Derek G. Sim

WITHIN‐HOST COMPETITION IN GENETICALLY DIVERSE MALARIA INFECTIONS: PARASITE VIRULENCE AND COMPETITIVE SUCCESS

Abstract Humans and animals often become coinfected with pathogen strains that differ in virulence. The ensuing interaction between these strains can, in theory, be a major determinant of the direction of selection on virulence genes in pathogen populations. Many mathematical analyses of this assume that virulent pathogen lineages have a competitive advantage within coinfected hosts and thus predict that pathogens will evolve to become more virulent where genetically diverse infections are common. Although the implications of these studies are relevant to both fundamental biology and medical science, direct empirical tests for relationships between virulence and competitive ability are lacking. Here we use newly developed strain‐specific real‐time quantitative polymerase chain reaction protocols to determine the pairwise competitiveness of genetically divergent Plasmodium chabaudi clones that represent a wide range of innate virulences in their rodent host. We found that even against their background of widely varying genotypic and antigenic properties, virulent clones had a competitive advantage in the acute phase of mixed infections. The more virulent a clone was relative to its competitor, the less it suffered from competition. This result confirms our earlier work with parasite lines derived from a single clonal lineage by serial passage and supports the virulencecompetitive ability assumption of many theoretical models. To the extent that our rodent model captures the essence of the natural history of malaria parasites, public health interventions which reduce the incidence of mixed malaria infections should have beneficial consequences by reducing the selection for high virulence.

Malaria-induced changes in host odors enhance mosquito attraction

Significance Recent research suggests that vector-borne pathogens may frequently manipulate host odors to influence vector attraction. Such effects could have implications for the use of olfactory lures or repellents to disrupt vector transmission, as well as for disease diagnosis. We observed enhanced attraction of mosquitoes to mice infected by the rodent malaria parasite Plasmodium chabaudii during a key period after the subsidence of acute malaria symptoms but during which mice remained highly infectious. This attraction appears to be mediated by an overall elevation of volatile emissions from infected individuals and characteristic changes in the levels of individual compounds. Furthermore, clear differences in the odor profiles of healthy and infected individuals persist throughout the course of infection. Vector-borne pathogens may alter traits of their primary hosts in ways that influence the frequency and nature of interactions between hosts and vectors. Previous work has reported enhanced mosquito attraction to host organisms infected with malaria parasites but did not address the mechanisms underlying such effects. Here we document malaria-induced changes in the odor profiles of infected mice (relative to healthy individuals) over the course of infection, as well as effects on the attractiveness of infected hosts to mosquito vectors. We observed enhanced mosquito attraction to infected mice during a key period after the subsidence of acute malaria symptoms, but during which mice remained highly infectious. This attraction corresponded to an overall elevation in the volatile emissions of infected mice observed during this period. Furthermore, data analyses—using discriminant analysis of principal components and random forest approaches—revealed clear differences in the composition of the volatile blends of infected and healthy individuals. Experimental manipulation of individual compounds that exhibited altered emission levels during the period when differential vector attraction was observed also elicited enhanced mosquito attraction, indicating that compounds being influenced by malaria infection status also mediate vector host-seeking behavior. These findings provide important insights into the cues that mediate vector attraction to hosts infected with transmissible stages of malaria parasites, as well as documenting characteristic changes in the odors of infected individuals that may have potential value as diagnostic biomarkers of infection.

CHEMOTHERAPY, WITHIN-HOST ECOLOGY AND THE FITNESS OF DRUG-RESISTANT MALARIA PARASITES

A major determinant of the rate at which drug‐resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug‐sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria model Plasmodium chabaudi, we found that low‐dose chemotherapy did reduce competitive release. A higher drug dose regimen exerted stronger positive selection on resistant parasites for no detectable clinical gain. We estimated instantaneous selection coefficients throughout the course of replicate infections to analyze the temporal pattern of the strength and direction of within‐host selection. The strength of selection on resistance varied through the course of infections, even in untreated infections, but increased immediately following drug treatment, particularly in the high‐dose groups. Resistance remained under positive selection for much longer than expected from the half life of the drug. Although there are many differences between mice and people, our data do raise the question whether the aggressive treatment regimens aimed at complete parasite clearance are the best resistance‐management strategies for humans.

Aggressive Chemotherapy and the Selection of Drug Resistant Pathogens

Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

The evolutionary consequences of blood-stage vaccination on the rodent malaria Plasmodium chabaudi.

A candidate malaria vaccine promoted the evolution of more virulent malaria parasites in mice.

The fitness of drug-resistant malaria parasites in a rodent model: multiplicity of infection

Malaria infections normally consist of more than one clonally replicating lineage. Within‐host interactions between sensitive and resistant parasites can have profound effects on the evolution of drug resistance. Here, using the Plasmodium chabaudi mouse malaria model, we ask whether the costs and benefits of resistance are affected by the number of co‐infecting strains competing with a resistant clone. We found strong competitive suppression of resistant parasites in untreated infections and marked competitive release following treatment. The magnitude of competitive suppression depended on competitor identity. However, there was no overall effect of the diversity of susceptible parasites on the extent of competitive suppression or release. If these findings generalize, then transmission intensity will impact on resistance evolution because of its effect on the frequency of mixed infections, not because of its effect on the distribution of clones per host. This would greatly simplify the computational problems of adequately capturing within‐host ecology in models of drug resistance evolution in malaria.

Virulence, drug sensitivity and transmission success in the rodent malaria, Plasmodium chabaudi

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.

Enhanced Transmission of Drug-Resistant Parasites to Mosquitoes following Drug Treatment in Rodent Malaria

The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasmodium chabaudi, co-infection with drug-sensitive parasites can prevent the transmission of initially rare resistant parasites to mosquitoes. Removal of drug-sensitive parasites following chemotherapy enabled resistant parasites to transmit to mosquitoes as successfully as sensitive parasites in the absence of treatment. We also show that the genetic composition of gametocyte populations in host venous blood accurately reflects the genetic composition of gametocytes taken up by mosquitoes. Our data demonstrate that, at least for this mouse model, aggressive chemotherapy leads to very effective transmission of highly resistant parasites that are present in an infection, the very parasites which undermine the long term efficacy of front-line drugs.

Rapid response to selection, competitive release and increased transmission potential of artesunate-selected Plasmodium chabaudi malaria parasites.

The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain infections.

Understanding genetic variation in in vivo tolerance to artesunate: implications for treatment efficacy and resistance monitoring.

Artemisinin‐based drugs are the front‐line weapon in the treatment of human malaria cases, but there is concern that recent reports of slow clearing infections may signal developing resistance to treatment. In the absence of molecular markers for resistance, current efforts to monitor drug efficacy are based on the rate at which parasites are cleared from infections. However, some knowledge of the standing variation in parasite susceptibility is needed to identify a meaningful increase in infection half‐life. Here, we show that five previously unexposed genotypes of the rodent malaria parasite Plasmodium chabaudi differ substantially in their in vivo response to treatment. Slower clearance rates were not linked to parasite virulence or growth rate, going against the suggestion that drug treatment will drive the evolution of virulence in this system. The level of variation observed here in a relatively small number of genotypes suggests existing ‘resistant’ parasites could be present in the population and therefore, increased parasite clearance rates could represent selection on pre‐existing variation rather than de novo resistance events. This has implications for resistance monitoring as susceptibility may depend on evolved traits unrelated to drug exposure.