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.

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.

The Plasmodium falciparum var gene switching rate, switching mechanism and patterns of parasite recrudescence described by mathematical modelling.

Recrudescing Plasmodium falciparum parasitaemia is attributed to the switching of PfEMP1, a variant antigen family encoded by the var gene repertoire, and the host's immune response. We have developed a mathematical model which incorporates var gene switching, and variant specific, non-variant specific and non-specific immunity. By conducting a sensitivity analysis of the model we have defined the parameter limits which produce chronic and recrudescing infections. We explore 3 switching mechanisms: ordered, random and uncoupled switching. We show that if var genes switch on and off independently at variable rates through the repertoire a chronic clinical infection is predicted. The fastest switching-on rate that produces a chronic infection is 0.03% per generation. The model predicts that non-variant specific immunity plays an important role in reducing disease severity. This work illustrates the complex relationship between the malaria parasite and its host and shows that var gene switching at rates substantially slower than 2% are essential for parasite survival.

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.

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.

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.

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.

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.

The emergence of drug-resistant malaria.

Stochastic processes play a vital role in the early stages of the evolution of drug-resistant malaria. We present a simple and flexible method for investigating these processes and understanding how they affect the emergence of drug-resistant malaria. Qualitatively different predictions can be made depending on the biological and epidemiological factors which prevail in the field. Intense intra-host competition between co-infecting clones, low numbers of genes required to encode resistance, and high drug usage all encourage the emergence of drug resistance. Drug-resistant forms present at the time drug application starts are less likely to survive than those which arise subsequently; survival of the former largely depends on how rapidly malaria population size stabilizes after drug application. In particular, whether resistance is more likely to emerge in areas of high or low transmission depends on malaria intra-host dynamics, the level of drug usage, the population regulation of malaria, and the number of genes required to encode resistance. These factors are discussed in relation to the practical implementation of drug control programmes.

Effects of thyroid hormone deficiency on mice selected for increased and decreased body weight and fatness.

A study was undertaken to test whether the elimination of metabolic pathways strongly involved in growth and fatness, comprising thyroid hormones (TH) and growth hormone (GH), is responsible for a substantial part of the genetic change produced by selection. Lines used in this study have been selected for about 50 generations for high (PH) and low (PL) body weight at 10 weeks and for high (F) and low fat content (L) at 14 weeks, producing a 3-fold difference in body weights and a 5-fold difference in fat content. Thyroid ablation was achieved by repeated backcrossing into the four selection lines of a transgene comprising the HSV1-tk gene coupled to the promoter of the thyroglobulin gene. Hemizygous pregnant dams were treated with ganciclovir leading to thyroid-ablated dams and offspring and therefore to a lack of TH and subsequently of GH. In the absence of TH and GH, lines still differ in body weight over the period studied (10 d to about 100 d; e.g. at the end PH = 32.1 g vs PL = 10.2 g) and in fat content (F = 16.2% vs L = 3.8%); the corresponding values for the wild-type controls were PH = 49.9 g vs PL = 17.4 g and F = 27.5% vs L = 4.8%. The effect of the transgene depended on the genetic background for body weights at most ages and for relative gonadal fat pad weights, but less for fat content. The L line showed the lowest growth depression. The lit gene, which causes GH but not TH deficiency, was also transferred by repeated backcrosses into three of these lines (PH, PL, F). The combined deficiency of TH and GH had bigger effects on body weights at earlier ages than did GH deprivation. The data show that changes in the TH- and GH-systems are not the only cause of line differences in growth and fatness resulting from long-term selection, but both are involved to a significant extent. The interactions between the effects of the transgene and of the lit gene and the genetic background were, nevertheless, relatively small and therefore these results support a polygenic model of selection response.

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.

Mutation and selection within the individual.

Selection within the individual may have played a critical and creative role in evolution, boosting the survival chances of mutations beneficial to the cell and the individual, hindering the spread of deleterious mutations, and reducing the genetic load imposed on the population. We review the literature and present new results to describe the effects of cell-lineage selection on the rate and fixation probability of new mutations. Cell-lineage selection can alter these quantities by several orders of magnitude. Cell-lineage selection is especially important in the case of rare recessive mutations, which are hidden from selection at the individual level but may be exposed to selection at cellular level. Because selection within the individual acts as a sieve eliminating deleterious mutations and increasing the frequency of beneficial ones, mutations observed among progeny will have been pre-selected and are more likely to increase cell proliferation than would randomly generated mutations. Although many authors have focused on the potential conflict between selection at the cellular and individual levels, it must be much more common that the two levels act concordantly. When selection at the cell and individual levels act in a cooperative manner, increased rather than decreased opportunity for germline selection will be favored by evolution.

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.

Effects of selection on food intake in the adult mouse.

SUMMARY: Replicated lines of mice were selected High and Low for adjusted food intake and contemporaneous control lines were maintained. The selection criterion was food intake between 8 and 10 weeks, adjusted by phenotypic regression on mean body weight at 8 and 10 weeks of age to reduce correlated changes in body weight. Responses are given for the first 23 generations of selection, after which adjusted food intake had diverged by a factor of 1.7-1.95. A small correlated response in body weight occurred and mice from the High line were slightly heavier: at 10 weeks of age body weight had diverged by a factor of 1.09-1.11. The realized within-family heritability varied between the replicates from 0.16-0.27 from which a mean estimated mass selection heritability (h(2) = 0.35±0.05) was derived. Mice from the Low line were fatter, however not significantly, because of a High between replicate variance (p > 0.05). Differences in growth over the selection period may account for around 5% of the divergence and increased maintenance costs associated with the larger lean mass of the high lines may explain a further 5%. Mice from the High lines spilled significantly (p < 0.05) more food which accounted for 23% of the divergence in apparent food intake. The heat increment of feeding, brown adipose tissue activity and locomotor activity all appear to be unchanged. ZUSAMMENFASSUNG: Auswirkungen der Selektion auf Futteraufnahme in der adulten Maus In einem Experiment mit Wiederholungen wurden Mäuselinien auf hohe und niedrige korrigierte Futteraufnahme selektiert und korrespondierende Konttrollen gehalten. Das Selektionskriterium war die Futteraufnahme im Alter von 8 bis 10 Wochen, die mittels phänotypischer Regression korrigiert wurde, um die Körpermasse möglichst konstant zu halten. Der direkte Selektionserfolg über die ersten 23 Generationen ist beschrieben. Die Linien divergierten zu diesem Zeitpunkt bezüglich des Selektionsmerkmals um 70 bis 95%. In der Körpermasse trat ein geringfügiger korrelierter Selektionserfolg auf. Die Tiere der 'high'-Linie waren im Alter von 8 bis Wochen ca. um 6 bis 11% schwerer. Die realisierte Intra-Familien-Heritabilität variierte zwischen den Wiederholungen zwischen 0.16 und 0.27, woraus sich eine mittlerer Heritabilitätskoeffizient von h(2) = 0.35±0.05 für die Massenselektion ergab. Mäuse der 'low'-Linie hatten mit 10 Wochen 2.4% (P > 0.05) und mit 17 Wochen ca. 7% (P < 0.05%) mehr Fett. Mit Unterschieden im Wachstum lassen sich weniger als 5% der Linienunterschiede in der Futteraufnahme erklären. Der höhere Erhaltungsbedarf, der aus einer höheren fett-freien Körpermasse in der 'high'-Linie resultiert, könnte weitere 5% erklären. Tiere der 'high'-Linie verstreuten deutlich (P < 0.05) mehr Futter, worauf sich 23% der Divergenz in der scheinbaren Futteraufnahme zurückführen ließen. Die Aktivät des braunen Fettgewebes, die lokomotorische Aktivität und die fütterungsbedingte Wàrmeproduktion sind scheinbar unverändert.

Sex, strains and virulence.

When are populations of infectious agents likely to evolve into distinct strains? Are they likely to differ in their virulence? Will genetically distinct strains or clones remain stable long enough to be useful as epidemiological markers? Sexual recombination can break down the genetic associations that define a strain structure, but if sex is rare or inbreeding is common, can strains persist? In this paper Ian Hastings and Bruce Wedgwood-Oppenheim show how some simple population genetic theory may provide a basis for addressing these questions.