Publications

Journal article

Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018,

Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

, Scientific Reports, Vol: 8, ISSN: 2045-2322

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

Journal article

Hellewell J, Walker P, Ghani A, Rao BV, Churcher Tet al., 2018,

Using ante-natal clinic prevalence data to monitor temporal changes in malaria incidence in a humanitarian setting in the Democratic Republic of Congo

, Malaria Journal, Vol: 17, ISSN: 1475-2875

BackgroundThe number of clinical cases of malaria is often recorded in resource constrained or conflict settings as a proxy for disease burden. Interpreting case count data in areas of humanitarian need is challenging due to uncertainties in population size caused by security concerns, resource constraints and population movement. Malaria prevalence in women visiting ante-natal care (ANC) clinics has the potential to be an easier and more accurate metric for malaria surveillance that is unbiased by population size if malaria testing is routinely conducted irrespective of symptoms. MethodsA suite of distributed lag non-linear models was fitted to clinical incidence time-series data in children under 5 years and ANC prevalence data from health centres run by Médecins Sans Frontières in the Democratic Republic of Congo, which implement routine intermittent screening and treatment (IST) alongside intermittent preventative treatment in pregnancy (IPTp). These statistical models enable the temporal relationship between the two metrics to be disentangled. ResultsThere was a strong relationship between the ANC prevalence and clinical incidence suggesting that both can be used to describe current malaria endemicity. There was no evidence that ANC prevalence could predict future clinical incidence, though a change in clinical incidence was shown to influence ANC prevalence up to 3 months into the future. ConclusionsThe results indicate that ANC prevalence may be a suitable metric for retrospective evaluations of the impact of malaria interventions and is a useful method for evaluating long-term malaria trends in resource constrained settings.

Journal article

White MT, Walker PGT, Karl S, Hetzel M, Freeman T, Waltzman A, Laman M, Robinson L, Ghani A, Mueller Iet al., 2018,

Mathematical modelling of the impact of expanding levels of malaria control interventions on Plasmodium vivax

, Nature Communications, Vol: 9, ISSN: 2041-1723

Plasmodium vivax poses unique challenges for malaria control and elimination, notably the potential for relapses to maintain transmission in the face of drug-based treatment and vector control strategies. We developed an individual-based mathematical model of P. vivax transmission calibrated to epidemiological data from Papua New Guinea (PNG). In many settings in PNG, increasing bed net coverage is predicted to reduce transmission to less than 0.1% prevalence by light microscopy, however there is substantial risk of rebounds in transmission if interventions are removed prematurely. In several high transmission settings, model simulations predict that combinations of existing interventions are not sufficient to interrupt P. vivax transmission. This analysis highlights the potential options for the future of P. vivax control: maintaining existing public health gains by keeping transmission suppressed through indefinite distribution of interventions; or continued development of strategies based on existing and new interventions to push for further reduction and towards elimination.

Journal article

Witmer K, Sherrard-Smith E, Straschil U, Tunnicliff M, Baum J, Delves Met al., 2018,

An inexpensive open source 3D printed membrane feeder for human malaria transmission studies

, Malaria Journal, Vol: 17, ISSN: 1475-2875

BackgroundThe study of malaria transmission requires the experimental infection of mosquitoes with Plasmodium gametocytes. In the laboratory, this is achieved using artificial membrane feeding apparatus that simulate body temperature and skin of the host, and so permit mosquito feeding on reconstituted gametocyte-containing blood. Membrane feeders either use electric heating elements or complex glass chambers to warm the infected blood; both of which are expensive to purchase and can only be sourced from a handful of specialized companies. Presented and tested here is a membrane feeder that can be inexpensively printed using 3D-printing technology.ResultsUsing the Plasmodium falciparum laboratory strain NF54, three independent standard membrane feeding assays (SMFAs) were performed comparing the 3D-printed feeder against a commercial glass feeder. Exflagellation rates did not differ between the two feeders. Furthermore, no statistically significant difference was found in the oocyst load nor oocyst intensity of Anopheles stephensi mosquitoes (mean oocyst range 1.3–6.2 per mosquito; infection prevalence range 41–79%).ConclusionsOpen source provision of the design files of the 3D-printed feeder will facilitate a wider range of laboratories to perform SMFAs in laboratory and field settings, and enable them to freely customize the design to their own requirements.

Journal article

Hogan AB, Winskill P, Verity R, Griffin J, Ghani Aet al., 2018,

Modelling population-level impact to inform target product profiles for childhood malaria vaccines

, BMC Medicine, Vol: 16, ISSN: 1741-7015

BackgroundThe RTS,S/AS01 vaccine for Plasmodium falciparum malaria demonstrated moderate efficacy in 5–17-month-old children in phase 3 trials, and from 2018, the vaccine will be evaluated through a large-scale pilot implementation program. Work is ongoing to optimise this vaccine, with higher efficacy for a different schedule demonstrated in a phase 2a challenge study. The objective of our study was to investigate the population-level impact of a modified RTS,S/AS01 schedule and dose amount in order to inform the target product profile for a second-generation malaria vaccine.MethodsWe used a mathematical modelling approach as the basis for our study. We simulated the changing anti-circumsporozoite antibody titre following vaccination and related the titre to vaccine efficacy. We then implemented this efficacy profile within an individual-based model of malaria transmission. We compared initial efficacy, duration and dose timing, and evaluated the potential public health impact of a modified vaccine in children aged 5–17 months, measuring clinical cases averted in children younger than 5 years.ResultsIn the first decade of delivery, initial efficacy was associated with a higher reduction in childhood clinical cases compared to vaccine duration. This effect was more pronounced in high transmission settings and was due to the efficacy benefit occurring in younger ages where disease burden is highest. However, the low initial efficacy and long duration schedule averted more cases across all age cohorts if a longer time horizon was considered. We observed an age-shifting effect due to the changing immunological profile in higher transmission settings, in scenarios where initial efficacy was higher, and the fourth dose administered earlier.ConclusionsOur findings indicate that, for an imperfect childhood malaria vaccine with suboptimal efficacy, it may be advantageous to prioritise initial efficacy over duration. We predict that a modified vaccine could outpe

Journal article

van Beek AE, Sarr I, Correa S, Nwakanma D, Brouwer MC, Wouters D, Secka F, Anderson STB, Conway DJ, Walther M, Levin M, Kuijpers TW, Cunnington AJet al., 2018,

Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity.

, Open Forum Infect Dis, Vol: 5, ISSN: 2328-8957

BACKGROUND: Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity. METHODS: We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P. falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity. RESULTS: FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not correlate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load. CONCLUSIONS: Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by P. falciparum might be useful for malaria prevention or treatment.

Journal article

Lee HJ, Georgiadou A, Walther M, Nwakanma D, Stewart L, Levin M, Otto T, Conway D, Coin L, Cunnington Aet al., 2018,

Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria

, Science Translational Medicine, Vol: 10, Pages: 1-17, ISSN: 1946-6234

The pathogenesis of infectious diseases depends on the interaction of host and pathogen. In Plasmodium falciparum malaria, host and parasite processes can be assessed by dual RNA-sequencing of blood from infected patients. Here we performed dual transcriptome analyses on samples from 46 malaria-infected Gambian children to reveal mechanisms driving the systemic pathophysiology of severe malaria. Integrating these transcriptomic data with estimates of parasite load and detailed clinical information allowed consideration of potentially confounding effects due to differing leukocyte proportions in blood, parasite developmental stage, and whole-body pathogen load. We report hundreds of human and parasite genes differentially expressed between severe and uncomplicated malaria, with distinct profiles associated with coma, hyperlactatemia, and thrombocytopenia. High expression of neutrophil granule-related genes was consistently associated with all severe malaria phenotypes. We observed severity-associated variation in the expression of parasite genes which determine cytoadhesion to vascular endothelium, rigidity of infected erythrocytes, and parasite growth rate. Up to 99% of human differential gene expression in severe malaria was driven by differences in parasite load, whereas parasite gene expression showed little association with parasite load. Co-expression analyses revealed interactions between human and P. falciparum, with prominent co-regulation of translation genes in severe malaria between host and parasite. Multivariate analyses suggested that increased expression of granulopoiesis and interferon-γ related genes, together with inadequate suppression of type-1 interferon signalling, best explained severity of infection. These findings provide a framework for understanding the contributions of host and parasite to the pathogenesis of severe malaria and identifying targets for adjunctive therapy.

Journal article

Routledge I, Chevez JER, Cucunubá ZM, Gomez Rogriguez M, Guinovart C, Gustafson K, Schneider K, Walker PGT, Ghani A, Bhatt Set al., 2018,

Estimating spatiotemporally varying malaria reproduction numbers in a near elimination setting

, Nature Communications, Vol: 9, Pages: 1-8, ISSN: 2041-1723

In 2016 the World Health Organization identified 21 countries that could eliminate malaria by 2020. Monitoring progress towards this goal requires tracking ongoing transmission. Here we develop methods that estimate individual reproduction numbers and their variation through time and space. Individual reproduction numbers, Rc, describe the state of transmission at a point in time and differ from mean reproduction numbers, which are averages of the number of people infected by a typical case. We assess elimination progress in El Salvador using data for confirmed cases of malaria from 2010 to 2016. Our results demonstrate that whilst the average number of secondary malaria cases was below one (0.61, 95% CI 0.55–0.65), individual reproduction numbers often exceeded one. We estimate a decline in Rc between 2010 and 2016. However we also show that if importation is maintained at the same rate, the country may not achieve malaria elimination by 2020.

Journal article

Sherrard-Smith E, Sala KA, Betancourt M, Upton LM, Angrisano F, Morin MJ, Ghani AC, Churcher TS, Blagborough AMet al., 2018,

Synergy in anti-malarial pre-erythrocytic and transmission-blocking antibodies is achieved by reducing parasite density

, eLife, Vol: 7, ISSN: 2050-084X

Anti-malarial pre-erythrocytic vaccines (PEV) target transmission by inhibiting human infection but are currently partially protective. It has been posited, but never demonstrated, that co-administering transmission-blocking vaccines (TBV) would enhance malaria control. We hypothesized a mechanism that TBV could reduce parasite density in the mosquito salivary glands, thereby enhancing PEV efficacy. This was tested using a multigenerational population assay, passaging Plasmodium berghei to Anopheles stephensi mosquitoes. A combined efficacy of 90.8% (86.7–94.2%) was observed in the PEV +TBV antibody group, higher than the estimated efficacy of 83.3% (95% CrI 79.1–87.0%) if the two antibodies acted independently. Higher PEV efficacy at lower mosquito parasite loads was observed, comprising the first direct evidence that co-administering anti-sporozoite and anti-transmission interventions act synergistically, enhancing PEV efficacy across a range of TBV doses and transmission intensities. Combining partially effective vaccines of differing anti-parasitic classes is a pragmatic, powerful way to accelerate malaria elimination efforts.

Journal article

Tadesse FG, Slater HC, Chali W, Teelen K, Lanke K, Belachew M, Menberu T, Shumie G, Shitaye G, Okell LC, Graumans W, van Gemert G-J, Kedir S, Tesfaye A, Belachew F, Abebe W, Mamo H, Sauerwein R, Balcha T, Aseffa A, Yewhalaw D, Gadisa E, Drakeley C, Bousema Tet al., 2018,

The relative contribution of symptomatic and asymptomatic Plasmodium vivax and Plasmodium falciparum infections to the infectious reservoir in a low-endemic setting in Ethiopia

, Clinical Infectious Diseases, Vol: 66, Pages: 1883-1891, ISSN: 1058-4838

Background: The majority of P. vivax and P. falciparum infections in low-endemic settings are asymptomatic. The relative contribution to the infectious reservoir of these infections, often of low-parasite-density, compared to clinical malaria cases, is currently unknown but important for malaria elimination strategies. Methods: We assessed infectivity of passively-recruited symptomatic malaria patients (n=41) and community-recruited asymptomatic individuals with microscopy- (n=41) and PCR-detected infections (n=82) using membrane feeding assays with Anopheles arabiensis mosquitoes in Adama, Ethiopia. Malaria incidence and prevalence data was used to estimate the contributions of these populations to the infectious reservoir. Results: Overall, 34.9% (29/83) of P. vivax and 15.1% (8/53) P. falciparum infected individuals infected ≥1 mosquitoes. Mosquito infection rates were strongly correlated with asexual parasite density for P. vivax (ρ = 0.63; P < .001) but not for P. falciparum (ρ = 0.06; P = .770). P. vivax symptomatic infections were more infectious to mosquitoes (infecting 46.5% of mosquitoes, 307/660) compared to asymptomatic microscopy-detected (infecting 12.0% of mosquitoes, 80/667; P = .005) and PCR-detected infections (infecting 0.8% of mosquitoes, 6/744; P < .001). Adjusting for population prevalence, symptomatic, asymptomatic microscopy- and PCR-detected infections were responsible for 8.0%, 76.2% and 15.8% of the infectious reservoir for P. vivax, respectively. For P. falciparum, mosquito infections were sparser and also predominantly from asymptomatic infections. Conclusions: In this low-endemic setting aiming for malaria elimination, asymptomatic infections are highly prevalent and responsible for the majority of onward mosquito infections. The early identification and treatment of asymptomatic infections might thus accelerate elimination efforts.

Journal article

Mcardle A, Turkova A, Cunnington A, 2018,

When do co-infections matter?

, Current Opinion in Infectious Diseases, Vol: 31, Pages: 209-215, ISSN: 0951-7375

Purpose of review: Advances in diagnostic methods mean that co-infections are increasingly being detected in clinical practice, yet their significance is not always obvious. In parallel, basic science studies are increasingly investigating interactions between pathogens to try to explain real-life observations and elucidate biological mechanisms. Recent findings: Co-infections may be insignificant, detrimental or even beneficial, and these outcomes can occur through multiple levels of interactions which include modulation of the host response, altering the performance of diagnostic tests and drug-drug interactions during treatment. The harmful effects of chronic co-infections such as tuberculosis or Hepatitis B and C in association with HIV are well established, and recent studies have focussed on strategies to mitigate these effects. However consequences of many acute co-infections are much less certain, and recent conflicting findings simply highlight many of the challenges of studying naturally acquired infections in humans. Summary: Tackling these challenges, using animal models or careful prospective studies in humans may prove to be worthwhile. There are already tantalising examples where identification and treatment of relevant co-infections seems to hold promise for improved health outcomes.

Journal article

Lee HJ, Georgiadou A, Otto T, Levin M, Coin L, Conway D, Cunnington Aet al., 2018,

Transcriptomic studies in malaria – a paradigm for investigation of systemic host-pathogen interactions

, Microbiology and Molecular Biology Reviews, Vol: 82, ISSN: 1092-2172

Transcriptomics, the analysis of genome-wide RNA expression, is a common approach to investigate host and pathogen processes in infectious diseases. Technical and bioinformatic advances have permitted increasingly thorough analysis of the association of RNA expression with fundamental biology, immunity, pathogenesis, diagnosis, and prognosis. Transcriptomic approaches can now be used to realize a previously unattainable goal, simultaneous study of RNA expression in host and pathogen, in order to better understand their interactions. This exciting prospect is not without challenges, especially as focus moves from interactions in vitro under tightly controlled conditions to tissue-and systemic-level interactions in animal models and natural and experimental infections in humans. Here we review the contribution of transcriptomic studies to the understanding of malaria, a parasitic disease which has exerted a major influence on human evolution and continues to cause a huge global burden of disease. We consider malaria as a paradigm for transcriptomic assessment of systemic host-pathogen interaction in humans, because much of the direct host-pathogen interaction occurs within the blood–a readily sampled compartment of the body. We illustrate lessons learned from transcriptomic studies of malaria, and how these may guide studies of host-pathogen interaction in other infectious diseases. We propose that the potential of transcriptomic studies to improve understanding of malaria as a disease remains partly untapped because of limitations in study design rather than as a consequence of technological constraints. Further advances will require integration of transcriptomic data with analytical approaches from other scientific disciplines including epidemiology and mathematical modelling.

Journal article

Marshall JM, Wu SL, Sanchez HMC, Kiware SS, Ndhlovu M, Ouedraogo AL, Toure MB, Sturrock HJ, Ghani AC, Ferguson NMet al., 2018,

Mathematical models of human mobility of relevance to malaria transmission in Africa

, Scientific Reports, Vol: 8, ISSN: 2045-2322

As Africa-wide malaria prevalence declines, an understanding of human movement patterns is essential to inform how best to target interventions. We fitted movement models to trip data from surveys conducted at 3–5 sites throughout each of Mali, Burkina Faso, Zambia and Tanzania. Two models were compared in terms of their ability to predict the observed movement patterns – a gravity model, in which movement rates between pairs of locations increase with population size and decrease with distance, and a radiation model, in which travelers are cumulatively “absorbed” as they move outwards from their origin of travel. The gravity model provided a better fit to the data overall and for travel to large populations, while the radiation model provided a better fit for nearby populations. One strength of the data set was that trips could be categorized according to traveler group – namely, women traveling with children in all survey countries and youth workers in Mali. For gravity models fitted to data specific to these groups, youth workers were found to have a higher travel frequency to large population centers, and women traveling with children a lower frequency. These models may help predict the spatial transmission of malaria parasites and inform strategies to control their spread.

Journal article

Ghani AC, 2018,

Can improving access to care help to eliminate malaria?

, Lancet, Vol: 391, Pages: 1870-1871, ISSN: 0140-6736

Journal article

White MT, Karl S, Koepfli C, Longley RJ, Hofmann NE, Wampfler R, Felger I, Smith T, Nguitragool W, Sattabongkot J, Robinson L, Ghani A, Mueller Iet al., 2018,

Plasmodium vivax and Plasmodium falciparum infection dynamics: re-infections, recrudescences and relapses

, Malaria Journal, Vol: 17, ISSN: 1475-2875

Background:In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation.Results:For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses.Conclusion:The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

Journal article

O'Brien A, Sherrard-Smith E, Sile B, Watts C, Simms Iet al., 2018,

Spatial clusters of gonorrhoea in England with particular reference to the outcome of partner notification: 2012 and 2013

, PLoS ONE, Vol: 13, ISSN: 1932-6203

Background:This study explored spatial-temporal variation in diagnoses of gonorrhoea to identify and quantify endemic areas and clusters in relation to patient characteristics and outcomes of partner notification (PN) across England, UK.Methods:Endemic areas and clusters were identified using a two-stage analysis with Kulldorff’s scan statistics (SaTScan).ResultsOf 2,571,838 tests, 53,547 diagnoses were gonorrhoea positive (positivity = 2.08%). The proportion of diagnoses in heterosexual males was 1.5 times that in heterosexual females. Among index cases, men who have sex with men (MSM) were 8 times more likely to be diagnosed with gonorrhoea than heterosexual males (p<0.0001). After controlling for age, gender, ethnicity and deprivation rank, 4 endemic areas were identified including 11,047 diagnoses, 86% of which occurred in London. 33 clusters included 17,629 diagnoses (34% of total diagnoses in 2012 and 2013) and spanned 21 locations, some of which were dominated by heterosexually acquired infection, whilst others were MSM focused. Of the 53,547 diagnoses, 14.5% (7,775) were the result of PN. The proportion of patients who attended services as a result of PN varied from 0% to 61% within different age, gender and sexual orientation cohorts. A third of tests resulting from PN were positive for gonorrhoea. 25% of Local Authorities (n = 81, 95% CI: 20.2, 29.5) had a higher than expected proportion for female PN diagnoses as compared to 16% for males (n = 52, 95% CI: 12.0, 19.9).Conclusions:The English gonorrhoea epidemic is characterised by spatial-temporal variation. PN success varied between endemic areas and clusters. Greater emphasis should be placed on the role of PN in the control of gonorrhoea to reduce the risk of onward transmission, re-infection, and complications of infection.

Journal article

Verity RJ, Hathaway N, Waltmann A, Doctor S, Watson O, Patel J, Mwandagalirwa K, Tshefu A, Bailey J, Ghani A, Juliano J, Meshnick Set al., 2018,

Plasmodium falciparum genetic variation of var2csa in the Democratic Republic of the Congo

, Malaria Journal, Vol: 17, ISSN: 1475-2875

Background: The Democratic Republic of the Congo (DRC) bears a high burden of malaria, which is exacerbated inpregnant women. The VAR2CSA protein plays a crucial role in pregnancy-associated malaria (PAM), and hence quantifyingdiversity at the var2csa locus in the DRC is important in understanding the basic epidemiology of PAM, and indeveloping a robust vaccine against PAM.Methods: Samples were taken from the 2013–14 Demographic and Health Survey conducted in the DRC, focusingon children under 5 years of age. A short subregion of the var2csa gene was sequenced in 115 spatial clusters, givingcountry-wide estimates of sequence polymorphism and spatial population structure.Results: Results indicate that var2csa is highly polymorphic, and that diversity is being maintained through balancingselection, however, there is no clear signal of phylogenetic or geographic structure to this diversity. Linear modellingdemonstrates that the number of var2csa variants in a cluster correlates directly with cluster prevalence, but not withother epidemiological factors such as urbanicity.Conclusions: Results suggest that the DRC fts within the global pattern of high var2csa diversity and little geneticdiferentiation between regions. A broad multivalent VAR2CSA vaccine candidate could beneft from targeting stableregions and common variants to address the substantial genetic diversity.

Journal article

Kaslow DC, Okumu F, Wells TNC, Rabinovich R, Bassat Q, Birkett A, Bompart F, Burt A, Chaccour C, Chitnis C, Culpepper J, Domingo G, Duffy P, Ghani A, Greenwood B, Hall BF, Hamon N, Jacobs-Lorena M, James S, Koram KA, Kremsner P, Kumar A, Leroy D, Leroy O, Lindsay S, Majambere S, Mbogo C, McCarthy J, Qi G, Rasgon J, Richardson J, Richie T, Sauerwein R, Slutsker L, Vekemans Jet al., 2017,

malERA: An updated research agenda for diagnostics, drugs, vaccines, and vector control in malaria elimination and eradication

, PLoS Medicine, Vol: 14, ISSN: 1549-1277

Since the turn of the century, a remarkable expansion has been achieved in the range andeffectiveness of products and strategies available to prevent, treat, and control malaria,including advances in diagnostics, drugs, vaccines, and vector control. These advanceshave once again put malaria elimination on the agenda. However, it is clear that even withthe means available today, malaria control and elimination pose a formidable challenge inmany settings. Thus, currently available resources must be used more effectively, and newproducts and approaches likely to achieve these goals must be developed. This paper considerstools (both those available and others that may be required) to achieve and maintainmalaria elimination. New diagnostics are needed to direct treatment and detect transmissionpotential; new drugs and vaccines to overcome existing resistance and protect against clinicaland severe disease, as well as block transmission and prevent relapses; and new vectorcontrol measures to overcome insecticide resistance and more powerfully interrupt transmission.It is also essential that strategies for combining new and existing approaches aredeveloped for different settings to maximise their longevity and effectiveness in areas withcontinuing transmission and receptivity. For areas where local elimination has been recentlyachieved, understanding which measures are needed to maintain elimination is necessaryto prevent rebound and the reestablishment of transmission. This becomes increasinglyimportant as more countries move towards elimination.

Journal article

Winskill P, Slater H, Griffin J, Ghani A, Walker Pet al., 2017,

The US President's Malaria Initiative, Plasmodium falciparum transmission and mortality: A modelling study

, PLoS Medicine, Vol: 14, ISSN: 1549-1277

BackgroundAlthough significant progress has been made in reducing malaria transmission globally inrecent years, a large number of people remain at risk and hence the gains made are fragile.Funding lags well behind amounts needed to protect all those at risk and ongoing contributionsfrom major donors, such as the President’s Malaria Initiative (PMI), are vital to maintainprogress and pursue further reductions in burden. We use a mathematical modellingapproach to estimate the impact of PMI investments to date in reducing malaria burden andto explore the potential negative impact on malaria burden should a proposed 44% reductionin PMI funding occur.Methods and findingsWe combined an established mathematical model of Plasmodium falciparum transmissiondynamics with epidemiological, intervention, and PMI-financing data to estimate the contributionPMI has made to malaria control via funding for long-lasting insecticide treated nets(LLINs), indoor residual spraying (IRS), and artemisinin combination therapies (ACTs). Weestimate that PMI has prevented 185 million (95% CrI: 138 million, 230 million) malariacases and saved 940,049 (95% CrI: 545,228, 1.4 million) lives since 2005. If funding is maintained,PMI-funded interventions are estimated to avert a further 162 million cases (95%CrI: 116 million, 194 million) cases, saving a further 692,589 (95% CrI: 392,694, 955,653)lives between 2017 and 2020. With an estimate of US$94 (95% CrI: US$51, US$166) perDisability Adjusted Life Year (DALY) averted, PMI-funded interventions are highly costeffective.We also demonstrate the further impact of this investment by reducing caseloadson health systems. If a 44% reduction in PMI funding were to occur, we predict that this lossof direct aid could result in an additional 67 million (95% CrI: 49 million, 82 million) cases and290,649 deaths (95% CrI: 167,208, 395,263) deaths between 2017 and 2020. We have notmodelled indirect impacts of PMI funding (such as health systems strengthening

Journal article

Challenger J, Bruxvoort K, Ghani AC, Okell LCet al., 2017,

Assessing the impact of imperfect adherence to artemether-lumefantrine on malaria treatment outcomes using within-host modelling

, Nature Communications, Vol: 8, ISSN: 2041-1723

Artemether-lumefantrine (AL) is the most widely-recommended treatment for uncomplicated Plasmodium falciparum malaria worldwide. Its safety and efficacy have been extensively demonstrated in clinical trials; however, its performance in routine health care settings, where adherence to drug treatment is unsupervised and therefore may be suboptimal, is less well characterised. Here we develop a within-host modelling framework for estimating the effects of sub-optimal adherence to AL treatment on clinical outcomes in malaria patients. Our model incorporates data on the human immune response to the parasite, and AL’s pharmacokinetic and pharmacodynamic properties. Utilising individual-level data of adherence to AL in 482 Tanzanian patients as input for our model predicted higher rates of treatment failure than were obtained when adherence was optimal (9% compared to 4%). Our model estimates that the impact of imperfect adherence was worst in children, highlighting the importance of advice to caregivers.

Journal article

Hammond AM, Kyrou K, Bruttini M, North A, Galizi R, Karlsson X, Kranjc N, Carpi FM, D'Aurizio R, Crisanti A, Nolan Tet al., 2017,

The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito

, PLoS Genetics, Vol: 13, ISSN: 1553-7390

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.

Journal article

Watson O, Slater HC, Verity R, Parr JB, Mwandagalirwa MK, Tshefu A, Meshnick SR, Ghani ACet al., 2017,

Modelling the drivers of the spread of Plasmodium falciparum hrp2 gene deletions in sub-Saharan Africa

, eLife, Vol: 6, ISSN: 2050-084X

Rapid diagnostic tests (RDTs) have transformed malaria diagnosis. The most prevalent P. falciparum RDTs detect histidine-rich protein 2 (PfHRP2). However, pfhrp2 gene deletions yielding false-negative RDTs, first reported in South America in 2010, have been confirmed in Africa and Asia. We developed a mathematical model to explore the potential for RDT-led diagnosis to drive selection of pfhrp2-deleted parasites. Low malaria prevalence and high frequencies of people seeking treatment resulted in the greatest selection pressure. Calibrating our model against confirmed pfhrp2-deletions in the Democratic Republic of Congo, we estimate a starting frequency of 6% pfhrp2-deletion prior to RDT introduction. Furthermore, the patterns observed necessitate a degree of selection driven by the introduction of PfHRP2-based RDT-guided treatment. Combining this with parasite prevalence and treatment coverage estimates, we map the model-predicted spread of pfhrp2-deletion, and identify the geographic regions in which surveillance for pfhrp2-deletion should be prioritised.

Journal article

Werther R, Hallinan JP, Lambert AR, Havens K, Pogson M, Jarjour J, Galizi R, Windbichler N, Crisanti A, Nolan T, Stoddard BLet al., 2017,

Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

, Nucleic Acids Research, Vol: 45, Pages: 8621-8634, ISSN: 0305-1048

The retargeting of protein–DNA specificity, outsideof extremely modular DNA binding proteins suchas TAL effectors, has generally proved to be quitechallenging. Here, we describe structural analysesof five different extensively retargeted variants of asingle homing endonuclease, that have been shownto function efficiently in ex vivo and in vivo applications.The redesigned proteins harbor mutationsat up to 53 residues (18%) of their amino acid sequence,primarily distributed across the DNA bindingsurface, making them among the most signifi-cantly reengineered ligand-binding proteins to date.Specificity is derived from the combined contributionsof DNA-contacting residues and of neighboringresidues that influence local structural organization.Changes in specificity are facilitated by theability of all those residues to readily exchange bothform and function. The fidelity of recognition is notprecisely correlated with the fraction or total numberof residues in the protein–DNA interface that areactually involved in DNA contacts, including directionalhydrogen bonds. The plasticity of the DNArecognitionsurface of this protein, which allows substantialretargeting of recognition specificity withoutrequiring significant alteration of the surroundingprotein architecture, reflects the ability of the correspondinggenetic elements to maintain mobility andpersistence in the face of genetic drift within potentialhost target sites.

Journal article

Bretscher MT, Griffin JT, Ghani AC, Okell LCet al., 2017,

Modelling the benefits of long-acting or transmission-blocking drugs for reducing Plasmodium falciparum transmission by case management or by mass treatment

, MALARIA JOURNAL, Vol: 16, ISSN: 1475-2875

BackgroundAnti-malarial drugs are an important tool for malaria control and elimination. Alongside their direct benefit in the treatment of disease, drug use has a community-level effect, clearing the reservoir of infection and reducing onward transmission of the parasite. Different compounds potentially have different impacts on transmission—with some providing periods of prolonged chemoprophylaxis whilst others have greater transmission-blocking potential. The aim was to quantify the relative benefit of such properties for transmission reduction to inform target product profiles in the drug development process and choice of first-line anti-malarial treatment in different endemic settings.MethodsA mathematical model of Plasmodium falciparum epidemiology was used to estimate the transmission reduction that can be achieved by using drugs of varying chemoprophylactic (protection for 3, 30 or 60 days) or transmission-blocking activity (blocking 79, 92 or 100% of total onward transmission). Simulations were conducted at low, medium or high transmission intensity (slide-prevalence in 2–10 year olds being 1, 10 or 40%, respectively), with drugs administered either via case management or mass drug administration (MDA).ResultsTransmission reductions depend strongly on deployment strategy, treatment coverage and endemicity level. Transmission-blocking was most effective at low endemicity, whereas chemoprophylaxis was most useful at high endemicity levels. Increasing the duration of protection as much as possible was beneficial. Increasing transmission-blocking activity from the level of ACT to a 100% transmission-blocking drug (close to the effect estimated for ACT combined with primaquine) produced moderate impact but was not as effective as increasing the duration of protection in medium-to-high transmission settings (slide prevalence 10–40%). Combining both good transmission-blocking activity (e.g. as achieved by ACT or ACT + primaquine) and a long durat

Journal article

Okell L, Griffin JT, Roper C, 2017,

Mapping sulphadoxine-pyrimethamine-resistant Plasmodium falciparum malaria in infected humans and in parasite populations in Africa

, Scientific Reports, Vol: 7, ISSN: 2045-2322

Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine in vulnerable populations reduces malaria morbidity in Africa, but resistance mutations in the parasite dhps gene (combined with dhfr mutations) threaten its efficacy. We update a systematic review to map the prevalence of K540E and A581G mutations in 294 surveys of infected humans across Africa from 2004-present. Interpreting these data is complicated by multiclonal infections in humans, especially in high transmission areas. We extend statistical methods to estimate the frequency, i.e. the proportion of resistant clones in the parasite population at each location, and so standardise for varying transmission levels. Both K540E and A581G mutations increased in prevalence and frequency in 60% of areas after 2008, highlighting the need for ongoing surveillance. Resistance measures within countries were similar within 300 km, suggesting an appropriate spatial scale for surveillance. Spread of the mutations tended to accelerate once their prevalence exceeded 10% (prior to fixation). Frequencies of resistance in parasite populations are the same or lower than prevalence in humans, so more areas would be classified as likely to benefit from IPT if similar frequency thresholds were applied. We propose that the use of resistance frequencies as well as prevalence measures for policy decisions should be evaluated.

Journal article

Patouillard E, Ghani ACH, Bhatt S, Griffin J, Cibulskis Ret al., 2017,

Global investment targets for malaria control and elimination between 2016 and 2030

, BMJ Global Health, Vol: 2, ISSN: 2059-7908

Background Access to malaria control interventions falls short of universal health coverage. The Global Technical Strategy for malaria targets at least 90% reduction in case incidence and mortality rates, and elimination in 35 countries by 2030. The potential to reach these targets will be determined in part by investments in malaria. This study estimates the financing required for malaria control and elimination over the 2016–2030 period.Methods A mathematical transmission model was used to explore the impact of increasing intervention coverage on burden and costs. The cost analysis took a public provider perspective covering all 97 malaria endemic countries and territories in 2015. All control interventions currently recommended by the WHO were considered. Cost data were sourced from procurement databases, the peer-reviewed literature, national malaria strategic plans, the WHO-CHOICE project and key informant interviews.Results Annual investments of $6.4 billion (95% uncertainty interval (UI $4.5–$9.0 billion)) by 2020, $7.7 billion (95% UI $5.4–$10.9 billion) by 2025 and $8.7 billion (95% UI $6.0–$12.3 billion) by 2030 will be required to reach the targets set in the Global Technical Strategy. These are equivalent to annual investment per person at risk of malaria of US$3.90 by 2020, US$4.30 by 2025 and US$4.40 by 2030, compared with US$2.30 if interventions were sustained at current coverage levels. The 20 countries with the highest burden in 2015 will require 88% of the total investment.Conclusions Given the challenges in increasing domestic and international funding, the efficient use of currently available resources should be a priority

Journal article

Brady OJ, Slater HC, Pemberton-Ross P, Wenger E, Maude RJ, Ghani AC, Penny MA, Gerardin J, White LJ, Chitnis N, Aguas R, Hay SI, Smith DL, Stuckey EM, Okiro EA, Smith TA, Okell LCet al., 2017,

Role of mass drug administration in elimination of Plasmodium falciparum malaria: a consensus modelling study

, The Lancet Global Health, Vol: 5, Pages: E680-E687, ISSN: 2214-109X

BackgroundMass drug administration for elimination of Plasmodium falciparum malaria is recommended by WHO in some settings. We used consensus modelling to understand how to optimise the effects of mass drug administration in areas with low malaria transmission.MethodsWe collaborated with researchers doing field trials to establish a standard intervention scenario and standard transmission setting, and we input these parameters into four previously published models. We then varied the number of rounds of mass drug administration, coverage, duration, timing, importation of infection, and pre-administration transmission levels. The outcome of interest was the percentage reduction in annual mean prevalence of P falciparum parasite rate as measured by PCR in the third year after the final round of mass drug administration.FindingsThe models predicted differing magnitude of the effects of mass drug administration, but consensus answers were reached for several factors. Mass drug administration was predicted to reduce transmission over a longer timescale than accounted for by the prophylactic effect alone. Percentage reduction in transmission was predicted to be higher and last longer at lower baseline transmission levels. Reduction in transmission resulting from mass drug administration was predicted to be temporary, and in the absence of scale-up of other interventions, such as vector control, transmission would return to pre-administration levels. The proportion of the population treated in a year was a key determinant of simulated effectiveness, irrespective of whether people are treated through high coverage in a single round or new individuals are reached by implementation of several rounds. Mass drug administration was predicted to be more effective if continued over 2 years rather than 1 year, and if done at the time of year when transmission is lowest.InterpretationMass drug administration has the potential to reduce transmission for a limited time, but is not an

Journal article

Koch M, Wright KE, Otto O, Herbig M, Salinas ND, Tolia NH, Satchwell TJ, Guck J, Brooks NJ, Baum Jet al., 2017,

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion.

, Proc Natl Acad Sci U S A, Vol: 114, Pages: 4225-4230

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

Journal article

Sundriyal S, Chen P, Lubin A, Lueg G, Lu F, White AJP, Malmquist N, Vedadi M, Scherf A, Fuchter MJet al., 2017,

Identification of diaminoquinazoline histone lysine methyltransferase structure activity relationships that allow for segregation of human G9a inhibition and anti-Plasmodium activity

, MedChemComm, Vol: 8, Pages: 1069-1092, ISSN: 2040-2511

Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti-Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon ‘Nle mimic’ at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.

Journal article

Wong W, Bai XC, Sleebs B, Triglia T, Brown A, Thompson JK, Jackson KE, Hanssen E, Marapana DS, Fernandez I, Ralph SA, Cowman AF, Scheres SHW, Baum Jet al., 2017,

Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis

, Nature Microbiology, Vol: 2, Pages: 1-9, ISSN: 2058-5276

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å electron cryo-microscopy structure of the Plasmodium falciparum 80S-ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated center. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-EM to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.

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