Persistent and multiclonal malaria parasite dynamics despite extended artemether-lumefantrine treatment in children.

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.

The Impact of Extended Treatment With Artemether-lumefantrine on Antimalarial Exposure and Reinfection Risks in Ugandan Children With Uncomplicated Malaria: A Randomized Controlled Trial.

BACKGROUND: Artemether-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. METHODS: We 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. RESULTS: A 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. CONCLUSIONS: Extending 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 REGISTRATION: ClinicalTrials.gov number NCT03453840.

Determination of unbound piperaquine in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry.

Piperaquine (PQ) is an antimalarial drug that is highly protein-bound. Variation in plasma protein contents may affect the pharmacokinetic (PK) exposure of unbound drug, leading to alteration of clinical outcomes. All published methods for determination of PQ in human plasma measure the total PQ including both bound and unbound PQ to plasma proteins. There is no published method for unbound PQ determination. Here we report an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determination of PQ in human plasma filtrate prepared by filtering human plasma through Millipore Microcon® centrifugal filters (10k NMWL). The filter cup had to be treated with 5% benzalkonium chloride to reduce non-specific binding to the filter devices before filtration of plasma samples. Multiple reactions monitoring (MRM) of the ion pairs m/z 535/288 for PQ and m/z 541/294 for the internal standard (IS) was selected for quantification. When electrospray ionization (ESI+) was used, paradoxical matrix effect was observed despite the structure similarity of the deuterated IS: Ion suppression for PQ versus ion enhancement for the PQ-d6, even though they were closely eluted: 0.62 min versus 0.61 min. Separation was achieved on Evo C18 column (50 × 2.1 mm, 1.7 µm, Phenomenex Inc.) eluted with 10 mM NH4OH and MeCN. When atmospheric pressure chemical ionization in positive mode (APCI+) was used for ion source, matrix effect diminished. Separation was achieved on a PFP column (30 × 2.1 mm, 1.7 µm, Waters, Corp.) eluted with aqueous 20 mM ammonium formate 0.14% trifluoroacetic acid (A) and methanol-acetonitrile (4:1, v/v) containing 0.1% trifluoroacetic acid (B) at 0.8 mL/min flow rate in a gradient mode: 30-30-80-80-30-30%B (0-0.1-1.0-1.40-1.41-1.50 min). The retention time was 0.67 min for both PQ and the IS. The method was validated with a linear calibration range from 20 to 5,000 pg/mL and applied to clinical samples.

Sodalis ligni Strain 159R Isolated from an Anaerobic Lignin-Degrading Consortium.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Identifying an optimal dihydroartemisinin-piperaquine dosing regimen for malaria prevention in young Ugandan children.

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.

Pharmacokinetics and Safety of Zidovudine, Lamivudine, and Lopinavir/Ritonavir in HIV-infected Children With Severe Acute Malnutrition in Sub-Saharan Africa: IMPAACT Protocol P1092.

BACKGROUND: Severe acute malnutrition (SAM) may alter the pharmacokinetics (PK), efficacy, and safety of antiretroviral therapy. The phase IV study, IMPAACT P1092, compared PK, safety, and tolerability of zidovudine (ZDV), lamivudine (3TC), and lopinavir/ritonavir (LPV/r) in children with and without SAM. MATERIALS AND METHODS: Children living with HIV 6 to <36 months of age with or without World Health Organization (WHO)-defined SAM received ZDV, 3TC, and LPV/r syrup for 48 weeks according to WHO weight band dosing. Intensive PK sampling was performed at weeks 1, 12, and 24. Plasma drug concentrations were measured using liquid chromatography tandem mass spectrometry. Steady-state mean area under the curve (AUC0-12h) and clearance (CL/F) for each drug were compared. Grade ≥3 adverse events were compared between cohorts. RESULTS: Fifty-two children were enrolled across 5 sites in Africa with 44% (23/52) female, median age 19 months (Q1, Q3: 13, 25). Twenty-five children had SAM with entry median weight-for-height Z-score (WHZ) -3.4 (IQR -4.0, -3.0) and 27 non-SAM had median WHZ -1.0 (IQR -1.8, -0.1). No significant differences in mean AUC0-12h or CL/F were observed (P ≥ 0.09) except for lower 3TC AUC0-12h (GMR, 0.60; 95% CI, 0.4-1.0; P = 0.047) at week 12, higher ZDV AUC0-12h (GMR, 1.52; 1.2-2.0; P = 0.003) at week 24 in the SAM cohort compared with non-SAM cohort. Treatment-related grade ≥3 events did not differ significantly between cohorts (24.0% vs. 25.9%). CONCLUSION: PK and safety findings for ZDV, 3TC, and LPV/r support current WHO weight band dosing of syrup formulations in children with SAM.

Combined Enzyme- and Transition Metal-Catalyzed Strategy for the Enantioselective Syntheses of Nitrogen Heterocycles: (-)-Coniine, DAB-1, and Nectrisine.

The enantioselective syntheses of (-)-coniine, DAB-1, and nectrisine have been developed, utilizing a complementary strategy of enzyme- and transition metal-catalyzed reactions. The initial stereocenter was set with >99% enantioselectivity via an enzyme-catalyzed hydrocyanation reaction. Substrate incompatibilities with the natural enzyme were overcome by tactical utilization of ruthenium-catalyzed olefin metathesis to functionalize an enzyme-derived (R)-allylic fragment. The piperidine and pyrrolidine alkaloid natural products were obtained by a route that leveraged regio- and stereoselective palladium-catalyzed 1,3-substitutive reactions.

Reduced Exposure to Piperaquine, Compared to Adults, in Young Children Receiving Dihydroartemisinin-Piperaquine as Malaria Chemoprevention.

Dihydroartemisinin (DHA)-piperaquine is being evaluated as intermittent preventive therapy for malaria, but dosing has not been optimized for children. We assessed exposure to DHA and piperaquine in Ugandan children at two ages during infancy. Intensive sampling was performed in 32 children at 32 weeks of age, 31 children at 104 weeks, and 30 female adult controls. Compared with adults, DHA area under the concentration-time curve (AUC0-8 hr ) was 52% higher at 32 weeks and comparable at 104 weeks. Compared with adults, piperaquine AUC0-21 d was 35% lower at 32 weeks and 53% lower at 104 weeks. Terminal piperaquine concentrations on days 7, 14, and 21 were lower in children compared with adults and lower at 104 compared with 32 weeks. Piperaquine exposure was lower in young children compared with adults, and lower at 104 compared with 32 weeks of age, suggesting a need for age-based DHA-piperaquine dose optimization for chemoprevention.

Complete Genome Sequence of Serratia quinivorans Strain 124R, a Facultative Anaerobe Isolated on Organosolv Lignin as a Sole Carbon Source.

The complete genome sequence of the gammaproteobacterial isolate Serratia quinivorans 124R consists of 5 Mb over 2 scaffolds and a G+C content of 52.85%. Genes relating to aromatic metabolism reflect its isolation on organosolv lignin as a sole carbon source under anoxic conditions as well as the potential for lignin biorefinery applications.

Comparative effectiveness of aerosolized versus oral ribavirin for the treatment of respiratory syncytial virus infections: A single-center retrospective cohort study and review of the literature.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of viral infections in immunocompromised hosts and is associated with significant morbidity and mortality. In January 2015, our institution switched from aerosolized to oral ribavirin (RBV) for primary treatment of RSV infection among high-risk immunocompromised adult patients. The objective of the study was to evaluate the clinical and economic outcomes associated with this switch. METHODS: Retrospective cohort analysis of adult patients diagnosed with RSV infection and treated with RBV between January 1, 2013, and May 31, 2016. RESULTS: Of 46 patients, 20 (43%) patients received oral RBV and 26 (57%) received aerosolized RBV. Underlying conditions included lung transplant (n = 22), hematopoietic cell transplant (n = 16), hematological malignancy (n = 5), and structural lung diseases (n = 4). At the time of RSV diagnosis, 42 (91%) were hospitalized, 36 (78%) had clinical and radiographic evidence of lower respiratory tract infection (RTI), 9 (20%) were admitted to the ICU, and 4 (9%) required intubation. There were no differences in clinical outcomes between the 2 groups with regard to adverse events, progression from upper to lower RTI, escalation of care, or 30-day mortality. Three (15%) in the oral group and 1 (4%) in the aerosolized group died within 30 days (P = .33). The cost avoidance attributable to the switch in therapy over 1 year is $1.2 million. CONCLUSION: Oral RBV appears to be a safe and cost-effective alternative to aerosolized RBV for the management of RSV infection in immunocompromised patients. Larger studies are needed to validate the safety and efficacy of this approach.

Impact of Intermittent Preventive Treatment During Pregnancy on Plasmodium falciparum Drug Resistance-Mediating Polymorphisms in Uganda.

Background: In 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. Methods: We 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. Results: Considering 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). Conclusions: IPTp 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.