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A strain of monkey malaria (Plasmodium cynomolgi) made resistant to proguanil (paludrine)
695 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 44. No. 6. June, 1951.
(PLASMODIUiV[ C YNOMOLGI) MADE R E S I S T A N T TO P R O G U A N I L
A S T R A I N OF M O N K E Y MALARIA
(PALUDRINE).* BY
FRANK HAWKING AND
JUNE P. THURSTON, National Institute for Medical Research, London.
This paper describes the production of resistance to proguanil (paludrine, chlorguanide) in a strain of Plasmodium cynomolgi, a parasite which closely resembles P. vivax. A preliminary account of this strain was given by HAWKING and PERRY (1948) but the slow development of full resistance and the distraction of other work have delayed the preparation of the full report. The work was initiated because BISHOP and BIRKETT (1947) and WILLIAMSON and LOURIE (1947) had shown that the avian parasite P. gallinaceum could be made highly resistant to this compound. METHODS.
The strain of P. cynomolgi was the one in common experimental use in England and America (HAWKING, PERRY and THURSTON, 1948). It was maintained in Macaca mulatta monkeys, in which it produces an acute primary attack, followed by several relapses at irregular intervals; when the infection Grateful a c k n o w l e d g e m e n t s are m a d e to Dr. W. L. M . PERRY a n d Dr. J. A.
MCFADZEANfor collaboration at various stages of this work, and to Mr. K. BROOMFIELD, Mr. E. C. ENGLANDand Mr. D. GARLICKfor technical assistance.
696
PROGUP2qIL RESISTANCE IN PLASMOD1UM CYSOMOLGI
has become latent, a further series of acute attacks can be initiated by splenectomy. Transmission was by syringe or mosquitoes (Anopheles maculipennis atroparvus). The density of malaria parasites in the blood was estimated by the examination of thin film~, stained with Giemsa ; the number of parasites was recorded per microscope field ( × 1,000), which usually contained about 200 red blood corpuscles. In the earlier work the drug was given by intramuscular injection ; large doses given by this method may cause much necrosis of the tissues and in later work the drugs were given by stomach tube in about 20 c.c. water. All doses refer to proguanil acetate (which contains 80 per cent. base), expressed as mg. per kg. body weight. Monkeys tolerate eight daily doses of 40 rag., or four doses of 80 mg. on alternate days, although these doses cause loss of weight and energy; they are probably the maximum dose-levels which can be given without toxic effects. INITIAL SENSITIVITY OF THE STRAIN. When a dose of proguanil is given, the parasites may continue to multiply for 24 hours and the therapeutic effect is usually not indicated until the 2nd day after treatment. This fact, together with the need for economy of monkeys, hindered precise determination of the initial sensitivity of the strain to proguanil. When the first animal (Baboon 98), which had been infected by sporozoites, was given 0.03 rag. subcutaneously on 2 successive days, the blood became negative after 2 days and remained so for 10 days; later 0.04 rag. was given on 3 successive days without definite effect ; at a later relapse 0.04 mg. was given on 6 successive days without any definite action. Subsequently a severe relapse was induced by splenectomy and doses of 0"04, 0-08, 0.1 and 0.2 mg. were given on successive days, and the blood became negative in 3 days after the last dose. In Rhesus Monkey 179, relapsing after splenectomy, oral doses of 0.03, 0"06, 0-13 and 0-25 rag. were given, before the parasitaemia began to diminish so that it disappeared (temporarily) on the 4th day after the last dose ; 11 days later, during a recrudescence of the infection, 0.12 mg. was given on 4 alternate days without reducing the parasitaemia, but 0.25 mg. given on 2 successive days gradually abolished the parasitaemia in 3 days. In Rhesus Monkey 182, experiencing a second relapse after splenectomy, 0.12 mg. given orally on 2 alternate days diminished the parasitaemia and a third dose abolished it temporarily. In another rhesus (186)experiencing the first attack after blood inoculation, two oral doses of 0.12 mg. on successive days cleared the blood of parasites for 10 days. It was concluded that the minimum effective oral dose of proguanil acetate is probably about 0.19. rag. if given on 4 successive days or 0.25 mg. if given on 4 alternate days. SCHMIDTet al. (1949) working with the same strain found that seven daily doses of 0.075 mg. base per kg. always suppressed parasitaemia although they did not sterilize the infection ; doses as low as 0.019 mg. reduced the parasitaemia significantly and in some instances temporarily abolished it.
FRANK
HAWKING
~ND
JUNE P. T H U R S T O N
697
PRODUCTION OF THE RESISTANT STRAIN.
The procedure adopted to produce a strain resistant to proguanil was to treat an infected monkey with repeated subeffective doses. In a few days, the parasites usually disappeared because of the host's immunity reactions or because the dose had been too high. When parasites reappeared after some days or weeks, treatment was resumed. This procedure was slow, but economical of monkeys. To save space, full protocols of the experiments are omitted, but the progress of the work may be indicated by the following notes : T h e experiment started with Baboon 98 infected by sporozoites of the normal strain on 17th June, 1947 ; t r e a t m e n t began on 3rd July. O n 1st January, 1948, 0-15 rag. intramuscularly was tolerated ; 1st March, 0"2 rag. ; 5th May, 0-75 rag. ; 24th May, 1-5 rag. ; 22nd June, 3-5 nag. ; 21st July, 6.0 rag. ; and 1st October, 25 rag. In October, four doses of 40 rag. per kg. were given intramuscularly in 8 days without affecting the parasitaemia. Baboon 100 was subinoculated from 98, 30th D e c e m b e r , 1947. O n 14th January, 1950, 0.45 rag. was tolerated ; 5th May, 3.0 rag. ; 16th June, 14 rag. ; 25th September, 29 rag. ; and 18th D e c e m b e r , 40 nag. ; after which the infection became latent. Baboon 99 was subinoculated from 100 on 4th May, 1948. O n 16th May, 1.5 mg. intramuscularly was tolerated ; 25th June, 7.2 rag. ; 22nd July, 14 rag. ; and 12th August, 29 mg. ; after which the infection became latent. T h e strain was carried on by subinoculation f r o m 99 into Rhesus 148 on 16th August, 1948. Monkey 118 was subinoculated from 99 on 16th August, 1948. O n 1st N o v e m b e r , 74 rag. intramuscularly was tolerated, and about 25th N o v e m b e r six daily doses of 55 rag. intramuscularly (the m o n k e y died from multiple muscle necroses). Monkey 155 subinoculated f r o m 148 on 1st December, 1948. A b o u t 23rd September, 1949, five oral doses of 83 rag. given during 6 days were tolerated.
Thus a significant increase of resistance was observed after 6 months and maximal resistance after 16 months ; probably resistance would have developed more quickly if the experimental conditions had been arranged differently so as to maintain a large population of actively multiplying parasites continuously exposed to sublethal concentrations of proguanil. Other relevant passages of the strain included the following : 173--blood inoculation f r o m 155. 1 7 6 - - b l o o d inoculation f r o m 173. 158--by inoculation of blood of 173 after preservation by deep-freeze for 3 weeks. 178--by mosquitoes fed on 173. 18~l--by blood from 178. 177--by mosquitoes fed on 173, 50 nag. proguanil being given on 8 successive days during the incubation period. 166--by mosquitoes which had fed on 173, 2-.21-houIs after it had received 40 rag. proguanil. CIIARACTERISTICS OF THE RESISTANT STRAIN.
1. Resistance to Proguanil Before and After Passage by Blood Inoculation. The measurement of resistance is complicated by the tendency of the parasitaemia to diminish spontaneously (due to immunity reactions) if treatment is prolonged ; consequently disappearance of parasites does not always indicate
698
PROGUANIL RESISTANCE
IN
PLASMOD1UM CYNOMOLG1
response to treatment. As mentioned above, the infection was not cleared by six daily intramuscular doses of 55 mg. in Monkey 148, nor by five oral doses of 83 mg. during 6 days in Monkey 155. In the other monkeys, e.g., 184, the strain usually resisted four oral doses of 80 mg. given on alternate days but sometimes the parasitaemia was apparently diminished by this treatment. Thus the resistant strain gave less response to repeated doses of 80 mg. than the normal strains did to dosesof 0"06 mg., i.e., the resistance had increased more than a thousand times. Resistance was not diminished during the above passages by syringe.
2. Resistance After Mosquito Passage. Monkey 178 was infected on 28th October, 1949, by inoculation with mosquitoes, which had fed on 173. Parasites appeared oi1 the 12th day. Next day, the monkey was given 80 mg. proguanil by mouth and the blood became negative for 18 days ; this apparent failure of resistance may have been due to the lightness of the infection when treated. On 22nd November the spleen was excised and a series of relapses followed. On 1st January, 1950, during a light relapse, 80 mg. proguanil was given and the parasites persisted for 2 days, when a second dose was given and the parasites disappeared. On 6th February, when the infection was heavier, 80 mg. was given again and was repeated twice on alternate days ; parasites persisted in scanty numbers for more than 3 weeks. Monkey 184 was subinoculated from 178 on 6th February and parasites appeared in the blood on 13th February when proguanil 80 mg. was given by mouth, and repeated 2 days later. The parasitaemia was controlled but not completely abolished. On 23rd February, when parasites were about one per 10 microscope fields, proguanil 80 mg. was given again and repeated three times on alternate days ; the parasites persisted in the blood. It is considered that there was no evidence in these two monkeys that the resistance of the parasites to proguanil was significantly less than in the other monkeys described.
3. Resistance of Pre-erythrocytic Stages. Monkey 177 was inoculated on 28th October with mosquitoes infected from Monkey 173. Shortly before inoculation, the monkey was given 50 mg. proguanil by mouth and this was repeated on 7 successive days. Parasites appeared in the blood on the 13th day, after a prepatent period of almost the same duration as in the untreated control (178). The infection developed normally and was resistant to 80 mg. proguanil given on several occasions. It is concluded that the pre-erythrocytic forms were resistant to proguanil.
4. Resistance of Gametocytes. On 7th October, when ripe gametocytes were present in the blood, Monkey 173 was given 40 mg. proguanil by mouth. Two and a half hours later, while
FRANK HAWKING AND JUNE P. THURSTON
699
anaesthetized wkh nembutal, it was exposed to mosquitoes. On 28th October, five mosquitoes were dissected but no sporozoites were seen in t h e glands. However, 50 mosquitoes were injected into Monkey 166, which showed parasites in the blood on the 16th day. The infection developed normally and during a relapse following splenectomy it was unaffected by three oral doses of 80 mg. given on alternate days. It is concluded that the gametocytes of this strain are proguanil-resistant and that the presence of proguanil in the blood does not prevent mosquitoes becoming infected.
5. Effect on Resistance of Preservation by Freezing. Parasitized blood from Monkey 173 was citrated and frozen in sealed ampoules at approximately -76 ° C. by immersion in a mixture of alcohol and solid carbon dioxide. Three weeks later, an ampoule was thawed and 0.5 c.c. of blood was injected intramuscularly into Monkey 158. Microscopic examination of the thawed blood showed that most of the erythrocytes had been lysed ; many parasites were present but they stained faintly and abnormally. Parasites appeared in the blood of the monkey after 20 days ; this is an unusually long incubation period and it suggests that only a few of the parasites had survived. The infection developed slowly. During a relapse following splenectomy, the parasitaemia was unaffected by three oral doses of 80 rag. given on alternate days. It is concluded that the resistance of the strain was unaffected by preservation in a frozen state, a fact which is important since this is a convenient method for the preservation of such strains.
6. Responseto Sulphadiazine. BISHOP and MCCONNACHIE (1950) reported that one of their proguanilresistant strains of P. gallinaceum was also resistant to sulphadiazine, but most other workers have stated that their proguanil-resistant strains showed no enhanced resistance to sulphonamides. The therapeutic effect of sulphadiazine on our strains was tested by giving monkeys four daily oral doses when the parasite count was rising, usually during relapses after spleneetomy. In the normal strain, gramme 0.125 per kg. (one monkey) caused the blood to become negative on the 4th day of treatment ; gramme 0.25 (four occasions) caused the parasites to become degenerate, and they disappeared in 2 to 4 days after the last dose; gramme 0.5 (one monkey) caused the parasites to disappear in 2 days after the last dose. With the proguanil-resistant strain, gramme 0.25 (Monkey 178) caused the parasites to disappear during treatment ; gramme 0.5 (Monkeys 158 and 176) caused the parasites to disappear during treatment; gramme 1.0 given only twice (Monkey 166) caused the blood to become negative soon after treatment. It is concluded that there was no evidence that the proguanil-resistant strain was significantly more resistant to sulphadiazine than the normal strain was. (For the degenerative changes in the parasites, caused by proguanil and sulphadiazine, see McFAOZ~AN, 1951.)
700
PROGUANIL RESISTANCE IN
PLASMODIUM CYNOMOLG[
DISCUSSION.
The development of resistance to proguanil has now been described in P. gallinaceum by BISHOP and BIRKETT(1948) ; WlLLIAMSONand LOURIE (1947) ; KNOPPERS (1947); and GREEN~ERC (1949a); in P. cynomolgi by SCHMIDT, GENTHER, FRADKIN and SQUIRES (1949) ; in P. vivax by SEATON and LOURIE (1949) ; and by COOPER, COATNEYand IMBODEN (1950) ; and in P. falciparum by SEATON and ADAMS (1949). Our results confirm the conclusions of these other workers. In the present instance, the resistance to proguanil persisted after passage through mosquitoes and after preservation by freezing, and it was present in the pre-erythrocytic forms and in the gametocytes and the early stages in the mosquito as well as in the ordinary erythrocytic forms. Obviously a fundamental and permanent change has been made in the metabolic system of the parasite which is sensitive to proguanil. As in all other reported cases, there was no detectable change in the morphology or the virulence of our strain when it became proguanil-resistant. The practical and theoretical implications of this development of resistance to proguanil have been fully discussed by previous workers especially by SCHMIDT and by BISHOP. Clearly proguanil-resistance can easily be developed in the laboratory, but the probability of this occurring in human malaria is still uncertain. The most significant evidence on the subject is the report by FIELD and EDESON (1949) that proguanil-resistance has actually appeared under field conditions in Malaya. BISHOP and MCCONNACHIE(1950) state that one of their proguanil-resistant strains of P. gallinaceum, which had been exposed to especially long treatment with oroguanit, was also resistant to sulphadiazine, and that all their strains which had been made resistant to sulphonamides were resistant to proguanil. Other workers who had made strains of P. gallinaceum resistant to proguanil found them normally sensitive to sulphadiazine; GREENBERG even found his strain hypersensitive. In the case of our own strain, no obvious change in the sensitivity to sulphadiazine has occurred. The reactions of our strain to other drugs such as chloroqnin were not investigated since there seemed already to be ample evidence that proguanil-resistant strains are normally sensitive to such drugs. The action of proguanil is fundamentally differem from that of the other known antimalarial drugs as is shown by (1) the ease with which proguanilresistant strains are developed; (2) the failure of proguanil to act upon snlphonamide-resistant strains; (3) the mutual potemiation of sulphonamides and proguanil (GREENBERG, 1949b) ; (4) the inhibition by p-aminobenzoic acid of the action of proguanil on P. berghei (THuRSTON, 1950). The last three facts indicate some relationship between proguanil and sulphonamides, although the nature of this relation is still obscure. SUMMARY.
(1) A high degree of resistance to proguanil (paludrine, chlorguanide) has been produced in P. cynomolgi by repeated subeffective treatment. Significant
FRANK HAWKING AND JUNE P. TKURS~ON
"
701
resistance was observed after 6 months and resista1~ce to m a x i m u m tolerated doses after 16 months. T h e resistance was a t h o u s a n d times as great as that ot the original normal strain. (2) Resistance was not diminished by passage t h r o u g h mosquitoes, or by preservation of the blood by freezing. (3) Resistance was shown by the pre-erythrocytic forms and by the gametocytes and early forms in the mosquito as well as by the endo-erythrocytic forms. (4) T h e proguanil-resistant strain was normally sensitive to sulphadiazine. REFERENCES.
A. & BIRKETT, ~. (1947). Nature, Lond., i59, 884. ---& - - - - - - . (1948). Parasitology, 39, 127. & McCONNACmE, E . W . (1950). Ibid., 40, 163. COOPER, W. C., COATNEY, G. R. & IMBODEN, C. A., Jr. (1950). ft. nat. Malaria Soc., 9, 59. FIELD, J. W. & EDESON, J. F . B . (1949). Trans. R. Soc. trop. ~fed. Hyg., 43, 233. GREENB•EC, J. (1949a). ft. nat. Malaria Soc., 8, 80. • (1949b). ft. Pharmacol., 97, 238. HAWKING, F. & PERRY, W. L. M. (1948). Lancet, 2, 859. , • THURSTON, J. P. (1948). Lancet, 1, 783. KNOPPERS, A. T. (1947). Nature, Lond., 160, 606• MCFADZEAN, J . A . (1951). Trans. R. Soc. trop. IVied. ttyg., 44, 707. SCHMIDT, L. H., GENTHER, C. S., FRADKIN, R. & SQUIRES, W. (1949). ft. Pharmacol., 95, 382. SEATON, D. R. & ADAMS,A. R . D . (1949). Lancet, 2, 323. ---& LoumE, E . M . (1949). Ibid., 1,394. THURSTON, J. (1950). Ibid., 2, 438. WILLIAMSON, J. & LOURIE, E . M . (1947). Ann. trop. d]4ed. Paras#., 41,278.
BISHOP,
(PLASMODIUiV[ C YNOMOLGI) MADE R E S I S T A N T TO P R O G U A N I L
A S T R A I N OF M O N K E Y MALARIA
(PALUDRINE).* BY
FRANK HAWKING AND
JUNE P. THURSTON, National Institute for Medical Research, London.
This paper describes the production of resistance to proguanil (paludrine, chlorguanide) in a strain of Plasmodium cynomolgi, a parasite which closely resembles P. vivax. A preliminary account of this strain was given by HAWKING and PERRY (1948) but the slow development of full resistance and the distraction of other work have delayed the preparation of the full report. The work was initiated because BISHOP and BIRKETT (1947) and WILLIAMSON and LOURIE (1947) had shown that the avian parasite P. gallinaceum could be made highly resistant to this compound. METHODS.
The strain of P. cynomolgi was the one in common experimental use in England and America (HAWKING, PERRY and THURSTON, 1948). It was maintained in Macaca mulatta monkeys, in which it produces an acute primary attack, followed by several relapses at irregular intervals; when the infection Grateful a c k n o w l e d g e m e n t s are m a d e to Dr. W. L. M . PERRY a n d Dr. J. A.
MCFADZEANfor collaboration at various stages of this work, and to Mr. K. BROOMFIELD, Mr. E. C. ENGLANDand Mr. D. GARLICKfor technical assistance.
696
PROGUP2qIL RESISTANCE IN PLASMOD1UM CYSOMOLGI
has become latent, a further series of acute attacks can be initiated by splenectomy. Transmission was by syringe or mosquitoes (Anopheles maculipennis atroparvus). The density of malaria parasites in the blood was estimated by the examination of thin film~, stained with Giemsa ; the number of parasites was recorded per microscope field ( × 1,000), which usually contained about 200 red blood corpuscles. In the earlier work the drug was given by intramuscular injection ; large doses given by this method may cause much necrosis of the tissues and in later work the drugs were given by stomach tube in about 20 c.c. water. All doses refer to proguanil acetate (which contains 80 per cent. base), expressed as mg. per kg. body weight. Monkeys tolerate eight daily doses of 40 rag., or four doses of 80 mg. on alternate days, although these doses cause loss of weight and energy; they are probably the maximum dose-levels which can be given without toxic effects. INITIAL SENSITIVITY OF THE STRAIN. When a dose of proguanil is given, the parasites may continue to multiply for 24 hours and the therapeutic effect is usually not indicated until the 2nd day after treatment. This fact, together with the need for economy of monkeys, hindered precise determination of the initial sensitivity of the strain to proguanil. When the first animal (Baboon 98), which had been infected by sporozoites, was given 0.03 rag. subcutaneously on 2 successive days, the blood became negative after 2 days and remained so for 10 days; later 0.04 rag. was given on 3 successive days without definite effect ; at a later relapse 0.04 mg. was given on 6 successive days without any definite action. Subsequently a severe relapse was induced by splenectomy and doses of 0"04, 0-08, 0.1 and 0.2 mg. were given on successive days, and the blood became negative in 3 days after the last dose. In Rhesus Monkey 179, relapsing after splenectomy, oral doses of 0.03, 0"06, 0-13 and 0-25 rag. were given, before the parasitaemia began to diminish so that it disappeared (temporarily) on the 4th day after the last dose ; 11 days later, during a recrudescence of the infection, 0.12 mg. was given on 4 alternate days without reducing the parasitaemia, but 0.25 mg. given on 2 successive days gradually abolished the parasitaemia in 3 days. In Rhesus Monkey 182, experiencing a second relapse after splenectomy, 0.12 mg. given orally on 2 alternate days diminished the parasitaemia and a third dose abolished it temporarily. In another rhesus (186)experiencing the first attack after blood inoculation, two oral doses of 0.12 mg. on successive days cleared the blood of parasites for 10 days. It was concluded that the minimum effective oral dose of proguanil acetate is probably about 0.19. rag. if given on 4 successive days or 0.25 mg. if given on 4 alternate days. SCHMIDTet al. (1949) working with the same strain found that seven daily doses of 0.075 mg. base per kg. always suppressed parasitaemia although they did not sterilize the infection ; doses as low as 0.019 mg. reduced the parasitaemia significantly and in some instances temporarily abolished it.
FRANK
HAWKING
~ND
JUNE P. T H U R S T O N
697
PRODUCTION OF THE RESISTANT STRAIN.
The procedure adopted to produce a strain resistant to proguanil was to treat an infected monkey with repeated subeffective doses. In a few days, the parasites usually disappeared because of the host's immunity reactions or because the dose had been too high. When parasites reappeared after some days or weeks, treatment was resumed. This procedure was slow, but economical of monkeys. To save space, full protocols of the experiments are omitted, but the progress of the work may be indicated by the following notes : T h e experiment started with Baboon 98 infected by sporozoites of the normal strain on 17th June, 1947 ; t r e a t m e n t began on 3rd July. O n 1st January, 1948, 0-15 rag. intramuscularly was tolerated ; 1st March, 0"2 rag. ; 5th May, 0-75 rag. ; 24th May, 1-5 rag. ; 22nd June, 3-5 nag. ; 21st July, 6.0 rag. ; and 1st October, 25 rag. In October, four doses of 40 rag. per kg. were given intramuscularly in 8 days without affecting the parasitaemia. Baboon 100 was subinoculated from 98, 30th D e c e m b e r , 1947. O n 14th January, 1950, 0.45 rag. was tolerated ; 5th May, 3.0 rag. ; 16th June, 14 rag. ; 25th September, 29 rag. ; and 18th D e c e m b e r , 40 nag. ; after which the infection became latent. Baboon 99 was subinoculated from 100 on 4th May, 1948. O n 16th May, 1.5 mg. intramuscularly was tolerated ; 25th June, 7.2 rag. ; 22nd July, 14 rag. ; and 12th August, 29 mg. ; after which the infection became latent. T h e strain was carried on by subinoculation f r o m 99 into Rhesus 148 on 16th August, 1948. Monkey 118 was subinoculated from 99 on 16th August, 1948. O n 1st N o v e m b e r , 74 rag. intramuscularly was tolerated, and about 25th N o v e m b e r six daily doses of 55 rag. intramuscularly (the m o n k e y died from multiple muscle necroses). Monkey 155 subinoculated f r o m 148 on 1st December, 1948. A b o u t 23rd September, 1949, five oral doses of 83 rag. given during 6 days were tolerated.
Thus a significant increase of resistance was observed after 6 months and maximal resistance after 16 months ; probably resistance would have developed more quickly if the experimental conditions had been arranged differently so as to maintain a large population of actively multiplying parasites continuously exposed to sublethal concentrations of proguanil. Other relevant passages of the strain included the following : 173--blood inoculation f r o m 155. 1 7 6 - - b l o o d inoculation f r o m 173. 158--by inoculation of blood of 173 after preservation by deep-freeze for 3 weeks. 178--by mosquitoes fed on 173. 18~l--by blood from 178. 177--by mosquitoes fed on 173, 50 nag. proguanil being given on 8 successive days during the incubation period. 166--by mosquitoes which had fed on 173, 2-.21-houIs after it had received 40 rag. proguanil. CIIARACTERISTICS OF THE RESISTANT STRAIN.
1. Resistance to Proguanil Before and After Passage by Blood Inoculation. The measurement of resistance is complicated by the tendency of the parasitaemia to diminish spontaneously (due to immunity reactions) if treatment is prolonged ; consequently disappearance of parasites does not always indicate
698
PROGUANIL RESISTANCE
IN
PLASMOD1UM CYNOMOLG1
response to treatment. As mentioned above, the infection was not cleared by six daily intramuscular doses of 55 mg. in Monkey 148, nor by five oral doses of 83 mg. during 6 days in Monkey 155. In the other monkeys, e.g., 184, the strain usually resisted four oral doses of 80 mg. given on alternate days but sometimes the parasitaemia was apparently diminished by this treatment. Thus the resistant strain gave less response to repeated doses of 80 mg. than the normal strains did to dosesof 0"06 mg., i.e., the resistance had increased more than a thousand times. Resistance was not diminished during the above passages by syringe.
2. Resistance After Mosquito Passage. Monkey 178 was infected on 28th October, 1949, by inoculation with mosquitoes, which had fed on 173. Parasites appeared oi1 the 12th day. Next day, the monkey was given 80 mg. proguanil by mouth and the blood became negative for 18 days ; this apparent failure of resistance may have been due to the lightness of the infection when treated. On 22nd November the spleen was excised and a series of relapses followed. On 1st January, 1950, during a light relapse, 80 mg. proguanil was given and the parasites persisted for 2 days, when a second dose was given and the parasites disappeared. On 6th February, when the infection was heavier, 80 mg. was given again and was repeated twice on alternate days ; parasites persisted in scanty numbers for more than 3 weeks. Monkey 184 was subinoculated from 178 on 6th February and parasites appeared in the blood on 13th February when proguanil 80 mg. was given by mouth, and repeated 2 days later. The parasitaemia was controlled but not completely abolished. On 23rd February, when parasites were about one per 10 microscope fields, proguanil 80 mg. was given again and repeated three times on alternate days ; the parasites persisted in the blood. It is considered that there was no evidence in these two monkeys that the resistance of the parasites to proguanil was significantly less than in the other monkeys described.
3. Resistance of Pre-erythrocytic Stages. Monkey 177 was inoculated on 28th October with mosquitoes infected from Monkey 173. Shortly before inoculation, the monkey was given 50 mg. proguanil by mouth and this was repeated on 7 successive days. Parasites appeared in the blood on the 13th day, after a prepatent period of almost the same duration as in the untreated control (178). The infection developed normally and was resistant to 80 mg. proguanil given on several occasions. It is concluded that the pre-erythrocytic forms were resistant to proguanil.
4. Resistance of Gametocytes. On 7th October, when ripe gametocytes were present in the blood, Monkey 173 was given 40 mg. proguanil by mouth. Two and a half hours later, while
FRANK HAWKING AND JUNE P. THURSTON
699
anaesthetized wkh nembutal, it was exposed to mosquitoes. On 28th October, five mosquitoes were dissected but no sporozoites were seen in t h e glands. However, 50 mosquitoes were injected into Monkey 166, which showed parasites in the blood on the 16th day. The infection developed normally and during a relapse following splenectomy it was unaffected by three oral doses of 80 mg. given on alternate days. It is concluded that the gametocytes of this strain are proguanil-resistant and that the presence of proguanil in the blood does not prevent mosquitoes becoming infected.
5. Effect on Resistance of Preservation by Freezing. Parasitized blood from Monkey 173 was citrated and frozen in sealed ampoules at approximately -76 ° C. by immersion in a mixture of alcohol and solid carbon dioxide. Three weeks later, an ampoule was thawed and 0.5 c.c. of blood was injected intramuscularly into Monkey 158. Microscopic examination of the thawed blood showed that most of the erythrocytes had been lysed ; many parasites were present but they stained faintly and abnormally. Parasites appeared in the blood of the monkey after 20 days ; this is an unusually long incubation period and it suggests that only a few of the parasites had survived. The infection developed slowly. During a relapse following splenectomy, the parasitaemia was unaffected by three oral doses of 80 rag. given on alternate days. It is concluded that the resistance of the strain was unaffected by preservation in a frozen state, a fact which is important since this is a convenient method for the preservation of such strains.
6. Responseto Sulphadiazine. BISHOP and MCCONNACHIE (1950) reported that one of their proguanilresistant strains of P. gallinaceum was also resistant to sulphadiazine, but most other workers have stated that their proguanil-resistant strains showed no enhanced resistance to sulphonamides. The therapeutic effect of sulphadiazine on our strains was tested by giving monkeys four daily oral doses when the parasite count was rising, usually during relapses after spleneetomy. In the normal strain, gramme 0.125 per kg. (one monkey) caused the blood to become negative on the 4th day of treatment ; gramme 0.25 (four occasions) caused the parasites to become degenerate, and they disappeared in 2 to 4 days after the last dose; gramme 0.5 (one monkey) caused the parasites to disappear in 2 days after the last dose. With the proguanil-resistant strain, gramme 0.25 (Monkey 178) caused the parasites to disappear during treatment ; gramme 0.5 (Monkeys 158 and 176) caused the parasites to disappear during treatment; gramme 1.0 given only twice (Monkey 166) caused the blood to become negative soon after treatment. It is concluded that there was no evidence that the proguanil-resistant strain was significantly more resistant to sulphadiazine than the normal strain was. (For the degenerative changes in the parasites, caused by proguanil and sulphadiazine, see McFAOZ~AN, 1951.)
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PROGUANIL RESISTANCE IN
PLASMODIUM CYNOMOLG[
DISCUSSION.
The development of resistance to proguanil has now been described in P. gallinaceum by BISHOP and BIRKETT(1948) ; WlLLIAMSONand LOURIE (1947) ; KNOPPERS (1947); and GREEN~ERC (1949a); in P. cynomolgi by SCHMIDT, GENTHER, FRADKIN and SQUIRES (1949) ; in P. vivax by SEATON and LOURIE (1949) ; and by COOPER, COATNEYand IMBODEN (1950) ; and in P. falciparum by SEATON and ADAMS (1949). Our results confirm the conclusions of these other workers. In the present instance, the resistance to proguanil persisted after passage through mosquitoes and after preservation by freezing, and it was present in the pre-erythrocytic forms and in the gametocytes and the early stages in the mosquito as well as in the ordinary erythrocytic forms. Obviously a fundamental and permanent change has been made in the metabolic system of the parasite which is sensitive to proguanil. As in all other reported cases, there was no detectable change in the morphology or the virulence of our strain when it became proguanil-resistant. The practical and theoretical implications of this development of resistance to proguanil have been fully discussed by previous workers especially by SCHMIDT and by BISHOP. Clearly proguanil-resistance can easily be developed in the laboratory, but the probability of this occurring in human malaria is still uncertain. The most significant evidence on the subject is the report by FIELD and EDESON (1949) that proguanil-resistance has actually appeared under field conditions in Malaya. BISHOP and MCCONNACHIE(1950) state that one of their proguanil-resistant strains of P. gallinaceum, which had been exposed to especially long treatment with oroguanit, was also resistant to sulphadiazine, and that all their strains which had been made resistant to sulphonamides were resistant to proguanil. Other workers who had made strains of P. gallinaceum resistant to proguanil found them normally sensitive to sulphadiazine; GREENBERG even found his strain hypersensitive. In the case of our own strain, no obvious change in the sensitivity to sulphadiazine has occurred. The reactions of our strain to other drugs such as chloroqnin were not investigated since there seemed already to be ample evidence that proguanil-resistant strains are normally sensitive to such drugs. The action of proguanil is fundamentally differem from that of the other known antimalarial drugs as is shown by (1) the ease with which proguanilresistant strains are developed; (2) the failure of proguanil to act upon snlphonamide-resistant strains; (3) the mutual potemiation of sulphonamides and proguanil (GREENBERG, 1949b) ; (4) the inhibition by p-aminobenzoic acid of the action of proguanil on P. berghei (THuRSTON, 1950). The last three facts indicate some relationship between proguanil and sulphonamides, although the nature of this relation is still obscure. SUMMARY.
(1) A high degree of resistance to proguanil (paludrine, chlorguanide) has been produced in P. cynomolgi by repeated subeffective treatment. Significant
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resistance was observed after 6 months and resista1~ce to m a x i m u m tolerated doses after 16 months. T h e resistance was a t h o u s a n d times as great as that ot the original normal strain. (2) Resistance was not diminished by passage t h r o u g h mosquitoes, or by preservation of the blood by freezing. (3) Resistance was shown by the pre-erythrocytic forms and by the gametocytes and early forms in the mosquito as well as by the endo-erythrocytic forms. (4) T h e proguanil-resistant strain was normally sensitive to sulphadiazine. REFERENCES.
A. & BIRKETT, ~. (1947). Nature, Lond., i59, 884. ---& - - - - - - . (1948). Parasitology, 39, 127. & McCONNACmE, E . W . (1950). Ibid., 40, 163. COOPER, W. C., COATNEY, G. R. & IMBODEN, C. A., Jr. (1950). ft. nat. Malaria Soc., 9, 59. FIELD, J. W. & EDESON, J. F . B . (1949). Trans. R. Soc. trop. ~fed. Hyg., 43, 233. GREENB•EC, J. (1949a). ft. nat. Malaria Soc., 8, 80. • (1949b). ft. Pharmacol., 97, 238. HAWKING, F. & PERRY, W. L. M. (1948). Lancet, 2, 859. , • THURSTON, J. P. (1948). Lancet, 1, 783. KNOPPERS, A. T. (1947). Nature, Lond., 160, 606• MCFADZEAN, J . A . (1951). Trans. R. Soc. trop. IVied. ttyg., 44, 707. SCHMIDT, L. H., GENTHER, C. S., FRADKIN, R. & SQUIRES, W. (1949). ft. Pharmacol., 95, 382. SEATON, D. R. & ADAMS,A. R . D . (1949). Lancet, 2, 323. ---& LoumE, E . M . (1949). Ibid., 1,394. THURSTON, J. (1950). Ibid., 2, 438. WILLIAMSON, J. & LOURIE, E . M . (1947). Ann. trop. d]4ed. Paras#., 41,278.
BISHOP,