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Chloroquine vs malaria
Parasitology Today, voL 4, no. I O, 1988
291 Table I. Malariacidal effects of chloroquine and iron-sequestering reagents Additions = 24
Chlorocluine vs Malaria Sir-A recent series of articles in Parasitology Today has described the different theories related to the mechanism of action of "12 chloroquine on P. falciparum malaria ' . The first theory suggeststhat the drug complexes with haematin and maintains its solubility in the host cell so that the haematinchloroquine complex can act as a Fenton catalyst in the generation of toxic oxygen products. The products eventually destroy the host red cell and therefore the parasite. The second theory suggeststhat chloroquine acts as a baseto modify the pH of a parasite vacuole where the host's haemoglobin is digested. The rise in pH of the digesting vacuole inactivatesa parasite haemoglobinase.The parasite is unable to derive nourishment and therefore starves. Prompted by these discussions, I have performed a series of experiments attempting to resolve the mechanism of action ofchloroquine in vitro. If the first theory is correct, then chloroquine-sensitive parasitesmight become chlorocluineresistant parasitesif there were no free iron available.If the second theory is correct, sequestration of free iron should make no difference to the proteolytic digestion of haemoglobin. I found that micromolar concentrations of chloroquine would kill the parasite in the presence of iron-sequestering reagents and oxygen radical scavengers.The ironsequestering reagents used were desferoxamine mesylate ('Desferal') and diethylenetriaminepentacetic acid (DTPA). Additional studies demonstrate that millimolar concentrations of the radical scavenger3dimethyl sulfoxide (DMSO) does not protect the parasite from the malariacidal action of micromolar concentrations of chloroquine. These two observations suggest that the malariacidalaction ofchloroquine is independent of the presence of iron, and of its ability to act as a Fenton catalyst4 in the production of peroxides and oxygen radicals. Table I and Fig. I illustrate the results. Desferal (or DTPA) demonstrated malariastasisin I 0, 20 and 40 llM concentrations s. Growth occurred when the sequestering agent was replaced with new media. Chloroquine alone was malariacidalat 20 llM (this concentration was chosen to ensure complete killing of the Honduras I strain ofP. falciparum). Only crisisforms6 were seen with the microscope and new growth was observed when drug-free media were added. Simultaneousaddition of equivalent (20 llM) concentrations of Desferal and chloroquine demonstrated malariacidaleffects. The malariacidaleffect of chloroquine was not reversed with the simultaneous addition of a mixture of 100 llM Desferal and 20 llM chloroquine. In a similar series of experiments using 1.3 mM DMSO, which stimulates the growth of the parasite
None 20 tim Chloroquine 20 llH Desferal 20 llM Desferal+ 20 llH chloroquine 100 LtM Desferal+ 20 FH chloroquine 1.3 mM DMSO 6.5 mM D M S O + 2 0 llM chloroquine
% Parasites/time (h) 48 96
3% 1% 3% 1% 1% 4% 2%
6% < 1% 3% 1% 1% 8% 3%
9% 0 6% 1% 1% I 1% 4%
aAdditiveswere replenishedevery 24 h. At 72 h mediawithout chloroquineor Desferalwere added;the final cultures were read at 96 h. Quantification of parasiteswas performed on a flow cytometer 7. All cultureswith chloroquineshowed crisisforms.
both at 3% and 20% oxygen (M.T. Makler, unpublished), there was, as expected, no growth with 20 llM chloroquine. In one experiment with a mixture of 6.5 mM DMSO and 20 laMchloroquine, morphological examination revealed rare morphologically normal parasites,and in vitro culture in drug-free media for 96 h demonstrated minimal growth(Fig, l e). In three additional experiments with this concentration of DMSO and chloroquine, no growth was recorded. The fluorescent photomicrographs (parasitesstained with thiazole orange7), in Fig. I illustrate that in RPMI media supplemented with 10% AB sera and grown at 3%02 and 6%COl 8, parasite numbers double every two days (Fig. I a); with addition of 20, 40 and 100 llM of Desferal there is
growth stasis(Fig. I b). The malariastaticeffect can be eliminated by removingthe sequestering reagent and then adding fresh media. (This observation suggeststhat micromolar concentrations of Desferal may be a useful chemical way of synchronizing malaria cultures.) With 20 llM of chloroquine there are only crisisforms (Fig. I c); with equivalent concentrations of chloroquine and Desferal there are still only crisisforms (Fig. I d); whereas with 6.5 mMof DSMO and 20 lirl of chloroquine (Fig. le)there are predominantly crisisforms and no significant growth compared to growth with DMSO alone. This study suggeststhat sincechloroquine remains malariacidal in the presence of both iron-sequestering agents and DMSO (a OH" radical scavenger)chloroquine must act to
Fig. h Fluorescence micrographs oferythrocytic Plasmoclium falciparum (originally stained with thiazole orange). (a) In m~dia supplemented with 10% AB sere and grown at 3% 02 and 6% C02. (b) After addison of Desferal. (c) After add~on of chloroquine. (d) After addition of Desferal and chloroquine. (e) After addition of DMSO and chloroquine.
Parasitology Today, vol. 4, no. I O, 1988
292 destroy the parasite by a mechanism other than that of the generation of toxic oxygen products. This mechanism may require a permease to transport the chloroquine to the digesting vacuole L2. In the vacuole a pH change is believed to occur, so that the malaria parasite proteases are unable to digest the host's haemoglobin and the parasite starves to death.
Density-dependence in Parasite Transmission Dynamics Sir- Professor Klaus Dietz has recently written two elegant accounts L2 of densitydependent regulation in parasite transmission dynamics, based on a generalization of the Ross malaria model. He shows in a variety of ways how, in principle, the outcome of vector-borne disease control can depend upon the strength of regulation in vertebrate and arthropod hosts. He concludes that it is crucially important to assessthe relative strengths of these regulatory forces. l am worried however, about the emphasis of Professor Dietz's article ~, and that its bottom line will cause unnecessary confusion over an issue which (within the scope of his article)is simpler than it has been made to seem. This kind of mathematical work can and should be identifying the probable amongthe possible. Many practitioners are already bewildered by the complexities of malaria control. When opportunities for simplification arise, they should be seized with both hands. Density-dependence arising from multiple infection ofanopheline mosquitoes (the kind described by Dietz) will usually be negligible because the fraction of anopheline mosquitoes which ever get infected is I o w eg. 10-20% as judged from delayed sporozoite rates in a highly endemic area of Tanzania3, where essentially every person is regularly infected. This reduces the number of reasonable malaria models, among the five Dietz discusses,to two (models iv and v). Models iv and v point to what may be the central question about density-dependence of Plasmodium in their hosts-that concerning human immunity. Vaccines are the subject of everyday speculation; there is a need to know much more about the epidemiological consequences of different notions of how immunity works. Recent ideas go far beyond what is embodied by the elderly models iv and v - transient immunity is an outstanding example. In contrast to multiple infection in the vector, this is a prominent area of uncertainty upon which further rigorous, quantitative analysis should now be focused. If malaria parasites are harmful to mosquitoes, two kinds of density-dependent processes (besides those described by Dietz) might help to regulate the parasite population in the vector population. The first arises because an increase in the prevalence of
References
I Fitch, C.D.; Warhurst, D.C. [Debate] (1986) Parasitology Today 2, 330-333 2 Ginsburg, H.; Warhurst, D.C. [Debate] (I 988) Parsitology Today 4, 209-213 3 Wasil, M. et ol. (1987) Biochem.]. 243,867-870 4 Fenton,H.J.H.(1894)]. Chem, Sac.65, 899-910 5 Raventos-Suarez,C.,Pollack,S. and Nagel, R.L. (1982)Am.]. Trap. Meal.Hyg. 3 I, 919-922 6 Jensen, J.B. et al. (1984) InfecC Immun. 45,
infection will increase the average death rate of mosquitoes. But this is likely to be a rather weak regulatory force for the same reason as multiple infection is, because the prevalence in mosquitoes is generally low. It is also possible that density-dependence in other parts of the mosquito's life cycle could compensate for it. The second process may be more significant. It occurs when the average intensity of infection (number of parasites), and consequently the average death rate of infected mosquitoes, increase with the proportion infected. Like human immunity, this is an area of current interest in which the search for appropriate data (the relation between intensity and prevalence of infection) will be conducted most efficiently from a firm theoretical base. C. Dye
London Schoolof Hygieneand Tropical Medicine KeppelStreet London WC I E7HT, UK
505-510 7 Makler, M.T., Lee, L.G. and Recktenwald, D. (1987) Cytometry 8,568-570 8 Jensen,J.B. and Trager, W. (1977)J. Porasitol. 63,883-886 M.T. Makler
Laboratory II 3C VAMC, Portland OR 9720 I, USA
clear that efforts in this direction will not lead to the simplifications that Dr Dye hopes for. In an earlier article in Parasitology Today Dr Dye asks the question "Must we measure all the components of vectorial capacity?''~. In the light of the analysis presented in the paper under discussion I the answer is clearly no. According to these models it would be sufficient to measure the two components 131= (Pl + ~ l ) R l and R2. If one knewthe density-dependence mechanisms in the vector and the vertebrate host one could derive estimates for R~ (rate of reproduction in the host) and R2(rate of reproduction in the vector) only from the prevalence of infection in the vector and in the vertebrate host without the use of entomological measurements on the individual components of vectorial capacity. This provides another reason to assessthe strength of densitydependent regulation in the arthropod and the vertebrate hosts. K. Dietz
References
I Dietz, K.(1988) Parasitology Today 4, 91-97 2 Dietz, K. in Textbook of Malaria (Wernsdorfer, W. and McGregor, I., eds), pp 1091-1133, Churchill Livingstone(in press) 3 Davidson,G. and Draper, C.C. (1953) Trans. R. Sac. Trap. Med. Hyg. 47,522-535 4 Dye, C. (1986) Parasitology Today 2, 203-209
Reply Sir- Dr Dye claims that density-dependence arising from multiple infection ofanopheline mosquitoes will usually be negligible because the fraction ofanopheline mosquitoes that ever gets infected is low, and concludes that models i-ill (see Ref. I)can be ruled out. But low infection prevalence in the vector- even for high infection prevalence in the vertebrate host-could be the consequence of strong density-dependence in the form of differential mortality of vectors (which is proportional to the density of infection) or it could be a consequence of heterogeneous contact rates of vectors due to different feeding rates on man. Neglecting these factors would lead to an underestimate of the basic reproduction rate derived from the low infection probability in the vector. I fully agree with Dr Dye that more realistic models are needed for density-dependence in the vertebrate host (taking into account immune mechanisms) and in the vector (taking into account differential mortality). The Garki model was a first but insufficient attempt to incorporate immunity. But it is
Institutfi~r MedizinischeBiometrie Universit~tTubingen,FRGermany
Dinner sans Pyrethroid Sir- I read with great interest and enjoyment your excellentJuly feature on the development of pyrethroid insecticides. I was bemused however, by the accompanying advertisement from ICI, showing two similar plates of food, one of which was apparently contaminated with insects. Surely ICI aren't suggesting that to combat possible insect contamination we should spray our dinner with one of their insecticides? I much prefer my Coq au Vin sans Pyrethroid! F. Leghorn
21 The Coops Cambridge CB2 I LA, UK
291 Table I. Malariacidal effects of chloroquine and iron-sequestering reagents Additions = 24
Chlorocluine vs Malaria Sir-A recent series of articles in Parasitology Today has described the different theories related to the mechanism of action of "12 chloroquine on P. falciparum malaria ' . The first theory suggeststhat the drug complexes with haematin and maintains its solubility in the host cell so that the haematinchloroquine complex can act as a Fenton catalyst in the generation of toxic oxygen products. The products eventually destroy the host red cell and therefore the parasite. The second theory suggeststhat chloroquine acts as a baseto modify the pH of a parasite vacuole where the host's haemoglobin is digested. The rise in pH of the digesting vacuole inactivatesa parasite haemoglobinase.The parasite is unable to derive nourishment and therefore starves. Prompted by these discussions, I have performed a series of experiments attempting to resolve the mechanism of action ofchloroquine in vitro. If the first theory is correct, then chloroquine-sensitive parasitesmight become chlorocluineresistant parasitesif there were no free iron available.If the second theory is correct, sequestration of free iron should make no difference to the proteolytic digestion of haemoglobin. I found that micromolar concentrations of chloroquine would kill the parasite in the presence of iron-sequestering reagents and oxygen radical scavengers.The ironsequestering reagents used were desferoxamine mesylate ('Desferal') and diethylenetriaminepentacetic acid (DTPA). Additional studies demonstrate that millimolar concentrations of the radical scavenger3dimethyl sulfoxide (DMSO) does not protect the parasite from the malariacidal action of micromolar concentrations of chloroquine. These two observations suggest that the malariacidalaction ofchloroquine is independent of the presence of iron, and of its ability to act as a Fenton catalyst4 in the production of peroxides and oxygen radicals. Table I and Fig. I illustrate the results. Desferal (or DTPA) demonstrated malariastasisin I 0, 20 and 40 llM concentrations s. Growth occurred when the sequestering agent was replaced with new media. Chloroquine alone was malariacidalat 20 llM (this concentration was chosen to ensure complete killing of the Honduras I strain ofP. falciparum). Only crisisforms6 were seen with the microscope and new growth was observed when drug-free media were added. Simultaneousaddition of equivalent (20 llM) concentrations of Desferal and chloroquine demonstrated malariacidaleffects. The malariacidaleffect of chloroquine was not reversed with the simultaneous addition of a mixture of 100 llM Desferal and 20 llM chloroquine. In a similar series of experiments using 1.3 mM DMSO, which stimulates the growth of the parasite
None 20 tim Chloroquine 20 llH Desferal 20 llM Desferal+ 20 llH chloroquine 100 LtM Desferal+ 20 FH chloroquine 1.3 mM DMSO 6.5 mM D M S O + 2 0 llM chloroquine
% Parasites/time (h) 48 96
3% 1% 3% 1% 1% 4% 2%
6% < 1% 3% 1% 1% 8% 3%
9% 0 6% 1% 1% I 1% 4%
aAdditiveswere replenishedevery 24 h. At 72 h mediawithout chloroquineor Desferalwere added;the final cultures were read at 96 h. Quantification of parasiteswas performed on a flow cytometer 7. All cultureswith chloroquineshowed crisisforms.
both at 3% and 20% oxygen (M.T. Makler, unpublished), there was, as expected, no growth with 20 llM chloroquine. In one experiment with a mixture of 6.5 mM DMSO and 20 laMchloroquine, morphological examination revealed rare morphologically normal parasites,and in vitro culture in drug-free media for 96 h demonstrated minimal growth(Fig, l e). In three additional experiments with this concentration of DMSO and chloroquine, no growth was recorded. The fluorescent photomicrographs (parasitesstained with thiazole orange7), in Fig. I illustrate that in RPMI media supplemented with 10% AB sera and grown at 3%02 and 6%COl 8, parasite numbers double every two days (Fig. I a); with addition of 20, 40 and 100 llM of Desferal there is
growth stasis(Fig. I b). The malariastaticeffect can be eliminated by removingthe sequestering reagent and then adding fresh media. (This observation suggeststhat micromolar concentrations of Desferal may be a useful chemical way of synchronizing malaria cultures.) With 20 llM of chloroquine there are only crisisforms (Fig. I c); with equivalent concentrations of chloroquine and Desferal there are still only crisisforms (Fig. I d); whereas with 6.5 mMof DSMO and 20 lirl of chloroquine (Fig. le)there are predominantly crisisforms and no significant growth compared to growth with DMSO alone. This study suggeststhat sincechloroquine remains malariacidal in the presence of both iron-sequestering agents and DMSO (a OH" radical scavenger)chloroquine must act to
Fig. h Fluorescence micrographs oferythrocytic Plasmoclium falciparum (originally stained with thiazole orange). (a) In m~dia supplemented with 10% AB sere and grown at 3% 02 and 6% C02. (b) After addison of Desferal. (c) After add~on of chloroquine. (d) After addition of Desferal and chloroquine. (e) After addition of DMSO and chloroquine.
Parasitology Today, vol. 4, no. I O, 1988
292 destroy the parasite by a mechanism other than that of the generation of toxic oxygen products. This mechanism may require a permease to transport the chloroquine to the digesting vacuole L2. In the vacuole a pH change is believed to occur, so that the malaria parasite proteases are unable to digest the host's haemoglobin and the parasite starves to death.
Density-dependence in Parasite Transmission Dynamics Sir- Professor Klaus Dietz has recently written two elegant accounts L2 of densitydependent regulation in parasite transmission dynamics, based on a generalization of the Ross malaria model. He shows in a variety of ways how, in principle, the outcome of vector-borne disease control can depend upon the strength of regulation in vertebrate and arthropod hosts. He concludes that it is crucially important to assessthe relative strengths of these regulatory forces. l am worried however, about the emphasis of Professor Dietz's article ~, and that its bottom line will cause unnecessary confusion over an issue which (within the scope of his article)is simpler than it has been made to seem. This kind of mathematical work can and should be identifying the probable amongthe possible. Many practitioners are already bewildered by the complexities of malaria control. When opportunities for simplification arise, they should be seized with both hands. Density-dependence arising from multiple infection ofanopheline mosquitoes (the kind described by Dietz) will usually be negligible because the fraction of anopheline mosquitoes which ever get infected is I o w eg. 10-20% as judged from delayed sporozoite rates in a highly endemic area of Tanzania3, where essentially every person is regularly infected. This reduces the number of reasonable malaria models, among the five Dietz discusses,to two (models iv and v). Models iv and v point to what may be the central question about density-dependence of Plasmodium in their hosts-that concerning human immunity. Vaccines are the subject of everyday speculation; there is a need to know much more about the epidemiological consequences of different notions of how immunity works. Recent ideas go far beyond what is embodied by the elderly models iv and v - transient immunity is an outstanding example. In contrast to multiple infection in the vector, this is a prominent area of uncertainty upon which further rigorous, quantitative analysis should now be focused. If malaria parasites are harmful to mosquitoes, two kinds of density-dependent processes (besides those described by Dietz) might help to regulate the parasite population in the vector population. The first arises because an increase in the prevalence of
References
I Fitch, C.D.; Warhurst, D.C. [Debate] (1986) Parasitology Today 2, 330-333 2 Ginsburg, H.; Warhurst, D.C. [Debate] (I 988) Parsitology Today 4, 209-213 3 Wasil, M. et ol. (1987) Biochem.]. 243,867-870 4 Fenton,H.J.H.(1894)]. Chem, Sac.65, 899-910 5 Raventos-Suarez,C.,Pollack,S. and Nagel, R.L. (1982)Am.]. Trap. Meal.Hyg. 3 I, 919-922 6 Jensen, J.B. et al. (1984) InfecC Immun. 45,
infection will increase the average death rate of mosquitoes. But this is likely to be a rather weak regulatory force for the same reason as multiple infection is, because the prevalence in mosquitoes is generally low. It is also possible that density-dependence in other parts of the mosquito's life cycle could compensate for it. The second process may be more significant. It occurs when the average intensity of infection (number of parasites), and consequently the average death rate of infected mosquitoes, increase with the proportion infected. Like human immunity, this is an area of current interest in which the search for appropriate data (the relation between intensity and prevalence of infection) will be conducted most efficiently from a firm theoretical base. C. Dye
London Schoolof Hygieneand Tropical Medicine KeppelStreet London WC I E7HT, UK
505-510 7 Makler, M.T., Lee, L.G. and Recktenwald, D. (1987) Cytometry 8,568-570 8 Jensen,J.B. and Trager, W. (1977)J. Porasitol. 63,883-886 M.T. Makler
Laboratory II 3C VAMC, Portland OR 9720 I, USA
clear that efforts in this direction will not lead to the simplifications that Dr Dye hopes for. In an earlier article in Parasitology Today Dr Dye asks the question "Must we measure all the components of vectorial capacity?''~. In the light of the analysis presented in the paper under discussion I the answer is clearly no. According to these models it would be sufficient to measure the two components 131= (Pl + ~ l ) R l and R2. If one knewthe density-dependence mechanisms in the vector and the vertebrate host one could derive estimates for R~ (rate of reproduction in the host) and R2(rate of reproduction in the vector) only from the prevalence of infection in the vector and in the vertebrate host without the use of entomological measurements on the individual components of vectorial capacity. This provides another reason to assessthe strength of densitydependent regulation in the arthropod and the vertebrate hosts. K. Dietz
References
I Dietz, K.(1988) Parasitology Today 4, 91-97 2 Dietz, K. in Textbook of Malaria (Wernsdorfer, W. and McGregor, I., eds), pp 1091-1133, Churchill Livingstone(in press) 3 Davidson,G. and Draper, C.C. (1953) Trans. R. Sac. Trap. Med. Hyg. 47,522-535 4 Dye, C. (1986) Parasitology Today 2, 203-209
Reply Sir- Dr Dye claims that density-dependence arising from multiple infection ofanopheline mosquitoes will usually be negligible because the fraction ofanopheline mosquitoes that ever gets infected is low, and concludes that models i-ill (see Ref. I)can be ruled out. But low infection prevalence in the vector- even for high infection prevalence in the vertebrate host-could be the consequence of strong density-dependence in the form of differential mortality of vectors (which is proportional to the density of infection) or it could be a consequence of heterogeneous contact rates of vectors due to different feeding rates on man. Neglecting these factors would lead to an underestimate of the basic reproduction rate derived from the low infection probability in the vector. I fully agree with Dr Dye that more realistic models are needed for density-dependence in the vertebrate host (taking into account immune mechanisms) and in the vector (taking into account differential mortality). The Garki model was a first but insufficient attempt to incorporate immunity. But it is
Institutfi~r MedizinischeBiometrie Universit~tTubingen,FRGermany
Dinner sans Pyrethroid Sir- I read with great interest and enjoyment your excellentJuly feature on the development of pyrethroid insecticides. I was bemused however, by the accompanying advertisement from ICI, showing two similar plates of food, one of which was apparently contaminated with insects. Surely ICI aren't suggesting that to combat possible insect contamination we should spray our dinner with one of their insecticides? I much prefer my Coq au Vin sans Pyrethroid! F. Leghorn
21 The Coops Cambridge CB2 I LA, UK