Consideration of some aspects of human malaria

145 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 59. No. 2. March, 1965.

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C O N S I D E R A T I O N OF S O M E A S P E C T S OF H U M A N M A L A R I A BY

I. A. McGREGOR Medical Research Council Laboratories, Gambia, West Africa

Knowledge of the behaviour of the malaria parasite within the human host has advanced notably over the past 16 years and it is perhaps timely now to consider how recent findings may influence our present concepts of the epidemiology of the disease. In 1948 SHORTTand GARNHAMdemonstrated the long-suspected existence of the preerythrocytic phase of development of Plasmodium vivax. Since then much information concerning this phase of the life cycle of human plasmodia has been gathered and has been reviewed by BRAY (1957, 1963). Recently COHEN, MCGREGORand CARRINGTON (1961), and COHEN and MCGREGOR (1963) have indicated that the basic mechanism of acquired malarial immunity in man is serological and is vested in a specific antibody associated with the 7S fraction of serum gamma globulin. Such immunity appears to be effective only against the late stages of asexual erythrocytic plasmodia and to have little or no effect upon developing or mature gametocytes (MCGREGOR, 1964a, 1964b). The investigations of earlier workers (BoYo and his colleagues, 1936a, 1936b, and Smxo~, 1940) indicated that acquired malarial immunity is singularly ineffective against other than the blood forms of human plasmodia. SHORTTand GARHmUa (1948) convincingly confirmed this point by demonstrating the successful establishment of pre-erythrocytic schizogony in a patient possessing effective acquired immunity. More recently, GARNHAMand BRAe (1956) reported that acquired simian immunity had no effect upon the sporozoite, the pre-erythrocytic phase or the first generation of asexual erythrocytic forms of homologous P. cynomolgi, but acted efficientlyupon the second and subsequent generations of asexual erythrocytic parasites. While similar precise studies in human malaria are strongly indicated, there seems at present no reason to believe that acquired immunity in man differs fundamentally from that in monkeys. If, therefore, the extreme specificity of naturally acquired malarial immunity is accepted, the following important sequels become discernible. 1. Inoculation of viable sporozoites will lead to successful penetration of hepatic cells even in the immune host. 2. Pre-erythrocytic development will proceed normally in the immune host. 3. The immediate progeny of the pre-erythrocytic forms will establish themselves in the blood of the immune subject. 4. Gametogenesis, provided this can stem from exo-erythrocytic or first-generation asexual erythrocytic forms, may not be directly inhibited in the immune host. It is now proposed briefly to consider how acquired immunity and plasmodial biology may combine to shape the pattern of malaria in individuals and in populations.

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The initiation and progress of asexual parasitaemia in the host

The morbid effects of malaria are broadly proportional to the degree of erythrocytic infection attained, and factors which influence the magnitude of the initial blood parasite density, or the subsequent rate of increase of parasitaemia, are consequently important. These are many and range from the little understood phenomenon of innate immunity to the complexities of haemoglobin chemistry. Only those, however, which may reasonably be expected to affect a normal host will be considered.

1. Quantum of sporozoite infection All sporozoites recovered from mosquitoes are not equally infective, and their presence in salivary glands cannot be regarded as evidence of viability. Yet, all things considered, the density of first-generation asexual parasites in the blood probably reflects the dose of sporozoites sustained by the host. Light inocula will give rise to scanty initial parasitaemia which may need several cycles of asexual development before achieving clinical significance. On the other hand, heavy successful inoculation will be followed by a correspondingly dense parasitaemia which may be of extreme clinical importance from the moment of first schizogony. JAMES,NICOL and SHUTE( 1 9 4 5 ) described how the number of sporozoites inoculated determines the nature of the ensuing clinical attack, and stressed the frequency with which abortive or "latent " infections follow light infection. The quantum of viable sporozoites inoculated may therefore be a matter of considerable importance to the host. SMITE(1945) found that in his laboratory-reared mosquitoes, infected from human subjects, the sporozoite content of salivary glands could vary greatly. In one insect he counted over 219,000 P. falciparum sporozoites. He also recorded that " i n heavily infected glands enormous numbers of sporozoites are situated in the salivary duct and it is presumably the case that these are discharged in the first few feeds." Unfortunately, this aspect of mosquito infection appears to have been neglected by the field entomologist and no information seems to exist on the range of sporozoite density in wild-caught mosquitoes for any region. It can only be hoped that some dedicated worker, undaunted by the technical problems involved, will ultimately rectify this defect. 2.

The reproductive potential of the pre-erythrocytic schizont The greater the ability to reproduce in its pre-erythrocytic stage, the more potentially dangerous will be the infecting plasmodial species. BRAY (1963) gives the merozoite content of the pre-erythrocytic schizont of the four species of human malaria as follows : P. malariae 2,000 ; P. ovale 15,000 ; P. vivax 10,000 ; and P. falciparum 40,000. Thus, given sporozoite inocula of equivalent density, and assuming uniform host reaction to infection, P. falciparum will produce an initial parasitaemia 20 times as great as P. malariae, 4 times as great as P. vivax, and about 3 times as great as P. ovale. Thus the long appreciated danger inherent in P. falciparum infections may, in no small measure, be due to its great capacity to multiply in its pre-erythrocytic stage, and to the apparent insusceptibility of this phase to immune influences. A seeming contradiction to the importance of pre-erythrocytic multiplication is the fact that P. ovale, despite the relatively high merozoite content of its hepatic schizont, is not noted for the severity of the clinical malaria it causes. This, however, may be partly explained by the relatively low capacity of this parasite to multiply in the erythrocytic stage. In our present state of knowledge it seems fitting to regard the pre-erythrocytic schizont as the force which imparts the initial momentum to erythrocytic infection. A brief example may be given.

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Example : Two identical infants possessing blood volumes of 800 ml. are infected with malaria. One receives 50,000 sporozoites of P. falciparum, and the other an equal dose of P. malariae sporozoites. Assuming full sporozoite viability, uniform parasite development, and totally absent host reaction, the following calculations may be made : Sporozoite dosage Pre-erythrocytic schizont density Total merozoite density of mature pre-erythrocytic schizonts

Initial erythrocytic parasitaemia

P. falciparum 50,000

P. malariae 50,000

50,000

50,000

50,000 × 40,000 = 2,000,000,000 ~ 2,000,000,000 per c.mm. 800 × 1,000 2,500 per c.mm. =

50,000 × 2,000 100,000,000 100,000,000 per c.mm. 800 × 1,000 125 per c.mm.

3.

The reproductive potential of erythrocytic schizogony P. falciparum and P. vivax each tend to produce erythrocytic schizonts with a mean merozoite content twice that of schizonts ofP. malariae and P. ovale and, in consequence, must be regarded as capable of advancing par0sitaemia beyond initial levels, in susceptible individuals, at twice the rate of the others. The time required for completion of each asexual cycle of development is also pertinent and in this respect P. falciparum, vivax, and ovale infections, with cycles of approximately 48 hours, may be expected to achieve more rapid multiplications than P. malariae infections (72 hours). Though the reproductive potential of erythrocytic schizogony is by itself important, a truer picture of the pathogenicity of each of the four human species of parasite is obtained if the combined potential of pre-erythrocytic and erythrocyt2c multiplication is considered. P. falciparum, with its 40,000-fold amplification in the liver, with the high merozoite content of its erythrocytic schizont, and with its 48-hour cycle of development, emerges as the most dangerous of the four species. P. vivax may rank next, for although its hepatic multiplication is less than that of P. ovale, this disadvantage is probably more than offset by its greater capacity for reproduction in the asexual erythrocytic stage. P. malariae, by those criteria, appears as the human parasite least likely to attain fearsome blood density. This pattern of possible aggression accords well with the accepted lethality of the various species. PAMPANA(1963) observes that, though a fatality rate as high as 25% may attend untreated primary infections with P. falciparum, and though P. vivax may kill in epidemics or if left untreated, infections with P. malariae and P. ovale are very rarely fatal. (The assessment of mortality from P. ovale has possibly been complicated by the frequent confusion of this parasite with the similar P. vivax.) WILSON (1936) and GARNHAM (1963) report that, in nature, effective communal immunity to P. falciparum takes longer to develop than immunity to the other human plasmodia. A partial explanation may be that this parasite, with its capacity for rapid multiplication, is capable of exploiting any weakness in immune defences. To be effective against it, acquired immunity must be of a high order and constantly maintained, and this may take time to achieve. 4.

The blood volume of the host The size of the blood volume of the host also plays a part in determining the initial

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density of parasitaemia and probably also its clinical importance. For example, sporozoke inoculations of equivalent dosage and viability may be expected to produco in an infant an initial parasitaemia some 6-8 times as great as in an adult of average size. For this reason young children constitute a section of the population singularly prone to develop severe, often lethal, parasitaemia.

5.

The degree of immunity possessed by the host

Ultimately, the outcome of any infection must depend, on one hand, on the size of the initial parasitaemia and its capacity to increase, and on the other, the ability of the host immediately, or after some delay, to curb parasitic multiplication.

Acquired immunity in the individual and in the community Immunity to malaria appears to be acquired in two broad stages. In the first, although an anti-parasitic element develops, the tendency is for the host to become increasingly tolerant of the pyrogenic products of erythrocytic schizogony, and to become capable of withstanding moderately dense parasitaemia with few clinical illeffects. The second stage is that in which immunity becomes increasingly directed towards eliminating the parasite. In the ensuing remarks references to the acquiskion of immunity relate mainly to this secondary, antiplasmodial, effect. (I) Acquired immunity in the individual As recent studies have shown that acquired immunity appears to act with considerable specificity against the late stages of asexual erythrocyfic development, it seems likely that antigen is formed or becomes accessible at this time. Thus, while density of parasitaemia may control the amount of antigen formed, the frequency of schizogony may determine the duration of the antigenic stimulus. SINTON (1939) observed that persistent parasitaemia of a relatively low grade induced better immunity than massive infections of short duration, thereby establishing the importance of duration of stimulus over quantity of antigen formed. An element of time may consequently be essential to the evocation of a satisfactory immune response, and in susceptible individuals the smoothest acquisition of immunity to primary infections may foUow relatively light sporozoite inoculations. These would give rise to initial parasitaemia of low density, which may even need one or more cycles of asexual development before producing clinical effects. This preliminary, asymptomatic, period could be important in priming the immune defences of the host and in ultimately ensuring the restriction of parasitic multiplication before grave illness supervenes. This possibility received some support from the studies, already quoted, of JAMEs and his colleagues. It should not be supposed that an immunity successfully elaborated towards a mild parasitaemia will be effective against further but much heavier challenges. The sudden appearance of a dense parasitaemia in a moderately immune individual could mop up all circulating antibody, and its subsequent progress could depend on the rate of multiplication and on the speed with which the host can synthesize adequate fresh supplies of antibody. Further, since neither the sporozoite nor the pre-erythrocytic phase appears to be susceptible to immune influences, the quantum of sporozoites inoculated by mosquitoes probably remains of significance throughout the lib of the " immune " individual. The role of a secondary exo-erythrocytic cycle in the acquisition and maintenace of immunity requires clarification. It is possible that, in those species postulated to posses such a phase (P. vivax, ovale and malariae), the periodic spillage of broods of asexual erythrocytic parasites supplies booster doses of antigen. In this way an effective

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immunity might be maintained after an isolated sporozoite infection for as long as the secondary exo-erythrocytic cycle persists. However, as no secondary exo-erythrocytic phase has been demonstrated in P. falciparum infections, the persistence of immunity towards this species would appear to depend upon repeated sporozoite infection or upon exacerbations of asexual activity stemming from persistent, refractory, erythrocytic forms. With regard to gametogenesis, it would seem likely that if this could arise from firstgeneration asexual parasites (or from their pre-erythrocytic precursors) then even highly immune individuals may retain the capacity to infect mosquitoes. Such a situation is suggested by the findings of MUIRHEAD THOMSON (1957), that in a Liberian population just as many mosquito infections originated from ostensibly "immune" individuals as from the main gametocyte reservoir--the young children under 5 years of age. However, if acquired immunity has little direct effect upon gametocytes, it probably has, as MACDONALD (1957) points out, a considerable indirect effect in limiting their numbers through the restriction of asexual parasitaemia. Thus, though immune and nonimmune individuals may both be capable of infecting mosquitoes, heavy infections originate almost exclusively from subjects with poor immunity. (2) Communal immunity MACDONALD (1957) has described the two epidemological extremes in which malaria can exist, namely the stable form and the epidemic. The former occurs wherever transmission is frequent and largely perennial, and is characterized by parasite rates which are highest in young children and which decline as age advances, until a state approaching commensalism is achieved in adult life. The latter occurs whenever transmission increases rapidly in a susceptible population and is characterized by the occurrence of severe clinical malaria in all age-groups. In stable circumstances young children constitute the recognizable gametocyte reservoir because of their inability to restrict asexual parasite density but, as Muirhead Thomson has shown, the older sections of the population give rise to as many mosquito infections even although their gametocyte-carrying capacity is difficult to establish by routine methods of blood examination. In an epidemic, because acquired immunity is low, individuals, irrespective of age, tend to develop heavy gametocytaemia. Thus an essential difference between the two types of malaria may be not so much the frequency with which mosquitoes become infected but rather the degree to which they do so. The basic distinction calling for emphasis is that in circumstances of hyperendemicity substantial acquired immunity in the population probably leads to a high frequency of low-density sporozoite inoculation, which may prove of advantage in promoting the relatively smooth acquisition of immunity in the young susceptible members, whereas in epidemic environments the low immune status of the entire population tends to favour the transmission of heavy sporozoite concentrations causing, eventually, blood infections of extreme hazard. This distinction may seem theoretical at first sight, but there is some evidence which may support it. MACDONALD(1950) demonstrated that the calculated rates of re-inoculation in some West African children were considerably lower than available entomological data indicated. DAVIDSONand DRAPER (1953) working in a hyperendemic area of East Africa, concurred and concluded that for some unknown reason many infections (estimated at between 9 in 10 and 99 in 100) failed to give rise to a persistent parasitaemia. Such a situation may perhaps be best explained by the hypothesis that in hyperendemic areas the range ef density of sporozoite inocula is such that, when sustained by infants possessing passive or actively acquired immunity in varying amount, only a small proportion result in persistenr parasitaemia detectable by field methods at present employed. PAMPANA(1963), however, has commented upon the

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high mortality that attends the second, but not the first, wave of morbidity in a malaria epidemic, and considers the phenomenon a sequel to mosquitoes becoming heavily infected from the gametocyte-rich blood of the early cases. Sporadic epidemics may occur in the indigenous populations of highly malarious regions as a result of the influx of non-immunes (TALIAFERRO, 1949) and an explanation may be that infection in the immigrants leads to heavy blood concentrations of gametocytes, to heavy mosquito infection, and eventually to the inoculation of the indigenous population with abnormally heavy doses of sporozoites. The dense initial parasitaemia resulting from such infections could well overcome established levels of immunity and produce a sharp epidemic. Were this indeed the true course of events, the questions arise as to whether the degree of immunity possessed by communities in stable environmerits differs from area to area, and whether an immunity acquired in one locality might fail, transiently at least, to protect against heavier prevalent sporozoite transmission in another. The size of sporozoite inoculum required to break the resistance of an "immune " African adult is not known and, although the phenomenon appears to occur but rarely ill nature, an attempt to relate peripheral blood parasite densities to sporozoite dosage may be of value. Example :--A P. falciparum density of 10 trophozoites per c.mm. occurring in an "immune " adult individual (blood volume = 6 litres) is considered to represent first-generation erythrocytic parasitaemia. Assuming uniform parasite dispersion in the blood, the total trophozoite density within the host is I0 × 6,000 × 1,000 ---- 60,000,000. Further, assuming that those parasites represent the total output of the pre-erythrocytic phase of development, the sporozoite dose which initiated this 60,000,000 degree of infection is -- 1,500. As few experienced malariologists would 40,000 consider a parasitaemia of 10 per c.mm. to be of any significance in an African adult resident in stable circumstances, and as it is unlikely that all sporozoites inoculated would successfully establish the pre-erythrocytic cycle, it seems necessary to multiply this number by a factor before a possible breakdown of immunity could be envisaged. Such a factor may be 100 or even greater. Any assessment of the importance of immunity possessed against weight of infection sustained is unfortunately bedevilled by the possible occurrence in different areas of variations in the antigenic composition of strains of the same species of parasite. However, with the development of new techniques, such as those employing fluorescent antibody (TOBIE et al., 1962 ; VOLL~-R,1962) and haemagglutination (DrsowiTz and STvm, 1962) principles, it may eventually prove possible to measure, with accuracy, differences in the levels of immunity of communities in stable regions, which show what, by present accepted malariometric standards, seem to be an identical pattern of resistance to malarial infection.

The strain specificity of acquired immunity From what has been said above it will be apparent that the author considers the quantum of sporozoite inoculation sustained by the host to be a factor of considerable epidemoilogical significance. Perhaps nowhere is this more important than in the study of strain specificity of acquired immunity. Much of the evidence which today supports the prevalent view that intraspecies variation in antigenicity is both frequent and widespread, rests upon the experimental challenge of acquired immunity by reinfection with the same species. Unfortunately in many instances the challenging infection was induced by sporozoites, and the investigations made at a time when precise knowledge of the reproductive capacity of pre-erythrocytic forms did not exist. Consequently,

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perusal of the results of many such studies does not permit a decision on whether the establishment of the challenging infection was due to difference in the antigenic composition of the parasite or to the strength of the challenge exceeding the measure of immunity possessed by the host. SINTON (1940), challenging with the homologous parasite, found difficulty in breaking acquired immunity by reinfection with trophozoites, but obtained a much higher success rate from inoculation of sporozoites. His results suggest that the success of sporozoite-induced challenges might be attributable to their giving rise to a sudden parasitaemia of a magnitude sufficient to absorb all circulating antibody. This would imply that the effectiveness of an acquired immunity is moulded by the nature of the infection(s) which produced it, and is governed by the availability of antibody in relation to the number of first-generation erythrocytic parasites which suddenly appear in the blood after reinfection. Experiments to test variation in parasitic antigenicity must be designed with scrupulous attention to the size of the challenging infection. This is, perhaps, more important in malaria than in many other diseases, for the battle of immunity is not fought at the point of invasion but at a later stage when the parasite has consolidated its position and has reinforced its strength. Apparently small fluctuations in the size of inoculations of viable sporozoites may result in important variations in the density of initial erythrocytic parasitaemia. Since West African immune serum recently proved surprisingly effective in the treatment of East African P. falciparum parasitaemia (McGREGOR et al., 1963), it would appear that, in Africa at any rate, antigenic variations in strains of this parasite are neither widespread nor frequent. The whole question of strain specificity of immunity should therefore be re-examined, and the possibility considered that the effectiveness of acquired immunity to reinfection may be largely determined by the size of the challenging infection.

Conclusions Advances in scientific knowledge frequently bring to light as many problems as they solve. So it is with our present understanding of the dynamics of malarial infection. In this communication the author has attempted to outline some of the questions that recent research in malaria appears to be posing. Some of the points are speculative but they have been made in an endeavour to attract the attention of the many competent malariologists to the need for a critical and dispassionate re-appraisal of the enormous volume of malariometric data compiled over the past half century. To achieve a better understanding of the progress of malaria in the individual and in the community, more information is required on the sporozoite concentrations that develop in the salivary glands of mosquitoes in different areas of the world, on the numbers of sporozoites that mosquitoes may inoculate in the course of a single feed, on the duration of both primary and secondary exo-erythrocytic phases within the host, on the uniformity with which such forms rupture, on the mechanism of acquired immunity, on the susceptibility of the various phases of the plasmodial life cycle to immune influences, on the levels of serological immunity achieved by different populations, and on the specificity of immunity to different strains of the same species of parasite.

Summary 1. Recent advances in knowledge concerning plasmodial biology and acquired immunity in malaria are examined, and their possible effects upon the epidemiology of malaria considered.

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2. The opinion is expressed that the specificity of immune processes and the reproductive capacity of pre-erythrocytic schizonts combine to indicate that the density of viable sporozoites inoculated may be a factor of importance in shaping the pattern of malaria in both the individual and the community. 3. Strain specificity of immunity is discussed and attention drawn to the need for careful control of the size of challenging infections in the course of studies designed to elucidate intra-species differences in plasmodial antigenicity. REFERENCES BOYD, M. F. & KITCHEN, S. F. (1936a). Amer. J. trop. Med., 16, 317. ~., STRATMAN-THOMAS,W. K. & KITCHEN, S. F. (1936b). Ibid., 16, 139. BRAY, R. S. (1957). Mem. Lond. Sch. Hyg. trop. Med., No. 12. (1963). Int. Rev. trop. Med., 2, 41. COHEN, S. & McGREGOR, I. A. (1963). Immunity to Protozoa. Oxford: Blackwell Scientific Publications. , - & CARRINGTON,S. P. (1961). Nature, Lond., 192, 733. DAVlDSON, G. & DRAPER, C. (1953). Trans. R. Soc. trop. Med. Hyg., 47, 522. DESOWlTZ, R. S. & STEIN, B. (1962). Ibid., 56, 257. GARNI-IAM,P. C. C. (1963). Immunity to Protozoa. Oxford : Blackwell Scientific Publications. & BRAY, R. S. (1956). Rev. bras. Malariol, 8, 151. JAMES, S. P., NICOL, W. D. & SHOTE, P. G. (1936). Proc. R. Soc. Med., 29, 27. MACDONALD, G. (1950). Trop. Dis. Bull., 47, 522. (1957). The Epidemiology and Control of Malaria. London : Oxford University Press. McGREGOR, I. A. (1964a). Trans. R. Soc. trop. Ailed. Hyg., 58, 80. (1964b). Amer. J. trop. Ailed. Hyg., 13, 237. ~., CARRINGTON,S. P., & COHEN, S. (1963). Trans. R. Soc. trop. Med. Hyg., 57, 170. MtlIRHEAD-THOMSON,R. C. (1957). Amer. J. trop. Med. Hyg., 6, 971. PAMPANA, E. (1963). A Textbook of Malaria Eradication. London : Oxford University Press. SHORTT,H. E. & GARNHAM,P. C. C. (1948). Trans. R. Soc. trop. Med. Hyg., 41, 785. SHUTE, P. G. (1945). Ibid., 38, 493. SINTON, J. A. (1939). J. Malar. Inst. India., 2, 191. - (1940). Trans. R. Soc. trop. Med. Hyg., 33, 439. TALIAFERRO,W. H. (1949). Malariology, Ed. M. F. Boyd, Philadelphia : W.B. Saunders. TomE, J. E., KUVlN, S. F., CONTACOS,P. G., COATNEY,G. R. & EVANS, C. B. (1962). Amer. J. trop. Med. Hyg., 11, 589. VOLLER,A. (1962). Bull. Worm Hlth. Org., 27, 283. WILSON, D. B. (1936). Trans. R. Soc. trop. Med. Hyg., 29, 583.