Improving Methods for Analyzing Antimalarial Drug Efficacy Trials: Molecular Correction Based on Length-Polymorphic Markers msp-1, msp-2, and glurp.

Drug efficacy trials monitor the continued efficacy of front-line drugs against falciparum malaria. Overestimating efficacy results in a country retaining a failing drug as first-line treatment with associated increases in morbidity and mortality, while underestimating drug effectiveness leads to removal of an effective treatment with substantial practical and economic implications. Trials are challenging: they require long durations of follow-up to detect drug failures, and patients are frequently reinfected during that period. Molecular correction based on parasite genotypes distinguishes reinfections from drug failures to ensure the accuracy of failure rate estimates. Several molecular correction "algorithms" have been proposed, but which is most accurate and/or robust remains unknown. We used pharmacological modeling to simulate parasite dynamics and genetic signals that occur in patients enrolled in malaria drug clinical trials. We compared estimates of treatment failure obtained from a selection of proposed molecular correction algorithms against the known "true" failure rate in the model. Our findings are as follows. (i) Molecular correction is essential to avoid substantial overestimates of drug failure rates. (ii) The current WHO-recommended algorithm consistently underestimates the true failure rate. (iii) Newly proposed algorithms produce more accurate failure rate estimates; the most accurate algorithm depends on the choice of drug, trial follow-up length, and transmission intensity. (iv) Long durations of patient follow-up may be counterproductive; large numbers of new infections accumulate and may be misclassified, overestimating drug failure rate. (v) Our model was highly consistent with existing in vivo data. The current WHO-recommended method for molecular correction and analysis of clinical trials should be reevaluated and updated.

Laboratory risk factors for hospital mortality in acutely admitted patients.

BACKGROUND: Many factors affecting hospital mortality in acutely admitted patients are poorly understood. Although scoring systems exist for critically ill patients, usually in intensive care units (ICUs), there are no specific mortality prediction systems for general acute admissions. AIM: To assess the relationship between simple admission laboratory variables on the risk of in-patient mortality. DESIGN: Retrospective analysis of hospital admissions and laboratory databases. METHODS: Where possible, all deceased patients in the 12-month period of study were matched with two surviving controls. The laboratory database was then analysed for admission investigations, including serum sodium, plasma glucose, and white blood cell (WCC) count. Abnormalities of these variables were then compared between cases (those who subsequently died), and controls (those who survived). RESULTS: There were 16 219 admissions, with an overall mortality of 7.6%. We investigated 602 cases and 1073 controls. Hyperglycaemia (glucose >11.0 mmol/l) (OR 2.0, p < 0.0001); severe hyponatraemia (sodium <125 mmol/l) (OR 4.0, p < 0.0001); and leukocytosis (WCC >10 x 10(9)/l) (OR 2.0, p < 0.001) were significantly associated with mortality. The respective associations on logistic regression analysis were: glucose, OR 1.7, p = 0.02; sodium, OR 4.4, p < 0.0001; WCC, OR 1.5, p = 0.006. Low glucose levels, high sodium levels, and low WCC levels were also associated with increased mortality, leading to 'U-shaped' mortality associations. The effect of more than one laboratory abnormality being present was cumulative, in a linear fashion. DISCUSSION: Plasma glucose, serum sodium and WCC are measured in most acutely admitted patients, and abnormalities of these variables have associations with in-hospital mortality. This may provide the basis for the development of a mortality risk scoring system.

Increased postpartum blood loss in pregnancies associated with placental malaria.

OBJECTIVE: To determine the relationship of placental malaria and parity with postpartum blood loss in a malarious area of Tanzania. METHODS: A total of 706 uncomplicated vaginal deliveries were studied at Muhimbili University Hospital, Dar es Salaam, Tanzania. Maternal age, parity, date of delivery, birth weight, presence of placental malaria, stillbirths, and delivery complications were noted. Collection and measurement of vaginal blood loss commenced immediately following birth using a plastic vinyl sheet placed underneath the mother. The bed was divided in the middle to allow the blood to drain into a bucket. Blood loss was measured for a period of 2 h following delivery. RESULTS: In singleton births the mean postpartum blood loss was 170 mL in nulliparas and 187 mL in multiparas (p=0.017). Blood loss was 400 mL or greater in 23 women (3.4%) and 500 mL or greater in 10 women (1.5%). Mean postpartum bleeding tended to increase with maternal age, parity, and birth weight. In logistic regression the odds ratio for a blood loss of 400 mL or greater was significantly increased for women with placental malaria (3.2; 95% confidence interval, 1.1-9.0; p=0.028), after adjusting for a birth weight greater than 4000 g. Placental malaria showed a marked seasonal pattern, which corresponded to the months of peak prevalence for a postpartum blood loss of 400 mL or greater (p=0.007). CONCLUSION: A postpartum blood loss of 400 mL or greater should be considered a possible complication of placental malaria.

Germline selection: population genetic aspects of the sexual/asexual life cycle.

Population geneticists make a distinction between sexual and asexual organisms depending on whether individuals inherit genes from one or two parents. When individual genes are considered, this distinction becomes less satisfactory for multicellular sexual organisms. Individual genes pass through numerous asexual mitotic cell divisions in the germline prior to meiosis and sexual recombination. The processes of mitotic mutation, mitotic crossing over, and mitotic gene conversion create genotypic diversity between diploid cells in the germline. Genes expressed in the germline whose products affect cell viability (such as many "housekeeping" enzymes) may be subjected to natural selection acting on this variability resulting in a non-Mendelian output of gametes. Such genes will be governed by the population genetics of the sexual/asexual life cycle rather than the conventional sexual/Mendelian life cycle. A model is developed to investigate some properties of the sexual/asexual life cycle. When appropriate parameter values were included in the model, it was found that mutation rates per locus per gamete may vary by a factor of up to 100 if selection acts in the germline. Sexual/asexual populations appear able to evolve to a genotype of higher fitness despite intervening genotypes of lower fitness, reducing the problems of underdominance and Wright's adaptive landscape encountered by purely sexual populations. As might be expected this ability is chiefly determined by the number of asexual mitotic cell divisions within the germline. The evolutionary consequences of "housekeeping" loci being governed by the dynamics of the sexual/asexual life cycle are considered.

Potential germline competition in animals and its evolutionary implications.

Mutation, mitotic crossing over and mitotic gene conversion can create genetic diversity in otherwise uniform diploid cell lineages. In the germline this diversification may result in competition between diploid germline phenotypes, with subsequent biases in the frequency of alleles transmitted to the offspring. Sperm competition is a well documented feature of many higher organisms and a model is developed to quantify this process. Competition, and hence selection, can also occur by differential survival of diploid lineages before meiosis. It is concluded that under certain circumstances germline selection is an efficient means of eliminating unfavorable alleles from the population. This does not require differences in adult fertility or viability which is the usual mechanism cited as causing changes in gene frequency in a population. It is proposed that such competition may play a role in maintaining the efficiency of basic metabolic pathways.

Monoclonal antibodies to human hypoglossal nucleus which stain neurons and astrocytes in normal brains and brains from cases of Alzheimer-type dementia.

Monoclonal antibodies were raised to membranes of hypoglossal nuclei from normal human post-mortem brain. Two of these clones were recloned to yield antibodies ES.18 and ES.19. Antibody ES.18 stained some, but not all, neuronal perikarya in the medulla oblongata and other brain areas. Neurons stained by this antibody did not have a common neurotransmitter or physiological function, although they tended to be large. Perikarya in the basal forebrain nucleus from a case of Alzheimer-type dementia were stained much more intensely by ES.18 than were these perikarya in a control brain. Antibody ES.19 did not stain neuronal perikarya but stained glial fibrillary acidic protein-positive processes below the pia, in the subependymal layer and in the molecular layer of the cerebellum of control and Alzheimer brains. This antibody also stained the numerous glial fibrillary acidic protein-positive astrocytes in Alzheimer cerebral cortex, but did not stain glial fibrillary acidic protein-positive astrocytes in the white matter of brains from controls or cases of Alzheimer-type dementia. The staining pattern of ES.19 suggests that fibrous astrocytes in Alzheimer cerebral cortex are antigenically different from fibrous astrocytes in white matter.

Manifestations of sexual selection may depend on the genetic bases of sex determination.

A variant of the 'handicap' model of sexual selection is described which predicts that the evolution of ornate male traits occurs more easily in species where females are the heterogametic sex. The process occurs even when the alleles conferring high paternal 'fitness' remain advantageous for only a short time due to a rapidly changing physical or biotic environment: the timescale of this advantage may approach the gestation time of the organism. This provides an explanation as to why sexual selection in species where females are heterogametic (such as birds) occurs mainly by the elaboration of ornate male secondary sexual characteristics, whereas in species where females are homogametic (such as mammals) sexual selection results predominantly in inter-male rivalry and the evolution of traits such as horns, antlers and large body size. An analogy between the evolution of elaborate male traits and the evolution of warning coloration is noted.

The evolution of multiple drug resistance in malaria parasites.

Forces determining the rate of spread of drug resistance in malaria were explored using a genetics transmission model which took account of the strong population structure of these parasites. The rate of change of frequency of drug resistant mutants in the parasite population is primarily a function of the proportion of hosts treated with drugs, and parasite transmission rates. With high transmission rates, selection by drugs is more effective than with lower rates because the resistant mutant passes on more copies of itself to the next generation of hosts. Thus reducing transmission rates, either at the overall population level or from drug-treated individuals, should be effective in curbing the spread of resistance. An exception to this is when 2 unlinked genes act jointly (not independently) to confer resistance, when the prevailing transmission rate is already low, drug use is minimal, and resistance genes are rare. Reductions in fitness of the mutant in the absence of drugs (i.e., a fitness cost to resistance) and the degree of epistasis and the mode of gene action of the drugs do not alter these conclusions.

American Society of Tropical Medicine and Hygiene--49th annual meeting.

This broad-based meeting covered all areas of tropical medicine, from basic science to clinical aspects, and focused on the perspectives from both Western travelers and public health strategies in developing countries. There was considerable interest in the development, assessment and implementation of antimalarial drug treatments, ranging from the most effective way to use existing compounds, to the strategic arguments associated with the desirability, feasibility and implementation of combination chemotherapy. Finally, attention was focused on the use of molecular markers to track and predict the emergence of antimicrobial drug resistance. Abstracts can be viewed on the ASTMH website, www.astmh.org, or obtained as a supplement to the American Journal of Tropical Medicine and Hygiene.

Complex dynamics and stability of resistance to antimalarial drugs.

A succession of antimalarial drugs has been deployed to treat human falciparum malaria but each has, in turn, been nullified by the spread of drug resistance. The consensus view has always been that, once present, resistance will inevitably rapidly increase to 100%. However, recent field evidence has shown this is not inevitable, and that drug resistance may initially spread and then stabilize at relatively low frequencies. It is proposed that intense competition between separate malaria clones co-infecting the same human can generate complex dynamics capable of explaining this observation. Standard population genetic analysis confirms this assertion. The dynamics underlying the evolution of antimalarial resistance may therefore be much more complex than previously realized, and can resolve the apparent paradox between field data and the underlying theory of the evolution of resistance. This explanation is novel and the results are equally applicable to other parasitic species where multiple infections of the same host are common.

Population genetic aspects of deleterious cytoplasmic genomes and their effect on the evolution of sexual reproduction.

A conflict of interest may arise between intra-cellular genomes and their host cell. The example explicitly investigated is that of a 'selfish' mitochondrion which increases its own rate of replication at the cost of reduced metabolic activity which is deleterious to the host cell. The results apply to deleterious cytoplasmic agents in general, such as intracellular parasites. Numerical simulation suggests that selfish mitochondria are able to invade an isogamous sexual population and are capable of reducing its fitness to below 5% of that prior to their invasion. Their spread is enhanced by decreasing the number of mitotic divisions between meioses, and this may constitute a significant constraint on the evolution of lifecycles. The presence of such deleterious cytoplasmic agents favours a nuclear mutation whose expression prevents cytoplasm from the other gamete entering the zygote at fertilization, resulting in uniparental inheritance of cytoplasm. Such a mutation appears physiologically plausible and can increase in frequency despite its deleterious effect in halving the amount of cytoplasm in the zygote. It is suggested that these were the conditions under which anisogamy evolved. These results have implications for the evolution of sexual reproduction. Standard theory suggests there is no immediate cost of sex, a twofold cost being incurred later as anisogamy evolves. The analysis described here predicts a large, rapid reduction in fitness associated with isogamous sexual reproduction, due to the spread of deleterious cytoplasmic agents with fitness only subsequently rising to a maximum twofold cost as uniparental inheritance of cytoplasm and anisogamy evolve.

The population genetics of alleles affecting enzyme activity.

It is possible to predict the population genetics of allozymes by assuming that fitness is proportional to flux through a biochemical pathway. The model presented here extends previous work by incorporating two additional features of biological realism. Firstly, that more than one biochemical route may exist between any two metabolites. The major routes have been identified as the classical biochemical pathways but in the event of a mutation blocking a major route, minor routes become significant. These minor routes are named "bypass fluxes" and have profound effects on the population genetics of allozymes. Secondly, recent work has suggested that a metabolic cost is associated with enzyme synthesis; this will constitute an additional selective pressure on alleles which affect the amount of enzyme synthesized. The model generates a fitness curve which predicts the fitness associated with any level of enzyme activity. It can utilize data on null or near-null, structural or regulatory, mutations in the presence or absence of bypass fluxes. When data from natural populations of Drosophila are investigated, it is concluded that selection pressures acting on enzyme variants may be much higher than previously thought.

Population genetics and the detection of immunogenic and drug-resistant loci in Plasmodium.

Host immunity will result in increased homozygosity at immunogenic loci if they encode products that elicit allele-specific immune responses. This increased homozygosity can be detected by comparison to 'neutral' (i.e. unselected) loci in the same genome which act as controls for the various epidemiological factors, such as biting rate, which affect homozygosity. Numerical results suggest that homozygosity should be 20 to 500% higher at immunogenic loci, a result which is robust to changes in host death rate and the degree of linkage between the immunogenic and neutral loci. The same logic applies to loci which encode drug resistance: treated individuals with resistant infections will transmit zygotes homozygous for the resistance allele. It is argued that this increased homozygosity at putative immunogenic loci can act as a diagnostic feature to test using field data. The method also serves as a potentially very powerful method of identifying immunogenic and drug-resistant loci in laboratory studies of species such as the murine malaria Plasmodium chabaudi.

Models of human genetic disease: how biased are the standard formulae?

Standard theory provides a simple prediction for the frequency of a recessive lethal allele conferring heterozygous protection against an infectious disease (the best-known example being sickle cell protection against malaria). This relationship allows historic disease mortality rates to be estimated. There are, however, hidden biases in this approach. Reproductively active human females in archaic societies normally produce children at intervals of around 4 years. If death of the fetus or young infant (less than around 3 years of age) occurs, then the mother re-enters oestrus and produces another child. This 'reproductive compensation' reduces selection against the agent causing early mortality (the recessive allele or infective agent) and biases our estimates of historic mortality rates. The magnitude of these biases is investigated. Re-conception also constitutes a demographic selective pressure acting alongside natural selection: lethal genetic diseases (or tightly linked loci) will be selected to become ever more virulent, killing at ever decreasing ages, to allow the mother to re-enter oestrus and re-conceive a (hopefully unaffected) sibling; this effect also invalidates statistical tests using the number of alleles to distinguish overdominance from drift as explanations for high allele frequency. The same bias affects calculations of mutation/selection balance: for any given mutation rate, syndromes which kill early in life will reach much higher frequencies than those killing at later ages. An intriguing consequence is that lethal recessive disorders in humans will increase in frequency by up to 45% as a consequence of the recent demographic transition to planned family size.

Selfish DNA as a method of pest control.

The inheritance of most genes is tightly controlled, governed by the rules of mendelian inheritance if nuclear or uniparental inheritance if cytoplasmic. A few notable genes and cytoplasmic genomes have escaped this regulation. Such genes may spread by increasing their own rate of transmission despite reducing host fitness and may be regarded as 'selfish'. Their population genetics are described and it appears they may impose a significant genetic load on the host population. Modern molecular techniques may enable similar loads to be imposed on pest species either by transferring selfish genes between species, or by linking deleterious genes to a selfish locus. Alternatively, 'modifier' genes that eliminate the virulent, or disease vectorial capacity, of the pest population may be introduced by linkage to a selfish locus. Selfish elements present in multiple copies may be preferable to single-copy elements as the former are capable of a larger reduction in host fitness. The practical application of these agents depends on five factors: (i) the rate of 'reversion' to a non-selfish form; (ii) the evolution of host repressor systems; (iii) their effect on host fitness, which determines their rate of invasion; (iv) the mechanism regulating host population size in the field; and (v) their ease of manipulation in the laboratory. The first two factors are the most uncertain in most systems, but should be amenable to experimental analysis. It is proposed that the development of such techniques may result in powerful new methods of population control which may be applied to both agricultural pests and disease vectors.

A model for the origins and spread of drug-resistant malaria.

A general method of investigating parasite population genetics is presented and used to investigate the evolution of drug resistance in Plasmodium. The most important biological factor is the nature of the control, presumably through host immunity, of the malarial infection. Two models are examined: a 'generalized immunity' (GI) model in which immunity regulates the overall level of infection, and a 'specific immunity' (SI) model in which each clone within the infection is regulated independently. These models are used to investigate 3 critical factors in the evolution of resistance: (i) the frequency of resistant alleles in the population prior to drug use, (ii) the dynamics of resistance following drug application and (iii) the magnitude of threshold frequencies below which resistance will not evolve. These analyses also identify the implicit assumptions made in several previous models, reconcile their differing conclusions and allow a more informed debate about the practical application of drugs.

Reproductive compensation and human genetic disease.

The effects of reproductive compensation on the population genetics of sex-linked recessive lethal mutations are investigated. Simple equations are presented which describe these effects, and so complement existing population genetic theory. More importantly, this type of mutation is responsible for several severe human genetic diseases such as Duchenne muscular dystrophy. It is argued that the applications of three modern reproductive technologies--effective family planning, in utero diagnosis with termination, and embryo sexing--will lead to reproductive compensation. The adoption of any of these technologies may rapidly elevate the frequencies of those mutations which are lethal in childhood. This increase is large, in the order of 33% upwards, and occurs rapidly over two to five generations. It also depends on the source of mutations, the effect being larger if most mutations are paternal. In utero diagnosis and/or embryo sexing increase the frequency of the mutation, but simultaneously decrease disease incidence by preventing the birth of affected offspring. In contrast, effective family planning may rapidly increase both mutation frequency and disease incidence.

Modelling parasite drug resistance: lessons for management and control strategies.

Mathematical models of the evolution of drug resistance in infectious diseases are predominantly concentrated in three main areas: antimalarial, antibiotic and anthelmintic resistance. There appears to be little or no cross-reference between them. This literature was examined to identify factors that influence the evolution of drug resistance irrespective of the species and drug under study. The aim is to provide non-technical readers with a basic qualitative understanding of the issues and pitfalls involved in designing drug treatment regimens to minimize the evolution of resistance. The principal factors determining the rate at which resistance evolves appear to be (i) the starting frequency of resistance, (ii) the level and pattern of drug use, (iii) the drug's pharmacokinetic properties, (iv) the number of genes required to encode resistance, (v) the level of sexual recombination in the parasite population, (vi) intrahost dynamics and, in particular, whether 'crowding' effects are present, (vii) the genetic basis of resistance and (viii) the number of individual parasites in an infection. The relative importance of these factors depends on the biology of the organisms under consideration and external factors such as the extent to which the infrastructure of health care delivery constrains the practicalities of drug regimens.

Modelling a predictable disaster: the rise and spread of drug-resistantmalaria.

The evolution of drug-resistant malaria is one of the most important factors thwarting the development of effective malaria disease control. Several mathematical models have been developed to try and understand the dynamics of this process and how it can be slowed or even avoided. Much of the mathematics describing the evolution of drug resistance in malaria focuses on the derivation and mechanics of the calculations, which can make it inaccessible to experimentalists and field workers. In this article, Ian Hastings and Umberto D'Alessandro describe general model results without recourse to mathematical details, identify the factors that should be considered in the design of drug control programmes, and discuss the crucial parameters that remain unknown and need to be measured in the field or laboratory.