Andreea Waltmann

Strategies for understanding and reducing the Plasmodium vivax and Plasmodium ovale hypnozoite reservoir in Papua New Guinean children: a randomised placebo-controlled trial and mathematical model

Background The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children. Methods and Findings From 17 August 2009 to 20 May 2010, 524 children aged 5–10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes. Conclusions These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission. Trial registration ClinicalTrials.gov NCT02143934

High Rates of Asymptomatic, Sub-microscopic Plasmodium vivax Infection and Disappearing Plasmodium falciparum Malaria in an Area of Low Transmission in Solomon Islands

Introduction Solomon Islands is intensifying national efforts to achieve malaria elimination. A long history of indoor spraying with residual insecticides, combined recently with distribution of long lasting insecticidal nets and artemether-lumefantrine therapy, has been implemented in Solomon Islands. The impact of these interventions on local endemicity of Plasmodium spp. is unknown. Methods In 2012, a cross-sectional survey of 3501 residents of all ages was conducted in Ngella, Central Islands Province, Solomon Islands. Prevalence of Plasmodium falciparum, P. vivax, P. ovale and P. malariae was assessed by quantitative PCR (qPCR) and light microscopy (LM). Presence of gametocytes was determined by reverse transcription quantitative PCR (RT-qPCR). Results By qPCR, 468 Plasmodium spp. infections were detected (prevalence = 13.4%; 463 P. vivax, five mixed P. falciparum/P. vivax, no P. ovale or P. malariae) versus 130 by LM (prevalence = 3.7%; 126 P. vivax, three P. falciparum and one P. falciparum/P. vivax). The prevalence of P. vivax infection varied significantly among villages (range 3.0–38.5%, p<0.001) and across age groups (5.3–25.9%, p<0.001). Of 468 P. vivax infections, 72.9% were sub-microscopic, 84.5% afebrile and 60.0% were both sub-microscopic and afebrile. Local residency, low education level of the household head and living in a household with at least one other P. vivax infected individual increased the risk of P. vivax infection. Overall, 23.5% of P. vivax infections had concurrent gametocytaemia. Of all P. vivax positive samples, 29.2% were polyclonal by MS16 and msp1F3 genotyping. All five P. falciparum infections were detected in residents of the same village, carried the same msp2 allele and four were positive for P. falciparum gametocytes. Conclusion P. vivax infection remains endemic in Ngella, with the majority of cases afebrile and below the detection limit of LM. P. falciparum has nearly disappeared, but the risk of re-introductions and outbreaks due to travel to nearby islands with higher malaria endemicity remains.

Uncovering the transmission dynamics of Plasmodium vivax using population genetics

Abstract Population genetic analysis of malaria parasites has the power to reveal key insights into malaria epidemiology and transmission dynamics with the potential to deliver tools to support control and elimination efforts. Analyses of parasite genetic diversity have suggested that Plasmodium vivax populations are more genetically diverse and less structured than those of Plasmodium falciparum indicating that P. vivax may be a more ancient parasite of humans and/or less susceptible to population bottlenecks, as well as more efficient at disseminating its genes. These population genetic insights into P. vivax transmission dynamics provide an explanation for its relative resilience to control efforts. Here, we describe current knowledge on P. vivax population genetic structure, its relevance to understanding transmission patterns and relapse and how this information can inform malaria control and elimination programmes.

An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection

Background Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both.

Comparison of three methods for detection of gametocytes in Melanesian children treated for uncomplicated malaria.

BackgroundGametocytes are the transmission stages of Plasmodium parasites, the causative agents of malaria. As their density in the human host is typically low, they are often undetected by conventional light microscopy. Furthermore, application of RNA-based molecular detection methods for gametocyte detection remains challenging in remote field settings. In the present study, a detailed comparison of three methods, namely light microscopy, magnetic fractionation and reverse transcriptase polymerase chain reaction for detection of Plasmodium falciparum and Plasmodium vivax gametocytes was conducted.MethodsPeripheral blood samples from 70 children aged 0.5 to five years with uncomplicated malaria who were treated with either artemether-lumefantrine or artemisinin-naphthoquine were collected from two health facilities on the north coast of Papua New Guinea. The samples were taken prior to treatment (day 0) and at pre-specified intervals during follow-up. Gametocytes were measured in each sample by three methods: i) light microscopy (LM), ii) quantitative magnetic fractionation (MF) and, iii) reverse transcriptase PCR (RTPCR). Data were analysed using censored linear regression and Bland and Altman techniques.ResultsMF and RTPCR were similarly sensitive and specific, and both were superior to LM. Overall, there were approximately 20% gametocyte-positive samples by LM, whereas gametocyte positivity by MF and RTPCR were both more than two-fold this level. In the subset of samples collected prior to treatment, 29% of children were positive by LM, and 85% were gametocyte positive by MF and RTPCR, respectively.ConclusionsThe present study represents the first direct comparison of standard LM, MF and RTPCR for gametocyte detection in field isolates. It provides strong evidence that MF is superior to LM and can be used to detect gametocytaemic patients under field conditions with similar sensitivity and specificity as RTPCR.

Dynamics of P. vivax clones in a cohort of children with or without primaquine treatment at baseline

P. vivax was detected by PCR in 45% of children aged 5-10 years from our study area in Papua New Guinea (PNG). 504 children were randomized into 2 arms according to Primaquine (PQ) treatment or not at baseline and actively and passively followed for 9 months. We genotyped all P. vivax infections, the majority of these being multi-clone infections. All blood samples positive for P. vivax by qPCR were tested for gametocyte carriage by targeting pvs25 transcripts. Primaquine reduced the risk of P. vivax infections by 80%. The multiplicity of infection and the density of asexual P. vivax stages were not significantly different in both treatment arms. The number of new clones (force of blood-stage infection) was 2.38 ± 0.17 per person per year-at-risk in the PQ-arm compared to 8.04 ± 0.41 in the Placebo arm (P < 0.05). The duration of infections did not differ between the treatment arms, with 73 days [95% CI: 33-849] and 68 days [95% CI: 40-247] in the PQ or Placebo arm, respectively. Detectability of P. vivax clones was low with 0.26 ± 0.06 and 0.24 ± 0.04 in the PQ and Placebo arms. PQ-treated children had a 75% lower risk of carrying gametocytes compared to Placebo recipients. P. vivax positive children in both arms were equally likely to show gametocyte positivity. We conclude that P. vivax relapses contribute significantly to the high burden of P. vivax infection and transmission in PNG. All other infection dynamics parameters were consistent between treatment arms and apparent relapses behave like new infections.

Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.STING is an intracellular DNA sensor that can alter response to infection, but in the case of malaria it is unclear how parasite DNA in red blood cells (RBCs) reaches DNA sensors in immune cells. Here the authors show that STING in human monocytes can sense P. falciparum nucleic acids transported from infected RBCs via parasite extracellular vesicles.

Gametocyte Clearance Kinetics Determined by Quantitative Magnetic Fractionation in Melanesian Children with Uncomplicated Malaria Treated with Artemisinin Combination Therapy

ABSTRACT Quantitative magnetic fractionation and a published mathematical model were used to characterize between-treatment differences in gametocyte density and prevalence in 70 Papua New Guinean children with uncomplicated Plasmodium falciparum and/or Plasmodium vivax malaria randomized to one of two artemisinin combination therapies (artemether-lumefantrine or artemisinin-naphthoquine) in an intervention trial. There was an initial rise in peripheral P. falciparum gametocyte density with both treatments, but it was more pronounced in the artemisinin-naphthoquine group. Model-derived estimates of the median pretreatment sequestered gametocyte population were 21/μl for artemether-lumefantrine and 61/μl for artemisinin-naphthoquine (P < 0.001). The median time for P. falciparum gametocyte density to fall to <2.5/μl (below which transmission becomes unlikely) was 16 days in the artemether-lumefantrine group and 20 days in artemisinin-naphthoquine group (P < 0.001). Gametocyte prevalence modeling suggested that artemisinin-naphthoquine-treated children became gametocytemic faster (median, 2.2 days) than artemether-lumefantrine-treated children (median, 5.3 days; P < 0.001) and had a longer median P. falciparum gametocyte carriage time per individual (20 versus 13 days; P < 0.001). Clearance of P. vivax gametocytes was rapid (within 3 days) in both groups; however, consistent with the reappearance of asexual forms in the main trial, nearly 40% of children in the artemether-lumefantrine group developed P. vivax gametocytemia between days 28 and 42 compared with 3% of children in the artemisinin-naphthoquine group. These data suggest that artemisinin is less active than artemether against sequestered gametocytes. Greater initial gametocyte release after artemisinin-naphthoquine increases the period of potential P. falciparum transmission by 4 days relative to artemether-lumefantrine, but the longer elimination half-life of naphthoquine than of lumefantrine suppresses P. vivax recurrence and consequent gametocytemia.

The complex relationship of exposure to new Plasmodium infections and incidence of clinical malaria in Papua New Guinea

The molecular force of blood-stage infection (molFOB) is a quantitative surrogate metric for malaria transmission at population level and for exposure at individual level. Relationships between molFOB, parasite prevalence and clinical incidence were assessed in a treatment-to-reinfection cohort, where P.vivax (Pv) hypnozoites were eliminated in half the children by primaquine (PQ). Discounting relapses, children acquired equal numbers of new P. falciparum (Pf) and Pv blood-stage infections/year (Pf-molFOB = 0–18, Pv-molFOB = 0–23) resulting in comparable spatial and temporal patterns in incidence and prevalence of infections. Including relapses, Pv-molFOB increased >3 fold (relative to PQ-treated children) showing greater heterogeneity at individual (Pv-molFOB = 0–36) and village levels. Pf- and Pv-molFOB were strongly associated with clinical episode risk. Yearly Pf clinical incidence rate (IR = 0.28) was higher than for Pv (IR = 0.12) despite lower Pf-molFOB. These relationships between molFOB, clinical incidence and parasite prevalence reveal a comparable decline in Pf and Pv transmission that is normally hidden by the high burden of Pv relapses. Clinical trial registration: ClinicalTrials.gov NCT02143934

Increasingly inbred and fragmented populations of Plasmodium vivax associated with the eastward decline in malaria transmission across the Southwest Pacific

The human malaria parasite Plasmodium vivax is more resistant to malaria control strategies than Plasmodium falciparum, and maintains high genetic diversity even when transmission is low. To investigate whether declining P. vivax transmission leads to increasing population structure that would facilitate elimination, we genotyped samples from across the Southwest Pacific region, which experiences an eastward decline in malaria transmission, as well as samples from two time points at one site (Tetere, Solomon Islands) during intensified malaria control. Analysis of 887 P. vivax microsatellite haplotypes from hyperendemic Papua New Guinea (PNG, n = 443), meso-hyperendemic Solomon Islands (n = 420), and hypoendemic Vanuatu (n = 24) revealed increasing population structure and multilocus linkage disequilibrium yet a modest decline in diversity as transmission decreases over space and time. In Solomon Islands, which has had sustained control efforts for 20 years, and Vanuatu, which has experienced sustained low transmission for many years, significant population structure was observed at different spatial scales. We conclude that control efforts will eventually impact P. vivax population structure and with sustained pressure, populations may eventually fragment into a limited number of clustered foci that could be targeted for elimination.