Experimental studies on the ecology and evolution of drug-resistant malaria parasites

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug

Relevance of Undetectably Rare Resistant Malaria Parasites in Treatment Failure: Experimental Evidence from Plasmodium chabaudi

Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants is extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance

Transmission stage investment of malaria parasites in response to in-host competition

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence

Phenotypic insecticide resistance in arbovirus mosquito vectors in Catalonia and its capital Barcelona (Spain)

A range of mosquito species that belong to the Culicidae family are responsible for the worldwide transmission of infectious arboviral diseases such as dengue fever, Zika, West Nile fever and Chikungunya fever. Spain is at risk of arbovirus outbreaks, as various arboviral diseases are frequently introduced and it has established competent vector populations. Autochthonous human cases of West Nile virus have been reported infrequently since 2004, and since October 2018 three autochthonous human case of dengue fever have been confirmed. In response to an outbreak of any arboviral disease, space spraying or fogging will be implemented to control adult mosquito populations. To ensure adulticiding is costeffective, the insecticide susceptibility status of vectors throughout Catalonia, an autonomous region in north-eastern Spain, was assessed through standardized WHO tube and CDC bottle bioassays. All Culex pipiens populations tested were resistant to at least one of the pyrethroids tested, whereas Aedes albopictus populations were susceptible to all pyrethroids tested. More detailed studies on the Cx. pipiens populations from the Barcelona area (the capital and largest city of Catalonia) revealed resistance to all four classes of public health insecticides available (pyrethroids, carbamates, organophosphates and organochlorides). All Ae. albopictus populations were susceptible to those classes, except for one of the tests performed with pirimiphos-methyl (an organophosphate). Pyrethroids are currently the first line chemical class to be used in space spray operations in response to an outbreak of an arboviral disease. While pyrethroids can be effective in reducing Ae. albopictus populations, this class may not be effective to control Cx. pipiens populations.info:eu-repo/semantics/publishedVersio

Long-lasting insecticidal nets and the quest for malaria eradication : a mathematical modeling approach

Recent dramatic declines in global malaria burden and mortality can be largely attributed to the large-scale deployment of insecticidal-based measures, namely long-lasting insecticidal nets (LLINs) and indoor residual spraying. However, the sustainability of these gains, and the feasibility of global malaria eradication by 2040, may be affected by increasing insecticide resistance among the Anopheles malaria vector. We employ a new differential-equations based mathematical model, which incorporates the full, weather-dependent mosquito lifecycle, to assess the population-level impact of the large-scale use of LLINs, under different levels of Anopheles pyrethroid insecticide resistance, on malaria transmission dynamics and control in a community. Moreover, we describe the bednet-mosquito interaction using parameters that can be estimated from the large experimental hut trial literature under varying levels of effective pyrethroid resistance. An expression for the basic reproduction number, R0, as a function of population-level bednet coverage, is derived. It is shown, owing to the phenomenon of backward bifurcation, that R0 must be pushed appreciably below 1 to eliminate malaria in endemic areas, potentially complicating eradication efforts. Numerical simulations of the model suggest that, when the baseline R0 is high (corresponding roughly to holoendemic malaria), very high bednet coverage with highly effective nets is necessary to approach conditions for malaria elimination. Further, while >50% bednet coverage is likely sufficient to strongly control or eliminate malaria from areas with a mesoendemic malaria baseline, pyrethroid resistance could undermine control and elimination efforts even in this setting. Our simulations show that pyrethroid resistance in mosquitoes appreciably reduces bednet effectiveness across parameter space. This modeling study also suggests that increasing pre-bloodmeal deterrence of mosquitoes (deterring them from entry into protected homes) actually hampers elimination efforts, as it may focus mosquito biting onto a smaller unprotected host subpopulation. Finally, we observe that temperature affects malaria potential independently of bednet coverage and pyrethroid-resistance levels, with both climate change and pyrethroid resistance posing future threats to malaria control.National Institute for Mathematical and Biological Synthesis (NIMBioS) is an Institute sponsored by the National Science Foundation, the U.S. Department of Homeland Security, and the U.S. Department of Agriculture through NSF Award #EF-0832858, with additional support from The University of Tennessee, Knoxville. ABG also acknowledges the support, in part, of the Simons Foundation (Award #585022).http://link.springer.com/journal/2852021-05-23hj2020Mathematics and Applied Mathematic

Empirical and theoretical investigation into the potential impacts of insecticide resistance on the effectiveness of insecticide-treated bed nets.

In spite of widespread insecticide resistance in vector mosquitoes throughout Africa, there is limited evidence that long-lasting insecticidal bed nets (LLINs) are failing to protect against malaria. Here, we showed that LLIN contact in the course of host-seeking resulted in higher mortality of resistant Anopheles spp. mosquitoes than predicted from standard laboratory exposures with the same net. We also found that sublethal contact with an LLIN caused a reduction in blood feeding and subsequent host-seeking success in multiple lines of resistant mosquitoes from the laboratory and the field. Using a transmission model, we showed that when these LLIN-related lethal and sublethal effects were accrued over mosquito lifetimes, they greatly reduced the impact of resistance on malaria transmission potential under conditions of high net coverage. If coverage falls, the epidemiological impact is far more pronounced. Similarly, if the intensity of resistance intensifies, the loss of malaria control increases nonlinearly. Our findings help explain why insecticide resistance has not yet led to wide-scale failure of LLINs, but reinforce the call for alternative control tools and informed resistance management strategies

Long-lasting insecticidal nets no longer effectively kill the highly resistant Anopheles funestus of southern Mozambique

BACKGROUND: Chemical insecticides are crucial to malaria control and elimination programmes. The frontline vector control interventions depend mainly on pyrethroids; all long-lasting insecticidal nets (LLINs) and more than 80% of indoor residual spraying (IRS) campaigns use chemicals from this class. This extensive use of pyrethroids imposes a strong selection pressure for resistance in mosquito populations, and so continuous resistance monitoring and evaluation are important. As pyrethroids have also been used for many years in the Manhica District, an area in southern Mozambique with perennial malaria transmission, an assessment of their efficacy against the local malaria vectors was conducted. METHODS: Female offspring of wild-caught Anopheles funestus s.s. females were exposed to deltamethrin, lambda-cyhalothrin and permethrin using the World Health Organization (WHO) insecticide-resistance monitoring protocols. The 3-min WHO cone bioassay was used to evaluate the effectiveness of the bed nets distributed or available for purchase in the area (Olyset, permethrin LLIN; PermaNet 2.0, deltamethrin LLIN) against An. funestus. Mosquitoes were also exposed to PermaNet 2.0 for up to 8 h in time-exposure assays. RESULTS: Resistance to pyrethroids in An. funestus s.s. was extremely high, much higher than reported in 2002 and 2009. No exposure killed more than 25.8% of the mosquitoes tested (average mortality, deltamethrin: 6.4%; lambda-cyhalothrin: 5.1%; permethrin: 19.1%). There was no significant difference in the mortality generated by 3-min exposure to any net (Olyset: 9.3% mortality, PermaNet 2.0: 6.0%, untreated: 2.0%; p = 0.2). Six hours of exposure were required to kill 50% of the An. funestus s.s. on PermaNet 2.0. CONCLUSIONS: Anopheles funestus s.s. in Manhica is extremely resistant to pyrethroids, and this area is clearly a pyrethroid-resistance hotspot. This could severely undermine vector control in this district if no appropriate countermeasures are undertaken. The National Malaria Control Programme (NMCP) of Mozambique is currently improving its resistance monitoring programme, to design and scale up new management strategies. These actions are urgently needed, as the goal of the NMCP and its partners is to reach elimination in southern Mozambique by 2020

Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum

Background: Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for P. falciparum resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance. Anopheles funestus were collected in southern Mozambique using CDC light traps and manual collec‑ tions. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of fve mosqui‑ toes. These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr1, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS. Results: Among the 846 samples screened for P. falciparum, 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for k13 and pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/ pfdhps mutations were near fxation and about half of the isolates contained the pfmdr184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing. Conclusions: Pooled deep sequencing of P. falciparum isolates extracted from mosquitoes is a promising, efcient and cost-efective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogene‑ ous geographical range

The importance of understanding fitness costs associated with drug resistance throughout the life cycle of malaria parasites

International audienceA recommendation – based on reviews by Sarah Reece and Marianna Szucs – of the article: Villa M., Berthomieu A., Rivero A. 2022. Fitness costs and benefits in response to artificial artesunate selection in Plasmodium. bioRxiv, 20220128478164, ver 3 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2022.01.28.47816

Experimental studies on the ecology and evolution of drug-resistant malaria parasites

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug.EThOS - Electronic Theses Online ServiceGBUnited Kingdo