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Evolutionary and Historical Aspects of the Burden of Malaria

University of Edinburgh, Division of Biological Sciences, ICAPB, Ashworth Laboratories, Edinburgh EH9 3JT, United Kingdom,¹ Roll Back Malaria Project, World Health Organization, Geneva 27, Switzerland²

SUMMARY

INTRODUCTION

MALARIA PARASITES OF HUMANS

EPIDEMIOLOGY OF THE BURDEN OF HUMAN MALARIA

Disease States of Malaria

Immunity to Malaria

Malarial Endemicity

How Endemicity and Immunity Interact To Determine the Effects of Malaria

Stable malaria.

Unstable and epidemic malaria.

Biological Basis of Stable, Unstable, and Epidemic Malaria

Indirect Mortality of Malaria

Nonlethal Health Consequences of Endemic Malaria

Effects of Malaria on Social and Economic Development

THE ''MALARIA HYPOTHESIS'' AND RECENT HUMAN EVOLUTION

Thalassemias

Glucose-6-Phosphate Dehydrogenase Deficiency

Sickle Cell Trait

Hemoglobin C

Hemoglobin E

Ovalocytosis

RBC Duffy Negativity

Some Points from the Malaria Hypothesis

How long does it take malaria to select for human polymorphisms?

Did malaria enter most human populations before or after their global dispersal?

Legacy of the impact of malaria on human populations.

ORIGINS AND EVOLUTION OF HUMAN MALARIA PARASITES

Origins of Malaria Parasites

Origins of Human Malaria Parasites

EXPANSIONS AND DISPERSALS OF HUMAN MALARIA

Out of Africa

The Agrarian Revolution in Africa

Adaptations of the vectors of malaria in Africa.

Parasite adaptations before and following the agrarian revolution.

(i) P. malariae, the ''gypsy'' parasite.

(ii) P. vivax, the great survivor, and P. ovale, the ''hothouse plant.''

(iii) P. falciparum, the parasite that ''did not exist''.

Beyond Africa

In the Old World.

In the New World.

In the Western Pacific.

MALARIA IN HISTORICAL TIMES

From the ''Dawn of History'' to the Nineteenth Century

In the Twentieth Century

Malaria in Europe and the Americas.

Malaria in Asia and the Western Pacific.

Malaria in Africa.

Death from Malaria in the Twentieth Century

The first half of the twentieth century.

The second half of the twentieth century.

STRATEGIES TO MANAGE THE BURDEN OF MALARIA TODAY

Lessons from the Past

The Malaria Situation Today

From ''global eradication'' to global gloom.

The problem at hand.

Current Global Approach to Controlling Malaria

Treating the sick.

Reducing the risk of malaria.

Managing epidemic malaria.

Global role in malaria management.

CONCLUSIONS

APPENDIX

Sources of Data and Approaches to Their Analysis

Limits of Uncertainty of the Estimates of Malaria-Related Mortality

Country Composition of the Six Global Regions

Sources used.

(i) Malaria mortality.

(ii) World and regional population sizes.

ADDENDUM IN PROOF

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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This article is about the nature and impact of malaria; it is not a review of scientific research on malaria. It explores how, when, and where malaria parasites, their mosquito vectors, and humans, may have interacted and with what effect on ourselves, their human hosts. This is not a straightforward task. It is hampered by difficulties of collecting appropriate and reliable data and by problems of their interpretation. Inferences or conclusions may vary from well-founded and widely accepted to tentative or controversial. But, while the picture remains blurred in a number of places, the many faces and features of malaria and its imprint on the human species emerge, usually clearly and unmistakably.

Many published discussions have dealt with the experiences of malaria, its nature, and its effects (see, e.g., references 5, 28, 29, 31, 35, 42-44, 47, 73, 91, 95, 106, 111, 112, 117, 123, 124, 139, 141, 165, 167, 173, 176, 192, 195, 200). They are informative and revealing, especially because each is a product of the medical and human health context, general outlook, and knowledge base of its time. Our present times also offer a unique perspective on this subject. It is only within the past half century that we have experienced malaria against a background of human health and health services which are, for the most part, vastly better than in all previous generations. In the same period, we have witnessed both the achievements of the first globally coordinated health delivery programs and also their problems. Within recent decades, and especially the last, biochemical and molecular genetic technologies to investigate distant events in the evolution and coevolution of malaria parasites and humans have become available.

The following, therefore, is a brief reconstruction of the evolution and history of malaria and its burdens as we may perceive them at the start of the 21st century.

MALARIA PARASITES OF HUMANS

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Malaria is due to blood infection by protozoan parasites of the genus Plasmodium, which are transmitted from one human to another by female Anopheles mosquitoes. Four species of malaria parasite infect humans (Table 1). The two which almost certainly achieved the widest global distribution are Plasmodium vivax and Plasmodium malariae. To the Europeans, these have been known and characterized since historically ancient times (5, 29, 95, 106) as the "benign tertian" (P. vivax) and "quartan" (P. malariae) periodic fevers. "Benign tertian" fevers were so named because they were not associated with the severe and often fatal manifestations of the "subtertian, malignant" periodic fevers (P. falciparum). "Tertian" and "quartan" refers to their characteristic feature of an acute febrile episode, or paroxysm, that returns respectively every third (P. vivax) or fourth (P. malariae) day. Tertian and quartan fevers are referred to with similar frequency in writings from northern Europe through much of the past millennium and from around the shores of the Mediterranean Sea from about the 5th century B.C. onward (29, 47, 95, 106).

The fourth human malaria parasite is Plasmodium ovale, which, like P. vivax, is the agent of a tertian malaria and which, also like P. vivax malaria today, carries a very low risk of fatal outcome. P. ovale has the most limited distribution of all the malaria parasites of humans. While it is prevalent throughout most of sub-Saharan Africa, it is otherwise known to be endemic only in New Guinea and the Philippines (122).

EPIDEMIOLOGY OF THE BURDEN OF HUMAN MALARIA

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Before we attempt to trace the passage and effects of malaria through evolutionary and historical time, we will review the principles which determine how malaria affects individuals and populations.

Repeated attacks of malaria due to any species of the parasites over several to many years severely debilitate body and mind. Cachexia, a wasting of body tissues, takes place, and splenic enlargement becomes a constant feature. Lethargic and with sunken and sallow features, spindly limbs, and hard swollen belly is the general description of the condition. In this state the affected individual succumbs to diseases or other hardships that would scarcely threaten a person in reasonable health. Under the burden of chronic malaria, both the quality and duration of life are greatly reduced.

An individual's experience of malaria at a particular time is, however, strongly governed by the type and degree of antimalarial immunity that he or she may have attained.

Two other forms of acquired immunity to malaria should be mentioned here. One is immunity which could protect against the parasites ever becoming established in a human host, in other words immunity against the stages which are introduced into the human body by a mosquito bite. These are the sporozoites and also the early, nonpathogenic stages as they develop in the liver. While immunity against sporozoites and liver stages may eventually develop following long exposure to intense malaria transmission, it is probably not very significant in the crucial early years of exposure. The other form of nonprotective immunity against malaria parasites is that against the sexual blood stages of the parasites. These are the stages which are infective to mosquitoes. Antibodies against antigens in gametocytes and gametes of malaria parasites develop readily during the course of natural infections and probably have some effect on the transmission dynamics of malaria in certain endemic situations. Anti-sexual-stage immunity, however, has no direct relevance to the status of an individual with respect to his or her experience of a current malarial infection.

The attainment of protective immunity against the disease-causing asexual blood stages of malaria parasites is a complex process. Not only is such immunity specific to each parasites species, but also there is certainly a major element of parasite "strain" specificity to antimalarial immunity (105, 130, 148, 182). Much of the slow acquisition of antiparasitic immunity occurs because an individual is usually subjected to a succession of inoculations of parasites which are genetically and antigenically distinct from each other. Therefore, to combat each infection, a human host must mount a new and "strain"-specific immune response to each antigenically distinct parasite inoculum. Only when a sufficiently wide spectrum of such parasite "strains" has been experienced is effective immunity achieved against all the parasites within a locality where infection is endemic.

However, not only are the parasites in different inoculations genetically different but also, during the course of each infection, the parasites from a single inoculation undergo clonal "antigenic variation" (130). To control an infection, the host must mount a new specific immune response to each new antigenic variant as it arises. Through this process, a single malarial infection can be prolonged over many months to years.

Because of the time taken to achieve effective immunity to malaria under conditions of endemic infection, antimalarial immunity is often said to be "age dependent." In the sense intended, however, it would be more accurate to say that it is "duration of exposure dependent." There are, nevertheless, truly age-dependent aspects both to the attainment of immunity and to the pathologic responses to malarial infection. Very young children appear to have a poor capacity to acquire effective protective antimalarial immunity of any sort, while older children and adults may so do more readily (9, 10). Infants and the very young are more prone to malarial anaemia, while cerebral damage due to P. falciparum malaria predominates in slightly older children. Yet other severe conditions, including renal, hepatic, and pulmonary failure, are most commonly seen in adults (6).

Every individual exposed to endemic malaria thus faces a long and dangerous battle to achieve protective immunity against the diversity of malaria parasites and their antigens to which he or she may become exposed. However, having been achieved at such cost, effective immunity is readily lost again. An interval of perhaps half a year to a year without reinfection appears to be sufficient to leave an individual vulnerable, once more, to the full impact of a malarial infection (158, 182, 186; A. Lukas, personal communication). Direct evidence for this statement is admittedly hard to find, yet it has long been attested by anecdote and expert opinion. Loss of protective immunity within a few months without reinfection also appears to hold in relation to infection-induced protective immunity against malaria in animals (R. Carter, unpublished observations).

If the above statement is indeed correct, then the number, type, and pattern of delivery of malarial inoculations, in other words the type of malarial endemicity, to which an individual is exposed must profoundly affect his or her immune status and hence disease status, as is now discussed.

The so-called age-dependent pattern of immunity to malaria in sub-Saharan Africa is often attributed to the intense malaria transmission rates in this region. However, we suggest that it is the stability of malaria transmission in tropical Africa, due to biological features of the Anopheles gambiae group of vectors of malaria, their ecology, and their environment (discussed below), that is the more important feature of African malaria transmission. Because, individuals rarely go more than a few months without a malaria challenge under conditions of stable transmission, there is little risk that immunity, once attained, will be lost again. This remains true even in the setting of quite low annual malarial inoculation rates (128). There is, nevertheless, a clear shift from severe malarial disease in younger children (younger than 5 years) toward relatively increased rates of both mild (191) and severe (179) disease in older age groups at entomological malarial inoculation rates probably below about 10 to 20 infectious bites per year. However, under conditions of stable malaria in Africa, the lifetime risk of severe disease, and hence of direct malaria mortality, due to P. falciparum seems to remain fairly constant across the spectrum of inoculation rates from very high to very low (179).

Unstable and epidemic malaria. By contrast, where transmission conditions are unstable, low to moderate mean malarial inoculation rates can be highly dangerous. This is because immunity to malaria, while slowly gained, is, as we have suggested, rapidly lost following perhaps half a year to a year without infection. Wherever the rate of delivery of malarial inoculations is both low and highly erratic, extended periods of a year or so without reinfection occur often, while at the same time the risk of eventual reinfection remains high. In these circumstances, individuals are vulnerable throughout life to clinically active malarial infection and are often affected by it. Furthermore, all species of malaria parasite, and not just P. falciparum, become dangerous under these conditions. Recurrent infections with any species of malaria parasite are eventually so debilitating that life expectancy can be reduced to half or less of that in a contemporary malaria-free and otherwise salubrious environment (47, 123). The situation is illustrated by the following quotation which refers to malarious districts around the shores of the Mediterranean in the early 19th century. "The most fertile portions of (Italy) are a prey to (malaria); the labourer wanders... the ghost of a man, a sufferer from his cradle to his grave; aged even in childhood, and laying down in misery that (brief) life which was but one disease... . Such also is Sicily, such Sardinia and such is classic Greece. To live a living death and to be cut off from even half that life" (123).

Wherever antimalarial immunity has declined, has been lost, or was never achieved in the first place, populations at the margins of malaria transmission zones are vulnerable to epidemic malaria. When P. falciparum is involved in an epidemic, death rates can become very high. A single untreated attack of P. falciparum malaria in a nonimmune individual carries a risk of death that may be anywhere from a few percent to at least 20 to 30% according to circumstances (3, 12, 21). These higher rates are the kinds of mortality rate that occur during P. falciparum malaria epidemics (30, 45a, 49, 67, 75, 219). The following quotation (67) is representative of these events. "In each house were... three or four patients who complained of chilling, severe headaches, sweating, pain in back and extremities... After four or five relapses, the headaches and pain became unbearable for many patients who then exhibited a muddling delirium with coma, ending in death. Most... were between the ages of 5 and 20 years. Since they are far away from even the simplest clinic, which means no possibility of saving their lives, they are dying like bees in a smoked hive" (from a Field Report from the 1958 malaria epidemic in Ethiopia [67]). In a season, this very typical malaria epidemic produced about 3.5 million cases of malaria among which both P. vivax and P. falciparum malaria would have been present; it took about 150,000 lives.

Similar and even higher malaria mortality rates were experienced by Europeans entering tropical regions before the introduction of quinine in the mid-19th century (20, 42-44, 156, 197). Indeed, the consequence of the introduction of nonimmune persons into a malaria-endemic region is, in effect, a form of epidemic malaria.

Biological Basis of Stable, Unstable, and Epidemic Malaria The differences in stability of malaria transmission, notably between tropical Africa and most other malarious regions, are due largely to the behaviors and other biological characteristics of the regional species and subspecies of Anopheles vectors and to their environment. The strong human-biting preferences and highly domestic habits of the tropical African vectors (17, 39) lead to very uniform contact between them and the human blood source in sub-Saharan Africa. The climatic conditions are also highly conducive to malaria transmission, being warm and humid with relatively few fluctuations. This supports longevity of the vector mosquitoes and rapid development of the parasites within them. All of these features combine to a recipe for stable and, indeed, generally intense malaria transmission.

Elsewhere, in the tropical, subtropical, and temperate worlds, the females of most Anopheles species have a preference for animal rather than human contact and have less domestic breeding, resting, and dispersal habits than the African vectors. The climates and especially the microclimates experienced by these vector mosquitoes are usually cooler, drier, and more variable than in tropical Africa, leading to more uncertain survival of the mosquitoes and less reliable development of the parasites within them. These are conditions that lead to highly erratic contact between potential vectors of human malaria and the human hosts and to very irregular delivery of malaria inoculations, in other words, to unstable malaria transmission.

Epidemic malaria arises in any situation in which new or elevated malaria transmission capacity is suddenly introduced into a population with inadequate levels of immunity with which to absorb it. The circumstances under which this can occur are extremely diverse (192). Typically they involve nonimmune populations in otherwise malaria-free locations adjacent to regions of endemic malaria. An epidemic occurs when atypical weather, e.g., drought, excess rainfall, and higher than usual temperatures, create conditions that transiently support malaria transmission. However, warfare, pioneering, and malaria control, among other human activities, have all been responsible for the creation of malaria epidemics.

Indirect Mortality of Malaria Under whatever condition of endemicity malaria occurs, much, and according to the evidence of a number of analyses, generally most (74, 134, 142, 219), of the malaria-related mortality has in the past been an indirect result of the effects of malarial infection combined with other infections and conditions. Wherever interventions have been conducted for the reduction of malaria transmission, reductions in total mortality rates have been several times greater than the malaria-related death rates as estimated prior to the interventions (3, 134). Some of this discrepancy may be due to failures to detect or account for all the deaths in which malaria would have been an apparent cause. However, most of the excess deaths associated with the presence of malaria, but not obviously due to malaria, can be attributed to delayed or indirect causes. These could include the nephropathy of long-term P. malariae infection (74). However, more generally, the deaths are due to the predisposing effects of malaria to death from other conditions such as respiratory infections (74) and malnutrition (219).

Nonlethal Health Consequences of Endemic Malaria In historical times, and as recently as the early to mid-20th century in southern Asia, continual malarial infection and reinfection had devastating effects on the mental, physical, social, and economic conditions of the individuals and communities affected. In addition to its toll in death and general morbidity, there are several features of the burden of malaria that warrant specific mention.

Malaria presents particular problems during pregnancy. This is probably due, at least in part, to the "immunosuppressed" status of the pregnant woman. Even in otherwise highly malaria-immune women, the risk of malarial infection in pregnancy is high, with increased risk of low birth weight, miscarriage, infant mortality (14), and morbidity and mortality in the pregnant woman. "Malaria exerts a considerable influence in reducing the births and increasing the number of still births in a community" (Bentley, 1911, quoted in reference 176).

Historically, "infantilism" in both sexes and impotence in males were associated with malarious areas (5). "One of the great evils is the impotency so commonly found in waterlogged villages (and) which results from the deterioration of the health produced by constant attacks of fever and the presence of an enlarged spleen" (Dyson, 1895, quoted in reference 176). Together with the problems of malaria in pregnancy and infant mortality, these added up to a large reduction in the fecundity of a population and, in the worst-affected locations, contributed greatly to depopulation under the impact of malaria. "In hyperendemic and severely endemic (for malaria) districts in India the tendency is for the population to decline" (91).

Among the most oppressive and, today, least appreciated of the effects of malaria is that on the mental state of the sufferer. Observers from many different times and places, including Hippocrates (95), Macculloch (123), Jones (106), Anderson (5), and Sinton (176), are remarkably consistent in their portrayals of the condition of the inhabitants of malarious regions especially in Europe or Asia. They depict individuals and communities in states of pronounced mental and psychological distress under the influence of endemic and invariably unstable or low-transmission malaria. The following are typical of these. "Anyone who has observed closely cases of malaria cannot fail to have noticed its effects upon the mentality of the sufferer—mental activity is dulled, irritability of temper is the rule, initiative is lacking, decisions are put off or reached with difficulty, ambition is lost and depression is a prominent symptom" (176); "School children in the Transvaal infected with chronic malaria were mentally classed as "feeble-minded"" (Leipoldt et al., 1921, quoted in reference 176). In today's much healthier world, situations matching their descriptions are conspicuous by their absence. Nevertheless, and not to be confused with the immediate consequences of malarial infection itself on the mental state, a significant proportion of the survivors of malarial infections still carry mental or neurological deficits (97).

Effects of Malaria on Social and Economic Development An adequate discussion of the economic effects of malaria is beyond the scope of this article. Populations exposed to the unremitting impact of malaria must always have lived and died in destitution of one degree or another. In historical times, economic enterprise has been difficult and often impossible in the presence of malaria (176). Malaria alone has often been sufficient to wreck efforts in pioneering, agriculture, and civil engineering, as illustrated by the following representative quotations. "The hyperendemic areas (for malaria), although sparsely inhabited, are often areas where large plantations and large industrial undertakings are situated and which are, therefore, often the site of a considerable immigrant populations that are quickly mown down." (Malaria Commission of the League of Nations, 1930, quoted in reference 176); "While there is good land in the Southern United States as in the North, the land in the North sells at about 12 to 20 times the price, the difference being mainly due to malaria" (Carter, 1922, quoted in reference 176); "Railway construction in the tropics is nearly always associated with fulminent epidemics of malaria—"a death a sleeper" is the generalisation on the happenings" (Senior-White, 1928, quoted in reference 176).

Throughout history (176) and to the present day (70), wherever and however it may have manifested itself, malaria has always imposed one of the severest of impediments to social and economic development.

THE "MALARIA HYPOTHESIS" AND RECENT HUMAN EVOLUTION

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It can be no surprise, therefore, that malaria parasites should have had a profound impact on recent human evolution (27). This is the proposition of the "malaria hypothesis," which posits that certain human genetic polymorphisms, especially those affecting red blood cells (RBCs), have been selected to high frequencies because they have protected against the effects of malarial infections. Indeed, a greater number of identified human genetic polymorphisms meet some or all of the expectations of the malaria hypothesis than can be attributed to selection under any other single agent (27).

The first statement of the malaria hypothesis was made in 1948 by J. B. S. Haldane (86), who proposed it as an explanation for the high frequencies of thalassemia around the shores of the Mediterranean Sea, where malaria had long been endemic. Probably unknown to Haldane at the time, a possible association between another inherited hemoglobinopathy, the sickle cell trait, and protection against malaria had already been noted. In 1946, in a study of inpatients at a regional hospital in Northern Rhodesia, now Zambia, E. A. Beet had recorded lower rates of malarial infection among carriers of the sickle cell trait than amongst nonsicklers (11).

In the following, we discuss the evidence for, and implications which arise from, the main candidate human polymorphisms to fall within the terms of the malaria hypothesis.

In Europe, high frequencies of thalassemias are found around the shores of the Mediterranean Sea, as Haldane had noted (65, 86). Elsewhere, thalassemias occur at elevated frequencies in populations through most of Africa, the Middle East and Central Asia, the Arabian peninsula, the Indian subcontinent, Southeast Asia, southern China, and the islands of the Western Pacific from the Philippines in the north to the Timor Sea in the south and to New Guinea and the islands of Melanesia in the east (27, 65, 119, 198). Up to about 2,000 years ago, these were the probable global limits within which malaria was endemic.

The basis of Haldane's original conjecture (86) has been confirmed and expanded in several studies which show the association of thalassemias with the long-standing exposure of a population to the presence of malaria (see, e.g., references 66 and 215). An approximately 50% reduction in the risk of malarial disease has recently been shown for both heretozygotes and homozygotes for certain alpha⁺ thalassemias (1).

Recent evidence indicates that the alleles for G6PD deficiency were selected in African populations between 4,000 and 12,000 years ago (188). This period coincides with some estimates for the time of emergence of P. falciparum as a major human pathogen at around 4,000 years ago (159, 194). Equally, however, the selection for G6PD deficiency may have preceded this event and overlapped a time when P. vivax malaria could still have been a major selective force in Africa (see below). So which parasite could have been responsible for the selection of G6PD deficiency in the African populations?

It may often appear that the malaria hypothesis should be taken to apply mainly, or solely, to P. falciparum. However, as we shall further explore, the hypothesis is equally valid for P. vivax and perhaps for other species of human malaria parasites as well. That P. vivax malaria may represent a force for the selection of G6PD deficiency is consistent with the presence of elevated frequencies of G6PD deficiency in a population in northern Holland (27), where P. vivax malaria, but almost certainly not P. falciparum malaria, was prevalent for at least 500 years. We suggest that either or both of P. falciparum and P. vivax could have supplied the force that first selected for G6PD deficiency in African populations.

The sickle cell trait is due to a single point mutation in the gene for the beta chain of hemoglobin in which the glutamate at position 6 is replaced with a valine (119). It results in a hemoglobin molecule, hemoglobin S, which, in the homozygote, gives rise to sickle cell anemia. Today in the United States, such individuals commonly reach middle adulthood (198). In the premodern world, however, those affected with sickle cell disease would probably rarely have survived to puberty (4). In reproductive terms, the cost to a hemoglobin S homozygote would have been a loss of fitness of nearly 100%. Such a mutation could have been sustained only if the heterozygotes, those carrying one hemoglobin S gene and one normal, hemoglobin A, gene, had an advantage great enough to balance the cost of the homozygotes to the population. It has been fully confirmed that this advantage is protection against the risk of death from infection with P. falciparum malaria. Children in West Africa who are heterozygous for the sickle cell gene are at approximately 1/10 of the risk of death from P. falciparum malaria as are children who are homozygous for the normal gene (76, 94).

Hemoglobin S clearly fulfils the conditions of the malaria hypothesis. In many parts of Africa, the frequency of the sickle gene exceeds 30% (4, 27, 119, 120). The reproductive losses which these frequencies impose through homozygosity for this gene affect at least 1 birth in 10. They reflect the cost of endemic P. falciparum malaria to a human population.

Hemoglobin C is found only within certain West African populations, where the frequency of the allele reaches, and sometimes even exceeds, 10 to 20% (119, 120). There is now little doubt that this polymorphism has been, and indeed is probably still being, selected under the effects of P. falciparum malaria in West Africa (133). However, the nature of the protection by hemoglobin C is very different from that associated with hemoglobin S. In the case of hemoglobin S, the heterozygous state confers a high level of protection against death from P. falciparum malaria. In that of hemoglobin C, similar high levels of protection, greater than 90% against infection and therefore against the risk of death by P. falciparum malaria, are achieved only in the homozygote (133). The protective effects of hemoglobin C in a heterozygous combination are, at about 30%, relatively weak.

Now, the rate of selection of an allele whose advantage is expressed mainly in the homogygous state, as is the case for hemoglobin C, is slow compared to that of an allele, such as that for hemoglobin S, which carries a similar advantage but in a heterozygous combination. This principle is discussed more fully in relation to selection for RBC Duffy negativity (see below). Under the influence of the same selective force, therefore, hemoglobin S would be expected at first to achieve higher frequencies than hemoglobin C. However, because the balancing cost of hemoglobin C is low and that of the allelic hemoglobin S is high, hemoglobin C should eventually replace hemoglobin S in populations exposed to selection by P. falciparum. In West Africa this has clearly not yet happened (4, 119, 120), and it suggests that P. falciparum malaria may have arrived only "recently" within the West African population.

When, therefore, might this "recent" arrival of P. falciparum in West Africa have taken place? The relative fitnesses of the different haemoglobin alleles, A, S, and C, in the West African situation can now be given (76, 94, 133), and the relevant calculations can be made (119). We suspect that they would show that the hemoglobin C allele should approach population equilibrium within several thousand years, and almost certainly within less than tens of thousands of years, of selection under P. falciparum malaria, as, indeed, has already been suggested (119, 120). Therefore, by this line of argument, because hemoglobin C has not yet reached its expected equilibrium frequency, P. falciparum has been a selective force in West Africa for less than this time, i.e., probably for less than a few tens of thousands of years.

Hemoglobin E has no obvious clinical effect except in combination with certain thalassemia mutations (65, 198). It does not, therefore, impose severe costs on a carrier population. While there appear to be no reports that directly demonstrate protection by hemoglobin E against P. falciparum, there is evidence suggesting that it may protect against P. vivax infection (157). It has also been reported that hemoglobin E trait patients clear infections of P. falciparum more rapidly than do others during treatment with artemisinin derivatives although not during treatment with other antimalarial drugs (100). The geographical region across which hemoglobin E is prevalent is one of the most malarious, both historically and today, and the burden of malaria in this region has therefore long been one of the heaviest in the world. The gene for hemoglobin E is certainly a good candidate to have been selected under the pressure of malaria in accordance with the malaria hypothesis, possibly under the influence of P. vivax as the principal agent of selection prior to the arrival of P. falciparum within these populations.

The malaria hypothesis almost certainly explains the high frequency of the ovalocytosis mutation in these populations. Neither the sickle cell gene nor hemoglobin E appears to be present among Melanesian populations. Other than ovalocytosis itself, only the moderately protective G6PD deficiency gene and some thalassemias are found (119). Ovalocytosis occurs at high frequencies (up to 20%) in the highly malarious lowlands of New Guinea but virtually disappears in the malaria-free highlands. Carriers of the trait are at reduced risk of infection with P. falciparum and, especially, P. vivax malaria (26).

Two studies of the frequency of the band 3 gene deletion mutation in children with P. falciparum malaria showed that none who carried the gene (in heterozygous combination) developed cerebral malaria (2, 72). Protection against malarial infection by ovalocytoses of unspecified genotypes has also been demonstrated in populations in nearby Southeast Asia (8, 68). Ovalocytic RBCs resist invasion by P. falciparum (110) and by Plasmodium knowlesi (85), a simian malaria parasite with similar RBC invasion characteristics to P. vivax. It is not known, however, if the ovalocytic conditions which protected against malaria in these studies were due to the same band 3 gene deletion that was shown to protect against cerebral malaria (2, 72). Given the apparent uniform in utero lethality of the homozygote form of the band 3 gene deletion mutation, its high frequency in the lowland populations of New Guinea once again reflects the magnitude of the reproductive cost of malaria to this population.

The molecular genetic basis for RBC Duffy negativity is a single-nucleotide substitution polymorphism (SNP) in the promoter region of the gene for the Duffy antigen. This promoter controls the expression of the Duffy antigen specifically in RBCs. The same SNP is associated with both the FY*A and the FY*B alleles (217), leading to the FY*A^null and FY*B^null RBC Duffy-negative alleles. Now, the promoter for a structural gene, such as that for the Duffy antigen, governs its expression on its own chromosome strand only. Therefore, in a heterozygous individual, expression of the Duffy antigen in RBCs should be suppressed only on the strand with an FY^null allele, while from the strand with a Duffy-positive allele, the Duffy antigen should be fully expressed.

In accordance with this expectation, Duffy antigen expression on RBCs from individuals from Papua New Guinea who were heterozygous for the FY*A^null allele was approximately half of that on RBCs from homozygous Duffy-positive individuals (217). In the same human population in Papua New Guinea, a 50%, but statistically insignificant, reduction was noted in the prevalence of P. vivax infections among heterozygous FY*A/FY*A^null individuals compared to those who were homozygous Duffy positive (217). The characteristics of human RBCs with genetically reduced RBC Duffy antigen expression (Fy^xFy^x, according to serotype nomenclature [126]) and weak reaction with Duffy antigen antisera was investigated using the simian malaria parasite P. knowlesi, which, like P. vivax, is absolutely dependent on the Duffy antigen for the ability to invade human RBCs. Merozoites of P. knowlesi invaded RBCs from such individuals with about half of the efficiency that they invaded RBCs from individuals with full Duffy antigen expression (126). However, RBCs from heterozygotes for RBC Duffy negativity were invaded as efficiently as were RBCs from normal RBC Duffy-positive individuals (J. Barnwell, personal communication). Susceptibility to infection with P. vivax malaria of FY^null heterozygotes may be partially reduced, but the effect is probably not very strong, certainly by comparison with the total refractoriness to P. vivax malaria of individuals who are homozygous for an FY^null allele.

Throughout West and Central Africa, the frequency of the FY*B^null allele is, as already noted, close to fixation. In most of these populations, its frequency exceeds 97% (27, 135), leading to almost universal RBC Duffy negativity in these populations. Not surprisingly, P. vivax malaria is very rare throughout the region (71, 131) (Table 1). It might seem natural to conclude that P. vivax malaria must have been the selective agent for the near fixation of the FY*B^null allele in these African populations, to the point that P. vivax was itself virtually eliminated. While this is our view, it is not universally held (217). Nor is the case, as we will now present it, straightforward.

In contrast to the frequently and directly lethal P. falciparum, it is often assumed that P. vivax exerts little or no selective pressure on a human population. On these grounds, therefore, and notwithstanding that the RBC Duffy-negative condition confers no evident disadvantage on a carrier (87, 121), P. vivax might be discounted as a selective force for the near fixation of RBC Duffy negativity in West and Central Africa. If, on the other hand, P. vivax can, and does, exert a significant selective force, we are confronted with a paradox. Why, in this case, has selection for RBC Duffy negativity not taken place in other parts of the world, in southern Asia and the Western Pacific rim (27, 135), where P. vivax has certainly been present for several thousand years? It has recently been suggested, in a report of the presence of an FY*A^null allele in a single population Papua New Guinea, that such selection may indeed be happening (217). However, no previous studies appear to have detected this allele in Papua New Guinea, where FY*A is otherwise at virtual fixation (27). Moreover, at around 2%, the FY*A^null allele in this single population in Papua New Guinea is still far below the frequencies of the FY*B^null allele, which, throughout tropical Africa, are rarely less than 50% and more often in excess of 95% (27).

Concerning the first of the points above, there is strong evidence that P. vivax has, in fact, often placed a heavy burden of mortality and loss of fecundity on the populations that it afflicted (47, 176). Its effects are greatest under conditions of relatively low and unstable malaria inoculation rates. These were the conditions that probably prevailed in Africa before 5,000 to 10,000 years ago (118) and possibly throughout much of the preceding 100,000 years. Had P. vivax been prevalent within human populations in West and Central Africa during this period, and we propose that it was, we would expect that the homozygous RBC Duffy-negative condition would have carried a considerable selective advantage and the heterozygous condition a slight one.

We would, however, expect that selection for raised frequencies of an FY^null allele in a population under P. vivax pressure should also take a "long" time. This expectation follows if mainly the FY^null homozygotes are refractory to P. vivax infection but rather little selective advantage is associated with the heterozygous condition. This, as we have just discussed, is quite likely to be the case.

Now, the homozygous combination of a rare allele, as the FY*B^null gene in Africa would at first have been, is itself almost vanishingly rare. Moreover, even when the homozygous combination of such an allele had arisen, it would, in the next generation, almost invariably have been diluted again to the weakly advantageous heterozygous state in combination with one of the high-frequency FY*B or FY*B RBC Duffy-positive alleles. Thus, a selection which involved mainly the persistently rare FY*B^null/FY*B^null homozygotes would inevitably proceed "slowly." It would proceed slowly, that is, compared, with the speed of selection for a gene such as the allele for hemoglobin S (Hb*S), in which the heterozygous condition, Hb*S/Hb*A, carries the main selective advantage and which, in the early stages of selection, arises as frequently as the Hb*S allele itself. Moreover, the speed of selection for Hb*S would be further increased above that for the FY*B^null allele, because P. falciparum is probably much more dangerous than P. vivax under any conditions of endemicity. Hb*S has been estimated to approach a balanced equilibrium after around 2,000 years of selection under P. falciparum malaria (119). The "longer" time expected for P. vivax malaria to select for the near fixation of the FY*B^null allele could, therefore, be in the order of 10,000 to several tens of thousands of years.

While we may infer that the process of selection for near fixation of the FY*B^null allele in Africa under P. vivax would have taken several tens of thousands of years, we cannot, from the above line of argument, even hazard a guess as to the times when this process may have begun or ended. These may, however, be partly estimated from other evidence.

Modern human migrations out of Africa are believed to have taken place largely, if not entirely, within the past 100,000 years. Now, RBC Duffy negativity is, as already noted, an apparently harmless genetic condition. Therefore, once an FY^null allele has been selected in a human population, there should be little or no loss of frequency of the gene, especially from a population in which it had become fixed. Outside Africa, RBC Duffy negativity is found in declining frequencies through the Arabian peninsular, across the Middle East, and to the edges of Central Asia, but beyond these areas it is, with rare exceptions, virtually absent (27). Had the high frequencies of the FY*B^null allele been selected before the main dispersals of modern humans began, this allele should be common in all human populations. Since it is not, its selection in Africa must have been completed only after these dispersals had taken place, i.e., within less than the past 100,000 years. Indeed, since human migrations out of Africa across the Old World and into the Western Pacific have probably continued into much more recent times and have carried almost no trace of the FY*B^null allele with them, we can probably reduce the time by which selection for African FY*B^null would have been completed to less than 50,000 years, which is the approximate period of the migrations to Melanesia and Australia. And if, as we do, we take it that the force for the selection of the FY*B^null allele in Africa was P. vivax malaria, then the fact that at least 5,000 years of exposure to P. vivax in southern Asia and China and the Western Pacific has not led to selection for high levels of RBC Duffy negativity anywhere in this region suggests that the process also takes longer than 5,000 years.

We now have a case that the selection for near fixation of RBC Duffy negativity takes at least 5,000 years and that it was completed in Africa less than 50,000 years ago. Unfortunately, we still cannot place an upper limit upon how long the process takes. The P. vivax pressure could, for example, have begun several hundred thousand years ago and reached completion in a final rapid spurt at any time within the past 50,000 years, or it could have begun in Africa only 6,000 years ago and been completed in the last few hundred years, just in time for us to record the outcome. However, we now have corroborating evidence for the rough period within which the beginning of the selection would have taken place. In a study of haplotypes associated with the FY*B^null allele in African and European populations, Hamblin and Di Rienzo (87) have proposed that a selective sweep towards the near fixation of RBC Duffy negativity in the African populations began between 97,200 and 6,500 years ago within 95% confidence limits.

We suggest that the most likely period for the selection of RBC Duffy negativity in Africa to have taken place, and therefore for strong selective pressure from P. vivax to have been still active on these populations, lies somewhere between perhaps 10,000 and 5,000 years ago. This falls at the height of the last major glacial period, when equatorial Africa would have been much cooler than today and when it would have been infested with Anopheles mosquitoes, which were at the time relatively inefficient vectors of malaria (39, 118). These are conditions which would have tended to support low levels of unstable, and, therefore, severely life-degrading, P. vivax transmission. They are the conditions which would have strongly favored selection for RBC Duffy negativity in the affected human populations in Africa.

Mutations which are moderately protective against malaria, which protect in heterozygous combination, and which carry a relatively low balancing cost to a population, namely, the thalassemias and G6PD deficiency, are generally the first to reach elevated frequency under selection by malaria. These are the malaria-selected polymorphisms which predominate in the parts of Europe, around the shores of the Mediterranean Sea, where malaria arrived between 2,500 and 2,000 years ago. In northern Europe, where it has arrived more recently, probably mainly between 1,000 and 500 years ago, even these polymorphisms are almost entirely absent.

In regions in which more than 2,000 to 3,000 years of selection by malaria has taken place, namely, in Africa, parts of the Middle East, southern Asia, and Melanesia, mutations are found which are highly protective against malaria in heterozygous combination but which also carry a high balancing cost in homozygous combination. These are the genes for sickle cell hemoglobin and ovalocytosis.

Longest of all, many thousands or tens of thousands of years, may be the selection for alleles whose protective effects against malaria are expressed only, or mainly, when they are present in homozygous combination. Such is the allele for hemoglobin C, which, in homozygous combination, protects strongly against P. falciparum malaria and which has probably not yet reached equilibrium frequencies even after several thousand years of selection in West Africa. Such, also, is the allele for RBC Duffy negativity, which, only in homozygous combination, protects solely and absolutely against P. vivax malaria. High frequencies of RBC Duffy negativity extend from West and Central Africa and, in declining frequency, toward East Africa and southern Africa as well. These regions, and especially West Africa, are the heartlands from which most, if not all, of today's populations of human malaria may have had their origin and in which, therefore, selection under both P. vivax and P. falciparum has been of longest duration.

Did malaria enter most human populations before or after their global dispersal? In each broadly grouped human population in which they occur, the malaria-protective polymorphisms are usually distinct from each other. Hemoglobin E occurs only in Southeast Asia, and ovalocytosis occurs in Melanesia and also, probably in several different forms, through Southeast Asia. Hemoglobin S is absent from these regions, and hemoglobin C occurs only in West Africa. The variety of different G6PD deficiency mutations found in, and characteristic of, different human populations is large, and for the thalassemias it is even greater (65). The G6PD deficiency mutations are associated with a number of different haplotypes in different human populations (188), and the sickle cell gene occurs in association with several different population haplotypes within Africa and with three distinct haplotypes in the Middle East and India (65). Thus, individual polymorphisms lie within haplotypes which are usually characteristic of the human populations within which the polymorphism is found.

These facts can be most easily explained if each indigenous human population had separated and settled before the selective agents, P. falciparum and P. vivax malaria, arrived within it. This, as will be analyzed in the following sections, is almost undoubtedly the general pattern of events.

Legacy of the impact of malaria on human populations. Regardless of its implications for our understanding of the evolution and history of malaria, the malaria hypothesis points to the public health reality that our past contacts with malaria have left a large burden of genetic diseases among us. Today this genetic burden, in the form of the thalassemias, sickle cell disease, G6PD deficiencies, and the ovalocytoses, is of the same order as the residual burden of malaria itself. About one-third of a million to half a million babies are born each year with severe forms of these inherited disorders (198).

ORIGINS AND EVOLUTION OF HUMAN MALARIA PARASITES

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The ancestors of the malaria parasites have probably led a parasitic existence almost since there were potential hosts to parasitize, at least half a billion years ago. Of course, the parasitic forms in this lineage must themselves have had a preparasitic ancestor. Molecular genetic evidence strongly suggests that this ancestor was a choroplast-containing, free-living protozoan which become adapted to live in the gut of a group of aquatic invertebrates (204). This single-celled organism probably had obligate sexual reproduction. Indeed, to the present time, all known members of the phylum Apicomplexa, to which the malaria parasites belong, have retained an obligate sexual stage in their life cycles (113). Moreover, the environment where gamete formation and fertilization takes place is still always within the midgut lumen of a host species.

At some relatively early stage in their evolution, these "premalaria parasites" acquired an asexual, and usually intracellular, form of reproduction called schizogony. By evolving this form of "vegetative" reproduction, the parasites greatly increased their proliferative potential. Schizogony may be defined as growth and subsequent nuclear and cellular division into numerous daughter cells from within the body of a single parent cell. It almost invariably takes place during the intracellular phases of parasite growth in the tissues of a host. The completion of each schizogonic event is usually followed by reinvasion of new host cells by the daughter cells or spores (designated sporozoites or merozoites), leading to another round of schizogonic development. In this way, numerous successive rounds of multiplication can take place. It is during schizogony in the RBCs of humans and other vertebrates that certain descendants of these ancestral parasites now cause the disease that we call malaria.

Among the invertebrates to which the ancestors of the malaria parasites became adapted were probably aquatic insect larvae, including those of early Dipterans, the taxonomic order to which mosquitoes and other blood-sucking flies belong. These insects first appeared around 150 million to 200 million years ago. During or following this period, certain lines of the ancestral malaria parasites achieved two-host life cycles which were adapted to the blood-feeding habits of the insect hosts. The malaria parasites of humans are typical products of this line of development since they alternate between human and the blood-feeding female Anopheles mosquito hosts (within whose midguts gamete formation and fertilization still, of course, take place).

In the 150 million years since the appearance of the early Diptera, many different lines of malaria and malaria-like parasites evolved and radiated. They came to parasitize members of most major groups of land vertebrates including reptiles, birds, and mammals. In the mammalian orders today, more species of malaria parasite have been identified among primates than in all other mammals combined (71). Over 25 distinct species of malaria parasites of primates have been named. Four of these are the recognized malaria parasites of humans: P. falciparum, P. vivax, P. malariae, and P. ovale.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Phylogeny of the malaria parasites of humans and of some other related malaria parasite species. See the text for sources on which this reconstruction is based.

The three remaining species of human malaria parasite, P. malariae, P. ovale, and P. vivax, fall within a single clade that includes all the mammalian malaria parasites, other than P. falciparum and P. reichenowi, on which molecular genetic analysis has been done (55, 56, 153) (Fig. 1). Within this clade, all of the primate malaria parasites belong to one or other of the lineages represented by P. malariae, P. ovale, or P. vivax. These three lines appear to have diverged over 100 million years ago (7) (Fig. 1), long before the emergence of the lines leading to the distinct mammalian, let alone primate, orders of today.

Among the primate malaria parasites, P. ovale is known only as an infection in humans and has no genetically confirmed close relative (55, 56, 153). P. ovale is therefore the sole known surviving representative of its line (7).

P. malariae, on the other hand, in addition to infecting humans, is found in apparently indistinguishable form as a natural parasite of chimpanzees in West Africa (71). Moreover, a morphologically indistinguishable parasite, P. brazilianum, infects New World monkeys in Central and South America (71). Molecular genetic analysis has failed to distinguish P. brazilianum and P. malariae (55, 56, 153). There can be no doubt that these are the same or almost the same parasite infecting different primate species, namely, humans and New World monkeys. We take the view that P. brazilianum is, in fact, a zoonotic form of P. malariae which has been introduced recently from humans into the New World monkey populations.

The converse of this, i.e., that P. malariae has been received by lateral transfer from New World monkeys to humans, has also been argued (7). However, a New World origin of this parasite does not reconcile with the common prevalence of P. malariae in the Mediterranean region more than 2,000 years ago (95). Virtually all, if not all, pre-Colombian human migrations of at least the past 50,000 years appear to have been from the Old to the New World, and none have been in the reverse direction. Therefore, had P. malariae originated in the Americas by lateral transfer from monkeys to humans, when, and by what route, could it have entered the Mediterranean region to have become prevalent there at least 2,000 years before any known or likely human traffic with the New World?

Except for P. brazilianum, no other primate malaria parasite is known to belong within the P. malariae lineage. Thus, whether as a human parasite or, in essentially identical form, as a parasite of New World monkeys, P. malariae/P. brazilianum, like P. ovale, is the only confirmed and extant representative of its line.

In contrast to these lonely survivors of ancient lines, P. vivax belongs within a large and closely related body of malaria parasites which today are found mainly infecting monkeys of southern and southeastern Asia and the Western Pacific rim (Fig. 1) (55, 56, 153). Among these parasites, P. cynomolgi and its subspecies have long been considered especially closely related to P. vivax (71). Because these close relatives of P. vivax are today found only infecting monkeys of the southern Asian regions, there is a view that P. vivax itself must have originated from within these regions and from nowhere else (see, e.g., reference 56). However, this conclusion may not necessarily be so simply reached. The most recent estimate for the time of the divergence of P. vivax and P. cynomolgi is in the range of two million to three million years ago (A. Escalante personal communication). At this time, the ancestral hosts of these parasites were probably spread throughout much of the tropical, subtropical, and, indeed, temperate regions of the combined Asian and African landmasses. Therefore, the region(s) where the P. vivax an