22 tant Vietnam Oak Knoll (F.v.o.) strains of P. falciparum.13-14 Both of these human isolates had been adapted to the owl monkey (Aotus trivirgatus) elsewhere,13 and a few old Aotus monkeys which had been in our laboratory for over 4 years were infected with these strains. During the course of infection about 50 µl of parasitised blood was collected. Chloroquine sensitivity was determined by measuring the extent to which normal maturation of trophozoites to schizonts was inhibited by various concentrations of chloroquine in the microtechnique described below.

Methods and Devices

DRUG SENSITIVITY OF PLASMODIUM FALCIPARUM An In-vitro Microtechnique K. H. RIECKMANN G. H. CAMPBELL

L. J. SAX J. E. MREMA

University of New Mexico, Albuquerque, New Mexico

87131, U.S.A.

THE resurgence of malaria in various areas of the world and the increased occurrence of chloroquine-resistant infections underline the importance of a simple in-vitro method for determining the sensitivity of Plasmodium falciparum to antimalarial drugs.1,2 An existing in-vitro drug-susceptibility test3-11 could be applied more widely if it were adapted to a technique requiring the collection of capillary instead of venous blood-samples. Aided by developments in the cultiva-

EFFECTS OF

I

I

CHLOROQUINE

ON

2

I

Microtechnique.-Various amounts of chloroquine diphosphate (0-1to 25.0 ng), dissolved in 25 µl of distilled water, were added to the flat-bottomed wells (diameter, 6.3 mm) of microtitre plates (Microtest II, Falcon Plastics, Oxnard, California) which were dried in an incubator and then stored at room temperature for future use. At the start of an experiment, 50 µl of culture medium was added to each well and stirred briefly to dissolve the chloroquine. The medium consists of RPMI 1640 powdered medium (Gibco, Grand Island, New York) supplemented with sodium bicarbonate (2 mg/ml),

buffer (6,mg/ml), and gentamicin sulphate (4 µg/ml). of parasitised blood was placed in each well by means of

HEPES

5

µl

an

Eppendorf pipette (Brinkmann Instruments, Westbury, York). The plate, covered with a lid (Falcon), was then

New

STRAINS OF P. FALCIPARUM IN VITRO

I

I

I

I

I

I

_.

I

I

I

I

*Parasites were obtained during the course of infection of 2 Aotus monkeys infected with the F.U.P. strain of P. falciparum and 3 Aotus monkeys infected with the F.v.o. strain of P. falciparum. tPercentage of asexual parasites, relative to the mean of 2 control replicates (100%), that matured to schizonts. §Mean of 2 replicates.

tion of P. falciparum,12 we have developed a microtechnique for measuring the susceptibility of malaria parasites to drugs. Methods

Investigations Uganda

sensitive

were carried out with the chloroquinePalo Alto (F.U.P.) and the chloroquine-resis-

8. Martinis, J., Bach, F. H. Transplantation (in the press). 9. Garrido, F., Schirrmacher, V., Festenstein, H. Nature, 1976, 259, 228. 10. Invernizzi, G., Parmiani, G. ibid. 1975, 254, 713. 11. Garrido, F., Schirrmacher, V., Festenstein, H. J. Immunogenet. 1977, 4, 15. 12. Invernizzi, G., Carbone, G., Meschini, A., Parmiani, G. ibid. 97. 13. Parmiani, G., Invernizzi, G. Int. J. Cancer, 1975, 16, 756. 14. Wrathmell, A. B., Gauci, G. L., Alexander, P. Br. J. Cancer, 1976, 33, 187. 15. Martin, W. J., Gipson, T. G., Martin, S. E., Rice, J. M. Science, 1976, 194, 532. 16. Martin, W. J., Gipson, T. G., Rice, J. M. Nature, 1977, 265, 738. 17. Lemonnier, F., Burakoff, S. J., Germain, R. N., Benacerraf, B. Proc. natn. Acad. Sci. U.S.A. 1977, 74, 1229. 18. Bodmer, W. F. Transpl. Proc. 1973, 5, 1471. 19. Silver, J., Hood, L. Contemp. Topics molec. Immun. 1976, 5, 35. 20. Sondel, P. M., Bach, F. H. J. exp. Med. 1975, 142, 1339. 21. Zarling, J. M., Bach, F. H. Unpublished.

shaken and placed in a vacuum glass desiccator containing a. paraffin candle. After the candle was lit, the desiccator lid was replaced and the fumes were allowed to escape through an open stopcock. The stopcock was closed just before the flame went out. The desiccator was then placed in an incubator at 38-39°C for 24-30 h. After incubation, thick blood films prepared from each well were dried thoroughly, stained for 20 min with Giemsa stain, and examined for parasite maturation. The number of schizonts per 500 white blood-cells was determined in control and drug samples. The values for samples containing drug were expressed as percentages of control samples. Results

Chloroquine was incubated with 4 samples of blood containing parasites of the F.U.P. strain and 5 samples of blood containing parasites of the F.v.o. strain (see accompanying table). When we measured the maturation of parasites of P. falciparum at different concentrations of chloroquine we found that the

strain was at least 10 times more sensitive to than the F.v.o. strain. Formation of schizonts in chloroquine the F.U.P. strain was completely inhibited at chloroquine concentrations of 1 to 2 ng per well, whereas at this concentration maturation of parasites of the F.v.o. strain was not affected. F.u.p.

23

Maturation of F.v.o. schizonts and completely inhibited by 20

was

to

partially inhibited by 10 ng chloroquine per well.

25 ng of

Requests for reprints should be addressed to K.H.R., Malaria Program, 282 Castetter Hall, University of New Mexico, Albuquerque, New Mexico, 87131, U.S.A. Research

Discussion

REFERENCES

This microtechnique, which only requires finger-tip specimens of blood, will simplify the in-vitro drug-susceptibility test, particularly in young children. Costs will be reduced substantially because the test requires less equipment, time, and effort than the standard in-vitro method. This will facilitate identification and treatment of drug-resistant falciparum infections, assessment of the prevalence or extension of chloroquine-resistant strains, screening of potential antimalarial agents, and classification of parasites of heterogeneous antigenic origins by their susceptibility to drugs.

1. 2.

Rieckmann, K. H., McNamara, J. V., Frischer, H., Stockert, T. A., Carson, P. E., Powell, R. D. Am. J. trop. Med. Hyg. 1968, 17, 661. Rieckmann, K. H., Lopez-Antuñano, F. J. Bull. Wld Hlth Org. 1971, 45,

157. 3. Rieckmann, K. H. J. Am. med. Ass. 1971, 217, 573. 4. Peters, W., Seaton, D. R. Ann. trop. Med. Parasit. 1971, 65, 2. 5. Colwell, E. J., Pung, P., Sadudee, N., Benjapong, W., Neopatimanondh, S. Am. J. trop. Med. Hyg. 1972, 21, 6. 6. Tech. Rep. Ser. Wld Hlth Org. no. 529, 1973. 7. Canfield, C. J., Rozman, R. S. Bull. Wld Hlth Org., 1974, 50, 203. 8. Kinnamon, K. E., Rothe, W. E. Am. J. trop. Med. Hyg. 1975, 24, 174. 9. Valéra, C. V., Shute, G. T. WHO/MAL/ 75.852 (W.H.O. report, unpub-

lished).

We thank Mrs R. Tufaro’ for her excellent technical assistance. Strains of P. falciparum were kindly forwarded to us by Colonel D. Davidson, Walter Reed Army Institute of Research, Washington, D.C., and Dr W. Siddiqui, University of Hawaii, Honolulu. This study was supported by contract no. AID/ta-C-1259 from the Agency for International Development, U.S. Department of State.

10. Palmer, T. T., Townley, L. B., Yigzaw, M., Armstrong, J. C. Am. J. trop. Med. Hyg. 1976, 25, 10. 11. Ebisawa, I., Fukuyama, T., Kawamura, Y. Trop. geogr. Med. 1976, 28, 349. 12. Trager, W., Jensen, J. B. Science, 1976, 193, 673. 13. Siddiqui, W. A., Schnell, J. V., Geiman, Q. M. Am. J. trop. Med. Hyg. 1972,

Reviews of Books

diagnostic reagents and the newer techniques, including immunoperoxidase enzyme immunoassays, radioimmunoassay, cell hybridisation, and immune electron microscopy. This book is attractively printed, and the pictures are well reproduced. It is perhaps invidious to single out individual reviews, but that on the diagnosis of viral hepatitis by Zuckerman and Howard is especially good. At first glance, the contents page may leave a physician, or even a clinical microbiologist, overawed, but

Pp.

78.

/;2.9S.

THE mechanisms involved in different types of heart-failure have not been well understood for many years but recently interest has been focused on the part played by derangements of the systemic venous capacitance vessels, the pulmonary venous capacitance bed, and pulmonary and systemic arteriolar resistance. Therapy is no longer only a matter of attempting to improve myocardial function, but should also be aimed at altering, where appropriate, the above-mentioned variables. Dr Bradley describes both the pathophysiology of heart-failure and the way in which different parts of the cardiovascular system can be manipulated in its treatment. He emphasises the way in which the right and left heart-systems may malfunction with respect to each other and out of this comes the appreciation that reliance on such parameters as the systemic venouspressure alone in the management of heart-failure may lead to many pitfalls. The importance of monitoring the function of both the right and left sides of the heart when treating acute

heart-failure is stressed;

measurement

of indirect left-atrial

pressure is

relatively simple by the technique of floating a balloon-tipped catheter to the pulmonary artery. This book is recommended to those who require a concise and detailed account of the pathophysiology of heart-failure and the way in which an understanding of this can lead to a more logical approach to therapy. Comparative Diagnosis of Viral Diseases Edited by EDOUARD KURSTAK and CHRISTINE KURSTAK. London and New York: Academic. 1977. Pp. 555. ;E27.70;$39.

THE editors are to be congratulated on their choice of subThe book labours under a cumbersome subtitle of Volume II-Human and Related Viruses, Part B, as though it was just a small part of a series. This may be so, but each one of the reviews touches upon a growing-point in virological research or a subject of practical importance. The subject of the first chapter, Marburg virus disease, may seem highly specialised, but recent events have shown that it is one we cannot afford to neglect. Among the other subjects covered are slowvirus infections, particularly of the central nervous system, and cancer viruses, especially welcome because they are discussed in the context of human medicine. Vaccines and chemotherapy also have a chapter each, and the last six chapters

jects.

21, 392. Schmidt, L. H.

Trans. R. Soc. trop. Med. Hyg.

1973, 67, 446.

cover

Studies in Acute Heart Failure R. D. BRADLEY. London: Arnold. 1977.

14.

the book contains the stuff of which future advances are made, and although there is no doubt that it will be added to, much of it will not be superseded. There is little in the way of pad’

ding. Treatment of Neuromuscular Diseases

.

Advances in Neurology: vol. XVII. Edited by R. C. GlttuGS and R. T. MOXLEY, III. New York: Raven. 1977. Pp. 370.$31.20.

Neurology

THIS volume in the worthwhile Advances in series is a "state of the art" discussion of therapeutic methods and of perspectives in human neuromuscular disorders; it also covers certain aspects of the clinical and laboratory evaluation of patients with neuromuscular disease and carries reviews of areas of research likely to lead to advances in the understanding and treatment of these conditions. The two introductory chapters by the editors provide an overview of clinical and research trends. The first of two chapters on techniques of clinical examination, by Brooke, emphasises the clues which may be derived from inspection, palpation, and so on, and the importance of assessment of function in the evaluation of patients, but the lack of illustrations is a severe test of the reader’s powers of imagination; the second, by Myers, deals with the evaluation of the floppy infant. There are three chapters on the application of laboratory techniques in the evaluation of patients with neuromuscular disease. That by Penn is a very good, up-to-date review of immunological aspects of myasthenia gravis, dermatomyositis, and polymyositis and of the experimental counterparts of the conditions. The other two chapters on clinical electromyography and pathology are more selective and are superficial in some areas. Similarly, in the chapter on genetic considerations, prenatal diagnosis is rightly considered in some depth but carrier-detection is dealt with summarily. The first of two chapters devoted to investigative techniques, by Engel, is a detailed discussion incorporating personal views on the nomenclature, classification, and other aspects of neuromuscular disease; it also raises some provocative and stimulating points for the clinical and laboratory in-

vestigator. Dyck, nerve

disease,

on

investigative approaches

concentrates on

the

use

to

peripheral-

of serial sections

to