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
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. When
,, ^~ 7 -
the infections were light, the count per mm' was arbitrarily assumed to
be the number of parasites per 100 microscopical fields multiplied by
five.
Procedure - After the inoculation of infected blood, a thick blood film
was taken daily until the first malaria parasite was demonstrated. Thick
blood films were then taken at 8 am, 4 pm and midnight and the temperature
was recorded four - hourly. As a general rule, leucocyte counts were made
once a week, oftener when there were special indications. Hematocrit
determinations were made daily during acute attacks and weekly thereafter,
more often in patients receiving pyrimethamine. The consultant in
internal medicine, Faculty of Medicine, was responsable for the care of
patients during intercurrent illness. The resources of the blood bank,
the laboratory, and X-ray department of the Hospital das Clínicas, were
freely available to, and used by, the Center.
Administration of drugs - For the first year of operation the drugs were
administered by the hospital staff. Thereafter, the treatments were given
by project personnel, usually under the direct supervision of one of us.
The dosage was usually based on a convenient number of tablets. Variations
in total dosage were deliberate, designed to furnish data on the relation
between dosage, cure rate, and possible resistance to the drugs. The
infections were treated at all stages, some when the levels of parasitemia
were high, others in the early stages of patency, a few before the first
appearance of parasites.
->-- -8-
Source of drugs - The drugs were supplied to the Center by the C.E.M.,
Sao Paulo, the P.A.H.O., Washington, or got by local purchase. The
sources were:
Chloroquine - Aralen (Winthrop) 150 mg tablets supplied by C.E.M., S. Paulo Pyrimethamine - Daraprim (Burroughs Wellcome) 25 mg tablets supplied by C.E.M., S. Paulo Sulphadiazine - (Parke Davis) 500 mg tablets supplied by Hosp. das Clinicas Rib. Preto ^-. Sulphadimethoxine - Madribbn (Roche) 500 mg tablets bought locally Sulphamethoxipyridazine - Kinex (Lederle) 500 mg tablets ~- supplied by PAHO, Washington Sulphorthomidine - Fanasil (Roche) 500 mg tablets supplied by Roche, Brazil Quinine - Old CEM stock of quinine sulphate tablets dispensed in capsules (400 and 500 mg) Mepacrine and Proguanil- C.E.M., Sao Paulo
Determination of chloroquine blood levela - Blood specimens for the de-
termination of serum or plasma levels were taken as a routine, usually 24
hours after the completion of treatment. For the determinations we are
indebted to the National Institutes of Health, Division of Parasitic
ahemotherapy , Bethesda, Md. From unforseen operational difficulties, some
specimens were unsuitable for examination. The results from 48 determi-
nations were available for analysis.
IV SENSITIVITY OF STRAINS TO DRUGS
A) CHLOROQUINE
The sensitivity to chloroquine of South American strains of
Plasmodium falciparum having become a problem of clinical and public health
importance, the main aim of the Center was to ascertain the clinical and
,a -9-
TABLE3 PARASITOLOGICAL AND CLINICAL RESPONSE TO TP=T.E?£r VITH CHLOROQUINE IN BLOOD INDUCED FALCIPAR7I, XIALARIA:SOUTH. ;.MRICAN STRIUNS.
3 Strain C"c Wt Att Chloroquino(1) Pateny Trophozoit¢s thousandn nm on day Fever Recurrence Cure(4) - day d C (3) ' r- KS .. oo/Is ol 9.treat. pig/L beagn 0 10 3 4 6 737'C 38'CIropa.C Fever
' RO¡DT-I 43 471 P1500/1 200 9 58.0 10-.0 11.0 2.0 0.2 0.1 2.2 1.9 2 I NC al 7 30>00/3 500 17 16.0 3.60.1 0 0 0 0 0 3 2 127 25 62 P19j50/ 14 13 14.0 17.0 o.1 o 0. - - - - 1 16 21 47 62 P2250/3 475 12 8.6 2.4
B.V.-I 2 65 P 2400/2 493 11 78.0 75.0 22.0 0.1 0 0 0 0 3 2 15 32 2 65 1l 3600/3 1230 5 19.0 48.0 0.7 .0.1 0 0 0 0 1 1 25 2 65P2[540C/3 970 4 25.0 4.0 0.1 0 0 0 0 01. 1 99 27 59 71500/p2 395 9 36.o 52.0 20.0 0.2 0.1 0.1.0.1 0.1 2 2 NC 10 13.04.00.1 0 0 0 02 1 30 33 7 60 p30/3 42211 102.0 4 7 6071o/2250/2 5 36.0 19.o 5.o o.01o.1 o 0 0 2 1 1e 39 13 75 P 4200/5 639 22 5.0 o0.1<0.10 0 0 0 0 2 1l 1i21 II 6 60P36001i/2 77810lo .0o14.0 0.1k0.1 o o 0 0°2 1 l04 Iin 17 6O P4950/3 1576 24 6.0 2.4o0.1 0 0 0 0 0 5 3 138 iv 3 65 24co/2 503 10 36.0 1.7 oiIÁ 38 60 P 2100/3 380 6 6.0 2.0 0.2 0 0 - 0 0 2 2 22 23 - -. 8 51 P1 Soo/3 30 14 83.0 42.0 3.0 0.1 O O O - 2 1 9 1- 8 51 R12250 15 16.o0 3.6 o.i O 0 0 0 10 0 0 21 29 49 P 2250/2 14 20.0 2.51i4.0o0.1.0. 1 0 0 0 3 2 20 23 0.2 0.1 0 0 0 3 2 121 41 48 7300/4 795 6 101.059.05.06 41 0 1 31 697 P2400/3 475 12 .0 .0 2.5 . 0 0 0 0°3 3 18 20 55722 -00/2 12 12.0 14.0 6.0 0.1 0 0 0 0 2 2 19 21 61g56 P2400/3 500 4 16.o 18.050 .í0.1 0 0 2 2 20 22 42412 R1I2203 15 2 <0.1 0.1.0.1 0 0 0 0 0 2 2 113 1 Go.ks 11 61 P24Co/4 4 7.0 g.o 2.0 . O.i 0..1 0 3 2 20 18 116 48 P24iC0/4 3 3.0 2.0 1.0 0.1 0 0 0 0 6 3 23 25 116 48 R1 2400/4 4 10-.0 3I0 1.010 0 0 0 0o . 1 g 125 55 P2400/4 2 .0.1.<0.1 0 0 0 0 0 0 2 2 24 25 121- 59 P 240/4 5 5.4 1.2 o0.10 0 0 0 0 4 2o 16 17 121 59 Pl 24c0/4 5 1.7 0.6 0.2 0 0 0 0 0 5 2 50h 50 7kR-I 50 56 P7240/2 270 9 70.0 22.0 7.0 0.1 0 0 0 0 2 2 22 24 61 60 P'2400/3 6 19.012.0 19o 0 03 O 0 0 0 3 334 34 11 48 67 P255/3 475 135.0 37.023.0 o.0'0.1 o0 0 0 3 2 17 20 67 557 2400/2 5 8.0 5.03.0 0 0 - 0 02 2 10 13 T, 60 587P2li00/3 8 3.020.0 2.30.1 0 0 0 0 3 2 36 Ece fowtnotea to t.ble next pcGe -v -3-0- s TABLE3 (continued) PARASITOLJGICAL AND CLINICAL,R2SPONSE TO TREAhT.MENTVIT! CHLOOQUII IN BLGCODIND;CED FALCPARiUM ?AIJsRIA: SOUTHA}'L ICAN STRAIiS. 4 h l q Cure( ) Strain C^e ?'tAtt C oro uinc(1) Patency Trophozoitet thousiids run3 on day Fever Recurrence dny days(2) (3) Er Kg ogQday3 Blood treat. I Fever ps/L began 0 1 2 3 4 5 6 737'C 38'C Troph. 5o P 240G/4 4 0.1 .0.1 1.2 0.1 0 0 0O 0 2 1 18 19 SAINT lO 9 127 62 P 2400/4 1 ,o.1 < o0.lo o 0 o 6 3 9 127 62 Rl 2400/4 1 ,0.1 0.5 O.1 O O 0 0.1 FP 0 7 FP 10 FP 127 62 R2 2700/J 2 II 26 58 P2250/2 570 13 30.0 17.0 0.2,0.1 0 0 - - 3 1 15 18 107 10 64 P 4350/5 356 9 .o.o .o0 0 o o0 o o 2 1 3 2 .91. 16 68 P 4500/3 1172 13 14.O 13.0 3.0[ 1.01 0 O O 23 20 58 P 5100/5 564 8 116.0 86.O081.o 6.0 0 IV 46 55 P2100/3 230 11 103.o01'7.013.-o .o.- 0 0 0 o 4 3 -125 ? YE? 66 56 P 1500/2 245 5 51.0 22.0 2.0 0. <0.1 0 0 0 3 3 8 6..0 2.0i0.1.0. 0 0 0 0 3 2 122 66 56R 212400/2 5 1 72 71 P 1500/2 330 13 22.0 2.01 4.0o,o.1 0.1 0 0 o 3 2 15 69 68 p 2400/2 6 5.0 22.01 2.01 0.l 0 0 o0 3 1 8 10 71 43 P 2400/2 8 0.3 6.0 6.0 ,.l1 0 0 0 0 3 1 23 26 "S 6 4 2L . 22 112. 61 P 2400/4 4 25.0 2.0 l0:1~t0.1O.-0.1 0 0 0 ?72 112 61 R112400/4 2 o0.1 0.1!0.1 o 0 0 0 0 '5 o 97 58 P 2400/4 1 .0.1 0O.1 0.1! 0l o 0 0 0 2 o0 1 22 85 103 52 R22400/4 2 0.1 .0.1.0.1 0 0 0 0 0 2 0 k P = prin;ary attack R1,R2,etc = first,econd recurrence,etc NC = blood not clered trophozoites F'P = fever persisted Day 0 = first day of treatnent Att = attack - = no blood examination (1) Total dosage vith nurber of days treatnent ia actual numbor · (2) Tne first figure gives the duration between daya 0 and 7 of fever exceeding 37 C: the ,econd the of days between days 0 and 7 on which a terperature exceeding 3S C vas recorded. (3) Days to recurrence counting from day 0 of last treatment. (4) Period in daya with trophozoites continously absent frcm blood films. 8 (c) received previous. (a) chloroquine repeated d.8 to 10 (b) received-pyrimethamine: 350 mg total d.6 to 19 d pyrimethamine: 300 ng total d.-12 to -10 (d) received quinine: 10800 ng total d.5 to 13 (e) blood not examined mg chloroquine veekly (f) received previous pyriLrethzLnine: 300 mg total d.-7 to -5 (g) also received suppressive 300 quir.ine 16500 mg X 5 fron d.10 (h) also received suppressive 300 mg chloroquine weekly X 5 from d.10 (i) received of quinine d. -13 to -5 total d.1 to 10 (J) received quinine 19550 :g total d. 4 to 12 (k) 4 suppressive doses - - ll - parasitological response to chloroquine treatment. The parasite strains tested were designated according to their preswned geographical origin viz - Rondonia I and II (Brazil), Para I and II (Brazil), Santarem (Brazil), Gurupi (Brazil), Boa Vista I, II, III and IV (Brazil), Goiania (Brazil), Goias (Brazil), Colombia I, II, III and IV and Venezuela. Variation in the stage at which the infections were treated may -S- have introduced an element of inconsistency in the response, a variable deliberately accepted to broaden the range of observation. Similarly varied was the dosage from a minimum of 1200 mg to a maximum of 5400 mg base. The extension to the upper limits of tolerance was made to determine how far the response to treatment might be improved by raising the dosage above the customary levels. The response is assessed from the initial impact of treatment on the asexual parasitemia and fever and from the incidence of recurrence. The effects of treatment having been defined, an attempt is made to interpret the findings in terms of sensitivity or resistance. Table 3 summarizes the essential data relating to chloroquine treated attacks, 65 primary and 25 recurrent, in 69 subjects. Figures 3 to 8 give a graphic presentation of the response to chloroquine in cases of special interest. Nine attacks treated with chloroquine are omitted, supplementary treatment with other drugs having confused the chloroquine respcnse. _y) The initial impact of chloroquine treatment on the asexual parasitemia In falciparum infections which are sensitive to chloroquine, the asexual parasites quickly disappear. The blood is cleared as a rule within - 12 - three days, seldom more than four days, from the start of treatment. Only with exceptionally heavy infections is the blood trophozoite-positive between the 4th and 7th days. Tables 4 and 5 show the rate of disappearance of asexual parasites in the infections studied. TABLE 4 CLEARANCE OF ASEXUAL PARASITES FROM BLOOD FILMS DURING CILOROQUINE TREATMENT IN RELATION TO PARASITE STRAIN Patients with trophozoites in blood films on day - * 0 1 2 3 4 5 6 7 Rondonia I 18 18 18 16 6 5 2 2 2 Boa Vista 13 13 12 12 9 2 1 1 1 Goiania 9 9 9 9 6 3 0 0 O Para I - II 5 5 5 5 4 i o o o Goias 6 6 6 5 2 1 O 1 0 Santarem 8 8 8 8 4 1 1 1 O Belem 2 2 2 2 2 2 2 2 2 Gurupi 2 2 2 2 2 2 O 0 O Colombia I 2 2 2 2 2 1 1 1 1 II 5 5 5 4 4 O 1 O O III 9 9 9 8 4 2 1 0 1 Venezuela 9 9 9 9 6 3 O 0 0 TOTAL 89* 89 87 82 51 23 9 8 7 - -1 Day O - first day of treatment i 1 case, Col-IV excluded TABLE 5 CLEARANCE OF ASEXUAL PARASITES FROM BLOOD FILMS DURING CHLOROQUINE TREATMENTS: PRIMARY AND RECURRENT ATTACKS Patients with trophozoites in Attack Cases blood films on day 0 1 2 3 4 5 6 7 Primary 65 65 65 62 47 24 11 9 7 Recurrent 25 25 24 21 8 1 0 O 1 - 13 - The number of cases observed in each series was usually too small for statistical analysis. Even so, conclusions which seem to be justified are: 1. The rate of clearance of the blood in most cases was within the range usually accepted as normal. 2. In about 10 per cent of cases the clearance was slow 3. Marked delay of the clearance of the blood occurred only in primary attacks. 4. There are no striking differences between strains, with the possible exception of the Belem strain (each of two infections slow to respond). 5. Infections induced by the same strain displayed a wide range of parasitological response. 6. Clearance of the blood in recurrent infections was within the normally acceptable range of variation. Recurrence of infection The view is widely held that chloroquine sensitive falciparum infections seldom recur after chloroquine treatment, and that recurrence after adequate doses indicates that the organisms are resistant. Tables 6 - 10 summarize data on the recurrence of infection in the 90 falciparum attacks treated with chloroquine. .·r _^~ - 14 - TABLE 6 RECURRENCE IN CHLOROQUINE - TREATED FALCIPARUM MALARIA IN 4, RELATION TO THE STRAIN OF PARASITE Strain Attacks treated Recurrent Recurrence per cent 7 Rondonia I 18 10 1 55 Boa Vista 13 6 46 Goiania 9 7 78 Goias 5 5 100 Para I - II 5 5 100 Santarem 8 8 100 Gurupl 2 2 Colombia I 2 2 Colombia II 5 2 40 Colombia III 9 9 100 Venezuela 9 6 66 With few exceptions, these infections were treated at a chloroquine dosage exceeding the standard of 1500 mg base given in three days. No -C1 strain was fully sensitive to chloroquine by the generally accepted criteria of sensitivity. Recurrence in relation to the. átge of treated infection The infections were treated at all stages, some in the early days of patency, some later or late in the course of primary or recurrent attacks. Tables 7 and 8 relate the stage at which treatment was given to the incidence of recurrence in the infections studied. TABLE 7 RECURRENCE IN PRIMARY FALCIPARUM ATTACKS IN RELATION TO THE DAY OF PATENCY WHEN TREATMENT BEGAN A-. Cases Days of patency u. 1 - 5 6 - 10 11 - 15 16 - 20 21 or more Treated * 20 20 14 2 4 Recurred 20 1 14 I 12 1 I 2 * 5 cases not cleared trophozoites are not included ; -L h .. - 15 - -r * _ 9 Few infections were cured by chloroquine treatment given during primary attacks, none when the treatment began before the sixth day of 'Y _ y patency. The significance of this behaviour lies in the bearing it may have on the assessment of base-line sensitivity to chloroquine, and in the -- ík possible distortions it may introduce when assessments are based on very - w early treatment. We may further recall that the high recurrence rates after chloroquine treatment sometimes recorded (e.g. American servicemen in Vietnam) were related to treatment begun very early in primary attacks. -r- The cure rate, as would be expected, was higher in recurrent than primary >-*i attacks (Table 8). -1 TABLE 8 RADICAL CURE IN CHLOROQUINE-TREATED PRIMARY AND RECURRENT ATTACKS w_, -- Y Primary attacks First recurrence Second or later recurrence -4 Treated Radical cure Treated Radical cure Treated Radical cure 58 9 (15%) 20 8 (409) 5 3 (6o%) _1 The difference in the rate of cure between primary and recurrent attacks has more than clinical significance*. It may influence assessment -r'J of sensitivity or resistance to chloroquine in long-term field trials. The .- 1 cure rate - and hence the incidence of infections presumed to be fully sensitive - may be related to the intensity of transmission, low when there .,, are frequent fresh infections, higher when transmission is at low ebb and the infectiona treated are mostly recurrent. -0 - 16 - -4l 4 Recurrence in relation to dosage The various regimens of chloroquine treatment present limited but suggestive data on the relationship between dosage and the rate of recurrence. Table 9 summarizes the evidence. The total dosage was given over periods -*- varying from six hours to six days, usually for three days. TABLE 9 RECURRENCE IN RELATION TO CHLOROQUINE DOSAGE IN FALCIPARUM MALARIA Chloroquine total dosagedosane Primary attacks Recurrent attacks mg base NO treated i Radical cures NO treated i Radical cures 1500 - 1950 8 o 2 0 2100 - 2700 41 i 2 11 4 30 00 6 3 3 2 42 -5400 9 4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~5 5 The number of subjects in three of the four series was small but with all due regard for random variation there can be little doubt that the prospects of permanent cure were better at the higher levels of dosage. To what extent and in what circumstances this advantage may be set against a lowered margin of safety is not clear. The limits of tolerance to chloroquine are not well known and until they are the possibilities of over- coming a mild resistance by an increase of dosage cannot be exploited with c-4 confidence. Moreover, with the highest dosage given there was still no assurance of a final cure. Recurrence and the rate of blood clearance Although recurrences of infections were recorded regardless of the rate at which trophozoites were cleared from the blood by treatment, they were more frequent when the blood clearance was delayed. (Table 10) -TW - 17 - -Y e TABLE 10 RECURRENCE IN RELATION TO THE RATE OF CLEARANCE OF TROPHOZOITES FROM BLOOD FILMS DURING CHLOROQUINE TREATMENT Days of first trophozoite-negative Cases Recurrence Cure blood films * -* 1 -3 35 22 13 -YY e, 4 - 5 43 36 7 6-7 4 4 0 -Ji L. . These observations do not support the assumption that a clearance of trophozoites from the blood by day 3 of chloroquine treatment is necessarily an indication of full sensitivity to the drug. Among the 35 infections with this early clearance, 22 recurred. -,w Early and late recurrence The interval between chloroquine treatment and the recurrence of asexual parasitemia fell with fair consistency between days 8 and 25, day O being the first day of treatment. Among 63 recurrences recorded, five only occurred later than day 30, and of the five, one had been delayed by -7 suppressive doses of chloroquine and one was uncertain since the patient was not examined for 19 days. (Fig. 2) An inference to be drawn from these observations is that with few ,-v exceptions the infections which do not recur within 30 days are likely to be cured, a finding with a practical bearing on the duration of long-term field trials for resistance. The remission of fever - z, The influence of drug treatment in malaria infections may be judged not only from the impact on the parasitemia but also from the relief of ---- - o 5 io 20 25 30 3o -lo Fig. 2 THE INTER-VAL BEST!I;EEN CLHLOROQUINi. TREATMEiTT AND PECURRF1ENT ASEXUAL PAPJRSITAEMIA Day O = first day of treatment as*rOnly two recurrences recordel after 40 days -- one _,. patient had received suppressive chloroquine X 5 weeks - a second vas not examined for 19 days. - 19 - symptoms -o the malaria patient, and often to his physician, the symptomatic relief is a reassuring indication that the drugs are active; and of all symptoms the most easily measured is the fever. The temperature of the patients at the Center was recorded four-hourly throughout the acute stage -- 4 of infection . Table 11 summarizes the data of (a) the duration of fever from the start of treatment until the temperature fell to and remained at or below 37QC, (b) the actual number of days with fever exceeding 38QC. -0. The two standards are given for comparison with records reported elsewhere. TABLE 11 DURATION OF FEVER DURING CHLOROQUINE THERAPY FOR ATTACKS OF FALCIPARUM MALARIA 'Days of fever after start of treatment Standard of Observation ¡Afebrile 1 2 3 4 5 6 7 - I> 1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Temperature exceeding 37QC 6 10 21 27 7 7 4 8 patients Temperature exceeding 38QC ! 16 28 31 10 4 0 0 1 " From this analysis it appears that: 1. Two thirds of the patients (58/84) were relieved of fever within four days and about a third (31/84) within three days. 2. A quarter (19/84) had a low grade fever beyond day 4 to day 7. --- 3- One patient only had a recorded temperature exceeding 38QC beyond day 4. The assessment of resistance Resistance to drugs in malaria infections can be measured only when -- wi there is an acceptable base-line of sensitivity. There would probably be general agreement that falciparum infections are fully sensitive to ,'l- chloroquine when a three-day course of 1500 mg base clears asexual parasites -- a c-1- -20- from the blood by day 5, with no return of the parasitemia, day O being the first day of treatment. Fever in sensitive infections rarely persists beyond five days. Both generalizations assume that the drug is absorbed and both exclude overwhelming infections. From this base-line, resistance to chloroquine may range from a mild loss of sensitivity manifest only by a recurrence of infection to a loss so marked that the drug can no longer be used with safety in severe infections. The bare statement that parasites are resistant is hence an over-simplification, even misleading. The grades and criteria of resistance adopted in this report are: Sensitive - Clearance of asexual parasites from the blood and remission of fever, usually within three and always within five days: infections permanently cured. Resistant I - Immediate impact on fever and asexual parasitemia within normal limits: infections recurrent. Resistant II - Asexual parasites persist in the blood for 5 days or more with or without later clearance, but numbers markedly reduced: remission of fever normal or delayed: infection recurrent. Resistant III - Little or no effect on parasites or fever. The infections studied at the Center usually recurred after chloroquine treatment and asexual parasites were sometimes slow to disappear from the blood. From this response we may surmise that the parasites were less sensitive to chloroquine than they were thought to be in past years° Table 12 analyses for each parasite strain the incidence and degree of f resistance by the criteria defined above. - 21 - TABLE 12. THE INCIDENCE AND DEGREE OF RESISTANCE TO CHLOROQUINE IN BLOOD- INDUCED FALCIPARUM MALARIA: SOUTH AMERICAN STRAINS* Parasite Infections 1 Infections Infections resistant strain treated fully sensitive I II III Rondonia 18 6 10 2 0 Boa Vista I 7 2 4 1 0 Boa Vista II 1 1 0 0 0 Boa Vista III 1 1 O 0 O Boa Vista IV 4 2 2 0 0 Goiania 9 2 7 0 0 -MI Goiás 5 0 5 0 0 Pará I 2 0 2 0 0 Pará II 3 O 3 0 0 Santarem 8 0 7 1 0 Colombia I 2 0 1 1 0 II 5 2 3 0 0 III 9 O '8 1 0 IV 1 1 0 0 0 Venezuela 9 3 6 0 0 Gurupi 2 O 2 0 0 Belém 2 0 0 2 0 TOTAL 88 20 60 8 0 --t *Two infections are excluded from the table: one had supplementary chloroquine suppression: a second displayed a response which was exceptional in that there was a permanent cure after a slow initial clearance of the blood. From the analysis presented in Table 3, the de- ductions we are disposed to draw are: lo By the criteria defined, a full sensitivity to doses of 1500 mg chloroquine base or more could be demonstrated in less than one quarter of the infections treated° -r^ - 22 - 2. The common o lder of resistance, oceurring in 69 per cent of the infections, as the mild resistance characterized by recurrence -L-4 after initia3 response within the normal range. was resistance -r· 3. In 11.3 per ent of the infections, there -4, of a higher rder, demonstrable by the presence of trophozoites in blood films cn day 5 or later. 4. There was no clear correlation between parasite strain and resistance to chloroquin e: from the same strain infections were induced which o,- were sensitive, mildly resistant, or even markedly resistant. 5. Differences t etween strains were probably within the limits of random variation. A consistent recurrence in the infections induced --T by the Goias, Par&, Santarem and Colombia III strains may have been related to treatment within the early days of patency. How the response to chloroquine in this series compares with that of South Americar falciparum infections in past years is not clear since no detailed data have been traced. Experience in clinical and field practice leaves ro doubt that the immediate clinical response was good but the impact on th parasitemia was ill-defined. Elsewhere in the tropics -,I. there is a simil1 r dearth of precise information except perhaps in Vietnam and Malaya where the immediate response had been assessed from daily blood examinations. TDble 13 presents comparisons which may perhaps put into perspective the esponse now reported. .1_ -;j, e - 23 - A., ·r TABLE 13 PARAS 1OLOGICAL RESPONSE TO CHLOROQUINE TREATMENT IN FALCIPARUM MALARIL: AMERICAN AND SOUTH-EAST ASIAN PARASITE STRAINS 1948-65 .14- Origin of Year Dosage Cases Patients with trophozoites SRecurrence strains on day erca(6 96 6 6 62 3 4 5 6 65 65 65 62 39 18 9 8 188 per cent r_ S.America(1) 1963-5 1500-2700 -- 3000-4200 13 13 13 13 11 4 0 0 53 " 4300-5400 ! 12 12 11 10 5 3 2 1 33 " " Vietnam(2) .1948 2100 120 120 120 91 29 5 1 0 Unknown Malaya(3) '1951-9 1500 60 60 58 39 12 1 O 0 S.Carolina( 1956 1500 46 46 46 36 18 9 2 0 2 per cent Panam&(4) -4 Day O - first da y of treatment (1) Induced inf ections in non-immune or partially immune subjects (present report) (2) Partially-i nmune hospital patients (Canet, 1948)* (3) Partially-i nmune hospital patients (Records Inst. Med. Res., Malaya, 195 L-9) (4) Induced pri nary infections in non-immune subjects (Jeffery et al., 195 D)** Figures 3 to 8, presenting graphically the course of selected infections 1-3· throughout the rhole period of observation, include data on chloroquine- -4 ---T treated attacks, The parasitemia is graded into ten arbitrary classes, 3 from class 1 wil ;h less than 100 parasites per mm to class 10 with 25,000 J-íw >_o or more. i~~~~~~~~-- * Canet J., 1i48 Soc. Path. Exot., Seance du 7 Juillet 1948 ** Jeffery G.M , Young M.D., Eyles D.E., (1956) - Amer. J. Hyg., 64:1-11 11 -0 - 21. - Case Nir 43 CCC 00oo 000 48 Kg; 000 i4 4 ,4 . lo c.-1 o r 4cs E 5 -30 d Pi i7 days Fig.3 WITSH 1500 íIG ?CQUINE .i.. QUIININE): CURTE WITH 3000 NiG iC1CqTiEJE. Case Nr 20 57 Kg CCCCc QCc 000 PYR 0.00 t"<;00_-D O 0o Ocuo ooOPNín ,D '- ' >cO atn0 4L' 1 4 ;¿} 4f 40 io 39 y n-' -o -, c4 -,I C) 37.c:E-( 3í 52 55 .172 days Fig, 4 ;URREiICE AFv.T;E, HTGI DOSAGE CLO.,RCQUII TI.2'E."iT: 'E AF'ER SSCOC'1D :',/CUR7U::CETIEJTED WITHi PY.Rii'vl'"I". -25- CASE l'ir 32 57 kg Pyí: cCC eZc CB oo 00 000 C-O _ _ (a C«) 0. eq 0. 0cQ a. (Os 4 4' k 000 CC:00 000 LCs! (n Loo oO o o O 006' OC 00 0000. f .zo lo Pi %- ,t 163 ti 417 72 85 90 97 days AFTER PYRIMiETH.IIi AilID CHLO:,OOQU',INE TRTPAT.iiNT: SPON`TAi'NEOUS F.ig. 5 IREC:UPRF2.:CE ANMD CLINICA., REMlITSSION: 10 RECURREICE AFTEJ? TRi:ITMENIT WITH QUININIE !YRIF!ETHAIHlIE, CASE Nr 127 CCCc CCCC c CCCC 64 Kg o000 cr 0000 o 0o 0000 oooo <> C, *rt .r1-E ii P4 v-- r 38 a, *c i r `' i A . .*k .. es 0 1,1 37¢C E- o sien-..-.T*.4~-K~2:--- `t - P~ 3 ....- Sff /I IO 17 23 29 45 days CURE AF-'TER PFig .ó6 RESE.D ER .R.'vPECNO. AlER, Ci:..O:.CUIU,, TR- J1,E,1.T: AFTER2 THiIPD RECU-:C: ¡i TREA'.TED WI.ITH QUINIINE. -26-- . CASE Nr 356 CC Qpyn 1 EP PR'o oo00 oc0o0 > 0C oOooo VOtoo ooo o OC)000 o0c5 Cti-c;i' rá; _ckn ( i i, , 4 4 k Z 4 z S$D 40 lo. c>o0oo oo o c cJ _ 54 5-4 :S or4 c0 i >l, . ; 30e \\ -. Uii.'L- \\ -' 4v 7. 204 dlays Fig 7 RECUPRE.CE ,AFTFER SUCCESSIVE TREATr.,E?'iTS WITH CHlI{ORO)QUIJNE, YRIlEiT-H.:MINE,, ;EPACINE AMI') IROGUA1i',L: CURE AYTE2RiHID ECURREiCE TREaTED WITii PYR!kIbETHAMiTINE 4 SULPHM.DIAZI!_E. -. 4 CASE Nr 69 68 Kg CC c. P,> . PRo oo 00 o000oo0c o oooooo _) _ 00000, %A 0> t'- t-->t-1 m,tl ,, Oe r-,t, .r4 , rr : 4 44 4d4 - 40 ib- -rl cS 4>t) -3S h ti n c- E-m 6 14 2. 36 L5 71 lt days Fig. 8 RPCUP CJ P:R" S UCC~ES^ TVif;iF Y.m WITH CiILOR0C B) PYRIMETHAMINE The terms of reference of the Center included the testing the sensitivity of South American strains of P. falciparum to pyrimethamine. The infections induced by various falciparum strains were treated with doses of pyrimethamine varying from 200 to 400 mg given in 2 to 4 days. Forty six attacks, 20 primary and 26 recurrent, were studied in 43 subjects° The response to treatment is presented as for chloroquine under three headings - the immediate impact in the parasitemia, the rate of permanent cure, and the remission of fever. Table 14 (next page) summarizes the essential data. The initial impact of pyrimethamine treatment on the asexual parasitemia The parasitemia in the first day of treatment ranged from less than 100 to 120,000 per mm3, and the day of patency from 3 to 24. Table 15 analyses the response in relation to the strain of parasites. TABLE 15 THE CLEARANCE OF ASEXUAL PARASITES IN BLOOD FILMS DURING PYRI- METHAMINE TREATMENT IN RELATION TO PARASITE STRAIN Strain |Cases j Pyrimethamine! Patients with trophozoites in ' i mg/days | blood films on day 0 1 2 3 4 5 6 7 -4 Goiania j 6 300 /3 6 6 6 6 5 5 5 5 * Rondonia I 10 300-400/3 -4 10 10 9 6 5 3 3 2 * Para I-II 4 300 /2-3 4 4 4 3 4 3 2 1 * Venezuela 3 200 /4 1 3 3 3 3 2 1 1 0 .- Santarem 1 1200 /4 1 1 1 1 1 1 1 1* Colombia III 5 200-300/3-4 5 5 5 5 2 1 1 0 I 4 200-300/2-3 4 4 3 0 0 0 0 0 II '5 200-300/2-3 5 5 3 1 0 0 0 0 Belem 6 200 /4 6 6 4 0 0 0 Oo Boa Vista 2 200-300/3 2 2 0 0 0 0 0 0 -_X. * Day of final clearance determined by other treatment. -28- TABLE 14 PAPASITOMIJICAI ANDCLINICAL RFSPO:NS' 10 TRFAT!IFNiT WITiI I'RIt'fiiAMINE IN BLCSD INDUCiD. FALCIPARUqi !'AL.PIA: SOUi1I J`iERICAN STRl.IlNS. Strn Cre V't tltteck PYR (1) Patencj l T oihozoite thsns r on da- Fcver crrene) Curc(4) dAY dtso(2) _ _, _-1--1--- T-- .. 0 1 2 3 4 5 61 7 37 38'C T1ro', ______~ ·· ~ - · · o · · ._- I F I _ ROND-I 25 62 30°/3 3.0 3.0 <0.1 0 0 35 35 36 50 350/3 29.0 12.0 <0.1 0.1 0.1 11 19 77 3co/3 10.0 6.0 17.0 3.0 !0.1 9 22 19 77 <0.1 0.1 O 0 O 14} 25 39 191 400/3 29.0 0.2 0.2 0 o0.1 13 15 18 64 200/2 16.0 30.o <0.1 0.1 0 19 25 18 64 300/3 8.o 1.4 BEL 78 51 200/4 9.0 141.0 0.1 O O 31 76 55 200/4 0.3 3.0 <0.1 0 0 119 84 42 200/4 15.0 32.0 <0.1 0 0 (12) 120 85 56 200/4 27.0 40.0 <0.1 0 0 oi ol 120 62 50 200/4 2.0 0.3 0 0 0 121 h 92 59 200oo/1 8,0 1.0 0 0o 0 o o o (11)(ii) 120 COL-I 21 69 200/2 37.0 9.0 '0.1 0 0 113 22 l,2 250/3 10.0 7.0 0 0 0 129 23 69 200/3 32.0 80.0 1.0 0 0 117 37 55 30D/3 24.o0o10.0o <0.1 O O0 120 0!o lo COL-II 2G 56 200/2 10.0 <0.1 0 0 O 0 95 30 70 2C)/3 29.01 1.5 <0.1 0 0 105 34 55 300/3 34.0 CO-III 35 49 300/3 19.0 <0.l!0.l 1 0.1 0 23 24 53 52 200o/4 2.0 2.0 0.210.1 <0.1 01 0.1 0 14 18 ssj Iol 0 49 55/ 200/4 3.0f 5.0 '0.1 <0.1 0 27 31 57 52 200/4 21,.0 0.7 4.0 .o.8 0.1 25 68 57 '0.1O 00 18 18 PAR-1 56. 3C0/3 61.0ol.o 5.0 3.0 0.1 8 12 61 68! 3C0/32CO/4 5 oo.00.' 0 . 0o<0.1 olo o 38 28 PAR-II 48 67i 2wc/4 36.0 170.o 4.0 j3.0 0.1 15 18 67 55 3Co/2 Nc '0.10 o .1.01 O0 VYN 72 71 1.0 20<0 1 <0.1 0 15 19 9.0 0.O3 0.3 1 O.1 69 6.3 22.0 3.0 0.3 0.1O, <0.10 8 9 71 431 15 SAhT 98 50i 2CO/!4 5.0 6. 2.0 0.2 L0.111.0 0.63.0 3 NC 7 - 1 ------I------ ------~------P = prim--r atea^: R1,R2,etc = irst,s The differences in response are striking. At one end of the sensitivity - resistance spectrum are the Belem, Boa Vista, and Colombia I strain infections which were commonly trophozoite-free by day 3; at the - _e, other were the Goiania strain infections in iwhich trophozoites persisted in 5 out of 6 until day 7 and beyond. From the over-riding influence of parasite strain on the apeed and completeness of the parasite response, the differences attributable to dosage, to the day of patency when treatment began, and to the initial weight of infection, could not be evaluated. _,, _st> Recurrence of infection The differences in the initial impact of treatment on the parasitemia were reflected in the rate of recurrence. Here again the over-riding _·- importance of parasite strain was evident (Table 16). TABLE 16 RECURRENCE IN PYRIMETHAMINE-TREATED FALCIPARUM MALARIA IN RELATION TO PARASITE STRAIN Strain I Attacks Trophozoites Recurrence 1 Recurrence treated not cleared , per cent by day 7* Goiania 6 1 3 3 100 Rondonia 10 2 8 100 Para I-II 4 1 i 3 100 -t Venezuela 3 0 1 3 100 -sr · Colombia III 5 0 5 100 Santarem i 1 1 Boa Vista 2 1- Belem 6 0 1 17 Colombia I 5 0 0O1 Colombia II 1 5 1 O0 0 * Subsequently treated by other drugs "D J, - 30 - The recurrence of infection appeared to be related to the rate of clearance of the blood after the start of treatment (Table 17). TABLE 17 RECURRENCE IN RELATION TO THE RATE OF CLEARANCE OF THE BLOOD IN PYRIMETHAMINE-TREATED FALCIPARUM MALARIA Days to blood clearance Attacks Recurrence Recurrence from the start of treated rate per treatment cent 1.- 3 18 4 22 4 - 5 12 11 92 6 - 9 8 8 100 * Not cleared 7 Other drug given - _) Remission of fever -"á Fever in the pyrimethamine-treated patients tended to be rather prolonged and to bear no close relation to the sensitivity to pyrimethamine of the parasite strain. Table 18 summarizes the data. Delayed episodes -Y. of fever, occurring when no parasites could be found and presumed to be a toxic effect of the drug, is considered in a later section of the report° (V) . , TABLE 18 THE DURATION OF FEVER IN RELATION TO PARASITE STRAIN AND TO PERMANENT CURE IN PYRIMETHAMINE-TREATED FALCIPARUM ATTACKS Sensitivity Attacks Permanent: Days of fever after start of of strain treated cure treatment ~i '.~o1 2 3 4 5 6 7 t - í Sensitive (1) 9 9 1 0 2 O 4 2 O 0 patients| Mildly resistant (2) 8 6 ! O 3 3 0 0 O 1 patients Markedly resistant (3) 29 1 !1 4 5 7 7 3 1 1 (1) Colombia I - II (2) Belem, Boa Vista (3) Rondonia I, Colombia III, Para I - II, Venezuela, Santarem -0 4-- 31 - A paradoxical feature of this response is that fever persisted for 4 days or more in 6 out of 9 of patients whose infections by parasitological criteria were highly sensitive, and that the fever in 17 out of 29 resistant infections was brought under control in 4 days or less. The duration of the fever after the start of pyrimethamine treatment may not, it seems, be a valid criterion of sensitivity or resistance to the drug. _~, The deductions to be drawn from this evidence are: 1. The sensitivity to pyrimethamine in the infections studied was sharply related to the strain of the parasites: three strains were highly sensitive, one mildly resistant and six moderately or markedly re- ' sistant to the drug. 2. The influence on the therapeutic response of dosage and stage of infection was masked by the influence of differences related to -1 parasite strain and could not be determined. 3. A close relation was evident between the rate of permanent cure and the rate of blood clearance after the start of treatment. -6> 4. No correlation was evident between the duration of fever after the start of treatment and the sensitivity or resistance of the parasites. C) PYRIMETHAMINE - SULPHONAMIDE The response to treatment with various combinations of pyrimethamine and sulphonamides was studied in 44 falciparum attacks, 23 primary and 21 recurrent, in 43 subjects. The sulpha drugs used were sulphadiazine, sulphadiniethoxine (Madribon, Roche), sulphamethoxipyridazine (Kinex, Lederle), and sulphorthomidine (Fanasil, Roche). 1- -32 - .-1 - l Ihe infections were induced by the parasite strains which have been q., named Colombia III, Venezuela, Goiania, Para II, Santarem and Belem. Table 19 (next page) summarizes the data on the parasitological and .- clinical response. d,. iF The initial im»act on the asexual -arasitemiq _`- ---- A _ ** _ W _ _ P .t,t The parasite count wihen treatment began varied from less than 100 _c to 69,O00 per mm3 blood. Tables 20 - 22 indicate the rate of clearance in -u relation to the strain of parasite, to the dosage of pyrimethamine, and to the type of sulphonamide. TABLE 20 THE CLEARANCE OF ASEXUAL PARASITES IN BLOOD FILMS DURING COMBINED PYRIMETHAMINE-SULPHONAMIDE TREATMENT IN RELATION TO THE STRAIN OF PARASITE Strain Cases Pyrimethamine' Sulphonamide Patients with asexual mg/days* mg/days* parasites in blood films on day 0 1 2 3 4 5 6 7 Colombia III, 18 50-300/1-4 1500-21000/2-8 18 16 6 2 1 0 1 0 Venezuela 13 50-200/1-4 2500- 3000/4-5 13 13 8 1 0 1 0 0 Goiania 2 300/3 !3000 /5 2 2 1 0 0 0 0 0 Para II 1 300/3 13000 /5 1 0 0 0 0 0 0 0 Rondonia I 3 300-350/3 L3000-15000/5 3 3 0 0 0 0 0 0 Santarem 5 50/1 ,3000 /5 5 4 2 0 0 0 0 0 Belem 2 1 200/4 2500 /4 2 1 1 0 0 0 0 0 TOTAL 144 44 39 i8 3 1 1 1 0 i i..4 * Total dosage: treatment with both drugs began on day O _;.I, The outstanding feature of the response was the rapid clearance of the blood. Three patients only had asexual parasites in blood films later -4 -A-Wo>> -33-- TBLE 19 PRtS!T0OiWICAL M;D CLINICAL RESPONSE TO VREATiE!iT%'I'iCO:I0?iZ PYRIM 3 4 Oooe¡ Dxv< doas' ae(1) Patcnc ropho¡oitc thoa idC m on0dai ' Yevr Recurrence(3) Cure( ) Strdr, wt Attac - - - dy dnyc.c (2) ?YR Is00 A treit.1V7 2 3 [5 6 7 37'C 3C Troh.',evcr _ og/day 1 v.,/dys 1 b.-.n 0o coD-II~~~~~~~~~~~~~~~ 03 050 2 0]Ž .... CO-11i 53 52 , 150/37s4 25001o 10 3 .4 6.0.i o.i 0 o o- 2 0 -- 74 75 R1 20oo/4 Sm 25¢o/4 8 50.0P.3o. 1 0 0o 0 0 2 0 1o20 49 55 20/4 Sl 2500/1 5 2. 6. O 0O0 0 0 0 0 4 O120 57 52 R2 200/4 SM 25(o/4 ?9 7.0 0.2 0.1 0 0 0 0 0 0 0 117 70 66 Rl 200/4 SM 2500/4 5 0o.9 0.1 0 0 0 0 0 0 2 1 120 Ž24,1 p 200/14 Si 2500/4 3 2.2 0 0 0 0 0 0 0 33 (11) 119 83 53 P 2oo/4 SM 2500/4 3 2.7 o. o o o0 1 1 (11) 119 87 4'8 P 200/4 SM 2500/4 3 24.0 <0.1 0 0 0 0 0 3 2 (19) 120 io5 48 p 200oo/4 SM 25, /4 4 2.5 2.1 2.0 0 0o 0 0 0oo 4 4 (7) 117 4 1.2 . 0 0 0 0 0 0 4 4 69 117 551 P 50/1 s. 13000/5200 4 53 52 R3 200/4 SD C/ 7 0.2 1.1 0 0 0 0 0 0 3 0 119 68 57 R2 3y0/4 SD 21000/8 6 2.0 <0.1 0 0 0 0 0 0 6 2 (8) 119 59 61 rli 200/4 SD 80,0/4 7 10.05.0 0 0 0 0 0 0 2 0 119 56 56 Rl 200/4 SD 12000/4 9 150o -1. O O O 0 0 0 3 0 122 115 68 P 50/1 SP 300/5 5 3.0 0.6 0 0 0 0 0 0 5 4 (39)' 81 12349 P 50/1 S 300o/5 3 3.4 0.91 o o o o0o 44 69 120o 58 P 50/1 SP 3000/503 0o.11 00.1 0.1 .1 0 .1 0 2 1 N 8 126 45 P 50/1 SO 1500/2 5 1~~~~~~3.0241<01 0 0 0 0 0 20 62 V S 72 71 R2 200/4 SM 2500/4 6 2.3 0.21 0 000 0 0 2 1 113 71 43 P2 200/y SM 2500/4 3 0.2 0.91i0 00,0 0 0 0 0 120 103 52; Rl 50/1 Sm 3000/5 3 <0.1 0.220.1 '0.1 0 1 0 0 3 2 15 8 90 51 p 220/4 SM4 2500/41 5 11.0 GNIA 55 72 R2 300/3 SD 13,00/5 2 0.o0.1 0o o0 0o 0 0o 0 3 0 121 b 51 46 p 300co/3 13000o/S 7 3.0 0.3<0.1o. o o o o 1 121 PAR-ii 483¡67 2R2 300/3 SD 1SO 0/5 3 0.1 0 0 0 0 0 0 3 0 121 c ROi-I '47 62 R2 350/3 SD 150C/5 9 0.2 <0.1 0 0 O 0 2 0 121 3949. 3 300/3 SD 150,00/5 6o 27.0 o.11 o 0 o0 0o 0 - 1 1o0 121 36 50 R3 1300/3 SD 130CO/5o 2 0.2 0.1 o10 0 00 o o o o 11 sANT 114 53 Rl 50/1 Si 3000/51 2 [<0.1 0.2 ,O.1 0 010 0 0 0 4) 63 101 50 Rl 50/1 S? 3000/51 5 2.02 4.0 0 00 0 10 0 4 2 81 102 53 P 50/1 ¡SP COO./5 2 .0o.1 o o o0 o00o o 3 2 83 n8 5o P 50/1 's? 3000/5 6 322.O.1: 1'o.lo o ' o o o o0 1 5og62 50/1 '0D0/_5 3 ¡.l230o 0 0 0 0 0 0 5 4 90 BE· 7851 2112 / 250 o. 4 11241 0 0o 'o 2 117 63602 20/4 M 2500/4 15 O l,0. 1 0ol 120 d fever rrsisted Day O = first day of t ter.t ?Y;C=yrim/than:ine S = zsulphadiazioa SM = sulmhadi thoxioe (MoiribonPoche) SP = snu1p'-cethoxipyridi--io (Kinex,L-derle) SO = su1ciorth?-idine (2aarsi1,,oche) - rno blocd exw.ir.ation 1 (1) Tetal dosFqe vith r.o:. er of days treatment (2) Toe firat fiaire rthe d,- .'ira:en ¿s O a:n 7 or fe.' r e-: than day 3, and of the three, two had received only 50 mg pyrimethamineo All strains appe:ared to be about equally sensitive to the combined drugs, although most were known to be moderately or markedly resistant to the r w pyrimethamine alone (Table 14). TABLE 21 THE CLEARANCE OF ASEXUAL PARASITES FROM BLOOD FILMS DURING COMBINED PYRIMETHAMINE-SULPHONAMIDE TREATMENT IN RELATION TO THE DOSAGE OF PYRIMETHAMINE Pyrimethamine t Cases Patients with asexual parasites in iRecurrent or mg/days 1 i blood films on da blood not cleared 1 2 4 5 6 7 days 50G /1 16 16 13 8 2 1 2 1 0 patients I 2 150 / 3 1 1 1 1 1 0 O 0 0 " 1 200 / 4 1 20 20 18 8 0 0 0 0 O " O 300-350/3-4 1 7 7 6 1 o o o o o 0 j~~~~ Although many infections were cured when the total pyrimethamine dosage was 50 mg, only at a total dosage of 200 mg or more, was there consistant cure. TABLE 22 THE CLEARANCE OF ASEXUAL PARASITES FROM BLOOD FILMS DURING COMBINED PYRIMETHAMINE-SULPHONAMIDE TREATMENT IN RELATION TO THE TYPE OF SULPHONAMIDE Sulphonamide i Cases Patients with asexual parasites! Recurrent in blood films on day or blood 'i 234567 !not cleared 1 2 3 4 5 6 7 not rd Sulphadiazine 10 101o 9 1 0 0 0 0 0¡ O Sulphadimethoxine 21 j21 19 11 2 1 1 0 0 2* Sulphamethoxipyridazine i 12 12 10 4 1 1 O 1 0 1 ** Sulphorthomidine 1 1 1 0 0 0 * Dosage pyrimethamine 5Omg/1 and 150 mg/3 ** , " 5SOmg/1 - 35 - The response to the combined treatment was good even when the parasites were mildly or markedly resistant to each drug given alone. Twelve Colombia III strain attacks were treated in three series, the first with pyrimethamine, the second with sulphadiazine, and the third with both drugs. The Colombia III was known to display resistance alike to pyri- methamine and to sulphadiazine. When, however the drugs were given together in Colombia III infections, the parasitological response was prompt and the infections were cured. (Table 23) TABLE 23 RESPONSE TO TREATMENT WITH PYRIMETHAMINE, SULPHADIAZINE AND PYRIMETHAMINE-SULPHADIAZINE COMBINATION: COLOMBIA III STRAIN Case Attack: Pyrimethamine i Sulphadiazine 1Days to first Recurrence Cure rmg/days mg/days negative blood ! (1) (2) (total) (total) film 1 35 P 300/3 - 4 23 53 Rl 200/3 - 7 14 ,- 49 R1 200/4 - 427 57 R1 200/4 - 6 25 68 R1 300/3 -i 4 18 _ 65 R1 15 000/6 i 121 .,y 59 P 12 000/3 5 26 56 P i 12 000/5 4 1 29 ,· 53 R3 200/4 12 000/4 2 119 68 I R2 300/4 21 000/8 2 I 119 59 i Rl 200/4 1 8 000/4 2 1 119 _-3 56 R1 200/4 12 000/4 2 122 P - primary attack R - recurrence (17 days from first day of treatment (2) days negative blood films There is a suggestion .L- from these findings of a potentiation in the effect of the drugs° For firm conclusions, however, the series were too small. __1 2* - 36 - Remission of fever Essential though the parasitological response to treatment may be in the appraisal of sensitivity or resistance to drugs in malaria infections, it is from the relief of symptoms that the patient is likely to judge the M value of a remedy. Table 24 records the duration of fever in the 44 tI- patients treated with combined pyrimethamine - sulphonamide. TABLE 24 REMISSION OF FEVER DURING TREATMENT WITH COMBINED PYRIMETHAMINE - SULPHONAMIDE IN RELATION TO STRAIN OF PARASITE Strain I Patients Days of fever exceeding 37QC after start of treatment Afebrile 1 2 3 4 5 6 7 days Colombia III 18 1 1 6 4 4 1 1 O patients Venezuela 13 1 0 1 4 4 1 O 2 " Goiania 2 O 0 1 1 0 0 0 0 " Para II I 0 0 0 1 O 0 O 0 " Rondonia I 3 1 1 1 O O 0 0 0 " -'-r Santarem 5 1 0 0 2 1 1 O 0 " Belem 2 1 0 O 1 0 0 0 0 " 4 5 2 9 13 9 3 1 2 TOTAL ! 44 i 5 2 9 13 9 3 I 2 From the analysis it seems that: 1. Two-thirds of the patients (24/39) were relieved of fever by the fourth day, and about a quarter (11/39) within three days. 2. Differences related to strains were probably not significant. -y? -4-- This fever response may be compared with the response to chloroquine in falciparum infections which display a mild or moderate chloroquine resistance (Table 11). The differences are slight. - 37 - D) QUININE A few attacks were treated with quinine (15), proguanil (4), mepacrine (3) or sulphadiazine (3). Table 25 summarizes the essential data on the parasitological and clinical response. With quinine only had the findings any statistical significance. And even in the quinine series, the variations - dosage of drug, duration of treatment, day of patency when -.s, treatment began, and initial parasite counts - were so marked that no firm conclusions can be drawn. Briefly, the findings were: 1. Four out of fifteen quinine-treated infections recurred, three after doses usually believed to be adequate. 2. Two patients had a low-grade parasitemia on day 5. 3. Three patients had fever continuing to day 5, though at a low level. It would be logical to deduce - by the criteria adopted in this report for chloroquine - that three of the infections were resistant to quinine. Whether this kind of response should be accepted as within a normal range, geographically flexible, or as evidence of resistance, is a problem of semantic difficulty beyond the scope of this report. Fig. 9 illustrates the failure of a course of 12,400 mg. quinine sulphate given during a fourth recurrence to cure the infection. It shows also, the successive failures of other drugs and the final cure with proguanil when the defenses of the host were making a decisive contribution. -7 -9,J70C> TAIBLE 25[PA2.SI1'TOI3IC'l A D CLINiICAL R.SPOYNSE T 'RiEATI:EhTI'IqTH QUINIEP,ES:PACRINIE, PFRUANIL OR SLPiiADIAZIt; IN P:IIc-!i IUC D EAI.CIFA: }U.-LA: U.L7IA:Ahi:RIC.AN FPA!AS IE STRAI!iS __II_ - i~~~~~~~~ ------I~--1 7-----T------i ------i - 1 - - 3 Strvlu CaIc Vt .tt; Tri,,trment (1) Trophoczoitez t!oulcnds :: o0ndry · 2ccurrence 37.Cs(2) (3) ?ír Kj trat. - d. :S bo,.i 1 4 5 6 7 37 C 13,5-i or H c--eor °O/ 1 ·-- "-t--·---1------2 COLI 11 68 Pl Q 159 31 0.2 '0.1 o o o 85 1oc>n/8 o o 24 59 Q '0.1 1 0O<0.1 0 119 P1 .O III 131 pl q O0.]. '0.1 o o 40 120 58 R2 9 21coo0/12 2.0 '0.1 o o o 62 35 49 Q 23.0 <0.1 '0.1 o o 119 '01 o o 25 62 Q 219t3,I/.7 17.0 3.0 6.o · ol0O ool o 27a 28 19 77 R2 Q 0.1 ,'0.1 o <0.1 '0.1Oo I.0.1 41 52 33 Q 5.0 1.2.0 <0.1 o '0. o 123 1< Q '0.1 o <0.1 127 62 23 Q 2!5i 0/15 0.2 o O o Q <0.1 O '0.1o 35 10!1 1<2 <0.1 '0.1 o 53 Q 147[c/12 <0,1 5.0 O ool 84 132 cl 265 \D/15 o o 41 Rl ,'0.1 o.10· QQl o GOIA5 55 21 Q 2.0 11.0 0.2 O.)O o 35 121 P2 Q 255X /15 1.01 <0.1 '0.1 O o o 114 59 '1 I1< Q o PAP-1 ('1 15->2x/10 <0.1 <0.1 0o O 118 a;a GU0IA 150 43 178Dw/17 10.0 19.2 2.3 o o 35 0 <0.1 I 31 69 P2 p z) 3<222/10 1.4 2.5 0.1 0 0 o 95 , 2 *O4 o o 0 39 '9 22 1.0 o o0 O 19 23 36 50 P .O 3/o/ <0,1 2.0 o. o o 21 o °l o PlO 3ooD/8 lO.O o0 o o 69 16 r3 0. 103 O GiIA 51 P2 -127 2 3-/7 4.7 0.2 o0.1 '0.1 Oo o 3 b 259 62 Itz'22 l' 2,GO;/7i 13.0 3.0 3.0 2.1, '0.1 0 o 75 19 77 .i,2 32G3/10o 0 o 18 19 5.4 '0.)io.0,1 Ri COL-III 65 64 R1 SD 15-X7/6 6.0 0.2,0.1 o o o 121 59 61 P Sr) L3]2C0/5 25.0 0o.0 0,2 0.1 10.1 o 26 29 5i P SD J1200W/5 0.2 1 .2 o0.1 '0.1 0 o 29 30 J__---- L_ --- ----- iiii iiiL ! _i L _I,¡_^i i____- i P rr--pr:- yr tt; R1i,1 2,ctc ficrst-,scondu recurrenretc 1;C = blocId not cleared of tro phczoite, iP =.'fever p`ro.ited Dly 0 D fi'zt d2y of treatorn t Q quinine ulph1ate hM? = rmepacrine PRO -= rpgutnil - SD D cJFlpihadia1n.í (1) TotRl da CASE ]'). 3-9 52 XgÓ CCCC CC PYR PYR 00 tl O tO0 C' <) e , ultr_1 '0 ooO 00 C O CoO,>-)t:., l_ o0 .r o <>o c .o e t . 0 ·ri - `, 1 .Í- d ~~~~~~~~~~~~~~~~~~~~39O 4tU> -ri t,) * 14 L~- days 3~' CASE llr 19 c olnt FEP C 0° Q ecicie c eC. c- 0O0 , c,c,0 c> . 0 oo0 oo c) e, ) _eC>)O m k't-r L" .. C) c, ¢.4 r~ el C-t C)'JC> _·_ zJ¡ _ _ Iz lf n, r1, ,. íq ,,) loí 1. t¿. -...... 4 $;4 4 t tl. t .E .1 rJ 43 *.(1 i:tc! P 1 .C EE- f.h ._ 1 t ;/1 0 - 9\ 97 102 t2. .155 - '~1 days CASE Nlr 1.9 PRo con% t0 ciO, O -42o .QQ o .39 5 < *rl°r( tE 43 r ! - 3 C)p, c$, r . . \ E. i .;,l o i 357°c E-l 2,----2 153 lbs 170 2,?2 days }>ig9...... -U.,' .. 'r:_... SUCC:~.S7VE Tr.;?AT,;"'TS ¥,'ITTi CIi,,OOUUI.~, P's.XII,:~?-.'i;I.:E, U .;IH.- A;~AND.i, r:,P;'.1.r.: .C .: C!?SZ . E.f..'f?-R C-!RE Sl.Tin vvml.',C, ' .. i-"4'"=': T-,R:- '....,O WiTl PPC, GU:.4.I. u;,e .tcext - 40 - V TOXICITY OF DRUGS Chloroquine Most patients treated with chloroquine received a total dosage of 2100 - 2700 mg base in 2 - 4 days, the upper limit of the normal range; some received the more usual doses of 1500 - 2000; others were given a total exceeding 3000 mg and a few, doses exceeding 5000 mg. The high v-74 plasma levels of chloroquine left little doubt on the absorption of the drug. The lower doses were inefficient, hence the increase to the higher levels. The patients were observed daily by one of us, often twice a day. There were few complaints during the treatment and no significant toxic reactions were observed. PvYrimethamine The high cure rate from combined pyrimethamine-sulphonamide treatment in the infections induced by South American strains of P. falciparun was clearly demonstrated in the course of the work. The toxicity of the pyrimethamine was the limiting factor. The minimal dose from which permanent cures had been consistent was 50 mg daily for 4 days. At this dosage, episodes of fever unrelated to the parasitemia were recorded and the white cell counts in some patients were low. The fall in the counts usually began a few days after the administration of pyrimethamine, the *?r fever after irregular intervals. Treatment for the leucopenia and fever included blood transfusions, folic acid, ascorbic acid, and citrovorum factor. Antibiotics, usually tetracyclin, were given for prophylaxis. Recovery -e - 41 - 4 was complete in all cases, usually within 2 - 3 weeks. Figure 10 shows the course of the fever and the changes in the white cell counts in five illustrative cases. The incidence of significant leucopenia in patients receiving pyrimethamine was not closely defined. Routine leucocyte counts were made in the early cases once a week only; later they were made daily. The known incidence of leucopenia is open to this distortion. In 9C! _ patients receiving pyrimethamine or combined pyrimethamine-sulphonamide, " ~ ~36 had daily counts from 1 to 3 weeks after treatment; among the 36, counts below 4000 mm3 were recorded in 17, an incidence of nearly 50 per cent. Fever occurring between days 4 and 19 after the start of treatment, at times when parasites were not found in blood films, was recorded in 12 patients among 90 who have been treated with pyrimethamine alone or in combination with sulphonamide. Nine have received 200 mg in 4 days, one 300 mg in 4 days, and two, single doses of 50 mg. Ten of the 12 had also received sulphonamide. From the uneven data, the relative contributions *· ~ to the episodes of fever of the pyrimethamine and of the sulphonamide could not be determined. It was clear, however, that most of the patients developing fever had received the higher pyrimethamine dosage (10/12). '`i VI SUMMARY AND CONCLUSIONS Malaria infections were induced by blood inoculation in 131 subjects, adult male inmates of the Santa Tereza Psychiatric Hospital, Riteir~o Preto, Sao Paulo. The original blood donors had been infected with Plasmodinum -i . - 'iL.- 4o .o_{0 ---23Case- 8;'1 $4 Hn 4 I .[D.- rCase 92.* oo 10o 20 days _ ~4o NO A OA _s~cT '' P : W:TrL * - .o - Case 82 -h~:.- 5.-*~~~~~~~~2DrjAdpyintmc D r,- + sull ---- r.>~ - SUoLPilOM§.MI DE TlE'Xri^ s il E..S jLUS "R..TII~¡ o'-3 ¡0o days 20~ 3' direthox 2 r/Id Dy O firt a of treatrn -i dir:i·=t-1 - nasexual parait¿ea . -days of treatrrier.t - 43 - In the infections so induced, 231 attacks, 128 primary and 103 recurrent, were treated with various drugs, mainly with chloroquine, pyrimethamine, or combined pyrimethamine - sulphonamide. The response to treatment was assessed by the effect on the asexual parasitemia, by the duration of fever, and by the rate of permanent cure. The patients were observed under conditions which excluded all chance of outside infection. Chloroquine The response to chloroquine treatment was studied in 90 attacks, ,- >65 primary and 25 recurrent. The initial impact on the parasitemia in most subjects was within the normal range of variation but slow in a signrificant proportion. The rate of recurrence was high. The cure rate was related to the day of patency when treatment began, to the rate of clearance of the parasitemia during treatment, and to the dosage of drug. Criteria for the assessment of resistance are defined. The parasitological and clinical response is discussed in terms of resistance to the drug. The infections were derived from 18 donors from 12 areas. No significant differences related to strain were recognized in the sensitivity to chloroquine. Pyrimethamine Forty-six pyrimethamine - treated attacks, 20 primary and 26 recurrent, were studied. The differences in responses were sharply related ·~-*i to parasite strain. Two strains, Colombia I and II were highly sensitive: six strains Goiania, Rondonia, Para I and II, Venezuela, and Colombia III, , . 7. .. 5-_ - 44 - were markedly resistant. The donor of the Colombia III strain, the only Col. strain shown to be pyrimethamine-resistant, had received two courses of pyrimethamine, each of 200 mg, 8 and 16 days before the blood was taken. A close correlation was noted between the rate of permanent cure and the rate of blood clearance after the start of treatment. Combined nvrimethamine - sulphonamide Forty-four attacks, 23 primary and 21 recurrent, were treated with combined pyrimethamine - sulphonamide. The blood was usually free from asexual parasites on the third day of treatment and the cure rate was high. Recurrences were observed only in patients receiving a pyrimethamine dosage of 150 mg or less: all of 27 infections treated with 200 mg or more were cured. The fever response tended to be slow. Toxic manifestations, leucopenia and irregular fever were common at the higher pyrimethamine dosage, alike in patients treated with the combined pyrimethamine-sulphonafmlide and with pyrimethamine alone. All patients with toxic symptoms recovered after specific treatment, usually within 1 - 3 weeks. The high potential for radical cure from the combined pyrimethamine - suiphonamide was subject to this limitation. Twelve infections induced by a single parasite strain (Colombia III) were treated in three series, one with pyrimetUamine alone, one with sulphadiazine alone, one with the combined drugs. A potentiation of effect seemed probable. - 45 - Quinine Fifteen attacks were treated with quinine. The variations in the amount of drug, in the duration of treatment, and in the stage of infection, were marked. Three patients were not cured by doses usually adequate. No conclusions are drawn on the propriety of regarding these failures as evidence of resistance to the drug. bol Miscellanea In appendices to the report, observations are recorded on the duration of the pre-patent period in relation to the number of parasites in the inoculum, on the infectivity of blood inocula containing less than 10 parasites, and on the marked fluctuations in parasitemia sometimes observed in primary infections. VII ACKNOWLEDGEMENTS We would like to mention the active participation and generous assistance on every occasion given by; Minister of Health; Health Secretary, State of Sao Paulo; Directing Staff of the Psychophatic Dept., Sao Paulo; under the direction of Dr. Milton Penha, Directing Staff of Santa Tereza Hospital; chiefs and staff of Depts. of Preventive Medicine, Pathology, Hematology, Parasitology and Microbiology of the Faculty of Medicine of Ribeirao Preto; Superintendent of the Eradication Campaign; Director, Chiefs of Laboratory and Zone IV of the Malaria Eradication Campaign of the State of Sao Paulo. We wish to make special mention of the effective cooperation of: Dr. Odair de Oliveira Director of the Santa , - 46- Tereza Hospital, for his constant willingness to help with whatever difficulty arose and his patience with the many minor emergencies which inevitably developed. Drs. Amadeo Fracon, Felix Montebello and Manoel Garcia for selecting the majority of patients for malaria therapy. Prof. José Lima Pedreira de Freitas (t) who was the administrative head of the project, gave technical and material help. Prof. Jose de Almeida provided the refrigeration and lyophilization facilities. Prof. Ernaldo de Freitas Menezes supplied the post-mortem diagnosis for three cases who Sdied of diseases unrelated with malaria after observation was finished. Prof. Cassio Bottura whose interest in toxicity of pyrimethamine was most helpful. We thank Dr. Astolpho Siqueira who made all Wasserman and Machado-Guerrero serology for the patients. The staff of the blood bank and particularly Dona Maura Silva Faria de Souza who typed the blood groups and gave many facilities for transfusions were most helpful. We -S are indebted to Dr. Mario Ferreira de Oliveira, Superintendent and Dr. José Aluisio da Fonseca, Epidemiologist, of Federal Campaign for their great help in making possible this experiment; to Dr. Octavio Alves Ferreira, Chief Zone IV, S.E.M., Sao Paulo for his continuous day-to-day assistance and for his sympathy and encouragement; and to Dr. Decio Camargo * Rodrigues for his valiant assistance in the maintenance of regular supply of dry-ice. We are grateful to the microscopists of the project and particularly to Geraldo R. Brandao who was the back-bone of rapid and efficient diagnosis. $> - s~Ci~~~ - 47 - In 1966, while on a world-wide mission for WHO, Geneva, to evaluate ·j what is known on resistance to chloroquine, Dr. John W. Field, retired director of the Institute for Medical Research, Kuala Lumpur, Malaysia, spent two weeks in Washington examining data from Brazil, including the -4- last report from the Ribeirao Preto Center and the records from which it was compiled. At that time, though concerned only with chloroquine, he showed interest in other aspects of the work, carried out with somewhat limited facilities, and expressed his willingness to return and explore these further. The present report has been prepared with his collaboration. We :-·t are grateful to him for reducing to a semblance of order a mass of seemingly unrelatable data and feel that it reflects our own impressions and somewhat diffident conclusions. We are, above all, grateful to the 150 patients who permitted all the discomforts of finger and venipuncture and the taking of often prolonged medication. A.J. Walker F.J. López Antuñano ,3 Washington, D.C., 31 March 1967 · t b. O-~ Appendix r History of donors Most of the original donors of infected blood had received chloroquine during the preceeding months. Table 26 summarizes the evidence of previous chloroquine treatment. TABLE 26 HISTORY OF CHLOROQUINE ADMINISTRATION IN THE ORIGINAL DONORS OF INFECTED BLOOD Parasite Donor iAge Chloroquine Pyrime- Date blood strain thamine í taken Date mg/days mg/days Colombia I ACJ 27 5/2/63 1950/16 5/3 1900/16 22/3 450/1 1/4 3000/3 6/5 2800/6 19/6/63 II TR 21 4/4/63 3000/3 6/5 1600/3 11/5 1050/6 19/6/63 III RA 17 14/1/64 200/3 22/1 200/3 30/1/64 IV MM 19 20/5/63 600/1 28/5 2250/3 18/6 1050/2 8/8 3000/3 28/1/64 Rondonia I JA 45 10/1/63 1800/4 3/3/63 II LG 42 24/10/63 1050/2(Amo) 3/11/63 Goiania ISS 16 8/10/63 Boa Vista I JM 42 24/11/62 1500/3 6/12 1500/3 25/12 1500/3 27/1/63 1500/3 24/4/63 II RPM 27 21/3/63 1740/4 10/4 1200/2 6/5/63 III LRS 42 18/4/63 1500/3 29/4 300/1 3/5 1800/ 20/5/63 IV WRP 23 28/1/63 1500/3 * 16/2 1500/3 17/3 1500/3 20/3 1500/3 30/4/63 Para I EML 43 25/1/64 2400/3 3/2 4200/7 8/2/64 II FSL 13 2/1/64 1050/2 8/2/64 Ai Belem MRA 8/9/64 Santarem JIR 22 -/8/64 3000/6 ? -/9/64 3000/6 ? -/10/64 3000 ? -/11/64 3000 ? 23/12/64 Gurupi SFR 58 5/8/64 1500/3 -/12/64 1500/3 18/1/65 Goias MMLS 15 14/12/64 2400/4 7/1/65 2400/4 ? 20/1/65 Venezuela OSF 24 8/6/64 300 x 5 ** 23/6/64 * Irregular chloroquine 300 mg weekly January-April 1963 ** Regular chloroquine 300 mg x 5 weekly May-June 1964 _i Appendix B Sequence of Passage The sequence of passage in the parasite strains is shown in Figure 14. The numbers in the circles identify the cases. Conservation of strains in the frozen state was attempted from the beginning but difficulties prevented the establishment of a continous supply of dry-ice. Specimens which survived were deposited with the Laboratory of Parasite Chemotherapy, National Institutes of Health, Bethesda, Md., and at the Gorgas Memorial Laboratory, Panama. -. 4s App.ncir L.i, l-,b'-2 [ioW u!i .-J, -J1 -J i· 1---I~~~~~~~~~1 ''i._.-á,, .. 42 0.30 00C1-_! 1 oe [=X1- 1[- 111 115 1~~~~~~~~~3 21) 12i _ : LGl 1I:,U PI '-1 Fi,.1Zt S.U.A;CIE:., PA.SSA.GE III '" SOT'iu. ;-,:.::,IC ,RN. FfCI'P:-~.'j:UI ST tR'AINS ST'U'DI ED Appendix C Pre-patent periods in relation to the number of parasites in the inoculum Estimations of the number of parasites injected were made in all cases. When the blood was chilled or frozen, the proportion of viable parasites was unknown. When, however, the inoculations were direct from donor to recipient, the viability of the organisms was probable. Fifty- six infections were induced by direct inoculation. Four patients received drugs before patency of the infections. Fig. 15 shows the relationship in the remaining 52 patients between the estimated number of parasites in the inoculumn and the first day of patency in the infections. FIG. 15 PRE-PATENT PERIOD IN RELATION TO THE NUMBER OF PARASITES IN THE INOCULUM (52 subjects) Number of parasites i Pre-patent Period in days injected 1 2 3 4 5 6 7 8 9 10 11 12 1 More than 10 o 000 000 0 00 000 0 6 o8 o 000o ,·-.i~ 10 - 10 ! 0 ° ío 4 ~~~6j000 000 000 O 104 - 10 00 0 0 00 00 Less than 20 0 0 o IL (4) (3) (12) Figures in brackets are the estimated numbers of parasites injected. o - 1 patient Apart from the well-known relationship between the number of parasites injected and the duration of the pre-patent period, the interesting feature of this analysis is the infectivity of blood containing an estimated 3, 4 and 12 parasites only. Appendix D A note of microscopic diagnosis In the course of the work, our attention was drawn to the abrupt changes in the level of parasitemia which sometimes took place during the primary attack. Although not new, the observation emphasized the possi- bilities of diagnostic error even in severe infections. The routine practice of examining the blood at 8-hour intervals allowed the phenomenon to be more clearly defined. Case 18, 32 and 43 were studied with blood specimens taken at one or two hour intervals. (Fig. 11-13). They showed surprising alteration in the counts within a short period. The parasitemia of case 32, for example, increased from 130 mm3 at noon to 25,000 mm 3 at 8 pm on the 10 day of patency. The implications are not always fully appreciated. A single blood examination may be unreliable and single daiiy examinations may not always reflect the true course of the asexual parasitemia. This point should be remembered in an assessment of the parasitological response to treatment. A rise in the parasite count 24 hours after the start of treatment does not necessarily mean that the drug is slow acting; it may express a normal behavior. ', tooo\o 4o A -4 0... - b. ... .1. Pi i L. . o-4 o o i~~~~~~ o o -37 ,1 4, c> .rL h So- P- E F4 · . i 7 10 DAYS OF 'fléTír4Cy Fig,ll. RAPID CIL.;NGES IN PAiRAS]SITF'IA: PRIM'EiRY ATTACK OF B,OOD)--INDUCED FALCIPARU INF'E;CION (Case 8) 120 -4 p hÁ\ Go-0 bi-' -40 hr : h So i. -39 P. ,-, o 60 D) -3 c c) o( o r- tI 40. -37 k +, 4, .45 ' i+ ¿~ 20 io 63 e fE Pi J I o 'E-E-¡c O P( 7 e LIpYS Fe-, EN .y DA-? ,F OFAI Ecy FJig,.2 RAPID Ci:3...,S IIl PARASI'E;.iIMi: Fig. K- C.PIARYA;TTACK (Ca.I> I2 PRI1,',':- ±iI*hCK (Case 413) PPI!ARYAI'TT'ACK (Cas.- 32) Appendix E Organization and staff Personnel - One Medical Director, 1 Medical Assistant, one Chief Microscopist, two Microscopists, two Drivers, 1 Messenger-janitor (full time) and 1 Medical Consultant, 1 Office helper and 6 Hospital Attendants (part time). At the Santa Tereza Hospital, a ward was designated exclusively for the inoculated persons, who remained there during the pre- patent period after inoculation and during acute attacks of malaria. All the routine manipulations of the patients were done either in this ward or in a small room specially adapted for the purpose. Most of the specimens taken from the patients at the Santa Tereza Hospital were stained, examined, recorded and interpreted in the laboratory of the Center, located at the Zone IV Headquarters of the Malaria Campaign in the State of Sao Paulo. Personnel, materials, and equipment, were provided by the Malaria Campaign, in collaboration with the Pan American Health Organization.