Describing the current status of Plasmodium falciparum population structure and drug resistance within mainland Tanzania using molecular inversion probes

High‐throughput Plasmodium genomic data is increasingly useful in assessing prevalence of clinically important mutations and malaria transmission patterns. Understanding parasite diversity is important for identification of specific human or parasite populations that can be targeted by control programmes, and to monitor the spread of mutations associated with drug resistance. An up‐to‐date understanding of regional parasite population dynamics is also critical to monitor the impact of control efforts. However, this data is largely absent from high‐burden nations in Africa, and to date, no such analysis has been conducted for malaria parasites in Tanzania countrywide. To this end, over 1,000 P. falciparum clinical isolates were collected in 2017 from 13 sites in seven administrative regions across Tanzania, and parasites were genotyped at 1,800 variable positions genome‐wide using molecular inversion probes. Population structure was detectable among Tanzanian P. falciparum parasites, approximately separating parasites from the northern and southern districts and identifying genetically admixed populations in the north. Isolates from nearby districts were more likely to be genetically related compared to parasites sampled from more distant districts. Known drug resistance mutations were seen at increased frequency in northern districts (including two infections carrying pfk13‐R561H), and additional variants with undetermined significance for antimalarial resistance also varied by geography. Malaria Indicator Survey (2017) data corresponded with genetic findings, including average region‐level complexity‐of‐infection and malaria prevalence estimates. The parasite populations identified here provide important information on extant spatial patterns of genetic diversity of Tanzanian parasites, to which future surveys of genetic relatedness can be compared.     

Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population

Avian malaria parasites (Plasmodium) occur commonly in wild birds and are an increasingly popular model system for understanding host–parasite co‐evolution. However, whether these parasites have fitness consequences for hosts in endemic areas is much debated, particularly since wild‐caught individuals almost always harbour chronic infections of very low parasite density. We used the anti‐malarial drug Malarone^TM to test experimentally for fitness effects of chronic malaria infection in a wild population of breeding blue tits (Cyanistes caeruleus). Medication caused a pronounced reduction in Plasmodium infection intensity, usually resulting in complete clearance of these parasites from the blood, as revealed by quantitative PCR. Positive effects of medication on malaria‐infected birds were found at multiple stages during breeding, with medicated females showing higher hatching success, provisioning rates and fledging success compared to controls. Most strikingly, we found that treatment of maternal malaria infections strongly altered within‐family differences, with reduced inequality in hatching probability and fledging mass within broods reared by medicated females. These within‐brood effects appear to explain higher fledging success among medicated females and are consistent with a model of parental optimism in which smaller (marginal) offspring can be successfully raised to independence if additional resources become available during the breeding attempt. Overall, these results demonstrate that chronic avian malaria infections, far from being benign, can have significant effects on host fitness and may thus constitute an important selection pressure in wild bird populations.     

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.     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

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.     

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.     

Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within‐host competition with wild‐type drug‐sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC₅₀ values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild‐type pfcrt in co‐culture competition assays. These three alleles mediated cross‐resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first‐line artemisinin‐based combination therapy. These data reveal ongoing region‐specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine‐resistant malaria.     

A Population Genetic Model for the Initial Spread of Partially Resistant Malaria Parasites under Anti-Malarial Combination Therapy and Weak Intrahost Competition

To develop public-health policies that extend the lifespan of affordable anti-malarial drugs as effective treatment options, it is necessary to understand the evolutionary processes leading to the origin and spread of mutations conferring drug resistance in malarial parasites. We built a population-genetic model for the emergence of resistance under combination drug therapy. Reproductive cycles of parasites are specified by their absolute fitness determined by clinical parameters, thus coupling the evolutionary-genetic with population-dynamic processes. Initial mutations confer only partial drug-resistance. Therefore, mutant parasites rarely survive combination therapy and within-host competition is very weak among parasites. The model focuses on the early phase of such unsuccessful recurrent mutations. This ends in the rare event of mutants enriching in an infected individual from which the successful spread of resistance over the entire population is initiated. By computer simulations, the waiting time until the establishment of resistant parasites is analysed. Resistance spreads quickly following the first appearance of a host infected predominantly by mutant parasites. This occurs either through a rare transmission of a resistant parasite to an uninfected host or through a rare failure of drugs in removing “transient” mutant alleles. The emergence of resistance is delayed with lower mutation rate, earlier treatment, higher metabolic cost of resistance, longer duration of high drug dose, and higher drug efficacy causing a stronger reduction in the sensitive and resistant parasites’ fitnesses. Overall, contrary to other studies’ proposition, the current model based on absolute fitness suggests that aggressive drug treatment delays the emergence of drug resistance.     

Environmental variation alters the fitness effects of rifampicin resistance mutations in Pseudomonas aeruginosa

The fitness effects of antibiotic resistance mutations in antibiotic‐free conditions play a key role in determining the long‐term maintenance of resistance. Although resistance is usually associated with a cost, the impact of environmental variation on the cost of resistance is poorly understood. Here, we test the impact of heterogeneity in temperature and resource availability on the fitness effects of antibiotic resistance using strains of the pathogenic bacterium Pseudomonas aeruginosa carrying clinically important rifampicin resistance mutations. Although the rank order of fitness was generally maintained across environments, fitness effects relative to the wild type differed significantly. Changes in temperature had a profound impact on the fitness effects of resistance, whereas changes in carbon substrate had only a weak impact. This suggests that environmental heterogeneity may influence whether the costs of resistance are likely to be ameliorated by second‐site compensatory mutations or by reversion to wild‐type rpoB. Our results highlight the need to consider environmental heterogeneity and genotype‐by‐environment interactions for fitness in models of resistance evolution.     

On the contribution of the rodent model Plasmodium chabaudi for understanding the genetics of drug resistance in malaria

Malaria is a devastating disease that still claims over half a million lives every year, mostly in sub–Saharan Africa. One of the main barriers to malaria control is the evolution and propagation of drug-resistant mutant parasites. Knowing the genes and respective mutations responsible for drug resistance facilitates the design of drugs with novel modes of action and allows predicting and monitoring drug resistance in natural parasite populations in real-time. The best way to identify these mutations is to experimentally evolve resistance to the drug in question and then comparing the genomes of the drug-resistant mutants to that of the sensitive progenitor parasites. This simple evolutive concept was the starting point for the development of a paradigm over the years, based on the use of the rodent malaria parasite Plasmodium chabaudi to unravel the genetics of drug resistance in malaria. It involves the use of a cloned parasite isolate (P. chabaudi AS) whose genome is well characterized, to artificially select resistance to given drugs through serial passages in mice under slowly increasing drug pressure. The end resulting parasites are cloned and the genetic mutations are then discovered through Linkage Group Selection, a technique conceived by Prof. Richard Carter and his group, and/or Whole Genome Sequencing. The precise role of these mutations can then be interrogated in malaria parasites of humans through allelic replacement experiments and/or genotype-phenotype association studies in natural parasite populations. Using this paradigm, all the mutations underlying resistance to the most important antimalarial drugs were identified, most of which were pioneering and later shown to also play a role in drug resistance in natural infections of human malaria parasites. This supports the use of P. chabaudi a fast-track predictive model to identify candidate genetic markers of resistance to present and future antimalarial drugs and improving our understanding of the biology of resistance.     

Variation in costs of parasite resistance among natural host populations

Organisms that can resist parasitic infection often have lower fitness in the absence of parasites. These costs of resistance can mediate host evolution during parasite epidemics. For example, large epidemics will select for increased host resistance. In contrast, small epidemics (or no disease) can select for increased host susceptibility when costly resistance allows more susceptible hosts to outcompete their resistant counterparts. Despite their importance for evolution in host populations, costs of resistance (which are also known as resistance trade‐offs) have mainly been examined in laboratory‐based host–parasite systems. Very few examples come from field‐collected hosts. Furthermore, little is known about how resistance trade‐offs vary across natural populations. We addressed these gaps using the freshwater crustacean Daphnia dentifera and its natural yeast parasite, Metschnikowia bicuspidata. We found a cost of resistance in two of the five populations we studied – those with the most genetic variation in resistance and the smallest epidemics in the previous year. However, yeast epidemics in the current year did not alter slopes of these trade‐offs before and after epidemics. In contrast, the no‐cost populations showed little variation in resistance, possibly because large yeast epidemics eroded that variation in the previous year. Consequently, our results demonstrate variation in costs of resistance in wild host populations. This variation has important implications for host evolution during epidemics in nature.     

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Effects of antagonistic coevolution on parasite‐mediated host coexistence

Parasites can promote diversity by mediating coexistence between a poorer and superior competitor, if the superior competitor is more susceptible to parasitism. However, hosts and parasites frequently undergo antagonistic coevolution. This process may result in the accumulation of pleiotropic fitness costs associated with host resistance, and could breakdown coexistence. We experimentally investigated parasite‐mediated coexistence of two genotypes of the bacterium Pseudomonas fluorescens, where one genotype underwent coevolution with a parasite (a virulent bacteriophage), whereas the other genotype was resistant to the evolving phages at all time points, but a poorer competitor. In the absence of phages, the resistant genotype was rapidly driven extinct in all populations. In the presence of the phages, the resistant genotype persisted in four of six populations and eventually reached higher frequencies than the sensitive genotype. The coevolving genotype showed a reduction in the growth rate, consistent with a cost of resistance, which may be responsible for a decline in its relative fitness. These results demonstrate that the stability of parasite‐mediated coexistence of resistant and susceptible species or genotypes is likely to be affected if parasites and susceptible hosts coevolve.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

Adaptive immunity is essential in preventing recrudescence of Plasmodium yoelii malaria parasites after artesunate treatment

Artemisinin‐based antimalarials, such as artesunate (ART), alone or in combination, are the mainstay of the therapy against malaria caused by Plasmodium falciparum. However, the emergence and spread of artemisinin resistance threatens the future success of its global malaria eradication. Although much of the reported artemisinin resistance can be attributed to mutations intrinsic to the parasite, a significant proportion of treatment failures are thought to be due to other factors such as the host's immune system. Exactly how the immune system participates in the clearance and elimination of malaria parasites during ART treatment is unknown. Here, we show that a developing primary immune response, involving both B and CD4⁺ T cells, is necessary for the complete elimination but not initial clearance, of Plasmodium yoelii YM parasites in mice treated with ART. Our study uncovers a dynamic interplay between ART and host adaptive immunity in Plasmodium sp. elimination.     

Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites

Pyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR-TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine-resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant DHFR-TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of >2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine-resistant allele has not been observed in the field. It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by >3.7% per generation, necessitating careful attention to background in head-to-head competition studies. A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.     

Competitiveness and life‐history characteristics of Daphnia with respect to susceptibility to a bacterial pathogen

Costs of resistance, i.e. trade‐offs between resistance to parasites or pathogens and other fitness components, may prevent the fixation of resistant genotypes and therefore explain the maintenance of genetic polymorphism for resistance in the wild. Using two approaches, the cost of resistance to a sterilizing bacterial pathogen were tested for in the crustacean Daphnia magna. First, groups of susceptible and resistant hosts from each of four natural populations were compared in terms of their life‐history characteristics. Secondly, we examined the competitiveness of nine clones from one population for which more detailed information on genetic variation for resistance was known. In no case did the results show that competitiveness or life history characteristics of resistant Daphnia systematically differed from susceptible ones. These results suggest that costs of resistance are unlikely to explain the maintenance of genetic variation in D. magna populations. We discuss methods for measuring fitness and speculate on which genetic models of host‐parasite co‐evolution may apply to the Daphnia‐microparasite system.     

Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

Effect of haemosporidian infections on host survival and recapture rate in the blue tit

Parasites are ubiquitous in the wild and by imposing fitness costs on their hosts they constitute an important selection factor. One of the most common parasites of wild birds are Plasmodium and Haemoproteus, protozoans inhabiting the blood, which cause avian malaria and malaria‐like disease, respectively. Although they are expected to cause negative effects in infected individuals, in many cases studies in natural populations failed to detect such effect. Using data from seven breeding seasons (2008–2014), we applied a multistate capture–mark–recapture approach to study the effect of infection with malaria and malaria‐like parasites, individual age and sex on the probability of survival and recapture rate in a small passerine, the blue tit Cyanistes caeruleus, inhabiting the island of Gotland, Sweden. We found no effect of infection on survival prospects. However, the recapture rate of infected individuals was higher than that of uninfected ones. Thus, while our data do not support the presence of infection costs in terms of host survival, it suggests that parasites from the genera Plasmodium and Haemoproteus may affect some aspects of host behaviour, which translates into biased estimation of infection frequency at the population level.