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Plasmodium vinckei: Production of chloroquine-resistant strain
EXPERIMENTAL PARASITOLOGY 26, 193-202 (1969)
~~as~odiu~ Kendall
G.
vinckei: Powers,
Production
Richard
of ~h~oroquine-Resjstant
1. Jacobs,
William
C. Good,
and
Louis
Strain C. Koontz
Laboratory of Parasite Chemotherapy, National Institute of Allergy and Infectious IXseases, ~at~naZ Znstitutes of realty, ~et~~~, gayer 20014 (Submitted for publication POWERS, KENDALL
16 April 1969)
G., JACOBS, RICHARD I.,., GOOD, WILLIAM
C., AND KOONTZ,
Lcnns C. 1969. Plasmodium uinckei: Production of chloroquine-resistant strain. Expe~m~tal Parasitology 26, 193-202. To date only two species of malarial parasites, P~a~~rn fa~~~~rn in man and P. berg& in mice, have demonstrated the ability to develop a high degree of resistance to chloroquine. A strain of P. uinckei which is resistant to the maximum tolerated dose ( MTD) of chloroquine in mice has been selected from a pyrimethamine-resistant parent strain. Groups of five or more mice were treated with varying doses of drug for 34 days starting the day after parasite inoculation. Subpassages were made from mice treated with the highest dose of drug that allowed for some development of parasites. Resistance developed gradually during 37 successive chloroqu~~~~t~ passages over a period of ahout 44 weeks. In subsequent passages, the parasite was resistant to the MTD of chloroquine for mice (200 mg/kg/day). The dose of chloroquine which suppressed the parent strain by 99.9% on Day 7 was 5 mg/kg/day for 4 days. Sixty percent of mice treated with 10 mg/kg/day of chloroquine and all mice treated with doses of 20 mg/kg/day and greater were cured. Associated with resistance to chloroquine were the parasite’s failure to produce detectable pigment in treated mice and the appearance of multiple vacuoles. The resistant strain in treated mice is characterized by slower developing, less fatal parasitemias while in untreated mice fulminating infections often result in death. This new species of chloroquine-resistant malaria may be of value as a laboratory model for the study of acquired drug resistance. INDEX DESCRIPTORS: Plasmodium uinckei; Chloroquine resistance; Chemotherapy; P~e~~ine resistance; Morphology; ~p~~r~o~ coccoides; Oxophenarsine hydrochloride; Quinine; Malaria; Treatment.
To date the only two species of malarial parasites which have demonstrated a high degree of resistance to chloroquine are P~c~~~rn berghe~ and P. fa~~~~T~~. In 1957 Ramakrishnan et al. first reported the selection of a chloroquine-resistant strain of I’. berghei in mice. Four years later, Moore and Lanier (1961) reported the first cases of chloroquine-resistant P. fu~~~u~nm. Subsequent to the appearance of resistant strains of 2’. fuZc+mrum in different areas of the world, chloroquine-resistant strains of P. berghei have been used extensively as a laboratory model for the study of chloroquine resistance. Although these
strains have been most useful in this purpose, there has been a continuing need for additional species of chloroquine-resistant plasmodia to provide additional test systems in which the mechanisms involved might be investigated. This report describes the selection and some characteristics of a strain of 2’. oinckei which is resistant to the maximum tolerated dose of chloroquine in mice.
193
MATIZRIALS
AND METHODS
Test Animals Twelve-week-old albino Swiss mice (NIH general purpose strain) weighing
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POWERS ET AL.
20-22 gm were maintained on wood shavings in stainless-steel cages. Purina Laboratory Chow and water were supplied ad &turn. Mice were examined for the presence of Eperythrozoon coccoides and when encountered this contaminant was eliminated from the strain using oxophenarsine hydrochloride ( Mapharsen) therapy described by Thompson and Bayles ( 1966). Parasite P. vinckei was originally isolated from the salivary glands of Anopheles dureni in Katanga by Rodhain ( 1952). The P. vinckei used in these studies was received through the courtesy of Professor P. C. C. Garnham, of the London School of Hygiene and Tropical Medicine, in 1964, and it has since been maintained in this Laboratory by blood-passage in mice. This parasite invades both mature and immature erythrocytes and is uniformly fatal in mice about 5-8 days after exposure. Some prominent morphologic characteristics are multiple chromatin dots of unequal size in the ring form, the tenuous nature of the cytoplasm, and the presence of very fine grains of pigment in trophozoites. Passages Passages were initiated using two strains of P. vinckei: (1) the normal strain sensitive to commonly used antimalarials and (2) a strain resistant to the maximum tolerated dose (MTD) of pyrimethamine (PR strain). Subpassages were made 7 days after inoculation except in instances where slow development of the parasite required some extension of this period. Donors were selected from groups of mice that had been treated with the highest dose of chloroquine that allowed for some development of parasites, in the previous passage. Single donor mice were used when more than 1% of erythrocytes were infected. Pooled blood from several mice was used to prepare in-
ocula when parasitemias were less than 1%. Unless otherwise indicated groups of five mice were routinely inoculated intraperitoneally with lo7 parasitized erythrocytes. The inocula were prepared by appropriate dilution of heart blood with physiological saline so that 0.2 ml contained the desired parasite dose. Thin blood films taken on Day 7 from the tail vein were fixed in methyl alcohol and stained with Giemsa stain. Parasite counts were made using a 10 X wide-field eyepiece fitted with a Howard disc and a 97X oil immersion objective. At least 200 cells were counted to determine percentage parasitemia and at least 100 fields were examined before a preparation was considered negative. Periodically during the treated passages samples of infected blood were preserved by freezing at -70°C. Drug Administration The diphosphate salts of chloroquine and primaquine were prepared in aqueous solution. Pyrimethamine (free base) was dissolved in 0.1 N HCl. All dosages are expressed in terms of the free base. Individual mice were weighed prior to treatment and the appropriate drug level was administered by stomach tube as single daily doses. The drug concentration was adjusted so that 0.01 ml of solution per gram body weight delivered the correct mg/kg dose of drug. During each passage a group of untreated controls was included and parasitemias compared with treated mice. PROTOCOLS AND REZXJLTS
Normal Parent Strain The sensitivity of the normal strain of P. vinckei to chloroquine was determined using groups of ten mice treated with varying doses of the drug. Oral doses were given daily for 4 days starting the day after
CHLOROQUINE
exposure (Day 1). Suppressive doses (SD) on Day 7 were computed by comparing parasitemias in treated and untreated mice and results were plotted on logarithmic probability paper as described by Jacobs et al. (1963). The SD 50 and SD 99.9 of the normal strain was 3.3 and 5 mg/kg chloroquine, respectively. The curative dose was determined using groups of mice treated with 10, 26, 40, and 80 mgkg. Negative mice were held for 21 days and heart blood (0.1 ml) from these mice was then subinoculated. Recipients were examined for 21 days after inoculation before considering the donors cured. Sixty percent of mice treated with 10 mg/kg of chloroquine and all mice treated with the higher doses were cured. Untreated infected controls died on Day 7. The first attempt to select a chloroquineresistant strain using the normal strain was unsuccessful in developing a high degree of resistance. The strain was first treated with three daily doses of either 5, 7.5, or 10 mg/kg of chloroquine. During the first 11 passages transfers were made only from the 5 mg/kg groups as the two higher doses completely suppressed development of parasitemias on Day 7. Starting with passage 12, the 7.5 mg/kg dose was tolerated and by Passage 23, parasites developed in mice treated with 10 mg/kg but not in mice treated with 15 mg/kg. An additional 27 passages were treated with these two doses. During 50 passages over 346 days the strain failed to tolerate doses greater than 10 mg/kg. A parallel series of treated passages was started beginning with treated Passage 32 in the series described above. Mice received one of three combinations for 3 or 4 days during 32 subsequent passages: 3.0 mg/kg of chloroquine and 0.3 mg/kg of primaquine, 6.0 mg/kg of chloroquine and 0.6 mg/kg of primaquine, or 12.0 mg/ kg of chloroquine and 1.2 mg/kg of prima-
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quine. The two lower doses allowed for some development of parasites while the higher dose was completely suppressive on Day 7. Finally the strain was treated during an additional I8 successive passages with 5, 10, or 15 mg/kg of chloroquine. In summary, after 82 successive passages under constant chloroquine pressure, including 32 chloroquine-primaquine-treated passages, the strain never tolerated more than four daily 10 mg,kg doses of chloroquine. This is approximately twice the dose required to completely suppress the parent strain on Day 7. At this point studies were discontinued on this strain, Pytimethamiw-Resistant
Parent Strain
A second attempt to select a chloroquineresistant strain of P. vinckei was initiated using a strain previously made resistant to pyrimethamine (PR strain), The PR strain was selected from the normal strain using increasing doses of pyrimethamine during consecutive passages. The strain iirst became resistant to the MTD of pyrimethamine in mice (50 mg/kg X 4) during treated Passage 8 and an additional 30 passages were treated with this regimen. The PR38 strain was found to be normally sensitive to chloroquine. After Passage 38, pyrimethamine therapy was discontinued and the strain was treated with chloroquine during each subsequent passage. Prior to treatment with chloroquine the PR strain was found to be contaminated with E. coccoides. This contaminant was eliminated during four blood passages in mice using daily 10 mg/kg parenteral doses of oxophenarsine hydrochloride. Figure 1 illustrates the development of chloroquine resistance during successive passages under chloroquine pressure. Groups of mice were first treated with 2.5 or 5 mg/kg of chloroquine for 3 days starting on Day 2. Doses were increased when the strain began toIerating the higher dose.
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POWERS
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FIG. 1. Development of resistance to chloroquine by P. vinckei during successive chloroquinetreated blood passages in mice. Chloroquine was administered as three daily doses during each of the first 55 passages. Thereafter four daily doses were given. A indicates that parasite transfer was delayed until Day 14. Oxophenarsine hydrochloride therapy_. was administered during Passages 40 through 45 to eliminate E. cocc&es.
Unless otherwise noted donor mice were selected on Day 7 from the group which received the highest doses of chloroquine which allowed for some development of parasites. After two passages the strain developed in mice treated with 5 mg/kg but was suppressed by 10 mg/kg. Occasionally during the next eight passages the time of transfer was delayed to Day 14 in an attempt to accelerate the development of resistance by using the mice receiving the higher drug dose as donors. By Passage 15, the parasites were routinely appearing on Day 7 in mice treated with 10 mg/kg. Starting with Passage 25 the strain tolerated 20 mg/kg and during the next 12 passages it rapidly became more tolerant to increasing doses of chloroquine. By Passage 37, which was reached after about 10 months, the strain developed in mice treated with the MTD of chloroquine (200 mg/kg) . During Passages 30 through 40 it was necessary several times to delay transfer of the strain until Day 14 as parasites could not be detected or did not ap-
pear in mice in sufficient numbers by Day 7. During Passage 39, mice were again observed to be infected with E. coccoides and oxophenarsine hydrochloride therapy was initiated during the next passage in addition to chloroquine and continued until the contaminant was eradicated by Passage 45. In general, parasitemias in mice treated with chloroquine and oxophenarsine were lower than seen in mice treated with similar doses of chloroquine alone. Starting with Passage 56 the number of daily doses was increased to 4 with the first dose given on Day 1. No apparent change in parasitemias on Day 7 was observed as a result of this regimen. Although parasites routinely appeared in mice treated with the MTD of chloroquine, the percentage parasitemias were usually quite low when transfers were made on Day 7. A total of 48 consecutive passages was treated with the MTD of chloroquine in an attempt to select for better parasite development on Day 7. No increase in the rate of development was seen throughout these additional passages.
CHLOROQUINE
In each passage a parallel group of untreated control mice was exposed to the same inooulum as the treated group to compare parasite morphology and rate of parasite development in these two groups. Parasite development in untreated mice was characterized by rapidly rising parasitemias resulting in death in a large percentage of infected mice on Days 6 through 9. Average parasitemias on Day 7 were usually greater than 80% and individual parasites appeared we11 pigmented and morphologically very similar to normal strain parasites. During treated Passage 84 (CR-84) groups of ten infected mice were each treated with 5, 10, 20, and 200 mg/kg of chloroquine for 4 days to obtain dose response information. On Day 7 these doses resulted in 5, 78, 86, and 97% suppression,
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RESISTANCE
respectively, of parasitemias in treated mice as compared to untreated controls. After 20, 40, 60, and 80 chloroquinetreated passages the strain was tested for continued resistance to pyrimethamine. In all instances the CR strain was found still to be resistant to the MTD of pyrimethamine in mice. Characteristics
of the CR Strain
Morphology. Giemsa-stained and fresh thin films of blood containing the CR strain were examined for pigment on Day 7 during each treated passage using bright- and dark-field microscopy. As the strain began to tolerate increasing doses of chloroquine there was an accompanying decrease in pigment production. By Passage 15, when tolerance of 10 mg/kg was first noted (Fig.
Morphological appearance of chloroquine-resistant (CR), normal (N) and pyrimethPLATE 1. blood films from infected mice. Notice amine-resistant (PR) strains of I’. oinckei in Giemsa-stained the absence of pigment and increased vacuolation in the various stages of development in the CR strain as compared to the normal strain. No obvious differences are seen between the PR and normal strain. All stages are drawn to scale. (Plate prepared by Gertrude H. Nicholson).
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POWERS
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FIG. 2. Graph illustrating the untreated course of infection of normal (N) and chloroquine-resistant (CR) strains of P. uinckei in groups of 20 mice. + indicates number of mice dying on a particular day. Eight mice (8t ) infected with the normal strain died before blood smears could be made on Day 6. Vertical lines represent the standard error for each mean value. Points are not shown after mice became negative.
1 ), the amount of pigment seen in the parasites had decreased by about one-half, Pigment was barely perceptible in the parasites starting with Passage 26, and from Passage 34 through 85 no pigment was detectable in any of the CR parasites examined on or before Day 7 (Plate 1). Another morphological feature accompanying the increase in resistance to chloroquine was multiple vacuoles in the cytoplasm of the parasite. Normal parasites usually contained one, or sometimes, two small vacuoles. In contrast, CR parasites developing in treated mice generally contained numerous vacuoles of various sizes. Morphologically, the PR strain did not appear to differ from the normal strain in pigmentation or vacuolation (Plate 1). Parasitemiu and mortality. Figure 2 shows death patterns and average percentage parasitemias in 2O-mouse groups exposed to the normal or the CR-85 strains
of P. uinckei. Each mouse received lo6 parasitized erythrocytes. Neither group was treated during the 28-day observation period. The normal strain infection was acute and deaths occurred 5 or 6 days after exposure. The CR strain developed very slowly during the first few days then the rise in parasitemia paralleled that seen for the normal strain. However, seven of 20 mice survived the acute infection and after a secondary increase of parasitemias, mice became negative on Day 23. Subinoculation of heart blood on Day 28 demonstrated parasites in four of the seven survivors. Very little pigment and considerable vacuolation were seen in CR strain parasites during the first 3 days after exposure. Thereafter parasites appeared well pigmented and quite similar to the normal strain. Both strains demonstrated a definite for predilection mature erythrocytes throughout the study.
iXLOROQUINE
RESISTANCE
199
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860b. L r ?Oc c 5 60P ? & 50-
DAYS
AFTER
EXPOSURE
FIG. 3. Graph illustrating the therapeutic activity of chloroquine against normal (N) and chloroquine-resistant (CR) strains of I’. uinckei in groups of 20 mice. Treatment (T) was started 5 days after inoculation during the period of rapidly increasing parasitemias; the 50 mg/kg dose was administered daily through Day 28 and the 200 mg/kg dose was given daily for 4 successive days. Mean parasitemias are represented by broken lines for the normal strain and by solid lines for the CR strain. Points are not shown after mice became negative.
Treatment of established infections. The therapeutic activity of chloroquine was evaluated against normal and chloroquineresistant (CR-85) strains of I’. vinckei in mice (Fig. 3). Each of 20 mice was inoculated with lo6 parasitized erythrocytes in the normal strain. These mice were then divided into two lo-mouse groups, one of which received 200 mg/kg of chloroquine for 4 consecutive days. The second group was treated with 50 mg/kg for 28 days. Initial treatments were carried out on postinoculation Day 5 when mean parasitemias were above 80%. Seven mice from the 50 mg/kg group and 4 mice from the 200 mg/kg group died during the first 3 days of treatment. No parasites were seen in the peripheral blood of survivors from Day 4 of treatment until the end of the experiment. On Day 28, heart blood from individual mice in each treatment group
was subinoculated into susceptible mice. No parasites were seen in the subinoculated mice during a 21-day observation period. Concurrently a second group of 20 mice was inoculated with lo6 parasitized erythrocytes of the CR strain and treated according to the above procedure. When treatment was initiated parasitemias were increasing rapidly. Although parasitemias continued to increase in treated mice through treatment Day 4, they were lower than would be expected when compared to untreated CR strain infections (Fig. 2). Thereafter, a rapid decline in parasitemias was observed with both treatment regimens; parasites kept under extended chloroquine pressure (50 mg/kg) disappeared from the blood on Day 1’7 while mice treated with 200 mg/kg for 4 days experienced a second parasite peak on Day
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POWERS ET AL.
16, but all were negative by Day 20. One mouse in the 200 mg/kg group died on Day 16. Heart blood from surviving mice was subinoculated on Day 23. All mice which received blood from the 200 mg/kg group and six of ten mice that received blood from the 50 mg/kg group became patent after subinoculation. Parasites in the CR group kept under extended chloroquine pressure (50 mg/kg/ day) were highly vacuolated and without detectable pigment throughout the course of visible parasitemia. Pigment was seen to reappear slowly in the 200 mg/kg group starting about 3 days after cessation of treatment and was approximately of normal intensity 7 days later. Stability of chloroquine resistance. During Passage 79 an untreated substrain was initiated to determine whether the resistance to chloroquine was a stable character in the absence of selective drug pressure. The strain was subpassaged twice weekly without treatment in mice. Untreated Passages 10 and 20 were tested for sensitivity to chloroquine. The strain retained resistance to the MTD of chloroquine throughout 20 successive drug-free passages over a period of 10 weeks. Susceptibility of the CR strain to quinine. During Passage 79 the CR strain was tested for sensitivity to quinine. Eight groups of 20 mice were inoculated with the normal or CR strain of P. vinckei and each strain was treated with 50,100, or 200 mg/kg oral doses of quinine (base) for 4 consecutive days starting the day after inoculation. Smears were made on Day 7 and percentage suppression of parasitemia by the various doses of quinine was calculated for both strains. The results indicated that the CR-79 strain was normally sensitive to quinine. DISCUSSION
Although there are numerous reports of the selection of strains of P. berghei re-
sistant to chloroquine, apparently no reports of resistance in P. vinclcei have appeared to date. The resistant strains of P. berghei generally have been selected from normally sensitive parent strains, using increasing doses of chloroquine during repeated blood passages in mice. Strains of P. berghei resistant to the MTD of chloroquine in mice were obtained by treated Passage 24 by Peters (1965) and as early as treated Passage 11 by Jacobs (1965). In our hands, the normal strain of P. vinckei failed to develop a high degree of resistance to chloroquine during 82 successive chloroquine-treated passages, including 32 passages treated with a chloroquineprimaquine combination. In 1966, Peters suggested the possible role of primaquine in inducing multiple drug resistance, particularly when combined with chloroquine. It became evident that a new approach to the problem was necessary in order to select a strain resistant to chloroquine. We, therefore, used a parent strain previously made resistant to pyrimethamine. For several reasons we decided to use this strain: ( 1) Chloroquine-resistant strains of falciparum malaria occurring in nature are also frequently resistant to pyrimethamine and other classes of antimalarials; and (2) a strain resistant to one compound might be altered in such a manner as to make it more prone to becoming resistant to additional compounds. However, we have no exact explanation as to how we were able to select a chloroquine-resistant strain from one already resistant to pyrimethamine while we were unable to select for the same character using the same methods with a normal strain parasite. A consistent characteristic of most strains of chloroquine-resistant P. berghei has been the lack of pigment production in parasites under chloroquine pressure. Release of this pressure has resulted in a
CHLOROQUINE
gradual loss of resistance to chloroquine accompanied by a return of pigment production. In the absence of drug pressure, Jacobs (1965) found that pigment production gradually reappeared during numerous subpassages or in recurrent parasitemias in mice surviving the initial infection. Peters (1964) saw the return of pigment formation in untreated mice infected with his chloroquine-resistant strain after 28 days and completely normal pigment occurred in parasites during recrudescence on Day 53. A lack of pigment was also observed in our CR strain of P. uinckei in mice treated with chloroquine. However, pigment started to reappear within 3 days after cessation of treatment in mice treated during a period of rising parasitemias ( Fig. 3). Pigment was approaching normal intensity during the second wave of parasitemias on Days 16 through 19. McNamara et al. ( 1967), and Kellett et u.?. ( 1968) observed pigment in chloroquine-resistant P. falcipr~rn both in the peripheral blood and after in vitro cultivation. McNamara’s group saw pigment in mature schizonts in blood from a patient who had received treatment with chloroquine 3 weeks previous to the observation and in parasites from two additional volunteers for whom th e presence or absence of chloroquine treatment was not reported. They concluded from these observations that “there is a major morphological difference in chloroquine-resistant P. berghei compared to chloroquine-resistant P. falciparum.” It should be pointed out that pigment does return both in chloroquine-resistant P. vinckei and P. berghei after chloroquine treatment has been discontinued. We are not aware that comparable studies have been done to show whether pigment is present or absent in chloroquine-resistant P. faZ&parmn under chloroquine pressure. The course of infection of the CR strain of P. uinckei in untreated mice appears
RESISTANCE
201
comparable to the normal strain with the exception of a short delay in the first wave of parasitemia and in the increased survival rate in mice infected with the CR strain, A typical course of infection in untreated mice is shown in Fig. 2. Mice infected with the normal strain usually die by Day 6 with 80-90% of the erythrocytes infected. High parasitemias are also common for the CR strain. Although average maximum parasitemias were 75%, 12 out of 20 mice developed peak parasitemias of 90% or greater. The course of infection of our CR strain of P. uinckei appears somewhat different from chloroquine-resistant P. berghei. Peters (1964) and Jacobs (1965) reported a much milder course of infection and higher survival rate in untreated mice infected with their chloroquine-resistant strains as compared to normal strain P. berghei. Peters (1964) observed in mice infected with his strain that about 10% of the erythrocytes were infected 7-10 days after exposure. Thereafter parasitemias increased to XL70% or more by the end of Week 3. About 30% of these mice recovered while the remainder experienced high parasitemias and died. We have observed a greater degree of suppression by chloroquine in our resistant P. uinckei than has been seen with numerous strains of chloroquine-resistant P. berghei [Hawking and Gammage, ( 1962) ; Peters, (1965); and Thompson et al., ( 1967 ) 1. The MTD of chloroquine given on Days 1 through 4 routinely suppressed parasitemias in mice infected with CR P. uinckei by 90% or more. However, parasites were not cleared from the blood of mice treated with MTD of chloroquine even for an extended period of time. Chloroquine also exhibited therapeutic activity against the resistant P. uinckei (Fig. 3). Although parasitemias continued to increase under chloroquine pressure, they were considerably lower than in untreated infections (Fig. 2) on comparable
202
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days after inoculation. Parasitemias decreased in both treated and untreated infections with the chloroquine-resistant strain, and. both infections became subpatent approximately the same number of days after inoculation. These trends were probably due to host immunity rather than to a chloroquine effect.
REFERENCES HAWKING, F., AND GAMMAGE, K. 1962. Chloroquine resistance produced in Plasmodium berghei. Transactions of the Royal Society of Tropical Medicine and Hygiene 56, 263. JACOBS, R. L., ALLING, D. W., AND CANTRELL, W. F. 1963. An evaluation of antimalarial combinations against Plasmodium berghei in the mouse. Journal of Parasitology 49, 920925. JACOBS, R. L. 1965. Selection of strains of Plasmodium berghei resistant to quinine, chloroquine, and pyrimethamine. Journal of Parasitology 51, 481-482. KELLETT, R. J., COWAN, G. O., AND PARRY, E. S. 1968. Chloroquine-resistant Plasmodium falciparum malaria in the United Kingdom. The Lancet 2, 946-948. J. V., RIECKMANN, K. H., AND MCNAMARA, POWELL, R. D. 1967. Pigment in asexual erythrocytic forms of chloroquine-resistant Plusmodium falciparum. Annals of Tropical Medicine and Parasitology 61, 125-127. MOORE, D. V., AND LANIER, J. E. 1961. Observations on two Plasmodium falciparum in-
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fections with an abnormal response to chloroquine. American Journal of Tropical Medicine
PETEzd y lo’ 5-g. > . 1964. Pigment
formation and nuclear division in chloroouine-resistant malaria parasites (Plasmodium Iberghei, Vincke and Lips, 1948), Nature 203, 1299-1291. PETERS, W. 1965. Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance. Experimental Parasitology 17, 89-89. 1966. The possible role of primaPETERS, W. quine in inducing multiple drug resistance in Plasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 149-141. RAMAKRISHNAN, S. P., PRAKASH, S., AND CHOUDHURY, D. S. 1957. Studies on Plasmodium berghei Vincke and Lips, 1948. XXIV. Selection of a chloroquine resistant strain. Indian Journal of Maluriology 11, 213-220. RODHAIN, J. 1952. Plasmodium vinckei n. sp. Un deuxieme Plasmodium parasite de rongeurs sauvages au Katanga. Annaks de lu Societe Beige de Medecine Tropicale 32, 275280. THOMPSON, P. E., AND BAYLES, A. 1966. Eradication of Eperythrozoon coccoides with oxophenarsine in normal and drug-resistant lines of Plasmodium berghei in mice. Journat of Pamsitology 52, 674-678. THOMPSON, P. E., OLSZEWSKI, B., BA~LES, A., AND WAITZ, J. A. 1967. Relations among antimalarial drugs: results of studies with cycloguanil-, sulfone-, or chloroquine-resistant Plasmodium berghei in mice. American Journal of Tropical Medicine and Hygiene 16, 133-145.
~~as~odiu~ Kendall
G.
vinckei: Powers,
Production
Richard
of ~h~oroquine-Resjstant
1. Jacobs,
William
C. Good,
and
Louis
Strain C. Koontz
Laboratory of Parasite Chemotherapy, National Institute of Allergy and Infectious IXseases, ~at~naZ Znstitutes of realty, ~et~~~, gayer 20014 (Submitted for publication POWERS, KENDALL
16 April 1969)
G., JACOBS, RICHARD I.,., GOOD, WILLIAM
C., AND KOONTZ,
Lcnns C. 1969. Plasmodium uinckei: Production of chloroquine-resistant strain. Expe~m~tal Parasitology 26, 193-202. To date only two species of malarial parasites, P~a~~rn fa~~~~rn in man and P. berg& in mice, have demonstrated the ability to develop a high degree of resistance to chloroquine. A strain of P. uinckei which is resistant to the maximum tolerated dose ( MTD) of chloroquine in mice has been selected from a pyrimethamine-resistant parent strain. Groups of five or more mice were treated with varying doses of drug for 34 days starting the day after parasite inoculation. Subpassages were made from mice treated with the highest dose of drug that allowed for some development of parasites. Resistance developed gradually during 37 successive chloroqu~~~~t~ passages over a period of ahout 44 weeks. In subsequent passages, the parasite was resistant to the MTD of chloroquine for mice (200 mg/kg/day). The dose of chloroquine which suppressed the parent strain by 99.9% on Day 7 was 5 mg/kg/day for 4 days. Sixty percent of mice treated with 10 mg/kg/day of chloroquine and all mice treated with doses of 20 mg/kg/day and greater were cured. Associated with resistance to chloroquine were the parasite’s failure to produce detectable pigment in treated mice and the appearance of multiple vacuoles. The resistant strain in treated mice is characterized by slower developing, less fatal parasitemias while in untreated mice fulminating infections often result in death. This new species of chloroquine-resistant malaria may be of value as a laboratory model for the study of acquired drug resistance. INDEX DESCRIPTORS: Plasmodium uinckei; Chloroquine resistance; Chemotherapy; P~e~~ine resistance; Morphology; ~p~~r~o~ coccoides; Oxophenarsine hydrochloride; Quinine; Malaria; Treatment.
To date the only two species of malarial parasites which have demonstrated a high degree of resistance to chloroquine are P~c~~~rn berghe~ and P. fa~~~~T~~. In 1957 Ramakrishnan et al. first reported the selection of a chloroquine-resistant strain of I’. berghei in mice. Four years later, Moore and Lanier (1961) reported the first cases of chloroquine-resistant P. fu~~~u~nm. Subsequent to the appearance of resistant strains of 2’. fuZc+mrum in different areas of the world, chloroquine-resistant strains of P. berghei have been used extensively as a laboratory model for the study of chloroquine resistance. Although these
strains have been most useful in this purpose, there has been a continuing need for additional species of chloroquine-resistant plasmodia to provide additional test systems in which the mechanisms involved might be investigated. This report describes the selection and some characteristics of a strain of 2’. oinckei which is resistant to the maximum tolerated dose of chloroquine in mice.
193
MATIZRIALS
AND METHODS
Test Animals Twelve-week-old albino Swiss mice (NIH general purpose strain) weighing
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POWERS ET AL.
20-22 gm were maintained on wood shavings in stainless-steel cages. Purina Laboratory Chow and water were supplied ad &turn. Mice were examined for the presence of Eperythrozoon coccoides and when encountered this contaminant was eliminated from the strain using oxophenarsine hydrochloride ( Mapharsen) therapy described by Thompson and Bayles ( 1966). Parasite P. vinckei was originally isolated from the salivary glands of Anopheles dureni in Katanga by Rodhain ( 1952). The P. vinckei used in these studies was received through the courtesy of Professor P. C. C. Garnham, of the London School of Hygiene and Tropical Medicine, in 1964, and it has since been maintained in this Laboratory by blood-passage in mice. This parasite invades both mature and immature erythrocytes and is uniformly fatal in mice about 5-8 days after exposure. Some prominent morphologic characteristics are multiple chromatin dots of unequal size in the ring form, the tenuous nature of the cytoplasm, and the presence of very fine grains of pigment in trophozoites. Passages Passages were initiated using two strains of P. vinckei: (1) the normal strain sensitive to commonly used antimalarials and (2) a strain resistant to the maximum tolerated dose (MTD) of pyrimethamine (PR strain). Subpassages were made 7 days after inoculation except in instances where slow development of the parasite required some extension of this period. Donors were selected from groups of mice that had been treated with the highest dose of chloroquine that allowed for some development of parasites, in the previous passage. Single donor mice were used when more than 1% of erythrocytes were infected. Pooled blood from several mice was used to prepare in-
ocula when parasitemias were less than 1%. Unless otherwise indicated groups of five mice were routinely inoculated intraperitoneally with lo7 parasitized erythrocytes. The inocula were prepared by appropriate dilution of heart blood with physiological saline so that 0.2 ml contained the desired parasite dose. Thin blood films taken on Day 7 from the tail vein were fixed in methyl alcohol and stained with Giemsa stain. Parasite counts were made using a 10 X wide-field eyepiece fitted with a Howard disc and a 97X oil immersion objective. At least 200 cells were counted to determine percentage parasitemia and at least 100 fields were examined before a preparation was considered negative. Periodically during the treated passages samples of infected blood were preserved by freezing at -70°C. Drug Administration The diphosphate salts of chloroquine and primaquine were prepared in aqueous solution. Pyrimethamine (free base) was dissolved in 0.1 N HCl. All dosages are expressed in terms of the free base. Individual mice were weighed prior to treatment and the appropriate drug level was administered by stomach tube as single daily doses. The drug concentration was adjusted so that 0.01 ml of solution per gram body weight delivered the correct mg/kg dose of drug. During each passage a group of untreated controls was included and parasitemias compared with treated mice. PROTOCOLS AND REZXJLTS
Normal Parent Strain The sensitivity of the normal strain of P. vinckei to chloroquine was determined using groups of ten mice treated with varying doses of the drug. Oral doses were given daily for 4 days starting the day after
CHLOROQUINE
exposure (Day 1). Suppressive doses (SD) on Day 7 were computed by comparing parasitemias in treated and untreated mice and results were plotted on logarithmic probability paper as described by Jacobs et al. (1963). The SD 50 and SD 99.9 of the normal strain was 3.3 and 5 mg/kg chloroquine, respectively. The curative dose was determined using groups of mice treated with 10, 26, 40, and 80 mgkg. Negative mice were held for 21 days and heart blood (0.1 ml) from these mice was then subinoculated. Recipients were examined for 21 days after inoculation before considering the donors cured. Sixty percent of mice treated with 10 mg/kg of chloroquine and all mice treated with the higher doses were cured. Untreated infected controls died on Day 7. The first attempt to select a chloroquineresistant strain using the normal strain was unsuccessful in developing a high degree of resistance. The strain was first treated with three daily doses of either 5, 7.5, or 10 mg/kg of chloroquine. During the first 11 passages transfers were made only from the 5 mg/kg groups as the two higher doses completely suppressed development of parasitemias on Day 7. Starting with passage 12, the 7.5 mg/kg dose was tolerated and by Passage 23, parasites developed in mice treated with 10 mg/kg but not in mice treated with 15 mg/kg. An additional 27 passages were treated with these two doses. During 50 passages over 346 days the strain failed to tolerate doses greater than 10 mg/kg. A parallel series of treated passages was started beginning with treated Passage 32 in the series described above. Mice received one of three combinations for 3 or 4 days during 32 subsequent passages: 3.0 mg/kg of chloroquine and 0.3 mg/kg of primaquine, 6.0 mg/kg of chloroquine and 0.6 mg/kg of primaquine, or 12.0 mg/ kg of chloroquine and 1.2 mg/kg of prima-
195
RESISTANCE
quine. The two lower doses allowed for some development of parasites while the higher dose was completely suppressive on Day 7. Finally the strain was treated during an additional I8 successive passages with 5, 10, or 15 mg/kg of chloroquine. In summary, after 82 successive passages under constant chloroquine pressure, including 32 chloroquine-primaquine-treated passages, the strain never tolerated more than four daily 10 mg,kg doses of chloroquine. This is approximately twice the dose required to completely suppress the parent strain on Day 7. At this point studies were discontinued on this strain, Pytimethamiw-Resistant
Parent Strain
A second attempt to select a chloroquineresistant strain of P. vinckei was initiated using a strain previously made resistant to pyrimethamine (PR strain), The PR strain was selected from the normal strain using increasing doses of pyrimethamine during consecutive passages. The strain iirst became resistant to the MTD of pyrimethamine in mice (50 mg/kg X 4) during treated Passage 8 and an additional 30 passages were treated with this regimen. The PR38 strain was found to be normally sensitive to chloroquine. After Passage 38, pyrimethamine therapy was discontinued and the strain was treated with chloroquine during each subsequent passage. Prior to treatment with chloroquine the PR strain was found to be contaminated with E. coccoides. This contaminant was eliminated during four blood passages in mice using daily 10 mg/kg parenteral doses of oxophenarsine hydrochloride. Figure 1 illustrates the development of chloroquine resistance during successive passages under chloroquine pressure. Groups of mice were first treated with 2.5 or 5 mg/kg of chloroquine for 3 days starting on Day 2. Doses were increased when the strain began toIerating the higher dose.
196
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FIG. 1. Development of resistance to chloroquine by P. vinckei during successive chloroquinetreated blood passages in mice. Chloroquine was administered as three daily doses during each of the first 55 passages. Thereafter four daily doses were given. A indicates that parasite transfer was delayed until Day 14. Oxophenarsine hydrochloride therapy_. was administered during Passages 40 through 45 to eliminate E. cocc&es.
Unless otherwise noted donor mice were selected on Day 7 from the group which received the highest doses of chloroquine which allowed for some development of parasites. After two passages the strain developed in mice treated with 5 mg/kg but was suppressed by 10 mg/kg. Occasionally during the next eight passages the time of transfer was delayed to Day 14 in an attempt to accelerate the development of resistance by using the mice receiving the higher drug dose as donors. By Passage 15, the parasites were routinely appearing on Day 7 in mice treated with 10 mg/kg. Starting with Passage 25 the strain tolerated 20 mg/kg and during the next 12 passages it rapidly became more tolerant to increasing doses of chloroquine. By Passage 37, which was reached after about 10 months, the strain developed in mice treated with the MTD of chloroquine (200 mg/kg) . During Passages 30 through 40 it was necessary several times to delay transfer of the strain until Day 14 as parasites could not be detected or did not ap-
pear in mice in sufficient numbers by Day 7. During Passage 39, mice were again observed to be infected with E. coccoides and oxophenarsine hydrochloride therapy was initiated during the next passage in addition to chloroquine and continued until the contaminant was eradicated by Passage 45. In general, parasitemias in mice treated with chloroquine and oxophenarsine were lower than seen in mice treated with similar doses of chloroquine alone. Starting with Passage 56 the number of daily doses was increased to 4 with the first dose given on Day 1. No apparent change in parasitemias on Day 7 was observed as a result of this regimen. Although parasites routinely appeared in mice treated with the MTD of chloroquine, the percentage parasitemias were usually quite low when transfers were made on Day 7. A total of 48 consecutive passages was treated with the MTD of chloroquine in an attempt to select for better parasite development on Day 7. No increase in the rate of development was seen throughout these additional passages.
CHLOROQUINE
In each passage a parallel group of untreated control mice was exposed to the same inooulum as the treated group to compare parasite morphology and rate of parasite development in these two groups. Parasite development in untreated mice was characterized by rapidly rising parasitemias resulting in death in a large percentage of infected mice on Days 6 through 9. Average parasitemias on Day 7 were usually greater than 80% and individual parasites appeared we11 pigmented and morphologically very similar to normal strain parasites. During treated Passage 84 (CR-84) groups of ten infected mice were each treated with 5, 10, 20, and 200 mg/kg of chloroquine for 4 days to obtain dose response information. On Day 7 these doses resulted in 5, 78, 86, and 97% suppression,
197
RESISTANCE
respectively, of parasitemias in treated mice as compared to untreated controls. After 20, 40, 60, and 80 chloroquinetreated passages the strain was tested for continued resistance to pyrimethamine. In all instances the CR strain was found still to be resistant to the MTD of pyrimethamine in mice. Characteristics
of the CR Strain
Morphology. Giemsa-stained and fresh thin films of blood containing the CR strain were examined for pigment on Day 7 during each treated passage using bright- and dark-field microscopy. As the strain began to tolerate increasing doses of chloroquine there was an accompanying decrease in pigment production. By Passage 15, when tolerance of 10 mg/kg was first noted (Fig.
Morphological appearance of chloroquine-resistant (CR), normal (N) and pyrimethPLATE 1. blood films from infected mice. Notice amine-resistant (PR) strains of I’. oinckei in Giemsa-stained the absence of pigment and increased vacuolation in the various stages of development in the CR strain as compared to the normal strain. No obvious differences are seen between the PR and normal strain. All stages are drawn to scale. (Plate prepared by Gertrude H. Nicholson).
198
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FIG. 2. Graph illustrating the untreated course of infection of normal (N) and chloroquine-resistant (CR) strains of P. uinckei in groups of 20 mice. + indicates number of mice dying on a particular day. Eight mice (8t ) infected with the normal strain died before blood smears could be made on Day 6. Vertical lines represent the standard error for each mean value. Points are not shown after mice became negative.
1 ), the amount of pigment seen in the parasites had decreased by about one-half, Pigment was barely perceptible in the parasites starting with Passage 26, and from Passage 34 through 85 no pigment was detectable in any of the CR parasites examined on or before Day 7 (Plate 1). Another morphological feature accompanying the increase in resistance to chloroquine was multiple vacuoles in the cytoplasm of the parasite. Normal parasites usually contained one, or sometimes, two small vacuoles. In contrast, CR parasites developing in treated mice generally contained numerous vacuoles of various sizes. Morphologically, the PR strain did not appear to differ from the normal strain in pigmentation or vacuolation (Plate 1). Parasitemiu and mortality. Figure 2 shows death patterns and average percentage parasitemias in 2O-mouse groups exposed to the normal or the CR-85 strains
of P. uinckei. Each mouse received lo6 parasitized erythrocytes. Neither group was treated during the 28-day observation period. The normal strain infection was acute and deaths occurred 5 or 6 days after exposure. The CR strain developed very slowly during the first few days then the rise in parasitemia paralleled that seen for the normal strain. However, seven of 20 mice survived the acute infection and after a secondary increase of parasitemias, mice became negative on Day 23. Subinoculation of heart blood on Day 28 demonstrated parasites in four of the seven survivors. Very little pigment and considerable vacuolation were seen in CR strain parasites during the first 3 days after exposure. Thereafter parasites appeared well pigmented and quite similar to the normal strain. Both strains demonstrated a definite for predilection mature erythrocytes throughout the study.
iXLOROQUINE
RESISTANCE
199
I oo90-
860b. L r ?Oc c 5 60P ? & 50-
DAYS
AFTER
EXPOSURE
FIG. 3. Graph illustrating the therapeutic activity of chloroquine against normal (N) and chloroquine-resistant (CR) strains of I’. uinckei in groups of 20 mice. Treatment (T) was started 5 days after inoculation during the period of rapidly increasing parasitemias; the 50 mg/kg dose was administered daily through Day 28 and the 200 mg/kg dose was given daily for 4 successive days. Mean parasitemias are represented by broken lines for the normal strain and by solid lines for the CR strain. Points are not shown after mice became negative.
Treatment of established infections. The therapeutic activity of chloroquine was evaluated against normal and chloroquineresistant (CR-85) strains of I’. vinckei in mice (Fig. 3). Each of 20 mice was inoculated with lo6 parasitized erythrocytes in the normal strain. These mice were then divided into two lo-mouse groups, one of which received 200 mg/kg of chloroquine for 4 consecutive days. The second group was treated with 50 mg/kg for 28 days. Initial treatments were carried out on postinoculation Day 5 when mean parasitemias were above 80%. Seven mice from the 50 mg/kg group and 4 mice from the 200 mg/kg group died during the first 3 days of treatment. No parasites were seen in the peripheral blood of survivors from Day 4 of treatment until the end of the experiment. On Day 28, heart blood from individual mice in each treatment group
was subinoculated into susceptible mice. No parasites were seen in the subinoculated mice during a 21-day observation period. Concurrently a second group of 20 mice was inoculated with lo6 parasitized erythrocytes of the CR strain and treated according to the above procedure. When treatment was initiated parasitemias were increasing rapidly. Although parasitemias continued to increase in treated mice through treatment Day 4, they were lower than would be expected when compared to untreated CR strain infections (Fig. 2). Thereafter, a rapid decline in parasitemias was observed with both treatment regimens; parasites kept under extended chloroquine pressure (50 mg/kg) disappeared from the blood on Day 1’7 while mice treated with 200 mg/kg for 4 days experienced a second parasite peak on Day
200
POWERS ET AL.
16, but all were negative by Day 20. One mouse in the 200 mg/kg group died on Day 16. Heart blood from surviving mice was subinoculated on Day 23. All mice which received blood from the 200 mg/kg group and six of ten mice that received blood from the 50 mg/kg group became patent after subinoculation. Parasites in the CR group kept under extended chloroquine pressure (50 mg/kg/ day) were highly vacuolated and without detectable pigment throughout the course of visible parasitemia. Pigment was seen to reappear slowly in the 200 mg/kg group starting about 3 days after cessation of treatment and was approximately of normal intensity 7 days later. Stability of chloroquine resistance. During Passage 79 an untreated substrain was initiated to determine whether the resistance to chloroquine was a stable character in the absence of selective drug pressure. The strain was subpassaged twice weekly without treatment in mice. Untreated Passages 10 and 20 were tested for sensitivity to chloroquine. The strain retained resistance to the MTD of chloroquine throughout 20 successive drug-free passages over a period of 10 weeks. Susceptibility of the CR strain to quinine. During Passage 79 the CR strain was tested for sensitivity to quinine. Eight groups of 20 mice were inoculated with the normal or CR strain of P. vinckei and each strain was treated with 50,100, or 200 mg/kg oral doses of quinine (base) for 4 consecutive days starting the day after inoculation. Smears were made on Day 7 and percentage suppression of parasitemia by the various doses of quinine was calculated for both strains. The results indicated that the CR-79 strain was normally sensitive to quinine. DISCUSSION
Although there are numerous reports of the selection of strains of P. berghei re-
sistant to chloroquine, apparently no reports of resistance in P. vinclcei have appeared to date. The resistant strains of P. berghei generally have been selected from normally sensitive parent strains, using increasing doses of chloroquine during repeated blood passages in mice. Strains of P. berghei resistant to the MTD of chloroquine in mice were obtained by treated Passage 24 by Peters (1965) and as early as treated Passage 11 by Jacobs (1965). In our hands, the normal strain of P. vinckei failed to develop a high degree of resistance to chloroquine during 82 successive chloroquine-treated passages, including 32 passages treated with a chloroquineprimaquine combination. In 1966, Peters suggested the possible role of primaquine in inducing multiple drug resistance, particularly when combined with chloroquine. It became evident that a new approach to the problem was necessary in order to select a strain resistant to chloroquine. We, therefore, used a parent strain previously made resistant to pyrimethamine. For several reasons we decided to use this strain: ( 1) Chloroquine-resistant strains of falciparum malaria occurring in nature are also frequently resistant to pyrimethamine and other classes of antimalarials; and (2) a strain resistant to one compound might be altered in such a manner as to make it more prone to becoming resistant to additional compounds. However, we have no exact explanation as to how we were able to select a chloroquine-resistant strain from one already resistant to pyrimethamine while we were unable to select for the same character using the same methods with a normal strain parasite. A consistent characteristic of most strains of chloroquine-resistant P. berghei has been the lack of pigment production in parasites under chloroquine pressure. Release of this pressure has resulted in a
CHLOROQUINE
gradual loss of resistance to chloroquine accompanied by a return of pigment production. In the absence of drug pressure, Jacobs (1965) found that pigment production gradually reappeared during numerous subpassages or in recurrent parasitemias in mice surviving the initial infection. Peters (1964) saw the return of pigment formation in untreated mice infected with his chloroquine-resistant strain after 28 days and completely normal pigment occurred in parasites during recrudescence on Day 53. A lack of pigment was also observed in our CR strain of P. uinckei in mice treated with chloroquine. However, pigment started to reappear within 3 days after cessation of treatment in mice treated during a period of rising parasitemias ( Fig. 3). Pigment was approaching normal intensity during the second wave of parasitemias on Days 16 through 19. McNamara et al. ( 1967), and Kellett et u.?. ( 1968) observed pigment in chloroquine-resistant P. falcipr~rn both in the peripheral blood and after in vitro cultivation. McNamara’s group saw pigment in mature schizonts in blood from a patient who had received treatment with chloroquine 3 weeks previous to the observation and in parasites from two additional volunteers for whom th e presence or absence of chloroquine treatment was not reported. They concluded from these observations that “there is a major morphological difference in chloroquine-resistant P. berghei compared to chloroquine-resistant P. falciparum.” It should be pointed out that pigment does return both in chloroquine-resistant P. vinckei and P. berghei after chloroquine treatment has been discontinued. We are not aware that comparable studies have been done to show whether pigment is present or absent in chloroquine-resistant P. faZ&parmn under chloroquine pressure. The course of infection of the CR strain of P. uinckei in untreated mice appears
RESISTANCE
201
comparable to the normal strain with the exception of a short delay in the first wave of parasitemia and in the increased survival rate in mice infected with the CR strain, A typical course of infection in untreated mice is shown in Fig. 2. Mice infected with the normal strain usually die by Day 6 with 80-90% of the erythrocytes infected. High parasitemias are also common for the CR strain. Although average maximum parasitemias were 75%, 12 out of 20 mice developed peak parasitemias of 90% or greater. The course of infection of our CR strain of P. uinckei appears somewhat different from chloroquine-resistant P. berghei. Peters (1964) and Jacobs (1965) reported a much milder course of infection and higher survival rate in untreated mice infected with their chloroquine-resistant strains as compared to normal strain P. berghei. Peters (1964) observed in mice infected with his strain that about 10% of the erythrocytes were infected 7-10 days after exposure. Thereafter parasitemias increased to XL70% or more by the end of Week 3. About 30% of these mice recovered while the remainder experienced high parasitemias and died. We have observed a greater degree of suppression by chloroquine in our resistant P. uinckei than has been seen with numerous strains of chloroquine-resistant P. berghei [Hawking and Gammage, ( 1962) ; Peters, (1965); and Thompson et al., ( 1967 ) 1. The MTD of chloroquine given on Days 1 through 4 routinely suppressed parasitemias in mice infected with CR P. uinckei by 90% or more. However, parasites were not cleared from the blood of mice treated with MTD of chloroquine even for an extended period of time. Chloroquine also exhibited therapeutic activity against the resistant P. uinckei (Fig. 3). Although parasitemias continued to increase under chloroquine pressure, they were considerably lower than in untreated infections (Fig. 2) on comparable
202
POWERS
days after inoculation. Parasitemias decreased in both treated and untreated infections with the chloroquine-resistant strain, and. both infections became subpatent approximately the same number of days after inoculation. These trends were probably due to host immunity rather than to a chloroquine effect.
REFERENCES HAWKING, F., AND GAMMAGE, K. 1962. Chloroquine resistance produced in Plasmodium berghei. Transactions of the Royal Society of Tropical Medicine and Hygiene 56, 263. JACOBS, R. L., ALLING, D. W., AND CANTRELL, W. F. 1963. An evaluation of antimalarial combinations against Plasmodium berghei in the mouse. Journal of Parasitology 49, 920925. JACOBS, R. L. 1965. Selection of strains of Plasmodium berghei resistant to quinine, chloroquine, and pyrimethamine. Journal of Parasitology 51, 481-482. KELLETT, R. J., COWAN, G. O., AND PARRY, E. S. 1968. Chloroquine-resistant Plasmodium falciparum malaria in the United Kingdom. The Lancet 2, 946-948. J. V., RIECKMANN, K. H., AND MCNAMARA, POWELL, R. D. 1967. Pigment in asexual erythrocytic forms of chloroquine-resistant Plusmodium falciparum. Annals of Tropical Medicine and Parasitology 61, 125-127. MOORE, D. V., AND LANIER, J. E. 1961. Observations on two Plasmodium falciparum in-
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fections with an abnormal response to chloroquine. American Journal of Tropical Medicine
PETEzd y lo’ 5-g. > . 1964. Pigment
formation and nuclear division in chloroouine-resistant malaria parasites (Plasmodium Iberghei, Vincke and Lips, 1948), Nature 203, 1299-1291. PETERS, W. 1965. Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance. Experimental Parasitology 17, 89-89. 1966. The possible role of primaPETERS, W. quine in inducing multiple drug resistance in Plasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 149-141. RAMAKRISHNAN, S. P., PRAKASH, S., AND CHOUDHURY, D. S. 1957. Studies on Plasmodium berghei Vincke and Lips, 1948. XXIV. Selection of a chloroquine resistant strain. Indian Journal of Maluriology 11, 213-220. RODHAIN, J. 1952. Plasmodium vinckei n. sp. Un deuxieme Plasmodium parasite de rongeurs sauvages au Katanga. Annaks de lu Societe Beige de Medecine Tropicale 32, 275280. THOMPSON, P. E., AND BAYLES, A. 1966. Eradication of Eperythrozoon coccoides with oxophenarsine in normal and drug-resistant lines of Plasmodium berghei in mice. Journat of Pamsitology 52, 674-678. THOMPSON, P. E., OLSZEWSKI, B., BA~LES, A., AND WAITZ, J. A. 1967. Relations among antimalarial drugs: results of studies with cycloguanil-, sulfone-, or chloroquine-resistant Plasmodium berghei in mice. American Journal of Tropical Medicine and Hygiene 16, 133-145.