Measuring the efficacy of anti-malarial drugs in vivo: quantitative PCR measurement of parasite clearance

BACKGROUND: Artemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs. Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy. This phenotype is likely to become a more important trial endpoint in studies of anti-malarial drug efficacy, but currently requires frequent, closely spaced blood sampling in hospitalized study participants, followed by quantitation of parasite density by microscopy. METHODS: A simple duplex quantitative PCR method was developed in which distinct fluorescent signals are generated from the human and parasite DNA components in each blood sample. The human amplification target in this assay is the β tubulin gene, and the parasite target is the unique methionine tRNA gene (pgmet), which exhibits perfect sequence identity in all six Plasmodium species that naturally infect humans. In a small series of malaria cases treated as hospital in-patients, the abundance of pgmet DNA was estimated relative to the human DNA target in daily peripheral blood samples, and parasite clearance times calculated. RESULTS: The qPCR assay was reproducibly able to replicate parasite density estimates derived from microscopy, but provided additional data by quantification of parasite density 24 hours after the last positive blood film. Robust estimates of parasite clearance times were produced for a series of patients with clinical malaria. CONCLUSIONS: Large studies, particularly in Africa where children represent a major proportion of treated cases, will require a simpler blood sample collection regime, and a method capable of high throughput. The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies

Chloroquine/Sulphadoxine-Pyrimethamine for Gambian Children with Malaria: Transmission to Mosquitoes of Multidrug-Resistant Plasmodium falciparum

OBJECTIVES: In the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes. DESIGN: We conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment. SETTING: The study took place in the town of Farafenni and surrounding villages in the Gambia. PARTICIPANTS: Participants were 500 children aged 6 mo to 10 y with uncomplicated P. falciparum malaria. INTERVENTIONS: Children were randomised to receive CQ, SP, or CQ/SP. OUTCOME MEASURES: Outcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes. RESULTS: After SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP. CONCLUSIONS: Parasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo

Artemisinin-Resistant Malaria: Research Challenges, Opportunities, and Public Health Implications

Artemisinin-based combination therapies are the most effective drugs to treat Plasmodium falciparum malaria. Reduced sensitivity to artemisinin monotherapy, coupled with the emergence of parasite resistance to all partner drugs, threaten to place millions of patients at risk of inadequate treatment of malaria. Recognizing the significance and immediacy of this possibility, the Fogarty International Center and the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health convened a conference in November 2010 to bring together the diverse array of stakeholders responding to the growing threat of artemisinin resistance, including scientists from malarious countries in peril. This conference encouraged and enabled experts to share their recent unpublished data from studies that may improve our understanding of artemisinin resistance. Conference sessions addressed research priorities to forestall artemisinin resistance and fostered collaborations between field- and laboratory-based researchers and international programs, with the aim of translating new scientific evidence into public health solutions. Inspired by this conference, this review summarizes novel findings and perspectives on artemisinin resistance, approaches for translating research data into relevant public health information, and opportunities for interdisciplinary collaboration to combat artemisinin resistance

Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence.

BACKGROUND: Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown. METHODS: We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequence-based amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment. RESULTS: Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.6-39.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.17-3.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.61-5.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.08-31.01; P < .001). CONCLUSIONS: Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia

The programming of sequences of saccades

Saccadic eye movements move the high-resolution fovea to point at regions of interest. Saccades can only be generated serially (i.e., one at a time). However, what remains unclear is the extent to which saccades are programmed in parallel (i.e., a series of such moments can be planned together) and how far ahead such planning occurs. In the current experiment, we investigate this issue with a saccade contingent preview paradigm. Participants were asked to execute saccadic eye movements in response to seven small circles presented on a screen. The extent to which participants were given prior information about target locations was varied on a trial-by-trial basis: participants were aware of the location of the next target only, the next three, five, or all seven targets. The addition of new targets to the display was made during the saccade to the next target in the sequence. The overall time taken to complete the sequence was decreased as more targets were available up to all seven targets. This was a result of a reduction in the number of saccades being executed and a reduction in their saccade latencies. Surprisingly, these results suggest that, when faced with a demand to saccade to a large number of target locations, saccade preparation about all target locations is carried out in paralle

Extended Malaria Parasite Clearance Time in African Children Following Artemisinincombination Therapy Enhances Transmission\ud to Anopheles Mosquitoes

Artemisinin resistance was recently shown to have spread or emerged on the Thailand/Myanmar border. Evidence is accumulating that the parasite clearance time after artemisinin-based combination therapy (ACT) is increasing in settings in Asia and Africa. It is currently unknown if an extended parasite clearance time after ACTs has consequences for the individual patient or confers a higher malaria transmission potential. 298 children in Mbita, Western Kenya, with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL, n = 153) ordihydroartemisinin-piperaquine (DP, n = 145). Parasite carriage post-treatment was determined by microscopy and qPCR, gametocyte carriage by quantitative nucleic acid sequence based amplication. Infectiousness to mosquitoes was determined by mosquito membrane feeding assays. Both drugs were efficacious as judged by standard trial outcomes. Sub-patent residual parasitaemia on day 3 was detected by qPCR in 36.11% (95% CI 25.11 - 48.29) of children treated with AL, and in 30.16% (95% CI 19.23 - 43.02) of children treated with DP. After adjustment for age, treatment arm and enrolment parasite density, children with an extended parasite clearance time were significantly more likely to have microscopically detected recurrent parasitaemia during follow-up (Odds Ratio: 19.51, 95% CI 5.24 - 72.71, p < 0.001). Children with an extended parasite clearance time were also more likely to be infectious to mosquitoes (Odds Ratio 2.76; 95% CI 1.14 - 6.67, p = 0.02) and gave rise to a higher oocyst load in mosquitoes (Incidence Rate Ratio 2.80, 95% CI 1.49 - 5.24, p = 0.001). Our findings indicate that an extended parasite clearance time after ACTs has consequences for the individual patient and for the population at large due to higher transmission potential. The high prevalence of residual subpatent parasitaemia after treatment may be due to novel parasite genotypes with reduced drug sensitivity, inadequate population-level immunity, or the higher sensitivity of qPCR for detection of persisting parasites.\u

The concurrent programming of saccades

Sequences of saccades have been shown to be prepared concurrently however it remains unclear exactly what aspects of those saccades are programmed in parallel. To examine this participants were asked to make one or two target-driven saccades: a reflexive saccade; a voluntary saccade; a reflexive then a voluntary saccade; or vice versa. During the first response the position of a second target was manipulated. The new location of the second saccade target was found to impact on second saccade latencies and second saccade accuracy showing that some aspects of the second saccade program are prepared in parallel with the first. However, differences were found in the specific pattern of effects for each sequence type. These differences fit well within a general framework for saccade control in which a common priority map for saccade control is computed and the influence of saccade programs on one another depends not so much on the types of saccade being produced but rather on the rate at which their programs develop

Defining Plasmodium falciparum Treatment in South West Asia: A Randomized Trial Comparing Artesunate or Primaquine Combined with Chloroquine or SP

INTRODUCTION: Antimalarial resistance has led to a global policy of artemisinin-based combination therapy. Despite growing resistance chloroquine (CQ) remained until recently the official first-line treatment for falciparum malaria in Pakistan, with sulfadoxine-pyrimethamine (SP) second-line. Co-treatment with the gametocytocidal primaquine (PQ) is recommended for transmission control in South Asia. The relative effect of artesunate (AS) or primaquine, as partner drugs, on clinical outcomes and gametocyte carriage in this setting were unknown. METHODS: A single-blinded, randomized trial among Afghan refugees in Pakistan compared six treatment arms: CQ; CQ+(single-dose)PQ; CQ+(3 d)AS; SP; SP+(single-dose)PQ, and SP+(3 d)AS. The objectives were to compare treatment failure rates and effect on gametocyte carriage, of CQ or SP monotherapy against the respective combinations (PQ or AS). Outcomes included trophozoite and gametocyte clearance (read by light microscopy), and clinical and parasitological failure. FINDINGS: A total of 308 (87%) patients completed the trial. Failure rates by day 28 were: CQ 55/68 (81%); CQ+AS 19/67 (28%), SP 4/41 (9.8%), SP+AS 1/41 (2.4%). The addition of PQ to CQ or SP did not affect failure rates (CQ+PQ 49/67 (73%) failed; SP+PQ 5/33 (16%) failed). AS was superior to PQ at clearing gametocytes; gametocytes were seen on d7 in 85% of CQ, 40% of CQ+PQ, 21% of CQ+AS, 91% of SP, 76% of SP+PQ and 23% of SP+AS treated patients. PQ was more effective at clearing older gametocyte infections whereas AS was more effective at preventing emergence of mature gametocytes, except in cases that recrudesced. CONCLUSIONS: CQ is no longer appropriate by itself or in combination. These findings influenced the replacement of CQ with SP+AS for first-line treatment of uncomplicated falciparum malaria in the WHO Eastern Mediterranean Region. The threat of SP resistance remains as SP monotherapy is still common. Three day AS was superior to single-dose PQ for reducing gametocyte carriage. TRIAL REGISTRATION: ClinicalTrials.gov NCT00959517

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD