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1.  Novel Endoperoxide-Based Transmission-Blocking Antimalarials with Liver- and Blood-Schizontocidal Activities 
ACS Medicinal Chemistry Letters  2013;5(2):108-112.
In a search for effective compounds against both the blood- and liver-stages of infection by malaria parasites with the ability to block the transmission of the disease to mosquito vectors, a series of hybrid compounds combining either a 1,2,4-trioxane or 1,2,4,5-tetraoxane and 8-aminoquinoline moieties were synthesized and screened for their antimalarial activity. These hybrid compounds showed high potency against both exoerythrocytic and erythrocytic forms of malaria parasites, comparable to representative trioxane-based counterparts. Furthermore, they efficiently blocked the development of the sporogonic cycle in the mosquito vector. The tetraoxane-based hybrid 5, containing an amide linker between the two moieties, effectively cleared a patent blood-stage P. berghei infection in mice after i.p. administration. Overall, these results indicate that peroxide-8-aminoquinoline hybrids are excellent starting points to develop an agent that conveys all the desired antimalarial multistage activities in a single chemical entity and, as such, with the potential to be used in malaria elimination campaigns.
doi:10.1021/ml4002985
PMCID: PMC4027774  PMID: 24900781
Antimalarials; endoperoxide; sporogonic cycle; P. berghei
2.  Use of Corticosteroids After Hepatoportoenterostomy for Bile Drainage in Infants With Biliary Atresia 
JAMA  2014;311(17):1750-1759.
IMPORTANCE
Biliary atresia is the most common cause of end-stage liver disease in children. Controversy exists as to whether use of steroids after hepatoportoenterostomy improves clinical outcome.
OBJECTIVE
To determine whether the addition of high-dose corticosteroids after hepatoportoenterostomy is superior to surgery alone in improving biliary drainage and survival with the native liver.
DESIGN, SETTING, AND PATIENTS
The multicenter, double-blind Steroids in Biliary Atresia Randomized Trial (START) was conducted in 140 infants (mean age, 2.3 months) between September 2005 and February 2011 in the United States; follow-up ended in January 2013.
INTERVENTIONS
Participants were randomized to receive intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks) followed by a tapering protocol for 9 weeks (n = 70) or placebo (n = 70) initiated within 72 hours of hepatoportoenterostomy.
MAIN OUTCOMES AND MEASURES
The primary end point (powered to detect a 25% absolute treatment difference) was the percentage of participants with a serum total bilirubin level of less than 1.5 mg/dL with his/her native liver at 6 months posthepatoportoenterostomy. Secondary outcomes included survival with native liver at 24 months of age and serious adverse events.
RESULTS
The proportion of participants with improved bile drainage was not statistically significantly improved by steroids at 6 months posthepatoportoenterostomy (58.6% [41/70] of steroids group vs 48.6% [34/70] of placebo group; adjusted relative risk, 1.14 [95% CI, 0.83 to 1.57]; P = .43). The adjusted absolute risk difference was 8.7% (95% CI, −10.4% to 27.7%). Transplant-free survival was 58.7% in the steroids group vs 59.4% in the placebo group (adjusted hazard ratio, 1.0 [95% CI, 0.6 to 1.8]; P = .99) at 24 months of age. The percentage of participants with serious adverse events was 81.4% [57/70] of the steroids group and 80.0% [56/70] of the placebo group (P > .99); however, participants receiving steroids had an earlier time of onset of their first serious adverse event by 30 days posthepatoportoenterostomy (37.2% [95% CI, 26.9% to 50.0%] of steroids group vs 19.0% [95% CI, 11.5% to 30.4%] of placebo group; P= .008).
CONCLUSIONS AND RELEVANCE
Among infants with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6 months, although a small clinical benefit could not be excluded. Steroid treatment was associated with earlier onset of serious adverse events in children with biliary atresia.
doi:10.1001/jama.2014.2623
PMCID: PMC4303045  PMID: 24794368
3.  Selection of Drug Resistance-Mediating Plasmodium falciparum Genetic Polymorphisms by Seasonal Malaria Chemoprevention in Burkina Faso 
Seasonal malaria chemoprevention (SMC), with regular use of amodiaquine plus sulfadoxine-pyrimethamine (AQ/SP) during the transmission season, is now a standard malaria control measure in the Sahel subregion of Africa. Another strategy under study is SMC with dihydroartemisinin plus piperaquine (DP). Plasmodium falciparum single nucleotide polymorphisms (SNPs) in P. falciparum crt (pfcrt), pfmdr1, pfdhfr, and pfdhps are associated with decreased response to aminoquinoline and antifolate antimalarials and are selected by use of these drugs. To characterize selection by SMC of key polymorphisms, we assessed 13 SNPs in P. falciparum isolated from children aged 3 to 59 months living in southwestern Burkina Faso and randomized to receive monthly DP or AQ/SP for 3 months in 2009. We compared SNP prevalence before the onset of SMC and 1 month after the third treatment in P. falciparum PCR-positive samples from 120 randomly selected children from each treatment arm and an additional 120 randomly selected children from a control group that did not receive SMC. The prevalence of relevant mutations was increased after SMC with AQ/SP. Significant selection was seen for pfcrt 76T (68.5% to 83.0%, P = 0.04), pfdhfr 59R (54.8% to 83.3%, P = 0.0002), and pfdhfr 108N (55.0% to 87.2%, P = 0.0001), with trends toward selection of pfmdr1 86Y, pfdhfr 51I, and pfdhps 437G. After SMC with DP, only borderline selection of wild-type pfmdr1 D1246 (mutant; 7.7% to 0%, P = 0.05) was seen. In contrast to AQ/SP, SMC with DP did not clearly select for known resistance-mediating polymorphisms. SMC with AQ/SP, but not DP, may hasten the development of resistance to components of this regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT00941785.)
doi:10.1128/AAC.02406-14
PMCID: PMC4068591  PMID: 24733476
4.  Pediatric liver transplant for urea cycle disorders and organic acidemias: United Network for Organ Sharing (UNOS) data 2002–2012 
Background
Decision-making about liver transplant is unique in children with urea cycle disorders (UCD) and organic acidemias (OA) because of immediate high priority on the waiting list, not related to severity of disease. There is limited national outcomes data on which to base recommendations about liver transplant for UCD or OA.
Methods
Retrospective analysis of UNOS data on liver recipients <18 years, transplanted 2002–12. Repeat transplants excluded.
Results
5.4% of pediatric liver transplants were liver-only for UCD/OA. UCD/OA increased from 4.3% of transplants in 2002–05 to 7.4% in 2010–12 (p<0.0001). 96% were deceased donor. Of these, 59% were transplanted at <2 years of age. Graft survival improved as age at transplant increased (p=0.04). By 5 years post-transplant, graft survival was 78% for children <2 years at transplant and 88% for those ≥2 years (p=0.06). Vascular thrombosis caused 45% of graft losses; 65% in children <2 years. Patient survival also improved as age at transplant increased; 5-year patient survival was 88% in UCD/OA children <2 years at transplant and 99% in those ≥2 years (p=0.006). At last-follow up (54 ± 34.4 months), children transplanted for UCD/OA were more likely to have cognitive and motor delay than those transplanted for other indications. Cognitive and motor delay in UCD/OA children were associated with metabolic disorder but not predicted by age or weight at transplant, gender, ethnicity, split vs. whole liver, or hospitalization at transplant in univariate and multivariate analysis.
Conclusion
Most liver transplants for UCD/OA occur in early childhood. Further research is needed on the benefits of early transplant for UCD/OA, as younger age may increase post-transplant morbidity.
doi:10.1002/lt.23765
PMCID: PMC3877181  PMID: 24136671
liver transplantation; children; metabolic liver disease; long-term outcomes
5.  Synthesis and in Vitro and in Vivo Pharmacological Evaluation of New 4-Aminoquinoline-Based Compounds 
ACS Medicinal Chemistry Letters  2013;4(12):1198-1202.
A new class of 4-aminoquinolines was synthesized and evaluated in vitro for antiplasmodial activity against both the chloroquine-sensitive (3D7) and -resistant (K1 and W2) strains. The most active compounds 3c–3e had acceptable cytotoxicity but showed strong inhibition toward a panel of cytochrome P450 enzymes in vitro. Pharmacokinetic studies on 3d and 3e in mice showed that they had moderate half-life (4–6 h) and low oral bioavailability. The front runner compound 3d exhibited moderate inhibition of the malaria parasite on P. berghei infected mice following oral administration (5 mg/kg), achieving reduction of parasitemia population by 47% on day 7.
doi:10.1021/ml400311r
PMCID: PMC4027225  PMID: 24900630
Aminoquinolines; antiplasmodial activity; pharmacokinetics; plasma protein binding
6.  Extra-hepatic anomalies in infants with biliary atresia: results of a large prospective North American multi-center study 
Hepatology (Baltimore, Md.)  2013;58(5):1724-1731.
Background and aims
The etiology of biliary atresia (BA) is unknown. Given that patterns of anomalies might provide etiopathogenetic clues, we utilized data from the North American Childhood Liver Disease Research and Education Network to analyze patterns of anomalies in infants with BA.
Methods
Two hundred eighty-nine infants who were enrolled into the prospective database prior to surgery at any of 15 centers participating were evaluated.
Results
Group 1 was non-syndromic, isolated BA (without major malformations) (n = 242, 84 %), Group 2 was BA and at least one malformation considered major as defined by the National Birth Defects Prevention Study but without laterality defects (n = 17, 6%). Group 3 was syndromic, with laterality defects (n = 30, 10%). In the population as a whole, anomalies (either major or minor) were most prevalent in the cardiovascular (16%) and gastrointestinal (14%) systems. Group 3 patients accounted for the majority of subjects with cardiac, gastrointestinal and splenic anomalies. Group 2 subjects also frequently displayed cardiovascular (71%) and gastrointestinal (24 %) anomalies; interestingly this group had genitourinary anomalies more frequently (47%) compared to Group 3 subjects (10%).
Conclusions
This study identified a group of BA (Group 2) that differed from the classical syndromic and non-syndromic groups and that was defined by multiple malformations without laterality defects. Careful phenotyping of the patterns of anomalies may be critical to the interpretation of both genetic and environmental risk factors associated with BA, allowing new insight into pathogenesis and/or outcome.
doi:10.1002/hep.26512
PMCID: PMC3844083  PMID: 23703680
birth defects; laterality defects; cholangiopathy; embryonic; nonsyndromic
7.  Health Related Quality of Life in Patients with Biliary Atresia Surviving with their Native Liver 
The Journal of pediatrics  2013;163(4):1052-7.e2.
Objectives
To quantify health related quality of life (HRQOL) of patients with biliary atresia with their native livers and compare them with healthy children and patients with biliary atresia post-liver transplant (LT) and to examine the relationship between HRQOL and medical variables.
Study design
A cross-sectional HRQOL study of patients with biliary atresia with their native livers (ages 2-25 years) was conducted and compared with healthy and post-LT biliary atresia samples using PedsQL™ 4.0 child self and parent proxy reports, a validated measure of physical/psychosocial functioning.
Results
221 patients with biliary atresia with native livers (54% female, 67% white) were studied. patient self and parent proxy reports showed significantly poorer HRQOL than healthy children across all domains (p < 0.001), particularly in emotional and psychosocial functioning. Child self and parent proxy HRQOL scores from patients with biliary atresia with their native livers and post-LT biliary atresia were similar across all domains (p=NS). Child self and parent proxy reports showed moderate agreement across all scales, except social functioning (poor to fair agreement). On multivariate regression analysis, black race and elevated total bilirubin were associated with lower Total and Psychosocial HRQOL summary scores.
Conclusions
HRQOL in patients with biliary atresia with their native livers is significantly poorer than healthy and similar to post-LT biliary atresia children. These findings identify significant opportunities to optimize the overall health of patients with biliary atresia.
doi:10.1016/j.jpeds.2013.04.037
PMCID: PMC4014354  PMID: 23746866
Biliary Atresia; Quality of Life; Health Related Quality of Life; Liver Transplant; PedsQL
8.  Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine 
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.
doi:10.4269/ajtmh.14-0031
PMCID: PMC4183414  PMID: 25048375
9.  GENETIC DEFECTS IN BILE ACID CONJUGATION CAUSE FAT-SOLUBLE VITAMIN DEFICIENCY 
Gastroenterology  2013;144(5):945-e15.
BACKGROUND & AIMS
The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis.
METHODS
We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples, and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-Co A ligase (SLC27A5).
RESULTS
Levels of urinary bile acids were increased (432±248 μmol/L) and predominantly excreted in unconjugated forms (79.4%±3.9%), and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile and serum. Unconjugated bile acids accounted for 95.7%±5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4%±5.5% of total. Duodenal bile acid concentrations were 12.1±5.9 mmol/L—a concentration too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested.
CONCLUSIONS
Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.
doi:10.1053/j.gastro.2013.02.004
PMCID: PMC4175397  PMID: 23415802
Chronic Liver Disease; Hepatic; Inherited; Nutrient; Bile acid conjugation; Bile acid-CoA amino acid N-acyltransferase; BAAT; Glycine; Taurine; Mass Spectrometry; Cholestasis; Fat-soluble vitamin deficiency
10.  An Artesunate-Containing Antimalarial Treatment Regimen Did Not Suppress Cytomegalovirus Viremia 
Background
Additional drugs are needed for the treatment of cytomegalovirus (CMV) infection. Artesunate is an antimalarial drug that has activity against CMV in vitro and in a rodent model. Only a small number of case reports are available describing the clinical effects of artesunate on CMV infection, and these yielded inconsistent results.
Objective
To evaluate the effect of artesunate on CMV infection, using blood samples collected from children who participated in malaria treatment trials.
Study design
Quantitative CMV DNA PCR was performed on dried blood spots collected from 494 Ugandan children, who were randomized either to artesunate plus amodiaquine or sulfadoxine-pyrimethamine plus amodiaquine for acute malaria infection. Poisson regression was used to compare treatment regimens with respect to the change in the frequency and quantity of CMV detected that occurred before and after treatment.
Results
CMV was detected in 11.4% of children immediately prior to treatment and 10.7% 3 days later (p=0.70). The average quantity of CMV was 0.30 log10 copies per million cells higher on day 3 than at treatment initiation (95% CI 0.01 to 0.58, p=0.041). There was no measurable difference in either the frequency or quantity of CMV detected in blood between children randomized to the two treatment arms.
Conclusions
A standard 3-day artesunate-containing antimalarial regimen had no detectable effect on CMV viremia in children with malaria. Longer treatment courses and/or higher doses of artesunate than those routinely used for malaria may be required for effective treatment of CMV infection.
doi:10.1016/j.jcv.2013.06.008
PMCID: PMC4036801  PMID: 23827788
11.  The interplay between drug resistance and fitness in malaria parasites 
Molecular microbiology  2013;89(6):1025-1038.
Summary
Controlling the spread of antimalarial drug resistance, especially resistance of Plasmodium falciparum to artemisinin-based combination therapies, is a high priority. Available data indicate that, as with other microorganisms, the spread of drug-resistant malaria parasites is limited by fitness costs that frequently accompany resistance. Resistance-mediating polymorphisms in malaria parasites have been identified in putative drug transporters and in target enzymes. The impacts of these polymorphisms on parasite fitness have been characterized in vitro and in animal models. Additional insights have come from analyses of samples from clinical studies, both evaluating parasites under different selective pressures and determining the clinical consequences of infection with different parasites. With some exceptions, resistance-mediating polymorphisms lead to malaria parasites that, compared to wild type, grow less well in culture and in animals, and are replaced by wild type when drug pressure diminishes in the clinical setting. In some cases, the fitness costs of resistance may be offset by compensatory mutations that increase virulence or changes that enhance malaria transmission. However, not enough is known about effects of resistance mediators on parasite fitness. A better appreciation of the costs of fitness-mediating mutations will facilitate the development of optimal guidelines for the treatment and prevention of malaria.
doi:10.1111/mmi.12349
PMCID: PMC3792794  PMID: 23899091
12.  Polymorphisms in K13 and Falcipain-2 Associated with Artemisinin Resistance Are Not Prevalent in Plasmodium falciparum Isolated from Ugandan Children 
PLoS ONE  2014;9(8):e105690.
The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006–7 (n = 49) and from 2010–12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.
doi:10.1371/journal.pone.0105690
PMCID: PMC4140830  PMID: 25144768
13.  Efficacy and Day 7 Plasma Piperaquine Concentrations in African Children Treated for Uncomplicated Malaria with Dihydroartemisinin-Piperaquine 
PLoS ONE  2014;9(8):e103200.
Background
One promising new Artemisinin-based combination therapies (ACTs) is dihydroartemisinin-piperaquine (DHA-PQ). However, the pharmacokinetics of piperaquine and the relationship between drug levels and clinical efficacy are incompletely characterized, particularly in children.
Methods
We performed a single-arm open-label trial in Bobo-Dioulasso, Burkina Faso. A total of 379 participants aged 6 months or more with uncomplicated falciparum malaria were enrolled. Each participant received daily dose of DHA-PQ for three days and followed for 42 days. Parasitological efficacy was analyzed, considering rates of recrudescence and overall recurrence. PK was an exploratory endpoint and a priori, no sample size had been determined. Day 7 capillary and venous plasma concentrations of piperaquine were measured in children aged 2–10 years.
Results
Of the 379 participants, 365 (96.3%) completed 42 days of follow-up. The median daily dose of PQ was 18.5 mg/kg [6.5–24]. Treatment with DHA-PQ was well tolerated with fever and parasitemia resolution within 48 hours in nearly all children. Recurrent malaria within 42 days of follow-up occurred in 31.3% (10/34) of children less than 2 years old, 16.0% (16/106) of those aged 2–5 years, 9.4% (15/160) of those aged 5–10 years, and none (0/68) of those over 10 years old. After genotyping, 3 of 41 recurrent episodes were recrudescence. An exploratory analysis shows that children with successful treatment outcomes had significantly higher median plasma concentrations of PQ compared to those with recurrent malaria within 42 days after therapy, considering either capillary samples (68 ng/ml [50–85] compared to 48 ng/ml [36–55], p<0.001) or venous samples (42 ng/ml [29–59] compared to 25 ng/ml [19–44], p<0.001).
Conclusion
DHA-PQ was effective for uncomplicated P. falciparum malaria treatment and offers an alternative to other ACTs. Recurrent malaria was mainly due to new infections after treatment and was correlated with low day 7 PQ concentration in the youngest patients.
Trial Registration
Controlled-Trials.com ISRCTN59761234
doi:10.1371/journal.pone.0103200
PMCID: PMC4136730  PMID: 25133389
14.  Protective Efficacy and Safety of Three Antimalarial Regimens for the Prevention of Malaria in Young Ugandan Children: A Randomized Controlled Trial 
PLoS Medicine  2014;11(8):e1001689.
Grant Dorsey and colleagues investigate the efficacy of three antimalarial drugs for preventing malaria in children living in Uganda, an area of high transmission intensity.
Please see later in the article for the Editors' Summary
Background
Chemoprevention offers a promising strategy for prevention of malaria in African children. However, the optimal chemoprevention drug and dosing strategy is unclear in areas of year-round transmission and resistance to many antimalarial drugs. To compare three available regimens, we conducted an open-label randomized controlled trial of chemoprevention in Ugandan children.
Methods and Findings
This study was conducted between June 28, 2010, and September 25, 2013. 400 infants were enrolled and 393 randomized at 6 mo of age to no chemoprevention, monthly sulfadoxine-pyrimethamine (SP), daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP). Study drugs were administered at home without supervision. Piperaquine (PQ) levels were used as a measure of compliance in the DP arm. Participants were given insecticide-treated bednets, and caregivers were encouraged to bring their child to a study clinic whenever they were ill. Chemoprevention was stopped at 24 mo of age, and participants followed-up an additional year. Primary outcome was the incidence of malaria during the intervention period. During the intervention, the incidence of malaria in the no chemoprevention arm was 6.95 episodes per person-year at risk. Protective efficacy was 58% (95% CI, 45%–67%, p<0.001) for DP, 28% (95% CI, 7%–44%, p = 0.01) for TS, and 7% for SP (95% CI, −19% to 28%, p = 0.57). PQ levels were below the detection limit 52% of the time when malaria was diagnosed in the DP arm, suggesting non-adherence. There were no differences between the study arms in the incidence of serious adverse events during the intervention and the incidence of malaria during the 1-y period after the intervention was stopped.
Conclusions
For preventing malaria in children living in an area of high transmission intensity, monthly DP was the most efficacious and safe, although adherence may pose a problem. Monthly SP and daily TS may not be appropriate in areas with high transmission intensity and frequent resistance to antifolates.
Trial registration
www.ClinicalTrials.gov NCT00948896
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Malaria is a parasitic disease that kills more than 600,000 people (mainly young children living in sub-Saharan Africa) every year. Malaria parasites, which are transmitted to people through the bites of night-flying mosquitoes, cause a characteristic fever that needs to be treated promptly with antimalarial drugs to prevent anemia and organ damage. Prompt treatment also helps to reduce malaria transmission and is a component of the Global Malaria Action Plan, which aims to control and eventually eliminate malaria. Other components of this plan include the provision of insecticide-treated bednets for people to sleep under to avoid mosquito bites and indoor residual spraying with insecticides. Widespread deployment of these preventative tools and the increased availability of effective antimalarial drugs have greatly reduced malaria-related deaths worldwide over the past decade, but new strategies are still urgently needed to reduce the burden of malaria among those most at risk—young children living in Africa.
Why Was This Study Done?
One promising strategy for the prevention of malaria in African children is the use of antimalarial drugs to prevent rather than treat malaria. In trials, giving infants sulfadoxine-pyrimethamine (SP) alongside routine vaccinations, for example, reduced the incidence of malaria (the number of new cases in the population in a year) by about 30% during the first year of life (a protective efficacy of 30%). However, the optimal chemoprevention drug and dosing strategy for children living in African regions where there is year-round transmission of malaria and where resistance to antimalarial drugs is common remains unclear. Here, the researchers undertake an open-label randomized controlled trial (RCT) of chemoprevention in infants in the Tororo District of eastern Uganda, an area with intense year-round malaria transmission. RCTs compare outcomes in groups of people chosen to receive different interventions through the play of chance; in open-label RCTs, both the researchers and the participants know which treatment is being administered.
What Did the Researchers Do and Find?
The researchers assigned 393 six-month-old infants to receive no chemoprevention, monthly SP, daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP) until they were 24 months old. SP and TS block the production of folic acid, which malaria needs for survival, whereas DP is a newer artemisinin-based combination therapy (ACT). All the drugs were given at home without supervision, and caregivers were asked to bring their children to a study clinic whenever they were ill. During the intervention, the incidence of malaria was 6.95 episodes per person-year at risk in the no chemoprevention arm but only three episodes per person-year at risk in the DP arm. That is, the protective efficacy of DP was 58%. By contrast, the protective efficacies of TS and SP were 28% and 7%, respectively. However, for SP the protective efficacy was not statistically different compared to the no chemoprevention arm. Notably, piperaquine levels on the day that malaria was diagnosed were below the detection limit in half of the malaria episodes in the DP arm, which suggests that a complete dose of DP had not been given to the infant in the previous month, despite caregivers reporting that they had administered virtually all the assigned doses. Finally, the incidence of serious adverse events was similar in all the study arms during the intervention, as was the incidence of malaria during the year after the intervention, which suggests that the chemoprevention strategies did not affect the development of naturally acquired immunity.
What Do These Findings Mean?
These findings show that, for children living in an area of intense malaria transmission, monthly DP was the most efficacious strategy for malaria chemoprevention but that adherence to the strategy may have been a problem. These findings also suggest that monthly SP and daily TS may not be appropriate chemoprevention strategies in areas of high transmission intensity, particularly those where resistance to antifolate drugs is common. The accuracy of these findings may be affected by drug administration being self-reported and by the number of comparisons included in the trial, which may have increased the risk of false-positive results. Moreover, the results of this trial may not be generalizable to other regions of sub-Saharan Africa. Overall, however, these results suggest that monthly DP is a strategy worth considering in regions in need of improved malaria control measures, with the important caveat that widespread ACT use for chemoprevention could compromise the efficacy of ACT when used for treatment.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001689.
Information is available from the World Health Organization on malaria (in several languages), including information on malaria chemoprevention; the World Malaria Report 2013 provides details of the current global malaria situation, including information on malaria in Uganda
The US Centers for Disease Control and Prevention provide information on malaria, including information on ways to reduce malaria cases and deaths; it also provides a selection of personal stories about malaria, including a story about malaria in a child in Africa
Information is available from the Roll Back Malaria Partnership on the global control of malaria and on the Global Malaria Action Plan (in English and French); its website includes fact sheets about malaria in Africa and about children and malaria
MedlinePlus provides links to additional information on malaria (in English and Spanish)
More information about this trial is available
doi:10.1371/journal.pmed.1001689
PMCID: PMC4122345  PMID: 25093754
15.  Synthesis, Antiplasmodial Activity, and β-Hematin Inhibition of Hydroxypyridone–Chloroquine Hybrids 
ACS Medicinal Chemistry Letters  2013;4(7):642-646.
A series of noncytotoxic 4-aminoquinoline-3-hydroxypyridin-4-one hybrids were synthesized on the basis of a synergistic in vitro combination of a precursor N-alkyl-3-hydroxypyridin-4-one with chloroquine (CQ) and tested in vitro against CQ resistant (K1 and W2) and sensitive (3D7) strains of Plasmodium falciparum. In vitro antiplasmodial activity of the precursors was negated by blocking the chelator moiety via complexation with gallium(III) or benzyl protection. None of the precursors inhibited β-hematin formation. Most hybrids were more potent inhibitors of β-hematin formation than CQ, and a correlation between antiplasmodial activity and inhibition of β-hematin formation was observed. Potent hybrids against K1, 3D7, and W2, respectively, were 8c (0.13, 0.004, and 0.1 μM); 8d (0.08, 0.01, and 0.02 μM); and 7g (0.07, 0.03, and 0.08 μM).
doi:10.1021/ml4001084
PMCID: PMC4027233  PMID: 24900724
4-Aminoquinoline; hydroxypyridinone; antiplasmodial; iron chelators
16.  Design, Synthesis, and Antiplasmodial Activity of Hybrid Compounds Based on (2R,3S)-N-Benzoyl-3-phenylisoserine 
ACS Medicinal Chemistry Letters  2013;4(7):637-641.
A series of hybrid compounds based on (2R,3S)-N-benzoyl-3-phenylisoserine, artemisinin, and quinoline moieties was synthesized and tested for in vitro antiplasmodial activity against erythrocytic stages of K1 and W2 strains of Plasmodium falciparum. Two hybrid compounds incorporating (2R,3S)-N-benzoyl-3-phenylisoserine and artemisinin scaffolds were 3- to 4-fold more active than dihydroartemisinin, with nanomolar IC50 values against Plasmodium falciparum K1 strain.
doi:10.1021/ml400164t
PMCID: PMC4027494  PMID: 24900723
(2R,3S)-N-Benzoyl-3-phenylisoserine; artemisinin; quinoline; hybrids; antiplasmodial activity
17.  Kaposi Sarcoma–Associated Herpesvirus (KSHV) Seroprevalence in Population-Based Samples of African Children: Evidence for At Least 2 Patterns of KSHV Transmission 
The Journal of infectious diseases  2009;200(3):430-438.
Background
Kaposi sarcoma–associated herpesvirus (KSHV) infection is endemic among adult populations in Africa. A prevailing view is that childhood transmission is primarily responsible for the high seroprevalence of KSHV among adults that is observed throughout the continent. However, few studies have directly examined children, particularly in locations where KS is not commonly endemic.
Methods
Participants were children aged 1.5−8.9 years, including 427 children from a population-based sample in South Africa, 422 from a population-based sample in Uganda, and 567 from a clinic-based sample in Uganda. All serum specimens were tested by the same laboratory for KSHV antibodies with use of 2 enzyme immunoassays (against K8.1 and ORF65) and 1 immunofluorescence assay.
Results
KSHV seroprevalence was 7.5%−9.0% among South African children and was not associated with age. In contrast, in the Ugandan population-based sample, KSHV seroprevalence increased from 10% among 2-year-old children to 30.6% among 8-year-old children (Ptrend < .001). In the Ugandan clinic-based sample, seroprevalence increased from 9.3% among 2-year-old children to 36.4% among 8-year-old children (Ptrend < .001).
Conclusion
Two distinct relationships between age and KSHV infection among children imply that KSHV transmission among children is not uniform throughout Africa and is therefore not always responsible for the high seroprevalence observed in adults. There are at least 2 patterns of KSHV transmission in Africa.
doi:10.1086/600103
PMCID: PMC3975590  PMID: 19534596
18.  Prevalence of Asymptomatic Parasitemia and Gametocytemia among HIV-Infected Ugandan Children Randomized to Receive Different Antiretroviral Therapies 
In a recent randomized controlled trial, the use of protease inhibitor (PI)-based antiretroviral therapy (ART) was associated with a significantly lower incidence of malaria compared with non-nucleoside reverse transcriptase inhibitor-based ART in a cohort of human immunodeficiency virus-infected Ugandan children living in an area of high malaria transmission intensity. In this report, we compared the prevalence of asymptomatic parasitemia and gametocytemia using data from the same cohort. The prevalence of asymptomatic parasitemia did not differ between the two ART treatment arms. The PI-based arm was associated with a lower risk of gametocytemia at the time of diagnosis of malaria (6.6% versus 14.5%, P = 0.03) and during the 28 days after malaria diagnosis (3.4% versus 6.5%, P = 0.04). Thus, in addition to decreasing the incidence of malaria, the use of PI-based ART may lower transmission, as a result of a decrease in gametocytemia, in areas of high malaria transmission intensity.
doi:10.4269/ajtmh.12-0658
PMCID: PMC3617863  PMID: 23358639
19.  Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target 
PLoS ONE  2014;9(4):e93008.
Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.
doi:10.1371/journal.pone.0093008
PMCID: PMC3974720  PMID: 24699522
20.  Intravenous N-acetylcysteine in Pediatric Patients with Non-Acetaminophen Acute Liver Failure: A Placebo-Controlled Clinical Trial 
Hepatology (Baltimore, Md.)  2013;57(4):1542-1549.
N-acetylcysteine (NAC) was found to improve transplantation-free survival in only those adults with non-acetaminophen (non-APAP) acute liver failure (ALF) and grade 1–2 hepatic encephalopathy (HE). Because non-APAP ALF differs significantly between children and adults, the Pediatric Acute Liver Failure (PALF) Study Group evaluated NAC in non-APAP PALF. Children from birth through age 17 years with non-APAP ALF enrolled in the PALF registry were eligible to enter an adaptively allocated, doubly masked, placebo-controlled trial using a continuous intravenous infusion of NAC (150 mg/kg/day in 5% dextrose in water [D5W]) or placebo (D5W) for up to 7 days. The primary outcome was 1-year survival. Secondary outcomes included liver transplantation-free survival, liver transplantation (LTx), length of ICU and hospital stays, organ system failure and maximum HE score. A total of 184 participants were enrolled in the trial with 92 in each arm. The 1-year survival did not differ significantly (p=0.19) between the NAC (73%) and placebo (82%) treatment groups. The 1-year LTx-free survival was significantly lower (p=0.03) in those who received NAC (35%) than those who received placebo (53%), particularly, but not significantly so, among those less than 2 years old with HE grade 0–1 (NAC 25%; placebo 60%; p=0.0493). There were no significant differences between treatment arms for hospital or ICU length of stay, organ systems failing, or highest recorded grade of HE.
Conclusion
NAC did not improve 1-year survival in non-APAP PALF. 1-year LTx-free survival was significantly lower with NAC, particularly among those < 2 years old. These results do not support broad use of NAC in non-APAP PALF and emphasizes the importance of conducting controlled pediatric drug trials, regardless of results in adults.
doi:10.1002/hep.26001
PMCID: PMC3509266  PMID: 22886633
Child; hepatic encephalopathy; liver transplantation; multi-organ system failure; treatment
21.  Estimation of malaria haplotype and genotype frequencies: a statistical approach to overcome the challenge associated with multiclonal infections 
Malaria Journal  2014;13:102.
Background
Reliable measures of anti-malarial resistance are crucial for malaria control. Resistance is typically a complex trait: multiple mutations in a single parasite (a haplotype or genotype) are necessary for elaboration of the resistant phenotype. The frequency of a genetic motif (proportion of parasite clones in the parasite population that carry a given allele, haplotype or genotype) is a useful measure of resistance. In areas of high endemicity, malaria patients generally harbour multiple parasite clones; they have multiplicities of infection (MOIs) greater than one. However, most standard experimental procedures only allow measurement of marker prevalence (proportion of patient blood samples that test positive for a given mutation or combination of mutations), not frequency. It is misleading to compare marker prevalence between sites that have different mean MOIs; frequencies are required instead.
Methods
A Bayesian statistical model was developed to estimate Plasmodium falciparum genetic motif frequencies from prevalence data collected in the field. To assess model performance and computational speed, a detailed simulation study was implemented. Application of the model was tested using datasets from five sites in Uganda. The datasets included prevalence data on markers of resistance to sulphadoxine-pyrimethamine and an average MOI estimate for each study site.
Results
The simulation study revealed that the genetic motif frequencies that were estimated using the model were more accurate and precise than conventional estimates based on direct counting. Importantly, the model did not require measurements of the MOI in each patient; it used the average MOI in the patient population. Furthermore, if a dataset included partially genotyped patient blood samples, the model imputed the data that were missing. Using the model and the Ugandan data, genotype frequencies were estimated and four biologically relevant genotypes were identified.
Conclusions
The model allows fast, accurate, reliable estimation of the frequency of genetic motifs associated with resistance to anti-malarials using prevalence data collected from malaria patients. The model does not require per-patient MOI measurements and can easily analyse data from five markers. The model will be a valuable tool for monitoring markers of anti-malarial drug resistance, including markers of resistance to artemisinin derivatives and partner drugs.
doi:10.1186/1475-2875-13-102
PMCID: PMC4004158  PMID: 24636676
Frequency versus prevalence; Statistical model; Anti-malarial resistance
22.  Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda 
Malaria Journal  2014;13:95.
Background
Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.
Methods
For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.
Results
SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K = 0.77) for pfcrt K76T, 79% (K = 0.55) for pfmdr1 N86Y, 83% (K = 0.65) for pfmdr1 Y184F, and 91% (K = 0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.
Conclusion
The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.
doi:10.1186/1475-2875-13-95
PMCID: PMC4004386  PMID: 24629020
Plasmodium falciparum; Malaria; Fluorescent microsphere assay; Drug resistance; Polymorphisms
23.  Impact of Intermittent Preventive Treatment With Dihydroartemisinin-Piperaquine on Malaria in Ugandan Schoolchildren: A Randomized, Placebo-Controlled Trial 
Dihydroartemisinin-piperaquine administered at monthly intervals, but not that dosed once a school term, is a remarkably effective measure for the prevention of incidence of malaria, prevalence of parasitemia, and prevalence of anemia in schoolchildren living in a high-transmission setting.
Background. Intermittent preventive treatment (IPT) in schoolchildren offers a promising option for malaria control. However, the optimal drug and dosing regimens for IPT remain to be determined.
Methods. We conducted a randomized, double-blind, placebo-controlled trial in 740 schoolchildren aged 6–14 years living in a setting of high malaria transmission in Uganda. Enrolled children were randomized to dihydroartemisinin-piperaquine (DP) given once a month (IPTm), DP given once a school term (4 treatments over 12 months, IPTst), or placebo and followed for 12 months. The primary outcome was the incidence of malaria over 12 months. Secondary outcomes included parasite prevalence and anemia over 12 months. Analyses were conducted on an intention-to-treat basis.
Results. In the placebo arm, the incidence of malaria was 0.34 episodes per person-year and the prevalence of parasitemia and anemia was 38% and 20%, respectively. IPTm reduced the incidence of malaria by 96% (95% confidence interval [CI], 88%–99%, P < .0001), the prevalence of asymptomatic parasitemia by 94% (95% CI, 92%–96%, P < .0001), and the prevalence of anemia by 40% (95% CI, 19%–56%, P < .0001). IPTst had no significant effect on the incidence of symptomatic malaria or the prevalence of anemia, but reduced the prevalence of asymptomatic parasitemia by 54% (95% CI, 47%–60%, P < .0001).
Conclusions. Monthly IPT with DP offered remarkable protection against clinical malaria, parasitemia, and anemia in schoolchildren living in a high-malaria-transmission setting.
Clinical Trials Registration.  NCT01231880.
doi:10.1093/cid/ciu150
PMCID: PMC4001293  PMID: 24621953
malaria; intermittent preventive treatment; schoolchildren
24.  Auto-antibodies and Autoimmune Disease during Treatment of Children with Chronic Hepatitis C 
Objectives
Auto-antibodies were studied in a well-characterized cohort of children with chronic hepatitis C (CHC) during treatment with PEG-IFN and ribavirin to assess the relationship to treatment and development of autoimmune disease.
Methods
114 children (5–17 years), previously screened for the presence of high titer autoantibodies, were randomized to Peg-IFN with or without ribavirin. Anti-nuclear (ANA), anti-liver-kidney-microsomal (LKM), anti-thyroglobulin (TG), anti-thyroid peroxidase (TPO), insulin (IA2), anti-glutamic acid decarboxylase (GAD) antibodies were measured after trial completion using frozen sera.
Results
At baseline,19% had auto-antibodies: ANA (8%), LKM (4%), and GAD (4%). At 24 and 72 weeks (24 weeks after treatment completion), 23% and 26% had auto-antibodies (p=0.50, 0.48 compared to baseline). One child developed diabetes and two hypothyroidism during treatment; none developed autoimmune hepatitis. At 24 weeks, the incidence of flu-like symptoms, gastrointestinal symptoms, and headaches were 42%, 8% and 19% in those with auto-antibodies vs. 52%, 17%, and 26% in those without (p=0.18, 0.36, and 0.20, respectively). In children with negative HCV PCR at 24 weeks, there was no difference in the rate of early virologic response /sustained virologic response respectively in those with auto-antibodies 76%/69%, vs 58%/65% in those without (p=0.48).
Conclusions
Despite screening, we found autoantibodies commonly at baseline, during treatment for CHC and after. The presence of antibodies did not correlate with viral response, side effects, or autoimmune hepatitis. Neither screening nor archived samples assayed for thyroid and diabetes-related antibodies identified the 3 subjects who developed overt autoimmune disease, diabetes (1) and hypothyroidism (2).
doi:10.1097/MPG.0b013e3182774cae
PMCID: PMC3583208  PMID: 23439301
Pediatrics; Viral hepatitis; Therapy; Complications; Diabetes; Hypothyroid; Auto-immune
25.  Fitness Consequences of Plasmodium falciparum pfmdr1 Polymorphisms Inferred from Ex Vivo Culture of Ugandan Parasites 
Polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene impact sensitivity to multiple antimalarials. In Africa, polymorphisms at N86Y and D1246Y are common and have various impacts on sensitivity to different drugs. To gain insight into the fitness consequences of these polymorphisms, we cultured parasites isolated from children with malaria in Tororo, Uganda, where the multiplicity of infection is high, and used pyrosequencing to follow polymorphism prevalences in culture over time. Of 71 cultures, parasites in 69 were successfully analyzed at N86Y and parasites in 68 were successfully analyzed at D1246Y over 3 to 36 days of culture. For position 86, the sequences of 39/69 (56.5%) parasites remained stable (>90% prevalence over 2 to 17 time points), with 82.1% of these being stable for the 86Y mutation. For position 1246, the sequences of 31/68 (45.6%) parasites remained stable, with 64.5% of these being stable for the wild-type D1246 sequence (P = 0.0002 for comparison of stable mutant genotypes for the two alleles). Defining allele selection as a ≥15% change in prevalence between the first and last samples assessed, for position 86, 11 samples showed selection, with selection toward 86Y occurring in 72.7% of alleles; for position 1246, 14 samples showed selection, with selection toward D1246 occurring in 64.3% of alleles (P = 0.11 for comparison of selection of mutations at the two alleles). Among the 7 samples with selection at both alleles, 5 showed selection for both 86Y and D1246. Overall, consistent trends in the direction of selection were seen, although differences were not statistically significant. Our results suggest fitness advantages for parasites with the pfmdr1 86Y mutation and wild-type D1246, highlighting the complex interplay between drug resistance and fitness in malaria parasites. (This study has been registered at ClinicalTrials.gov under registration no. NCT00948896 and NCT00993031.)
doi:10.1128/AAC.00161-13
PMCID: PMC3754360  PMID: 23796921

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