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In vitro cultivation of malaria parasites
Editorial ii
In Vitro Cultivation of Malaria Parasites
Robert E. Desjardins, M.D. Head, Anti-lnfectives Section Medical Division IVellcome Research Laboratories Research Triangle Park, North Carolina 27709 Jean H. Bowdre, Ph.D. Department of Hospital Laboratories North Carolina Memorial Hospital Chapel Hill, North Carolina 27514 Interest in cultivation of the causative agent of human malaria must have followed soon after Laveran's historic observation of the living parasite in a fresh blood specimen. The first recorded report of in vitro culture of Plasmodiumfalciparun~ was made in 1912 by Bass and Johns (2). Since that time there have been many attempts to maintain the erythrocytic cycle of the parasite in cultures, primarily with avian, simian or murine species of Plasmodiunt. It was not until 1976 that the final essential elements of a successful method for the continuous cultivation of Plasmodium falciparum in human erythrocytes were discovered and reported (4, 10). The new technique primarily differs from the many earlier reported attempts in that it employs dilute cultures with low initial parasite densities and an atmosphere with reduced oxygen tension (4, 10). Successful in vitro cultivation of P. falciparum and other species of Plasmodium has now been reported by many investigators in various laboratories. The culture medium most commonly employed consists of buffered RPMI 1640 supplemented with 10-15070 human serum or plasma (10). Frequent dilution with a fresh red blood cell suspension and daily changes of the culture medium are essential. In this way a continuous but asynchronous intraerythrocytic cycle can be maintained indefinitely with parasites infecting 10070 or more of the erythrocytes. Several isolates of P. falciparum with varying susceptibility to antimalarial
agents have now been successfully adapted to continuous in vitro culture (3, 5), providing an unparalleled resource for research on the biology, metabolism, immunology and chemotherapy of human malaria. The most immediate impact of this new technology has been in the field of antimalarial chemotherapy. A resurgence of interest in antimalarial drug research was triggered in the early 1960s by the initial appearance of chloroquine-resistant falciparum malaria in Colombia. Resistance to other antimalarial drugs such as pyrimethamine and chloroguanide was also then widespread. By the early 1970s chloroquine-resistant isolates of P. falciparunl had been reported throughout Southwest Asia, Central and South America and in India. Recently, the first chloroquineresistant isolate on the African continent was reported from Kenya (6). The treatment and prophylaxis of falciparum malaria has again become a complex and difficult problem (1). It is of great epidemiologic interest and therapeutically helpful to rapidly determine the susceptibility of a strain of P. falciparum to chloroquine and other antimalarial drugs. The use of short-term cultures of P. falciparum directly from clinical specimens to evaluate chloroquine susceptibility was described by Rieckmann in 1971 (8). This simple but extremely useful method was a modification of the technique originally
described by B.ass and Johns (2). In a defibrinated whole blood specimen supplemented with glucose and incubated at 38°C, intraerythrocytic maturation of the parasites from trophozoites to multinucleated schizonts occurs in vitro within 24 hours. Chloroquine (10 ng/ml) will inhibit this maturation if the isolate is susceptible. Resistant isolates will undergo normal maturation even at 5-10 times higher concentrations of the drug. This in vitro method is useful for epidemiologic studies and is the World Health Organization standard test to determine chloroquine susceptibility. Use of the method is limited by its requirement for blood specimens that contain parasites at the proper stage of development for initiation of the culture. Because of poor maturation of the parasites, the results are sometimes unreliable when the parasite density in the specimen is high. While other 4-aminoquinolines and quinine could also be evaluated readily by this technique (8), other drugs such as pyrimethamine, which requires more than a single intraerythrocytic cycle to exert a suppressive effect, could not. Rieckmann et al. (9) more recently described a modification of the earlier method that has proved more reliable. The new method uses microtitration plates that are prepared in advance by the addition of solutions of chloroquine to selected wells of the plates, which are then dried and stored. Small
Editors: Donna J. Blazevic, L. R. McCarthy, and Josephine A. Morello Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; $2.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume I (1979), 24 issues $42.50. Volume 2 (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regarding subscription to: Clinical Microbiology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical Microbiology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited. Editorials and letters printed in this Newsletter are published for the interest of the readers and do not necessarily reflect the opinions of the editors.
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aliquots (5 ~u1) o f the infected blood specimen are added to each well along with 50/al of bicarbonate buffered RPMI 1640 culture medium. The inoculated plate is covered and incubated in a candle extinction jar at 37°C for 24 hours. Drug effect is assessed at the end o f the incubation period by the same morphologic criteria described earlier. A similar technique described by Nguyen-Dinh and Trager (7) employs parasites that are diluted in fresh red blood cells to a density o f / ~ 1.0°7o and then added to the wells o f a multiwell plate (Linbro, Hamden, Conn.) that contains sufficient culture medium to dilute the blood to a 2% hematocrit. The plates are then incubated in a candle extinction jar at 37°C for 48 hours. Parasite density in wells without chloroquine increases 3-7 times during the incubation period; the increase is proportionately less in the presence of increasing concentrations of chloroquine. Multiplication of known susceptible strains o f P. falciparmn (FCR-8/West Africa and FCN-1/Nigeria) was completely inhibited at 10 ng/ml of chloroquine, whereas a chloroquine-resistant isolate required I00 ng/ml o f chloroquine for complete inhibition (6). This technique has also been used successfully to identify pyrimethaminesensitive and resistant isolates of P. falciparum. * A semiautomated microdilution *P. Nguyen-Dinh. Personal communication.
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technique for assessing potential antimalarial activity by inhibiting uptake of radiolabeled metabolic precursors was described by Desjardins et al. (3). This technique uses continuous cultures as a source of parasites. It is capable o f screening large numbers o f compounds and provides a quantitative concentration-response analysis suitable for detailed structure-activity determinations. Integration of this in vitro model with existing animal models should greatly accelerate the process of identification and development o f potential new antimalarial drugs. The speed and precision o f this assay may also make it especially useful for susceptibility testing. Experience with bacterial susceptibility testing has shown that the ability to determine a precise endpoint is valuable for predicting clinical response from an in vitro assay, particularly when strains of intermediate susceptibility exist. In such cases a precise endpoint is crucial for establishing correlation o f the assay with clinical experience. In addition to its immediate diagnostic and epidemiologic value, the new in vitro technology for cultivation of P. falcipartun provides an invaluable resource for antimalarial drug research. With the amply demonstrated capacity of this protozoan parasite to become resistant to all existing antimalarial drugs, the search for effective new drugs must be a continuing effort.
Refereltces 1. Barrett-Connor, E. 1978. Chemoprophylaxis of malaria. Ann. Intern. Med. 89:417-418. 2. Bass, C. C., and F. M. Johns. 1912. The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparttm) in vitro. J. Exp. Med. 16:567-579. 3. Desjardins, R. E., et al. 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16:710-718. 4. Haynes, J. D., et al. 1976. Culture of human malaria parasites Plasmodittm falciparum. Nature 263:767-769. 5. Jensen, J. B., and W. Tragcr. 1978. Plasmodittmfalciparttm in culture: Establishment of additional strains. Am. J. Trop. Med. Hyg. 27:743746. 6. Kean, B. 11. 1979. Chloroquineresistant falciparum malaria from Africa. JAMA 241:395. 7. Nguyen-Dinh, P., and W. Trager. 1980. Plasmodittmfalciparttm in vitro: Determination of chloroquine sensitivity of three new strains of a modified 48-hour test. Am. J. Trop. Med. Hyg. 29:339-342. 8. Rieekmann, K. 11. 1971. Determination of the drug sensitivity of Plasmodittmfalciparttnl. JAMA. 217:573578. 9. Reickmann, K. i!., et al. 1978. Drugsensitivity of Plasmodiumfalciparttm. An in vitro microtechnique. Lancet 1:22-23. 10. Trager, E., andJ. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675.
In the past several years, clinical microbiology services have significantly decreased the time needed to identify pathogenic microorganisms from clinical sources. Counterimmunoelectrophoresis (CIE) is one o f the current procedures designed to contribute to rapid diagnosis while maintaining high specificity. This technique has been used to detect antigens in cerebrospinal fluid, pleural fluid, urine and other clinical specimens (1, 6). CIE has also been adapted for use
in the identification o f organisms in blood cultures (3, 4, 7). This has contributed to the rapid presumptive identification of agents involved in significant bacteremia. This report describes several spurious results encountered in our laboratory with the use o f CIE in the rapid identification of organisms from blood cultures. Fifteen blood culture broth samples were tested for pneumococcal antigen by CIE, and the results were compared to those obtained by standard culture methods (Table 1). Seven broth sam-
Laboratory M e t h o d s
Limitations of Rapid Identification of Streptococcus pneumoniae from Blood Cultures by Counterimmunoeleclrophoresis Allan L. Truant, Ph.D. James A. Buckner, M.T. (ASCP) Elaine Tacquard, M.T. (ASCP) Michael T. Kelly, M.D., Ph.D. Clinical Microbiology Division Clinical Laboratories Department of Pathology The Universityof Texas Medical Branch Galveston, Texas 77550
In Vitro Cultivation of Malaria Parasites
Robert E. Desjardins, M.D. Head, Anti-lnfectives Section Medical Division IVellcome Research Laboratories Research Triangle Park, North Carolina 27709 Jean H. Bowdre, Ph.D. Department of Hospital Laboratories North Carolina Memorial Hospital Chapel Hill, North Carolina 27514 Interest in cultivation of the causative agent of human malaria must have followed soon after Laveran's historic observation of the living parasite in a fresh blood specimen. The first recorded report of in vitro culture of Plasmodiumfalciparun~ was made in 1912 by Bass and Johns (2). Since that time there have been many attempts to maintain the erythrocytic cycle of the parasite in cultures, primarily with avian, simian or murine species of Plasmodiunt. It was not until 1976 that the final essential elements of a successful method for the continuous cultivation of Plasmodium falciparum in human erythrocytes were discovered and reported (4, 10). The new technique primarily differs from the many earlier reported attempts in that it employs dilute cultures with low initial parasite densities and an atmosphere with reduced oxygen tension (4, 10). Successful in vitro cultivation of P. falciparum and other species of Plasmodium has now been reported by many investigators in various laboratories. The culture medium most commonly employed consists of buffered RPMI 1640 supplemented with 10-15070 human serum or plasma (10). Frequent dilution with a fresh red blood cell suspension and daily changes of the culture medium are essential. In this way a continuous but asynchronous intraerythrocytic cycle can be maintained indefinitely with parasites infecting 10070 or more of the erythrocytes. Several isolates of P. falciparum with varying susceptibility to antimalarial
agents have now been successfully adapted to continuous in vitro culture (3, 5), providing an unparalleled resource for research on the biology, metabolism, immunology and chemotherapy of human malaria. The most immediate impact of this new technology has been in the field of antimalarial chemotherapy. A resurgence of interest in antimalarial drug research was triggered in the early 1960s by the initial appearance of chloroquine-resistant falciparum malaria in Colombia. Resistance to other antimalarial drugs such as pyrimethamine and chloroguanide was also then widespread. By the early 1970s chloroquine-resistant isolates of P. falciparunl had been reported throughout Southwest Asia, Central and South America and in India. Recently, the first chloroquineresistant isolate on the African continent was reported from Kenya (6). The treatment and prophylaxis of falciparum malaria has again become a complex and difficult problem (1). It is of great epidemiologic interest and therapeutically helpful to rapidly determine the susceptibility of a strain of P. falciparum to chloroquine and other antimalarial drugs. The use of short-term cultures of P. falciparum directly from clinical specimens to evaluate chloroquine susceptibility was described by Rieckmann in 1971 (8). This simple but extremely useful method was a modification of the technique originally
described by B.ass and Johns (2). In a defibrinated whole blood specimen supplemented with glucose and incubated at 38°C, intraerythrocytic maturation of the parasites from trophozoites to multinucleated schizonts occurs in vitro within 24 hours. Chloroquine (10 ng/ml) will inhibit this maturation if the isolate is susceptible. Resistant isolates will undergo normal maturation even at 5-10 times higher concentrations of the drug. This in vitro method is useful for epidemiologic studies and is the World Health Organization standard test to determine chloroquine susceptibility. Use of the method is limited by its requirement for blood specimens that contain parasites at the proper stage of development for initiation of the culture. Because of poor maturation of the parasites, the results are sometimes unreliable when the parasite density in the specimen is high. While other 4-aminoquinolines and quinine could also be evaluated readily by this technique (8), other drugs such as pyrimethamine, which requires more than a single intraerythrocytic cycle to exert a suppressive effect, could not. Rieckmann et al. (9) more recently described a modification of the earlier method that has proved more reliable. The new method uses microtitration plates that are prepared in advance by the addition of solutions of chloroquine to selected wells of the plates, which are then dried and stored. Small
Editors: Donna J. Blazevic, L. R. McCarthy, and Josephine A. Morello Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; $2.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume I (1979), 24 issues $42.50. Volume 2 (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regarding subscription to: Clinical Microbiology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical Microbiology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited. Editorials and letters printed in this Newsletter are published for the interest of the readers and do not necessarily reflect the opinions of the editors.
I ¸
aliquots (5 ~u1) o f the infected blood specimen are added to each well along with 50/al of bicarbonate buffered RPMI 1640 culture medium. The inoculated plate is covered and incubated in a candle extinction jar at 37°C for 24 hours. Drug effect is assessed at the end o f the incubation period by the same morphologic criteria described earlier. A similar technique described by Nguyen-Dinh and Trager (7) employs parasites that are diluted in fresh red blood cells to a density o f / ~ 1.0°7o and then added to the wells o f a multiwell plate (Linbro, Hamden, Conn.) that contains sufficient culture medium to dilute the blood to a 2% hematocrit. The plates are then incubated in a candle extinction jar at 37°C for 48 hours. Parasite density in wells without chloroquine increases 3-7 times during the incubation period; the increase is proportionately less in the presence of increasing concentrations of chloroquine. Multiplication of known susceptible strains o f P. falciparmn (FCR-8/West Africa and FCN-1/Nigeria) was completely inhibited at 10 ng/ml of chloroquine, whereas a chloroquine-resistant isolate required I00 ng/ml o f chloroquine for complete inhibition (6). This technique has also been used successfully to identify pyrimethaminesensitive and resistant isolates of P. falciparum. * A semiautomated microdilution *P. Nguyen-Dinh. Personal communication.
I
technique for assessing potential antimalarial activity by inhibiting uptake of radiolabeled metabolic precursors was described by Desjardins et al. (3). This technique uses continuous cultures as a source of parasites. It is capable o f screening large numbers o f compounds and provides a quantitative concentration-response analysis suitable for detailed structure-activity determinations. Integration of this in vitro model with existing animal models should greatly accelerate the process of identification and development o f potential new antimalarial drugs. The speed and precision o f this assay may also make it especially useful for susceptibility testing. Experience with bacterial susceptibility testing has shown that the ability to determine a precise endpoint is valuable for predicting clinical response from an in vitro assay, particularly when strains of intermediate susceptibility exist. In such cases a precise endpoint is crucial for establishing correlation o f the assay with clinical experience. In addition to its immediate diagnostic and epidemiologic value, the new in vitro technology for cultivation of P. falcipartun provides an invaluable resource for antimalarial drug research. With the amply demonstrated capacity of this protozoan parasite to become resistant to all existing antimalarial drugs, the search for effective new drugs must be a continuing effort.
Refereltces 1. Barrett-Connor, E. 1978. Chemoprophylaxis of malaria. Ann. Intern. Med. 89:417-418. 2. Bass, C. C., and F. M. Johns. 1912. The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparttm) in vitro. J. Exp. Med. 16:567-579. 3. Desjardins, R. E., et al. 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16:710-718. 4. Haynes, J. D., et al. 1976. Culture of human malaria parasites Plasmodittm falciparum. Nature 263:767-769. 5. Jensen, J. B., and W. Tragcr. 1978. Plasmodittmfalciparttm in culture: Establishment of additional strains. Am. J. Trop. Med. Hyg. 27:743746. 6. Kean, B. 11. 1979. Chloroquineresistant falciparum malaria from Africa. JAMA 241:395. 7. Nguyen-Dinh, P., and W. Trager. 1980. Plasmodittmfalciparttm in vitro: Determination of chloroquine sensitivity of three new strains of a modified 48-hour test. Am. J. Trop. Med. Hyg. 29:339-342. 8. Rieekmann, K. 11. 1971. Determination of the drug sensitivity of Plasmodittmfalciparttnl. JAMA. 217:573578. 9. Reickmann, K. i!., et al. 1978. Drugsensitivity of Plasmodiumfalciparttm. An in vitro microtechnique. Lancet 1:22-23. 10. Trager, E., andJ. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675.
In the past several years, clinical microbiology services have significantly decreased the time needed to identify pathogenic microorganisms from clinical sources. Counterimmunoelectrophoresis (CIE) is one o f the current procedures designed to contribute to rapid diagnosis while maintaining high specificity. This technique has been used to detect antigens in cerebrospinal fluid, pleural fluid, urine and other clinical specimens (1, 6). CIE has also been adapted for use
in the identification o f organisms in blood cultures (3, 4, 7). This has contributed to the rapid presumptive identification of agents involved in significant bacteremia. This report describes several spurious results encountered in our laboratory with the use o f CIE in the rapid identification of organisms from blood cultures. Fifteen blood culture broth samples were tested for pneumococcal antigen by CIE, and the results were compared to those obtained by standard culture methods (Table 1). Seven broth sam-
Laboratory M e t h o d s
Limitations of Rapid Identification of Streptococcus pneumoniae from Blood Cultures by Counterimmunoeleclrophoresis Allan L. Truant, Ph.D. James A. Buckner, M.T. (ASCP) Elaine Tacquard, M.T. (ASCP) Michael T. Kelly, M.D., Ph.D. Clinical Microbiology Division Clinical Laboratories Department of Pathology The Universityof Texas Medical Branch Galveston, Texas 77550