- Email: [email protected]
The in vitro antimalarial activity of chloramphenicol against Plasmodium falciparum
Acta Tropica, 56(1994)51-54
51
© 1994 Elsevier Science B.V. All rights reserved 0001-706X/94/$07.00 ACTROP 00344
The in vitro antimalarial activity of chloramphenicol against Plasmodiumf alciparum Anthony E.T. Yeo* and Karl H. Rieckmann Army Malaria Research Unit, University of Sydney, Ingleburn, NSW 2174, Australia (Received 12 July 1993; accepted 15 September 1993)
The minimum inhibitory concentrations, MIC, of chloramphenicol were determined for two isolates of Plasmodiumfalciparum at 48, 96 and 144 h. The MIC decreased from values greater than 100 p,g/ml at 48 h to 10.7-12.5 p.g/ml at 96 h. During 144 h of incubation, concentrations of 0.8-1.6 ~tg/ml were effective in suppressing parasite growth. These results indicate that the multiplication of malaria parasites can be inhibited by clinically achievable concentrations of chloramphenicol provided that exposure to the drug is prolonged over several asexual life cycles. They suggest that undiagnosed falciparum infections may be cured when patients with fever of doubtful origin are treated with 10 to 14 day courses of chloramphenicol. They also raise the possibility that this antibiotic may eventually be used, in combination with a rapidly acting but non-curative drug regimen, to treat patients with falciparum infections in whom the use of tetracyclines is contraindicated, e.g., young children. Key words: Chloramphenicol; Antibiotic activity; Antimalarial activity, in vitro; Plasmodiumfalciparum; Malaria
Introduction
About 40 years ago, some antibiotics including chloramphenicol were reported to be active against Plasmodium vivax (Imboden et al., 1950) and against Plasmodium falciparum (Ruiz-Sanchez et al., 1952). However, since the clearance of fever and parasitaemia was slower than that observed with other antimalarials such as chloroquine, antibiotics were not considered to be of practical value in the treatment of malaria infections (WHO, 1961). Antibiotics only started to be used for the treatment and prophylaxis of malaria after Rieckmann et al. (1971, 1972) showed that tetracycline was effective against the pre-erythrocytic and erythrocytic stages of chloroquineresistant strains of P. falciparum. Although the tetracyclines, in combination with quinine, are now the main antimalarials used to cure and prevent malaria in areas where parasites have become resistant to mefloquine and other drugs, they cannot be used during early childhood and pregnancy. In many tropical countries with limited health resources, chloramphenicol is the second most commonly used antibiotic, after penicillin, for treating infectious diseases in children and adults. Chloramphenicol is often administered to severely ill patients for several days when there is some doubt as to the cause of the febrile *Corresponding author. SSDI 0 0 0 1 - 7 0 6 X ( 9 3 ) E 0 0 7 7 - Y
52 illness, a situation that does not exclude malaria either as a principal or concurrent causative agent. Under such circumstances, it would be useful to know to what extent, if any, chloramphenicol might be able to control a malaria infection. Because of these considerations, this study was initiated to determine the activity of chloramphenicol against P. falciparum in vitro.
Materials and Methods
Chloramphenicol base was kindly supplied by Parke Davis, Sydney. It was dissolved in 100% ethanol (0.5 g/L) and various amounts of this solution were added to 96well microtitre plates (FLOW). The plates were dried overnight at 37°C and were used to determine the effects of chloramphenicol against the FC and K1 isolates of P. falciparum. The FC isolate is very sensitive to chloroquine and antifolate drugs and was originally obtained from Madang, Papua New Guinea (Chen et al., 1980) while the K1 isolate is highly resistant to both chloroquine and antifolate drugs and was originally obtained from Kanchanaburi, Thailand (Thaithong and Beale, 1981). Parasites were maintained in continuous culture (Trager and Jensen, 1976) using 10% O-positive human serum, erythrocytes obtained from the Red Cross Blood Bank and RPMI 1640 medium containing folic acid 0.01 mg/L and para-aminobenzoic acid 0.0005 mg/L (Gibco, New York, USA). This was supplemented with 24 mM Hepes, 32 mM NaHCO3, glucose (2 g/L) and gentamicin (40 mg/L). Prior to use, parasites were synchronised with sorbitol (Lambros and Vanderberg, 1979). The antimalarial activity of chloramphenicol was determined by using two in vitro assays. In assay 1, parasites were incubated for 48 to 96 h. In assay 2, parasites were incubated for 144 h. Incubation was prolonged because antibiotics tend to act slowly against malaria parasites. In assay 1, in vitro antimalarial activity was determined by the Rieckmann method using Linbro 96-well (flat bottom) microtitre plates (Flow, McLean, VA, USA). The inoculum for each well consisted of 50 p.1 of an 4% suspension of 0.1% parasitised erythrocytes ( > 9 0 % ring stage) in supplemented RPMI-1640 solution and 50 ~tl of human serum. Parasites were incubated at 37.5°C in a gas mixture of 5% carbon dioxide, 5% oxygen and 90% nitrogen. During the period of incubation, there was no replacement of the gas mixture or culture medium. At the end of each experiment, thick films were made from each well, stained with Giemsa's stain and examined for the presence of rings, trophozoites and schizonts. Such a qualitative analysis allows for an assessment of the effect of these drugs on the various stages of parasite development. Additionally, the minimum inhibitory concentration, MIC, was determined by noting the drug concentrations at which at least 95% of the parasites were inhibited from invading erythrocytes (IC95). In assay 2, antimalarial activity was determined using Linbro 24 well (flat bottom) tissue culture plates (Flow, McLean, Virginia, USA). The inoculum in each well consisted of 250 ~tl of serum containing various amounts of chloramphenicol and 250 ~tl of an 8% suspension of 0.5% parasitised erythrocytes in supplemented RPMI 1640 culture medium. The drug containing serum and culture medium were changed at 48 and 96 h, and the gas mixture was changed at 48, 72, 96 and 120 h. Additionally, 100 ~tl of a 50% erythrocyte suspension was added to the cultures at 48 and 96 h
53 to supply a sufficient number of cells for replicating parasites. Blood slides were made at 48, 72, 96 and 120 h for observation of the effect of the drug on parasite growth and morphology.
Results
Incubation for 48 h did not prevent parasite re-invasion even at the highest concentration of chloramphenicol - 100 lag/ml (n = 12). At 72 h, inhibition of parasite growth was again not observed in any of the drug concentrations (0.8-100 gg/ml). However, at 96 h, trophozoites failed to mature to schizonts at concentrations between 12.5 and 100 gg/ml, indicating that, at these concentrations, chloramphenicol inhibits the formation of schizonts during the 2nd asexual life cycle of the parasite. At this time, the re-invasion of parasites, to start the third asexual cycle, was also prevented. At 96 h, the MIC for parasites of the FC isolate was 10.7_+3.4 gg/ml (mean _+standard deviation; n = 12) and for the K1 isolate, it was 12.5_+6.0 lag/ml (n = 14). After incubation for 144 h, parasites of the K1 isolate were inhibited by chloramphenicol concentrations of 0.8 gg/ml (n =2), and those of the FC isolate were inhibited by concentrations of 0.8 to 1.6 gg/ml (n = 3).
Discussion
Our findings show that the in vitro activity of chloramphenicol is related to the period of time that parasites are exposed to the drug. During the first 48 h of incubation, parasite growth was not inhibited by drug concentrations greater than 100 lag/ml. Since serum concentrations of chloramphenicol after administration of 1.0 g of the drug are of the order of 3 to 13 ~tg/ml (Sande and Mandell, 1980), it is unlikely that chloramphenicol would be effective if it is administered for only 2 to 3 days. However, the marked reduction of the MIC at 96 h, approaching clinically achievable peak concentrations of chloramphenicol, indicates that exposure of parasites to the drug for 4 days may achieve partial suppression of parasitaemia. Further reduction of the MIC at 144 h to concentrations of the order of 1 lag/ml suggests that 6 days of drug administration may prevent the replication of parasites completely in vivo. In view of the shortage of alternative drugs for the treatment of multidrug resistant malaria, further studies should be carried out to determine the antimalarial properties of chloramphenicol. Such studies are especially relevant since chloramphenicol is already being used quite extensively in many malarious countries for the treatment of illnesses such as meningitis, severe pneumonia, pyrexia of unknown origin, etc. During such investigations, it should be kept in mind that chloramphenicol will probably act slowly against the malaria parasite and that, as with the tetracyclines, it will have to be taken for at least a week. For acute infections of malaria, it will probably also have to be taken in conjunction with a rapidly acting but non-curative drug regimen, such as a 3-day course of chloroquine, quinine or one of the arteminisin derivatives. The very slight risk of developing fatal aplastic anaemia following
54 t r e a t m e n t with c h l o r a m p h e n i c o l (1:25000 to 1:100000) should also be taken into consideration. The practical value of c h l o r a m p h e n i c o l as an a n t i m a l a r i a l drug could be quite significant if f o r t h c o m i n g clinical studies indicate that it is able to cure m a l a r i a infections. In c o m b i n a t i o n with a rapidly acting b u t n o n - c u r a t i v e antimalarial, c h l o r a m p h e n i c o l could be used to treat febrile patients in w h o m diagnostic u n c e r t a i n ties exist. It could also be very useful in achieving radical cure of falciparum infections, particularly in patients who are u n a b l e to be treated with the tetracyclines, e.g., d u r i n g early childhood.
References Chen, P., Lamont, G., Elliot, T., Kidson, C., Brown, G., Mitchell, G., Stace, J. and Alpers, M. (1980) Plasmodium falciparum strains from Papua New Guinea: culture characteristics and drug sensitivity. S.E. Asian J. Trop. Med. Pub. Hlth. 11,435-440. Imboden, C.A., Cooper, W.C., Coatney, G.R. and Jeffrey, G.M. (1950) Studies in human malaria. XXIX. Trials of aureomycin, chloramphenicol, penicillin, and dihydrostreptomycin against the Chesson strain of P. vivax. J. Nat. Mal. Soc. 9, 377 380. Lambros, C. and Vanderberg, J.P. (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65, 418-420. Rieckmann, K.H., Powell, R.D., McNamara, J.V., Willerson, D., Kass, C., Frischer, H. and Carson, P.E. (1971) Effect of tetracycline against chloroquine resistant and chloroquine sensitive Plasmodium falciparum. Am. J. Trop. Med. Hyg. 20, 811-815. Rieckmann, K.H., Willerson, W.D., Carson, P.E., Frischer, H. (1972) Effects of tetracycline against drug-resistant malaria. Proc. Helminthol. Soc. Wash. 39 (Special Issue), 339-347. Ruiz-Sanchez, F., Quezada, M., Paredes, M.E., Casillas, J. and Riebling, R. (1952) Chloramphenicol in malaria. Am. J. Trop. Med. Hyg. 1,936-940. Sande, M.A. and Mandell, G.L. (1980) Tetracyclines and chloramphenicoi. In: The Pharmacological Basis of Therapeutics, Gilman, A.G., Goodman, L.S. and Gilman, A. (eds.). Macmillan, New York, pp. 1181-1199. Thaithong, S. and Beale, G.H. (1981) Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75, 271-273. Trager, W. and Jensen, J.B. (1976) Human malaria parasites in continuous culture. Science 193,673-675. World Health Organization. (1961) Chemotherapy of malaria. WHO Tech. Rep. Ser. 226, 16.
51
© 1994 Elsevier Science B.V. All rights reserved 0001-706X/94/$07.00 ACTROP 00344
The in vitro antimalarial activity of chloramphenicol against Plasmodiumf alciparum Anthony E.T. Yeo* and Karl H. Rieckmann Army Malaria Research Unit, University of Sydney, Ingleburn, NSW 2174, Australia (Received 12 July 1993; accepted 15 September 1993)
The minimum inhibitory concentrations, MIC, of chloramphenicol were determined for two isolates of Plasmodiumfalciparum at 48, 96 and 144 h. The MIC decreased from values greater than 100 p,g/ml at 48 h to 10.7-12.5 p.g/ml at 96 h. During 144 h of incubation, concentrations of 0.8-1.6 ~tg/ml were effective in suppressing parasite growth. These results indicate that the multiplication of malaria parasites can be inhibited by clinically achievable concentrations of chloramphenicol provided that exposure to the drug is prolonged over several asexual life cycles. They suggest that undiagnosed falciparum infections may be cured when patients with fever of doubtful origin are treated with 10 to 14 day courses of chloramphenicol. They also raise the possibility that this antibiotic may eventually be used, in combination with a rapidly acting but non-curative drug regimen, to treat patients with falciparum infections in whom the use of tetracyclines is contraindicated, e.g., young children. Key words: Chloramphenicol; Antibiotic activity; Antimalarial activity, in vitro; Plasmodiumfalciparum; Malaria
Introduction
About 40 years ago, some antibiotics including chloramphenicol were reported to be active against Plasmodium vivax (Imboden et al., 1950) and against Plasmodium falciparum (Ruiz-Sanchez et al., 1952). However, since the clearance of fever and parasitaemia was slower than that observed with other antimalarials such as chloroquine, antibiotics were not considered to be of practical value in the treatment of malaria infections (WHO, 1961). Antibiotics only started to be used for the treatment and prophylaxis of malaria after Rieckmann et al. (1971, 1972) showed that tetracycline was effective against the pre-erythrocytic and erythrocytic stages of chloroquineresistant strains of P. falciparum. Although the tetracyclines, in combination with quinine, are now the main antimalarials used to cure and prevent malaria in areas where parasites have become resistant to mefloquine and other drugs, they cannot be used during early childhood and pregnancy. In many tropical countries with limited health resources, chloramphenicol is the second most commonly used antibiotic, after penicillin, for treating infectious diseases in children and adults. Chloramphenicol is often administered to severely ill patients for several days when there is some doubt as to the cause of the febrile *Corresponding author. SSDI 0 0 0 1 - 7 0 6 X ( 9 3 ) E 0 0 7 7 - Y
52 illness, a situation that does not exclude malaria either as a principal or concurrent causative agent. Under such circumstances, it would be useful to know to what extent, if any, chloramphenicol might be able to control a malaria infection. Because of these considerations, this study was initiated to determine the activity of chloramphenicol against P. falciparum in vitro.
Materials and Methods
Chloramphenicol base was kindly supplied by Parke Davis, Sydney. It was dissolved in 100% ethanol (0.5 g/L) and various amounts of this solution were added to 96well microtitre plates (FLOW). The plates were dried overnight at 37°C and were used to determine the effects of chloramphenicol against the FC and K1 isolates of P. falciparum. The FC isolate is very sensitive to chloroquine and antifolate drugs and was originally obtained from Madang, Papua New Guinea (Chen et al., 1980) while the K1 isolate is highly resistant to both chloroquine and antifolate drugs and was originally obtained from Kanchanaburi, Thailand (Thaithong and Beale, 1981). Parasites were maintained in continuous culture (Trager and Jensen, 1976) using 10% O-positive human serum, erythrocytes obtained from the Red Cross Blood Bank and RPMI 1640 medium containing folic acid 0.01 mg/L and para-aminobenzoic acid 0.0005 mg/L (Gibco, New York, USA). This was supplemented with 24 mM Hepes, 32 mM NaHCO3, glucose (2 g/L) and gentamicin (40 mg/L). Prior to use, parasites were synchronised with sorbitol (Lambros and Vanderberg, 1979). The antimalarial activity of chloramphenicol was determined by using two in vitro assays. In assay 1, parasites were incubated for 48 to 96 h. In assay 2, parasites were incubated for 144 h. Incubation was prolonged because antibiotics tend to act slowly against malaria parasites. In assay 1, in vitro antimalarial activity was determined by the Rieckmann method using Linbro 96-well (flat bottom) microtitre plates (Flow, McLean, VA, USA). The inoculum for each well consisted of 50 p.1 of an 4% suspension of 0.1% parasitised erythrocytes ( > 9 0 % ring stage) in supplemented RPMI-1640 solution and 50 ~tl of human serum. Parasites were incubated at 37.5°C in a gas mixture of 5% carbon dioxide, 5% oxygen and 90% nitrogen. During the period of incubation, there was no replacement of the gas mixture or culture medium. At the end of each experiment, thick films were made from each well, stained with Giemsa's stain and examined for the presence of rings, trophozoites and schizonts. Such a qualitative analysis allows for an assessment of the effect of these drugs on the various stages of parasite development. Additionally, the minimum inhibitory concentration, MIC, was determined by noting the drug concentrations at which at least 95% of the parasites were inhibited from invading erythrocytes (IC95). In assay 2, antimalarial activity was determined using Linbro 24 well (flat bottom) tissue culture plates (Flow, McLean, Virginia, USA). The inoculum in each well consisted of 250 ~tl of serum containing various amounts of chloramphenicol and 250 ~tl of an 8% suspension of 0.5% parasitised erythrocytes in supplemented RPMI 1640 culture medium. The drug containing serum and culture medium were changed at 48 and 96 h, and the gas mixture was changed at 48, 72, 96 and 120 h. Additionally, 100 ~tl of a 50% erythrocyte suspension was added to the cultures at 48 and 96 h
53 to supply a sufficient number of cells for replicating parasites. Blood slides were made at 48, 72, 96 and 120 h for observation of the effect of the drug on parasite growth and morphology.
Results
Incubation for 48 h did not prevent parasite re-invasion even at the highest concentration of chloramphenicol - 100 lag/ml (n = 12). At 72 h, inhibition of parasite growth was again not observed in any of the drug concentrations (0.8-100 gg/ml). However, at 96 h, trophozoites failed to mature to schizonts at concentrations between 12.5 and 100 gg/ml, indicating that, at these concentrations, chloramphenicol inhibits the formation of schizonts during the 2nd asexual life cycle of the parasite. At this time, the re-invasion of parasites, to start the third asexual cycle, was also prevented. At 96 h, the MIC for parasites of the FC isolate was 10.7_+3.4 gg/ml (mean _+standard deviation; n = 12) and for the K1 isolate, it was 12.5_+6.0 lag/ml (n = 14). After incubation for 144 h, parasites of the K1 isolate were inhibited by chloramphenicol concentrations of 0.8 gg/ml (n =2), and those of the FC isolate were inhibited by concentrations of 0.8 to 1.6 gg/ml (n = 3).
Discussion
Our findings show that the in vitro activity of chloramphenicol is related to the period of time that parasites are exposed to the drug. During the first 48 h of incubation, parasite growth was not inhibited by drug concentrations greater than 100 lag/ml. Since serum concentrations of chloramphenicol after administration of 1.0 g of the drug are of the order of 3 to 13 ~tg/ml (Sande and Mandell, 1980), it is unlikely that chloramphenicol would be effective if it is administered for only 2 to 3 days. However, the marked reduction of the MIC at 96 h, approaching clinically achievable peak concentrations of chloramphenicol, indicates that exposure of parasites to the drug for 4 days may achieve partial suppression of parasitaemia. Further reduction of the MIC at 144 h to concentrations of the order of 1 lag/ml suggests that 6 days of drug administration may prevent the replication of parasites completely in vivo. In view of the shortage of alternative drugs for the treatment of multidrug resistant malaria, further studies should be carried out to determine the antimalarial properties of chloramphenicol. Such studies are especially relevant since chloramphenicol is already being used quite extensively in many malarious countries for the treatment of illnesses such as meningitis, severe pneumonia, pyrexia of unknown origin, etc. During such investigations, it should be kept in mind that chloramphenicol will probably act slowly against the malaria parasite and that, as with the tetracyclines, it will have to be taken for at least a week. For acute infections of malaria, it will probably also have to be taken in conjunction with a rapidly acting but non-curative drug regimen, such as a 3-day course of chloroquine, quinine or one of the arteminisin derivatives. The very slight risk of developing fatal aplastic anaemia following
54 t r e a t m e n t with c h l o r a m p h e n i c o l (1:25000 to 1:100000) should also be taken into consideration. The practical value of c h l o r a m p h e n i c o l as an a n t i m a l a r i a l drug could be quite significant if f o r t h c o m i n g clinical studies indicate that it is able to cure m a l a r i a infections. In c o m b i n a t i o n with a rapidly acting b u t n o n - c u r a t i v e antimalarial, c h l o r a m p h e n i c o l could be used to treat febrile patients in w h o m diagnostic u n c e r t a i n ties exist. It could also be very useful in achieving radical cure of falciparum infections, particularly in patients who are u n a b l e to be treated with the tetracyclines, e.g., d u r i n g early childhood.
References Chen, P., Lamont, G., Elliot, T., Kidson, C., Brown, G., Mitchell, G., Stace, J. and Alpers, M. (1980) Plasmodium falciparum strains from Papua New Guinea: culture characteristics and drug sensitivity. S.E. Asian J. Trop. Med. Pub. Hlth. 11,435-440. Imboden, C.A., Cooper, W.C., Coatney, G.R. and Jeffrey, G.M. (1950) Studies in human malaria. XXIX. Trials of aureomycin, chloramphenicol, penicillin, and dihydrostreptomycin against the Chesson strain of P. vivax. J. Nat. Mal. Soc. 9, 377 380. Lambros, C. and Vanderberg, J.P. (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65, 418-420. Rieckmann, K.H., Powell, R.D., McNamara, J.V., Willerson, D., Kass, C., Frischer, H. and Carson, P.E. (1971) Effect of tetracycline against chloroquine resistant and chloroquine sensitive Plasmodium falciparum. Am. J. Trop. Med. Hyg. 20, 811-815. Rieckmann, K.H., Willerson, W.D., Carson, P.E., Frischer, H. (1972) Effects of tetracycline against drug-resistant malaria. Proc. Helminthol. Soc. Wash. 39 (Special Issue), 339-347. Ruiz-Sanchez, F., Quezada, M., Paredes, M.E., Casillas, J. and Riebling, R. (1952) Chloramphenicol in malaria. Am. J. Trop. Med. Hyg. 1,936-940. Sande, M.A. and Mandell, G.L. (1980) Tetracyclines and chloramphenicoi. In: The Pharmacological Basis of Therapeutics, Gilman, A.G., Goodman, L.S. and Gilman, A. (eds.). Macmillan, New York, pp. 1181-1199. Thaithong, S. and Beale, G.H. (1981) Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75, 271-273. Trager, W. and Jensen, J.B. (1976) Human malaria parasites in continuous culture. Science 193,673-675. World Health Organization. (1961) Chemotherapy of malaria. WHO Tech. Rep. Ser. 226, 16.