Plasmodium Research Paper

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Abstract

Background

Although significant progress has been made in reducing malaria transmission globally in recent 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 contributions from major donors, such as the President’s Malaria Initiative (PMI), are vital to maintain progress and pursue further reductions in burden. We use a mathematical modelling approach to estimate the impact of PMI investments to date in reducing malaria burden and to explore the potential negative impact on malaria burden should a proposed 44% reduction in PMI funding occur.

Methods and findings

We combined an established mathematical model of Plasmodium falciparum transmission dynamics with epidemiological, intervention, and PMI-financing data to estimate the contribution PMI has made to malaria control via funding for long-lasting insecticide treated nets (LLINs), indoor residual spraying (IRS), and artemisinin combination therapies (ACTs). We estimate that PMI has prevented 185 million (95% CrI: 138 million, 230 million) malaria cases 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 (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) per Disability Adjusted Life Year (DALY) averted, PMI-funded interventions are highly cost-effective. We also demonstrate the further impact of this investment by reducing caseloads on health systems. If a 44% reduction in PMI funding were to occur, we predict that this loss of direct aid could result in an additional 67 million (95% CrI: 49 million, 82 million) cases and 290,649 (95% CrI: 167,208, 395,263) deaths between 2017 and 2020. We have not modelled indirect impacts of PMI funding (such as health systems strengthening) in this analysis.

Conclusions

Our model estimates that PMI has played a significant role in reducing malaria cases and deaths since its inception. Reductions in funding to PMI could lead to large increases in the number of malaria cases and deaths, damaging global goals of malaria control and elimination.

Author summary

Why was this study done?

  • The United States contributes a significant proportion of the global budget for malaria control in the form of foreign aid through the President’s Malaria Initiative (PMI).
  • Due to proposed cuts to US foreign aid and PMI funding, it is important to demonstrate the impact and cost-effectiveness of PMI.

What did the researchers do and find?

  • We used an established malaria transmission model to investigate the impact of PMI funding for malaria control.
  • We estimated the past impact of PMI funding on malaria-related cases and deaths and the potential future impact if PMI funding were to be cut.
  • PMI funding is highly cost-effective, averting an estimated 185 million cases and saving 940,049 lives since it was set up in 2005.
  • A reduction in funding of 44% would lead to an additional 67 million cases and 290,649 deaths over the next 4 years.

What do these findings mean?

  • Ongoing support from PMI is critical to maintain recent advances in malaria control and progress towards malaria elimination goals.
  • PMI has proven to be a highly cost-effective means by which US foreign aid can be invested to reduce malaria burden.

Citation: Winskill P, Slater HC, Griffin JT, Ghani AC, Walker PGT (2017) The US President's Malaria Initiative, Plasmodium falciparum transmission and mortality: A modelling study. PLoS Med 14(11): e1002448. https://doi.org/10.1371/journal.pmed.1002448

Academic Editor: Lorenz von Seidlein, Mahidol-Oxford Tropical Medicine Research Unit, THAILAND

Received: June 9, 2017; Accepted: October 18, 2017; Published: November 21, 2017

Copyright: © 2017 Winskill et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: PW and ACG acknowledge research grant support from the Bill & Melinda Gates Foundation URL: http://www.gatesfoundation.org/ (OPP1068440). PGTW acknowledges funding from an MRC Population Health Scientist Fellowships (MR/LO12189/1). HCS acknowledges support from an Imperial College Junior Research Fellowship (F24101). PW, HCS, ACG, and PGTW acknowledge Centre support from the Medical Research Council (MRC) URL: https://www.mrc.ac.uk/ and the Department for International Development (DFID) URL: https://www.gov.uk/government/organisations/department-for-international-development. JTG received no specific funding for this work.

Competing interests: PW discloses his consultancy services to the Global Fund to support investment case and allocation modelling and country planning support. ACG discloses financial consultancy services to the Global Fund to support investment case and allocation modelling and country planning support and unrestricted research grants from a range of funders, including BMGF, UK Medical Research Council, The Wellcome Trust, NIH, Malaria Vaccine Initiative, Medicines for Malaria Venture, Integrated Vector Control Consortium, and Gavi. She is also a member of WHO Malaria Policy Advisory Committee and of WHO Global Technical Strategy for Malaria Scientific Committee. HCS discloses her consultancy from the Global Fund for country support planning and modelling impact of national malaria elimination strategies.

Abbreviations: ACT, artemisinin combination therapy; DALY, Disability Adjusted Life Year; DFID, Department for International Development; DHS, Demographic and Health Survey; GDP, gross domestic product; GMS, Greater Mekong Subregion; GTS, Global Technical Strategy; IPTp, intermittent preventive treatment in pregnancy; IRS, indoor residual spraying; LLIN, long-lasting insecticide treated net; MICS, Multiple Indicator Cluster Surveys; NGO, nongovernmental organisation; NMCP, National Malaria Control Programme; PMI, President’s Malaria Initiative; SMC, seasonal malaria chemoprevention; USAID, United States Agency for International Development

Introduction

Unprecedented effort has seen the global burden of malaria halve since the turn of the 21st century due to the widespread distribution of highly effective preventative interventions such as long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS) and the provision of highly efficacious treatment with artemisinin combination therapies (ACTs) [1]. However, funding for malaria control has plateaued, falling well behind what is necessary to expand protection to all those in need [2,3]. The continued high level of support for foreign aid contributions in a fluid global political landscape is not guaranteed and gains in malaria control can be fragile if intervention coverage, which is largely dependent on donor funding, is not maintained [4].

The US is the world’s largest donor of foreign aid for malaria control [5] and therefore a mainstay in global malaria efforts. The President’s Malaria Initiative (PMI), established in 2005 and funded by the United States Agency for International Development (USAID), has been particularly influential in investing in malaria control over the past 12 years [6]. PMI provides support to malaria control programmes in 19 African focus countries and the Greater Mekong Subregion (GMS) and is the largest bilateral funder of malaria prevention and treatment [5,7]. In the 12 years since its inception, PMI has procured 197 million LLINs and 378 million courses of ACTs, provided over 215 million person-years of protection with IRS, and distributed 35.7 million courses of preventative therapy for pregnant women [6]. In 2015, PMI funding represented over one-fifth of the global malaria budget envelope [5,6]. In a recent statistical analysis, the influence of PMI funding has been estimated to have had significant impact on under-5 mortality in sub-Saharan Africa, with an estimated reduction of 16% [8]. The US’s commitment to overseas aid has been threatened in recent months [9], highlighting the fragility of global funding for malaria control and a reliance on global political stability. In May 2017, Congress published the Congressional Budget Justification [9], which outlined a commitment to malaria control for 2018 of US$424 million. This is equivalent to a 44% reduction relative to commitments reported for 2017 [10].

To quantify the importance of the PMI contribution to global malaria efforts, we combined data on PMI commodity contributions over time and by country [6] with a mathematical model of the impact of interventions on malaria transmission, morbidity, and mortality parameterised at the subnational level [11] and previously used to inform the Global Technical Strategy (GTS) for malaria [5]. We used this to estimate the global health impact of past PMI funding and the potential implications that a reduction in funding from a key stakeholder and donor in the near term could have on malaria globally.

Methods

We linked data on PMI financing, historical intervention coverage, and the underlying epidemiology in modelled countries with estimates of the potential effect of reduction in PMI funding on the coverage of interventions nationally. These estimates were then used as inputs for an established transmission model of P. falciparum malaria [11,12] to project the impact of reductions in funding on cases, deaths, and Disability Adjusted Life Years (DALYs) (Fig 1).

Fig 1. Schematic of the modelling process.

Data inputs and sources (left column) are combined and linked to estimate the contribution of PMI and the impact of funding cuts on national-level intervention coverage (middle column). These estimates are then used as inputs in a dynamic transmission model to estimate the impact of changes in intervention coverage on epidemiological outcomes (right column). ACT, artemisinin combination therapy; DALY, Disability Adjusted Life Year; DHS, Demographic and Health Survey; IRS, indoor residual spraying; LLIN, long-lasting insecticide treated net; MICS, Multiple Indicator Cluster Surveys; NMCP, National Malaria Control Programme; PMI, President’s Malaria Initiative.

https://doi.org/10.1371/journal.pmed.1002448.g001

Mathematical model

We used an established individual-based malaria transmission model that incorporates a full dynamic mosquito-vector element to allow vector-control interventions to be accurately represented [13]. We briefly describe the model structure below. Full mathematical details can be found in S1 Appendix, Text A-I, and associated references [11,12].

Modelled humans are initially susceptible and may become infected, with a given probability, via the bite of an infectious mosquito. Upon infection, following a period reflecting liver-stage infection, an individual may become symptomatic and seek treatment. Successfully treated individuals benefit from a period of drug-dependent prophylaxis before returning to the susceptible compartment. Symptomatic individuals who do not receive treatment experience a period of symptomatic disease (which has high onward infectivity) before recovering to an asymptomatic state. These individuals, along with those who experience asymptomatic infection, move from being patently asymptomatic to subpatent before natural clearance moves them back into the susceptible compartment. Superinfection can occur from all asymptomatic and subpatent states. Those who experience clinical disease are considered at risk from severe disease and its associated mortality [14].

Naturally acquired immunity is incorporated at several stages of the infection process [12]. Clinical immunity is developed earliest, protecting individuals against severe disease and then clinical disease, and is exposure driven with an age-dependent component to the severe disease pathology and associated mortality rate. Antiparasite immunity develops later, driven by both age and exposure to infection, and reduces the detectability of infections through the control of parasite density. A degree of anti-infection immunity develops later in life, reducing the probability that an infectious bite results in patent infection. The parameters determining the acquisition of immunity were estimated through fitting to severe disease incidence, clinical incidence, and parasite prevalence data stratified by age across a range of transmission settings [12,15].

All infection states are assumed to be onwardly infectious to mosquitoes, with infectivity correlated with parasite density (i.e., highest for clinical disease, intermediate for patent asymptomatic infection, and lowest for subpatent infection), with the parameters estimated by fitting to mosquito feeding studies [12,14,15].

Vectors are modelled as a stochastic compartmental formulation incorporating the larval stages of infection and adult female infection stages [10, 13].

Geographically specific data inputs

We modelled each first administrative unit (first administrative level below national) in all countries with stable malaria transmission, totalling 1,020 administrative units. Prior scale-up of interventions (LLINs and IRS) was estimated from World Malaria Report data [16], which are based on reports from National Malaria Control Programmes (NMCPs). Demographic and Health Survey (DHS) and Multiple Indicator Cluster Surveys (MICS) for within Africa [17] and World Malaria Report [18] estimates for elsewhere were used to estimate treatment coverage. It was assumed there was no prior scale-up of seasonal malaria chemoprevention (SMC). Each administrative unit was assigned a seasonal pattern that determined the seasonal fluctuation in the carrying capacity of the environment. Seasonality was estimated using Fourier transformations of daily rainfall data from 2002–2009 from Garske et al. (2013) [19]. The carrying capacity was then fitted to 2015 estimates of prevalence (within Africa) [1] or cases (outside of Africa) [16,20] using a root-finding algorithm. Data on populations were compiled from the Gridded Population of the World dataset, adjusted for United Nations estimates of country-level populations [21]. Estimates of the spatial limits of P. falciparum transmission [20] were used to delimit populations at risk.

PMI intervention data

To estimate the impact of PMI funding, we firstly estimate the proportion of intervention coverage that is attributable to PMI funding in each location. This is then subtracted from the total intervention coverage estimated. The number of LLINs procured and distributed, the number of people protected by IRS, and the number of ACTs procured and distributed stratified by year and country were all obtained from PMI’s 10th Annual Report to Congress [6]. Absolute numbers were converted to coverage using the appropriate denominators: the estimated population at risk for LLINs and IRS and estimates of the total number of ACT treatment courses delivered [5] for ACT in each country. Examples of this process are detailed in Box 1 (and S1 Table). Throughout, we assumed that 1 LLIN covered 1.8 people (in line with WHO methodology [5]). To estimate the relationships between net delivery, coverage, and usage, we follow an approach by Bhatt et al. (2015) relating distribution data (i.e., procurement as reported by PMI) to household ownership and usage, accounting for household size [22]. The coverage estimates in the model relate to usage and also incorporate wear and tear and decay of insecticide over time. We make an optimistic assumption that ACTs delivered are efficiently used (i.e., reach the health clinics and are effectively employed to treat malaria). In Senegal and Mali, where PMI funds support SMC, we assumed that SMC coverage attributable to PMI was 20%, supporting and complementing SMC implementation by NMCPs and other nongovernmental organisations (NGOs) in these countries [6]. These estimates are then used to simulate malaria trajectories, both retrospectively and prospectively, assuming varying levels of PMI funding.

Box 1. Example of estimating future coverage attributable to PMI funding: Uganda.

Estimated population at risk (2015): 37,913,546

LLINs:

Number of LLINs distributed (2015) by PMI: 747,320

People covered by LLINs distributed: 747,320 × 1.8 = 1,345,176

LLIN coverage attributable to PMI: 1,345,176 / 37,913,546 = 3.5%

IRS:

Number of people protected (2015) by PMI: 3,086,789

IRS coverage attributable to PMI: 3,086,789 / 37,913,546 = 8.1%

ACTs:

Number of ACT treatment courses distributed (2015) by PMI: 1,616,130

WHO estimate of total ACT courses delivered: 30,166,620

Estimate of the proportion of treatments attributable to PMI: 1,616,130 / 30,166,620 = 5.3%

Budget scenarios for 2017 onwards

We considered 3 budget scenarios, one in which PMI funding was kept constant to 2017 levels, one in which 100% of the PMI budget was removed, and a third in which the budget was reduced by 44% (applied uniformly across PMI-supported countries) to reflect the difference in budget attributed to malaria control detailed in the 2017 financial omnibus [10] and the proposed budget for 2018 onwards [9]. The relationship between PMI’s budget and intervention coverage was assumed to be linear, whereby an assumed budget cut of 44% was associated with a proportional decrease in the PMI-attributable intervention coverage. We also ran a scenario with a less drastic reduction in funding of 20%. We assume no mitigation through alternative funding routes or reallocation of reduced budgets. Extra savings and benefits to the health system of PMI funding were also estimated. The savings to the health system of cases averted due to PMI-funded interventions were calculated as the costs of case management and drug commodity costs of the cases averted. In addition, we calculated the additional deaths that may occur if PMI-funded interventions were removed and a national health system did not have the capacity to absorb and adequately treat the additional severe cases.

All scenarios were run multiple times in a sensitivity analysis using 20 separate sets of parameters drawn from the posterior of the modelling fitting [15]. Associated outputs are presented as the median and 95% credible intervals.

Results

To date, PMI has allocated over US$5 billion to 19 PMI focus countries in sub-Saharan Africa as well as the GMS [23] (Fig 2). We attribute increases in coverage of 8.13% for LLINs, 4.18% for IRS, and 12.9% for ACTs to PMI funding in supported countries in 2015. We estimate that in the 12 years since its inception, PMI has prevented 185 million malaria cases (95% CrI: 138 million, 230 million) (Fig 3A) and saved 940,049 lives (95% CrI: 545,228, 1.4 million) (Fig 3B), the majority of which (77%, 95% CrI: 75%, 81%) would have occurred in children under the age of 5. In sub-Saharan Africa, we estimate that PMI investment has led to an 11.6% (95% CrI: 9.5%, 13.0%) reduction in incidence and an 18.3% (95% CrI: 16.3%, 20.4%) reduction in under-5 malaria-mortality rates in 2015. We estimate the biggest impact in terms of absolute cases averted to have occurred in long-term supported countries with the highest burden. For example, Nigeria, the country with the highest burden globally [5], has received approximately US$345 million from PMI since 2010 [6], leading to an estimated 13.8 million cases (95% CrI: 8.7 million, 17.0 million) averted and 128,861 lives (95% CrI: 75,852, 200,075) saved. Angola has benefitted from continuous support since 2005, seeing investments totalling US$248 million dollars [6], leading to an estimated 8.7 million cases (95% CrI: 6.3 million, 10.4 million) averted and 43,752 lives (95% CrI: 24,946, 61,433) saved.

Fig 2. Map of PMI activities.

Individual countries and regions that have received PMI-funding and support are highlighted to reflect the level of funding from PMI in (A) sub-Saharan Africa and (B) the GMS over the period 2013–2015. The total regional assignment to the 6 GMS countries over this period is US$9.5 million. Estimated funding per population at risk over this period ranged from US$0.54 (Myanmar) to US$8.08 (Liberia). GMS, Greater Mekong Subregion; PMI, President’s Malaria Initiative.

https://doi.org/10.1371/journal.pmed.1002448.g002

Fig 3. The projected impact of PMI funding on past and future global malaria trends.

The (A) past trends (median estimates) in the global incidence of P. falciparum malaria given funding as occurred (black line) and estimate of the counterfactual trend had PMI support not existed (light blue line). The shaded area represents the cases averted due to PMI funding and (B) shows the associated estimates of death averted each year due to PMI funding. Projected estimates of the additional cumulative numbers of (C) cases (and 95% CrI) and (D) deaths (and 95% CrI) that would occur if PMI funding was reduced by 100% (dark green bars) or 44% (light green bars) over the 4-year period 2017–2020. PMI, President’s Malaria Initiative.

https://doi.org/10.1371/journal.pmed.1002448.g003

We estimate that a 44% cut in PMI funding would lead to an additional 67 million cases (95% CrI: 49 million, 82 million) (Fig 3B) and 290,649 deaths (95% CrI: 167,208, 395,263) (Fig 3C; S1 Appendix, Table F) from malaria compared to maintaining current levels of funding from 2017 to 2020. A 20% reduction in funding was associated with an additional 31 million cases (95% CrI: 21 million, 38 million) and 127,799 deaths (95% CrI: 73,313, 178,234) over the same period. If PMI-funded coverage of interventions can be maintained over the next 4 years, PMI support will be responsible for averting an estimated total of 162 million cases (95% CrI: 116 million, 194 million) (Fig 3B) and 692,589 deaths (95% CrI: 392,694, 955,653) (Fig 3C) in the 4-year period from 2017 to 2020, compared to no PMI support.

The impact on malaria burden will be focussed in high-burden countries receiving significant financial support in sub-Saharan Africa. Additionally, with ongoing concern surrounding the emergence and spread of ACT drug resistance [24], support for the GMS is also contributing to the malaria elimination goals in that region.

We estimate that PMI support would avert an additional US$174 million dollars (95% CrI: 121 million, 224 million) of national health system spending through averted malaria cases from 2017 to 2020 (Fig 4A). In the absence of PMI funding, a failure of health systems to absorb the extra caseload (through lack of capacity, finances, or both) would lead to an estimated 69,314 extra deaths (95% CrI: 39,102, 94,888) over this period (Fig 4B), in addition to the 692,589 deaths estimated to be directly caused by reductions in intervention coverage. These impact estimates are likely conservative, not accounting for the indirect impacts of increased transmission.

Fig 4. The health-system benefits associated with PMI funding.

PMI investment in malaria interventions reduces caseloads of national health systems with resulting (A) averted spending due to reduced treatments of clinical and severe cases by country. Without PMI investment, these health system gains are lost, potentially resulting in (B) the estimated cumulative malaria-related deaths in addition to those caused directly by removal of interventions due to health systems not being able to respond to increased caseloads. PMI, President’s Malaria Initiative.

https://doi.org/10.1371/journal.pmed.1002448.g004

Over the period 2013–2015, when the PMI programme was fully scaled to current levels, PMI reported that spending in the 19 focus countries in sub-Saharan Africa was approximately US$1.7 billion. Translating the modelled epidemiological impact into system-wide cost-effectiveness, we estimate a cost of US$20.6 per malaria case averted (95% CrI: US$15.2, US$31.4), US$4,081 per death averted (95% CrI: US$2,084, US$7,435), and US$94 per DALY averted (95% CrI: US$51, US$166) (Table 1). This represents a range of 2%–57% as a proportion of per-capita gross domestic product (GDP) in these countries. Cost-effectiveness estimates are driven by the intervention mix and national-level differences in the cost of treating clinical and severe cases. Differences between cases and deaths averted are primarily driven by the intervention mix, especially the proportion of funding that went towards treatment (treatment contribution is positively associated with the proportion of deaths to cases averted, linear model coefficient = 0.012, p = 0.035).

Discussion

Here, we have produced modelled estimates of the programme-wide effectiveness of PMI in terms of the impact it has had upon malaria morbidity and mortality since its inception in 2005. We estimate that PMI has averted 185 million cases and 940,049 deaths to date. If funding for PMI is maintained, we predict that a further 162 million cases and 692,589 deaths could be averted over the next 4 years, compared to no PMI funding. However, in comparison to continued full PMI support, a 44% cut in the PMI budget, as indicated in the May 2017 Congressional Budget Justification, could result in an additional 67 million cases and 290,649 deaths in the next 4 years.

Our results highlight the fragility of the gains in malaria control that have been made to date, particularly given the changing geopolitical landscape [25]. International funding, including that from governments, such as from PMI, the United Kingdom’s Department for International Development (DFID), the Global Fund, and others, accounts for a large proportion (approximately 68% [5]) of the funds available for malaria control worldwide. Malaria control is therefore reliant on sustained long-term investment from foreign donors. Without continued commitment to support programmes, recent gains in the control of malaria will be difficult to sustain and potential rebound epidemics likely [4].

Prudent investment of foreign aid relies on being able to effectively implement cost-effective interventions to maximise health gains. PMI has proven to be a capable mediator of this process for malaria. The estimates of cost per DALY averted here are significantly below the WHO threshold for cost-effectiveness of less than 300% of a country’s per-capita GDP [26]. Even among highly cost-effective interventions, malaria control compares favourably as a means by which to improve global health [27]. Between-country variation in cost-effectiveness is pronounced. The effect is driven by the intervention mix and underlying epidemiological variation (such as the intrinsic transmission potential). Costs are driven by the intervention mix and, specifically, the impact of PMI support on treatment costs, which varies between countries. Whilst the past and current positive health impacts of PMI-funded interventions is very apparent, there remains much debate as to the impact that foreign aid has on recipient countries [28].

In addition to its direct impact on cases and malaria-attributable mortality, investment in malaria control brings about substantial further potential health gains by alleviating the burden that malaria places on health systems in affected countries [29]. Supporting vector control interventions is expected to decrease caseloads, freeing up health system capacity and reducing costs incurred from treating clinical and severe cases of malaria. A recent PMI-supported study demonstrated reductions in malaria-related inpatient and outpatient admissions and hospital costs after the scale-up of interventions in Southern Province in Zambia [30]. Funding cuts lead to increased caseloads due to the negative impacts of reduced intervention coverage, the stress of which will be borne by the national health systems of malaria-endemic countries. Lack of health-system capacity was a critical factor in the recent Ebola epidemic in West Africa [31], the impact of which reverberated globally. Those countries worst affected are highly malaria endemic and had health systems already dealing with the challenges of a high malaria burden [32,33]. A redistribution of emergency funds earmarked for the Ebola epidemic [9] could potentially help to mitigate budget cuts for malaria control. However, this is a finite fund that would only serve as a very near-term solution to budget reductions.

Our results provide a conservative estimate of the overall impact of PMI funding, as we do not capture the impact of all PMI-associated activities, notably intermittent preventive treatment in pregnancy (IPTp), which we have not modelled but is one of the most cost-effective malaria interventions [34,35]. PMI presence in a country further catalyses and facilitates the procurement, distribution, and implementation of interventions from other funders with the initiative distributing 80 million LLINs and 34 million ACT courses procured by other donors in the period 2006–2015 [6]. Furthermore, PMI is involved with a number of capacity and health system-strengthening initiatives, such as training health workers in malaria diagnosis and treatment [6], the loss of which would compound issues of increased caseload if PMI support were reduced. Our estimates of reductions in under-5 mortality attributable to PMI funding are lower when compared with estimated reductions of a similar magnitude in all-cause mortality in a recently published difference-in-differences analysis of PMI impact [7]. Whilst our estimates of intervention coverage attributable to PMI funding are similar, the additional impact estimated by Jakubowski et al. may be ascribed to indirect impacts of PMI funding on nonmalaria outcomes (through, for example, health systems strengthening), although considerable uncertainties also impact both analyses. We also do not capture the wider societal costs of the disease, such as missed workdays by carers, reduced education, or impact on future lifetime earnings, nor the economic effects of endemic malaria on factors such as migration, trade, tourism, or foreign investment within a country [36]. It is likely that, when facing cuts, PMI and NMCPs would reallocate existing funds to cover those interventions seen as vital. However, in an already budget-restricted environment, a limit to the potentially mitigating effects of such reallocations would quickly be reached. There are a number of difficulties associated with estimating accurate coverage estimates and uptake for interventions with a wide range of definitions and methodologies adopted. We have assumed that PMI-reported contribution and interventions figures, taken from their 10th Annual Report to Congress [6] and building upon a well-established monitoring and evaluation strategy, are representative and accurate. We also are including assumptions that the PMI-delivered interventions are reaching required recipients in an efficient manner. Whilst we know inefficiencies do exist, for example in LLIN distribution [22], these are difficult to attribute to specific sources. Furthermore, due to the nonlinear impact of interventions such as LLINs, it is difficult to split contributions from different funding sources (i.e., should an X% funding contribution be linked to the first N% or last N% of observed LLIN coverage?). We do account for falloff between coverage and usage as well as deterioration of insecticide and wear and tear of LLINs in this analysis. Similarities to empirical estimates [8] indicate that we are accurately capturing broad trends in intervention coverage due to PMI funding.

As malaria transmission is brought to low levels, increased efforts are needed to target hard-to-reach populations as well as increase surveillance efforts, and hence the programmatic costs are likely to increase [7]. In such circumstances, investment decisions need to take into account the potential for permanent gains that would be accrued if an area or country can achieve elimination. However, there still remain large, extremely cost-effective gains that can be obtained by investing further to reduce the burden of malaria in areas of high endemicity. WHO GTS for malaria has set targets of achieving of at least 90% reductions in global case incidence and mortality rates by 2030 compared to levels in 2015, with vector control, chemoprevention, diagnosis and treatment, and surveillance being key pillars of the outlined strategy [37]. Based on the estimates of our model, PMI’s ongoing support of these activities in countries of high burden or strategic importance is vital in order to avoid a rapid erosion of the progress made in the last 15 years on the road towards malaria eradication.

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Introduction

Half of the world population is at risk of malaria (World Health Organization, 2013), the most common, and severe parasitic mosquito-borne disease (White et al., 2014). Five species of the protozoan genus Plasmodium infect humans, with Plasmodium falciparum and P. vivax causing over 200 million cases/year and P. falciparum inflicting virtually all the 6–700,000 annual deaths (2013) recorded mainly in children of Sub-Saharan Africa.

The malaria parasite exhibits a complex life cycle involving an Anopheles mosquito and a vertebrate host (Figure 1). When an infected female mosquito bites a human, the Plasmodium sporozoites travel to the liver and invade hepatocytes, where parasites replicate as hepatic schizonts until several thousand merozoites are produced and released in the bloodstream. In P. vivax, but not in P. falciparum, some liver parasites remain instead quiescent (hypnozoites), resuming replication, and infection after several weeks or months. Upon erythrocyte invasion in the bloodstream Plasmodium parasites undergo asexual replication forming mature schizonts whose rupture releases merozoites that invade new erythrocytes. Some blood stage parasites differentiate instead into male and female gametocytes that, when ingested in the mosquito blood meal, are activated to produce gametes. Gamete fusion in the insect midgut produces a zygote which develops into a motile ookinete, traversing the gut wall, and transforming into an oocyst, where 1000s of sporozoites are produced. The life cycle is closed when sporozoites, migrated from the ruptured oocyst to the mosquito salivary glands, are injected in a new human host by the insect bite.

FIGURE 1. Transgenic bioluminescent malaria parasites at different stages of their development in the vertebrate and mosquito hosts. The central diagram represents the Plasmodium life cycle, showing the progression through the developmental stages of the parasites in the mosquito vector and in the vertebrate host. (A) Bioluminescence imaging (BLI) of individual Plasmodium falciparum gametocytes expressing a click beetle luciferase under a sexual stage-specific promoter. The bright field image shows immobilized gametocytes, highlighted in green, amongst uninfected erythrocytes; the dark field shows the bioluminescence signal of the gametocytes incubated with D-luciferin. Magnification bar: 15 μm. (Adapted from Cevenini et al., 2014). (B) BLI of a mouse infected with asexual P. berghei parasites expressing a firefly luciferase-green fluorescent protein (GFP) fusion. Heatmap of the bioluminescent signal identifies the sites of accumulation of the parasites (Reproduced with permission from Claser et al., 2011). (C) Fluorescence of a firefly luciferase-GFP fusion protein expressed in P. falciparum sporozoites contained in a oocyst and (D) obtained from the dissection of infected mosquito salivary glands. Magnification bar: 5 μm. (E)In vivo bioluminescent signal obtained by transgenic P. falciparum liver stage parasites developing in the chimeric liver of a humanized mouse (C–E) are reproduced with permission from Vaughan et al. (2012).

The pathogenesis of malaria is caused by the asexual blood stages. In the clinical manifestations of P. falciparum malaria, the ability of parasites to sequester in the microvasculature of several organs, including the brain, is a major cause of disease severity, and of a fatal outcome (Miller et al., 2002; Milner et al., 2014; Sack et al., 2014). Consequently, the need to cure symptomatic patients traditionally drove efforts toward finding drugs targeting the asexual blood stage parasites, often underestimating the importance of eliminating also the sporozoite, and gametocyte transmission stages or, in P. vivax, the hypnozoites. The recent concerning reports from South East Asia of a decreased sensitivity of some P. falciparum infections to frontline combination therapies based on artemisinin derivatives is now calling for renewed efforts to address this emergency in the frame of a global strategy to control malaria and eventually eradicate this deadly parasite.

It is possible to cultivate all asexual and sexual blood stages of P. falciparum in vitro, unlike P. vivax. Plasmodium species infecting rodents have been also intensely studied as mouse models of aspects of malaria, with P. berghei particularly exploited for its amenability to genetic manipulation. In contrast, transgenesis technology has been comparatively more troublesome in P. falciparum. This review aims to highlight the importance of Plasmodium transgenic parasites, particularly those engineered with bioluminescent reporters, both in the study of the fundamental biology of Plasmodium and in developing effective antimalarial treatments.

Luciferase enzymes catalyze the light-producing chemical reactions of bioluminescent organisms, in which a luminogenic substrate (e.g., D-luciferin) is oxidized in the presence of ATP, yielding photons. These can be accurately measured by a luminometer with a sensitivity and a virtual absence of background that made bioluminescent reporters potent and versatile tools in biology (Smale, 2010). Luciferases hold a special place in the history of Plasmodium transgenesis: the first plasmid construct to be successfully transfected in malaria parasites contained a firefly (Photynus pyralis) luciferase gene whose expression, driven by the promoter of a parasite sexual stage-specific gene, was measured in ookinetes of the bird parasite P. gallinaceum (Goonewardene et al., 1993). Subsequently, luciferase reporters have been used to optimize transfection techniques in Plasmodium parasites (Epp et al., 2008; Hasenkamp et al., 2012), including the introduction of the luciferase from the sea pansy Renilla reniformis, where use of different substrates (D-luciferin and coelenterazine) enabled simultaneous detection of the two parasite produced reporters (Militello and Wirth, 2003; Helm et al., 2010). Since the 1990s, with the stable genetic transformation of different species of Plasmodium (Waters et al., 1997), luciferase reporter genes greatly contributed to elucidate key aspects of malaria infection, from the parasite cellular biology, protein trafficking, gene function, and drug resistance, in several developmental stages throughout the Plasmodium life cycle (Figure 1).

The Plasmodium Life Cycle Marked by Bioluminescent Parasite Developmental Stages

Plasmodium Mosquito Stages

Parasite sexual stage development in the mosquito vector is crucial for the transmission of Plasmodium, and elucidating the biology of this process may therefore lead to design novel malaria transmission-blocking strategies. Some studies with bioluminescent parasites highlighted the importance of post-transcriptional regulation acting on stability and translation of several mRNAs, including those encoding major proteins of the gamete and ookinete surface (Mair et al., 2006). Assays with luciferase reporters were for instance fundamental to identify regulatory elements in the transcripts of the P25 and P28 surface proteins of P. gallinaceum and P. falciparum (Golightly et al., 2000; Oguariri et al., 2006).

Plasmodium parasites expressing luciferases also improved tool development for applied studies. A powerful bioassay to determine parasite ability to infect mosquitoes is based on feeding cultured Plasmodium gametocytes to mosquitoes, and it is used to measure effect of transmission blocking drugs or antibodies. This assay is, however, technically demanding and time consuming as the resulting oocysts need to be individually counted in dissected insects. After improvements by using P. berghei parasites expressing a green fluorescent protein (GFP) in mosquito stages (Delves and Sinden, 2010), a transgenic line of the human parasite P. falciparum line expressing the firefly luciferase in oocysts was developed. In the resulting luminescence-based standard membrane feeding assay (SMFA) the mean luminescence intensity of individual and pooled mosquitoes accurately quantified mean oocyst intensity, eliminating the need for mosquito dissection, and putting the basis for significant SMFA scalability (Stone et al., 2014).

Toward the end of parasite development in the mosquito, the sporozoites produced in the oocyst migrate to the insect salivary glands. Number of salivary gland sporozoites, the only mosquito stages infectious to a mammalian host, is an important index of Plasmodium mosquito development. The construction of a P. berghei line where a GFP-luciferase fusion is specifically expressed in sporozoites enabled establishment of a simple and fast assay of sporozoite loads from whole mosquitoes (Ramakrishnan et al., 2012).

Transmission from Mosquitoes: Sporozoites and Liver Stages

Plasmodium sporozoites injected from an infected mosquito to a human or rodent host start their intracellular development into the liver hepatocytes. This clinically silent stage is the target for prophylactic or vaccine strategies, particularly against P. vivax long lasting hypnozoites.

Plasmodium liver stage development has been poorly explored compared to that of blood stages partly because the in vivo and in vitro analyses, respectively, in mouse models and in cultured liver cells, are constrained by the necessity to sacrifice high numbers of mice or by inefficiency of sporozoite infection of cultured liver cells. Transgenic luciferase-expressing sporozoites improved detection strategies introducing bioluminescence imaging (BLI) and in vivo imaging system (IVIS) in the analysis of parasite liver stage development in live mice and in cultured hepatocytes. Real-time BLI requires injection of the luciferin substrate in the mouse or in the dissected organ and an intensified charge-coupled photon counting video camera to measure photon emission (Franke-Fayard et al., 2006; Braks et al., 2013). BLI and IVIS using firefly or sea pansy luciferases have been used for real-time, live monitoring of the progression of rodent parasitic infection in the whole animal or in specific organs (Ploemen et al., 2009; Annoura et al., 2013; Manzoni et al., 2014) and to test activity of drugs targeting liver stage infection, using P. yoelii and P. berghei transgenic sporozoites in human liver HepG2 or Huh-7 cells and in whole mice (Mwakingwe et al., 2009; Ramalhete et al., 2011, 2014; Derbyshire et al., 2012; Lacrue et al., 2013; Li et al., 2014; Marcsisin et al., 2014; Zuzarte-Luis et al., 2014). To improve these approaches, identification of parasite promoters specifically activated in liver development was achieved in P. berghei, also in this case relying on use of transgenic luciferase-promoter fusions (Helm et al., 2010).

The ability to reliably quantify parasite infection in hepatocytes is essential in the development of malaria vaccines. To overcome limitations of qRT-PCR-based quantification, P. berghei parasites expressing a GFP-luciferase fusion were introduced to evaluate antimalarial immunity both in vivo, in mice where this was induced by sporozoites unable to proliferate after irradiation or chloroquine prophylaxis, and in vitro in Huh-7 human liver hepatoma cells (Ploemen et al., 2011; Miller et al., 2013). Luciferase expressing P. berghei and P. falciparum sporozoites were also used to assess adequacy of sporozoite attenuation, obtained this time by genetic mutation, respectively, in in vivo murine malaria model and in primary human hepatocytes (Annoura et al., 2012; van Schaijk et al., 2014). These studies highlighted the role of cell mediated immunity mounting against the multiplication-deficient sporozoites. A role for antibody mediated immunity was instead shown by BLI of luciferase-expressing sporozoites of the human parasite P. falciparum in mice with a humanized liver, showing that infection in this organ was reduced by passive transfer of a monoclonal antibody targeting the sporozoite surface protein CSP (Sack et al., 2014). Finally, P. berghei and P. yoelii luciferase transgenic parasites were instrumental to evaluate modes of sporozoite administration, a critical bottleneck in immunization, and challenge protocols (Ploemen et al., 2013).

From the Liver to the Blood: the Asexual Erythrocytic Stages

Maturation of the liver schizont releases 1000s of merozoites that invade blood stream erythrocytes and starts the asexual, symptomatic blood stage infection. In P. falciparum the blood stage schizonts disappear from circulation as they adhere to host ligands on endothelial cells of the microvasculature in several organs, especially in the brain and in the placenta, through parasite proteins expressed on the infected erythrocyte surface, leading to severe pathogenesis such as cerebral malaria or adverse effects during pregnancy. As parasites are observed to accumulate in several organs, including the brain, also in the mouse malaria model, real-time BLI in whole mice or in dissected organs were conducted with P. berghei transgenic lines expressing luciferase under a constitutive or a schizont-specific promoter to identify the involved components of the immune system (Franke-Fayard et al., 2005; Amante et al., 2007; Spaccapelo et al., 2010; Claser et al., 2011; Pasini et al., 2013; Imai et al., 2014).

The need to elucidate the mechanisms of malaria pathogenesis directed research on the fundamental biology of parasite asexual development, one important aspect being how the parasite regulates its gene expression. The extremely high A+T content of the Plasmodium genomes however, prevented homology based identification of promoters, regulatory elements, and parasite transcription factors, whereas luciferase reporters proved to be of paramount importance in functionally identifying gene promoters and regulatory regions (Horrocks and Kilbey, 1996; Porter, 2002; Militello et al., 2004; Hasenkamp et al., 2013a). This work identified sequences functioning as bi-directional promoters, like the intergenic region of the P. berghei elongation factor-1α (ef-1α) gene (de Koning-Ward et al., 1999; Fernandez-Becerra et al., 2003) or the intron of the P. falciparum var genes (Epp et al., 2008), or evaluated whether specific promoters from one Plasmodium species were able (Fernandez-Becerra et al., 2003; Ozwara et al., 2003), or unable (Azevedo and del Portillo, 2007) to recruit the transcriptional machinery of a different malaria species. Importantly, luciferase expressing parasites were used to identify regulatory regions governing the expression of the P. falciparum polymorphic var genes encoding the parasite sequestration ligands, whose expression switch is responsible for parasite antigenic variation, and immune evasion (Deitsch et al., 1999; Calderwood et al., 2003; Frank et al., 2006; Muhle et al., 2009). In summary, luciferase reporters not only contributed to identify functional elements involved in parasite gene regulation (Bischoff et al., 2000; Militello et al., 2004; López-Estraño et al., 2007; Gopalakrishnan and López-Estraño, 2010; Zhang et al., 2011; Patakottu et al., 2012), but also were essential to select specific promoters in the development of Plasmodium inducible expression systems (de Azevedo et al., 2012; Kolevzon et al., 2014) and to test new regulatory regions in chromosomally integrated luciferase cassettes (Ekland et al., 2011; Weiwer et al., 2011; Che et al., 2012; Khan et al., 2012; Hasenkamp et al., 2013b).

A major effort in the fight against malaria, particularly P. falciparum, has been the screening for new antimalarial drugs, an endeavor that the appearance of artemisinin resistance in South East Asia makes dramatically urgent. In the past decades, in vitro methods measuring the incorporation of [3H]-labeled hypoxanthine and ethanolamine or the activity of parasite Lactate Dehydrogenase have been the standard for P. falciparum cell based assays and used in large drug screenings (Fidock, 2010). The demand for high-throughput, non-radioactive assays prompted to exploit also in Plasmodium the high sensitivity and virtual absence of background of luciferase reporters, until recently used in this field only to study expression of the P. falciparum multidrug resistance gene pfmdr1 in drug treated parasites (Myrick et al., 2003; Waller et al., 2003). To this aim a P. falciparum line expressing the firefly luciferase under the heat shock protein 86 (pfhsp86) gene promoter in asexual stages enabled establishment of a cell-based luciferase drug screening assay in 96w plates (Cui et al., 2008), subsequently adapted to 384w plate using 105–106 parasites per well (Lucumi et al., 2010). Also P. berghei parasites expressing a firefly luciferase-GFP fusion were used for in vitro and in vivo bioluminescence drug assay, enabling use of animal models to test new drugs in vivo (Franke-Fayard et al., 2008; Lin et al., 2013).

Preparing Departure from the Blood: the Gametocytes

Plasmodium gametocytes are the parasite sexual stages responsible for the transmission from the vertebrate host to the mosquito. Male and female gametocytes are formed in the bloodstream and, in P. falciparum, they mature in 10 days through five developmental stages. Upon ingestion in the mosquito gut, mature gametocytes promptly differentiate into gametes and fertilization ensures parasite infection in the insect vector. A key priority in the present goal to globally eliminate malaria is to identify new drugs targeting in the bloodstream both the asexual and the sexual stages of the parasite. However, the non-replicative nature of gametocytes imposed to develop specific cell based screening assays, different from those used for asexual stages. One problem is for instance that of false negative signals due to the persistence of fluorescent reporter or of parasite enzyme activities in unhealthy or dying gametocytes. P. falciparum lines expressing a GFP-firefly luciferase gene under gametocyte specific promoters were established (Adjalley et al., 2011) and used in high-throughput screening assays of compounds with anti-asexual stage activity (Lucantoni et al., 2013). Recently, luciferase-based gametocyte assays have been improved by replacing the commonly used commercial luciferase substrates with an ATP-free, non-lysing D-luciferin formulation, yielding assay readouts that more reliably monitored viability and sensitivity to compounds of the treated gametocytes (Cevenini et al., 2014). In this work, absence of parasite cell lysis and the introduction in P. falciparum of the use of a potent luciferase from Pyrophorus plagiophthalamus under a gametocyte promoter enabled to perform for the first time BLI at the level of single parasite cells, individually distinguishing live and dead P. falciparum gametocytes (Cevenini et al., 2014).

Multiplexing, Subcellular Localization, Imaging: the Future in the Use of Bioluminescent Malaria Parasites

Virtually all stages of the complex life cycle of malaria parasites have been enlightened by the use in several studies of luciferase reporters. These engineered parasites provided key answers to fundamental biological questions and now represent important tools for drug screening. Novel potent reporters have already expanded the luciferase repertoire used in P. falciparum beyond the P. pyralis and Renilla enzymes (Azevedo et al., 2014; Cevenini et al., 2014), increasing sensitivity and enabling to further reduce parasite numbers in high-throughput screening assays, a non-trivial improvement when using specific stages (e.g., the gametocytes) whose cultivation is technically demanding. Nevertheless exploitation of the full potential of bioluminescent reporters in malaria research is just moving the first steps.

The possibility to tune luciferase emission properties, such as emission wavelength, kinetics or termo- and pH-stability, via random or site-directed mutagenesis or use of enzyme natural variants, led to introduce multicolor bioluminescence in antimalarial drug screening. A green and a red light emitting luciferase from P. plagiophthalamus were expressed in P. falciparum immature and mature gametocytes, providing for the first time the possibility to simultaneously measure differential, stage specific effects of drugs in a dual-color luciferase assay, and opening the possibility to apply multicolor bioluminescence to any parasite stage in fundamental and applied studies. A dual expression system with distinct luciferases would for instance be valuable in cell based high-throughput screenings to readily identify and discard compounds active against the reporter rather than the target cell (Thorne et al., 2012), as they will most likely affect only one luciferase type.

Another promising application of luciferase reporters is through their fusion to specific signals used by the parasite to traffic proteins in different extra cellular compartments of the infected erythrocyte. As protein export is uniquely regulated in the parasite and is essential for its survival, use of such fusions may be invaluable to screen for compounds targeting this process. Preliminary studies were conducted with the P. pyralis enzyme (Burghaus and Lingelbach, 2001) and more recent work established P. falciparum lines which express a brighter deep-sea shrimp luciferase equipped with sequences driving the reporter in the parasite cytoplasm or in erythrocyte compartments (Azevedo et al., 2014).

In another field of application, the achievement of single parasite cell BLI and the availability of luciferases whose red-shifted light emission is more efficiently detectable from blood and tissues are paving the road to significant progress in analyses of the host-parasite interplay. Co-cultures of different P. falciparum stages and human cell types in vitro can provide new insights of the physiology of asexual and sexual stage parasite sequestration. The increased sensitivity achieved in in vivo mouse imaging with a red-shifted luciferase expressed by the unicellular protozoan parasite Trypanosoma brucei (Van Reet et al., 2014) is promising in view of use also in Plasmodium infected mice. Importantly, the increasing availability of humanized mouse models for P. falciparum and P. vivax infections, supporting development of asexual, and sexual blood stages and of liver stages (Kaushansky et al., 2014) and the use of P. falciparum transgenic lines with a luciferase expressed constitutively (Vaughan et al., 2012) or under stage-specific promoters are expected to answer many unsolved questions.

The wealth of biological information provided by the use of engineered bioluminescent malaria parasites, not to mention those not reviewed here expressing a variety of fluorescent reporters, has been and will most likely continue to be enormous. The confined use of these whole cell biosensors in laboratory settings does not pose regulatory concerns on environmental release. From their aseptic sites of utilization, these genetically modified parasites will nevertheless have the most significant impact in the real world, contrasting the unbearable burden of a worldwide devastating disease.

Author Contribution

GS drafted and PA edited the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

Authors are indebted to Drs Luca Cevenini, Elisa Michelini, and Aldo Roda, University of Bologna, Bologna, Italy and Dr. Bruce B. Branchini, Connecticut College, New London, CT, USA, for several insightful discussions along our collaborations, and thank Mr. Cosimo M. Curianò for the figure artwork. Research in the authors’ laboratory is supported from grants from the Bill & Melinda Gates Foundation and the EU EVIMalaR Network of Excellence.

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