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Antimicrobial Agents and Chemotherapy, April 2002, p. 958-965, Vol. 46, No. 4
0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.4.958-965.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Malaria Parasites Giving Rise to Recrudescence In Vitro
Shusuke Nakazawa,1* Takashi Maoka,2 Haruki Uemura,1 Yoshihiro Ito,2 and Hiroji Kanbara1Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523,1 Japan Foundation of Research Institute for Production Development, Shimogamo-Morimoto 15, Kyoto 606-0805, Japan2
Received 21 September 2000/ Returned for modification 21 December 2000/ Accepted 4 January 2002
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Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.
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Malaria remains one of the most serious threats to human beings in the world. Every year 300 million to 500 million people are infected with malaria parasites and 1 million people die of malaria (17). The appearance and expansion of drug-resistant Plasmodium falciparum make the chemotherapy of malaria more difficult. Recrudescence is a sign of treatment failure and has been thought to occur due to the selection of drug-resistant parasites. However, there have been reports of discrepancies between clinical findings and laboratory test results; that is, recrudescences occurred after drug treatment, but the recrudescent parasites were sensitive in laboratory drug sensitivity tests (2, 3, 4, 9, 10). Drug-sensitive parasites sometimes recrudesced despite adequate drug treatment (16). The accepted explanation for such a phenomenon was either that the concentration of the drug in plasma was insufficient to suppress parasites or that the concentration was not maintained above the effective value long enough to eliminate the parasites (5, 11, 12).
In a previous study we used an in vitro culture to demonstrate recrudescences after D-sorbitol and pyrimethamine treatment in culture (6). The culture was free of host factors, such as variabilities in pharmacodynamics and pharmacokinetics, specific immunity against the parasites, and possible hiding places for the parasites. It was suggested that the dormant parasites survived the treatments. However, the behaviors of the parasites that recrudesced remain to be clarified. We therefore attempted to examine the frequency of recrudescence and the relationship between recrudescence and drug concentration.
We show in this paper that the incidence of recrudescence in vitro is not related to the drug concentration at which the drug was predicted to eradicate the parasites but is dependent both on the length of treatment duration and on the initial number of parasites. The proportions of parasites in the initial parasite population that recrudesced might be invariable.
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Parasite culture and cloning. The FCR-3 strain of P. falciparum parasites was maintained in standard medium (13) in a 5% CO2-5% O2-90% N2 atmosphere at 37°C. The medium was changed daily, and the level of parasitemia was counted by Giemsa staining of thin blood films.
Parasite clones were obtained by the limiting dilution method. A ring-form-infected erythrocyte suspension containing 2 parasites per ml at 2% hematocrit was dispensed into a 96-well culture plate at 100 µl per well. On day 6, the blood in the 96-well plate was transferred to 24-well culture plates and supplemented with 400 µl of fresh erythrocyte suspension at 2% hematocrit per well. Four hundred microliters of the fresh erythrocyte suspension was added to each well on days 12 and 18. The level of parasitemia was examined on day 20 and day 24. Three wells were selected on the basis of the parasitemia levels on day 20, and the parasitized erythrocytes from these wells were subjected to two other rounds of the same cloning procedures. The rates of positivity for wells in the first, second, and third rounds of cloning were 11.5, 13.3, and 14.2%, respectively.
Pyrimethamine and mefloquine sensitivity tests. Mefloquine (Mepha, Aesch- Basel, Switzerland) was dissolved in pure water at a concentration of 10-3 M and was stored at 4°C for 2 weeks. Pyrimethamine (Sigma, St. Louis, Mo.) was dissolved in 0.5% lactic acid (8) at a concentration of 10-2 M and was stored at 4°C for 2 weeks. All recrudescent parasites in this study were examined for their sensitivities to drug treatment when the level of recrudescent parasitemia was more than 2%. Briefly, 0.25 ml of a 20% suspension of parasitized erythrocytes was mixed with aliquots of serial 10-fold dilutions from 2 x 10-6 to 2 x 10-10 M pyrimethamine or mefloquine in standard medium in a 24-well tissue culture plate (final suspension, 10%; final drug concentration, a serial 10-fold dilution from 1 x 10-10 to 1 x 10-6 M). As a control, parasitized erythrocytes were incubated with the medium. On days 2 and 3 of incubation, the drug-containing medium was changed and thin blood films were made. Growth inhibition is presented as the percent parasitemia at each concentration of drug on day 3 compared to the percent parasitemia without drug on the same day. The 50% inhibitory concentration (IC50) of FCR-3 was determined on the basis of the results of sensitivity tests, in which the parasites were treated with serial twofold dilutions of drug covering a range from 10-9 M to 10-7 M. The IC50s of mefloquine and pyrimethamine were 4 x 10-9 and 6 x 10-9 M, respectively.
Examination of the proportion of recrudescent parasites. An aliquot of the erythrocytes containing 109 parasitized erythrocytes was obtained from the culture with 5% parasitemia. The aliquot was diluted with standard medium to yield 108 parasitized erythrocytes per ml. The cell suspension was diluted with a fresh erythrocyte suspension in standard medium at 10% hematocrit to make 10-fold serial dilutions of parasitized erythrocytes from 107 to 104 per ml. The aliquot containing 109 parasitized erythrocytes was resuspended in 20 ml of drug-containing medium and inoculated into a 200-ml culture flask. Aliquots containing 108, 107, 106, 105, and 104 parasitized erythrocytes were resuspended in 5 ml of drug-containing medium, and the suspension was inoculated into 20-ml culture flask. The drug-containing medium was changed every day. On the day when treatment ceased, the cultures were washed extensively with standard medium, supplemented with 0.1 ml of packed fresh erythrocytes, and inoculated into fresh flasks. The level of parasitemia was examined daily from 7 days after the end of treatment. One-tenth and 0.2 ml of the packed fresh erythrocytes were added to a 20-ml culture flask and a 200-ml culture flask, respectively, every 7 days after treatment until parasites reappeared.
Examination of relationship between recrudescence and drug concentration. Equal numbers of parasitized erythrocytes were treated with 10-fold serial dilutions of pyrimethamine (from 10-9 to 10-4 M) or mefloquine (from 10-9 to 10-5 M) for 4 days with daily changes of the drug-containing medium. After treatment, the parasitized erythrocytes were washed, supplemented with fresh erythrocytes, and maintained in the same way as described above. The time from the cessation of treatment to the day on which recrudescent parasitemia levels reached or surpassed 1% was recorded.
Comparison of recrudescences in parasites treated with 10-6 and 10-5 M pyrimethamine. Erythrocytes parasitized with clone FCR-3 were diluted with fresh erythrocytes to obtain 107, 106, 105, and 104 parasitized erythrocytes. They were then treated with 10-6 or 10-5 M pyrimethamine for 4 days. After treatment, they were washed, supplemented with fresh erythrocytes, and inoculated into standard medium in the same way as described above.
Repetition of recrudescence in cloned parasites. Twenty million parasitized erythrocytes of clone FCR-3 were treated with 10-6 M pyrimethamine for 4 days, washed, supplemented with fresh erythrocytes, and returned to standard medium. The recrudescent parasites at a parasitemia level of more than 2% were again treated in the same way as in the first recrudescence experiment. In this way recrudescences were allowed to occur four more times. The duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and parasite growth were compared among six recrudescence experiments.
Measurement of residual drug levels. Six hundred million normal erythrocytes were incubated with standard medium containing pyrimethamine or mefloquine at 10-8, 10-6, and 10-4 M for 4 days with daily changes of the drug-containing medium. As a control, erythrocytes were incubated with standard medium. On day 4, erythrocytes were washed four times with standard medium and lysed by subjecting them to repeated changes of osmotic pressure. Erythrocyte membranes were washed extensively with phosphate-buffered saline.
Six hundred million parasitized erythrocytes at 10% parasitemia were incubated with standard medium containing pyrimethamine at 10-8, 10-6, and 10-4 M and standard medium alone as a control. To harvest the membranes and the water-insoluble sediment, these cells were treated in the same way as the normal erythrocytes.
The sediments were suspended in 200 µl of methanol, mixed vigorously, and centrifuged at 1,800 x g for 5 min at 10°C. The supernatants were analyzed with a high-performance liquid chromatography (HPLC) system (Hitachi, Tokyo, Japan). The solvent for mefloquine detection was a mixture of phosphate-buffered 50 mM sodium sulfate (pH 2.84) and acetonitrile (67:33; vol/vol). The solvent for pyrimethamine detection was a mixture of phosphate-buffered 37.5 mM sodium sulfate (pH 2.84) and acetonitrile (3:1; vol/vol). The flow rate was 1.0 ml/min. The limits of detection for mefloquine and pyrimethamine were less than 10-7 M. The peak area ratios in the calibration standard curves for mefloquine and pyrimethamine were linear over the range from 10-6 to 10-4 M, respectively.
Statistical analysis. Variations in the duration from the cessation of treatment to 1% parasitemia among the various treatments tested in the study were analyzed by two-way analysis of variance, while multiple comparisons of means were done by use of the Bonferroni method. Statistical calculations were conducted with Systat statistical software.
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Proportion of parasites that recrudesced. To evaluate the number of parasites that survived treatment, serial dilutions of parasitized erythrocytes were exposed to 10-6 M pyrimethamine treatment for various lengths of time. No recrudescence was observed for 104, 105, 107, and 108 parasitized erythrocytes for 2 months after 4, 6, 8, and 10 days of pyrimethamine treatment, respectively (Table 1). In contrast, recrudescence occurred in 105 and 106 parasitized erythrocytes after 4 days of treatment. With a longer duration of treatment, larger numbers of parasitized erythrocytes were needed to observe recrudescence.
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TABLE 1. Recrudescences after pyrimethamine treatment
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TABLE 2. Recrudescences after mefloquine treatment
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FIG. 1. Recrudescent parasites showed the same patterns of sensitivity to pyrimethamine (PYR) as the control parasites did. (A) Control parasites; (B to H) recrudescent parasites: 105 for 4 days (B), 106 for 4 days (C), 106 for 6 days (D), 107 for 6 days (E), 108 for 8 days (F), 109 for 8 days (G), and 109 for 10 days (H).
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FIG. 2. Recrudescent parasites showed the same pattern of sensitivity to mefloquine (MEF) as the control parasites did. (A) Control parasites; (B to E) recrudescent parasites: 106 for 4 days (B), 107 for 4 days (C), 107 for 6 days (D), 108 for 6 days (E).
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FIG. 3. (A) Parasites recrudesce independently of the pyrimethamine concentration. Fifty million parasitized erythrocytes (7% parasitemia) were exposed to the indicated concentrations of pyrimethamine for 4 days, washed, and returned to standard medium. Experiments were carried out in triplicate. Mean ± standard deviation levels of parasitemia and mean ± standard deviation durations from the day when treatment ceased until the day when the recrudescent parasitemia level reached or surpassed 1% are shown. The data are representative of three separate experiments. Means followed by the same letter were not significantly different (P = 0.744). (B) Recrudescent parasites showed the same pattern of sensitivity to pyrimethamine (PYR) as the parasites tested before treatment did.
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FIG. 4. (A) Parasites recrudesce independently of the mefloquine concentration. Fifty million parasitized erythrocytes (6.5% parasitemia) were treated with the indicated concentrations of mefloquine. Experiments were carried out in triplicate. Mean ± standard deviation levels of parasitemia and mean ± standard deviation durations from the day when treatment ceased until the day when the recrudescent parasitemia level reached or surpassed 1% are shown. Data shown are representative of three separate experiments. Means followed by the same letter were not significantly different (P = 0.565). (B) Recrudescent parasites showed the same pattern of sensitivity to mefloquine (MEF) as the parasites tested before treatment did.
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No mefloquine was detected in the membrane fractions of normal erythrocytes which had been treated with 10-8 M mefloquine for 4 days or those that had been incubated in standard medium. On the other hand, mefloquine remained in the erythrocytes treated with 10-6 or 10-4 M (7.28 and 762 ng/mg of protein, respectively). Pyrimethamine was not detected in normal erythrocytes, which had been treated with the same concentrations of pyrimethamine as mefloquine (Table 3). These data indicated that mefloquine remained in culture by binding to the cell membrane. Chevli and Fitch (1) showed that mefloquine had a high affinity for phospholipids in the erythrocyte membrane.
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TABLE 3. Detection of residual pyrimethamine both in erythrocyte membranes and in the insoluble fractions from parasitized erythrocytesa
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Effect of residual mefloquine on parasite growth. Parasites were cultured in erythrocytes treated with mefloquine to examine whether the residual mefloquine affected the multiplication of parasites. The level of parasitemia decreased for the first 2 days and then increased when the parasites were cultured in erythrocytes that had been treated with 10-6 M mefloquine for 4 days and washed with standard medium (Fig. 5). After being treated and washed as described above, the erythrocytes which were reincubated for a week in standard medium with daily medium changes supported parasite growth in the same manner that normal erythrocytes did.
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FIG. 5. Residual mefloquine suppressed parasite growth, and its effect was reduced during the incubation. Parasitized erythrocytes at 18% parasitemia were diluted with fresh erythrocytes that had been treated as follows: by treatment with 10-6 M mefloquine for 4 days and washing (filled circles); by treatment with mefloquine, washing, and incubation in standard medium for 7 days (open triangles); or by incubation in standard medium (open circles). Parasite culture was carried out in triplicate. Mean ± standard deviation levels of parasitemia are shown. The data are representative of two separate experiments.
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TABLE 4. Duration from cessation of treatment to 1% after treatment with parasitemia between 10-6 and 10-5 M pyrimethaminea
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FIG. 6. Recrudescent parasites showed the same pattern of sensitivity to pyrimethamine (PYR) as parasites tested before treatment did.
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TABLE 5. Duration from cessation of treatment to 1% parasitemia and growth periods from 0.01 to 1% parasitemia in a series of six successive recrudescencesa
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Recrudescences were produced in cultures. The recrudescent parasites in these recrudescence experiments were as sensitive to drugs as the parasites tested before treatment, as shown in Fig. 1, 2, 3B, 4B, and 6. The results suggest that drug-sensitive parasites survived drug treatment for several days in culture.
No parasites in culture inoculated with 104, 105, 107, and 108 parasites survived pyrimethamine treatment for 4, 6, 8, and 10 days, respectively, as shown in Table 1. Despite the use of the same treatment for the same periods, recrudescences occurred in the cultures that contained 10 times more parasites than those amounts mentioned above. A simple explanation for this result may be incomplete eradication. However, parasites seemed to have survived drug treatment for several days by escaping the effect of the drug. Assuming that treatment with pyrimethamine at 10-6 M reduces the number of parasites 100-fold per one cycle (killing rate, 100), treatment for 4, 6, 8, and 10 days eliminated parasites 104-, 106-, 108-, and 1010-fold, respectively. Recrudescence may occur in 105, 107, and 109 parasites after treatment for 4, 6, and 8 days, respectively. These results are comparable to those seen in Table 1. As shown in Fig. 3A and Table 4, recrudescence occurred independently of the pyrimethamine concentration at concentrations of 10-6 and 10-5 M. Besides, the killing rate obtained microscopically with pyrimethamine at both 10-6 and 10-5 M is actually 1,000 (range, 870 to 1,330) per one cycle in our hands. It is not likely that recrudescence arose because of a low killing rate.
To analyze the relationship between killing of parasites by treatment and growth of surviving parasites, we have replotted the data for the logarithmic kill and growth phases from Table 1, as described by White (15) (Fig. 7). If the killing rate remains constant with succeeding cycles (15), it is predicted that treatment with pyrimethamine at 10-6 M for 4 days will completely kill 104 and 105 parasites (Fig. 7). Similarly, 6 days of treatment is enough to eradicate up to 108 parasites. A total of 109 parasites are eradicated far before the end of treatment for 8 and 10 days. This analysis suggests that the experimental treatments were adequate to eradicate the parasites and that the parasites recrudesced despite the adequate treatment.
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FIG. 7. Killing by pyrimethamine treatment and growth of recrudescent parasites. The figure was prepared with the data from Table 1. All pyrimethamine treatment durations except the 6-day treatment against 106 parasites eradicated within the treatment duration. Broken lines, killing rate if the killing rate remains constant with succeeding cycles; dotted lines, numbers of surviving parasites extrapolated from a growth rate above the detection limit.
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Wernsdorfer (14) referred to the possibility that parasites stop passing through their cell cycles in order to repair the damage induced by drug treatment. If recrudescence occurred by this mechanism, treatment with a higher concentration of drug might cause damage in a larger number of parasites. Those damaged parasites might then repair the damage by spending various lengths of time without undergoing cell cycles. As shown in Table 4, the durations from the time of cessation of treatment to 1% parasitemia were equal for the 10-6 and 10-5 M treatments, and no recrudescences arose in assays with 104 parasitized erythrocytes. Drug treatment might not be likely to induce parasites to stop passing through the cell cycle in order to undergo repair.
It is also likely that drug-sensitive parasites escape the effects of drugs by remaining in the ring-form stage during the treatment period. Ring-form stages of parasites are not susceptible to drug treatment. In a population of parasites there may be parasites with various lengths of ring-form stages. As indicated in Table 1, a larger population shows a larger variation in the length of the ring-form stage. This may be the reason that recrudescence does not depend on the drug concentration (Fig. 3A and Table 4) but does depend on the initial number of parasites and the duration of treatment. Differences in the lengths of the ring-form stage may be produced at the merozoite stage, as different rhoptry proteins are expressed in merozoites originating from a single schizont (7).
Recrudescences occurred in the same manner in six consecutive recrudescence experiments with cloned parasites, as shown in Table 5. These results suggest that the proportion of inactive parasites in a parasite population may be unchanged in each experiment or that similar numbers of parasites in each recrudescence experiment may slow their growth after exposure to drug.
Treatment with mefloquine at 10-6 M reduces the number of parasites more than 200-fold per one cycle. As shown in Fig. 5, the residual mefloquine in uninfected erythrocytes delayed parasite growth by about 4 days. Residual mefloquine reduces the number of parasites 100-fold even after treatment. If the killing rate is constant, as is assumed for pyrimethamine treatment, in our study the mefloquine treatment was adequate to eradicate parasites. Despite the adequate mefloquine treatment, recrudescence was observed in 106 parasites treated for 4 days and in 107 parasites treated for 6 days, as shown in Table 2. It is likely that the recrudescence that occurred after mefloquine treatment was caused by parasites in a lag phase or parasites in an inactive ring form, as was conceived to be the cause of recrudescence after pyrimethamine treatment.
Although the killing and growth of parasites that occur below the detectable levels of parasitemia remain to be verified, this study provides evidence that drug-sensitive parasites that escape the effect of a drug may cause recrudescence.
We thank Gloria Lumbera Enriquez for kind help.
This work was partially supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.
* Corresponding author. Mailing address: Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan. Phone: 81-95-849-7838. Fax: 81-95-849-7805. E-mail: nakazawa{at}net.nagasaki-u.ac.jp.
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Antimicrobial Agents and Chemotherapy, April 2002, p. 958-965, Vol. 46, No. 4
0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.4.958-965.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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