CHLOROQUJNE-RESISTANT

PORTO
EDITH

D. BOX,* QUELLIN

PLASMODIUM
FALCIPARUM
VELHO, BRAZIL
T. BOX,t AND MARTIN

FROM

D. YOUNG@

Chioroquine has been an exceedingly useful

drug for eradicating the erythrocytic stages of
human malaria, thereby achieving temporary

scientific
meetingat Lima,Peru.From October
28toNovember2,before
returning
toGalveston,
theyvisited
PortoVetho.Malariawas common

clinical cure of infections with all species and

radical cure of Plasmodium falcipar-um infections.
Although resistance of human plasmodia to

in the area at the time, and they were liberally

pyrimethamine

exposed to mosquito bites both in town and

duringa 3-daystayata ranchoutside
ofPorto
Velho.Bothpatients
took225mg ofchloroquine
base each on October 27 and a week later on

and proguanil has been known

for many years in various parts of the world,

resistance of human plasmodia to chioroquine

was not indicated until 1961 when Moore and
Lather' described two cases of P. falcipar-um
originating in Colombia, South America, which
did not respond to the usual doses of chioroquine.

November

A strain derived from one of these cases was

further tested by inoculation of neurosyphilitic

patients and proved to be resistant, not only to

thought to be P. vivax, particularly

the usual doses of chioroquine,2 but also to other

These cases were in United States citizens whose

South American itinerary included only this one
malarious region. Chioroquine taken by these
patients was in the form of 250 mg tablets of the

on

when epi

sodes of chills and fever continued to occur after

courses of therapy considered adequate for radical
cure of P. falciparum. CEll experienced his first
chills and fever on November 12 and parasites
were foundin hisbloodfilmsat the time his
wife's
symptomswerediagnosed
asbeingdue to
malaria.
A summary oftheexperiences
ofthese
two patients
with malariaand the treatment
administered
fromNovemberthroughJanuaryis
giveninTable1.Thisinformation
was obtained

4-arninoquinolines.3 Later in 1961, Rodrigues4

observed two cases of P. falciparum in workers
employed in construction of the Brazilia-Acre
Road whose infections were not eradicated by
curative doses of chioroquine. Although the
workers lived in Sao Paulo on the east coast of
Brazil, both became ill of malaria in Porto Velho
near the Bolivian border late in 1960 and had
returned to Sao Paulo for treatment.
The following report documents the resistance
of two cases of P. fat ciparum apparently acquired
in the vicinity of Porto Vetho, Brazil, in 1961.

4. They returned to Galveston

November 6. OH first experienced chills and

fever on November 9 and was hospitalized on
November 15. Malaria was diagnosed on Novem
ber 18 in the hospital hematology laboratory and
chioroquine was administered. The malaria was

from the patients and from the hospital records

of OH.

The unusual aspects of these cases came to our

attention when we borrowed blood films from the
patients prepared by the Clinical Pathological
Laboratories in November. These slides were
used for demonstration
of current cases of
parasitic infections in a parasitology course for

diphosphate salt each of which contained 150 mg

medical students. They showed what appeared

of the base. Chloroquine dosages are expressed in

to be ring stages of P. falcipar'um rather than P.

terms of the base throughout the text.

In October of 1961, a faculty member, CEll,

vivax. When these patients were contacted on

February 1, 1962, they had just taken, on the

and his wife, OH, from the University of Texas

Medical Branch, Galveston, Texas, attended a

day before, a single dose of 45 mg of primaquine

and 150 mg of chioroquine. Blood films, taken
on February 1 and 2 after this combined dose of

* Department

of

Microbiology,

University

of

drugs, showed gametocytes of P. fakipar-urn in

tDepartment of l@'ediatrics,
Universityof the blood of both patients.
Texas Medical Branch Galveston, Texas.
In view of the history of repeated attacks of
Department of health, Education, and
Welfare, Public Health Service, Laboratory of malaria in both patients with repeated treatment
Parasite Chemotherapy, National Institute of which should have eradicated the usual P.
Texas Medical Branch Galveston, Texas.

Allergy and Infectious Diseases, National

stitutes of Health, Bethesda, Maryland.

In

falciparum infection, it was considered probable

300

CHLOROQUINE-RESISTANTP. FALCIPARUM FROM BRAZIL

301

TABLE 1
Malarial attacks and therapy before evaluation for chloroquine resistance
baseOH
DateMalarial

and dosage

attacks and symptomsDrugs

1981

10-27
10-28 through 11-2
11-4
11-9
11-18
12-2 through 12-15

mg chioroquine
to malaria)
225 mg chloroquine
1 Chills & fever
Parasites found in blood

1.5 g chioroquine in 3 days

15 mg primaquine/day X 14 days +

150 mg chloroquine/wk
12-25
1962
1-21
131(Exposed
roquineCEH

X 2

2 Chills & fever

1.5 g chloroquine in 3 days

3 Chills & fever225

900 mg chloroquine
45 mg primaquine

in 1 day
+ 150 mg chlo

1961

mg chloroquine

10-27
10-28 through 11-2
11-4
11-12
11-18

to malaria)

225 mg chloroquine
Chills & fever
1 Parasites found

1.5 g chloroquine in 3 days

15 mg primaquine/day X 14 days +

11-25 through 12-8

150 mg chloroquine/wk X 2
12-21
1962
1-31(Exposed

1.5 g chloroquine in 3 days

2 Chills & fever225

45 mg primaquine
roquine

that the strain was chloroquine-resistant. (At

this time we were unaware of the report of two
similar cases from this area by Rodriqu&). In
reports of drug resistance, it should be considered

possible that for some reason the drug is not

reaching the blood and fixed tissues of the patient,
i.e, the drug is not taken by the patient as pre
scribed or that the individual has a physiological
idiosyncrasy which prevents the drug from reach
ing the blood and tissues in adequate levels.
Therefore, our procedure consisted of determining

determinations

were made

150 mg chlo

by the

method

of

Brodie et al.6
The patients very kindly agreed to cooperate
in the study as outlined, and, since they were
continuing with their normal activities, it was

designed to cause them as little inconvenience as

possible.
RESULTS

The results of this study are summarized in

Table 2. OH had an episode of chills and fever on

parasitemias associated with chioroquine adminis

February

tration and ascertaining the presence of resultant

approximately

chioroquine in the blood and urine. Parasitemias

were estimated by counting the number of para

samples were obtained as soon as possible after

sites per 200 or more leucocytes

quine treatment totaling 2.1 g was started im

mediately. The plasma level of chioroquine was

in a Giemsa

stained thick film. Presence or absence of chioro

quine in the urine was determined by the Haskins'
tests.6 To determine chioroquine levels, plasma
or sera were obtained, deep frozen, and sent to
the National Institutes of Health where the

12 associated with a parasitemia

of

5,400 per cmm. Blood and urine

the blood smear on that day and aS day chioro

47 pg/L before treatment and 436 @sg/L24 hours

after the first dose. Her temperature, which had

been 101.5F orally, fell to normal by the next
day, and by 72 hours after the first dose of the

302

BOX, BOX, AND YOUNG

Parasitemia
@

TABLE 2
and chloroquine levels during evaluation for drug resistance

no. asexual
Date (1962)AttaCkParasiteinia;
forms per cmm
taken)OH
bloodSymptomsChloro@uine

2-1
2-12

5406

2-13
2-14
245
246
2-17
3-2
3-5
3-9
3-10
3-12
3-14
3-15

and fever

6678

levels
ime

blood

(5 p.m.)
436 (5 p.m.)

101
0
0
0
0
>0
20
10
60

300 (1 p.m.)

230

g bus

test
(time
sampledflChloroquine(time
@Haskins'

(5 p.m.)
0.3
0.3
0.3
0.3
0.3

+
+
+
+
+
+
+
+
+
+

(6 p.m.)
(12p.m.)
(6 p.m.)
(6 p.m.)
(6 p.m.)
(6 p.m.)

5(G)t 2250

Chills and fever

42 (11 a.m.)+

+0.6

Quinine

(3 p.m.)

1.8 g/day
+0.6

gCEH
3-16

400
0
0Chills

3-20
5-23
12-194

2-1
2-12
2-14
2-16
2-19

X 10

None
None47

1296
973
805
1349 (G)

(2 p.m.)

(2 p.m.)

(5 p.m.)

0.45 (11 p.m.)

2-20

234

221

2-22
2-23
2-24
6-1
12-193(G)t
*

(G)

392 (2 p.m.

0
0 (G)
0 (G)
0 (G)
0None54

gametocytes

415 (12 N)

+
+
+
+
+0.9

0.45 (5 p.m.)

0.45 (5 p.m.)
0.45 (5 p.m.)
0.45 (5 p.m.)

present.

t Thin ifim.
+ = positive for chioroquine.

drug, parasites could not be found on a thick

film. Blood smears taken 3 times a week after
the

chioroquine

was

discontinued

remained

negative until March 5, when a single parasite

was found. The parasitemia gradually increased
until 2,250 parasites per cmm were estimated to

was 42 pg per liter. She was then placed on a 10

day treatment regimen of 1.8 g of quinine sul
fate daily. The day after quinine was begun, her

parasitemia had declined to 400 per cmm. A

total of 8 thick blood films spaced approximately

be present on March 15. At this time chills and

a week apart during the 8 weeks subsequent to

treatment were negative for parasites. To date

fever recurred. The plasma level of chloroquine

(December 19) she has had no recurrence of

303

CULOROQUINE-RESISTANT P. FALCIPARUM FROM BRAZIL

symptoms. Daily urine samples were consistently

and definitely positive by the Haskins' test from
the February relapse through the March relapse.
CEH had a parasitemia of approximately 1,000
rings per cmm from February 12 through Febru
ary 19 without appreciable symptoms other

weight was approximately 56 kg, this amount is

equivalent on a body weight basis to 37.5 mg/kg,
or almost double the 21 mg/kg recommended for
eradication of P. fakiparum in a 70 K subject
(1.5 g in 3 days). Chioroquine blood levels of OH

than night sweats. It was his preference to be

pg/L) were higher than the average of those

reported by Coatney et at.9 for individuals re

treated without waiting for a clinical relapse to

occur. His dose of chioroquine was adjusted so
that he would receive an amount roughiy com
parable on a body weight basis to that taken by

OH. By 72 hours after the initiation of this course

of chloroquine, his thick smears became negative
for parasites except for gametocytes which were

present for about 10 days after completion of

treatment. Blood smears taken two or three
times per week for a month after therapy and
once a week for the next 8 weeks were negative
for parasites. The Haskins' test became com
pletely negative between 7 and 9 weeks after his

last dose of chloroquine. Citrated

blood from

patient OH on February 12 and March 15, and

from patient CEH on February 19 was frozen in
a dry ice-acetone bath and forwarded to the
National Institutes of Health for further study.

during the treatment period (436 pg/L and 300

ceiving a total of 2.8 g base of the drug over a

6-day period. This indicates adequate absorption
of chioroquine by OH. The blood levels observed
for OH during the treatment period were also
several times the levels found by Earle et at.@to
eradicate P. fakiparum infections.

CEll, whose weight was 80 K, took a total of

3.15 g chioroquine base over a 5-day period during

his third attack. This amounted to approximately

39 mg/kg and eradicated his infection. Young
and Moore2 reported that one of the neuro

syphilitics experimentally
chioroquine-resistant

inoculated

Colombian

with the

strain

of P.

falciparum was cured of the infection by a total

In August this blood was injected intravenously

into two volunteers, but failed to produce an

amount of drug only slightly higher on a body

weight basis (41.6 mg/kg).
The fact that the P. fakiparum infections
reported above apparently were acquired in the
same place and at the same time by two in

infection.

dividuals in both of whom the infection responded

DISCUSSION

It appears that the P. fatcipar-um strain en

countered in these patients was relatively resist
ant to chioroquine. During the period of approxi
mately 3 months immediately preceding the

beginning of our study, OH had taken a total of

4.8 g chioroquine base and CEH 3.9 g without
elimination

of the infections;

these totals

in

chided two each of the usual 1.5 g regimens.

Jeffery et at.7 found experimentally that one

abnormally to chloroquine makes the case for

chioroquine resistance especially strong. The
presence of such a strain in western Brazil is
reinforced by the report of Rodriques4 since the
two cases described by him were working in the

Porto Vetho area when they became ill.

SUMMARY

Two cases of Plasinodiumfaki par-urninfections,

apparently originating in Porto Vetho, Brazil,
which were not eliminated by the usual curative

course of 1.5 g eradicated P. fakiparum in all

but one of 87 neurosyphilitic patients. Lower

doses of chloroquine are documented. One patient

doses will eradicate the parasite in many cases,

including four regimens varying from 0.9 to 2.1

as summarized by Young and Moore.2 A second

indication of resistance in the strain studied here
is that both patients had relapses with the para

grams each, without

sites reaching a density of a thousand or more

per cmm in the blood when chioroquine blood

3.675 grams chioroquine base, including two

courses of 1.5 grams each, without eradicating

levels were still approximately

50 pg/L.

Earle

et aL8 reported that plasma levels averaging 30

pg/L

for 6 days

eradicated

falciparum infections. Thirdly,

experimental

P.

parasitemia re

curred in OH after administration of a 5-day

regimen of 2.1 g of chloroquine base. Since her

received a total of 6.675 grams chioroquine base,

eradicating

the parasites.

She was cured subsequently by a 10-day quinine

treatment. The second patient received a total of

the infection. He was cured by a 5-day treatment

totaling 3.1 grams of chioroquine. This study

confirms the presence of chioroquine-resistant

P.

fakiparum in Porto Vetho, Brazil, the second

area in South America where chioroquine-re
sistant strains have been reported.

304

BOX, BOX, AND YOUNG

mento

ACKNOWLEDGMENTS

Appreciation is expressed to Dr. Leonard

Friedman and Dr. Richard Jacobs of the Labora
tory of Parasite Chemotherapy, National Insti

tutes of Health, for determining the chioroquine

levels. We also wish to acknowledge the valuable

aid and support of Dr. Haig Najarian of the

Department of Microbiology of the University
Texas Medical Branch during this study.

of

1. MooRE, D. V. AND LANIER, J. E., 1961. Ob

on two

infections

with an abnormal

Plasmodium

Arq. Hig.

falciparum

response

to

chloroquine. Am. .1. Trop. Med. & Hyg., 10:

59.
2. YOUNG,M. D. ANDMOORE,D. V., 1961. Chloro
quine resistance in Plasmodium falciparum.
Am. 1. Trop. Med. & Hyg.1 10: 317320.

3. YOUNG,M. D., 1961. Amodiaquine and hy

Saude

5. HASKINS, W. T., 1958. A simple qualitative

test for chloroquine in urine. Am. J. Trop.

Med. & Hyg., 7: 199-200.

6. BRODIE,B. B. UDENFRIEND,S., DILL, W. AND

CHENKIN, 1@., 1947. The estimation
organic compounds
in biological

of Iasic
material.

III. Estimation by conversion to fluorescent

compounds. J. Biol. Chem., 168: 319-325.
7. JEFFERY, G. M. YOUNG, M. D. AND EYLES,
D. E., 1956. @i'hetreatment o! Plasmodium
falciparum infection with chloroquine, with
a note on infectivity

REFERENCES
servations

pela chloroquina.

Pub., 26: 231-235.

to mosq@uitoes of prima

quine-pyrimethamine-treated cases. Am. .1.

Hyg., 64: 111.
8. EARLE,D. P., JR., BERLINER,R. W. TAGGART,
J. V., ZUBROD,C. G., WELCH W'. S., BIGE
LOW, F. S., KENNEDY, T. J. JR., AND Saaw
NON

J. A.,

1948.

Studies

of the human malarias.

antimalarial
effect of

on the

chemotherapy

X. The suppressive
paludrine.
.1. Clin.

Invest., 27(No. 3, Part 2): 130-133.

9. COATNEY,G. R., RUHE, D. S., COOPERW. C.,

droxychioroquine resistance in Plasmodium

falciparum. Am. J. Trop. Med. & Hyg., 10:

JOSEPHSON E. S., AND YOUNG M. i3., 1949.

689693.

and therapeutic

4. RoDmouxs, D. C., 1961. Casos de malaria por

Plasmodium falciparum resistentes ao trata

Studies in human malaria. X.

7618) against

action

@heprotective

of chloroquine

St. Elizabeth

strain

malaria. Am. .1. Hyg., 49: 49-59.

(SN

vivax