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Pan American Health Organization ADVISORY COMMITTEE on MEDICAL RESEARCH Sixth Meeting Washington, Doco, 12-16 June 1967 Item Pan American Health Organization ADVISORY COMMITTEE ON MEDICAL RESEARCH Sixth Meeting Washington, DoCo, 12-16 June 1967 Item 5.2 of the Agenda STUDIES ON THE RESPONSE TO DRUGS OF BLOOD-INDUCED FALCIPARUM MALARIA: SOUTH AMERICAN PARASITE STRAINS Ref: RES 6/2 14 April 1967 PAN AMERICAN HEALTH ORGANIZATION Pan American Sanitary Bureau, Regional Office of the WORLD HEALTH ORGANIZATION Washington, D.C. STUDIES CN THE RESPONSE TO DLRUGS OF BLOOD-INDUCED FALCI- PARUM MALARIA. SOUTH ATERICAN PARASITE STRAINS Final Report of the Ribeirao Preto Screening Center Sao Paulo, Brazil by A.J. Walker --y and . -Y -1 F.J. López-Antuñano A-> - --la PREFACE A preliminary report on the work of the Ribeirao Preto Center was prepared in May 1965, covering the period January 1963 to March 1965 (AMRO Project 0212). The report, hurriedly assembled -7. from a complex mass of data, failed to give the complete picture. In March 1967, the Pan American Health Organization requested the authors to re- examine with more deliberation the material and its implications. This report, prepared with the help of Dr. John W. Field, STC/WHO, is the result. -I rt. CONTENTS -I Iritroduction II Origin of strains III Methods and standards IV Sensitivity of strains to drugs A) Chloroquine B) Pyrimethamine C) Combined pyrimethanine - sulphonamides D) Quinine *1- V Toxicity of drugs VI Summary and conclusions VII Acknowledgements Appendices A) History of donors B) Sequence of passage C) Pre-patent periods in relation to the number , -. of parasites in the inoculum. D) A note of microscopic diagnosis -. ;i E) Organization and staff -Jo _-.s- RESPONSE TO DRUGS IN BLOOD-INDUCED FALCIPARUM MALARIA: -$ SOUTH AMERICAN PARASITE STRAINS - I INTRODUCTION The history of malaria chemotherapy over the second quarter of the present century is one of sustained progress in the synthesis of active remedies. Plasmoquine, the first successful synthetic remedy, was introduced in 1927. Mepacrine appeared in 1932, proguanil, pyrimethamine, and the 4-aminoquinolines, during or soon after the second world war° Highly active and relatively non-toxic, the synthetic compounds opened 4--+ up a new era in the chemotherapy of malaria. Everywhere the case mor- tality fell, blackwater fever almost disappeared, and a powerful supple- ment to the insecticides in the eradication of malaria seemed assured. But in 1947 there were disquieting reports of malaria infections which resisted proguanil and within a few years it was clear that all the malaria parasites of man might become resistant not only to proguanil but also to pyrimethamine, a compound of similar plasmodicidal action. The 4-aminoquinolines, compounds to which the parasites displayed no loss of sensitivity, were accorded an increasingly important role. Attempts to induce resistance in the laboratory had usually failed and there was little reason to think that these important remedies would ever pose a clinical or public health problem. From South America in 1961 came the first hint of a loss of sensitivity to the 4-aminoquinolines in the malaria infections of man. Reports from Colombia and Brazil drew attention to relapses in falciparum .- y~~~~~~~~~~~~2 - - infections treated with chloroquine, best-knovm of the 4-aminoquinolineso Standard courses of chloroquine failed to cure the infections induced by a falciparum strain of Colombian origin; and similar failures were re- ported from field trials in Colombia and northern Brazil. Alert to the possibility of an emerging problem and to the need for fuller information on the sensitivity to drugs of the malaria parasites of Brazil, the Brazilian Government in co-operation with the Pan American Sanitary Bureau established a screening center for the investigation of possible resistance malaria. Under an agreement signed in 1962 by the Minister of Health, Brazil and the Secretary of Health and Social Assistance, State of Sao Paulo, the center was located at the Santa Tereza Hospital, a branch of the Psychopatic Department of the Public Health and Social Assistance, and operated in conjunction with the malaria campaign of the Health Ministry. The center was opened on 28 January 1963. From this date until operations were suspended on 31 March 1965 the response to treatment with various drugs was studied in the infections induced by falciparum strains from Brazil, Colombia and Venezuela. This report presents an account of the work. II ORIGIN OF STRAINS The strains of Plasmodium falciDarum studied at the center were recovered from donors who had lived in various parts of Brazil, Colombia or Venezuela where they had presumably acquired their infections. The donors were malaria patients whose infections had been recognized either in the field or at the Zone IV Office of the CEM in Ribeirao Preto. -3- Treatment with chloroquine had failed in most of the donors to cure the infections, a possible indication of resistance to the drugo A few, selected with a hope of recovering a strain fully sensitive to chloroquine, had had little or no treatment. The strains, designated according to their place of presumed origin with no implication of special biological signi.fi- cance, were recovered from widely scattered areas, thirteen from northern, central or western Brazil, four from the Magdalena Valley, Colombia and one from the Orinoco Valley, Venezuela. Fig. 1 lists the localities ,where the infections are thought to have been acquired and Table 1 briefly records data relating to the donors. Fuller details appear in Appendix A. TABLE 1 STRAINS OF PLASMODIUM FALCIPARUM STUDIED AT RIBEIRAO PRETO CENTER, WITH PRESUMED SOURCE AND DATA RELATING TO DONORS Designation Data relating to donors of strain .Where Chloroquine ---k blood within previous .taken six months .n Rondonia I Center 1800 mg base II Center 1050 (Amod) Goiania Center -- Boa Vista I Field 6000 II Field 2940 III Field 3600 IV Field 9410 Para I Field 6600 II Field 2560 Belem Center - Santarem Center 12000 (?) Gurupi Center 3000 Goias Center 4800 (?) él, Colombia I Field 9600 II Field 5600 -- qk III Field - IV Field 7100 Venezuela Field 1500 Center - blood taken at Ribeirao Preto Center from patients appearing at Zone IV office of CEM .. w -4- o o 14 13 6 5 15 7 Fig.1 PRESUMIED GEOGRAPHICAL ORIGIN OF FALCIPARUM STRAINS STUDIED. Nr Strain Probable lace of infection 1 Colombia I Vereda El Pescadolpio.Pto.BoyacA 2 " II Puerto Niño " " "t 3 " . III Vereda El:LTbano,MFpio.Ro de Oro 1 IV 4 El Cruce,1 Spio.RIo de Oro. 5 Rondonia I Rio do !Nachado(MatoGrosso border) 6 " II Near Porto Velho 7 Goiania Crixas 8 Boa Vista Boa VistaTerritorio de Rorairma- 9 Para 1 Igarapemirim 10 " II Barcarena 11 Belem Bélem-Brasilia Road (?) 12 Venezuela Puerto Ayacucho,Terr. Amazonas 13 Santar-em RIo Cripori(Afluente do Alto Tapa'jos).. 14 Gurupí Rio Gurupl (?) 15 Goias AruanA,Goias. r --5- TII METHODS AND STANDARDS Selection of patients - The subjects of the trial were psychiatric patients at the Santa Tereza Hospital. They were adults males of uriknown immunity to malaria. Some had probably had malaria in childhood; none gave a ~, history of recent infection. Immunity to malaria was hence likely to have 15-* been slight. The subjects were initially selected by the Medical Director of the Santa Tereza Hospital. From this list, a final selection was made by one of us after a clinical screening which included a routine physical examination, Machado-Guerrero and Wasserman tests, leucocyte counts and blood group and hematocrit determinations. Blood inoculation - All infections were induced by the intravenous r' inoculation of blood collected by one of us in the field or at the Center, or sent to us by workers in the field. For the early inoculations, the blood was usually chilled or frozen, the preservation of the strains at this stage having been an important consideration. Later inoculations were made direct from patient to patient. The methods were: (a) Chilled blood - From 3 to 9 ml blood, collected in a sterile vial v· with anticoagulant, was stored with ice in a vacuum flask. Kept in this manner, the blood did not remain infective for more than 12 days. (b) Frozen blood - Five ml serum bottles of resistant glass with tight rubber stopper were used dry or with 3.3 ml of glucose-citrate mixture added. One to 1.5 ml venous blood was quickly added and the whole "shell-frozen" within 60 seconds in an ethanol-dry-ice mixture. The vials were stored in contact with dry-ice. When required the O, -6- bloods were quickly melted in warm water and injected without delay With this procedure blood could be used after months of storageo (c) Direct inoculation - Blood from the donor was mixed with glucose- citrate at the proportion of 1.0:0.3 and passed to the recipient direct, or else placed in a vial and injected within 30 minutes. The amount of blood in the direct inoculum varied from 0.00001 to 90.0 ml. The high dilutions were made in physiologic saline with constant agitation. The estimated number of parasites injected varied from 3 to 5 x 10 A total of 131 candidates were inoculated on 169 occasions. Table 2 summarizes the data. TABLE 2 NUMBER AND TYPE OF INOCULA, WITH NLUMBEF: SUCCESSFUL Blood injected! Successfuli Unsuccessful! Total Chilled 64 i 21 85 --4-4 Frozen 1 15 i 26 Direct 56 2 58 1-ii Total 131 i 38 169 _, Blood examinations - All microscopical exaiuinations of the blood were -- o made from thick blood films stained by the method of Walker. During the acute phase of infection, films were taken three times a day; later -4 examinations were made daily for 30 days, twice a week from 31 to 60 days, .r then once a week. Parasite counts were based on differential parasite/ _.L leucocyte counts and on total leucocyte counts made twice a week.
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