- Email: [email protected]
Epidemiological evidence for an association between chloroquine resistance of Plasmodium falciparum and its immunological properties
105
Parasitology Today, vol. 9, no. 3, 1993
Toxoplasma 5. The t w o clusters distinguished in E. histolytica 4, which correspond to the pathogenic and nonpathogenic zymodemes 13, present an obvious medical relevance, and hence, would deserve a specific status. Similarly, the 'virulent' clonal lineage found in T. gondii s could also be made a species, when its individuality and medical relevance are confirmed, and if specialists of the study of this parasite consider it desirable. Lastly, the striking genetic dissimilarities shown within G. duodenalis 3 could serve as a guide to look for significant biological and medical differences among the putative species, which would confirm their value as separate taxa. The new Linnean taxa built in this way could still be too broad for medical purposes. They can be usefully supplemented with the notion of 'natural clones '~,d7 brought by the clonal model. If the clones, in certain cases, have to be taken as taxonomic units, instead of the species, two problems appear: (I) the number of clones that comprise a given species is potentially unlimited; and (2) the clonal variability recorded
in a given study is highly dependent upon the level of resolution of the genetic markers employed, To solve these problems, the notion of 'clonet' (all the isolates in a clonal species that appear to be genetically identical to one another on the basis of a particular set of markers) 6,2° can be used as a taxonomic unit. Widespread clonets (major clones) 17 call for priority studies to deal with issues such as virulence, resistance to drugs and host specificity. The ubiquitous Giardia genotype referred to as zymodeme M4 (Ref. 7), or as zymodeme I (Ref. 3), as well as the 'virulent' ubiquitous T gondii clone s are probably analogous to the major clonets identified in T cruzi 17. References
I Tibayrenc, M., Kjellber~, F. and Ayala, F.J. (1990) Proc. Natl Acad. Sci. USA 87, 2414 2418 2 Tibayrenc, M. et el. ( 1991) Proc. Natl Acad. Sci USA 88,5129 5133 3 Andrews, R.H. et al. (I 989) Int.J. Parasitol. 19. 183 190 4 Blanc,D.S. (1992)J. Protozool.39, 471 479 5 Sibley, LD. and Boothroyd, J.C. (1992) Nature 359, 82 85 6 Tibayrenc,M. and Ayala, F.J.( 1991) Parasitology
Today 7, 228 232 7 Meloni, B.P., Lymbery, A.j. and Thompson, R.C.A. (1988) Am. J. Trap Mad Hyg. 38, 65 73 8 Meloni, B.P. et al. (1989) Am. J. Trap. Meal Hyg. 40, 629 637 9 Sargeaunt, P.G. (1987) Parasitology Today 3, 40 43 10 Proctor, E.M. et al. (1987) Am. J. Trap. Med. Hyg. 37, 296 301 tl Dard~, M.L. et aL (1988) Am. J. Trap. Mad Hyg. 39, 551 558 12 Dard6, M.L. et al. (1990) Bull. Sac. F~ Parasitol. 8, 238 13 Sargeaunt, P.G. et al. (1982) Trans R. Sac Trap. Mad Hyg. 76, 401 402 14 Grimont, P.A.D. (1988) Can.J. Microbial. 34, 541 547 15 Nei, M. (1972) Am. Nat. 106,283 292 16 Ready, P.D. and Miles, M.A. (1980) Trans. R. Sac Trap. Med. Hyg. 74, 238 242 r7 Tibayrenc, M. and Ayala, F.J.(1988) Evolution 42, 277 292 J8 Jenni, L. et al. (1986) Nature 322, 173 175 19 Sargeaunt, P.G. (1985) Trans. R. Sac. Trap. Meal Hyg. 79, 86 89 20 Tibayrenc, M., Kjellberg, F. and Ayala, F.J. ( 1991) Biascience4 I, 767 774 21 Rannala,B.H. (t990)J. Parasitol. 76, 929 930 Michel Tibayrenc is at the UMR CNRS/ORSTOM 9926: G~.n~tique Mol~culaire des Parasites et des Vecteurs, ORSTOM, BP 5045, 34032 Montpellier Cedex 0 t, France.
Epidemiological Evidence for an Association Between Chloroquine Resistance of Plasmodium falciparum
and its Immunological Properties J.C. Koella The appearance of chloroquine resistant genotypes 0fPlasmodium falciparum has thwarted the goal of global eradication of malaria. Although much effort has been put into understanding the molecular mechanisms of chloroquine resistance, many questions about its distribution remain open: Why, some 30 years after the emergence of chloroquine resistance, have resistant genotypes not taken over the population? Why have many parasites remained sensitive? Why, after its first appearance in Africa, has chloroquine resistance spread so rapidly through sub-Saharan Africa? In this paper Jacob Koella reviews epidemiological data that suggest that an answer to these questions may involve an association between chforoquine resistance and immunological properties of malaria parasites. © 1993, Elsevier Science Pubhshers Ltd, (UK)
Chloroquine resistance first appeared in Thailand and South America in the late 1950s (Re£ I; T. Harinasuta, S. Migasen and D. Boonag, abstract*), and has since spread to most parts of the world where malaria is endemic 2,3. Several observations on the distribution of resistance suggest that selection on resistance is frequency dependent, so that when resistant parasites are rare they have a large advantage over sensitive parasites, but as they become more common, they have a disadvantage. Such frequency-dependent selection would arise if chloroquine resistance of * UNESCO First Regional Symposium on Scien tific Knowledge of Tropical Parasites (1962) Singapore
a parasite is associated with its immunological properties, as has been suggested by Clyde 4 and Peters S. This idea relies on the generally accepted assumption6, 7 that immunity against malaria is strain specific. Then, if resistance is associated with immunological properties, resistant parasites will encounter little immunity and thus be favoured shortly after invading a population. As resistance becomes more common, the advantage of resistance will be balanced by high levels of immunity, so that sensitive parasites will be maintained in the population. Thus, different immunological properties of resistant and sensitive parasites would explain why it is that, in areas with intense transmission, resistance initially spreads very rapidly, but then remains fairly stable 8.
106
Parasitology Today, vol. 9. no. 3, 1993
immunity. This prediction is made by any hypothesis that involves a cost of resistance. These patterns should be observed only in areas of intense transmission, because little immunity is developed in areas where transmission rates are low. In testing these predictions, this paper will discuss only resistance, in vitro, which is measured as the EC50, ie. the concentration of chloroquine that kills 50% of the parasites, in vitro, and will neglect the better known association between in vivo resistance and immunity 9. Resistance, in vitro, is determined in defined laboratory conditions and reflects the intrinsic properties of the parasite more closely than does resistance, in vivo, which is determined by the synergism between resistance of the parasite and the immune system9. The ECs0 was estimated with the method advocated by Hills I°. The tests of the predictions rely on two assumptions. (I) Following most epidemiological studies (eg. the Kilombero Malaria Project pi), the level of immunity in humans is not measured explicitly, but is approximated with age. This is possible because immunity against malaria increases only slowly with age ~2. (2) Chloroquine is continually used. In Tanzania, for example, more than 600 million tablets of chloroquine were used every year during a mass drug administration (MDA) program 07V.H. Wemsdorfer, pets. commun.),
Epidemiological Predictions
An association between resistance and immunogenic properties would ideally be tested experimentally, but epidemiological patterns of resistance can also give an indication of the validity of the idea The major prediction of an association is that, in areas of intense transmission, the time since emergence of resistance as well as the immune status of the patients are important factors in determining chloroquine resistance. Thus, during the initial stages of the spread of resistance, because resistant parasites will encounter little immunity but sensitive parasites will be killed by drug treatment, fewer sensitive isolates will be obtained from immune than from non-immune patients and the average level of resistance of the parasites obtained from the immune patients will be higher. Where resistance has been established for some time, and immunity has been developed against the resistant parasites, the opposite pattern will appear. A second, though not exclusive, prediction is that, in areas with intense transmission, where most adults have developed a high degree of immunity against malaria, the frequency of resistant parasites will not reach 100%, but rather will be maintained at an intermediate equilibrium level because of the high level of
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1987
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Perhaps the most complete data that allow the predictions of an association between resistance and immunological properties to be tested have been collected by the Ifakara Centre in a repeated cross-sectional study near Ifakara, Kilombero District, Tanzania I
Box I. Areas of Differing Intensity of Transmission of Malaria Low transmission West Asia: Afghanistan
Iran Nepal Oman Pakistan Saudi Arabia Yemen Intermediate transmission Southeast Asia: Bangladesh Laos Malaysia Thailand Vietnam Southeast Asian Islands: Indonesia Philippines Vanuatu Intense transmission East Africa: Burundi Kenya Madagaskar Malawi Mozambique Somalia Sudan Tanzania Uganda Zambia
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and even up to 1990, several years after the MDA was phased out, chloroquine can be found in nearly 50% of a population ~3.
I
1989
Year Fig. I. The ECso values from each isolate obtained from children in the MCH clinic (+) and from schoolchildren(diamonds) in Ifakara, Tanzania. The curves show the regressionsof log (ECso) against year. The slopes of the lines were O. 126 (P <0.001) for MCH (broken curve) and -0.233 (P < 0.001) for schoolchildren(solid curve). (Adapted from Ref. 15.)
West Africa: Angola Cameroon Central African Republic CSte d'lvoire Gabon The Gambia Ghana Guinea Guinea-Bassau Liberia Mall Nigeria Zaire
Parasitology Today, vol. 9, no. 3, i 993
In this area of very intense transmission, parasites were obtained from children attending the Mother and Child Health (MCH) clinic every May between 1982 and 1989, and from 10-15-year-old children attending a local school between 1986 and 1989. The study showed that, after the initial emergence of resistance in 1981, parasites isolated from non-immune children attending the MCH clinic became increasingly resistant up to 1989 (Fig. I). Conversely, parasites isolated from schoolchildren, who generally have high levels of immunity in this area ~i.i6, had, on average, a high ECs0 in 1986, but became more sensitive in subsequent years. Further evidence stems from the W H O database on drug resistance, which contains the data of all reported microtests. The analysis shown here used the ECs0 for the samples obtained between 1981 and 1989 from children who were less than 15 years old, with the data grouped according to geographical area (Box I) to get an impression of intensity of transmission. Those combinations of area and year that had more than ten samples were
107
used. The effects of age and year on the logarithm of ECs0 were estimated with an analysis of variance that took account of the uptake of chloroquine by the children. The results of this analysis (Table I) were consistent with the predictions of the model: in areas with low transmission rates (West Asia), age had no effect on resistance; in areas of more intense transmission, year and age interacted to determine resistance, and the interaction depended on whether or not resistance was increasing. In those areas where, on average, resistance was increasing (Africa, Asian Islands), ECs0 initially increased with the age of the children from whom the isolates were obtained, but later decreased with age. Where, on average, resistance was decreasing (Asia), no interaction was found. Thus, the repeated cross-sectional study in Ifakara and the patterns found in the W H O database are consistent with an association between chloroquine resistance and immunogenic properties of the parasites. How can such an association arise? The answer to this question is important, because different mechanisms will lead to differ-
ent predictions about the spread of resistance. How
Can an Association
Arise?
Chloroquine resistance and immunogenic properties could be associated because the two traits are correlated genetically. On the one hand, genes encoding proteins involved in resistance could be closely linked to those encoding proteins responsible for antigenic properties, so that the association would reflect a linkage disequilibnum between resistance and antigenic properties, arising when resistant parasites introduced into a population carry novel antigens. Linkage could arise if the genes that encode antigens are on the same chromosome as are those involved in resistance, or if most parasite zygotes are created by selffertilization. This explanation seems unlikely for several reasons. First, even a slight degree of recombination between genes would soon lead to linkage equilibrium, resulting in the disappearance of an initial association after only a few parasite generations, ie.
Table I. Analysis of variance of chloroquine resistance with time a Source Asia CQ Year Age Year* age
D e g r e e s of f r e e d o m
S u m of squares ( T y p e III)
F value
2 I I -
1.220 0.307 1.287 -
0.76 0.38 1.60
A s i a n Islands CQ Year Age Year* age
4 I I I
7.059 I 1.866 6.432 6.255
1.89 12.70 6.88 6.70
0.44 <0.001 0.009 0.01
0.267 2.35 I -0.028
East A f r i c a CQ Year Age Year* age
2 I I I
17.329 42.272 28.252 28.21 I
8.95 43.69 29.20 29.16
<0.001 <0.001 <0.001 <0.001
0.314 2.67 I -0.031
West Africa CQ Year Age Year* age
4 I I I
7.801 22.211 5.573 5.741
3.14 35.72 8.96 9.23
0.01 <0.001 0.003 0.002
0.279 2.076 -0.025
West Asia CQ Year Age Year* age
4 I I -
2.809 2.546 0.228 -
1.64 5.95 0.53 -
0.17 0.02 0.47 ns
-0.073 -0.013 -
-
P
Estimate
0.47 0.54 0.20
-0.024 -0.024
ns
b
_
•For each area, the effects of chloroquine (CQ) usage, the number of years since 1980, age of the children involved and the interaction of year and age (*) on the estimate of the ECs0 are shown. Where the interaction was insignificant, the table shows the results of an analysis done without the interaction term. Abbreviations: ns, not significant. The hypothesis char resistance is associated with immunogenic properties predicts char the estimate of the interaction is zero in areas with low intensity of transmission (West Asia), that the interaction is negative in areas with intense transmission where resistance is increasing (Asian Islands, Africa), and that the resistance decreases with age where resistance is close to constant (Asia).
108
Parasitology Today, vat. 9, no. 3, 1993
after several months. This is contrary to the data shown, where an association was observed for several years. Furthermore, none of the genes that are assumed to be involved in chloroquine resistance w,18 are found on the same chromosome as are those genes known to code for antigens. Finally, though the degree of self-fertilization is debatable~9 2~, evidence suggests that mating between different genotypes and genetic recombination are fairly common in many parasite populations. On the other hand, resistance could be directly associated with immunogenic properties, if the genes coding for resistance have a pleiotropic effect on the immunogenic properties of the parasite. Resistance is thought to arise by overproduction of a membranebound glycoprotein functioning as an export pump 22. The differential expression of a membrane-bound protein might lead to differential antigen properties. However, none of the genes known to code for antigenic properties has an effect on resistance, and none of the three genes currently thought to be involved in chloroquine resistance has a known effect on immunogenic properties. A direct association between resistance and immunogenic properties would also arise if the presence of chloroquine itself altered the antigenic properties of a parasite. Because resistant parasites, by definition, can have longer contact with chloroquine within the human host than can sensitive parasites, the immune system experiences the chloroquine-induced properties of resistant parasites more often, but their chloroquine-free properties less often, than those of sensitive parasites. Therefore, immunity is more rapidly acquired against resistant parasites. This hypothesis is made plausible by the property of chloroquine to
modify the structure of cell membranes, though no direct evidence is known. Concluding Remarks
An association between chloroquine resistance and antigenic properties has many implications for control strategies. At the very least, an association makes comparisons of resistance, in vitro, between areas with different patterns of transmission and immunity difficult. Only if parasites that have been isolated from non-immune patients are used do we get an impression of the resistance in the population of parasites. More importantly, a direct association would ensure that resistance, because of its cost of enhanced immunity, cannot increase continuously in a population, but will evolve towards a stable level. Conversely, if resistance has no other cost, resistance will not disappear from the population as drug pressure is relaxed, because the cost imposed on resistance due to more intense immunity as well as the benefit of resistance are relaxed when chloroquine is no longer used. Because such conclusions rely not only on the presence of an association between resistance and immunogenic properties, but also on the mechanism of the association, controlled laboratory studies of the associations of resistance to chloroquine and other drugs with immunogenic properties should be able to enhance our understanding of the spread and distribution of drug resistance. Acknowledgements I thank the malaria teams of the Swiss Tropical Institute and of Imperial College for many inspiring discussions on chloroquine resistance, and Walther Wernsdorfer
Recent
and an anonymous reviewer for detailed comments on the manuscript. This work was supported in part by a grant from the Wellcome Trust. References I Moore, D.V. and Lanier, J.E. (1961) Am. J. Trap. Meal Parasitol. 74, 243 244 2 Wernsdorfer, W.H. ( 1991 ) Parasitology Today 7, 297 303 3 Anon. (1990) WHO Stat. Q. 43, 68 79 4 Clyde, D.F. (1958) Riv. Malarial. 37, 263 276 5 Peters, W. (1987) Chemotherapy and Drug Resistance in Malaria (Vol. 2), Academic Press 6 Forsyth, K.P. et al. (1989) Am. J. Trap. Meal Hyg. 42, 259 265 7 Mendis, K.M., David, P.H. and Carter, R. (I 99 I) in Immunoparasitology Today (Ash, C. and Gallagher, R.B., eds), pp 34 37, Elsevier Trends Journals, Cambridge 8 Bj~rkman, A. (1991) in Malaria: Waiting for the Vaccine (-rar~ett, G.A.T., ed.), pp 105 120, John Wiley & Sons 9 TargetS, G.A.T. (1984) Antimalarial Drugs (Vol. I) (Peters, W. and Richards, W.H.G., eds), Springe~Verlag I0 Hills, M., Hudson, C. and Smith, P.G. (1986) Working Paper No. 2.8.5, WH O II Kilombero Malaria Project (1992) Trans. R. Sac. Trap. Meal Hyg. 86, 499 504 12 Molineaux, L and Gramiccia, G. (1980) The Garki Project: Research on the Epidemiology and Control of Malaria in the Sudan Savanna of West Afnca, WHO, Geneva 13 Kilombero Malaria ProJect Mere. Inst. Oswaldo Cruz (in press) 14 Tanner, M. et at. (1987) East Af~ Meal J. 64, 464470 15 Koella, J.C. et al. (1990) Trans. R. Sac. Trap. Meal Hyg. 84, 662 665 16 Tanner, M. et al. (1986) Mere. Ins~ Oswaldo Cruz 81 (Suppl II), 199 205 17 Wellems, T.E., Walker-Jonah, A. and Panton, L.J. (1991) Proc. Natl Acad Sci. USA 88, 3382 3386 18 Foote, S.J.etal. (1990) Nature 345, 255 258 19 Tibayrenc, M. and Ayala, F.J. (1991) Parasitology Today 7, 228 232 20 Walliker, D. ( 1991 ) Parasitology Today 7, 232 235 21 Read, A.F. et al. (1992) Parasitology 104, 387 395 22 Warhurst, D.C. (1988) Parasitology Today 4, 211 213 Jacob Koella is at the Institute of Terrestrial Ecology, ETH ZE~rich, Grabenstrasse 3, CH8952 Sehlieren, Switzerland
a r t i c l e s in t h e Trends J o u r n a l s . . .
The adaptive significance of self-medication, D.H. Clayton and N.D. Wolfe (I 993) Trends in Ecology and Evolution 8, 60-63 Transcription factor based therapeutics: drugs of the future, M.G. Peterson and V.R. Baichwal (1993) Trends in Biotechnology I I, I 1-18 Colony stimulating factors, cytokines and monocyte-macrophages - some controversies, J.A. Hamilton (1993) Immunology Today 14, 18-24
Multiple levels of peripheral tolerance, B. Arnold, G. SchSnrich and G.J. H~immerling (1993) ImmunologyToday 14, 12-14 The role of individual human cytochromes P450 in drug metabolism and clinical response, S. Cholerton, A.K. Daly and J.R. Idle (1992) Trends in PharmacologicalSciences 13, 434-439 The role of CD45 in T-cell activation - resolving the paradoxes? D. Alexander, M. Shiroo, A. Robinson, M. Biffen and E. Shivan (I 992) ImmunologyToday 13, 477-48 I A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport, M.D. Marger and M.H. Saier, Jr (I 993) Trends in BiochemicalSciences 18, 20-25
Parasitology Today, vol. 9, no. 3, 1993
Toxoplasma 5. The t w o clusters distinguished in E. histolytica 4, which correspond to the pathogenic and nonpathogenic zymodemes 13, present an obvious medical relevance, and hence, would deserve a specific status. Similarly, the 'virulent' clonal lineage found in T. gondii s could also be made a species, when its individuality and medical relevance are confirmed, and if specialists of the study of this parasite consider it desirable. Lastly, the striking genetic dissimilarities shown within G. duodenalis 3 could serve as a guide to look for significant biological and medical differences among the putative species, which would confirm their value as separate taxa. The new Linnean taxa built in this way could still be too broad for medical purposes. They can be usefully supplemented with the notion of 'natural clones '~,d7 brought by the clonal model. If the clones, in certain cases, have to be taken as taxonomic units, instead of the species, two problems appear: (I) the number of clones that comprise a given species is potentially unlimited; and (2) the clonal variability recorded
in a given study is highly dependent upon the level of resolution of the genetic markers employed, To solve these problems, the notion of 'clonet' (all the isolates in a clonal species that appear to be genetically identical to one another on the basis of a particular set of markers) 6,2° can be used as a taxonomic unit. Widespread clonets (major clones) 17 call for priority studies to deal with issues such as virulence, resistance to drugs and host specificity. The ubiquitous Giardia genotype referred to as zymodeme M4 (Ref. 7), or as zymodeme I (Ref. 3), as well as the 'virulent' ubiquitous T gondii clone s are probably analogous to the major clonets identified in T cruzi 17. References
I Tibayrenc, M., Kjellber~, F. and Ayala, F.J. (1990) Proc. Natl Acad. Sci. USA 87, 2414 2418 2 Tibayrenc, M. et el. ( 1991) Proc. Natl Acad. Sci USA 88,5129 5133 3 Andrews, R.H. et al. (I 989) Int.J. Parasitol. 19. 183 190 4 Blanc,D.S. (1992)J. Protozool.39, 471 479 5 Sibley, LD. and Boothroyd, J.C. (1992) Nature 359, 82 85 6 Tibayrenc,M. and Ayala, F.J.( 1991) Parasitology
Today 7, 228 232 7 Meloni, B.P., Lymbery, A.j. and Thompson, R.C.A. (1988) Am. J. Trap Mad Hyg. 38, 65 73 8 Meloni, B.P. et al. (1989) Am. J. Trap. Meal Hyg. 40, 629 637 9 Sargeaunt, P.G. (1987) Parasitology Today 3, 40 43 10 Proctor, E.M. et al. (1987) Am. J. Trap. Med. Hyg. 37, 296 301 tl Dard~, M.L. et aL (1988) Am. J. Trap. Mad Hyg. 39, 551 558 12 Dard6, M.L. et al. (1990) Bull. Sac. F~ Parasitol. 8, 238 13 Sargeaunt, P.G. et al. (1982) Trans R. Sac Trap. Mad Hyg. 76, 401 402 14 Grimont, P.A.D. (1988) Can.J. Microbial. 34, 541 547 15 Nei, M. (1972) Am. Nat. 106,283 292 16 Ready, P.D. and Miles, M.A. (1980) Trans. R. Sac Trap. Med. Hyg. 74, 238 242 r7 Tibayrenc, M. and Ayala, F.J.(1988) Evolution 42, 277 292 J8 Jenni, L. et al. (1986) Nature 322, 173 175 19 Sargeaunt, P.G. (1985) Trans. R. Sac. Trap. Meal Hyg. 79, 86 89 20 Tibayrenc, M., Kjellberg, F. and Ayala, F.J. ( 1991) Biascience4 I, 767 774 21 Rannala,B.H. (t990)J. Parasitol. 76, 929 930 Michel Tibayrenc is at the UMR CNRS/ORSTOM 9926: G~.n~tique Mol~culaire des Parasites et des Vecteurs, ORSTOM, BP 5045, 34032 Montpellier Cedex 0 t, France.
Epidemiological Evidence for an Association Between Chloroquine Resistance of Plasmodium falciparum
and its Immunological Properties J.C. Koella The appearance of chloroquine resistant genotypes 0fPlasmodium falciparum has thwarted the goal of global eradication of malaria. Although much effort has been put into understanding the molecular mechanisms of chloroquine resistance, many questions about its distribution remain open: Why, some 30 years after the emergence of chloroquine resistance, have resistant genotypes not taken over the population? Why have many parasites remained sensitive? Why, after its first appearance in Africa, has chloroquine resistance spread so rapidly through sub-Saharan Africa? In this paper Jacob Koella reviews epidemiological data that suggest that an answer to these questions may involve an association between chforoquine resistance and immunological properties of malaria parasites. © 1993, Elsevier Science Pubhshers Ltd, (UK)
Chloroquine resistance first appeared in Thailand and South America in the late 1950s (Re£ I; T. Harinasuta, S. Migasen and D. Boonag, abstract*), and has since spread to most parts of the world where malaria is endemic 2,3. Several observations on the distribution of resistance suggest that selection on resistance is frequency dependent, so that when resistant parasites are rare they have a large advantage over sensitive parasites, but as they become more common, they have a disadvantage. Such frequency-dependent selection would arise if chloroquine resistance of * UNESCO First Regional Symposium on Scien tific Knowledge of Tropical Parasites (1962) Singapore
a parasite is associated with its immunological properties, as has been suggested by Clyde 4 and Peters S. This idea relies on the generally accepted assumption6, 7 that immunity against malaria is strain specific. Then, if resistance is associated with immunological properties, resistant parasites will encounter little immunity and thus be favoured shortly after invading a population. As resistance becomes more common, the advantage of resistance will be balanced by high levels of immunity, so that sensitive parasites will be maintained in the population. Thus, different immunological properties of resistant and sensitive parasites would explain why it is that, in areas with intense transmission, resistance initially spreads very rapidly, but then remains fairly stable 8.
106
Parasitology Today, vol. 9. no. 3, 1993
immunity. This prediction is made by any hypothesis that involves a cost of resistance. These patterns should be observed only in areas of intense transmission, because little immunity is developed in areas where transmission rates are low. In testing these predictions, this paper will discuss only resistance, in vitro, which is measured as the EC50, ie. the concentration of chloroquine that kills 50% of the parasites, in vitro, and will neglect the better known association between in vivo resistance and immunity 9. Resistance, in vitro, is determined in defined laboratory conditions and reflects the intrinsic properties of the parasite more closely than does resistance, in vivo, which is determined by the synergism between resistance of the parasite and the immune system9. The ECs0 was estimated with the method advocated by Hills I°. The tests of the predictions rely on two assumptions. (I) Following most epidemiological studies (eg. the Kilombero Malaria Project pi), the level of immunity in humans is not measured explicitly, but is approximated with age. This is possible because immunity against malaria increases only slowly with age ~2. (2) Chloroquine is continually used. In Tanzania, for example, more than 600 million tablets of chloroquine were used every year during a mass drug administration (MDA) program 07V.H. Wemsdorfer, pets. commun.),
Epidemiological Predictions
An association between resistance and immunogenic properties would ideally be tested experimentally, but epidemiological patterns of resistance can also give an indication of the validity of the idea The major prediction of an association is that, in areas of intense transmission, the time since emergence of resistance as well as the immune status of the patients are important factors in determining chloroquine resistance. Thus, during the initial stages of the spread of resistance, because resistant parasites will encounter little immunity but sensitive parasites will be killed by drug treatment, fewer sensitive isolates will be obtained from immune than from non-immune patients and the average level of resistance of the parasites obtained from the immune patients will be higher. Where resistance has been established for some time, and immunity has been developed against the resistant parasites, the opposite pattern will appear. A second, though not exclusive, prediction is that, in areas with intense transmission, where most adults have developed a high degree of immunity against malaria, the frequency of resistant parasites will not reach 100%, but rather will be maintained at an intermediate equilibrium level because of the high level of
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1985
1987
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Epidemiological S u p p o r t
Perhaps the most complete data that allow the predictions of an association between resistance and immunological properties to be tested have been collected by the Ifakara Centre in a repeated cross-sectional study near Ifakara, Kilombero District, Tanzania I
Box I. Areas of Differing Intensity of Transmission of Malaria Low transmission West Asia: Afghanistan
Iran Nepal Oman Pakistan Saudi Arabia Yemen Intermediate transmission Southeast Asia: Bangladesh Laos Malaysia Thailand Vietnam Southeast Asian Islands: Indonesia Philippines Vanuatu Intense transmission East Africa: Burundi Kenya Madagaskar Malawi Mozambique Somalia Sudan Tanzania Uganda Zambia
<>
.
and even up to 1990, several years after the MDA was phased out, chloroquine can be found in nearly 50% of a population ~3.
I
1989
Year Fig. I. The ECso values from each isolate obtained from children in the MCH clinic (+) and from schoolchildren(diamonds) in Ifakara, Tanzania. The curves show the regressionsof log (ECso) against year. The slopes of the lines were O. 126 (P <0.001) for MCH (broken curve) and -0.233 (P < 0.001) for schoolchildren(solid curve). (Adapted from Ref. 15.)
West Africa: Angola Cameroon Central African Republic CSte d'lvoire Gabon The Gambia Ghana Guinea Guinea-Bassau Liberia Mall Nigeria Zaire
Parasitology Today, vol. 9, no. 3, i 993
In this area of very intense transmission, parasites were obtained from children attending the Mother and Child Health (MCH) clinic every May between 1982 and 1989, and from 10-15-year-old children attending a local school between 1986 and 1989. The study showed that, after the initial emergence of resistance in 1981, parasites isolated from non-immune children attending the MCH clinic became increasingly resistant up to 1989 (Fig. I). Conversely, parasites isolated from schoolchildren, who generally have high levels of immunity in this area ~i.i6, had, on average, a high ECs0 in 1986, but became more sensitive in subsequent years. Further evidence stems from the W H O database on drug resistance, which contains the data of all reported microtests. The analysis shown here used the ECs0 for the samples obtained between 1981 and 1989 from children who were less than 15 years old, with the data grouped according to geographical area (Box I) to get an impression of intensity of transmission. Those combinations of area and year that had more than ten samples were
107
used. The effects of age and year on the logarithm of ECs0 were estimated with an analysis of variance that took account of the uptake of chloroquine by the children. The results of this analysis (Table I) were consistent with the predictions of the model: in areas with low transmission rates (West Asia), age had no effect on resistance; in areas of more intense transmission, year and age interacted to determine resistance, and the interaction depended on whether or not resistance was increasing. In those areas where, on average, resistance was increasing (Africa, Asian Islands), ECs0 initially increased with the age of the children from whom the isolates were obtained, but later decreased with age. Where, on average, resistance was decreasing (Asia), no interaction was found. Thus, the repeated cross-sectional study in Ifakara and the patterns found in the W H O database are consistent with an association between chloroquine resistance and immunogenic properties of the parasites. How can such an association arise? The answer to this question is important, because different mechanisms will lead to differ-
ent predictions about the spread of resistance. How
Can an Association
Arise?
Chloroquine resistance and immunogenic properties could be associated because the two traits are correlated genetically. On the one hand, genes encoding proteins involved in resistance could be closely linked to those encoding proteins responsible for antigenic properties, so that the association would reflect a linkage disequilibnum between resistance and antigenic properties, arising when resistant parasites introduced into a population carry novel antigens. Linkage could arise if the genes that encode antigens are on the same chromosome as are those involved in resistance, or if most parasite zygotes are created by selffertilization. This explanation seems unlikely for several reasons. First, even a slight degree of recombination between genes would soon lead to linkage equilibrium, resulting in the disappearance of an initial association after only a few parasite generations, ie.
Table I. Analysis of variance of chloroquine resistance with time a Source Asia CQ Year Age Year* age
D e g r e e s of f r e e d o m
S u m of squares ( T y p e III)
F value
2 I I -
1.220 0.307 1.287 -
0.76 0.38 1.60
A s i a n Islands CQ Year Age Year* age
4 I I I
7.059 I 1.866 6.432 6.255
1.89 12.70 6.88 6.70
0.44 <0.001 0.009 0.01
0.267 2.35 I -0.028
East A f r i c a CQ Year Age Year* age
2 I I I
17.329 42.272 28.252 28.21 I
8.95 43.69 29.20 29.16
<0.001 <0.001 <0.001 <0.001
0.314 2.67 I -0.031
West Africa CQ Year Age Year* age
4 I I I
7.801 22.211 5.573 5.741
3.14 35.72 8.96 9.23
0.01 <0.001 0.003 0.002
0.279 2.076 -0.025
West Asia CQ Year Age Year* age
4 I I -
2.809 2.546 0.228 -
1.64 5.95 0.53 -
0.17 0.02 0.47 ns
-0.073 -0.013 -
-
P
Estimate
0.47 0.54 0.20
-0.024 -0.024
ns
b
_
•For each area, the effects of chloroquine (CQ) usage, the number of years since 1980, age of the children involved and the interaction of year and age (*) on the estimate of the ECs0 are shown. Where the interaction was insignificant, the table shows the results of an analysis done without the interaction term. Abbreviations: ns, not significant. The hypothesis char resistance is associated with immunogenic properties predicts char the estimate of the interaction is zero in areas with low intensity of transmission (West Asia), that the interaction is negative in areas with intense transmission where resistance is increasing (Asian Islands, Africa), and that the resistance decreases with age where resistance is close to constant (Asia).
108
Parasitology Today, vat. 9, no. 3, 1993
after several months. This is contrary to the data shown, where an association was observed for several years. Furthermore, none of the genes that are assumed to be involved in chloroquine resistance w,18 are found on the same chromosome as are those genes known to code for antigens. Finally, though the degree of self-fertilization is debatable~9 2~, evidence suggests that mating between different genotypes and genetic recombination are fairly common in many parasite populations. On the other hand, resistance could be directly associated with immunogenic properties, if the genes coding for resistance have a pleiotropic effect on the immunogenic properties of the parasite. Resistance is thought to arise by overproduction of a membranebound glycoprotein functioning as an export pump 22. The differential expression of a membrane-bound protein might lead to differential antigen properties. However, none of the genes known to code for antigenic properties has an effect on resistance, and none of the three genes currently thought to be involved in chloroquine resistance has a known effect on immunogenic properties. A direct association between resistance and immunogenic properties would also arise if the presence of chloroquine itself altered the antigenic properties of a parasite. Because resistant parasites, by definition, can have longer contact with chloroquine within the human host than can sensitive parasites, the immune system experiences the chloroquine-induced properties of resistant parasites more often, but their chloroquine-free properties less often, than those of sensitive parasites. Therefore, immunity is more rapidly acquired against resistant parasites. This hypothesis is made plausible by the property of chloroquine to
modify the structure of cell membranes, though no direct evidence is known. Concluding Remarks
An association between chloroquine resistance and antigenic properties has many implications for control strategies. At the very least, an association makes comparisons of resistance, in vitro, between areas with different patterns of transmission and immunity difficult. Only if parasites that have been isolated from non-immune patients are used do we get an impression of the resistance in the population of parasites. More importantly, a direct association would ensure that resistance, because of its cost of enhanced immunity, cannot increase continuously in a population, but will evolve towards a stable level. Conversely, if resistance has no other cost, resistance will not disappear from the population as drug pressure is relaxed, because the cost imposed on resistance due to more intense immunity as well as the benefit of resistance are relaxed when chloroquine is no longer used. Because such conclusions rely not only on the presence of an association between resistance and immunogenic properties, but also on the mechanism of the association, controlled laboratory studies of the associations of resistance to chloroquine and other drugs with immunogenic properties should be able to enhance our understanding of the spread and distribution of drug resistance. Acknowledgements I thank the malaria teams of the Swiss Tropical Institute and of Imperial College for many inspiring discussions on chloroquine resistance, and Walther Wernsdorfer
Recent
and an anonymous reviewer for detailed comments on the manuscript. This work was supported in part by a grant from the Wellcome Trust. References I Moore, D.V. and Lanier, J.E. (1961) Am. J. Trap. Meal Parasitol. 74, 243 244 2 Wernsdorfer, W.H. ( 1991 ) Parasitology Today 7, 297 303 3 Anon. (1990) WHO Stat. Q. 43, 68 79 4 Clyde, D.F. (1958) Riv. Malarial. 37, 263 276 5 Peters, W. (1987) Chemotherapy and Drug Resistance in Malaria (Vol. 2), Academic Press 6 Forsyth, K.P. et al. (1989) Am. J. Trap. Meal Hyg. 42, 259 265 7 Mendis, K.M., David, P.H. and Carter, R. (I 99 I) in Immunoparasitology Today (Ash, C. and Gallagher, R.B., eds), pp 34 37, Elsevier Trends Journals, Cambridge 8 Bj~rkman, A. (1991) in Malaria: Waiting for the Vaccine (-rar~ett, G.A.T., ed.), pp 105 120, John Wiley & Sons 9 TargetS, G.A.T. (1984) Antimalarial Drugs (Vol. I) (Peters, W. and Richards, W.H.G., eds), Springe~Verlag I0 Hills, M., Hudson, C. and Smith, P.G. (1986) Working Paper No. 2.8.5, WH O II Kilombero Malaria Project (1992) Trans. R. Sac. Trap. Meal Hyg. 86, 499 504 12 Molineaux, L and Gramiccia, G. (1980) The Garki Project: Research on the Epidemiology and Control of Malaria in the Sudan Savanna of West Afnca, WHO, Geneva 13 Kilombero Malaria ProJect Mere. Inst. Oswaldo Cruz (in press) 14 Tanner, M. et at. (1987) East Af~ Meal J. 64, 464470 15 Koella, J.C. et al. (1990) Trans. R. Sac. Trap. Meal Hyg. 84, 662 665 16 Tanner, M. et al. (1986) Mere. Ins~ Oswaldo Cruz 81 (Suppl II), 199 205 17 Wellems, T.E., Walker-Jonah, A. and Panton, L.J. (1991) Proc. Natl Acad Sci. USA 88, 3382 3386 18 Foote, S.J.etal. (1990) Nature 345, 255 258 19 Tibayrenc, M. and Ayala, F.J. (1991) Parasitology Today 7, 228 232 20 Walliker, D. ( 1991 ) Parasitology Today 7, 232 235 21 Read, A.F. et al. (1992) Parasitology 104, 387 395 22 Warhurst, D.C. (1988) Parasitology Today 4, 211 213 Jacob Koella is at the Institute of Terrestrial Ecology, ETH ZE~rich, Grabenstrasse 3, CH8952 Sehlieren, Switzerland
a r t i c l e s in t h e Trends J o u r n a l s . . .
The adaptive significance of self-medication, D.H. Clayton and N.D. Wolfe (I 993) Trends in Ecology and Evolution 8, 60-63 Transcription factor based therapeutics: drugs of the future, M.G. Peterson and V.R. Baichwal (1993) Trends in Biotechnology I I, I 1-18 Colony stimulating factors, cytokines and monocyte-macrophages - some controversies, J.A. Hamilton (1993) Immunology Today 14, 18-24
Multiple levels of peripheral tolerance, B. Arnold, G. SchSnrich and G.J. H~immerling (1993) ImmunologyToday 14, 12-14 The role of individual human cytochromes P450 in drug metabolism and clinical response, S. Cholerton, A.K. Daly and J.R. Idle (1992) Trends in PharmacologicalSciences 13, 434-439 The role of CD45 in T-cell activation - resolving the paradoxes? D. Alexander, M. Shiroo, A. Robinson, M. Biffen and E. Shivan (I 992) ImmunologyToday 13, 477-48 I A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport, M.D. Marger and M.H. Saier, Jr (I 993) Trends in BiochemicalSciences 18, 20-25