581G) compared to DP (90.8% vs 60.0%, P = .001).ConclusionsIPTp selected for genotypes associated with decreased sensitivity to treatment regimens, but genotypes associated with clinically relevant DP resistance in Asia have not emerged in Uganda.

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

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.).

A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h(-1)) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

ObjectiveTrimethoprim-sulfamethoxazole prophylaxis is recommended for HIV-exposed infants until breastfeeding ends and HIV infection has been excluded. Extending prophylaxis with a focus on preventing malaria may be beneficial in high transmission areas. We investigated three regimens for the prevention of malaria in young HIV-exposed children.DesignAn open-label, randomized controlled trial.SettingTororo, Uganda, a rural area with intense, year-round, malaria transmission.ParticipantsTwo hundred infants aged 4-5 months enrolled and 186 randomized after cessation of breastfeeding and confirmed to be HIV uninfected (median 10 months of age).InterventionNo chemoprevention, monthly sulfadoxine-pyrimethamine, daily trimethoprim-sulfamethoxazole or monthly dihydroartemisinin-piperaquine given from randomization to 24 months of age.Main outcome measuresThe primary outcome was the incidence of malaria during the intervention period. Secondary outcomes included the incidence of hospitalization, diarrhoeal illness, or respiratory tract infection; prevalence of anaemia and asymptomatic parasitemia; measures of safety; and incidence of malaria over 1 year after the intervention was stopped.ResultsDuring the intervention, the incidence of malaria in the no chemoprevention group was 6.28 episodes per person-year at risk. Protective efficacy was 69% [95% confidence interval (95% CI) 53-80, P

ObjectiveAfrican potato (Hypoxis obtusa) is commonly used in Sub-Saharan Africa as a complementary herbal remedy for HIV-infected patients. It is unknown whether or not co-administration of African potato alters the pharmacokinetics of protease inhibitor antiretrovirals. The objective of this study was to investigate the impact of the African potato on the steady-state pharmacokinetics of ritonavir-boosted lopinavir (LPV/r).MethodsSixteen adult volunteers were administered LPV/r 400/100 mg twice a day for 14 days, followed by concomitant administration with African potato given once daily for 7 days. Lopinavir plasma exposure as estimated by the area under the concentration-time curve over the 12-h dosing interval (AUC(0-12h), AUCτ) was determined on day 14 and again on day 21. Lopinavir in plasma was analyzed using a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. Steady-state AUCτ and the maximum concentration following dose administration (C(max)) were determined using non-compartmental methods using WinNonlin Professional version 5.2.1. Statistical analyses were performed using Stata version 12.1.ResultsCo-administration of African potato was not associated with any change in lopinavir AUCτ, C(max), or C(trough).ConclusionsAfrican potato when taken concomitantly with LPV/r is well-tolerated and not associated with clinically significant changes in lopinavir pharmacokinetics.

BackgroundNumerous methods have been reported for the determination of artemether (ARM) and its metabolite dihydroartemisinin (DHA) in plasma. However, stability issues in patient plasma have not received enough attention.ResultsAn LC-MS/MS method for simultaneous determination of ARM and DHA in human plasma (K3EDTA) turned out to be problematic: ARM and DHA were degraded partially or completely in some patient plasma samples as indicated by the stable isotope-labeled internal standards. We postulated iron II (Fe(2+)) in hemoglobin or its derived products from malaria patients causes degradation of the drugs, and found that hydrogen peroxide (H2O2) protected the drugs from degradation. Acidifying plasma increased recovery of ARM significantly. Using only 50 µl of plasma sample, the method has a LLOQ at 0.5 ng/ml for both ARM and DHA.ConclusionH2O2 is a stabilizing agent for artemisinin derivatives. The modified method is reliable and sensitive.

Antiretroviral therapy (ART) significantly reduced the CD8 cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8(+) cells from long-term survivors were cocultured with HIV-infected CD4(+) cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8(+) cells from 4 of the 14 LTSs was reduced with exposure to 10 μM of nevirapine (P < 0.05). The antiviral activity of CD8(+) cells from 2 LTSs was inhibited by 5 μM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8(+) cells and thus could influence the effectiveness of antiretroviral therapy.

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.