Randomized trials of artemisinin-piperaquine, dihydroartemisinin-piperaquine phosphate and artemether-lumefantrine for the treatment of multi-drug resistant falciparum malaria in Cambodia-Thailand border area

<p>Abstract</p> <p>Background</p> <p>Drug resistance of falciparum malaria is a global problem. Sulphadoxine/pyrimethamine-resistant and mefloquine-resistant strains of falciparum malaria have spread in Southeast Asia at lightning speed in 1980s-1990s, and the Cambodia-Thailand border is one of the malaria epidemic areas with the most severe forms of multi-drug resistant falciparum malaria.</p> <p>Methods</p> <p>Artemisinin-piperaquine (AP), dihydroartemisinin-piperaquine phosphate (DHP) and artemether-lumefantrine (AL) were used to treat 110, 55 and 55 uncomplicated malaria patients, respectively. The total dosage for adults is 1,750 mg (four tablets, twice over 24 hours) of AP, 2,880 mg (eight tablets, four times over two days) of DHP, and 3,360 mg (24 tablets, six times over three days) of AL. The 28-day cure rate, parasite clearance time, fever clearance time, and drug tolerance of patients to the three drugs were compared. All of the above methods were consistent with the current national guidelines.</p> <p>Results</p> <p>The mean parasite clearance time was similar in all three groups (66.7 ± 21.9 hrs, 65.6 ± 27.3 hrs, 65.3 ± 22.5 hrs in AP, DHP and AL groups, respectively), and there was no remarkable difference between them; the fever clearance time was also similar (31.6 ± 17.7 hrs, 34.6 ± 21.8 hrs and 36.9 ± 15.4 hrs, respectively). After following up for 28-days, the cure rate was 95.1%(97/102), 98.2%(54/55) and 82.4%(42/51); and the recrudescence cases was 4.9%(5/102), 1.8%(1/55) and 17.6%(9/51), respectively. Therefore, the statistical data showed that 28-day cure rate in AP and DHP groups was superior to AL group obviously.</p> <p>The patients had good tolerance to all the three drugs, and some side effects (anoxia, nausea, vomiting, headache and dizziness) could be found in every group and they were self-limited; patients in control groups also had good tolerance to DHP and AL, there was no remarkable difference in the three groups.</p> <p>Conclusions</p> <p>AP, DHP and AL all remained efficacious treatments for the treatment of falciparum malaria in Cambodia-Thailand border area. However, in this particular setting, the AP regimen turned out to be favourable in terms of efficacy and effectiveness, simplicity of administration, cost and compliance.</p> <p>Trial Registration</p> <p>The trial was registered at <it>Chinese Clinical Trial Register </it>under identifier 2005L01041.</p

Spread of artemisinin resistance in Plasmodium falciparum malaria.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.)

Evaluation of cutaneous immune response in a controlled human in vivo model of mosquito bites

International audienceMosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito ‘bite’, eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes -endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to T h 2-driven responses and CD8 + T cell activation. Stimulation of participants’ bitten skin cells with Aedes aegypti salivary gland extract results in reduced pro-inflammatory cytokine production. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease

Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia.

BackgroundWe established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins.MethodsChildren aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing.ResultsFrom 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01).ConclusionsHigh levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children.Clinical trials registrationNCT03534245

Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

International audienceBACKGROUND: Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections. METHODS: We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test. FINDINGS: In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0-3 h ring-stage parasites (10*88% vs 0*23%; p=0*007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0*74, 95% CI 0*50-0*87; p<0*0001). INTERPRETATION: The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed. FUNDING: Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH

Independent origin and global distribution of distinct plasmodium vivax duffy binding protein gene duplications

BACKGROUND:Plasmodium vivax causes the majority of malaria episodes outside Africa, but remains a relatively understudied pathogen. The pathology of P. vivax infection depends critically on the parasite's ability to recognize and invade human erythrocytes. This invasion process involves an interaction between P. vivax Duffy Binding Protein (PvDBP) in merozoites and the Duffy antigen receptor for chemokines (DARC) on the erythrocyte surface. Whole-genome sequencing of clinical isolates recently established that some P. vivax genomes contain two copies of the PvDBP gene. The frequency of this duplication is particularly high in Madagascar, where there is also evidence for P. vivax infection in DARC-negative individuals. The functional significance and global prevalence of this duplication, and whether there are other copy number variations at the PvDBP locus, is unknown. METHODOLOGY/PRINCIPAL FINDINGS:Using whole-genome sequencing and PCR to study the PvDBP locus in P. vivax clinical isolates, we found that PvDBP duplication is widespread in Cambodia. The boundaries of the Cambodian PvDBP duplication differ from those previously identified in Madagascar, meaning that current molecular assays were unable to detect it. The Cambodian PvDBP duplication did not associate with parasite density or DARC genotype, and ranged in prevalence from 20% to 38% over four annual transmission seasons in Cambodia. This duplication was also present in P. vivax isolates from Brazil and Ethiopia, but not India. CONCLUSIONS/SIGNIFICANCE:PvDBP duplications are much more widespread and complex than previously thought, and at least two distinct duplications are circulating globally. The same duplication boundaries were identified in parasites from three continents, and were found at high prevalence in human populations where DARC-negativity is essentially absent. It is therefore unlikely that PvDBP duplication is associated with infection of DARC-negative individuals, but functional tests will be required to confirm this hypothesis