Malaria associated anaemia,drug resistance and antimalarial combination therapy

Malaria associated anaemia represents a major cause of childhood mortality in sub-Saharan Africa. Prevention of severe anaemia necessitates rapid treatment of symptomatic high density parasitaemia, as well as reduction of asymptomatic parasite prevalence to provide recovery period to restore production of erythrocytes. Both interventions are being increasingly impaired by reduced efficacy of antimalarial treatment due to parasite drug resistance. A new treatment strategy, including combinations of antimalarial drugs with optimal pharmacodynamic and kinetic properties may respond to the need of rapid and radical parasite clearance, temporary protection to re-infection, and prevention of drug resistance.     

Standing genetic variation and the evolution of drug resistance in HIV

Drug resistance remains a major problem for the treatment of HIV. Resistance can occur due to mutations that were present before treatment starts or due to mutations that occur during treatment. The relative importance of these two sources is unknown. Resistance can also be transmitted between patients, but this process is not considered in the current study. We study three different situations in which HIV drug resistance may evolve: starting triple-drug therapy, treatment with a single dose of nevirapine and interruption of treatment. For each of these three cases good data are available from literature, which allows us to estimate the probability that resistance evolves from standing genetic variation. Depending on the treatment we find probabilities of the evolution of drug resistance due to standing genetic variation between 0 and 39%. For patients who start triple-drug combination therapy, we find that drug resistance evolves from standing genetic variation in approximately 6% of the patients. We use a population-dynamic and population-genetic model to understand the observations and to estimate important evolutionary parameters under the assumption that treatment failure is caused by the fixation of a single drug resistance mutation. We find that both the effective population size of the virus before treatment, and the fitness of the resistant mutant during treatment, are key-arameters which determine the probability that resistance evolves from standing genetic variation. Importantly, clinical data indicate that both of these parameters can be manipulated by the kind of treatment that is used.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic-producing strains, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Spread and evolution of Plasmodium falciparum drug resistance

Worldwide spread of Plasmodium falciparum drug resistance to conventional antimalarials, chloroquine and sulfadoxine/pyrimethamine, has been imposing a serious public health problem in many endemic regions. Recent discovery of drug resistance-associated genes, pfcrt, pfmdr1, dhfr, and dhps, and applications of microsatellite markers flanking the genes have revealed the evolution of parasite resistance to these antimalarials and the geographical spread of drug resistance. Here, we review our recent knowledge of the evolution and spread of parasite resistance to chloroquine and sulfadoxine/pyrimethamine. In both antimalarials, resistance appears to be largely explained by the invasion of limited resistant lineages to many endemic regions. However, multiple, indigenous evolutionary origins of resistant lineages have also been demonstrated. Further molecular evolutionary and population genetic approaches will greatly facilitate our understanding of the evolution and spread of parasite drug resistance, and will contribute to developing strategies for better control of malaria.     

Population dynamics and the evolution of antifungal drug resistance in Candida albicans

Candida albicans is an important human fungal pathogen. Resistance to all major antifungal agents has been observed in clinical isolates of Candida spp. and is a major clinical challenge. The rise and expansion of drug-resistant mutants during exposure to antifungal agents occurs through a process of adaptive evolution, with potentially complex population dynamics. Understanding the population dynamics during the emergence of drug resistance is important for determining the fundamental principles of how fungal pathogens evolve for resistance. While few detailed reports that focus on the population dynamics of C. albicans currently exist, several important features on the population structure and adaptive landscape can be elucidated from existing evolutionary studies in in vivo and in vitro systems.     

Modeling within-host HIV-1 dynamics and the evolution of drug resistance: trade-offs between viral enzyme function and drug susceptibility

There are many biological steps between viral infection of CD4(+) T cells and the production of HIV-1 virions. Here we incorporate an eclipse phase, representing the stage in which infected T cells have not started to produce new virus, into a simple HIV-1 model. Model calculations suggest that the quicker infected T cells progress from the eclipse stage to the productively infected stage, the more likely that a viral strain will persist. Long-term treatment effectiveness of antiretroviral drugs is often hindered by the frequent emergence of drug resistant virus during therapy. We link drug resistance to both the rate of progression of the eclipse phase and the rate of viral production of the resistant strain, and explore how the resistant strain could evolve to maximize its within-host viral fitness. We obtained the optimal progression rate and the optimal viral production rate, which maximize the fitness of a drug resistant strain in the presence of drugs. We show that the window of opportunity for invasion of drug resistant strains is widened for a higher level of drug efficacy provided that the treatment is not potent enough to eradicate both the sensitive and resistant virus.     

The evolution of host resistance: tolerance and control as distinct strategies

In response to parasitic infection, hosts may evolve defences that reduce the deleterious effects on survivorship. This may be interpreted as a form of resistance, as long as infected hosts are able to either recover or reproduce. Here we distinguish two important routes to this form of resistance. An infected host may either: (1) tolerate pathogen damage, or (2) control the pathogen by inhibiting its growth. A model is constructed to examine the evolutionary dynamics of tolerance and control to a free-living microparasite, where both forms of resistance are costly in terms of other life-history traits. We do not observe polymorphism of tolerant genotypes. In contrast, the evolution of control may lead to disruptive selection, and ultimately dimorphism of extreme strains. The optimal host genotype also varies with the type of resistance-individuals invest more in tolerance and pay a greater cost. The free-living framework used makes the distinction between tolerance and control explicit but the distinction applies equally to directly transmitted parasites. Due to the evolutionary differences exhibited, it is important to design experiments that distinguish between the two forms of resistance.     

Malaria control: achievements, problems and strategies

Even if history has not always been the Magistra vitae, Cicero expected it to be, it should provide, as Baas said, a mirror in which to observe and compare the past and present in order to draw therefrom well-grounded conclusions for the future. Based on this belief, this paper aims to provide an overview of the foundations and development of malaria control policies during the XX century. It presents an analysis of the conflicting tendencies which shaped the development of these policies and which appear to have oscillated between calls for frontal attack in an all-out campaign and calls for sustainable gains, even if slow. It discusses the various approaches to the control of malaria, their achievements and their limitations, not only to serve as a background to understand better the foundations of current policies, but also to prevent that simplistic generalisations may again lead to exaggerated expectations and disillusion. The first part of the paper is devoted to the development of malaria control during the first half of the century, characterised by the ups and downs in the reliance on mosquito control as the control measure applicable everywhere. The proliferation of "man-made-malaria", which accompanied the push for economic development in most of the endemic countries, spurred the need for control interventions and, while great successes were obtained in many specific projects, the general campaigns proposed by the enthusiasts of vector control faced increasing difficulties in their practical implementation in the field. Important events, which may be considered representative of this period are, on the campaign approach, the success of Gorgas in the Panama Canal, but also the failure of the Mian Mir project in India; while on the developmental approach, the Italian and Dutch schools of malariology, the Tennessee Valley and the development of malaria sanitation, included the so called species sanitation. The projection of these developments to a global scale was steered by the Malaria Commission of the League of Nations and greatly supported by the Rockefeller Foundation. Perhaps the most important contribution of this period was the development of malaria epidemiology, including the study of the genesis of epidemics and their possible forecasting and prevention. Although the great effectiveness of DDT was perhaps the main determinant for proposing the global eradication of the disease in the 1950s, it was the confidence in the epidemiological knowledge and the prestige of malariology, which gave credibility to the proposal at the political level. The second part deals with the global malaria eradication campaign of the 1950s and 1960s. It recognises the enormous impact of the eradication effort in the consolidation of the control successes of the first half of the century, as well as its influence in the development of planning of health programmes. Nevertheless, it also stresses the negative influence that the failure to achieve its utopian expectations had on the general disappointment and slow progress of malaria control, which characterised the last third of the century. The paper then analyses the evolution of malaria control funding, which often appears out of tune with political statements. The fourth part is devoted to the search for realistic approaches to malaria control, leading to the adoption of the global malaria control strategy in Amsterdam in 1992, and the challenge, at the end of the century, to rally forces commensurate with the magnitude of the problem, while aiming at realistic objectives. After discussing the conflicting views on the relations between malaria and socio-economic development and the desirable integration of malaria control into sustainable development, the paper ends with some considerations on the perspectives of malaria control, as seen by the author in early 1998, just before the launching of the current Roll Back Malaria initiative by WHO.     

Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.     

Malaria control priorities and constraints

Each year, there are still between 300-500 million clinical cases of malaria and over one million deaths due to the disease, 90% of which occur in Africa south of the Sahara. In all continents, malaria risk is highest in remote rural areas where poverty abounds, population densities are low and the quality and coverage of the health services are poor or in existent. A sustained impact on the malaria burden can only be achieved through the cost-effective use of current tools, by including malaria in health sector development and inter-sectorial action, by mobilizing malaria control within communities and by investing in new and more effective tools. This paper highlights some of the constraints faced by countries in controlling malaria and outlines the priority activities that are being carried out to address these constraints within both communities and the health services. It aims to be set the scene for the papers of this Centenary book which address some of these issues in more detail.     

Emergence of drug resistance: implications for antiviral control of pandemic influenza

Given the danger of an unprecedented spread of the highly pathogenic avian influenza strain H5N1 in humans, and great challenges to the development of an effective influenza vaccine, antiviral drugs will probably play a pivotal role in combating a novel pandemic strain. A critical limitation to the use of these drugs is the evolution of highly transmissible drug-resistant viral mutants. Here, we develop a mathematical model to evaluate the potential impact of an antiviral treatment strategy on the emergence of drug resistance and containment of a pandemic. The results show that elimination of the wild-type strain depends crucially on both the early onset of treatment in indexed cases and population-level treatment. Given the probable delay of 0.5-1 day in seeking healthcare and therefore initiating therapy, the findings indicate that a single strategy of antiviral treatment will be unsuccessful at controlling the spread of disease if the reproduction number of the wild-type strain (R0s) exceeds 1.4. We demonstrate the possible occurrence of a self-sustaining epidemic of resistant strain, in terms of its transmission fitness relative to the wild-type, and the reproduction number R0s. Considering reproduction numbers estimated for the past three pandemics, the findings suggest that an uncontrollable pandemic is likely to occur if resistant viruses with relative transmission fitness above 0.4 emerge. While an antiviral strategy is crucial for containing a pandemic, its effectiveness depends critically on timely and strategic use of drugs.     

Malaria vector analysis and control

Antivector measures in malaria control should aim for a cost-effective reduction of the transmission potential ideally to below the critical level for sustained transmission. The available measures include those that decrease vector abundance, vector-human contact and vector survival rate or that increase the length of the sporogonic cycle. These have widely different impact on malaria transmission, as shown by epidemiological modelling. Direct modification of vector receptivity to Plasmodium is also hypothetically attainable by the use of transmission-blocking vaccines or by genetic manipulation and replacement of the vector population. Vector analysis constitutes the essential prerequisite for basic malaria epidemiology as well as for the development, planning and evaluation of antivector measures. The rationale, the problems and the perspectives of vector analysis are reviewed here by Mario Coluzzi, on the basis of his experience with Afrotropical and Mediterranean malaria vectors.     

Antiviral resistance and the control of pandemic influenza: the roles of stochasticity, evolution and model details

Antiviral drugs, most notably the neuraminidase inhibitors, are an important component of control strategies aimed to prevent or limit any future influenza pandemic. The potential large-scale use of antiviral drugs brings with it the danger of drug resistance evolution. A number of recent studies have shown that the emergence of drug-resistant influenza could undermine the usefulness of antiviral drugs for the control of an epidemic or pandemic outbreak. While these studies have provided important insights, the inherently stochastic nature of resistance generation and spread, as well as the potential for ongoing evolution of the resistant strain have not been fully addressed. Here, we study a stochastic model of drug resistance emergence and consecutive evolution of the resistant strain in response to antiviral control during an influenza pandemic. We find that taking into consideration the ongoing evolution of the resistant strain does not increase the probability of resistance emergence; however, it increases the total number of infecteds if a resistant outbreak occurs. Our study further shows that taking stochasticity into account leads to results that can differ from deterministic models. Specifically, we find that rapid and strong control cannot only contain a drug sensitive outbreak, it can also prevent a resistant outbreak from occurring. We find that the best control strategy is early intervention heavily based on prophylaxis at a level that leads to outbreak containment. If containment is not possible, mitigation works best at intermediate levels of antiviral control. Finally, we show that the results are not very sensitive to the way resistance generation is modeled.     

Occurrence and properties of composite transposon Tn2672: evolution of multiple drug resistance transposons.

We found Tn2671 (the 23-kb long IS1-flanked r-determinant of NR1-Basel) inserted into the ampicillin resistance gene bla of the Tn3-related transposon Tn902. The resulting 28-kilobase-long composite transposon Tn2672 (= Tn902 bla::Tn2671) is stable, and it translocates as a unit into various loci including IS1 of the resistance transfer factor of R100-1. These results are discussed with respect to the evolution of R plasmids providing multiple drug resistance.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.